Validation of a Low-Cost Human Papillomavirus Genotyping Assay Based on PGMY PCR and Reverse Blotting Hybridization with Reusable Membranes.

The genotyping of human papillomaviruses (HPV) is essential for the surveillance of HPV vaccines. We describe and validate a low-cost PGMY-based PCR assay (PGMY-CHUV) for the genotyping of 31 HPV by reverse blotting hybridization (RBH). Genotype-specific detection limits were 50 to 500 genome equivalents per reaction. RBH was 100% specific and 98.61% sensitive using DNA sequencing as the gold standard (n = 1,024 samples). PGMY-CHUV was compared to the validated and commercially available linear array (Roche) on 200 samples. Both assays identified the same positive (n = 182) and negative samples (n = 18). Seventy-six percent of the positives were fully concordant after restricting the comparison to the 28 genotypes shared by both assays. At the genotypic level, agreement was 83% (285/344 genotype-sample combinations; κ of 0.987 for single infections and 0.853 for multiple infections). Fifty-seven of the 59 discordant cases were associated with multiple infections and with the weakest genotypes within each sample (P < 0.0001). PGMY-CHUV was significantly more sensitive for HPV56 (P = 0.0026) and could unambiguously identify HPV52 in mixed infections. PGMY-CHUV was reproducible on repeat testing (n = 275 samples; 392 genotype-sample combinations; κ of 0.933) involving different reagents lots and different technicians. Discordant results (n = 47) were significantly associated with the weakest genotypes in samples with multiple infections (P < 0.0001). Successful participation in proficiency testing also supported the robustness of this assay. The PGMY-CHUV reagent costs were estimated at $2.40 per sample using the least expensive yet proficient genotyping algorithm that also included quality control. This assay may be used in low-resource laboratories that have sufficient manpower and PCR expertise.

First consumption ever of multiple substances: applying an expert-based taxonomy to a Swiss national sample of adolescents.

The use of multiple legal and illegal substances by adolescents is a growing concern in all countries, but since no consensus about a taxonomy did emerge yet, it is difficult to understand the different patterns of consumption and to implement tailored prevention and treatment programs directed towards specific subgroups of the adolescent population. Using data from a Swiss survey on adolescent health, we analyzed the age at which ten legal and illegal substances were consumed for the first time ever by applying a method combining the strength of both automatic clustering and use of substance experts. Results were then compared to 30 socio-economic factors to establish the usefulness of and to validate our taxonomy. We also analyzed the succession of substance first use for each group. The final taxonomy consists of eight groups ranging from non-consumers to heavy drug addicts. All but four socio-economic factors were significantly associated with the taxonomy, the strongest associations being observed with health, behavior, and sexuality factors. Numerous factors influence adolescents in their decision to first try substances or to use them on a regular basis, and no factor alone can be considered as an absolute marker of problematic behavior regarding substance use. Different processes of experimentation with substances are associated with different behaviors, therefore focusing on only one substance or only one factor is not efficient. Prevention and treatment programs can then be tailored to address specific issues related to different youth subgroups.

One naive T cell, multiple fates in CD8+ T cell differentiation.

The mechanism by which the immune system produces effector and memory T cells is largely unclear. To allow a large-scale assessment of the development of single naive T cells into different subsets, we have developed a technology that introduces unique genetic tags (barcodes) into naive T cells. By comparing the barcodes present in antigen-specific effector and memory T cell populations in systemic and local infection models, at different anatomical sites, and for TCR-pMHC interactions of different avidities, we demonstrate that under all conditions tested, individual naive T cells yield both effector and memory CD8+ T cell progeny. This indicates that effector and memory fate decisions are not determined by the nature of the priming antigen-presenting cell or the time of T cell priming. Instead, for both low and high avidity T cells, individual naive T cells have multiple fates and can differentiate into effector and memory T cell subsets.

Direct three-dimensional myocardial strain tensor quantification and tracking using zHARP.

Images of myocardial strain can be used to diagnose heart disease, plan and monitor treatment, and to learn about cardiac structure and function. Three-dimensional (3D) strain is typically quantified using many magnetic resonance (MR) images obtained in two or three orthogonal planes. Problems with this approach include long scan times, image misregistration, and through-plane motion. This article presents a novel method for calculating cardiac 3D strain using a stack of two or more images acquired in only one orientation. The zHARP pulse sequence encodes in-plane motion using MR tagging and out-of-plane motion using phase encoding, and has been previously shown to be capable of computing 3D displacement within a single image plane. Here, data from two adjacent image planes are combined to yield a 3D strain tensor at each pixel; stacks of zHARP images can be used to derive stacked arrays of 3D strain tensors without imaging multiple orientations and without numerical interpolation. The performance and accuracy of the method is demonstrated in vitro on a phantom and in vivo in four healthy adult human subjects.

Identification and characterization of novel classes of macrophage migration inhibitory factor (MIF) inhibitors with distinct mechanisms of action.

Macrophage migration inhibitory factor (MIF), a proinflammatory cytokine, is considered an attractive therapeutic target in multiple inflammatory and autoimmune disorders. In addition to its known biologic activities, MIF can also function as a tautomerase. Several small molecules have been reported to be effective inhibitors of MIF tautomerase activity in vitro. Herein we employed a robust activity-based assay to identify different classes of novel inhibitors of the catalytic and biological activities of MIF. Several novel chemical classes of inhibitors of the catalytic activity of MIF with IC(50) values in the range of 0.2-15.5 microm were identified and validated. The interaction site and mechanism of action of these inhibitors were defined using structure-activity studies and a battery of biochemical and biophysical methods. MIF inhibitors emerging from these studies could be divided into three categories based on their mechanism of action: 1) molecules that covalently modify the catalytic site at the N-terminal proline residue, Pro(1); 2) a novel class of catalytic site inhibitors; and finally 3) molecules that disrupt the trimeric structure of MIF. Importantly, all inhibitors demonstrated total inhibition of MIF-mediated glucocorticoid overriding and AKT phosphorylation, whereas ebselen, a trimer-disrupting inhibitor, additionally acted as a potent hyperagonist in MIF-mediated chemotactic migration. The identification of biologically active compounds with known toxicity, pharmacokinetic properties, and biological activities in vivo should accelerate the development of clinically relevant MIF inhibitors. Furthermore, the diversity of chemical structures and mechanisms of action of our inhibitors makes them ideal mechanistic probes for elucidating the structure-function relationships of MIF and to further determine the role of the oligomerization state and catalytic activity of MIF in regulating the function(s) of MIF in health and disease.

Islands of euchromatin-like sequence and expressed polymorphic sequences within the short arm of human chromosome 21.

The goals of the human genome project did not include sequencing of the heterochromatic regions. We describe here an initial sequence of 1.1 Mb of the short arm of human chromosome 21 (HSA21p), estimated to be 10% of 21p. This region contains extensive euchromatic-like sequence and includes on average one transcript every 100 kb. These transcripts show multiple inter- and intrachromosomal copies, and extensive copy number and sequence variability. The sequencing of the "heterochromatic" regions of the human genome is likely to reveal many additional functional elements and provide important evolutionary information.

Parasites and sociality in ants

SUMMARY : Parasites and sociality in ants This thesis investigates the complex relationships between sociality, defences against parasites and the regulation of social structures. We studied how fungal parasites influenced colony organization, collective defences and social immunity in the ant Formica selysi. We first describe the diversity and prevalence of fungal pathogens associated with ant nests. The richness of fungal parasites community may increase the risk of multiple infections and select for a diversification of anti-parasitic defences in ants. Collective defences are powerful means to combat parasites, but can also increase the risk of disease transmission. Here, we showed that allo-grooming (mutual cleaning) was directed towards every returning individuals, be they contaminated or not. This collective behaviour removed conidia more efficiently than self-grooming but did not improve the survival of contaminated individuals. This suggests that allo-grooming may rather protect the group than cure contaminated individuals. It may also permit "social vaccination" if a contact with contaminated ants protects groomers frorn a second fungal exposure. Social transfer of immunity is an emerging theme in insect immunology. Here, we showed that ants in contact with an ant from a different genetic lineage had a higher disease resistance. We also found that naïve ants had a higher resistance after a contact with an immunized ant. This suggests that a transfer of resistance is possible and that "social vaccination" may improve the resistance of the group. However, it remains unclear whether repeated exposure to parasites may also increase the resistance of infected individuals themselves. lmmune memory in invertebrates is still debated. We tested whether immune priming against fungal parasite arose in ants and whether it was strain-specific. We found no evidence of immune priming. Naïve and immunized ants had a similar survival when infected. Together with our previous results, this suggests that ants have evolved efficient collective anti-fungal defences but that these defences aim at protecting the group rather than the contaminated individuals. ln colonies of our study population, there is a strong variation in the number of breeders. This is associated with important changes in life-history traits like demography or queen and worker body size. In the second part of the thesis, we investigated how social structures evolved and were maintained. We showed that queens from monogyne and polygyne colonies were able to found new colonies both alone or in association. We also found that there was no difference between monogyne and polygyne colonies in the acceptance of additional queens. These results suggest that a high plasticity has been maintained in this population, which may permit to adapt rapidly to changing environmental conditions. RESUME : Parasites et socialité chez les fourmis Durant cette thèse, nous avons étudié comment la socialité apporte de nouvelles réponses a des problèmes complexes telle que la défense contre les parasites ou l'organisation de la vie en groupe. Nous avons choisi comme modèle la fourmi Formica selysi et ses champignons pathogènes. Nous avons d'abord montré que la diversité et la prévalence de champignons pathogènes associés aux nids de fourmis étaient très élevées. Cela a pu pousser les fourmis à diversifier le champ de leur défenses anti-parasitaires afin d'éviter les infections multiples, La socialité a en particulier permis l'évolution de défenses collectives qui pourraient être plus efficaces que les défenses individuelles. Nous nous sommes donc intéressés de plus près aux défenses collectives et avons étudié quels en étaient les coûts et les bénéfices pour le groupe et pour ses membres. Nous avons trouvé que les fourmis nettoyaient tous les individus entrant dans la colonie, qu'ils soient contaminés ou non. Cela permettait d'ôter plus de spores que le nettoyage individuel et n'augmentait pas la transmission de maladie. Cependant, le nettoyage mutuel n'augmentait pas non plus la survie des individus contaminés. ll se pourrait donc que ce comportement serve plutôt a éviter une dissémination de la maladie qu'à soigner les individus contaminés. Le nettoyage mutuel pourrait aussi permettre aux individus sains d'avoir un premier contact non-létal avec un parasite et d'être vaccinés contre une future exposition. Cette hypothèse a été soutenue par une expérience dans laquelle nous avons montré que le contact avec une fourmi immunisée permettait d'augmenter la résistance d'individus naïfs. Les fourmis avaient aussi une meilleure résistance lorsqu'elles étaient en contact avec une fourmi provenant d'une autre lignée génétique. Cette "vaccination sociale" pourrait permettre d'une part d'augmenter le nombre d'espèce de parasites contre lesquelles le groupe serait protégé et d'autre part de faire l'économie d'autres défenses individuelles telles que la réponse immunitaire. Nous avons testé si les fourmis étaient elles-mêmes "vaccinées", c'est-à-dire, si elles exprimaient une mémoire immunitaire après un premier contact avec un champignon parasite. Nous n'avons trouvé aucune différence de survie entre les individus naïfs et immunisés ce qui suggère les fourmis favorisent d'autres défenses que la mémoire immunitaire contre les champignons entomopathogènes. Cela suggère également que les comportements coopératifs anti-parasitaires pourraient compléter, voire remplacer les défenses individuelles. La socialité telle qu'elle est pratiquée par les fourmis pose un autre problème de poids qui est celui de savoir combien d'individus se reproduisent. En effet, si les ouvrières sont stériles, le nombre de reines assurant la reproduction peut varier considérablement. Dans la population de E sebrsi étudiée, les colonies monogynes (une reine) co-existent avec des colonies polygynes (plusieurs reines) dans le même habitat. Nous nous sommes demandés si ces structures sociales étaient fixes ou si un changement de l'une à l'autre était possible. Pour cela nous avons comparé la fondation de nouvelles colonies par les jeunes reines issues de colonies monogynes et polygynes. Nous avons également observé si l'acceptation de nouvelles reines était possible dans les deux types de colonies. Nous n'avons trouvé aucune différence entre les deux types de colonies. Cela suggère qu'un changement est possible et que l'évolution des structures sociales est un processus dynamique. Cela pourrait être dû à l'habitat particulièrement changeant dans lequel se trouve notre population qui exigerait d'être capable de s'adapter très rapidement a de nouvelles conditions.

Molecular dating, evolutionary rates, and the age of the grasses.

Many questions in evolutionary biology require an estimate of divergence times but, for groups with a sparse fossil record, such estimates rely heavily on molecular dating methods. The accuracy of these methods depends on both an adequate underlying model and the appropriate implementation of fossil evidence as calibration points. We explore the effect of these in Poaceae (grasses), a diverse plant lineage with a very limited fossil record, focusing particularly on dating the early divergences in the group. We show that molecular dating based on a data set of plastid markers is strongly dependent on the model assumptions. In particular, an acceleration of evolutionary rates at the base of Poaceae followed by a deceleration in the descendants strongly biases methods that assume an autocorrelation of rates. This problem can be circumvented by using markers that have lower rate variation, and we show that phylogenetic markers extracted from complete nuclear genomes can be a useful complement to the more commonly used plastid markers. However, estimates of divergence times remain strongly affected by different implementations of fossil calibration points. Analyses calibrated with only macrofossils lead to estimates for the age of core Poaceae ∼51-55 Ma, but the inclusion of microfossil evidence pushes this age to 74-82 Ma and leads to lower estimated evolutionary rates in grasses. These results emphasize the importance of considering markers from multiple genomes and alternative fossil placements when addressing evolutionary issues that depend on ages estimated for important groups.

Multiple introductions boosted genetic diversity in the invasive range of black cherry (Prunus serotina; Rosaceae).

BACKGROUND AND AIMS: Black cherry (Prunus serotina) is a North American tree that is rapidly invading European forests. This species was introduced first as an ornamental plant, then it was massively planted by foresters in many countries, but its origins and the process of invasion remain poorly documented. Based on a genetic survey of both native and invasive ranges, the invasion history of black cherry was investigated by identifying putative source populations and then assessing the importance of multiple introductions on the maintenance of gene diversity. METHODS: Genetic variability and structure of 23 populations from the invasive range and 22 populations from the native range were analysed using eight nuclear microsatellite loci and five chloroplast DNA regions. KEY RESULTS: Chloroplast DNA diversity suggests there were multiple introductions from a single geographic region (the north-eastern United States). A low reduction of genetic diversity was observed in the invasive range for both nuclear and plastid genomes. High propagule pressure including both the size and number of introductions shaped the genetic structure in Europe and boosted genetic diversity. Populations from Denmark, The Netherlands, Belgium and Germany showed high genetic diversity and low differentiation among populations, supporting the hypothesis that numerous introduction events, including multiple individuals and exchanges between sites, have taken place during two centuries of plantation. CONCLUSIONS: This study postulates that the invasive black cherry has originated from east of the Appalachian Mountains (mainly the Allegheny plateau) and its invasiveness in north-western Europe is mainly due to multiple introductions containing high numbers of individuals.

Abnormal social behaviors and altered gene expression rates in a mouse model for Potocki-Lupski syndrome.

The Potocki-Lupski syndrome (PTLS) is associated with a microduplication of 17p11.2. Clinical features include multiple congenital and neurobehavioral abnormalities and autistic features. We have generated a PTLS mouse model, Dp(11)17/+, that recapitulates some of the physical and neurobehavioral phenotypes present in patients. Here, we investigated the social behavior and gene expression pattern of this mouse model in a pure C57BL/6-Tyr(c-Brd) genetic background. Dp(11)17/+ male mice displayed normal home-cage behavior but increased anxiety and increased dominant behavior in specific tests. A subtle impairment in the preference for a social target versus an inanimate target and abnormal preference for social novelty (the preference to explore an unfamiliar mouse versus a familiar one) was also observed. Our results indicate that these animals could provide a valuable model to identify the specific gene(s) that confer abnormal social behaviors and that map within this delimited genomic deletion interval. In a first attempt to identify candidate genes and for elucidating the mechanisms of regulation of these important phenotypes, we directly assessed the relative transcription of genes within and around this genomic interval. In this mouse model, we found that candidates genes include not only most of the duplicated genes, but also normal-copy genes that flank the engineered interval; both categories of genes showed altered expression levels in the hippocampus of Dp(11)17/+ mice.

Persistent Candida albicans colonization and molecular mechanisms of azole resistance in autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED) patients.

Objectives: Patients with autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED, APS-I) suffer from chronic candidosis caused mainly by Candida albicans, and repeated courses of azole antifungals have led to the development of resistance in the APECED patient population in Finland. The aim of our study was to address whether the patients are persistently colonized with the same or genetically closely related strains, whether epidemic strains are present and which molecular mechanisms account for azole resistance. Methods: Sets of C. albicans (n?=?19) isolates from nine APECED patients reported with decreased susceptibility to fluconazole isolated up to 9 years apart were included. The strains were typed by multilocus sequence typing. CDR1/2, MDR1 and ERG11 mRNA expression was analysed by northern blotting and Cdr1, Cdr2 and Mdr1 protein expression by western blotting, and TAC1 and ERG11 genes were sequenced. Results: All seven patients with multiple C. albicans isolates analysed were persistently colonized with the same or a genetically closely related strain for a mean of 5 years. All patients were colonized with different strains and no epidemic strains were found. The major molecular mechanisms behind the azole resistance were mutations in TAC1 contributing to overexpression of CDR1 and CDR2. Six new TAC1 mutations were found, one of which (N740S) is likely to be a gain-of-function mutation. Most isolates were found to have gained multiple TAC1 and ERG11 point mutations. Conclusions: Despite clinically successful treatment leading to relief of symptoms, colonization by C. albicans strains is persistent within APECED patients. Microevolution and point mutations occur within strains, leading to the development of azole-resistant isolates.

Leri's pleonosteosis, a congenital rheumatic disease, results from microduplication at 8q22.1 encompassing GDF6 and SDC2 and provides insight into systemic sclerosis pathogenesis.

OBJECTIVES: Leri's pleonosteosis (LP) is an autosomal dominant rheumatic condition characterised by flexion contractures of the interphalangeal joints, limited motion of multiple joints, and short broad metacarpals, metatarsals and phalanges. Scleroderma-like skin thickening can be seen in some individuals with LP. We undertook a study to characterise the phenotype of LP and identify its genetic basis. METHODS AND RESULTS: Whole-genome single-nucleotide polymorphism genotyping in two families with LP defined microduplications of chromosome 8q22.1 as the cause of this condition. Expression analysis of dermal fibroblasts from affected individuals showed overexpression of two genes, GDF6 and SDC2, within the duplicated region, leading to dysregulation of genes that encode proteins of the extracellular matrix and downstream players in the transforming growth factor (TGF)-β pathway. Western blot analysis revealed markedly decreased inhibitory SMAD6 levels in patients with LP. Furthermore, in a cohort of 330 systemic sclerosis cases, we show that the minor allele of a missense SDC2 variant, p.Ser71Thr, could confer protection against disease (p<1×10(-5)). CONCLUSIONS: Our work identifies the genetic cause of LP in these two families, demonstrates the phenotypic range of the condition, implicates dysregulation of extracellular matrix homoeostasis genes in its pathogenesis, and highlights the link between TGF-β/SMAD signalling, growth/differentiation factor 6 and syndecan-2. We propose that LP is an additional member of the growing 'TGF-β-pathies' group of musculoskeletal disorders, which includes Myhre syndrome, acromicric dysplasia, geleophysic dysplasias, Weill-Marchesani syndromes and stiff skin syndrome. Identification of a systemic sclerosis-protective SDC2 variant lays the foundation for exploration of the role of syndecan-2 in systemic sclerosis in the future.

Implementing evidence-based patient and family education on oral anticoagulation therapy: a community-based participatory project.

AIMS AND OBJECTIVES: This study aimed at developing and implementing evidence-based patient and family education on oral anticoagulation therapy. BACKGROUND: The number of persons with chronic diseases who live at home is increasing. They have to manage multiple diseases and complex treatments. One such treatment is oral anticoagulation therapy, a high risk variable dose medication. Adherence to oral anticoagulation therapy is jeopardised by limited information about the medications, their risk and complications, the impact of individual daily routine and the limited inclusion of family members in education. Hence, improved and tailored education is essential for patients and families to manage oral anticoagulation therapy at home. DESIGN AND METHODS: A community-based participatory research design combined with the Precede-Proceed model was used including a systematic literature review, posteducation analysis, an online nurse survey, a documentation analysis and patient/family interviews. The study was conducted between April 2010-December 2012 at a department of general internal medicine in a teaching hospital in Switzerland. Participants were the department's nursing and medical professionals including the patients and their families. RESULTS: The evidence-based patient and family education on oral anticoagulation therapy emerged comprising a learning assessment, teaching units, clarification of responsibilities of nurse professionals and documentation guidelines. CONCLUSION AND CLINICAL RELEVANCE: The inclusion of the whole department has contributed to the development and implementation of this evidence-based patient family education on oral anticoagulation therapy, which encompasses local characteristics and patient preferences. This education is now being used throughout the department.

Combined CSL and p53 downregulation promotes cancer-associated fibroblast activation.

Stromal fibroblast senescence has been linked to ageing-associated cancer risk. However, density and proliferation of cancer-associated fibroblasts (CAFs) are frequently increased. Loss or downmodulation of the Notch effector CSL (also known as RBP-Jκ) in dermal fibroblasts is sufficient for CAF activation and ensuing keratinocyte-derived tumours. We report that CSL silencing induces senescence of primary fibroblasts from dermis, oral mucosa, breast and lung. CSL functions in these cells as a direct repressor of multiple senescence- and CAF-effector genes. It also physically interacts with p53, repressing its activity. CSL is downmodulated in stromal fibroblasts of premalignant skin actinic keratosis lesions and squamous cell carcinomas, whereas p53 expression and function are downmodulated only in the latter, with paracrine FGF signalling as the probable culprit. Concomitant loss of CSL and p53 overcomes fibroblast senescence, enhances expression of CAF effectors and promotes stromal and cancer cell expansion. The findings support a CAF activation-stromal co-evolution model under convergent CSL-p53 control.

Rare genomic structural variants in complex disease: lessons from the replication of associations with obesity.

The limited ability of common variants to account for the genetic contribution to complex disease has prompted searches for rare variants of large effect, to partly explain the 'missing heritability'. Analyses of genome-wide genotyping data have identified genomic structural variants (GSVs) as a source of such rare causal variants. Recent studies have reported multiple GSV loci associated with risk of obesity. We attempted to replicate these associations by similar analysis of two familial-obesity case-control cohorts and a population cohort, and detected GSVs at 11 out of 18 loci, at frequencies similar to those previously reported. Based on their reported frequencies and effect sizes (OR≥25), we had sufficient statistical power to detect the large majority (80%) of genuine associations at these loci. However, only one obesity association was replicated. Deletion of a 220 kb region on chromosome 16p11.2 has a carrier population frequency of 2×10(-4) (95% confidence interval [9.6×10(-5)-3.1×10(-4)]); accounts overall for 0.5% [0.19%-0.82%] of severe childhood obesity cases (P = 3.8×10(-10); odds ratio = 25.0 [9.9-60.6]); and results in a mean body mass index (BMI) increase of 5.8 kg.m(-2) [1.8-10.3] in adults from the general population. We also attempted replication using BMI as a quantitative trait in our population cohort; associations with BMI at or near nominal significance were detected at two further loci near KIF2B and within FOXP2, but these did not survive correction for multiple testing. These findings emphasise several issues of importance when conducting rare GSV association, including the need for careful cohort selection and replication strategy, accurate GSV identification, and appropriate correction for multiple testing and/or control of false discovery rate. Moreover, they highlight the potential difficulty in replicating rare CNV associations across different populations. Nevertheless, we show that such studies are potentially valuable for the identification of variants making an appreciable contribution to complex disease.