Structural evolution drives diversification of the large LRR-RLK gene family

Cells are continuously exposed to chemical signals that they must discriminate between and respond to appropriately. In embryophytes, the leucine‐rich repeat receptor‐like kinases (LRR‐RLKs) are signal receptors critical in development and defense. LRR‐RLKs have diversified to hundreds of genes in many plant genomes. Although intensively studied, a well‐resolved LRR‐RLK gene tree has remained elusive. To resolve the LRR‐RLK gene tree, we developed an improved gene discovery method based on iterative hidden Markov model searching and phylogenetic inference. We used this method to infer complete gene trees for each of the LRR‐RLK subclades and reconstructed the deepest nodes of the full gene family. We discovered that the LRR‐RLK gene family is even larger than previously thought, and that protein domain gains and losses are prevalent. These structural modifications, some of which likely predate embryophyte diversification, led to misclassification of some LRR‐RLK variants as members of other gene families. Our work corrects this misclassification. Our results reveal ongoing structural evolution generating novel LRR‐RLK genes. These new genes are raw material for the diversification of signaling in development and defense. Our methods also enable phylogenetic reconstruction in any large gene family

Comparative studies on the pathogenicity and tissue distribution of three virulence variants of classical swine fever virus, two field isolates and one vaccine strain, with special regard to immunohistochemical investigations

<p>Abstract</p> <p>Background</p> <p>The aim of this study was to compare the tissue distribution and pathogenicity of three virulence variants of classical swine fever virus (CSFV) and to investigate the applicability of various conventional diagnostic procedures.</p> <p>Methods</p> <p>64 pigs were divided into three groups and infected with the highly virulent isolate ISS/60, the moderately virulent isolate Wingene'93 and the live attenuated vaccine strain Riems, respectively. Clinical signs, gross and histopathological changes were compared in relation to time elapsed post infection. Virus spread in various organs was followed by virus isolation, by immunohistochemistry, applying monoclonal antibodies in a two-step method and by <it>in situ </it>hybridisation using a digoxigenin-labelled riboprobe.</p> <p>Results</p> <p>The tissue distribution data are discussed in details, analyzing the results of the various diagnostic approaches. The comparative studies revealed remarkable differences in the onset of clinical signs as well as in the development of the macro- and microscopical changes, and in the tissue distribution of CSFV in the three experimental groups.</p> <p>Conclusion</p> <p>The present study demonstrates that in the case of highly and moderately virulent virus variants the virulence does not affect the pattern of the viral spread, however, it influences the outcome, the duration and the intensity of the disease. Immunohistochemistry has the advantage to allow the rapid detection and localisation of the virus, especially in cases of early infection, when clinical signs are still absent. Compared to virus isolation, the advantage of this method is that no cell culture facilities are required. Thus, immunohistochemistry provides simple and sensitive tools for the prompt detection of newly emerging variants of CSFV, including the viruses of very mild virulence.</p

Random Matrix Theory for the Hermitian Wilson Dirac Operator and the chGUE-GUE Transition

We introduce a random two-matrix model interpolating between a chiral Hermitian (2n+nu)x(2n+nu) matrix and a second Hermitian matrix without symmetries. These are taken from the chiral Gaussian Unitary Ensemble (chGUE) and Gaussian Unitary Ensemble (GUE), respectively. In the microscopic large-n limit in the vicinity of the chGUE (which we denote by weakly non-chiral limit) this theory is in one to one correspondence to the partition function of Wilson chiral perturbation theory in the epsilon regime, such as the related two matrix-model previously introduced in refs. [20,21]. For a generic number of flavours and rectangular block matrices in the chGUE part we derive an eigenvalue representation for the partition function displaying a Pfaffian structure. In the quenched case with nu=0,1 we derive all spectral correlations functions in our model for finite-n, given in terms of skew-orthogonal polynomials. The latter are expressed as Gaussian integrals over standard Laguerre polynomials. In the weakly non-chiral microscopic limit this yields all corresponding quenched eigenvalue correlation functions of the Hermitian Wilson operator.Comment: 27 pages, 4 figures; v2 typos corrected, published versio

Finite Element Analysis of Osteosynthesis Screw Fixation in the Bone Stock: An Appropriate Method for Automatic Screw Modelling

The use of finite element analysis (FEA) has grown to a more and more important method in the field of biomedical engineering and biomechanics. Although increased computational performance allows new ways to generate more complex biomechanical models, in the area of orthopaedic surgery, solid modelling of screws and drill holes represent a limitation of their use for individual cases and an increase of computational costs. To cope with these requirements, different methods for numerical screw modelling have therefore been investigated to improve its application diversity. Exemplarily, fixation was performed for stabilization of a large segmental femoral bone defect by an osteosynthesis plate. Three different numerical modelling techniques for implant fixation were used in this study, i.e. without screw modelling, screws as solid elements as well as screws as structural elements. The latter one offers the possibility to implement automatically generated screws with variable geometry on arbitrary FE models. Structural screws were parametrically generated by a Python script for the automatic generation in the FE-software Abaqus/CAE on both a tetrahedral and a hexahedral meshed femur. Accuracy of the FE models was confirmed by experimental testing using a composite femur with a segmental defect and an identical osteosynthesis plate for primary stabilisation with titanium screws. Both deflection of the femoral head and the gap alteration were measured with an optical measuring system with an accuracy of approximately 3 µm. For both screw modelling techniques a sufficient correlation of approximately 95% between numerical and experimental analysis was found. Furthermore, using structural elements for screw modelling the computational time could be reduced by 85% using hexahedral elements instead of tetrahedral elements for femur meshing. The automatically generated screw modelling offers a realistic simulation of the osteosynthesis fixation with screws in the adjacent bone stock and can be used for further investigations

Capacitative calcium influx and proliferation of human osteoblastic-like MG-63 cells

Adult bone tissue is continuously being remodelled and bone mass is maintained by a balance between osteoclastic bone resorption and osteoblastic bone formation. Alteration of osteoblastic cell proliferation may account in part for lack of balance between these two processes in bone loss of osteoporosis. There is calcium (Ca2+) control in numerous cellular functions; however, involvement of capacitative Ca2+ entry (CCE) in proliferation of bone cells is less well investigated. OBJECTIVES: The study described here was aimed to investigate roles of CCE in the proliferation of osteoblast-like MG-63 cells. MATERIALS AND METHODS: Pharmacological characterizations of CCE were undertaken in parallel, with evaluation of the expression of transient receptor potential canonical (TRPC) channels and of cell proliferation. RESULTS: Intracellular Ca2+ store depletion by thapsigargin induced CCE in MG-63 cells; this was characterized by a rapid transient increase of intracellular Ca2+ followed by significant CCE, induced by conditions that stimulated cell proliferation, namely serum and platelet-derived growth factor. Inhibitors of store-operated Ca2+ channels (2-APB and SKF-96365) prevented CCE, while voltage-dependent Ca2+ channel blockers had no effect. Expression of various TRPC channels was shown in the cells, some having been shown to be responsible for CCE. Voltage-dependent Ca2+ channel blockers had no effect on osteoblast proliferation while thapsigargin, 2-APB and SKF-96395, inhibited it. Cell cycle analysis showed that 2-APB and SKF-96395 lengthen the S and G2/M phases, which would account for the reduction in cell proliferation. CONCLUSIONS: Our results indicate that CCE, likely attributed to the activation of TRPCs, might be the main route for Ca2+ influx involved in osteoblast proliferation

Modelling the Evolution and Spread of HIV Immune Escape Mutants

During infection with human immunodeficiency virus (HIV), immune pressure from cytotoxic T-lymphocytes (CTLs) selects for viral mutants that confer escape from CTL recognition. These escape variants can be transmitted between individuals where, depending upon their cost to viral fitness and the CTL responses made by the recipient, they may revert. The rates of within-host evolution and their concordant impact upon the rate of spread of escape mutants at the population level are uncertain. Here we present a mathematical model of within-host evolution of escape mutants, transmission of these variants between hosts and subsequent reversion in new hosts. The model is an extension of the well-known SI model of disease transmission and includes three further parameters that describe host immunogenetic heterogeneity and rates of within host viral evolution. We use the model to explain why some escape mutants appear to have stable prevalence whilst others are spreading through the population. Further, we use it to compare diverse datasets on CTL escape, highlighting where different sources agree or disagree on within-host evolutionary rates. The several dozen CTL epitopes we survey from HIV-1 gag, RT and nef reveal a relatively sedate rate of evolution with average rates of escape measured in years and reversion in decades. For many epitopes in HIV, occasional rapid within-host evolution is not reflected in fast evolution at the population level

Investigation of Gamma-aminobutyric acid (GABA) A receptors genes and migraine susceptibility

Background Migraine is a neurological disorder characterized by recurrent attacks of severe headache, affecting around 12% of Caucasian populations. It is well known that migraine has a strong genetic component, although the number and type of genes involved is still unclear. Prior linkage studies have reported mapping of a migraine gene to chromosome Xq 24–28, a region containing a cluster of genes for GABA A receptors (GABRE, GABRA3, GABRQ), which are potential candidate genes for migraine. The GABA neurotransmitter has been implicated in migraine pathophysiology previously; however its exact role has not yet been established, although GABA receptors agonists have been the target of therapeutic developments. The aim of the present research is to investigate the role of the potential candidate genes reported on chromosome Xq 24–28 region in migraine susceptibility. In this study, we have focused on the subunit GABA A receptors type ε (GABRE) and type θ (GABRQ) genes and their involvement in migraine. Methods We have performed an association analysis in a large population of case-controls (275 unrelated Caucasian migraineurs versus 275 controls) examining a set of 3 single nucleotide polymorphisms (SNPs) in the coding region (exons 3, 5 and 9) of the GABRE gene and also the I478F coding variant of the GABRQ gene. Results Our study did not show any association between the examined SNPs in our test population (P > 0.05). Conclusion Although these particular GABA receptor genes did not show positive association, further studies are necessary to consider the role of other GABA receptor genes in migraine susceptibility

A Common CNR1 (Cannabinoid Receptor 1) Haplotype Attenuates the Decrease in HDL Cholesterol That Typically Accompanies Weight Gain

We have previously shown that genetic variability in CNR1 is associated with low HDL dyslipidemia in a multigenerational obesity study cohort of Northern European descent (209 families, median  = 10 individuals per pedigree). In order to assess the impact of CNR1 variability on the development of dyslipidemia in the community, we genotyped this locus in all subjects with class III obesity (body mass index >40 kg/m2) participating in a population-based biobank of similar ancestry. Twenty-two haplotype tagging SNPs, capturing the entire CNR1 gene locus plus 15 kb upstream and 5 kb downstream, were genotyped and tested for association with clinical lipid data. This biobank contains data from 645 morbidly obese study subjects. In these subjects, a common CNR1 haplotype (H3, frequency 21.1%) is associated with fasting TG and HDL cholesterol levels (p = 0.031 for logTG; p = 0.038 for HDL-C; p = 0.00376 for log[TG/HDL-C]). The strength of this relationship increases when the data are adjusted for age, gender, body mass index, diet and physical activity. Mean TG levels were 160±70, 155±70, and 120±60 mg/dL for subjects with 0, 1, and 2 copies of the H3 haplotype. Mean HDL-C levels were 45±10, 47±10, and 48±9 mg/dL, respectively. The H3 CNR1 haplotype appears to exert a protective effect against development of obesity-related dyslipidemia

A Platform-Independent Method for Detecting Errors in Metagenomic Sequencing Data: DRISEE

We provide a novel method, DRISEE (duplicate read inferred sequencing error estimation), to assess sequencing quality (alternatively referred to as “noise” or “error”) within and/or between sequencing samples. DRISEE provides positional error estimates that can be used to inform read trimming within a sample. It also provides global (whole sample) error estimates that can be used to identify samples with high or varying levels of sequencing error that may confound downstream analyses, particularly in the case of studies that utilize data from multiple sequencing samples. For shotgun metagenomic data, we believe that DRISEE provides estimates of sequencing error that are more accurate and less constrained by technical limitations than existing methods that rely on reference genomes or the use of scores (e.g. Phred). Here, DRISEE is applied to (non amplicon) data sets from both the 454 and Illumina platforms. The DRISEE error estimate is obtained by analyzing sets of artifactual duplicate reads (ADRs), a known by-product of both sequencing platforms. We present DRISEE as an open-source, platform-independent method to assess sequencing error in shotgun metagenomic data, and utilize it to discover previously uncharacterized error in de novo sequence data from the 454 and Illumina sequencing platforms