Mutation screening of multiple genes in Spanish patients with autosomal recessive retinitis pigmentosa by targeted resequencing.

Retinitis Pigmentosa (RP) is a heterogeneous group of inherited retinal dystrophies characterised ultimately by the loss of photoreceptor cells. RP is the leading cause of visual loss in individuals younger than 60 years, with a prevalence of about 1 in 4000. The molecular genetic diagnosis of autosomal recessive RP (arRP) is challenging due to the large genetic and clinical heterogeneity. Traditional methods for sequencing arRP genes are often laborious and not easily available and a screening technique that enables the rapid detection of the genetic cause would be very helpful in the clinical practice. The goal of this study was to develop and apply microarray-based resequencing technology capable of detecting both known and novel mutations on a single high-throughput platform. Hence, the coding regions and exon/intron boundaries of 16 arRP genes were resequenced using microarrays in 102 Spanish patients with clinical diagnosis of arRP. All the detected variations were confirmed by direct sequencing and potential pathogenicity was assessed by functional predictions and frequency in controls. For validation purposes 4 positive controls for variants consisting of previously identified changes were hybridized on the array. As a result of the screening, we detected 44 variants, of which 15 are very likely pathogenic detected in 14 arRP families (14%). Finally, the design of this array can easily be transformed in an equivalent diagnostic system based on targeted enrichment followed by next generation sequencing.

Clinical pharmacogenomic testing of KRAS, BRAF and EGFR mutations by high resolution melting analysis and ultra-deep pyrosequencing

BACKGROUND: Epidermal growth factor receptor (EGFR) and its downstream factors KRAS and BRAF are mutated in several types of cancer, affecting the clinical response to EGFR inhibitors. Mutations in the EGFR kinase domain predict sensitivity to the tyrosine kinase inhibitors gefitinib and erlotinib in lung adenocarcinoma, while activating point mutations in KRAS and BRAF confer resistance to the anti-EGFR monoclonal antibody cetuximab in colorectal cancer. The development of new generation methods for systematic mutation screening of these genes will allow more appropriate therapeutic choices. METHODS: We describe a high resolution melting (HRM) assay for mutation detection in EGFR exons 19-21, KRAS codon 12/13 and BRAF V600 using formalin-fixed paraffin-embedded samples. Somatic variation of KRAS exon 2 was also analysed by massively parallel pyrosequencing of amplicons with the GS Junior 454 platform. RESULTS: We tested 120 routine diagnostic specimens from patients with colorectal or lung cancer. Mutations in KRAS, BRAF and EGFR were observed in 41.9%, 13.0% and 11.1% of the overall samples, respectively, being mutually exclusive. For KRAS, six types of substitutions were detected (17 G12D, 9 G13D, 7 G12C, 2 G12A, 2 G12V, 2 G12S), while V600E accounted for all the BRAF activating mutations. Regarding EGFR, two cases showed exon 19 deletions (delE746-A750 and delE746-T751insA) and another two substitutions in exon 21 (one showed L858R with the resistance mutation T590M in exon 20, and the other had P848L mutation). Consistent with earlier reports, our results show that KRAS and BRAF mutation frequencies in colorectal cancer were 44.3% and 13.0%, respectively, while EGFR mutations were detected in 11.1% of the lung cancer specimens. Ultra-deep amplicon pyrosequencing successfully validated the HRM results and allowed detection and quantitation of KRAS somatic mutations. CONCLUSIONS: HRM is a rapid and sensitive method for moderate-throughput cost-effective screening of oncogene mutations in clinical samples. Rather than Sanger sequence validation, next-generation sequencing technology results in more accurate quantitative results in somatic variation and can be achieved at a higher throughput scale.

A new approach for potential drug target discovery through in silico metabolic pathway analysis using Trypanosoma cruzi genome information

The current drug options for the treatment of chronic Chagas disease have not been sufficient and high hopes have been placed on the use of genomic data from the human parasite Trypanosoma cruzi to identify new drug targets and develop appropriate treatments for both acute and chronic Chagas disease. However, the lack of a complete assembly of the genomic sequence and the presence of many predicted proteins with unknown or unsure functions has hampered our complete view of the parasite's metabolic pathways. Moreover, pinpointing new drug targets has proven to be more complex than anticipated and has revealed large holes in our understanding of metabolic pathways and their integrated regulation, not only for this parasite, but for many other similar pathogens. Using an in silicocomparative study on pathway annotation and searching for analogous and specific enzymes, we have been able to predict a considerable number of additional enzymatic functions in T. cruzi. Here we focus on the energetic pathways, such as glycolysis, the pentose phosphate shunt, the Krebs cycle and lipid metabolism. We point out many enzymes that are analogous to those of the human host, which could be potential new therapeutic targets.

Two approaches to discovering and developing new drugs for Chagas disease

This review will focus on two general approaches carried out at the Sandler Center, University of California, San Francisco, to address the challenge of developing new drugs for the treatment of Chagas disease. The first approach is target-based drug discovery, and two specific targets, cytochrome P450 CYP51 and cruzain (aka cruzipain), are discussed. A "proof of concept" molecule, the vinyl sulfone inhibitor K777, is now a clinical candidate. The preclinical assessment compliance for filing as an Investigational New Drug with the United States Food and Drug Administration (FDA) is presented, and an outline of potential clinical trials is given. The second approach to identifying new drug leads is parasite phenotypic screens in culture. The development of an assay allowing high throughput screening of Trypanosoma cruzi amastigotes in skeletal muscle cells is presented. This screen has the advantage of not requiring specific strains of parasites, so it could be used with field isolates, drug resistant strains or laboratory strains. It is optimized for robotic liquid handling and has been validated through a screen of a library of FDA-approved drugs identifying 65 hits.

Exposure to Trihalomethanes through Different Water Uses and Birth Weight, Small for Gestational Age, and Preterm Delivery in Spain

BACKGROUND Evidence associating exposure to water disinfection by-products with reduced birth weight and altered duration of gestation remains inconclusive. OBJECTIVE We assessed exposure to trihalomethanes (THMs) during pregnancy through different water uses and evaluated the association with birth weight, small for gestational age (SGA), low birth weight (LBW), and preterm delivery. METHODS Mother-child cohorts set up in five Spanish areas during the years 2000-2008 contributed data on water ingestion, showering, bathing, and swimming in pools. We ascertained residential THM levels during pregnancy periods through ad hoc sampling campaigns (828 measurements) and regulatory data (264 measurements), which were modeled and combined with personal water use and uptake factors to estimate personal uptake. We defined outcomes following standard definitions and included 2,158 newborns in the analysis. RESULTS Median residential THM ranged from 5.9 μg/L (Valencia) to 114.7 μg/L (Sabadell), and speciation differed across areas. We estimated that 89% of residential chloroform and 96% of brominated THM uptakes were from showering/bathing. The estimated change of birth weight for a 10% increase in residential uptake was -0.45 g (95% confidence interval: -1.36, 0.45 g) for chloroform and 0.16 g (-1.38, 1.70 g) for brominated THMs. Overall, THMs were not associated with SGA, LBW, or preterm delivery. CONCLUSIONS Despite the high THM levels in some areas and the extensive exposure assessment, results suggest that residential THM exposure during pregnancy driven by inhalation and dermal contact routes is not associated with birth weight, SGA, LBW, or preterm delivery in Spain.

Codeine and breastfeeding: cases and STIS position

Codeine is commonly used in North America in the postpartum period for pain associated with episotomyand caesarean section. Analgesic properties of codeine are mainly due to its metabolisation intomorphine (5-10%) via CYP2D6. This enzyme is subject to genetic variability, which can alter theamount of active narcotic excreted into breastmilk. A recent case report highlighted this issue, reportingfatal consequences in a newborn whose mother was taking codeine for episiotomy-related pain (1-2). New-born's blood (post-mortem) and mother's milk showed high morphine concentrations. Genotypeanalysis classified the mother as a CYP2D6 ultrarapid metabolizer, a genotype associated withenhanced formation of morphine from codeine. The authors concluded "clinical and laboratory picturewas consistent with opioid toxicity leading to neonatal death". Subsequent comments expressed reasonnabledoubts on this conclusion, though (3-4). Since, anxiety increased about the safety of codeineduring breastfeeding and genetic screening was proposed as a prevention strategy.STIS position:? Codeine with paracetamol is not a usual pain prescription in the postpartum period in Switzerland.This markedly reduces codeine use during lactation in our country, and may partly explain why webarely collected 3 codeine exposures through breastmilk in 15 years at the STIS (all reported afterabove case's publication and without side effects).? Other centrally acting analgesics are not considered safer (5) than codeine during lactation andrequire close observation for somnolence in both the mother and the infant in case of repeated maternaldosage. A lack of monitoring was salient in the case reported above (1).? If the incidence of CYP2D6 polymorphism (1-10% of individuals in Western Europe) (6) can beconsidered of clinical significance, it is not the exclusive predisposing factor to toxic effects. Healthynewborns can be particularly sensitive to even usual doses of narcotic analgesics because of immaturedrug disposition (7). Conditions leading to impaired clearance or increased susceptibility inthe infant (e.g. preterm birth, metabolic diseases) represent further risk factors for opioid toxicity,regardless of the molecule.In conclusion, when prescribed on a large scale, codein can be rarely associated with adverse drugreactions in breastfed infants (8-9). However, other central acting analgesics cannot be considered asinvariably safer. Therefore, paracetamol and well documented NSAID should be used in 1st choiceduring lactation. In case of severe pain, codeine (with paracetamol) remains an acceptable choice butrequires close monitoring, and breastfeeding mothers should be educated regarding risks related toaccumulation in the newborn. Finally, it is doubtful whether CYP2D6 genetic screening would preventall toxic effects, as other risk factors exist for opioids toxicity

Increased soluble Fas plasma levels in subjects at high cardiovascular risk - Atorvastatin on inflammatory markers (AIM) study, a substudy of ACTFAST

OBJECTIVE Increasing evidence indicates that the Fas/Fas ligand interaction is involved in atherogenesis. We sought to analyze soluble Fas (sFas) and soluble Fas ligand (sFasL) concentrations in subjects at high cardiovascular risk and their modulation by atorvastatin treatment. METHODS AND RESULTS ACTFAST was a 12-week, prospective, multicenter, open-label trial which enrolled subjects (statin-free or statin-treated at baseline) with coronary heart disease (CHD), CHD-equivalent, or 10-year CHD risk > 20%. Subjects with LDL-C between 100 to 220 mg/dL (2.6 to 5.7 mmol/L) and triglycerides < or = 600 mg/dL (6.8 mmol/L) were assigned to a starting dose of atorvastatin (10 to 80 mg/d) based on LDL-C at screening. Of the 2117 subjects enrolled in ACTFAST, AIM sub-study included the 1078 statin-free patients. At study end, 85% of these subjects reached LDL-C target. Mean sFas levels were increased and sFasL were reduced in subjects at high cardiovascular risk compared with healthy subjects. Atorvastatin reduced sFas in the whole population as well as in patients with metabolic syndrome or diabetes. Minimal changes were observed in sFasL. CONCLUSIONS sFas concentrations are increased and sFasL are decreased in subjects at high cardiovascular risk, suggesting that these proteins may be novel markers of vascular injury. Atorvastatin reduces sFas, indicating that short-term treatment with atorvastatin exhibits antiinflammatory effects in these subjects.

Optimising translational oncology in clinical practice: Strategies to accelerate progress in drug development.

Despite intense efforts, the socioeconomic burden of cancer remains unacceptably high and treatment advances for many common cancers have been limited, suggesting a need for a new approach to drug development. One issue central to this lack of progress is the heterogeneity and genetic complexity of many tumours. This results in considerable variability in therapeutic response and requires knowledge of the molecular profile of the tumour to guide appropriate treatment selection for individual patients. While recent advances in the molecular characterisation of different cancer types have the potential to transform cancer treatment through precision medicine, such an approach presents a major economic challenge for drug development, since novel targeted agents may only be suitable for a small cohort of patients. Identifying the patients who would benefit from individual therapies and recruiting sufficient numbers of patients with particular cancer subtypes into clinical trials is challenging, and will require collaborative efforts from research groups and industry in order to accelerate progress. A number of molecular screening platforms have already been initiated across Europe, and it is hoped that these networks, along with future collaborations, will benefit not only patients but also society through cost reductions as a result of more efficient use of resources. This review discusses how current developments in translational oncology may be applied in clinical practice in the future, assesses current programmes for the molecular characterisation of cancer and describes possible collaborative approaches designed to maximise the benefits of translational science for patients with cancer.

High host-plant nitrogen content: a prerequisite for the evolution of ant-caterpillar mutualism?

The amount of nitrogen required to complete an insect's life cycle may vary greatly among species that have evolved distinct life history traits. Myrmecophilous caterpillars in the Lycaenidae family produce nitrogen-rich exudates from their dorsal glands to attract ants for protection, and this phenomenon has been postulated to shape the caterpillar's host-plant choice. Accordingly, it was postulated that evolution towards myrmecophily in Lycaenidae is correlated with the utilization of nitrogen-rich host plants. Although our results were consistent with the evolutionary shifts towards high-nutrient host plants serving as exaptation for the evolution of myrmecophily in lycaenids, the selection of nitrogen-rich host plants was not confined to lycaenids. Butterfly species in the nonmyrmecophilous family Pieridae also preferred nitrogen-rich host plants. Thus, we conclude that nitrogen is an overall important component in the caterpillar diet, independent of the level of myrmecophily, as nitrogen can enhance the overall insect fitness and survival. However, when nitrogen can be obtained through alternative means, as in socially parasitic lycaenid species feeding on ant brood, the selective pressure for maintaining the use of nutrient-rich host plants is relaxed, enabling the colonization of nitrogen-poor host plants.

Co-infections associated with human immunodeficiency virus type 1 in pregnant women from southern Brazil: high rate of intraepithelial cervical lesions

Human immunodeficiency virus type 1 (HIV-positive) pregnant women require specific prophylactic and therapeutic approaches. The efficacy of established approaches is further challenged by co-infection with other sexually transmitted diseases (STDs). The objective of this study was to determine the prevalence of co-infections in pregnant women infected with different HIV-1 subtypes and to relate these findings, together with additional demographic and clinical parameters, to maternal and infant outcomes. Blood samples from pregnant women were collected and tested for syphilis, hepatitis B virus (HBV) and hepatitis C virus (HCV). Human papillomavirus (HPV) diagnosis was evaluated by the presence of alterations in the cervical epithelium detected through a cytopathological exam. Medical charts provided patient data for the mothers and children. Statistical analyses were conducted with STATA 9.0. We found a prevalence of 10.8% for HCV, 2.3% for chronic HBV, 3.1% for syphilis and 40.8% for HPV. Of those co-infected with HPV, 52.9% presented high-grade intraepithelial lesions or in situ carcinoma. Prematurity, birth weight, Apgar 1' and 5' and Capurro scores were similar between co-infected and non-co-infected women. The presence of other STDs did not impact maternal and concept outcomes. More than half of the patients presenting cervical cytology abnormalities suggestive of HPV had high-grade squamous intraepithelial lesions or cervical cancer, evidencing an alarming rate of these lesions.

Zebrafish Based Small Molecule Screening For Retinoblastoma Treatment.

2320 composés chimiques ont été screenés à l'aide d'une lignée transgénique de zébrafish. Cette lignée comportait un gène humain fortement exprimé très tôt dans le développement et la croissance de différentes tumeurs, dont celle du rétinoblastome. L'activation de ce gène induisait la mort des embryons de lâ lignée transgénique. Nous avons donc pu identifier des composés agissant sur l'effet létal de ce gène. Cette étude a permis d'isoler plusieurs composés dont 1 très intéressant, l'Amitriptyline. Ce composé induit une inhibition de la prolifération et une induction d'apoptose dans les cellules humaines de rétinoblastome mais également dans d'autres cellules cancéreuses dont les ostéosarcomes connus. L'ostéosarçome est connu pour faire partie des cancers secondaires dû au rétinoblastome dans la forme héréditaire notamment. Ce composé induit la survie des embryons et réduit également le niveau d'expression de la protéine humaine intégrée dans la lignée de poisson zèbre transgénique de 50%. Le niveau d'expression de ce gène est également réduit de 50 à 60% dans des cultures cellulaire de rétinoblastome humain. L'inhibition de la prolifération a été démontrée par la réduction d'ATP dans plusieurs lignées cellulaires lorsque celles-ci sont traitées avec ce composé. L'induction d'apoptose a été démontrée par induction 10 fois plus élevée des éléments pro-apoptotiques caspase-3 et caspase-7 ainsi que par l'augmentation 10 fois plus élevée d'un élément anti-apoptotique bcl-2. Ces résultats permettent de croire que ce composé pourrait être utilisé pour traiter le rétinoblastome humain. -- Background: Retinoblastoma is a rare malignant tumor. This disease is the most prevalent intraocular cancer in childhood with an incidence of 1 in 15,000 live births. Many therapies are available to treat retinoblastoma, but best treatments are individually selected according to cases. Cryotherapy, thermotherapy, laser therapy including brachytherapy, radiation therapy and chemotherapy are some examples. New drug and new treatments discovery is essential for cancer therapy including retinoblastoma. Purpose: A vertebrate model as zebrafish for retinoblastoma would provide many advantages especially to perform drug screening. The high number of fertilized eggs per mating, the rapidity of extra¬utero development, the available genetic manipulation, the easy manipulations under a microscope, the ability to dispense embryos in 96-well plates and the direct incubation of chemical compounds in fish water are some examples of the advantages. Therefore, we design a transgenic zebrafish carrying a human gene implicated in retinoblastoma development and maintenance. Results: With the small compounds screening, several compounds were isolated. One of these compounds, Amitriptyline demonstrated proliferation inhibition and apoptosis in human retinoblastoma cells, U20S osteosarcoma cells and MBA-231 breast cancer cells. Osteosarcoma is known as secondary cancer due to retinoblastoma. Amitriptyline induced survival in our zebrafish transgenic line and 50% reduction of the integrated gene expression. In retinoblastoma cultured cells, the expression of this gene was also reduced in a range of 50-60 %. Proliferation inhibition was demonstrated by ATP luminescence assay. Apoptosis was demonstrated by a 10-fold induction of caspase-3 and caspase-7, two pro-apoptotic elements and by a 10-fold reduction of bcl-2 anti-apoptotic element. Conclusion: The results suggest that Amitriptyline could be used to treat human retinoblastoma in the near future.

Validation of a modified fluorimetric assay for the screening of trichomonacidal drugs

A fluorimetric microassay that uses a redox dye to determine the viability of the flagellate Trichomonas vaginalis has been optimised to provide a more sensitive method to evaluate potential trichomonacidal compounds. Resazurin has been used in recent years to test drugs against different parasites, including trichomonadid protozoa; however, the reproducibility of these resazurin-based methods in our laboratory has been limited because the flagellate culture medium spontaneously reduces the resazurin. The objective of this work was to refine the fluorimetric microassay method previously developed by other research groups to reduce the fluorescence background generated by the media and increase the sensitivity of the screening assay. The experimental conditions, time of incubation, resazurin concentration and media used in the microtitre plates were adjusted. Different drug sensitivity studies against T. vaginalis were developed using the 5-nitroimidazole reference drugs, new 5-nitroindazolinones and 5-nitroindazole synthetic derivatives. Haemocytometer count results were compared with the resazurin assay using a 10% solution of 3 mM resazurin dissolved in phosphate buffered saline with glucose (1 mg/mL). The fluorimetric assay and the haemocytometer counts resulted in similar percentages of trichomonacidal activity in all the experiments, demonstrating that the fluorimetric microtitre assay has the necessary accuracy for high-throughput screening of new drugs against T. vaginalis.

Characteristics of leprosy diagnosed through the surveillance of contacts: a comparison with index cases in Rio de Janeiro, 1987-2010

Contact surveillance is an important strategy to ensure effective early diagnosis and control of leprosy; passive detection may not be as efficient because it is directly tied to the ready availability of heath care services and health education campaigns. The aim of this study was to reinforce that contact surveillance is the most effective strategy for the control of leprosy. The analysed data were obtained from a cohort of contacts and cases diagnosed through a national referral service for leprosy. We analysed data from patients diagnosed between 1987-2010 at the Souza Araújo Ambulatory in Rio de Janeiro. Epidemiological characteristics of leprosy cases diagnosed through contact surveillance and characteristics of passively detected index cases were compared using a conditional logistic regression model. Cases diagnosed by contact surveillance were found earlier in the progression of the disease, resulting in less severe clinical presentations, lower levels of initial and final disability grades, lower initial and final bacterial indices and a lower prevalence of disease reaction. In this respect, contact surveillance proved to be an effective tertiary prevention strategy, indicating that active surveillance is especially important in areas of high endemicity, such as Brazil.

Saccharomyces cerevisiae, a tool to study the β-oxidation through the synthesis of polyhydroxyalkanoates

Summary Polyhydroxyalkanoates (PHAs) represent a family of polyesters naturally synthesized by a wide variety of bacteria. Through their thermoplastic and elastomeric qualities, together with their biodegradable and renewable properties, they are predicted to be a good alternative to the petroleum- derived plastics. Nevertheless, as PHA production costs using bacteria fermentation are still too high, PHA synthesis within eukaryotic systems, such as plants, has been elaborated. Although the costs were then efficiently lowered, the yield of PHAs produced remained low. In this study, Saccharomyces cerevisae has been used as another eukaryotic model in order to reveal the steps which limit PHA production. These cells express the PHA synthase of Pseudomonas aeruginosa and the PHAs obtained were analyzed to understand the flux of fatty acids towards and through the peroxisomal β-oxidation core cycle, generating the main substrate of the PHA synthase. When S. cerevisiae wild-type cells are grown in a media containing glucose as carbon source as well as fatty acids, the PHA monomer composition is largely influenced by the nature of the external fatty acid used. Thus, even-chain PHA monomers are generated from oleic acid (18:1Δ9cis) and odd- chain PHA monomers are generated from heptadecenoic acid (17:1Δ. 10 cis). Moreover, PHA synthesis is dependent on the first two enzymes of the 0-oxidation core cycle, the acyl-CoA oxidase and the multifunctional enzyme enoyl-CoA hydratase II / R-3-hydroxyacyl-CoA dehydrogenase. S. cerevisiae mutant cells growing on oleic or heptadecenoic acid and deficient in either the R-3- hydroxyacyl-CoA dehydrogenase or in the 3-ketothiolase activity, the last β-oxidation cycle steps, surprisingly contained PHAs of predominantly even-chain monomers. This is also noticed in wild- type and mutants grown on glucose or raffinose, indicating that the substrate used for PHA synthesis is generated from the degradation of intracellular short- and medium-chain fatty acids by the 3- oxidation cycle. Inhibition of fatty acid biosynthesis by cerulenin blocks the synthesis of PHAs from intracellular fatty acids but still enables the use of extracellular fatty acids for polymer production. Together, these results uncovered the existence of a substantial futile cycle whereby short- and medium-chain intermediates of the cytoplasmic fatty acid biosynthetic pathway are directed towards the peroxisomal β-oxidation pathway. In this thesis, no increase of the yield of PHA produced could be obtained. But the PHA synthesis confirmed the carbon flux into and through the β-oxidation core cycle and unveiled the existence of novel mechanisms. It is thus a good tool to study in vivo the flux of carbons in S. cerevisiae cells. Résumé Les polyhydroxyalkanoates (PHAs) sont une famille de polyesters naturellement synthétisés par un grand nombre de bactéries. Ayant des propriétés de thermoplastiques, d'élastomères et étant des ressources biodégradables et renouvelables, les PHAs représentent une bonne alternative aux plastiques dérivés du pétrole. Pour pallier aux coûts considérables de la production de PHAs par fermentation bactérienne, la synthèse de PHAs par des systèmes eucaryotes telles les plantes a été élaborée. Les coûts ont ainsi efficacement été diminués, mais le rendement de PHAs produits reste faible. Dans cette étude, Saccharomyces cerevisiae a été utilisé comme autre modèle eucaryote pour révéler les étapes limitantes de la production de PHAs. Les PHAs obtenus dans les cellules exprimant la F'HA synthase de Pseudomonas aeruginosa ont été analysés afin de comprendre le flux d'acides gras vers et à travers le cycle péroxisomal de la β-oxidation, principal producteur du substrat de la PHA synthase. Lorsque la souche S. cerevisiae de type sauvage se développe dans un milieu contenant du glucose et des acides gras, la composition des monomères de PHAs est influencée par la nature des acides gras extracellulaires. Ainsi, les monomères pairs sont générés par l'acide oléique (18:1Δ9cis), tandis que les impairs le sont par l'acide heptadécénoïque (17:1Δ10cis). La synthèse de PHAs est dépendante des deux premières enzymes de la β-oxidation; l'acyl-CoA oxidase et l'enzyme multifonctionnelle enoyl-CoA hydratase II / R-3-hydroxyacyl-CoA déshydrogénase. Les souches mutantes ne possédant pas les activités de la R-3-hydroxyacyl-CoA déshydrogénase ou de la 3- ketothiolase contiennent, en présence d'acide oléique ou heptadécénoïque, des PHAs composés essentiellement de monomères pairs. Cela a également été observé en présence de glucose ou de raffinose uniquement. Le substrat utilisé pour la synthèse de PHAs a ainsi été généré par la dégradation d'acides gras intracellulaires à chaîne courte et moyenne via le cycle de la β-oxidation. L'inhibition de la synthèse d'acides gras par la cérulénine a bloqué la synthèse de PHAs par les acides gras internes. Ces résultats ont révélés l'existence d'un cycle futile par lequel des intermédiaires à chaîne courte et moyenne de la synthèse cytoplasmique d'acides gras sont dirigés vers le cycle péroxisomal de la β-oxidation. Dans cette étude, le rendement de PHAs produits reste inchangé, mais l'analyse des PHAs permet de confirmer le flux de carbones vers et à travers le cycle péroxisomal de la β-oxidation et l'existence de nouveaux méchanismes a été dévoilée. Cette synthèse s'avère être un bon outil pour étudier in vivo le flux de carbones dans les cellules de S. cerevisiae.

Medical geography and phthisic immunity in the high altitudes: The origins of a Therapeutic hypothesis

Medical geography expanded considerably in the 19 th century. Its expansion was aided by a Neo-Hippocratic trend in medical thinking, progress in statistics and hygiene, and an overall vision of geography formulated early in the century by French and German geographers inspired by Alexander von Humboldt. By tracing out the process that prompted certain « doctor-geographers » to put forth the hypothesis of immunity phthisis in elevated regions, this article seeks to show how various trends in medical geography led to the establishment of the « altitude cure » as a treatment for tuberculosis.