Etude de l'activité antivirale d'extraits d'Euphorbia de Corse : recherche de nouveaux diterpènes d'intérêt biologique

Chikungunya fever is caused by an arthropod-borne virus that is associated with massive epidemics and severe morbidity (virus-induced arthralgia, fever, myalgia and rashes). Worldwide expansion of the mosquito vectors, such as Aedes albopictus ("Tiger moquito) is responsible for the spread of Chikungunya virus (CHIKV) throughout the world. A. albopictus has spread throughout Mediterranean areas, which could lead to epidemics outbreaks. Currently, no antiviral drugs or vaccines are available for the treatment or prevention of CHIKV infection. Since ten years, however, recent results showed that diterpene esters from Trignostemon (Euphorbiaceae) possess inhibiting activity of CHIKV replication.With the objective to discover new compounds with antiviral activities, 45 extracts from various plant parts of 11 Euphorbiaceae species native to Corsica were evaluated for selective inhibition of CHIKV replication. In collaboration with Dr. Leyssen (KU Leuven, Belgium), several extracts made from 10 Euphorbia species exhibited significant and selective anti-CHIKV activity in a virus-cell-based assay. The antiviral activities of 29 commercially available phorboïds were studied. Some phorboïds were potent inhibitors of CHIKV and human immunodeficiency virus (VIH) replication. Results allowed drawing new structure-activity relationships, which supported the hypothesis that PKC may be an important target in CHIKV replication. In order to confirm or infirm the presence of phorboïds with anti-CHIKV activity in Euphorbia extracts, a liquid chromatography (LC) coupled to linear ion trap mass spectrometry (MSn) method was developed using standard compounds. Application of this methodology indicated that none anti-CHIKV phorboïds was present in Euphorbia extracts. A second LC-MSn procedure was developed to profile untargeted phorboïdes. Results suggested that numerous other diterpene esters were present in the Euphorbia extracts. The species Euphorbia amygdaloides ssp. semiperfoliata was selected to perform a bioassay-guided purification procedure, which led to the isolation and identification of 14 jatrophane esters, including eight new components. Among them, antiviral evaluation indicated that one jatrophane ester was possessing anti-CHIKV and anti-HIV activities. Furthermore, the structure of an atypical jatrophane ester derivative, jatrohemiketal, was determined unambiguously through an original strategy combining NMR spectroscopy and molecular modeling. Finally, an original tandem mass spectrometry (MS/MS)-targeted supercritical fluid chromatography (SFC) method was developed and used to study bioactive fractions of E. amygdaloïdes ssp. semiperfoliata. The MS/MS data were analyzed by molecular networking. Thanks to this approach, four 4-deoxyphorbol esters and two new jatrophane esters were targeted, isolated and identified. Collaboration with the group of Pr. Alcami (CNM, Espagne) was started to explore the anti-HIV properties of the isolated diterpene esters. Thus, one compound derived from 4-deoxyphorbol esters proved to possess a potent inhibiting activity of HIV-1 replication (IC50 = 8 nM, and selectivity index > 6250). The investigation of the mechanism of this component indicated that it acted like prostratin, but with antiviral effect more than 28-fold. Furthermore, the evaluation of the anti-CHIKV activity indicated that another 4-deoxyphorbol derivative was one of the strongest inhibitor of CHIKV replication isolated up to date (EC50 = 0.34 ± 0.12 µM and selectivity index > 638).Le chikungunya est une maladie transmise par des moustiques du genre Aedes (dont A. albopictus, dit "moustique tigre"). Cette maladie provoque d'intenses fièvres et des douleurs articulaires chroniques fortement invalidantes. Les moustiques potentiellement vecteurs du virus du chikungunya (CHIKV) sont des espèces invasives qui, à la faveur du réchauffement climatique, se sont récemment implantés dans plusieurs régions du monde, dont la région méditerranéenne. Sa présence constitue un terreau favorable à la survenue d'épidémie. A l'heure actuelle, il n'existe ni vaccin, ni traitement médicamenteux efficace. Toutefois, des articles scientifiques ont récemment rapporté que des esters de diterpène isolés du genre Trigonostemon (Euphorbiaceae), avaient une activité inhibitrice de la réplication du CHIKV.Dans le cadre de ces travaux de thèse, des extraits de plantes du genre Euphorbia de Corse ont été étudiés dans le but d'isoler de nouvelles molécules douées d'activité antivirale sur la réplication du CHIKV. En collaboration avec le Dr. P. Leyssen (KU Leuven, Belgique), l'évaluation de l'activité anti-CHIKV de 45 extraits, obtenus à partir de 11 Euphorbiaceae de Corse, a permis de mettre en évidence la forte activité inhibitrice et sélective de des extraits d'espèces du genre Euphorbia in cellulo. L'activité antivirale d'une série de 27 diterpènes de type phorboïde, disponibles commercialement, a également été étudiée. Les résultats ont montré que certains dérivés avaient une forte activité inhibitrice de la réplication du CHIKV, mais aussi sur celle du virus de l'immunodéficience humaine (VIH). Ces études ont permis d'une part, de déduire des relations structure-activité inédites et d'autre part, de soutenir l'hypothèse d'un mécanisme d'action anti-CHIKV impliquant la modulation des protéines kinases C (PKCs) par les phorboïdes. Dans le but de confirmer ou d'infirmer la présence des phorboïdes dans les extraits d'Euphorbia, une première méthode utilisant la chromatographie liquide (LC) haute performance couplée à un spectromètre de masse à trappe d'ions (MSn), a été développée à partir des composés standards. L'application de cette méthodologie a révélé qu'aucun des phorboïdes ciblés n'était présent dans les extraits d'Euphorbia. Ainsi, une seconde procédure LC-MSn a été mise en œuvre afin de détecter - de manière non ciblée - différents types d’esters diterpéniques. L'utilisation de cette approche a révélé que de nombreux diterpènes, non-apparentés aux phorboïdes, étaient présents dans les extraits. Un extrait de l'espèce Euphorbia amygdaloides subsp. semiperfoliata a été sélectionné pour réaliser un fractionnement bio-guidé, aboutissant à l'isolement et l'identification de 14 esters de jatrophane, dont neuf nouveaux composés. Parmi eux, l'un s'est avéré inhiber la réplication du CHIKV et du VIH. Par ailleurs, la structure d'un ester de jatrophane atypique, le jatrohémicétal, a été élucidée grâce à une approche originale combinant modélisation moléculaire et spectroscopie par résonance magnétique nucléaire (RMN). Enfin, une nouvelle procédure de purification ciblée par spectrométrie de masse tandem (MS/MS) en chromatographie en phase fluide supercritique (SFC) a été développée et appliquée sur des fractions bioactives d'E. amygdaloïdes subsp. semiperfoliata. L’interprétation des données MS/MS s’est appuyée sur la génération de réseaux moléculaires. Par cette méthodologie, quatre nouvelles molécules ont pu être détectées, purifiées et identifiées ; il s’agit de deux nouveaux esters de jatrophane et de quatre esters dérivés du 4-déoxyphorbol. L'activité anti-VIH des constituants isolés a également pu être explorée dans le cadre d’une collaboration avec l'équipe du Pr. Alcami (CNM, Espagne). Ainsi, l’un des esters de 4-deoxyphorbol s’est révélé être doué d'un exceptionnel pouvoir inhibiteur de la réplication du VIH-1 (IC50 = 8 nM et index de sélectivité > 6250). Son mécanisme d'action semble s'apparenter à celui de la prostratine (molécule antivirale de référence) mais avec des propriétés antivirales environ 28 fois supérieur. Un deuxième ester de 4-déoxyphorbol s’est avéré être un des plus puissants inhibiteurs du CHIKV isolé à ce jour (EC50 = 0,34 ± 0,12 µM and SI > 638). Mots clés : Euphorbia, activité antivirale, chikungunya, diterpène, spectrométrie de mass

Performance and Carcass Characteristics of Feedlot Steers: Effects of Delayed Implanting and Programmed Feeding During the Growing Period

This experiment was conducted to determine the effect of programming the rate of gain and delaying the first implant in feedlot steers on feedlot performance and carcass characteristics. Ninety-six growing steers (269 ± 16.2 kg) were assigned to 12 pens in a completely randomized design. Treatments were implant (Synovex-S®; 20 mg estradiol benzoate and 200 mg progesterone; Fort Dodge Animal Health, Overland Park, KS) on d 1 or no implant and programmed feeding to gain at a slow (0.68 kg/d) or fast (1.14 kg/d) rate during the growing period; these treatments were randomly assigned (n = 8) to pens of steers in a 2 × 2 factorial arrangement. Steers were fed a growing diet and after 88 and 60 d (for steers fed to gain at a slow or fast rate, respectively), steers were transitioned to ad libitum consumption of a high concentrate finishing diet. Growing period implant treatments did not affect ADG but did affect (P\u3c0.01) gain efficiency during the finishing period. Feeding steers for a slow rate of BW gain during the growing period improved (P=0.062) gain efficiency in the finishing period (169 vs 145 g gain/kg feed). Correlation coefficients between fat thickness and marbling score obtained via ultrasound and fat thickness and marbling score measured at harvest were greater the closer the ultrasound measurements were made to the final harvest date. These data indicate that feeding level prior to the start of the finishing period may affect BW gain efficiency during the finishing period

Lynch Syndrome from a surgeon perspective: retrospective study of clinical impact of mismatch repair protein expression analysis in colorectal cancer patients less than 50 years old.

BACKGROUND: In clinical practice, unexpected diagnosis of colorectal cancer in young patients requires prompt surgery, thus genetic testing for Lynch Syndrome is frequently missed, and clinical management may result incorrect. METHODS: Patients younger than 50 years old undergoing colorectal resection for cancer in the period 1994-2007 were identified (Group A, 49 cases), and compared to a group of randomly selected patients more than 50 (Group B, 85 cases). In 31 group A patients, immunohistochemical expression analysis of MLH1, MSH2 and MSH6 was performed; personal and familial history of patients with defective MMR proteins expression was further investigated, searching for synchronous and metachronous tumors in probands and their families. RESULTS: Fifty-one percent of patients did not express one or more MMR proteins (MMR-) and should be considered Lynch Syndrome carriers (16 patients, group A1); while only 31.2% of them were positive for Amsterdam criteria, 50% had almost another tumor, 37.5% had another colorectal tumor and 68% had relatives with colorectal tumor. This group of patients, compared with A2 group (< 50 years old, MMR+) and B group, showed typical characteristics of HNPCC, such as proximal location, mucinous histotype, poor differentiation, high stage and shorter survival. CONCLUSIONS: The present study confirms that preoperative knowledge of MMR proteins expression in colorectal cancer patients would allow correct staging, more extended colonic resection, specific follow-up and familial screening

WHAT IS THE DIGESTATE?

As anaerobic digestion (AD) is quickly being harnessed in Italy and in other European countries, there is a need for a more in-depth description of the main by-product of the process, the digestate. Little information on digestate characteristics and composition is available and unclear legislation causes problems in biogas plant management. In this work, the organic matter (OM) of this matrix was described through chemical, biological, spectroscopic, and statistical approaches. It was shown that AD results in a strong reduction of the easily degradable fraction of the OM and an accumulation of recalcitrant molecules (possible humus precursors). This contributes to a relatively high biological stability of the residual OM content in the digestate and may lead to good amendment properties. Besides, the observed relative accumulation and the high mineralisation of nitrogen and phosphorus may point to the digestate as a readily available liquid fertiliser for agronomic use. Moreover, xenobiotics and pathogens respected limits for both biosolids and compost in Italian and European legislation

Development of a tomato pomace biorefinery based on a CO2-supercritical extraction process for the production of a high value lycopene product, bioenergy and digestate

Tomato peels and seeds (TP) are the most abundant canning industry waste actually used to produce biogas. TP is rich in lycopene (lyc) and represent a more sustainable feedstock than tomato fruits actually employed. It was therefore chosen as feedstock together with supercritical CO2 extraction (SFE-CO2) technology to develop a TP-SFE-CO2 biorefinery, topic scarcely investigated. Two TP were tested and although TP-SFE-CO2 parameters were the same, lyc recoveries depended by peel structure changes occurred during pre -SFE-CO2 drying step. Higher moisture (102.7 g kg-1 wet weight) permitted 97 % lyc recovery and gave a water-in-oil emulsion as extract. Mass balance confirmed that lyc isomerisation did not cause lyc losses. After a significant oil extraction, exhaust TP showed a biodegradability 64% higher than the raw one, attributable to fibre structure disruption. The biorefinery proposed (SFE_CO2+anaerobic digestion) determined positive economic revenue (+787.9 \u20ac t-1 TP) on the contrary of the actual TP management

tRNAdb 2009: compilation of tRNA sequences and tRNA genes

One of the first specialized collections of nucleic acid sequences in life sciences was the ‘compilation of tRNA sequences and sequences of tRNA genes’ (http://www.trna.uni-bayreuth.de). Here, an updated and completely restructured version of this compilation is presented (http://trnadb.bioinf.uni-leipzig.de). The new database, tRNAdb, is hosted and maintained in cooperation between the universities of Leipzig, Marburg, and Strasbourg. Reimplemented as a relational database, tRNAdb will be updated periodically and is searchable in a highly flexible and user-friendly way. Currently, it contains more than 12 000 tRNA genes, classified into families according to amino acid specificity. Furthermore, the implementation of the NCBI taxonomy tree facilitates phylogeny-related queries. The database provides various services including graphical representations of tRNA secondary structures, a customizable output of aligned or un-aligned sequences with a variety of individual and combinable search criteria, as well as the construction of consensus sequences for any selected set of tRNAs

Inhibition of StearoylCoA Desaturase Activity Blocks Cell Cycle Progression and Induces Programmed Cell Death in Lung Cancer Cells

Lung cancer is the most frequent form of cancer. The survival rate for patients with metastatic lung cancer is ∼5%, hence alternative therapeutic strategies to treat this disease are critically needed. Recent studies suggest that lipid biosynthetic pathways, particularly fatty acid synthesis and desaturation, are promising molecular targets for cancer therapy. We have previously reported that inhibition of stearoylCoA desaturase-1 (SCD1), the enzyme that produces monounsaturated fatty acids (MUFA), impairs lung cancer cell proliferation, survival and invasiveness, and dramatically reduces tumor formation in mice. In this report, we show that inhibition of SCD activity in human lung cancer cells with the small molecule SCD inhibitor CVT-11127 reduced lipid synthesis and impaired proliferation by blocking the progression of cell cycle through the G1/S boundary and by triggering programmed cell death. These alterations resulting from SCD blockade were fully reversed by either oleic (18:1n-9), palmitoleic acid (16:1n-7) or cis-vaccenic acid (18:1n-7) demonstrating that cis-MUFA are key molecules for cancer cell proliferation. Additionally, co-treatment of cells with CVT-11127 and CP-640186, a specific acetylCoA carboxylase (ACC) inhibitor, did not potentiate the growth inhibitory effect of these compounds, suggesting that inhibition of ACC or SCD1 affects a similar target critical for cell proliferation, likely MUFA, the common fatty acid product in the pathway. This hypothesis was further reinforced by the observation that exogenous oleic acid reverses the anti-growth effect of SCD and ACC inhibitors. Finally, exogenous oleic acid restored the globally decreased levels of cell lipids in cells undergoing a blockade of SCD activity, indicating that active lipid synthesis is required for the fatty acid-mediated restoration of proliferation in SCD1-inhibited cells. Altogether, these observations suggest that SCD1 controls cell cycle progression and apoptosis and, consequently, the overall rate of proliferation in cancer cells through MUFA-mediated activation of lipid synthesis

The oldest record of Panochthus (Xenarthra, Glyptodontidae)

Panochthus is one of the largest sized and most frequently recorded genera of Pleistocene South American Glyptodontidae. A recent taxonomic revision shows that the genus includes six species: P. intermedius and P. subintermedius (early Pleistocene-middle Pleistocene), P. tuberculatus and P. frenzelianus (middle Pleistocene-late Pleistocene), whereas P. greslebini and P. jaguaribensis only can be referred to the Pleistocene sensu lato. In this contribution the oldest record of the genus Panochthus (MMP 5171) is presented and described. The remains came from the late Pliocene (Playa Los lobos Aloformation, Chapadmalal Formation; Chapadmalalan; Paraglyptodon chapalmalensis Biozone) of the Mar del Plata, Buenos Aires province, Argentina. The material is represented by a considerable fragment of dorsal carapace and some associated osteoderms, which are assignable to an indeterminate species of Panochthus. One of the most significant characters in the exposed surface of the osteoderms is the presence of a clear "reticular" pattern, a character only observed in Panochthus. From a biostratigraphic viewpoint, it is remarkable the absence of records of Panochthus in the Marplatan Age/Stage (late Pliocene-early Pleistocene).Facultad de Ciencias Naturales y Muse

The oldest record of Panochthus (Xenarthra, Glyptodontidae)

Panochthus is one of the largest sized and most frequently recorded genera of Pleistocene South American Glyptodontidae. A recent taxonomic revision shows that the genus includes six species: P. intermedius and P. subintermedius (early Pleistocene-middle Pleistocene), P. tuberculatus and P. frenzelianus (middle Pleistocene-late Pleistocene), whereas P. greslebini and P. jaguaribensis only can be referred to the Pleistocene sensu lato. In this contribution the oldest record of the genus Panochthus (MMP 5171) is presented and described. The remains came from the late Pliocene (Playa Los lobos Aloformation, Chapadmalal Formation; Chapadmalalan; Paraglyptodon chapalmalensis Biozone) of the Mar del Plata, Buenos Aires province, Argentina. The material is represented by a considerable fragment of dorsal carapace and some associated osteoderms, which are assignable to an indeterminate species of Panochthus. One of the most significant characters in the exposed surface of the osteoderms is the presence of a clear "reticular" pattern, a character only observed in Panochthus. From a biostratigraphic viewpoint, it is remarkable the absence of records of Panochthus in the Marplatan Age/Stage (late Pliocene-early Pleistocene).Facultad de Ciencias Naturales y Muse

Chemical Characterization and Nematicidal Activity of the Essential Oil of Nepeta nuda L. ssp. pubescens and Nepeta curviflora Boiss. from Lebanon

The chemical characterization and the nematicidal activity of the essential oils from Nepeta nuda L. ssp. pubescens and Nepeta curviflora Boiss. growing wild in Lebanon are reported. A comparative study was carried out as, to the best of our knowledge, no information is available on Nepeta nuda L. ssp. pubescens. In addition, both Nepeta species were collected in the same geographical area in order to rule out the environmental factors influencing essential oil composition and bioactivity. The most abundant (> 5 %) components of N. nuda ssp. pubescens essential oil were pinene (12.89 %), 1-ethyl-1H-pyrrole (12.67 %), 1-cycloethyl- 1-(2-methylenecyclohexyl) ethanol (10.37 %), 3-methyl-2-cyclohexen-1-one (9.17 %) and 2,3-dimethyl-3- hexanol (5.88 %). Among oxygenated monoterpenes, two nepetalactones were identified, i.e. (E, Z)nepetalactone (2.24 %) and (Z, E)-nepetalactone (0.31 %). The major constituents (> 5 %) of N. curviflora essential oil were 2-isopropyl-5-methyl-3-cyclohexen-1-one (12.51%), (-)-spathulenol (11.73%), cis-Z-alpha-bisabolene epoxide (8.07 %), widdrol (7.0 %), (E, Z)-5,7-dodecadiene (6.93 %), dihydronepetalactone (5.57 %) and 4-propyl-cyclohexene (5.43 %). The essential oil of N. curviflora was more active than the N. nuda ssp. pubescens one against the nematode Panagrolaimus rigidus. According to the motility assay, LD50 was 0.5 mg/mL and 2.5 mg/mL 24 h after treatment with N. curviflora and N. nuda ssp. pubescens essential oil, respectively