Evolutionary histories of expanded peptidase families in Schistosoma mansoni

Schistosoma mansoni is one of the three main causative agents of human schistosomiasis, a major health problem with a vast socio-economic impact. Recent advances in the proteomic analysis of schistosomes have revealed that peptidases are the main virulence factors involved in the pathogenesis of this disease. In this context, evolutionary studies can be applied to identify peptidase families that have been expanded in genomes over time in response to different selection pressures. Using a phylogenomic approach, we searched for expanded endopeptidase families in the S. mansoni predicted proteome with the aim of contributing to the knowledge of such enzymes as potential therapeutic targets. We found three endopeptidase families that comprise leishmanolysins (metallopeptidase M8 family), cercarial elastases (serine peptidase S1 family) and cathepsin D proteins (aspartic peptidase A1 family). Our results suggest that the Schistosoma members of these families originated from successive gene duplication events in the parasite lineage after its diversification from other metazoans. Overall, critical residues are conserved among the duplicated genes/proteins. Furthermore, each protein family displays a distinct evolutionary history. Altogether, this work provides an evolutionary view of three S. mansoni peptidase families, which allows for a deeper understanding of the genomic complexity and lineage-specific adaptations potentially related to the parasitic lifestyle.

Role of the HCF-1 basic region in sustaining cell proliferation.

BACKGROUND: The human herpes simplex virus-associated host cell factor 1 (HCF-1) is a conserved human transcriptional co-regulator that links positive and negative histone modifying activities with sequence-specific DNA-binding transcription factors. It is synthesized as a 2035 amino acid precursor that is cleaved to generate an amino- (HCF-1(N)) terminal subunit, which promotes G1-to-S phase progression, and a carboxy- (HCF-1(C)) terminal subunit, which controls multiple aspects of cell division during M phase. The HCF-1(N) subunit contains a Kelch domain that tethers HCF-1 to sequence-specific DNA-binding transcription factors, and a poorly characterized so called "Basic" region (owing to a high ratio of basic vs. acidic amino acids) that is required for cell proliferation and has been shown to associate with the Sin3 histone deacetylase (HDAC) component. Here we studied the role of the Basic region in cell proliferation and G1-to-S phase transition assays. METHODOLOGY/PRINCIPAL FINDINGS: Surprisingly, much like the transcriptional activation domains of sequence-specific DNA-binding transcription factors, there is no unique sequence within the Basic region required for promoting cell proliferation or G1-to-S phase transition. Indeed, the ability to promote these activities is size dependent such that the shorter the Basic region segment the less activity observed. We find, however, that the Basic region requirements for promoting cell proliferation in a temperature-sensitive tsBN67 cell assay are more stringent than for G1-to-S phase progression in an HCF-1 siRNA-depletion HeLa-cell assay. Thus, either half of the Basic region alone can support G1-to-S phase progression but not cell proliferation effectively in these assays. Nevertheless, the Basic region displays considerable structural plasticity because each half is able to promote cell proliferation when duplicated in tandem. Consistent with a potential role in promoting cell-cycle progression, the Sin3a HDAC component can associate independently with either half of the Basic region fused to the HCF-1 Kelch domain. CONCLUSIONS/SIGNIFICANCE: While conserved, the HCF-1 Basic region displays striking structural flexibility for controlling cell proliferation.

Differential in vitro activity of the DNA topoisomerase inhibitor idarubicin against Trypanosoma rangeli and Trypanosoma cruzi

In this study the effect of eight DNA topoisomerase inhibitors on the growth Trypanosoma rangeli epimastigotes in cell culture was investigated. Among the eight compounds tested, idarubicin was the only compound that displayed promising trypanocidal activity with a half-maximal growth inhibition (GI50) value in the sub-micromolar range. Fluorescence-activated cell sorting analysis showed a reduction in DNA content in T. rangeli epimastigotes when treated with idarubicin. In contrast to T. rangeli, against Trypanosoma cruzi epimastigotes idarubicin was much less effective exhibiting a GI50 value in the mid-micromolar range. This result indicates that idarubicin displays differential toxic effects in T. rangeli and T. cruzi. Compared with African trypanosomes, it seems that American trypanosomes are generally less susceptible to DNA topoisomerase inhibitors.

Nontuberculous mycobacteria in respiratory samples from patients with pulmonary tuberculosis in the state of Rondonia, Brazil

The main cause of pulmonary tuberculosis (TB) is infection with Mycobacterium tuberculosis (MTB). We aimed to evaluate the contribution of nontuberculous mycobacteria (NTM) to pulmonary disease in patients from the state of Rondônia using respiratory samples and epidemiological data from TB cases. Mycobacterium isolates were identified using a combination of conventional tests, polymerase chain reaction-based restriction enzyme analysis of hsp65 gene and hsp65 gene sequencing. Among the 1,812 cases suspected of having pulmonary TB, 444 yielded bacterial cultures, including 369 cases positive for MTB and 75 cases positive for NTM. Within the latter group, 14 species were identified as Mycobacterium abscessus, Mycobacterium avium, Mycobacterium fortuitum, Mycobacterium intracellulare, Mycobacterium gilvum, Mycobacterium gordonae, Mycobacterium asiaticum, Mycobacterium tusciae, Mycobacterium porcinum, Mycobacterium novocastrense, Mycobacterium simiae, Mycobacterium szulgai, Mycobacterium phlei and Mycobacterium holsaticum and 13 isolates could not be identified at the species level. The majority of NTM cases were observed in Porto Velho and the relative frequency of NTM compared with MTB was highest in Ji-Paraná. In approximately half of the TB subjects with NTM, a second sample containing NTM was obtained, confirming this as the disease-causing agent. The most frequently observed NTM species were M. abscessus and M. avium and because the former species is resistant to many antibiotics and displays unsatisfactory cure rates, the implementation of rapid identification of mycobacterium species is of considerable importance.

Characterization of a plant-derived peptide displaying water clarifying and antimicrobial activities

SUMMARY Drinking water is currently a scarce world resource, the preparation of which requires complex treatments that include clarification of suspended particles and disinfection. Seed extracts of Moringa oleifera Lam., a tropical tree, have been proposed as an environment- friendly alternative, due to their traditional use for the clarification of drinking water. However, the precise nature of the active components was unknown. Here, we show that recombinant or synthetic forms of a cationic seed polypeptide mediate efficient sedimentation of suspended mineral particles and bacteria. Unexpectedly, the polypeptide was also found to possesses a bactericidal activity capable of disinfecting heavily contaminated water. Furthermore, the polypeptide has been shown to efficiently kill several pathogenic bacteria, including antibiotic-resistant isolates of Pseudomona, Streptococcus and Legionella species. Structural modeling of the peptide coupled to the functional analysis of synthetic peptide derivatives delineated distinct structural determinants for the flocculation and antibacterial activities. Our results suggest that a glutamine-rich portion of the polypeptide is involved in the sedimentation process; alternatively, the antibacterial activity depends on a amphiphilic loop. Assembly of multiple copies of this loop into a branched peptide derivative strongly enhances antibacterial activity without displaying hemolytic effect. In conclusion, this polypeptide displays the unprecedented feature of combining efficient water purification and disinfectant properties indicating different molecular mechanisms involved in each case. This work not only identified the features responsible for these activities but also provides useful information that has implications for the further development of this cationic polypeptide as a potent antibacterial agent. RESUME L'eau potable est actuellement une ressource limitée dans le monde. La production d'eau propre à la consommation exige des traitements complexes, incluant la clarification des particules en suspension ainsi que sa désinfection par des additifs chimiques. Les extraits de la graine d'un arbre tropical, Moringa oleifera, sont utilisés traditionnellement en Afrique afin de clarifier l'eau. Quoique la nature exacte des composants actifs était inconnue, on a pu mettre en évidence un polypeptide cationique contenu dans ces graines, capable de sédimenter de manière efficace des particules minérales en suspension ainsi que des bactéries. Ce travail a aussi mis en évidence que ce polypeptide a une activité bactéricide, permettant une désinfection d'eau fortement contaminée. De plus, nous avons démontré que ce polypeptide est efficace contre de nombreuses souches bactériennes pathogènes, également celles résistantes aux antibiotiques comme Pseudomonas, Streptococcus et Legionella. L'analyse de la structure moléculaire de ce polypeptide, couplée à son analyse fonctionnelle a mis en évidence deux domaines structuraux distinct, un pour l'activité de floculation et l'autre pour l'activité antibactérienne. Nos résultats suggèrent que le domaine riche en glutamine est impliqué dans le processus de sédimentation et que l'activité antimicrobienne dépend d'un domaine formant une boucle amphiphilique. En ramifiant plusieurs copies de cette boucle on a pu augmenter de manière significative l'activité antibactérienne. En conclusion, nous avons pu démontrer que ce polypeptide à la capacité unique de combiner des propriétés de purification et de désinfection de l'eau, ce qui implique des mécanismes moléculaires distincts pour ces deux activités. Ce travail a permis d'identifier les domaines du polypeptide qui sont responsables de ses activités et offre une perspective pour le développement d'un nouvel agent antimicrobien.

Performing masculinity, influencing health: a qualitative mixed-methods study of young Spanish men

Background: The literature shows how gender mandates contribute to differences in exposure and vulnerability to certain health risk factors. This paper presents the results of a study developed in the south of Spain, where research aimed at understanding men from a gender perspective is still limited. Objective: The aim of this paper is to explore the lay perceptions and meanings ascribed to the idea of masculinity, identifying ways in which gender displays are related to health. Design: The study is based on a mixed-methods data collection strategy typical of qualitative research. We performed a qualitative content analysis focused on manifest and latent content. Results: Our analysis showed that the relationship between masculinity and health was mainly defined with regard to behavioural explanations with an evident performative meaning. With regard to issues such as driving, the use of recreational drugs, aggressive behaviour, sexuality, and body image, important connections were established between manhood acts and health outcomes. Different ways of understanding and performing the male identity also emerged from the results. The findings revealed the implications of these aspects in the processes of change in the identity codes of men and women. Conclusions: The study provides insights into how the category ‘man’ is highly dependent on collective practices and performative acts. Consideration of how males perform manhood acts might be required in guidance on the development of programmes and policies aimed at addressing gender inequalities in health in a particular local context.

Lactotransferrin gene functional polymorphisms do not influence susceptibility to human immunodeficiency virus-1 mother-to-child transmission in different ethnic groups

Lactotransferrin, also known as lactoferrin, is an iron binding glycoprotein that displays antiviral activity against many different infectious agents, including human immunodeficiency virus (HIV)-1. Lactotransferrin is present in the breast milk and in the female genitourinary mucosa and it has been hypothesised as a possible candidate to prevent mother-to-child HIV-1 transmission. To verify if two functional polymorphisms, Thr29Ala and Arg47Lys, in the lactotransferrin encoding gene (LTF) could affect HIV-1 infection and vertical transmission, a preliminary association study was performed in 238 HIV-1 positive and 99 HIV-1 negative children from Brazil, Italy, Africa and India. No statistically significant association for the Thr29Ala and Arg47Lys LTF polymorphisms and HIV-1 susceptibility in the studied populations was found. Additionally LTF polymorphisms frequencies were compared between the four different ethnic groups.

Clinicopathologic analysis of acute myeloid leukemia arising from chronic myelomonocytic leukemia.

Acute myeloid leukemia arising from chronic myelomonocytic leukemia is currently classified as acute myeloid leukemia with myelodysplasia-related changes, a high-risk subtype. However, the specific features of these cases have not been well described. We studied 38 patients with chronic myelomonocytic leukemia who progressed to acute myeloid leukemia. We compared the clinicopathologic and genetic features of these cases with 180 patients with de novo acute myeloid leukemia and 34 patients with acute myeloid leukemia following myelodysplastic syndromes. We also examined features associated with progression from chronic myelomonocytic leukemia to acute myeloid leukemia by comparing the progressed chronic myelomonocytic leukemia cases with a cohort of chronic myelomonocytic leukemia cases that did not transform to acute myeloid leukemia. Higher white blood cell count, marrow cellularity, karyotype risk score, and Revised International Prognostic Scoring System score were associated with more rapid progression from chronic myelomonocytic leukemia to acute myeloid leukemia. Patients with acute myeloid leukemia ex chronic myelomonocytic leukemia were older (P<0.01) and less likely to receive aggressive treatment (P=0.02) than de novo acute myeloid leukemia patients. Most cases showed monocytic differentiation and fell into the intermediate acute myeloid leukemia karyotype risk group; 55% had normal karyotype and 17% had NPM1 mutation. Median overall survival was 6 months, which was inferior to de novo acute myeloid leukemia (17 months, P=0.002) but similar to post myelodysplastic syndrome acute myeloid leukemia. On multivariate analysis of all acute myeloid leukemia patients, only age and karyotype were independent prognostic variables for overall survival. Our findings indicate that acute myeloid leukemia following chronic myelomonocytic leukemia displays aggressive behavior and support placement of these cases within the category of acute myeloid leukemia with myelodysplasia-related changes. The poor prognosis of these patients may be related to an older population and lack of favorable-prognosis karyotypes that characterize many de novo acute myeloid leukemia cases.

A combination of ascorbic acid and α-tocopherol to test the effectiveness and safety in the fragile X syndrome: study protocol for a phase II, randomized, placebo-controlled trial.

BACKGROUND Fragile X syndrome (FXS) is an inherited neurodevelopmental condition characterised by behavioural, learning disabilities, physical and neurological symptoms. In addition, an important degree of comorbidity with autism is also present. Considered a rare disorder affecting both genders, it first becomes apparent during childhood with displays of language delay and behavioural symptoms.Main aim: To show whether the combination of 10 mg/kg/day of ascorbic acid (vitamin C) and 10 mg/kg/day of α-tocopherol (vitamin E) reduces FXS symptoms among male patients ages 6 to 18 years compared to placebo treatment, as measured on the standardized rating scales at baseline, and after 12 and 24 weeks of treatment.Secondary aims: To assess the safety of the treatment. To describe behavioural and cognitive changes revealed by the Developmental Behaviour Checklist Short Form (DBC-P24) and the Wechsler Intelligence Scale for Children-Revised. To describe metabolic changes revealed by blood analysis. To measure treatment impact at home and in an academic environment. METHODS/DESIGN A phase II randomized, double-blind pilot clinical trial. SCOPE male children and adolescents diagnosed with FXS, in accordance with a standardized molecular biology test, who met all the inclusion criteria and none of the exclusion criteria. INSTRUMENTATION clinical data, blood analysis, Wechsler Intelligence Scale for Children-Revised, Conners parent and teacher rating scale scores and the DBC-P24 results will be obtained at the baseline (t0). Follow up examinations will take place at 12 weeks (t1) and 24 weeks (t2) of treatment. DISCUSSION A limited number of clinical trials have been carried out on children with FXS, but more are necessary as current treatment possibilities are insufficient and often provoke side effects. In the present study, we sought to overcome possible methodological problems by conducting a phase II pilot study in order to calculate the relevant statistical parameters and determine the safety of the proposed treatment. The results will provide evidence to improve hyperactivity control and reduce behavioural and learning problems using ascorbic acid (vitamin C) and α-tocopherol (vitamin E). The study protocol was approved by the Regional Government Committee for Clinical Trials in Andalusia and the Spanish agency for drugs and health products. TRIAL REGISTRATION ClinicalTrials.gov Identifier: NCT01329770 (29 March 2011).

Disentangling reasons for low Y chromosome variation in the greater white-toothed shrew (Crocidura russula).

Y chromosome variation is determined by several confounding factors including mutation rate, effective population size, demography, and selection. Disentangling these factors is essential to better understand the evolutionary properties of the Y chromosome. We analyzed genetic variation on the Y chromosome, X chromosome, and mtDNA of the greater white-toothed shrew, a species with low variance in male reproductive success and limited sex-biased dispersal, which enables us to control to some extent for life-history effects. We also compared ancestral (Moroccan) to derived (European) populations to investigate the role of demographic history in determining Y variation. Recent colonization of Europe by a small number of founders (combined with low mutation rates) is largely responsible for low diversity observed on the European Y and X chromosomes compared to mtDNA. After accounting for mutation rate, copy number, and demography, the Y chromosome still displays a deficit in variation relative to the X in both populations. This is possibly influenced by directional selection, but the slightly higher variance in male reproductive success is also likely to play a role, even though the difference is small compared to that in highly polygynous species. This study illustrates that demography and life-history effects should be scrutinized before inferring strong selective pressure as a reason for low diversity on the Y chromosome.

Didactique de la géographie et formation initiale des enseignants spécialistes : conception et première évaluation du nouveau dispositif de formation initiale des enseignants de géographie du Secondaire supérieur à la HEP Vaud

Geography as a school subject is specifically thought for and by the schools. The contents of the school subject, nowadays, do not reflect the concerns and the evolution of the discipline as such. Nevertheless, official curricula set school objectives that address issues affecting the world and people's lives. These issues are coherent with the ones addressed by geography as a social science, that is to say the study of how people and their environment interact and how societies are interconnected through space. On an every day basis, Geography as a school subject is most of the time reduced to accumulating knowledge outside any given context. This knowledge may even be partially untrue or old and the related activities focus on low cognitive tensions. These practices do not contribute to the learners' understanding of the world because it does not allow them to build a geographical competence, which they. will need as future citizens in order to make responsible choices when they are confronted to questions related to how the locations of human and physical features are influenced by each other and how they interact across space. The central part of the text relies on the ideas and the processes discussed in the publications, which constitute the published file; it is divided into two parts. The first part (chapter 4) presents a didactic approach, which gives meaningful insights into Geography as a school subject and shows a brief account of the theoretical background that supports it. This socio-constructivist approach relies on the main following features: a priming stage (élément déclencheur), which presents geographical knowledge as an issue to be explored, discussed or solved; the issue is given to learners;. the planning of the teaching-learning sequence in small units launched by the main issue in the priming stage ; the interconnections of geographical knowledge with integrative concepts ; the synthetic stage or reporting stage where final concepts and knowledge are put together in order to be learned. Such an approach allows learners to re-invest the knowledge they have built themselves. This knowledge is organised by geographical integrative concepts, which represent true thinking operative tools and with which key issues in the geographical thinking are associated. The second part of the text (chapter 5) displays the didactic principles that governed the conception of the new initial training course for the future upper secondary school teachers at the HEP Vaud. The ambition of this course is to prepare future teachers to plan and realize the teaching of geography that provides pupils with the tools to understand better how people and their environment interact and how societies are interconnected through space. One of the tools for the teachers is the conceptual framework, whose most salient interest is to be relevant at every stage of the preparation and planning of the teaching, including the necessary epistemological reflection that should always be present. The synthesis of the text starts with a short account of the first evaluation of the new course. Various reflections on the future concerns and issues, that the didactics and methodology of Geography will be confronted with, constitute the synthesis.

Interindividual variability of the clinical pharmacokinetics of methadone: implications for the treatment of opioid dependence

Methadone is widely used for the treatment of opioid dependence. Although in most countries the drug is administered as a racemic mixture of (R)- and (S)- methadone, (R)-methadone accounts for most, if not all, of the opioid effects. Methadone can be detected in the blood 15-45 minutes after oral administration, with peak plasma concentration at 2.5-4 hours. Methadone has a mean bioavailability of around 75% (range 36-100%). Methadone is highly bound to plasma proteins, in particular to alpha(1)-acid glycoprotein. Its mean free fraction is around 13%, with a 4-fold interindividual variation. Its volume of distribution is about 4 L/kg (range 2-13 L/kg). The elimination of methadone is mediated by biotransformation, followed by renal and faecal excretion. Total body clearance is about 0.095 L/min, with wide interindividual variation (range 0.02-2 L/min). Plasma concentrations of methadone decrease in a biexponential manner, with a mean value of around 22 hours (range 5-130 hours) for elimination half-life. For the active (R)-enantiomer, mean values of around 40 hours have been determined. Cytochrome P450 (CYP) 3A4 and to a lesser extent 2D6 are probably the main isoforms involved in methadone metabolism. Rifampicin (rifampin), phenobarbital, phenytoin, carbamazepine, nevirapine, and efavirenz decrease methadone blood concentrations, probably by induction of CYP3A4 activity, which can result in severe withdrawal symptoms. Inhibitors of CYP3A4, such as fluconazole, and of CYP2D6, such as paroxetine, increase methadone blood concentrations. There is an up to 17-fold interindividual variation of methadone blood concentration for a given dosage, and interindividual variability of CYP enzymes accounts for a large part of this variation. Since methadone probably also displays large interindividual variability in its pharmacodynamics, methadone treatment must be individually adapted to each patient. Because of the high morbidity and mortality associated with opioid dependence, it is of major importance that methadone is used at an effective dosage in maintenance treatment: at least 60 mg/day, but typically 80-100 mg/day. Recent studies also show that a subset of patients might benefit from methadone dosages larger than 100 mg/day, many of them because of high clearance. In clinical management, medical evaluation of objective signs and subjective symptoms is sufficient for dosage titration in most patients. However, therapeutic drug monitoring can be useful in particular situations. In the case of non-response trough plasma concentrations of 400 microg/L for (R,S)-methadone or 250 microg/L for (R)-methadone might be used as target values.

Hierarchical analyses of genetic differentiation in a hybrid zone of Sorex araneus (Insectivora : Soricidae)

Microsatellites are used to unravel the fine-scale genetic structure of a hybrid zone between chromosome races Valais and Cordon of the common shrew (Sorex araneus) located in the French Alps. A total of 269 individuals collected between 1992 and 1995 was typed for seven microsatellite loci. A modified version of the classical multiple correspondence analysis is carried out. This analysis clearly shows the dichotomy between the two races. Several approaches are used to study genetic structuring. Gene flow is clearly reduced between these chromosome races and is estimated at one migrant every two generations using X-statistics and one migrant per generation using F-statistics. Hierarchical F- and R-statistics are compared and their efficiency to detect inter- and intraracial patterns of divergence is discussed. Within-race genetic structuring is significant, but remains weak. F-ST displays similar values on both sides of the hybrid zone, although no environmental barriers are found on the Cordon side, whereas the Valais side is divided by several mountain rivers. We introduce the exact G-test to microsatellite data which proved to be a powerful test to detect genetic differentiation within as well as among races. The genetic background of karyotypic hybrids was compared with the genetic background of pure parental forms using a CRT-MCA. Our results indicate that, without knowledge of the karyotypes, we would not have been able to distinguish these hybrids from karyotypically pure samples.

Natural genetic variation of root system architecture from Arabidopsis to Brachypodium: towards adaptive value.

Root system architecture is a trait that displays considerable plasticity because of its sensitivity to environmental stimuli. Nevertheless, to a significant degree it is genetically constrained as suggested by surveys of its natural genetic variation. A few regulators of root system architecture have been isolated as quantitative trait loci through the natural variation approach in the dicotyledon model, Arabidopsis. This provides proof of principle that allelic variation for root system architecture traits exists, is genetically tractable, and might be exploited for crop breeding. Beyond Arabidopsis, Brachypodium could serve as both a credible and experimentally accessible model for root system architecture variation in monocotyledons, as suggested by first glimpses of the different root morphologies of Brachypodium accessions. Whether a direct knowledge transfer gained from molecular model system studies will work in practice remains unclear however, because of a lack of comprehensive understanding of root system physiology in the native context. For instance, apart from a few notable exceptions, the adaptive value of genetic variation in root system modulators is unknown. Future studies should thus aim at comprehensive characterization of the role of genetic players in root system architecture variation by taking into account the native environmental conditions, in particular soil characteristics.

The Fanconi anemia protein FANCM can promote branch migration of Holliday junctions and replication forks.

Fanconi anemia (FA) is a genetically heterogeneous cancer-prone disorder associated with chromosomal instability and cellular hypersensitivity to DNA crosslinking agents. The FA pathway is suspected to play a crucial role in the cellular response to DNA replication stress. At a molecular level, however, the function of most of the FA proteins is unknown. FANCM displays DNA-dependent ATPase activity and promotes the dissociation of DNA triplexes, but the physiological significance of this activity remains elusive. Here we show that purified FANCM binds to Holliday junctions and replication forks with high specificity and promotes migration of their junction point in an ATPase-dependent manner. Furthermore, we provide evidence that FANCM can dissociate large recombination intermediates, via branch migration of Holliday junctions through 2.6 kb of DNA. Our data suggest a direct role for FANCM in DNA processing, consistent with the current view that FA proteins coordinate DNA repair at stalled replication forks.