Application of Sequence-Dependent Electrophoresis Fingerprinting in Exploring Biodiversity and Population Dynamics of Human Intestinal Microbiota: What Can Be Revealed?

Sequence-dependent electrophoresis (SDE) fingerprinting techniques such as denaturing gradient gel electrophoresis (DGGE) have become commonplace in the field of molecular microbial ecology. The success of the SDE technology lays in the fact that it allows visualization of the predominant members of complex microbial ecosystems independent of their culturability and without prior knowledge on the complexity and diversity of the ecosystem. Mainly using the prokaryotic 16S rRNA gene as PCR amplification target, SDE-based community fingerprinting turned into one of the leading molecular tools to unravel the diversity and population dynamics of human intestinal microbiota. The first part of this review covers the methodological concept of SDE fingerprinting and the technical hurdles for analyzing intestinal samples. Subsequently, the current state-of-the-art of DGGE and related techniques to analyze human intestinal microbiota from healthy individuals and from patients with intestinal disorders is surveyed. In addition, the applicability of SDE analysis to monitor intestinal population changes upon nutritional or therapeutic interventions is critically evaluated

Elk-1 a Transcription Factor with Multiple Facets in the Brain

The ternary complex factor (TCF) Elk-1 is a transcription factor that regulates immediate early gene (IEG) expression via the serum response element (SRE) DNA consensus site. Elk-1 is associated with a dimer of serum response factor (SRF) at the SRE site, and its phosphorylation occurs at specific residues in response to mitogen-activated protein kinases (MAPKs), including c-Jun-N terminal kinase (JNK), p38/MAPK, and extracellular-signal regulated kinase (ERK). This phosphorylation event is critical for triggering SRE-dependent transcription. Although MAPKs are fundamental actors for the instatement and maintenance of memory, and much investigation of their downstream signaling partners have been conducted, no data yet clearly implicate Elk-1 in these processes. This is partly due to the complexity of Elk-1 sub-cellular localization, and hence functions, within neurons. Elk-1 is present in its resting state in the cytoplasm, where it colocalizes with mitochondrial proteins or microtubules. In this particular sub-cellular compartment, overexpression of Elk-1 is toxic for neuronal cells. When phosphorylated by the MAPK/ERK, Elk-1 translocates to the nucleus where it is implicated in regulating chromatin remodeling, SRE-dependent transcription, and neuronal differentiation. Another post-translational modification is the conjugation to SUMO (Small Ubiquitin-like MOdifier), which relocalizes Elk-1 in the cytoplasm. Thus, Elk-1 plays a dual role in neuronal functions: pro-apoptotic within the cytoplasm, and pro-differentiation within the nucleus. To address the role of Elk-1 in the brain, one must be aware of its multiple facets, and design molecular tools that will shut down Elk-1 expression, trafficking, or activation, in specific neuronal compartments. We summarize in this review the known molecular functions of Elk-1, its regulation in neuronal cells, and present evidence of its possible implication in model systems of synaptic plasticity, learning, but also in neurodegenerative diseases

The non-receptor tyrosine kinase Pyk2 modulates acute locomotor effects of cocaine in D1 receptor-expressing neurons of the nucleus accumbens

The striatum is critical for cocaine-induced locomotor responses. Although the role of D1 receptor-expressing neurons is established, underlying molecular pathways are not fully understood. We studied the role of Pyk2, a non-receptor, calcium-dependent protein-tyrosine kinase. The locomotor coordination and basal activity of Pyk2 knock-out mice were not altered and major striatal protein markers were normal. Cocaine injection increased Pyk2 tyrosine phosphorylation in mouse striatum. Pyk2-deficient mice displayed decreased locomotor response to acute cocaine injection. In contrast, locomotor sensitization and conditioned place preference were normal. Cocaine-activated ERK phosphorylation, a signaling pathway essential for these late responses, was unaltered. Conditional deletion of Pyk2 in the nucleus accumbens or in D1 neurons reproduced decreased locomotor response to cocaine, whereas deletion of Pyk2 in the dorsal striatum or in A2A receptor-expressing neurons did not. In mice lacking Pyk2 in D1-neurons locomotor response to D1 agonist SKF-81297, but not to an anticholinergic drug, was blunted. Our results identify Pyk2 as a regulator of acute locomotor responses to psychostimulants. They highlight the role of tyrosine phosphorylation pathways in striatal neurons and suggest that changes in Pyk2 expression or activation may alter specific responses to drugs of abuse, or possibly other behavioral responses linked to dopamine action

Dysbiosis of bifidobacteria and Clostridium cluster XIVa in the cystic fibrosis fecal microbiota

BACKGROUND: Recurrent antimicrobial interventions and disease-related intestinal dysfunction are suspected to contribute to the dysbiosis of the gastrointestinal microbial ecosystem in patients with cystic fibrosis (CF). The present study set out to detect and identify microbial discriminants in the gut microbiota composition that are associated with CF-related intestinal dysbiosis. METHODS: An in-depth description of CF-associated gut dysbiosis was obtained by screening denaturing gradient gel electrophoresis (DGGE) fingerprints for potentially discriminating bacterial species, and quantification by means of real-time PCR analyses using group-specific primers. RESULTS: A total of 8 DGGE band-classes assigned to the genus Bifidobacterium (n=3), and members of Clostridium clusters XIVa (n=3) and IV (n=2), were significantly (p<0.05) underrepresented in samples of patients with CF. Real-time PCR analyses confirmed a significantly lower abundance and temporal stability of bifidobacteria and Clostridium cluster XIVa in the faecal microbiota of patients with CF. CONCLUSION: This study is the first to report specific microbial determinants of dysbiosis in patients with CF

Modulation and functions of dopamine receptor heteromers in drugs of abuse-induced adaptations

Drug addiction is a chronic and relapsing disorder that leads to compulsive drug intake despite deleterious consequences. By increasing dopamine (DA) in the mesolimbic system, drugs of abuse hijack the brain reward circuitry, which is critical for the development of enduring behavioral alterations. DA mainly acts onto DA D1 (D1R) and D2 (D2R) receptor subtypes, which are positively and negatively coupled to adenylyl cyclase, respectively. Extensive research has aimed at targeting these receptors for the treatment of addiction, however this often results in unwanted side-effects due to the implication of DA receptors in numerous physiological functions. A growing body of evidence indicates that the physical interaction of DA receptors with other receptors can finely tune their function, making DA receptor heteromers promising targets for more specific treatment strategies. An increasing number of articles highlighted the ability of both D1R and D2R to form heteromers, however, most studies carried out to date stem from observations in heterologous systems and the biological significance of DA receptor heteromers in vivo is only emerging. We focused this review on studies that were able to provide insights into functions on D1R and D2R heteromers in drug-evoked adaptations and discuss the limitations of current approaches to study receptor heteromers in vivo. This article is part of the Special Issue entitled 'Receptor heteromers and their allosteric receptor-receptor interactions'.Rôle des heteromères formés par les récepteurs dopamine-glutamate et de signalisation dépendante du calcium nucléaire associée dans l'addictionImpact de la composition lipidique membranaire sur la transmission dopaminergique dépendante du récepteur D2 et la motivationProgram Initiative d’Excellenc

Разработка и исследование асинхронного электропривода с наблюдателем состояния

Выпускная квалификационная работа 109 с., 35 рис., 18 табл., 47 источников, 5 прил. Объектом исследования является дискретная математическая модель наблюдателя состояния полного порядка асинхронного двигателя. Цель работы – Разработка и исследование асинхронного электропривода с наблюдателем состояния В процессе исследования проводилось имитационное моделирование разработанной дискретной математической модели асинхронного двигателя и разработанной дискретной математической модели наблюдателя состояния полного порядкаFinal qualifying work 109 p., 35 fig., 18 tab., 47 sources, 5 adj. The object of research is a discrete mathematical model of the observer status of full order of the induction motor. Objective - Development and research of the asynchronous electric drive with observer status The study was conducted simulations developed discrete mathematical model of the induction motor and the developed mathematical model of discrete observer of full order stat

De dubbele waterput uit het laat-Romeinse castellum van Oudenburg (prov. West-Vlaanderen): tafonomie, chronologie en interpretatie

This article focuses on a remarkable well structure that was brought to light by the Flemish Heritage Institute during recent archaeological research at the south-western corner of the Saxon Shore fort at Oudenburg (2001-2005). The site of Oudenburg is situated 8 km from the Flemish coastline, in the polder area between Bruges and Ostend. During Roman times however, positioned strategically on an elevated sandy ridge, the site overlooked the coastal plain consisting of mudflats and marshes intersected by natural gullies. The remains of the fort at Oudenburg were discovered in 1956-1957 by J. Mertenslater excavation campaigns in 1960 and 1970 on the western defence area revealed a sequence of three successive forts. The 1960s excavations on two late Roman military cemeteries more than 400 m to the west of the castellum revealed burials of 4th-century fort inhabitants with rich grave goods. During archaeological research within the fort walls in 1976-1977 the first information was collected about the inner organisation of the fort and the remains of a stone building of late 3rd-century date were excavated. It was only in 2001 that new excavations could take place on the fort area. This systematic research resulted in a finer chronology for the occupation of the castellum. A succession of five main fort periods was revealed, dating between ca. 200 and the beginning of the 5th century AD. These excavations yielded insight into the spatial organisation of the south west area of the fort, which had different functions in each successive fort period. The first three phases belonged to wood and earthen fortstemporary installations in times of trouble and Germanic threat. Probably in the later 3rd century AD, a more permanent fort measuring 153 by 176 m was built in stonethis was renovated and reoccupied during the second quarter of the 4th century AD. The characteristics of the ground plan, its topographical position and several finds pointing to a close link with the Saxon Shore forts on the coast of south Britannia, suggest that Oudenburg was probably part of the Litus Saxonicum. In this paper the so-called double well, a context of the fifth fort period (4th centurybeginning 5th century), is analysed. During this period the south-western area of the castellum was dominated by a stone bath building with hypocaust system. Later in the 4th century, long fences were constructed to divide the area into yards, a timber-framed construction with simple plan may be identified as a stable structure, and a large oak basin was probably a reservoir for drinking water. The double well, which received feature number OS 2562, seems to be a key context for this fort period

Cocaine increases dopaminergic connectivity in the nucleus accumbens.

The development of addictive behavior is associated with functional and structural plasticity in the mesocorticolimbic pathway. Increased connectivity upon cocaine administration has been inferred from increases in dendritic spine density, but without observations of presynaptic elements. Recently, we established a method that enables analyses of both dendritic spines and glutamatergic boutons and presented evidence that cocaine induces changes in striatal connectivity. As the pharmacological and behavioral effects of cocaine directly implicate dopaminergic neurons and their afferents, a remaining question is whether dopaminergic striatal innervations also undergo structural plasticity. To address this issue, we generated transgenic mice in which the fluorophore tdTomato is expressed under the promoter of the dopamine transporter gene. In these mice, specific labeling of dopaminergic boutons was observed in the striatum. Of note, the accordance of our results for control mice with previous electron microscopy studies confirms that our method can be used to decipher the spatial organization of boutons in relation to dendritic elements. Following repeated cocaine administration that led to behavioral locomotor sensitization, an increased density of dopaminergic boutons was observed 1 day later in the nucleus accumbens shell specifically, and not in other striatal regions. Combined labeling of dopaminergic boutons and striatal dendrites showed that cocaine significantly increased the percentage of dendritic spines associated with a dopaminergic bouton. Our results show that chronic cocaine administration induces structural plasticity of dopaminergic boutons that could participate in dopamine-dependent neuronal adaptations in the striatum.This work was supported by Centre National pour la Recherche Scientifique (CNRS), Institut National de la Santé et de la Recherche Médicale (INSERM), University Pierre and Marie Curie (UPMC), Agence Nationale de la Recherche (ANR), Fondation pour la Recherche Médicale (FRM) and the Labex Bio-Psy cluster of excellence. M.D.S. was a recipient of a fellowship from French Ministry of Research and Labex Bio-Psy. E.N.C. was supported by the Ecole de Neuroscience de Paris (ENP) and FRM

Absence of SPARC results in increased cardiac rupture and dysfunction after acute myocardial infarction

The matricellular protein SPARC (secreted protein, acidic and rich in cysteine, also known as osteonectin) mediates cell–matrix interactions during wound healing and regulates the production and/or assembly of the extracellular matrix (ECM). This study investigated whether SPARC functions in infarct healing and ECM maturation after myocardial infarction (MI). In comparison with wild-type (WT) mice, animals with a targeted inactivation of SPARC exhibited a fourfold increase in mortality that resulted from an increased incidence of cardiac rupture and failure after MI. SPARC-null infarcts had a disorganized granulation tissue and immature collagenous ECM. In contrast, adenoviral overexpression of SPARC in WT mice improved the collagen maturation and prevented cardiac dilatation and dysfunction after MI. In cardiac fibroblasts in vitro, reduction of SPARC by short hairpin RNA attenuated transforming growth factor β (TGF)–mediated increase of Smad2 phosphorylation, whereas addition of recombinant SPARC increased Smad2 phosphorylation concordant with increased Smad2 phosphorylation in SPARC-treated mice. Importantly, infusion of TGF-β rescued cardiac rupture in SPARC-null mice but did not significantly alter infarct healing in WT mice. These findings indicate that local production of SPARC is essential for maintenance of the integrity of cardiac ECM after MI. The protective effects of SPARC emphasize the potential therapeutic applications of this protein to prevent cardiac dilatation and dysfunction after MI