Hepatitis B virus surface proteins accelerate cholestatic injury and tumor progression in Abcb4-knockout mice

Understanding of the pathophysiology of cholestasis associated carcinogenesis could challenge the development of new personalized therapeutic approaches and thus improve prognosis. Simultaneous damage might aggravate hepatic injury, induce chronic liver disease and even promote carcinogenesis. We aimed to study the effect of Hepatitis B virus surface protein (HBsAg) on cholestatic liver disease and associated carcinogenesis in a mouse model combining both impairments. Hybrids of Abcb4-/- and HBsAg transgenic mice were bred on fibrosis susceptible background BALB/c. Liver injury, serum bile acid concentration, hepatic fibrosis, and carcinogenesis were enhanced by the combination of simultaneous damage in line with activation of c-Jun N-terminal kinase (JNK), proto-oncogene c-Jun, and Signal transducer and activator of transcription 3 (STAT3). Activation of Protein Kinase RNA-like Endoplasmic Reticulum Kinase (PERK) and Eukaryotic translation initiation factor 2A (eIF2a) indicated unfolded protein response (UPR) in HBsAg-expressing mice and even in Abcb4-/- without HBsAg-expression. CONCLUSION: Cholestasis-induced STAT3- and JNK-pathways may predispose HBsAg-associated tumorigenesis. Since STAT3- and JNK-activation are well characterized critical regulators for tumor promotion, the potentiation of their activation in hybrids suggests an additive mechanism enhancing tumor incidence

(2R)-4-[(9H-Fluoren-9-ylmeth­oxy)carbon­yl]-2-methyl­piperazin-1-ium chloride

The synthesis of the title salt, C20H23N2O2 +·Cl−, was carried out with a precursor of known absolute configuration (R) and the X-ray analysis confirmed that the product retained the absolute configuration. In the crystal, the dominant packing motif is a chain running along [010] generated by N—H⋯Cl hydrogen bonding. C—H⋯O and C—H⋯Cl inter­actions are also observed

LIPAD (LRRK2/Luebeck International Parkinson's Disease) Study Protocol:Deep Phenotyping of an International Genetic Cohort

Background: Pathogenic variants in the Leucine-rich repeat kinase 2 (LRRK2) gene are the most common known monogenic cause of Parkinson's disease (PD). LRRK2-linked PD is clinically indistinguishable from idiopathic PD and inherited in an autosomal dominant fashion with reduced penetrance and variable expressivity that differ across ethnicities and geographic regions.Objective: To systematically assess clinical signs and symptoms including non-motor features, comorbidities, medication and environmental factors in PD patients, unaffected LRRK2 pathogenic variant carriers, and controls. A further focus is to enable the investigation of modifiers of penetrance and expressivity of LRRK2 pathogenic variants using genetic and environmental data.Methods: Eligible participants are invited for a personal or online examination which comprises completion of a detailed eCRF and collection of blood samples (to obtain DNA, RNA, serum/plasma, immune cells), urine as well as household dust. We plan to enroll 1,000 participants internationally: 300 with LRRK2-linked PD, 200 with LRRK2 pathogenic variants but without PD, 100 PD patients with pathogenic variants in the GBA or PRKN genes, 200 patients with idiopathic PD, and 200 healthy persons without pathogenic variants.Results: The eCRF consists of an investigator-rated (1 h) and a self-rated (1.5 h) part. The first part includes the Movement Disorder Society Unified Parkinson's Disease Rating, Hoehn &Yahr, and Schwab & England Scales, the Brief Smell Identification Test, and Montreal Cognitive Assessment. The self-rating part consists of a PD risk factor, food frequency, autonomic dysfunction, and quality of life questionnaires, the Pittsburgh Sleep Quality Inventory, and the Epworth Sleepiness as well as the Hospital Anxiety and Depression Scales. The first 15 centers have been initiated and the first 150 participants enrolled (as of March 25th, 2021).Conclusions: LIPAD is a large-scale international scientific effort focusing on deep phenotyping of LRRK2-linked PD and healthy pathogenic variant carriers, including the comparison with additional relatively frequent genetic forms of PD, with a future perspective to identify genetic and environmental modifiers of penetrance and expressivityClinical Trial Registration:ClinicalTrials.gov, NCT04214509

The use of isothermal titration calorimetry for the assay of enzyme activity: Application in higher education practical classes

Determination of enzyme activity is crucial for discovery, research, and development in life sciences. The activity of enzymes is routinely determined using spectrophotometric assays that measure rates of substrate consumption or product formation. Though colorimetric-based detection systems are simple, rapid, and economical to perform, the majority of enzymes are unsuitable for this technique as their substrates/products do not absorb in the UV or visible range. This limitation can be addressed by the use of coupled-enzyme assays or artificial chromogenic substrates; however these approaches have their own drawbacks. Here, we describe a method based on the use of an isothermal titration calorimeter (ITC) to measure the heat produced or absorbed during any enzyme-catalyzed reaction. The concept of calorimetric enzyme assays was demonstrated for the determination of enzyme hexokinase activity, which cannot be monitored colorimetrically without first coupling it to another enzymatic reaction. The assay is suitable for incorporation into undergraduate laboratory classes, providing students with an appreciation for; the versatility and ease of use of ITC assays; ITC as a flexible generic method for exploring the functional characteristics of uncharacterized enzymes; an activity detection parameter suitable for enzymes that either have no straightforward colorimetric methods available or require the use of nonartificial chromogenic substrates

Evaluating Enzymatic Productivity—The Missing Link to Enzyme Utility

Kinetic productivity analysis is critical to the characterization of enzyme catalytic performance and capacity. However, productivity analysis has been largely overlooked in the published literature. Less than 0.01% of studies which report on enzyme characterization present productivity analysis, despite the fact that this is the only measurement method that provides a reliable indicator of potential commercial utility. Here, we argue that reporting productivity data involving native, modified, and immobilized enzymes under different reaction conditions will be of immense value in optimizing enzymatic processes, with a view to accelerating biotechnological applications. With the use of examples from wide-ranging studies, we demonstrate that productivity is a measure of critical importance to the translational and commercial use of enzymes and processes that employ them. We conclude the review by suggesting steps to maximize the productivity of enzyme catalyzed reactions

CIGESMED : Coralligenous based Indicators to evaluate and monitor the "Good Environmental Status" of the Mediterranean coastal waters

Final reportCoralligenous is a hard-bottom mainly biogenic habitat, produced by the agglomeration of calcareous encrusting algae growing in dim-light conditions. It is characterized by high structural complexity and spatial heterogeneity, thus supporting rich biodiversity and a variety of sessile assemblages, shaping a typical and one of the most important habitats of the Mediterranean Sea. It produces goods (e.g. food, raw material) and services in several domains (e.g. CO2 sequestration, aesthetics and education). Pollution, smothering and abrasion from a variety of human activities may cause its degradation at a broad scale, whilst fishing and collection of organisms mainly affect target species. Its high aesthetic value may also induce frequentation by SCUBA divers, an additional cause of degradation. Coralligenous is also susceptible to invasive alien species. This habitat, which is of great ecological, socio-economic and cultural importance, is also under the pressures linked to global warming.CIGESMED’s GOAL was to understand links between natural and anthropogenic pressures and coralligenous habitats as well as the effects on their functioning to define the Good Environmental Status (GES) of the coastal Mediterranean Sea and propose solutions for maintaining good environmental conditions.Coralligenous specific indices have been constructed and tested by scientists, marine natural parks and reserves managers, also through the implementation of a “citizen science” pilot network. The use of the newest data mining techniques and the development of visualization tools to sort, organize and illustrate very large heterogeneous sets of data constitute an original but complex approach. It permitted to mobilize, visualize and share large data collections, and to manage knowledge to study these habitats.The OUTCOME consists of: i) experimentation and results of new methods to build survey at large scales (testing operating process and materials during dive, photo analyses, population genetics, phylogenetic and metabarcoding approaches) ii) tools to diffuse new methods (website, services, training and field tools for scientists and citizen science, publications) and avoid indices misun-derstanding, iii) tools, methods and prototypes to provide datamining usable for an integrative assessment of the GES within the Framework of the Marine Strategy Directive (for this part, CIGESMED members initiated a new consortium using CIGESMED metadata and dataset to build graph representation, mine graphs and provide tools for environmental decision making). All the outcomes are freely accessible online on websites with open access, open source and open data.The overall achievement was to to bring together researchers (in ecology, economics, sociology, law, etc.) and managers in order to (i) identify the needs and to better address them, (ii) to determine interdisciplinary areas of research concerning the development and management of the coralligenous that could be the subject of a new [multidisciplinary / European / Mediterranean] research project.CIGESMED gathered scientists from France, Greece and Turkey, making it possible to assess the coralligenous habitat in a number of sites in both the northwestern Mediterranean basin and the Aegean-Levantine, under a common approach. Members of ten highly experienced marine ecology laboratories were involved.A total of 10 stations in France (in the Gulf of Lions), Greece (in the Ionian and the Aegean Seas) and Turkey (in the Aegean and the Levantine Seas) were selected to study coralligenous assemblages across the Mediterranean Sea. Analyses of photoquadrats (50x50 cm) and in situ visual observations revealed 313 species, belonging to 15 higher taxa. A total of 204 species were found in Turkey, 192 species in France and 109 species in Greece. Only fifty species were common in all sites. The abundance of the taxonomic groups in coralligenous habitats vary among sites and countries. The multivariate analysis revealed five main assemblages across the Mediterra-nean Sea. A number of important threats were withnessed to have an important impact on coralligenous, with the settlement of invasive alien species (e.g. Caulerpa cylindracea, Womersleyella setacea), sedimentation and factors causing algal bleaching being the most important ones.A new method and index were applied and tested in France (60 stations in Gulf of Lions and Provence) and Greece (4 stations in the Gulf of Corinth) to evaluate the health condition of coralligenous assemblages. This method (INDEX-COR), based on images analyses (60x40cm) and in situ observations, takes into account three metrics giving different levels of information: (i) the ratio "Sensitive-Tolerante Species", (ii) the observable taxonomic richness of the assemblages and (iii) the structural complexity. The global index combining these metrics was tested according to a global index of pressure. Reference conditions were defined in France to propose an interpretation grid to evaluate the status of coralligenous assemblages. This grid applied in Greece demonstrated the necessity to collecte multiple and complete dataset in order to define the reference conditions for the different Mediterranean sectors (e.g. Ionian Sea, Egean Sea and Cretan Sea). Finally, additional indices (CAI, COARSE and ESCA) were tested on the datasets obtained in France and Greece. The comparison of the results is still in progress. The first ones show the advantages and the limits of each index. They underline the need to achieve precisions on (i) their degree of sensitivity in the evaluation of the coralligenous assemblages and (ii) the impact of the different images analysis techniques.Although the global set of samples is still under analysis, the metabarcoding pilot study already gave very promising results for the assessment of coralligenous community species composition: many more species were identified than by eye, predicting a higher resolution than traditional approaches for monitoring and comparing coralligenous assemblages.A dedicated Citizen Science initiative was designed and launched in the course of the project, aiming to engage enthusiast divers in the study and monitoring of coralligenous assemblages through the gathering of basic information regarding spatial occurrence, assemblage structure and associated pressures and threats. The implementation platform comprises a data collection protocol and a multilingual website which serve both educational and data submission purposes. Online and paper educational documentation, as well as observation protocol guidelines are essential tools developed to train volunteer divers. Underwater slates based on the princi-ples of rapid visual assessment have also been developed and distributed to all participants for data collection. Geo-referenced data reporting focuses on: (a) basic topographic and abiotic features for the preliminary description of each site, and the creation of data series for sites receiving multiple visits; (b) presence and relative abundance of typical conspicuous species, as well as, (c) existence of pressures and imminent threats, for the characterization and assessment of coralligenous assemblages. More than 100 observation sets from across the Mediterranean have been registered to date by approximately 30 divers while 75 members have registered to the website, which remain active after the conclusion of the project.Metadata and data produced by the CIGESMED project have a high potential for use by several stakeholders involved in environmental management. Mapping this information needed to share common definitions on coralligenous components and allows starting building a micro thesaurus. The methodology is now developed and the first part of the thesaurus is online. A new consortium called IndexMed whose task is to index Mediterranean biodiversity data, makes it possible to build graphs in order to analyse the CIGESMED data and develop new solutions for coralligenous data mining.The outreach activity of the project included participation of the Consortium to an impressive number of Conferences, Symposia and Workshops, which made it possible to achieve not only the communication of the main objectives of the project but also the development of links with other projects, targeting Marine Biodiversity [e.g. EMBOS (The European Marine Biodiversity Observa-tory), LifeWatch (ESFRI Research Infrastruc-ture), DEVOTES (DEVelopment Of innovative Tools for understanding marine biodiversity and assessing good Environmental Status), VECTORS (VECTORS of Change in European Marine Ecosystems and their Environmental and Socio-Economic Impacts) and EU BON (Building the European Biodiversity Observation Net-work)]. A flyer was developed to provide the basic information on the project. The project was also advertised during much larger events, in the premises of the participating institutes, such as open days (e.g. exhibitions for the Climate change, TEDx events). The target audience for the outreach activity consisted of researchers and scientists, students, educators, environmental managers, policy makers and stakeholders from all the economic sectors including industry. The means which were used were: (a) the project website, which until now shows a high number of visits recorded for a specialized one: 250,000 hits; (b) the production of 353 articles for the scientific audience and for the society at large. Five websites have been created from which information on the project is broadcasted.The methods and datasets produced by CIGESMED are disseminated to the STIC community, as free tools for studies to be used for any type of data sciences (data mining, data representation ...), particularly through the means provided by IndexMed and through data qualification processes (which will need to be continuously improved to keep them relevant). This reusability of the data will be improved in particular by the production of data papers and future animations planned within the framework of the IndexMed consortiumThe Steering Committee consisting of all the WP leaders and the coordinator was responsible for all practical decision making, strategic planning and implementation.A Committee of External Advisors met at an annual basis, and aimed at providing advice on all aspects of the execution of the project to ensure CIGESMED to meet its objectives

CIGESMED project: evidence-based management for coralligenous habitats in the Mediterranean Sea

International audienceCIGESMED’s (2013-2016) goal is to understand the links and consequences of natural and anthropogenic pressures to the functioning of these habitats and to define and maintain their Good Environmental Status (GES) in the Mediterranean Sea.

Versatile peroxidase degradation of humic substances: Use of isothermal titration calorimetry to assess kinetics, and applications to industrial wastes

The kinetic constants of a hybrid versatile-peroxidase (VP) which oxidizes complex polymeric humic substances (HS) derived from lignin (humic and fulvic acids) and industrial wastes were determined for the first time using isothermal titration calorimetry (iTC). The reaction conditions were manipulated to enable manganese-peroxidase (MnP) and/or lignin-peroxidase (Lip) activities to be evaluated. The peroxidase reactions exhibited varying degrees of product inhibition or activation; properties which have not previously been reported for VP enzymes. In contrast to previous work (Ertan et al., 2012) on small non-polymeric substrates (MnSO4, veratryl alcohol and dyes), all kinetic plots for polymeric HS were sigmoidal, lacked Michaelis-Menten characteristics, and were indicative of positive cooperativity. Under conditions when both LiP and MnP were active, the kinetic data fitted to a novel biphasic Hill Equation, and the rate of enzymatic reaction was significantly greater than the sum of individual LiP plus MnP activities implying synergistic activation. By employing size-exclusion chromatography and electrospray ionization mass spectrometry, the characteristics of the oxidative degradation products of the HS were also monitored. Our study showed that the allosteric behaviour of the VP enzyme promotes a high level of regulation of activity during the breakdown of model and industrial ligninolytic substrates. The work was extended to examine the kinetics of breakdown of industrial wastes (effluent from a pulp and paper plant, and fouled membrane solids extracted from a ground water treatment membrane) revealing unique, VP-mediated, kinetic responses. This work demonstrates that iTC can be successfully employed to study the kinetic properties of VP enzymes in order to devise reaction conditions optimized for oxidative degradation of HS present in materials used in a wide range of industries. Crown Copyright (c) 2014 Published by Elsevier B.V. All rights reserved

CIGESMED: Coralligenous based indicators to evaluate and monitor the "good environmental statut" of the Mediterranean coastal waters, a SeasEra project

International audienceCoralligenous is one the main shallow Mediterranean milieu generating structural complexity and biodiversity. It produces goods and services for several sectors. Pollution, anchors and trawling may cause its degradation, whilst traditional fishing as well as angling mainly affect target species. Diver frequentation is another cause of degradation. Coralligenous may also be susceptible to invasive alien species. These habitats, which are of great ecological, socioeconomic and patrimonial importance, are also under the pressures caused by the global warming. CIGESMED's (2013-2016) goal is to understand the links and consequences of natural and anthropogenic pressures to the functioning of these habitats and to define and maintain their Good Environmental Status (GES) in the Mediterranean Sea. Indexes, specific to coralligenous habitat, will be co-constructed and collectively tested by scientists, marine natural parks and reserves, through the implementation of a " citizen science " network. Among other methods, trees of knowledge will be experimented as tools to sort, organize and illustrate very large heterogeneous sets of data. CIGESMED outcome will be an integrative assessment of the GES within the Marine Strategy Framework Directive. CIGESMED gathers scientists from France, Greece and Turkey, making it possible to access to sites and to work on the same issues in both the northwestern Mediterranean basin and the Aegean-Levantine one. Ten trained (scientific diving and ROV) laboratories of marine ecology are involved. A Committee of External Advisors (scientists, stakeholders and policy-makers), meeting at an annual basis, and aiming at providing advice on all aspects of the execution of the project is helping the scientific steering committee and is ensuring CIGESMED to meet its objectives