Workgroup report: incorporating in vitro alternative methods for developmental neurotoxicity into international hazard and risk assessment strategies.

This is the report of the first workshop on Incorporating In Vitro Alternative Methods for Developmental Neurotoxicity (DNT) Testing into International Hazard and Risk Assessment Strategies, held in Ispra, Italy, on 19-21 April 2005. The workshop was hosted by the European Centre for the Validation of Alternative Methods (ECVAM) and jointly organized by ECVAM, the European Chemical Industry Council, and the Johns Hopkins University Center for Alternatives to Animal Testing. The primary aim of the workshop was to identify and catalog potential methods that could be used to assess how data from in vitro alternative methods could help to predict and identify DNT hazards. Working groups focused on two different aspects: a) details on the science available in the field of DNT, including discussions on the models available to capture the critical DNT mechanisms and processes, and b) policy and strategy aspects to assess the integration of alternative methods in a regulatory framework. This report summarizes these discussions and details the recommendations and priorities for future work.

Anaerobically controlled expression system derived from the arcDABC operon of Pseudomonas aeruginosa: application to lipase production.

The anaerobically inducible arcDABC operon encodes the enzymes of the arginine deiminase pathway in Pseudomonas aeruginosa. Upon induction, the arcAB mRNAs and proteins reach high intracellular levels, because of a strong anaerobically controlled promoter and mRNA processing in arcD, leading to stable downstream transcripts. We explored the usefulness of this system for the construction of expression vectors. The lacZ gene of Escherichia coli was expressed to the highest levels when fused close to the arc promoter. Insertion of lacZ further downstream into arcA or arcB did not stabilize the intrinsically unstable lacZ mRNA. On the contrary, lacZ mRNA appeared to be a vulnerable endonuclease target destabilizing arcAB mRNAs in the 5'-to-3' direction in P. aeruginosa. The native arc promoter was modified for optional expression in the -10 sequence and in the -40 region, which is a binding site for the anaerobic regulator ANR. In P. aeruginosa grown either anaerobically or with oxygen limitation in unshaken cultures, this promoter was stronger than the induced tac promoter. The P. aeruginosa lipAH genes, which encode extracellular lipase and lipase foldase, respectively, were fused directly to the modified arc promoter in an IncQ vector plasmid. Semianaerobic static cultures of P. aeruginosa PAO1 carrying this recombinant plasmid overproduced extracellular lipase 30-fold during stationary phase compared with the production by strain PAO1 without the plasmid. Severe oxygen limitation, in contrast, resulted in poor lipase productivity despite effective induction of the ANR-dependent promoter, suggesting that secretion of active lipase is blocked by the absence of oxygen. In conclusion, the modified arc promoter is useful for driving the expression of cloned genes in P. aeruginosa during oxygen-limited growth and stationary phase.

HIV-1 superinfection with a triple-class drug-resistant strain in a patient successfully controlled with antiretroviral treatment.

We report a case of HIV-1 superinfection (HSI) with a clade B, triple-class resistant virus in a patient successfully controlling viremia with continuous combination antiretroviral therapy started 8 years earlier during primary HIV infection. The course of HIV infection prior to HSI was monitored in both the source partner and recipient (8 and 11 years, respectively) and 4 years following HSI. This case report demonstrates re-infection with HIV-1 despite effective combination antiretroviral therapy.

When information can save lives: the duty to warn relatives about sudden cardiac death and environmental risks.

In certain cases of sudden death, forensic experts may discover during an investigation or autopsy that family members of the deceased are also at risk of harm-from genetic disease, for instance. But do they have a duty to warn them? Looking at similar duties of physicians and researchers to warn third parties of risk suggests they do.

Environmental sex reversal, Trojan sex genes, and sex ratio adjustment: conditions and population consequences.

Abstract The great diversity of sex determination mechanisms in animals and plants ranges from genetic sex determination (GSD, e.g. mammals, birds, and most dioecious plants) to environmental sex determination (ESD, e.g. many reptiles) and includes a mixture of both, for example when an individual's genetically determined sex is environmentally reversed during ontogeny (ESR, environmental sex reversal, e.g. many fish and amphibia). ESD and ESR can lead to widely varying and unstable population sex ratios. Populations exposed to conditions such as endocrine-active substances or temperature shifts may decline over time due to skewed sex ratios, a scenario that may become increasingly relevant with greater anthropogenic interference on watercourses. Continuous exposure of populations to factors causing ESR could lead to the extinction of genetic sex factors and may render a population dependent on the environmental factors that induce the sex change. However, ESR also presents opportunities for population management, especially if the Y or W chromosome is not, or not severely, degenerated. This seems to be the case in many amphibians and fish. Population growth or decline in such species can potentially be controlled through the introduction of so-called Trojan sex genes carriers, individuals that possess sex chromosomes or genes opposite from what their phenotype predicts. Here, we review the conditions for ESR, its prevalence in natural populations, the resulting physiological and reproductive consequences, and how these may become instrumental for population management.

Small RNAs controlled by two-component systems.

Two-component systems (TCSs) allow bacteria to monitor diverse environmental cues and to adjust gene expression accordingly at the transcriptional level. It has been recently recognized that prokaryotes also regulate many genes and operons at a posttranscriptional level with the participation of small, noncoding RNAs which serve to control translation initiation and stability of target mRNAs, either directly by establishing antisense interactions or indirectly by antagonizing RNA-binding proteins. Interestingly, the expression of a subset of these small RNAs is regulated by TCSs and in this way, the small RNAs expand the scope of genetic control exerted by TCSs. Here we review the regulatory mechanisms and biological relevance ofa number of small RNAs under TCS control in Gram-negative and -positive bacteria. These regulatory systems govern, for instance, porin-dependent permeability of the outer membrane, quorum-sensing control of pathogenicity, or biocontrol activity. Most likely, this emerging and rapidly expanding field of molecular microbiology will provide more and more examples in the near future.

Environmental pollution promotes selection of microbial degradation pathways

Astonishing as it may seem, one organism's waste is often ideal food for another. Many waste products generated by human activities are routinely degraded by microorganisms under controlled conditions during waste-water treatment. Toxic pollutants resulting from inadvertent releases, such as oil spills, are also consumed by bacteria, the simplest organisms on Earth. Biodegradation of toxic or particularly persistent compounds, however, remains problematic. What has escaped the attention of many is that bacteria exposed to pollutants can adapt to them by mutating or acquiring degradative genes. These bacteria can proliferate in the environment as a result of the selection pressures created by pollutants. The positive outcome of selection pressure is that harmful compounds may eventually be broken down completely through biodegradation. The downside is that biodegradation may require extremely long periods of time. Although the adaptation process has been shown to be reproducible, it remains very difficult to predict.

ppGpp controlled by the Gac/Rsm regulatory pathway sustains biocontrol activity in Pseudomonas fluorescens CHA0.

In Pseudomonas fluorescens CHA0 and other fluorescent pseudomonads, the Gac/Rsm signal transduction pathway is instrumental for secondary metabolism and biocontrol of root pathogens via the expression of regulatory small RNAs (sRNAs). Furthermore, in strain CHA0, an imbalance in the Krebs cycle can affect the strain's ability to produce extracellular secondary metabolites, including biocontrol factors. Here, we report the metabolome of wild-type CHA0, a gacA-negative mutant, which has lost Gac/Rsm activities, and a retS-negative mutant, which shows strongly enhanced Gac/Rsm-dependent activities. Capillary electrophoresis-based metabolomic profiling revealed that the gacA and retS mutations had opposite effects on the intracellular levels of a number of central metabolites, suggesting that the Gac/Rsm pathway regulates not only secondary metabolism but also primary metabolism in strain CHA0. Among the regulated metabolites identified, the alarmone guanosine tetraphosphate (ppGpp) was characterized in detail by the construction of relA (for ppGpp synthase) and spoT (for ppGpp synthase/hydrolase) deletion mutants. In a relA spoT double mutant, ppGpp synthesis was completely abolished, the expression of Rsm sRNAs was attenuated, and physiological functions such as antibiotic production, root colonization, and plant protection were markedly diminished. Thus, ppGpp appears to be essential for sustaining epiphytic fitness and biocontrol activity of strain CHA0.

Environmental changes in the aftermath of Neoproterozoic glaciations: a biogeochemical study of sediments from SW-Gondwana

Abstract The late Neoproterozoic or Ediacaran period, (635 to -543 Ma) is a primordial time in the Earth history corresponding to the beginning of animal life and the most extreme ice ages on Earth. In this dissertation, palaeoenvironmental conditions were reconstructed for Ediacaran, post-Gaskiers shelf deposits in SW- Gondwana and their changes were evaluated according to the diversity of organisms. The present study addresses the question of interactions between biodiversity and environmental change by using the elemental and isotopic geochemistry of sedimentary rocks and associated organic matter, as well as the distribution of hydrocarbon biomarkers. The studied sedimentary sequences are from a large basin extended from the Paraguay belt to the Rio de la Plata craton, including the Corumbâ Group in SW-Brazil (Paraguay belt), the Arroyo del Soldado Group in Uruguay and the Sierras Bayas Group in Argentina (both in the Rio de la Plata craton). Several geochemical signatures of the sediments from Corumbâ and Sierras Bayas groups provides evidence for an euxinic setting in the Ediacaran Ocean: 1) The occurrence of syngenetic pyrite in the Corumbâ Group together with hydrocarbon biomarkers of an anoxic microbial consortium including traces of gammacerane, a distribution of hopanes with maxima at C29 as well as a low pristane/phytane (Pr/Ph) ratio; 2) the occurrence of 34S enrichments within sulfides of the Sierras Bayas Group exceeding the sulfur isotopic composition of coeval carbonate-associated sulfate. In the Arroyo del Soldado Group, an event of reducing conditions was revealed by higher concentrations of redox-sensitive trace elements and negative 513Ccar shifts in all sections. This event is extended to the whole unit in the deepest section and is restricted to tempestites in the two other shallow sections. The persistent negative. ôl3Ccar values recorded at the basinal setting implies strong isotopic gradient between shallow and deep water environments and therefore, a locus of deposition below the redox chemocline. In all studied sections, the excursions, the strong enrichment of authigenic trace-elements, the occurrence of longer chain «-alkanes, gammacerane and low Pr/Ph and Ph/>;-C]a ratios, combined with the previous sedimentological and paleontological observations indicate that the chemistry of the ocean was strongly controlled by the oxygen availability; waters being moderately oxic at the surface and anoxic at depth for much of the Neoproterozoic. This water column stratification was favourable to the storage of large amounts of nutrients in the deep ocean. During upwelling periods, the export of these nutrient-rich waters may have triggered an important bioproductivity in surface waters. Drops in Al3Cc,,.](Cr and positive ôl3Ccllr excursions highlight the increase in primary productivity. Preservation of organic carbon was ensured by reducing conditions at the bottom. The Al3ccar.kcr excursions could also reflect changes in the composition of the primary biomass. New geochemical evidence from SW-Gondwana sections supports a stratified Ediacaran ocean, outside restricted or hypersaline environments, in the aftermath of glaciations. The association of ocean stratification and the appearance of metazoans support the model that the evolution of eukaryotic life was related to the increase of oxygen levels in surface environments due to an efficient recycling of nutrients in the anoxic deep ocean. Résumé Le Néoprotérozoïque terminal, ou Édiacarien (635 à -543 Ma), est un période de première importance dans l'histoire de la Terre, car elle correspond a l'apparition des métazoaires pendant un intervalle de glaciations extrêmes. Le présent mémoire se propose de reconstituer les conditions paléoenvironnementales des dépôts de plateforme mis en place durant l'Édiacarien, au sud-ouest du Gondwana. Les interactions entre changements environnementaux et biodiversité sont évaluées en s'appuyant d'une part sur la composition élémentaire et isotopique des roches sédimentaires et de leur matière organique, et d'autre part sur la distribution moléculaire de biomarqueurs hydrocarbonés. Les séquences sédimentaires étudiées proviennent d'un grand bassin qui s'étend de la chaîne du Paraguay jusqu'au craton du Rio de la Plata. La séquence du Groupe Corumbâ au Sud Ouest du Brésil se situe dans la chaîne du Paraguay, tandis que le Groupe Arroyo del Soldado en Uruguay et le Groupe Sierras Bayas en Argentine sont situés sur le craton du Rio de la Plata. L'étude géochimique des sédiments des groupes Corumbâ et Sierras Bayas révèle de façon claire des conditions euxiniques dans l'océan édiacarien. On trouve ainsi, dans le Groupe Corumbâ, les biomarqueurs d'un cortège microbien anoxique et sulfurique comprenant des bactéries sulfato-réductrices, et dans les sulfures du Groupe Sierras Bayas, des enrichissements en Î4S excédant les rapports isotopiques du soufre dans le sulfate cogénétique associé aux carbonates. Dans la séquence de l'Arroyo del Soldado, un événement réducteur est mis en évidence par des teneurs plus élevées en éléments traces sensibles aux conditions redox et par des excursions négatives du 613Ccardans toutes les coupes. Cet événement affecte la totalité de la section la plus profonde et n'apparaît que dans les tempestites dans les sections les moins profondes. La persistance de valeurs négatives du ô13Ccarau large implique un gradient isotopique prononcé entre les environnements superficiels et profonds, et donc, ta présence d'une chémocline redox. Les excursions du. ôBCcar, l'enrichissement authigène en éléments traces, la présence de gammacérane et de rt-alcanes à longue chaîne, ainsi que de faibles rapports Pr/Ph et Ph/«-Cl8, viennent s'ajouter aux observations préliminaires sur la sédimentologie et la paléontologie pour indiquer que la chimie de l'océan était fortement contrôlée par la disponibilité d'oxygène, les eaux étant modérément oxiques à la surface et anoxiques en profondeur pendant la plus grande partie du Néoprotérozoïque. La stratification de la colonne d'eau était favorable au stockage de grandes quantités de nutriments dans l'océan profond. Dans les zones d'upwelling, la migration d'eaux profondes riches en nutriment vers la surface a pu provoquer une bioproductivité prononcée dans les eaux de surface. La conservation du carbone organique était assurée par les conditions anoxiques prévalant au fond. Les excursions du A13Ccar.kt.r pourraient aussi refléter des changements dans la biomasse primaire. Le présent travail apporte donc de nouvelles preuves qu'un océan stratifié s'est maintenu à la suite des glaciations néoprotérozoïques dans le Sud Ouest du Gondwana. L'association d'un océan stratifié et de l'apparition de la vie animale est en accord avec le modèle stipulant que l'évolution de la vie est associée à une meilleure oxygénation des environnements de surface. Résumé pour le grand public La période Ediacarienne (635 à -543 Ma) à la fin du Précambrien est l'une de plus énigmatiques dans l'histoire de la Terre, car elle est caractérisée par la diversification de la vie multicellulaire (eucaryote) pendant un intervalle de glaciations extrêmes. Dans ce travail de thèse, nous cherchons à déceler l'existence éventuelle d'un lien entre ces changements environnementaux et l'évolution de la vie eucaryote à travers une étude biogéochimique. La biogéochimie est l'étude des activités biologiques dans la géosphère, telles que celles intervenant dans les cycles des éléments chimiques (y compris les isotopes stables) et celles de production de composés carbonés caractérisant certains groups d'organismes ou taxons. La recherche des signatures paléoenvironnementales dans les roches précambriennes a été fortement facilitée par l'utilisation des biomarqueurs ou fossiles moléculaires. Ces composés, provenant des lipides biologiques (molécules avec des fonctions spécifiques dans les organismes), peuvent être reliés à des taxons spécifiques ou à des voies métaboliques. La transformation d'un biolipide en fossile moléculaire intervient lorsque des restes organiques déposés dans un substrat subissent un enfouissement et une augmentation de la pression (diagenèse). Ce processus mène à la formation de kérogène, un grand agrégat chimique de matière organique insoluble dans des solvants organiques, et de bitume ou fraction soluble (extractible) de la matière organique. L'analyse intégrée du kérogène et du bitume fournit des indications précieuses pour les reconstitutions paléoenvironnementales. Des conditions paléoenvironnementales ont ainsi été déterminées pour une plateforme marine Ediacarienne située dans la partie sud-américaine du bloc occidental du paléocontinent Gondwana. Les séquences sédimentaires étudiées appartiennent au même bassin qui s'étend de la ceinture du Paraguay (Groupe Corumbâ, Brésil) au craton du Rio de la Plata (Groupes Arroyo del Soldado, Uruguay et Sierras Bayas, Argentina). Nous nous sommes intéressés aux isotopes stables de carbonates et de la matière organique associée (kérogène et bitume), aux éléments majeurs et traces, ainsi qu'aux biomarqueurs caractérisant ces roches. Les résultats de cette dissertation suggèrent qu'au cours de l'Édiacarien, suite aux glaciations néoprotérozoïques dans le bloc occidental du Gondwana, l'océan était stratifié en zones spécifiques d'eaux riches en sulfures et dépourvues d'oxygène (euxiniques). L'association d'un océan stratifié et de l'apparition de la vie animale est en accord avec le modèle stipulant que l'évolution de la vie est associée à une meilleure oxygénation des environnements de surface. Les excursions isotopiques (tendance à des valeurs positives ou négatives) en constante fluctuation pour le carbone et très positives pour le soufre des sulfures, l'enrichissement en éléments trace et la présence de certains composés (e.g. gammacerane; Pr/Ph et Ph/«-Ci8 en basse proportion) conjugués aux observations sédimentologiques et paléontologiques des différents profils étudiés indiquent que la chimie de l'océan était fortement contrôlée par la disponibilité d'oxygène, avec des eaux modérément oxygénées en surface et euxiniques en profondeur pour la plupart du Néoprotérozoïque.

A quorum sensing regulated small volatile molecule reduces acute virulence and promotes chronic infection phenotypes.

A significant number of environmental microorganisms can cause serious, even fatal, acute and chronic infections in humans. The severity and outcome of each type of infection depends on the expression of specific bacterial phenotypes controlled by complex regulatory networks that sense and respond to the host environment. Although bacterial signals that contribute to a successful acute infection have been identified in a number of pathogens, the signals that mediate the onset and establishment of chronic infections have yet to be discovered. We identified a volatile, low molecular weight molecule, 2-amino acetophenone (2-AA), produced by the opportunistic human pathogen Pseudomonas aeruginosa that reduces bacterial virulence in vivo in flies and in an acute mouse infection model. 2-AA modulates the activity of the virulence regulator MvfR (multiple virulence factor regulator) via a negative feedback loop and it promotes the emergence of P. aeruginosa phenotypes that likely promote chronic lung infections, including accumulation of lasR mutants, long-term survival at stationary phase, and persistence in a Drosophila infection model. We report for the first time the existence of a quorum sensing (QS) regulated volatile molecule that induces bistability phenotype by stochastically silencing acute virulence functions in P. aeruginosa. We propose that 2-AA mediates changes in a subpopulation of cells that facilitate the exploitation of dynamic host environments and promote gene expression changes that favor chronic infections.

Epilepsy priorities in Europe: A report of the ILAE-IBE Epilepsy Advocacy Europe Task Force.

The European Forum on Epilepsy Research (ERF2013), which took place in Dublin, Ireland, on May 26-29, 2013, was designed to appraise epilepsy research priorities in Europe through consultation with clinical and basic scientists as well as representatives of lay organizations and health care providers. The ultimate goal was to provide a platform to improve the lives of persons with epilepsy by influencing the political agenda of the EU. The Forum highlighted the epidemiologic, medical, and social importance of epilepsy in Europe, and addressed three separate but closely related concepts. First, possibilities were explored as to how the stigma and social burden associated with epilepsy could be reduced through targeted initiatives at EU national and regional levels. Second, ways to ensure optimal standards of care throughout Europe were specifically discussed. Finally, a need for further funding in epilepsy research within the European Horizon 2020 funding programme was communicated to politicians and policymakers participating to the forum. Research topics discussed specifically included (1) epilepsy in the developing brain; (2) novel targets for innovative diagnostics and treatment of epilepsy; (3) what is required for prevention and cure of epilepsy; and (4) epilepsy and comorbidities, with a special focus on aging and mental health. This report provides a summary of recommendations that emerged at ERF2013 about how to (1) strengthen epilepsy research, (2) reduce the treatment gap, and (3) reduce the burden and stigma associated with epilepsy. Half of the 6 million European citizens with epilepsy feel stigmatized and experience social exclusion, stressing the need for funding trans-European awareness campaigns and monitoring their impact on stigma, in line with the global commitment of the European Commission and with the recommendations made in the 2011 Written Declaration on Epilepsy. Epilepsy care has high rates of misdiagnosis and considerable variability in organization and quality across European countries, translating into huge societal cost (0.2% GDP) and stressing the need for cost-effective programs of harmonization and optimization of epilepsy care throughout Europe. There is currently no cure or prevention for epilepsy, and 30% of affected persons are not controlled by current treatments, stressing the need for pursuing research efforts in the field within Horizon 2020. Priorities should include (1) development of innovative biomarkers and therapeutic targets and strategies, from gene and cell-based therapies to technologically advanced surgical treatment; (2) addressing issues raised by pediatric and aging populations, as well as by specific etiologies and comorbidities such as traumatic brain injury (TBI) and cognitive dysfunction, toward more personalized medicine and prevention; and (3) translational studies and clinical trials built upon well-established European consortia.

Management of Fulminant Multiple Sclerosis With Rituximab: A Case Report.

INTRODUCTION: Malignant variant is a rare subtype of multiple sclerosis (MS) that is rapidly progressive and may lead to significant disability or even death. No consensus exists on best management of this disorder, although corticosteroids and plasmapheresis are commonly used in the acute phase, followed either by MS-specific disease-modifying therapy or an immunosuppressant. CASE REPORT: The patient is a 30-year-old man with relapsing-remitting MS previously well controlled with natalizumab, who has developed fulminant disease activity upon natalizumab cessation. In the acute phase, patient had a suboptimal response to multiple corticosteroid treatments but responded very well to plasmapheresis. Patient continued to have worsening disease activity despite fingolimod treatment. Disease control has been eventually achieved by switching to rituximab. CONCLUSION: Rituximab treatment should be considered for a patient with fulminant MS who responded well to plasmapheresis.