Nanopores: maltoporin channel as a sensor for maltodextrin and lambda-phage

BACKGROUND: To harvest nutrition from the outside bacteria e.g. E. coli developed in the outer cell wall a number of sophisticated channels called porins. One of them, maltoporin, is a passive specific channel for the maltodextrin uptake. This channel was also named LamB as the bacterial virus phage Lambda mis-uses this channel to recognise the bacteria. The first step is a reversible binding followed after a lag phase by DNA injection. To date little is known about the binding capacity and less on the DNA injection mechanism. To elucidate the mechanism and to show the sensitivity of our method we reconstituted maltoporin in planar lipid membranes. Application of an external transmembrane electric field causes an ion current across the channel. Maltoporin channel diameter is around a few Angstroem. At this size the ion current is extremely sensitive to any modification of the channels surface. Protein conformational changes, substrate binding etc will cause fluctuations reflecting the molecular interactions with the channel wall. The recent improvement in ion current fluctuation analysis allows now studying the interaction of solutes with the channel on a single molecular level. RESULTS: We could demonstrate the asymmetry of the bacterial phage Lambda binding to its natural receptor maltoporin. CONCLUSION: We suggest that this type of measurement can be used as a new type of biosensors

Fabrication and functionalization of nanochannels by electron-beam-induced silicon oxide deposition

We report on the fabrication and electrical characterization of functionalized solid-state nanopores in low stress silicon nitride membranes. First, a pore of approximately 50 nm diameter was drilled using a focused ion beam technique, followed by the local deposition of silicon dioxide. A low-energy electron beam induced the decomposition of adsorbed tetraethyl orthosilicate resulting in site-selective functionalization of the nanopore by the formation of highly insulating silicon oxide. The deposition occurs monolayer by monolayer, which allows for control of the final diameter with subnanometer accuracy. Changes in the pore diameter could be monitored in real time by scanning electron microscopy. Recorded ion currents flowing through a single nanopore revealed asymmetry in the ion conduction properties with the sign of the applied potential. The low-frequency excess noise observed at negative voltage originated from stepwise conductance fluctuations of the open pore

Functional asymmetry of transmembrane segments in nicotinic acetylcholine receptors

Nicotinic acetylcholine receptors are heteropentameric ion channels that open upon activation to a single conducting state. The second transmembrane segments of each subunit were identified as channel-forming elements, but their respective contribution in the gating process remains unclear. Moreover, the detailed impact of variations of the membrane potential, such as occurring during an action potential, on the transmembrane domains, is unknown. Residues at the 12' position, close to the center of each second transmembrane segment, play a key role in channel gating. We examined their functional symmetry by substituting a lysine to that position of each subunit and measuring the electrical activity of single channels. For 12' lysines in the alpha, gamma and delta subunits rapid transitions between an intermediate and large conductance appeared, which are interpreted as single lysine protonation events. From the kinetics of these transitions we calculated the pK (a) values of respective lysines and showed that they vary differently with membrane hyperpolarization. Respective mutations in beta or epsilon subunits gave receptors with openings of either intermediate or large conductance, suggesting extreme pK (a) values in two open state conformations. The results demonstrate that these parts of the highly homologous transmembrane domains, as probed by the 12' lysines, sense unequal microenvironments and are differently affected by physiologically relevant voltage changes. Moreover, observation of various gating events for mutants of alpha subunits suggests that the open channel pore exists in multiple conformations, which in turn supports the notion of functional asymmetry of the channel

Local solid-state modification of nanopore surface charges

The last decade, nanopores have emerged as a new and interesting tool for the study of biological macromolecules like proteins and DNA. While biological pores, especially alpha-hemolysin, have been promising for the detection of DNA, their poor chemical stability limits their use. For this reason, researchers are trying to mimic their behaviour using more stable, solid-state nanopores. The most successful tools to fabricate such nanopores use high energy electron or ions beams to drill or reshape holes in very thin membranes. While the resolution of these methods can be very good, they require tools that are not commonly available and tend to damage and charge the nanopore surface. In this work, we show nanopores that have been fabricated using standard micromachning techniques together with EBID, and present a simple model that is used to estimate the surface charge. The results show that EBID with a silicon oxide precursor can be used to tune the nanopore surface and that the surface charge is stable over a wide range of concentrations.Comment: 10 pages, 6 figure

Micro- and nanostructured devices for investigating biomolecular interactions

Cell membrane receptors and ion channels are essential in many different cellular processes. To analyse the activity of membrane proteins in vesicles and biological cells, we fabricated micro- nanostructured chips, enabling the application of electrophysiology and fluorescence-based techniques. A SU-8 biochip was developed for simultaneously micromanipulating and investigating optically and electrically individual vesicles in a microfluidic channel. Lipid vesicles were transported, positioned by electrophoretic movement on a micrometer sized aperture, and fused to form a planar suspended membrane, which is suited to study ion channel activity. Aiming to investigate G protein-coupled receptor signaling pathways in native-like environment, we developed a method for producing well-oriented planar cell membrane sheets on silicon films containing nanoaperture arrays. The accessibility of extracellular and cytosolic surfaces was demonstrated by targeting membrane constituents side-specifically with fluorescent markers. Our approach can be applied for studying membrane proteins from a large variety of cells and cellular organelles using chip-based screening assays

Immune activation of the p75 neurotrophin receptor: implications in neuroinflammation

Despite structural similarity with other tumor necrosis factor receptor superfamily (TNFRSF) members, the p75 neurotrophin receptor (p75NTR, TNFR16) mediates pleiotropic biological functions not shared with other TNFRs. The high level of p75NTR expression in the nervous system instead of immune cells, its utilization of co-receptors, and its interaction with soluble dimeric, rather than soluble or cell-tethered trimeric ligands are all characteristics which distinguish it from most other TNFRs. Here, we compare these attributes to other members of the TNFR superfamily. In addition, we describe the recent evolutionary adaptation in B7-1 (CD80), an immunoglobulin (Ig) superfamily member, which allows engagement to neuronally-expressed p75NTR. B7-1-mediated binding to p75NTR occurs in humans and other primates, but not lower mammals due to specific sequence changes that evolved recently in primate B7-1. This discovery highlights an additional mechanism by which p75NTR can respond to inflammatory cues and trigger synaptic elimination in the brain through engagement of B7-1, which was considered to be immune-restricted. These observations suggest p75NTR does share commonality with other immune co-modulatory TNFR family members, by responding to immunoregulatory cues. The evolution of primate B7-1 to bind and elicit p75NTR-mediated effects on neuronal morphology and function are discussed in relationship to immune-driven modulation of synaptic actions during injury or inflammation

The porin and the permeating antibiotic: A selective diffusion barrier in gram-negative bacteria

Gram-negative bacteria are responsible for a large proportion of antibiotic resistant bacterial diseases. These bacteria have a complex cell envelope that comprises an outer membrane and an inner membrane that delimit the periplasm. The outer membrane contains various protein channels, called porins, which are involved in the influx of various compounds, including several classes of antibiotics. Bacterial adaptation to reduce influx through porins is an increasing problem worldwide that contributes, together with efflux systems, to the emergence and dissemination of antibiotic resistance. An exciting challenge is to decipher the genetic and molecular basis of membrane impermeability as a bacterial resistance mechanism. This Review outlines the bacterial response towards antibiotic stress on altered membrane permeability and discusses recent advances in molecular approaches that are improving our knowledge of the physico-chemical parameters that govern the translocation of antibiotics through porin channel

Bibliometric analysis of themes competitive intelligence, knowledge management and organizational knowledge in Institutional Repository University of Brasília

Trata da análise dos temas Inteligência Competitiva (IC), Gestão do Conhecimento (GC) e Conhecimento Organizacional (CO), no Repositório Institucional da Universidade da Brasília (RIUnB), que está disponível em acesso aberto desde setembro de 2008. O objetivo é verificar a produção científica sobre as áreas de pesquisa, a partir dos documentos que constam no RIUnB. O método de trabalho consistiu nos estudos bibliométricos, a fim de se inteirar sobre a produção de trabalhos referentes ao conhecimento resultante do desenvolvimento de pesquisas no âmbito institucional, para conhecer a comunidade. Os resultados apontam para um crescimento notório desses temas na ciência da informação, com perspectiva de crescimento em outras áreas: administração, engenharia elétrica, educação e gestão social e trabalho, demonstrando sua potencial interdisciplinaridade; houve maior concentração de publicação nos temas gestão do conhecimento, conhecimento organizacional e inteligência competitiva, respectivamente, e existe uma concentração dessas publicações em três pesquisadores na comunidade da Faculdade de Ciência da Informação (FCI). Conclui-se que os temas são mais frequentes na área de ciência da informação, entretanto, nota-se o seu aparecimento em outras áreas do conhecimento, na UnB: administração, engenharia elétrica, educação e gestão social e do trabalho. Esses dados podem revelar uma tendência de consolidação dos temas de pesquisa em outras áreas. ________________________________________________________________________________________ ABSTRACTThis analysis of topics Competitive Intelligence (CI), Knowledge Management (KM) and Organizational Knowledge (CO) in the Institutional Repository of the University of Brasília (RIUnB) which is available in open access since September 2008. The aim is to check the scientific literature on the research areas from the documents listed in RIUnB. The working method consisted in bibliometric studies, in order to learn about the production of works relating to knowledge resulting from research development at the institutional level to know the community. The results indicate a remarkable growth of these themes in information science, with growth prospects in other areas: administration, electrical engineering, management and education and social work, demonstrating its potential interdisciplinarity, there was a higher concentration of publishing on issues of knowledge management, organizational knowledge and competitive intelligence, respectively, and there is a concentration of such publications in three community researchers in the Faculty of Information Science (FCI). We conclude that the issues are more frequent in the area of information science, however, note its appearance in other areas of knowledge in UNB: administration, electrical engineering, education and social management and labor. These data may reveal a trend of consolidation of research topics in other areas

How β-Lactam Antibiotics Enter Bacteria: A Dialogue with the Porins

BACKGROUND:Multi-drug resistant (MDR) infections have become a major concern in hospitals worldwide. This study investigates membrane translocation, which is the first step required for drug action on internal bacterial targets. beta-lactams, a major antibiotic class, use porins to pass through the outer membrane barrier of Gram-negative bacteria. Clinical reports have linked the MDR phenotype to altered membrane permeability including porin modification and efflux pump expression. METHODOLOGY/PRINCIPAL FINDINGS: Here influx of beta-lactams through the major Enterobacter aerogenes porin Omp36 is characterized. Conductance measurements through a single Omp36 trimer reconstituted into a planar lipid bilayer allowed us to count the passage of single beta-lactam molecules. Statistical analysis of each transport event yielded the kinetic parameters of antibiotic travel through Omp36 and distinguishable translocation properties of beta-lactams were quantified for ertapenem and cefepime. Expression of Omp36 in an otherwise porin-null bacterial strain is shown to confer increases in the killing rate of these antibiotics and in the corresponding bacterial susceptibility. CONCLUSIONS/SIGNIFICANCE: We propose the idea of a molecular "passport" that allows rapid transport of substrates through porins. Deciphering antibiotic translocation provides new insights for the design of novel drugs that may be highly effective at passing through the porin constriction zone. Such data may hold the key for the next generation of antibiotics capable of rapid intracellular accumulation to circumvent the further development MDR infections

Arnica montana : évaluation des ressources génétiques françaises en vue du développement de la culture en plaine et en montagne

Ce volume regroupe les textes issus du programme Casdar "Innovation et Partenariat" et "Recherche finalisée et innovation" de 2013. Le colloque de restitution s’est déroulé le 6 février 2019 sous l’égide du GIS Relance AgronomiqueArnica montana is a major medicinal specie, which is now mainly produced from wild harvesting,especially in mainland France. As the wild resource is decreasing, and in order to maintain or even Gourlin L. et al. 68 Innovations Agronomiques 71 (2019), 67-80 develop the French production, cultivation is a good option, that is still very limited, because of its difficulty. Finding the right plant material could help to enhance cultivation programs. This project aimed at growing 24 wild populations, which were collected in mainland France, and to compare them with 2 commercial varieties, ‘Arbo’ and ‘Arnimed’. This was set on 4 experimentation spots, chosen for their potential match for Arnica cultivation. The experiment lasted 3 years, and morphological andagronomical subjects were studied on the populations. Sesquiterpene lactones and flavonoidsanalyzed, and a new methodology of evaluation was developed. The results showed extreme variabilityof phenotypic and chemical expression of the different populations. An important death rate has beennoticed on wild populations, but the causes are still unknown. On the set of variables chosen,commercial varieties ‘Arbo’ and ‘Arnimed’ were particularly competitive, and two wild populations standout with promising results. One seems appropriate for starting selection works on a variety that would besuitable for loaw altitude, and the other one could be a local (French) alternative to the cultivation ofselected varieties (‘Arbo’ and ‘Arnimed’ are from Swiss and German selection work).L’arnica des montagnes est une espèce médicinale importante dont la production est principalementissue de la cueillette à l’état sauvage, notamment sur le territoire métropolitain. La ressource étant enrégression, le maintien, voire le développement de la production française passe donc par la mise enculture, actuellement anecdotique car difficile. Certains freins pourraient être levés par la mise enévidence de matériel végétal adapté à la production. L’objectif de ce projet était de mettre en culture 24populations d’origines sauvages (prospectées en France métropolitaine) et de les comparer à deuxvariétés commerciales témoins ‘Arbo’ et ‘Arnimed’, sur 4 sites d’expérimentation aux contextespédoclimatiques variés mais a priori adaptés à la culture de l’espèce. Durant les 3 années d’essai, unsuivi morphologique et agronomique des populations a été réalisé. Des analyses des sesquiterpèneslactones et flavonoïdes ont été effectuées, et une nouvelle méthodologie de dosage de ces composés aété développée. Les résultats mettent en exergue la forte variabilité de l’expression phénotypique etchimique des différentes souches testées. Une forte mortalité globale a pu être constatée surl’ensemble des populations sauvages étudiées sans que les causes aient pu en être identifiées. Surl’ensemble des variables suivies, les variétés commerciales ‘Arbo’ et ‘Arnimed’ sont particulièrementperformantes, et deux populations sauvages se démarquent par leurs résultats intéressants : l’uneparait pertinente pour démarrer des travaux de sélection d’une variété adaptée à la basse altitude,tandis que l’autre, originaire du Massif central, pourrait se proposer comme une alternative d’originelocale (française) à la culture de variétés commerciales sélectionnées (suisse et allemande)