The trypanosomatid evolution workshop London School of Hygiene and Tropical Medicine

The trypanosome evolution workshop, a joint meeting of the University of Exeter and the London School of Hygiene and Tropical Medicine, focused on topics relating to trypanosomatid and vector evolution. The meeting, sponsored by The Wellcome Trust, The Special Programme for Research and Training in Tropical Disease of World Health Organization and the British Section of the Society of Protozoologists, brought together an international group of experts who presented papers on a wide range of topics including parasite and vector phylogenies, molecular methodology and relevant biogeographical data.

Chagas disease: current epidemiological trends after the interruption of vectorial and transfusional transmission in the Southern Cone countries

Chagas disease, named after Carlos Chagas who first described it in 1909, exists only on the American Continent. It is caused by a parasite, Trypanosoma cruzi, transmitted to humans by blood-sucking triatomine bugs and by blood transfusion. Chagas disease has two successive phases, acute and chronic. The acute phase lasts 6 to 8 weeks. After several years of starting the chronic phase, 20% to 35% of the infected individuals, depending on the geographical area will develop irreversible lesions of the autonomous nervous system in the heart, esophagus, colon and the peripheral nervous system. Data on the prevalence and distribution of Chagas disease improved in quality during the 1980's as a result of the demographically representative cross-sectional studies carried out in countries where accurate information was not available. A group of experts met in Brasília in 1979 and devised standard protocols to carry out countrywide prevalence studies on human T. cruzi infection and triatomine house infestation. Thanks to a coordinated multi-country program in the Southern Cone countries the transmission of Chagas disease by vectors and by blood transfusion has been interrupted in Uruguay in1997, in Chile in 1999, and in 8 of the 12 endemic states of Brazil in 2000 and so the incidence of new infections by T. cruzi in the whole continent has decreased by 70%. Similar control multi-country initiatives have been launched in the Andean countries and in Central America and rapid progress has been recorded to ensure the interruption of the transmission of Chagas disease by 2005 as requested by a Resolution of the World Health Assembly approved in 1998. The cost-benefit analysis of the investments of the vector control program in Brazil indicate that there are savings of US$17 in medical care and disabilities for each dollar spent on prevention, showing that the program is a health investment with good return. Since the inception in 1979 of the Steering Committee on Chagas Disease of the Special Program for Research and Training in Tropical Diseases of the World Health Organization (TDR), the objective was set to promote and finance research aimed at the development of new methods and tools to control this disease. The well known research institutions in Latin America were the key elements of a world wide network of laboratories that received - on a competitive basis - financial support for projects in line with the priorities established. It is presented the time line of the different milestones that were answering successively and logically the outstanding scientific questions identified by the Scientific Working Group in 1978 and that influenced the development and industrial production of practical solutions for diagnosis of the infection and disease control.

Combination of enteral and parenteral nutrition in intensive care patients: A new concept of optimization

Undernutrition is a widespread problem in intensive care unit and is associated with a worse clinical outcome. A state of negative energy balance increases stress catabolism and is associated with increased morbidity and mortality in ICU patients. Undernutrition-related increased morbidity is correlated with an increase in the length of hospital stay and health care costs. Enteral nutrition is the recommended feeding route in critically ill patients, but it is often insufficient to cover the nutritional needs. The initiation of supplemental parenteral nutrition, when enteral nutrition is insufficient, could optimize the nutritional therapy by preventing the onset of early energy deficiency, and thus, could allow to reduce morbidity, length of stay and costs, shorten recovery period and, finally, improve quality of life. (C) 2009 Elsevier Masson SAS. All rights reserved.

Effect of acute and chronic exercise on fat oxidation and hormonal and plasma metabolite kinetics in obese and severely obese men

Acute exercise increases energy expenditure (EE) during exercise and post-exercise recovery [excess post-exercise oxygen consumption (EPOC)] and therefore may be recommended as part of the multidisciplinary management of obesity. Moreover, chronic exercise (training) effectively promotes an increase in insulin sensitivity, which seems to be associated with increased fat oxidation rates (FORs). The main purpose of this thesis is to investigate 1) FORs and extra-muscular factors (hormones and plasma metabolites) that regulate fat metabolism during acute and chronic exercise; and 2) EPOC during acute post-exercise recovery in obese and severely obese men (class II and III). In the first study, we showed that obese and severely obese men present a lower exercise intensity (Fatmax) eliciting maximal fat oxidation and a lower reliance on fat oxidation at high, but not at low and moderate, exercise intensities compared to lean men. This was most likely related to an impaired muscular capacity to oxidize non-esterified fatty acids (NEFA) rather than decreased plasma NEFA availability or a change in the hormonal milieu during exercise. In the second study, we developed an accurate maximal incremental test to correctly and simultaneously evaluate aerobic fitness and fat oxidation kinetics during exercise in this population. This test may be used for the prescription of an appropriate exercise training intensity. In the third study, we demonstrated that only 2 wk of exercise training [continuous training at Fatmax and adapted high-intensity interval training (HIIT)], matched with respect to mechanical work, may be effective to improve aerobic fitness, FORs during exercise and insulin sensitivity, which suggest that FORs might be rapidly improved and that adapted HIIT is feasible in this population. The increased FORs concomitant with the lack of changes in lipolysis during exercise suggest an improvement in the mismatching between NEFA availability and oxidation, highlighting the importance of muscular (oxidative capacity) rather than extra-muscular (hormones and plasma metabolites) factors in the regulation of fat metabolism after a training program. In the fourth study, we observed a positive correlation between EE during exercise and EPOC, suggesting that a chronic increase in the volume or intensity of exercise may increase EE during exercise and during recovery. This may have an impact in weight management in obesity. In conclusion, these findings might have practical implications for exercise training prescriptions in order to improve the therapeutic approaches in obesity and severe obesity. -- L'exercice aigu augmente la dépense énergétique (DE) pendant l'exercice et la récupération post-exercice [excès de consommation d'oxygène post-exercise (EPOC)] et peut être utilisé dans la gestion multidisciplinaire de l'obésité. Quant à l'exercice chronique (entraînement), il est efficace pour augmenter la sensibilité à l'insuline, ce qui semble être associé à une amélioration du débit d'oxydation lipidique (DOL). Le but de cette thèse est d'étudier 1) le DOL et les facteurs extra-musculaires (hormones et métabolites plasmatiques) qui régulent le métabolisme lipidique pendant l'exercice aigu et chronique et 2) l'EPOC lors de la récupération aiguë post-exercice chez des hommes obèses et sévèrement obèses (classe II et III). Dans la première étude nous avons montré que les hommes obèses et sévèrement obèses présentent une plus basse intensité d'exercice (Fatmax) correspondant au débit d'oxydation lipidique maximale et un plus bas DOL à hautes, mais pas à faibles et modérées, intensités d'exercice comparé aux sujets normo-poids, ce qui est probablement lié à une incapacité musculaire à oxyder les acides gras non-estérifiés (AGNE) plutôt qu'à une diminution de leur disponibilité ou à un changement du milieu hormonal pendant l'exercice. Dans la deuxième étude nous avons développé un test maximal incrémental pour évaluer simultanément l'aptitude physique aérobie et la cinétique d'oxydation des lipides pendant l'exercice chez cette population. Dans la troisième étude nous avons montré que seulement deux semaines d'entraînement (continu à Fatmax et intermittent à haute intensité), appariés par la charge de travail, sont efficaces pour améliorer l'aptitude physique aérobie, le DOL pendant l'exercice et la sensibilité à l'insuline, ce qui suggère que le DOL peut être rapidement amélioré chez cette population. Ceci, en absence de changements de la lipolyse pendant l'exercice, suggère une amélioration de la balance entre la disponibilité et l'oxydation des AGNE, ce qui souligne l'importance des facteurs musculaires (capacité oxydative) plutôt que extra-musculaires (hormones et métabolites plasmatiques) dans la régulation du métabolisme lipidique après un entraînement. Dans la quatrième étude nous avons observé une corrélation positive entre la DE pendant l'exercice et l'EPOC, ce qui suggère qu'une augmentation chronique du volume ou de l'intensité de l'exercice pourrait augmenter la DE lors de l'exercice et lors de la récupération post-exercice. Ceci pourrait avoir un impact sur la gestion du poids chez cette population. En conclusion, ces résultats pourraient avoir des implications pratiques lors de la prescription des entraînements dans le but d'améliorer les approches thérapeutiques de l'obésité et de l'obésité sévère.

Immunological and non-immunological effects of cytokines and chemokines in the pathogenesis of chronic Chagas disease cardiomyopathy

The pathogenesis of Chagas disease cardiomyopathy (CCC) is not well understood. Since studies show that myocarditis is more frequent during the advanced stages of the disease, and the prognosis of CCC is worse than that of other dilated cardiomyopathies of non-inflammatory aetiology, which suggest that the inflammatory infiltrate plays a major role in myocardial damage. In the last decade, increasing evidence has shown that inflammatory cytokines and chemokines play a role in the generation of the inflammatory infiltrate and tissue damage. CCC patients have an increased peripheral production of the inflammatory Th1 cytokines IFN-³ and TNF-± when compared to patients with the asymptomatic/indeterminate form. Moreover, Th1-T cells are the main producers of IFN-³ and TNF-± and are frequently found in CCC myocardial inflammatory infiltrate. Over the past several years, our group has collected evidence that shows several cytokines and chemokines produced in the CCC myocardium may also have a non-immunological pathogenic effect via modulation of gene and protein expression in cardiomyocytes and other myocardial cell types. Furthermore, genetic polymorphisms of cytokine, chemokine and innate immune response genes have been associated with disease progression. We will review the molecular and immunological mechanisms of myocardial damage in human CCC in light of recent findings.

A MODEL OF CREATINE DEFICIENCY SYNDROMES IN 3D BRAIN CELL CULTURES BY KNOCKDOWN OF GAMT AND SLC6A8 GENES

La créatine joue un rôle essentiel dans le métabolisme cellulaire par sa conversion, par la creatine kinase, en phosphocreatine permettant la régénération de l'ATP. La synthèse de créatine, chez les mammifères, s'effectue par une réaction en deux étapes impliquant Γ arginine: glycine amidinotransférase (AGAT) et la guanidinoacétate méthyltransférase (GAMT). L'entrée de créatine dans les cellules s'effectue par son transporteur, SLC6A8. Les déficiences en créatine, dues au déficit en GAMT, AGAT ou SLC6A8, sont fréquentes et caractérisées par une absence ou une forte baisse de créatine dans le système nerveux central. Alors qu'il est connu que AGAT, GAMT et SLC6A8 sont exprimés par le cerveau, les conséquences des déficiences en créatine sur les cellules nerveuses sont peu comprises. Le but de ce travail était de développer de nouveaux modèles expérimentaux des déficiences en Cr dans des cultures 3D de cellules nerveuses de rat en agrégats au moyen de l'interférence à l'ARN appliquée aux gènes GAMT et SLC6A8. Des séquences interférentes (shRNAs) pour les gènes GAMT et SLC6A8 ont été transduites par des vecteurs viraux AAV (virus adéno-associés), dans les cellules nerveuses en agrégats. Nous avons ainsi démontré une baisse de l'expression de GAMT au niveau protéique (mesuré par western blot), et ARN messager (mesuré par qPCR) ainsi qu'une variation caractérisitique de créatine et guanidinoacétate (mesuré par spectrométrie de masse). Après avoir validé nos modèles, nous avons montré que les knockdown de GAMT ou SLC6A8 affectent le développement des astrocytes et des neurones ou des oligodendrocytes et des astrocytes, respectivement, ainsi qu'une augmentation de la mort cellulaire et des modifications dans le pattern d'activation des voies de signalisation impliquant caspase 3 et p38 MAPK, ayant un rôle dans le processus d'apoptose. - Creatine plays essential roles in energy metabolism by the interconversion, by creatine kinase, to its phosphorylated analogue, phosphocreatine, allowing the regeneration of ATP. Creatine is synthesized in mammals by a two step mechanism involving arginine:glycine amidinotransferase (AGAT) and guanidinoacetate methyltransferase (GAMT). Creatine is taken up by cells by a specific transporter, SLC6A8. Creatine deficiency syndromes, due to defects in GAMT, AGAT and SLC6A8, are among the most frequent inborn errors of metabolism, and are characterized by an absence or a severe decrease of creatine in central nervous system, which is the main tissue affected. While it is known that AGAT, GAMT and SLC6A8 are expressed in CNS, many questions remain on the specific effects of AGAT, GAMT and SLC6A8 deficiencies on brain cells. Our aim was to develop new experimental models of creatine deficiencies by knockdown of GAMT and SLC6A8 genes by RNAi in 3D organotypic rat brain cell cultures in aggregates. Specific shRNAs for the GAMT and SLC6A8 genes were transduced in brain cell aggregates by adeno-associated viruses (AAV). The AAV-transduced shRNAs were able to efficiently knockdown the expression of our genes of interest, as shown by a strong decrease of protein by western blotting, a decrease of mRNA by qPCR or characteristic variations of creatine and guanidinoacetate by tandem mass spectrometry. After having validated our experimental models, we have also shown that GAMT and SLC6A8 knockdown affected the development of astrocytes and neurons or oligodendrocytes and astrocytes, respectively. We also observed an increase of cell death and variations in activation pattern of caspase 3 and p38 MAPK pathways, involved in apoptosis, in our experimental model.

Impact of thermal proofing of a church on its indoor air-quality - The combustion of candles and incense as a source of pollution

Some years ago, a parish in Geneva decided to reduce heating costs by insulating its church to make it more energy efficient. Three years after the last renovations, it was observed that the internal surfaces of the naves had already become dusty compared with the customary frequency of 10-12 years. Dust even deposited on various surfaces during religious services. Our investigation showed that nearly all the dust found inside the church may in fact be soot from incense and candle combustion. Incense appears to be a significant source of polycyclic aromatic hydrocarbons. With a mechanical ventilation system and petrol lamps resembling candles the problem can be resolved.

Construction and Quantitative Evaluation of a Dual Specific Promoter System for Monitoring the Expression Status of Stra8 and c-kit Genes.

Applications of genetic constructs with multiple promoters, which are fused with reporter genes and simultaneous monitoring of various events in cells, have gained special attention in recent years. Lentiviral vectors, with their distinctive characteristics, have been considered to monitor the developmental changes of cells in vitro. In this study, we constructed a novel lentiviral vector (FUM-M), containing two germ cell-specific promoters (Stra8 and c-kit), fused with ZsGreen and DsRed2 reporter genes, and evaluated its efficiency in different cells following treatments with retinoic acid and DMSO. Several cell lines (P19, GC-1 spg and HEK293T) were transduced with this vector, and functional capabilities of the promoters were verified by flow cytometry and quantitative RT-PCR. Our results indicate that FUM-M shows dynamic behavior in the presence and absence of extrinsic factors. A correlation was also observed between the function of promoters, present in the lentiviral construct and the endogenous level of the Stra8 and c-kit mRNAs in the cells. In conclusion, we recommend this strategy, which needs further optimization of the constructs, as a beneficial and practical way to screen chemical inducers involved in cellular differentiation toward germ-like cells.

Surface-expressed enolases of Plasmodium and other pathogens

Enolase is the eighth enzyme in the glycolytic pathway, a reaction that generates ATP from phosphoenol pyruvate in cytosolic compartments. Enolase is essential, especially for organisms devoid of the Krebs cycle that depend solely on glycolysis for energy. Interestingly, enolase appears to serve a separate function in some organisms, in that it is also exported to the cell surface via a poorly understood mechanism. In these organisms, surface enolase assists in the invasion of their host cells by binding plasminogen, an abundant plasma protease precursor. Binding is mediated by the interaction between a lysine motif of enolase with Kringle domains of plasminogen. The bound plasminogen is then cleaved by specific proteases to generate active plasmin. Plasmin is a potent serine protease that is thought to function in the degradation of the extracellular matrix surrounding the targeted host cell, thereby facilitating pathogen invasion. Recent work revealed that the malaria parasite Plasmodium also expresses surface enolase, and that this feature may be essential for completion of its life cycle. The therapeutic potential of targeting surface enolases of pathogens is discussed.

Separating the contribution of glucocorticoids and wakefulness to the molecular and electrophysiological correlates of sleep homeostasis.

Study Objectives: The sleep-deprivation-induced changes in delta power, an electroencephalographical correlate of sleep need, and brain transcriptome profiles have importantly contributed to current hypotheses on sleep function. Because sleep deprivation also induces stress, we here determined the contribution of the corticosterone component of the stress response to the electrophysiological and molecular markers of sleep need in mice. Design: N/A Settings: Mouse sleep facility. Participants: C57BL/6J, AKR/J, DBA/2J mice. Interventions: Sleep deprivation, adrenalectomy (ADX). Measurements and Results: Sleep deprivation elevated corticosterone levels in 3 inbred strains, but this increase was larger in DBA/2J mice; i.e., the strain for which the rebound in delta power after sleep deprivation failed to reach significance. Elimination of the sleep-deprivation-associated corticosterone surge through ADX in DBA/2J mice did not, however, rescue the delta power rebound but did greatly reduce the number of transcripts affected by sleep deprivation. Genes no longer affected by sleep deprivation cover pathways previously implicated in sleep homeostasis, such as lipid, cholesterol (e.g., Ldlr, Hmgcs1, Dhcr7, -24, Fkbp5), energy and carbohydrate metabolism (e.g., Eno3, G6pc3, Mpdu1, Ugdh, Man1b1), protein biosynthesis (e.g., Sgk1, Alad, Fads3, Eif2c2, -3, Mat2a), and some circadian genes (Per1, -3), whereas others, such as Homer1a, remained unchanged. Moreover, several microRNAs were affected both by sleep deprivation and ADX. Conclusions: Our findings indicate that corticosterone contributes to the sleep-deprivation-induced changes in brain transcriptome that have been attributed to wakefulness per se. The study identified 78 transcripts that respond to sleep loss independent of corticosterone and time of day, among which genes involved in neuroprotection prominently feature, pointing to a molecular pathway directly relevant for sleep function.

Genome-Wide Analysis of Salicylate and Dibenzofuran Metabolism in Sphingomonas Wittichii RW1.

Sphingomonas wittichii RW1 is a bacterium isolated for its ability to degrade the xenobiotic compounds dibenzodioxin and dibenzofuran (DBF). A number of genes involved in DBF degradation have been previously characterized, such as the dxn cluster, dbfB, and the electron transfer components fdx1, fdx3, and redA2. Here we use a combination of whole genome transcriptome analysis and transposon library screening to characterize RW1 catabolic and other genes implicated in the reaction to or degradation of DBF. To detect differentially expressed genes upon exposure to DBF, we applied three different growth exposure experiments, using either short DBF exposures to actively growing cells or growing them with DBF as sole carbon and energy source. Genome-wide gene expression was examined using a custom-made microarray. In addition, proportional abundance determination of transposon insertions in RW1 libraries grown on salicylate or DBF by ultra-high throughput sequencing was used to infer genes whose interruption caused a fitness loss for growth on DBF. Expression patterns showed that batch and chemostat growth conditions, and short or long exposure of cells to DBF produced very different responses. Numerous other uncharacterized catabolic gene clusters putatively involved in aromatic compound metabolism increased expression in response to DBF. In addition, only very few transposon insertions completely abolished growth on DBF. Some of those (e.g., in dxnA1) were expected, whereas others (in a gene cluster for phenylacetate degradation) were not. Both transcriptomic data and transposon screening suggest operation of multiple redundant and parallel aromatic pathways, depending on DBF exposure. In addition, increased expression of other non-catabolic genes suggests that during initial exposure, S. wittichii RW1 perceives DBF as a stressor, whereas after longer exposure, the compound is recognized as a carbon source and metabolized using several pathways in parallel.

Impact of the gut microbiota on the development of obesity and type 2 diabetes mellitus.

Obesity and its associated disorders are a major public health concern. Although obesity has been mainly related with perturbations of the balance between food intake and energy expenditure, other factors must nevertheless be considered. Recent insight suggests that an altered composition and diversity of gut microbiota could play an important role in the development of metabolic disorders. This review discusses research aimed at understanding the role of gut microbiota in the pathogenesis of obesity and type 2 diabetes mellitus (TDM2). The establishment of gut microbiota is dependent on the type of birth. With effect from this point, gut microbiota remain quite stable, although changes take place between birth and adulthood due to external influences, such as diet, disease and environment. Understand these changes is important to predict diseases and develop therapies. A new theory suggests that gut microbiota contribute to the regulation of energy homeostasis, provoking the development of an impairment in energy homeostasis and causing metabolic diseases, such as insulin resistance or TDM2. The metabolic endotoxemia, modifications in the secretion of incretins and butyrate production might explain the influence of the microbiota in these diseases.

The combination of social and personal contexts affects dominance hierarchy development in shore crabs, Carcinus maenas

Many animals that live in groups maintain competitive relationships, yet avoid continual fighting, by forming dominance hierarchies. We compare predictions of stochastic, individual-based models with empirical experimental evidence using shore crabs to test competing hypotheses regarding hierarchy development. The models test (1) what information individuals use when deciding to fight or retreat, (2) how past experience affects current resource-holding potential, and (3) how individuals deal with changes to the social environment. First, we conclude that crabs assess only their own state and not their opponent's when deciding to fight or retreat. Second, willingness to enter, and performance in, aggressive contests are influenced by previous contest outcomes. Winning increases the likelihood of both fighting and winning future interactions, while losing has the opposite effect. Third, when groups with established dominance hierarchies dissolve and new groups form, individuals reassess their ranks, showing no memory of previous rank or group affiliation. With every change in group composition, individuals fight for their new ranks. This iterative process carries over as groups dissolve and form, which has important implications for the relationship between ability and hierarchy rank. We conclude that dominance hierarchies emerge through an interaction of individual and social factors, and discuss these findings in terms of an underlying mechanism. Overall, our results are consistent with crabs using a cumulative assessment strategy iterated across changes in group composition, in which aggression is constrained by an absolute threshold in energy spent and damage received while fighting.

A nuclear ribosomal DNA pseudogene in triatomines opens a new research field of fundamental and applied implications in Chagas disease

A pseudogene, designated as "ps(5.8S+ITS-2)", paralogous to the 5.8S gene and internal transcribed spacer (ITS)-2 of the nuclear ribosomal DNA (rDNA), has been recently found in many triatomine species distributed throughout North America, Central America and northern South America. Among characteristics used as criteria for pseudogene verification, secondary structures and free energy are highlighted, showing a lower fit between minimum free energy, partition function and centroid structures, although in given cases the fit only appeared to be slightly lower. The unique characteristics of "ps(5.8S+ITS-2)" as a processed or retrotransposed pseudogenic unit of the ghost type are reviewed, with emphasis on its potential functionality compared to the functionality of genes and spacers of the normal rDNA operon. Besides the technical problem of the risk for erroneous sequence results, the usefulness of "ps(5.8S+ITS-2)" for specimen classification, phylogenetic analyses and systematic/taxonomic studies should be highlighted, based on consistence and retention index values, which in pseudogenic sequence trees were higher than in functional sequence trees. Additionally, intraindividual, interpopulational and interspecific differences in pseudogene amount and the fact that it is a pseudogene in the nuclear rDNA suggests a potential relationships with fitness, behaviour and adaptability of triatomine vectors and consequently its potential utility in Chagas disease epidemiology and control.

Comparative review of vertebroplasty and kyphoplasty.

The aim of this review is to compare the effectiveness of percutaneous vertebroplasty and kyphoplasty to treat pain and improve functional outcome from vertebral fractures secondary to osteoporosis and tumor conditions. In 2009, two open randomized controlled trials published in the New England Journal of Medicine questioned the value of vertebroplasty in treating vertebral compression fractures. Nevertheless, the practice of physicians treating these conditions has barely changed. The objective of this review is to try to clarify the most important issues, based on our own experience and the reported evidence about both techniques, and to guide towards the most appropriate choice of treatment of vertebral fractures, although many questions still remain unanswered.