A species delimitation approach in the Trochulus sericeus/hispidus complex reveals two cryptic species within a sharp contact zone.

BACKGROUND: Mitochondrial DNA sequencing increasingly results in the recognition of genetically divergent, but morphologically cryptic lineages. Species delimitation approaches that rely on multiple lines of evidence in areas of co-occurrence are particularly powerful to infer their specific status. We investigated the species boundaries of two cryptic lineages of the land snail genus Trochulus in a contact zone, using mitochondrial and nuclear DNA marker as well as shell morphometrics. RESULTS: Both mitochondrial lineages have a distinct geographical distribution with a small zone of co-occurrence. In the same area, we detected two nuclear genotype clusters, each being highly significantly associated to one mitochondrial lineage. This association however had exceptions: a small number of individuals in the contact zone showed intermediate genotypes (4%) or cytonuclear disequilibrium (12%). Both mitochondrial lineage and nuclear cluster were statistically significant predictors for the shell shape indicating morphological divergence. Nevertheless, the lineage morphospaces largely overlapped (low posterior classification success rate of 69% and 78%, respectively): the two lineages are truly cryptic. CONCLUSION: The integrative approach using multiple lines of evidence supported the hypothesis that the investigated Trochulus lineages are reproductively isolated species. In the small contact area, however, the lineages hybridise to a limited extent. This detection of a hybrid zone adds an instance to the rare reported cases of hybridisation in land snails.

PPARgamma but not PPARalpha ligands are potent repressors of major histocompatibility complex class II induction in atheroma-associated cells.

Peroxisome proliferator-activated receptors (PPARs) are essential in glucose and lipid metabolism and are implicated in metabolic disorders predisposing to atherosclerosis, such as diabetes and dyslipidemia. Conversely, antidiabetic glitazones and hypolipidemic fibrate drugs, known as PPARgamma and PPARalpha ligands, respectively, reduce the process of atherosclerotic lesion formation, which involves chronic immunoinflammatory processes. Major histocompatibility complex class II (MHC-II) molecules, expressed on the surface of specialized cells, are directly involved in the activation of T lymphocytes and in the control of the immune response. Interestingly, expression of MHC-II has recently been observed in atherosclerotic plaques, and it can be induced by the proinflammatory cytokine interferon-gamma (IFN-gamma) in vascular cells. To explore a possible role for PPAR ligands in the regulation of the immune response, we investigated whether PPAR activation affects MHC-II expression in atheroma-associated cells. In the present study, we demonstrate that PPARgamma but not PPARalpha ligands act as inhibitors of IFN-gamma-induced MHC-II expression and thus as repressors of MHC-II-mediated T-cell activation. All different types of PPARgamma ligands tested inhibit MHC-II. This effect of PPARgamma ligands is due to a specific inhibition of promoter IV of CIITA and does not concern constitutive expression of MHC-II. Thus, the beneficial effects of antidiabetic PPARgamma activators on atherosclerotic plaque development may be partly explained by their repression of MHC-II expression and subsequent inhibition of T-lymphocyte activation.

Modeling of the TCR-MHC-peptide complex.

The methodology for generating a homology model of the T1 TCR-PbCS-K(d) class I major histocompatibility complex (MHC) class I complex is presented. The resulting model provides a qualitative explanation of the effect of over 50 different mutations in the region of the complementarity determining region (CDR) loops of the T cell receptor (TCR), the peptide and the MHC's alpha(1)/alpha(2) helices. The peptide is modified by an azido benzoic acid photoreactive group, which is part of the epitope recognized by the TCR. The construction of the model makes use of closely related homologs (the A6 TCR-Tax-HLA A2 complex, the 2C TCR, the 14.3.d TCR Vbeta chain, the 1934.4 TCR Valpha chain, and the H-2 K(b)-ovalbumine peptide), ab initio sampling of CDR loops conformations and experimental data to select from the set of possibilities. The model shows a complex arrangement of the CDR3alpha, CDR1beta, CDR2beta and CDR3beta loops that leads to the highly specific recognition of the photoreactive group. The protocol can be applied systematically to a series of related sequences, permitting the analysis at the structural level of the large TCR repertoire specific for a given peptide-MHC complex.

Hybridization and Population Structure of the Culex Pipiens Complex in the Islands of Macaronesia

The Culex pipiens complex includes two widespread mosquito vector species, Cx. pipiens and Cx. quinquefasciatus. The distribution of these species varies in latitude, with the former being present in temperate regions and the latter in tropical and subtropical regions. However, their distribution range overlaps in certain areas and interspecific hybridization has been documented. Genetic introgression between these species may have epidemiological repercussions for West Nile virus (WNV) transmission. Bayesian clustering analysis based on multilocus genotypes of 12 microsatellites was used to determine levels of hybridization between these two species in Macaronesian islands, the only contact zone described in West Africa. The distribution of the two species reflects both the islands’ biogeography and historical aspects of human colonization. Madeira Island displayed a homogenous population of Cx. pipiens, whereas Cape Verde showed a more intriguing scenario with extensive hybridization. In the islands of Brava and Santiago, only Cx. quinquefasciatus was found, while in Fogo and Maio high hybrid rates (~40%) between the two species were detected. Within the admixed populations, second-generation hybrids (~50%) were identified suggesting a lack of isolation mechanisms. The observed levels of hybridization may locally potentiate the transmission to humans of zoonotic arboviruses such as WNV.

Identification of naturally processed hepatitis C virus-derived major histocompatibility complex class I ligands.

Fine mapping of human cytotoxic T lymphocyte (CTL) responses against hepatitis C virus (HCV) is based on external loading of target cells with synthetic peptides which are either derived from prediction algorithms or from overlapping peptide libraries. These strategies do not address putative host and viral mechanisms which may alter processing as well as presentation of CTL epitopes. Therefore, the aim of this proof-of-concept study was to identify naturally processed HCV-derived major histocompatibility complex (MHC) class I ligands. To this end, continuous human cell lines were engineered to inducibly express HCV proteins and to constitutively express high levels of functional HLA-A2. These cell lines were recognized in an HLA-A2-restricted manner by HCV-specific CTLs. Ligands eluted from HLA-A2 molecules isolated from large-scale cultures of these cell lines were separated by high performance liquid chromatography and further analyzed by electrospray ionization quadrupole time of flight mass spectrometry (MS)/tandem MS. These analyses allowed the identification of two HLA-A2-restricted epitopes derived from HCV nonstructural proteins (NS) 3 and 5B (NS3₁₄₀₆₋₁₄₁₅ and NS5B₂₅₉₄₋₂₆₀₂). In conclusion, we describe a general strategy that may be useful to investigate HCV pathogenesis and may contribute to the development of preventive and therapeutic vaccines in the future.

The inflammasome, autoinflammatory diseases, and gout.

IL-1beta is a cytokine with major roles in inflammation and innate immune responses. IL-1beta is produced as an inactive proform that must be cleaved within the cell to generate biologically active IL-1beta. The enzyme caspase-1 catalyzes the reaction. Recent work showed that caspase-1 must be activated by a complex known as the inflammasome. The inflammasome comprises NALP, which is an intracellular receptor involved in innate immunity, and an ASC adapter that ensures caspase-1 recruitment to the receptor. The most extensively described inflammasome to date is formed by the NALP3 receptor within monocytes. Mutations involving the NALP3 gene cause hereditary periodic fever syndromes in humans. Increased inflammasome activity responsible for uncontrolled IL-1beta production occurs in these syndromes. Inhibition of the IL-1beta pathway by IL-1 receptor antagonist (anakinra) is a highly effective treatment for inherited periodic fever syndromes. A major role for inflammasome activity in the development of gout attacks was established recently. Urate monosodium crystals are specifically detected via the NALP3 inflammasome, which results in marked IL-1beta overproduction and initiation of an inflammatory response. This finding opens up new possibilities for the management of gouty attacks.

Purification and ligand binding of a soluble class I major histocompatibility complex molecule consisting of the first three domains of H-2Kd fused to beta 2-microglobulin expressed in the baculovirus-insect cell system.

A recombinant baculovirus encoding a single-chain murine major histocompatibility complex class I molecule in which the first three domains of H-2Kd are fused to beta 2-microglobulin (beta 2-m) via a 15-amino acid linker has been isolated and used to infect lepidopteran cells. A soluble, 391-amino acid single-chain H-2Kd (SC-Kd) molecule of 48 kDa was synthesized and glycosylated in insect cells and could be purified in the absence of detergents by affinity chromatography using the anti-H-2Kd monoclonal antibody SF1.1.1.1. We tested the ability of SC-Kd to bind antigenic peptides using a direct binding assay based on photoaffinity labeling. The photoreactive derivative was prepared from the H-2Kd-restricted Plasmodium berghei circumsporozoite protein (P.b. CS) peptide 253-260 (YIPSAEKI), a probe that we had previously shown to be unable to bind to the H-2Kd heavy chain in infected cells in the absence of co-expressed beta 2-microglobulin. SC-Kd expressed in insect cells did not require additional mouse beta 2-m to bind the photoprobe, indicating that the covalently attached beta 2-m could substitute for the free molecule. Similarly, binding of the P.b. CS photoaffinity probe to the purified SC-Kd molecule was unaffected by the addition of exogenous beta 2-m. This is in contrast to H-2KdQ10, a soluble H-2Kd molecule in which beta 2-m is noncovalently bound to the soluble heavy chain, whose ability to bind the photoaffinity probe is greatly enhanced in the presence of an excess of exogenous beta 2-m. The binding of the probe to SC-Kd was allele-specific, since labeling was selectively inhibited only by antigenic peptides known to be presented by the H-2Kd molecule.

Audit report on the Iowa Water Pollution Control Works Financing Program (Clean Water Program) and the Iowa Drinking Water Facilities Financing Program (Drinking Water Program), joint programs of the Iowa Finance Authority and the Iowa Department of Natural Resources, for the year ended June 30, 2006

Audit report on the Iowa Water Pollution Control Works Financing Program (Clean Water Program) and the Iowa Drinking Water Facilities Financing Program (Drinking Water Program), joint programs of the Iowa Finance Authority and the Iowa Department of Natural Resources, for the year ended June 30, 2006

Microcrystals As Damps And Their Role In Joint Inflammation

The concept of danger signals as important triggers of inflammation and immune activation has passed from fanciful hypothesis to a widely accepted biological process with receptors, signalling cascades and mediators. Products of cell death or cell stress are prime examples of DAMPs. They interact with receptors on the cell surface such as TLRs as well as cytoplasmic proteins such as the NLRs to modulate cellular metabolism and activation. Recently, the identification of the inflammasomes and their role in processing IL-1b and IL18 provided further insights into how DAMPs provoke inflammation. A class of substances that are potent activators of the inflammasome is microcrystals. All three microcrystals associated with joint disease in man : urate, CPP and hydroxyapatite require the NLRP3 inflammasome to process and release IL-1b from leucocytes. This mechanism most probably explain the inflammatory phase of acute crystal arthritis. However, microcrystals can also induce apoptosis, cell death as well as cell activation, depending on the cell type they are in contact with. These different cellular effects could well explain the role crystals can play in degenerative joint diseases, where inflammation is not as prominent. Our understanding of the intracellular pathways linking microcrystals to inflammation and cell activation is currently still very sketchy, and we hope that the detailed analysis of these pathways may lead to better comprehension and treatment of microcrystal induced joint diseases.Disclosures : The author has declared no conflicts of interest.

Need of reduced and harmonized bureaucracy in multi-centre clinical research - a case-report from a Swiss trial

Introduction: We launched an investigator-initiated study (ISRCTN31181395) to evaluate the potential benefit of pharmacokinetic-guided dosage individualization of imatinib for leukaemiapatients followed in public and private sectors. Following approval by the research ethics committee (REC) of the coordinating centre, recruitment throughout Switzerland necessitatedto submit the protocol to 11 cantonal RECs.Materials and Methods: We analysed requirements and evaluation procedures of the 12 RECs with associated costs.Results: 1-18 copies of the dossier, in total 4300 printed pages, were required (printing/posting costs: ~300 CHF) to meet initial requirements. Meeting frequencies of RECs ranged between 2 weeks and 2 months, time from submission to fi rst feedback took 2-75 days. Study approval was obtained from a chairman, a subor the full committee, the evaluation work being invoiced by0-1000 CHF (median: 750 CHF, total: 9200 CHF). While 5 RECs gave immediate approval, the other 6 rose in total 38 queries before study release, mainly related to wording in the patient information, leading to 7 different fi nal versions approved. Submission tasks employed an investigator half-time over about 6 months.Conclusion: While the necessity of clinical research evaluation by independent RECs is undisputed, there is a need of further harmonization and cooperation in evaluation procedures. Current administrative burden is indeed complex, time-consuming and costly. A harmonized electronic application form, preferably compatible with other regulatory bodies and European countries, could increase transparency, improve communication, and encourage academic multi-centre clinical research in Switzerland.

Effective Shoulder Design and Maintenance, 2007

Granular shoulders are an important element of the transportation system and are constantly subjected to performance problems due to wind- and water-induced erosion, rutting, edge drop-off, and slope irregularities. Such problems can directly affect drivers’ safety and often require regular maintenance. The present research study was undertaken to investigate the factors contributing to these performance problems and to propose new ideas to design and maintain granular shoulders while keeping ownership costs low. This report includes observations made during a field reconnaissance study, findings from an effort to stabilize the granular and subgrade layer at six shoulder test sections, and the results of a laboratory box study where a shoulder section overlying a soft foundation layer was simulated. Based on the research described in this report, the following changes are proposed to the construction and maintenance methods for granular shoulders: • A minimum CBR value for the granular and subgrade layer should be selected to alleviate edge drop-off and rutting formation. • For those constructing new shoulder sections, the design charts provided in this report can be used as a rapid guide based on an allowable rut depth. The charts can also be used to predict the behavior of existing shoulders. • In the case of existing shoulder sections overlying soft foundations, the use of geogrid or fly ash stabilization proved to be an effective technique for mitigating shoulder rutting.

Recurrent dominant mutations affecting two adjacent residues in the motor domain of the monomeric kinesin KIF22 result in skeletal dysplasia and joint laxity.

Spondyloepimetaphyseal dysplasia with joint laxity, leptodactylic type (lepto-SEMDJL, aka SEMDJL, Hall type), is an autosomal dominant skeletal disorder that, in spite of being relatively common among skeletal dysplasias, has eluded molecular elucidation so far. We used whole-exome sequencing of five unrelated individuals with lepto-SEMDJL to identify mutations in KIF22 as the cause of this skeletal condition. Missense mutations affecting one of two adjacent amino acids in the motor domain of KIF22 were present in 20 familial cases from eight families and in 12 other sporadic cases. The skeletal and connective tissue phenotype produced by these specific mutations point to functions of KIF22 beyond those previously ascribed functions involving chromosome segregation. Although we have found Kif22 to be strongly upregulated at the growth plate, the precise pathogenetic mechanisms remain to be elucidated.

Research joint ventures in an international economy

D'Aspremont and Jacquemin's (1988) model is extended to studyalternative configurations of research agreements in a two--country integratedworld economy. Under unambiguous conditions on spillovers we show that:1) Allowing national firms to cooperate in R\&D confers them an advantageover foreign rivals, an effect similar to R\&D subsidies. 2) In a policygame, each government would allow national cooperative agreements. 3) Contraryto other trade policies which lead to a ``prisoners' dilemma'' result,welfare in both countries increases when they both allow R\&D cooperation.4) Welfare is even higher if a generalized (international) coalition isformed.

A new approach to accurate measurement of uniaxial joint angles based on a combination of accelerometers and gyroscopes.

A new method of measuring joint angle using a combination of accelerometers and gyroscopes is presented. The method proposes a minimal sensor configuration with one sensor module mounted on each segment. The model is based on estimating the acceleration of the joint center of rotation by placing a pair of virtual sensors on the adjacent segments at the center of rotation. In the proposed technique, joint angles are found without the need for integration, so absolute angles can be obtained which are free from any source of drift. The model considers anatomical aspects and is personalized for each subject prior to each measurement. The method was validated by measuring knee flexion-extension angles of eight subjects, walking at three different speeds, and comparing the results with a reference motion measurement system. The results are very close to those of the reference system presenting very small errors (rms = 1.3, mean = 0.2, SD = 1.1 deg) and excellent correlation coefficients (0.997). The algorithm is able to provide joint angles in real-time, and ready for use in gait analysis. Technically, the system is portable, easily mountable, and can be used for long term monitoring without hindrance to natural activities.