The bacterial antitoxin HipB establishes a ternary complex with operator DNA and phosphorylated toxin HipA to regulate bacterial persistence

Nearly all bacteria exhibit a type of phenotypic growth described as persistence that is thought to underlie antibiotic tolerance and recalcitrant chronic infections. The chromosomally encoded high-persistence (Hip) toxin-antitoxin proteins HipA(SO) and HipB(SO) from Shewanella oneidensis, a proteobacterium with unusual respiratory capacities, constitute a type II toxin-antitoxin protein module. Here we show that phosphorylated HipA(SO) can engage in an unexpected ternary complex with HipB(SO) and double-stranded operator DNA that is distinct from the prototypical counterpart complex from Escherichia coli. The structure of HipB(SO) in complex with operator DNA reveals a flexible C-terminus that is sequestered by HipA(SO) in the ternary complex, indicative of its role in binding HipA(SO) to abolish its function in persistence. The structure of HipA(SO) in complex with a non-hydrolyzable ATP analogue shows that HipA(SO) autophosphorylation is coupled to an unusual conformational change of its phosphorylation loop. However, HipA(SO) is unable to phosphorylate the translation factor Elongation factor Tu, contrary to previous reports, but in agreement with more recent findings. Our studies suggest that the phosphorylation state of HipA is an important factor in persistence and that the structural and mechanistic diversity of HipAB modules as regulatory factors in bacterial persistence is broader than previously thought

Antioxidant Activity and Phenolic Content of Marine Dissolved Organic Matter and Their Relation to Molecular Composition

The potential of marine dissolved organic matter (DOM) for free radical scavenging has been extensively evaluated, however, the quantitative assessment of the antioxidant potential has been recently measured for the first time. The linkage of the DOM antioxidant potential to its molecular composition has not yet been examined. Following this line, this article takes a step forward by assessing, throughout a polarity-mediated fractionation, (1) the antioxidant capacity and phenolic content and (2) the molecular characterization of DOM in a more exhaustive manner. (3) The DOM antioxidant potential and phenolic content was linked to the molecular composition of DOM, which was molecularly characterized using ultrahigh resolution Fourier transform Ion Cyclotron Resonance mass spectrometry (FT-ICR MS). Antioxidant activity and phenolic content were quantified by the free radical 2,2’-azino-bis-3-ethylbenzothiazoline-6-sulfonic acid (ABTS⋅) and the Folin-Ciocalteu methods, respectively. We considered three types of different natural DOM samples: the deep North Pacific Ocean, the oligotrophic surface of the North Pacific Ocean and porewater from the sulfidic tidal flats of the Wadden Sea. Bulk porewater and its individual polarity fractions presented the highest antioxidant activity and phenolic content. DOM from the water column samples had lower antioxidant activity and phenolic content than porewater, but exceeded what it is commonly found in macroalgae, microalgae, fruits and vegetables with cosmeceutical purposes. Our values were similar to published values for terrestrial DOM. The variations in bioactivity were dependent on polarity and molecular composition. The high resolution and high mass accuracy used to determine the molecular composition of marine DOM and the chemometric and multistatistical analyses employed have allowed to distinguish molecular categories that are related to the bioactive potential. As a future perspective, we performed cytotoxicity tests with human cells and propose marine DOM as a natural ingredient for the development of cosmeceutical products

Experimentelle Untersuchung des Neuronenverlusts bei fokaler zerebraler Ischämie im Rattenhirn nach Applikation von Gewebeplasminogenaktivator und hyperbarem Sauerstoff

Die systemische Thrombolyse mit rekombinantem Gewebeplasminogenaktivator (rtPA) stellt das einzige pharmakologisch evidente und kausale Therapiekonzept für die Behandlung der akuten zerebralen Ischämie dar. Dieses Be-handlungskonzept ist nur in einem kurzen therapeutischen Zeitfenster anwendbar und birgt das Risiko unerwünschter zum Teil lebensbedrohlicher Nebenwirkungen. Darüber hinaus ist rtPA potenziell neurotoxisch. In der vorliegenden Arbeit sollten am thrombembolischen Schlaganfallmodell der Ratte die Effekte der hyperbaren Sauerstofftherapie (HBO) nach induzierter fokaler zerebraler Ischämie in Kombination mit rtPA erstmals im Langzeitverlauf von 4 Wochen untersucht werden. Grundlage hierfür waren die in früheren Studien nachgewiesenen potenziell neuroprotektiven Effekte der HBO. In 4 verschiedenen Infarkt-assoziierten Arealen wurden durch Immunperoxidasemarkierung Neuronale Nuklei (NeuN) im Gehirn von Ratten detektiert und durch den Vergleich mit der kontralateralen Hemisphäre der Neuronenverlust erfasst. Mithilfe einer Dreifach-immunfluoreszenzfärbung und konfokaler Laserscanningmikroskopie wurden neben dem Neuronenverlust sowohl Astrogliose als auch Mikrogliaaktivierung, als wesentliche Bestandteile der neurovaskulären Einheit (NVU), qualitativ analysiert. Für die Ischämie-assoziierten Gebiete ergab sich ein über den gesamten Beobachtungszeitraum konstantes und mit dem Penumbramodell korres-pondierendes schalenförmiges Muster des Neuronenverlusts. Die Hypothese einer Neuroprotektion durch HBO in Kombination mit rtPA konnte im Vergleich zu früheren experimentellen Studienergebnissen nicht bestätigt werden.:Bibliografische Beschreibung I Abkürzungsverzeichnis II Inhaltsverzeichnis V 1. Einleitung 1 1.1 Sozioökonomische Bedeutung des Schlaganfalls 1 1.2 Pathophysiologische Grundlagen der zerebralen Ischämie 1 1.2.1 Neuronale Zellen und Peri-Infarkt-Depolarisation 2 1.2.2 Oxidativer Stress und Stickstoffmonoxid 4 1.2.3 Nekrose und Apoptose 5 1.2.4 Inflammatorische Prozesse 5 1.2.5 Wechselwirkungen zwischen Neuronen, Astroglia und Mikroglia 7 2. Therapiekonzepte bei zerebraler Ischämie 8 2.1 Thrombolyse mit rekombinantem Gewebeplasminogenaktivator 8 2.2 Experimentelle Therapieansätze 10 2.3 Physiologische Grundlagen der hyperbaren Sauerstofftherapie 10 2.4 Nebenwirkungen der hyperbaren Sauerstofftherapie 12 3. Zielstellung 13 4. Material und Methoden 14 4.1 Gewebeproben 14 4.2 Studiendesign 14 4.3 Material 15 4.3.1 Verwendete Chemikalien 15 4.3.2 Verwendete Lösungen 16 4.4 Methoden 17 4.4.1 Operativ induzierte thrombembolische Ischämie 17 4.4.2 Applikation von rtPA und hyperbarem Sauerstoff 19 4.4.3 Gewebeaufbereitung 19 4.4.4 Auswahl der Hirnschnitte 19 4.4.5 Erfassung der Infarktlokalisation im Rattenhirn 20 4.4.6 NeuN-Immunperoxidase Färbung 21 4.4.7 Immunfluoreszenzmarkierung von Neuronen, Astro- und Mikroglia 22 4.4.8 Kontrollfärbungen 24 4.4.9 Mikroskopie und Bildgebung 24 4.4.10 Semiquantitative Auswertung 25 5. Ergebnisse 27 5.1 Lokalisation der fokal zerebralen Ischämie im Rattenhirn 29 5.2 Neuronenverlust in Abhängigkeit von zuvor definierten „Regions of Interests“ 30 5.3 Interventionsabhängiger Verlauf des Neuronenverlusts 31 5.4 Aktivierung von Astro- und Mikroglia in Abhängigkeit vom Neuronenverlust 35 6. Diskussion 41 6.1 Translationale Aspekte 42 6.2 Die Penumbra als potenziell rettbares Hirngewebe 45 6.3 Neuronenverlust in Abhängigkeit von zeitlichen Verlauf und Interventionskonzept 47 6.4 Interaktionen von Neuronen, Astro- und Mikroglia nach fokaler zerebraler Ischämie 51 7. Zusammenfassung 56 8. Literaturverzeichnis 59 Tabellenverzeichnis VII Abbildungsverzeichnis VIII Lebenslauf X Publikationen XI Erklärung über die eigenständige Abfassung der Arbeit XII Danksagung XII

Effect of the (n +1) residue on peptide deamidation

Deamidation occurs spontaneously in peptides and proteins [1]. Many reports show that deamidation has a substantial effect on protein structure, function and stability. An experimental study on pentapeptides [2] shows that Asn deamidation rates are directly related to primary structure near Asn, with a prominent effect from the carboxyl-side (Yyy). We explore the correlation between experimental deamidation rates and primary structure for two pentapeptides (Yyy=Gly, Ile), to identify the factors causing this dependence. A pentapeptide in a water box is subject to Replica-Exchange Molecular Dynamics (REMD) calculations performed with GROMACS 4 software. Furthermore, a succinimide-mediated reaction [3] is investigated with QM/MM calculations utilizing the CP2K code using Nudged-Elastic-Band (NEB) calculations.[p] [p] [p][1] Robinson, NE, Robinson, AB Proc. Natl. Acad. Sci. USA 2001 , 98, 944.[p][2] Robinson, NE J. Peptide Res. 2004 , 63, 426.[p][3] Catak, S., Monard, G., Aviyente, V., Ruiz-López, MF J. Phys. Chem. A 2009 , 113, 1111

Approach for tuberculosis-associated immune reconstitution inflammatory syndrome in an HIV-negative patient

A male refugee from the Middle East was diagnosed with pulmonary tuberculosis and Pott’s disease with paravertebral abscess. After starting the standard regimen, the sputum culture converted to negative and the patient’s general condition improved. Six weeks later, the patient presented with clinical worsening of known symptoms, new appearance of focal neurological deficits and progress of radiological features showing progression of the paravertebral abscess. Immune reconstitution inflammatory syndrome with Mycobacterium tuberculosis (TB-IRIS) was presumed, and treatment with high-dose steroids was started. Due to recurrent relapses while tapering, corticosteroids had to be given over a prolonged period. After treatment completion, the patient was in a good general condition, abscesses had decreased and neurological deficits were in complete remission. This case presents the rare manifestation of TB-IRIS in HIV-negative patients and its management in a high-income country

Strongly non-linear interaction between non-classical light and a blockaded Rydberg atomic ensemble

We investigate the interaction between non-classical light with a tunable multiphoton component and a highly nonlinear medium based on cold Rydberg atoms. The non-classical field emitted by a DLCZ quantum memory is stored using Rydberg electromagnetically induced transparency, experiencing strong nonlinear response due to the dipole blockade. We show that the storage efficiency in the Rydberg ensemble decreases as function of the multiphoton strength of the input field, as a result of the nonlinearity. We also show that the autocorrelation function $g^{(2)}(0)$ of the retrieved field after storage in the Rydberg state is considerably reduced, leading to the first demonstration of single photon filtering with non-classical input light. Finally, we develop a simple simulation that allows us to model the effect of our medium on the input state. This work is a step towards matter-mediated photon-photon interactions with non-classical light

Central ions and lateral asparagine/glutamine zippers stabilize the post-fusion hairpin conformation of the SARS coronavirus spike glycoprotein

AbstractThe coronavirus spike glycoprotein is a class I membrane fusion protein with two characteristic heptad repeat regions (HR1 and HR2) in its ectodomain. Here, we report the X-ray structure of a previously characterized HR1/HR2 complex of the severe acute respiratory syndrome coronavirus spike protein. As expected, the HR1 and HR2 segments are organized in antiparallel orientations within a rod-like molecule. The HR1 helices form an exceptionally long (120 Å) internal coiled coil stabilized by hydrophobic and polar interactions. A striking arrangement of conserved asparagine and glutamine residues of HR1 propagates from two central chloride ions, providing hydrogen-bonding “zippers” that strongly constrain the path of the HR2 main chain, forcing it to adopt an extended conformation at either end of a short HR2 α-helix

New energy carriers in vehicles and their impact on confined infrastructures Overview of previous research and research needs

International audienceThe global warming debate forces the vehicle industry to come up with new environmentally friendly solutions. In 10 years time, or even faster depending on the pressure from different governments in particular in Europe, vehicles will not only use gasoline, diesel and LPG, but also CNG, Hydrogen, ethanol, DME and other bio-fuels, as well as batteries and fuel cells. This quick development and the diversity of new energy carriers can jeopardize the safety in underground infrastructures such as tunnels or car parks. This can cause a major drawback in the adoption of new energy carriers as regulators or operators may prohibit use of these vehicles in underground systems if no new relevant measures will be taken. Unclear situation will also affect the implementation of international policies aiming at reducing the environmental footprint and especially CO2 emission in road traffic. The problem became clear after a workshop with the vehicle industry, tunnel operators, authorities, and safety experts organised in November 2008 by L-surF Services with the support of ITA-COSUF, ECTP and HYSAFE. This workshop demonstrated that the construction sector lacks appropriate design data and tools as well as knowledge to build safe underground infrastructure compatible with a diversity of new and alternative energy carriers. Vehicle industry, infrastructure operators and regulators have not yet addressed this problem. In a first part, an overview of the regulatory situation regarding safety and security of the admission of new energy carriers for vehicles in underground infrastructures is presented. Then, a detailed review of previous relevant research projects performed makes it possible to formulate recommendations in terms of a strategic research & development agenda. The overview shows that it is necessary to develop an integrated risk assessment and management method specific for underground transport systems, metros and hubs in confined spaces taking into account the "emerging risk" aspects