Crystal Structure of Thermotoga maritima α-Glucosidase AglA Defines a New Clan of NAD+-dependent Glycosidases

Glycoside hydrolase family 4 represents an unusual group of glucosidases with a requirement for NAD(+), divalent metal cations, and reducing conditions. The family is also unique in its inclusion of both alpha- and beta-specific enzymes. The alpha-glucosidase A, AglA, from Thermotoga maritima is a typical glycoside hydrolase family 4 enzyme, requiring NAD(+) and Mn2+ as well as strongly reducing conditions for activity. Here we present the crystal structure of the protein complexed with NAD(+) and maltose, refined at a resolution of 1.9 Angstrom. The NAD(+) is bound to a typical Rossman fold NAD(+)-binding site, and the nicotinamide moiety is localized close to the maltose substrate. Within the active site the conserved Cys-174 and surrounding histidines are positioned to play a role in the hydrolysis reaction. The electron density maps indicate that Cys-174 is oxidized to a sulfinic acid. Most likely, the strongly reducing conditions are necessary to reduce the oxidized cysteine side chain. Notably, the canonical set of catalytic acidic residues common to other glucosidases is not present in the active site. This, combined with a high structural homology to NAD-dependent dehydrogenases, suggests an unusual and possibly unique mechanism of action for a glycoside-hydrolyzing enzyme

Thermomechanical material modelling based on a hybrid free energy density depending on pressure, isochoric deformation and temperature

AbstractIn order to represent temperature-dependent mechanical material properties in a thermomechanical consistent manner it is common practice to start with the definition of a model for the specific Helmholtz free energy. Its canonical independent variables are the Green strain tensor and the temperature. But to represent calorimetric material properties under isobaric conditions, for example the exothermal behaviour of a curing process or the dependence of the specific heat on the temperature history, the temperature and the pressure should be taken as independent variables. Thus, in the field of calorimetry the Gibbs free energy is usually used as thermodynamic potential whereas in continuum mechanics the Helmholtz free energy is normally applied. In order to simplify the representation of calorimetric phenomena in continuum mechanics a hybrid free energy density is introduced. Its canonical independent variables are the isochoric Green strain tensor, the pressure and the temperature. It is related to the Helmholtz free energy density by a Legendre transformation. In combination with the additive split of the stress power into the sum of isochoric and volumetric terms this approach leads to thermomechanical consistent constitutive models for large deformations. The article closes with applications of this approach to finite thermoelasticity, curing adhesives and the glass transition

Spatially resolved sampling for untargeted metabolomics: a new tool for salivomics

Saliva is a complex bodily fluid composed of metabolites secreted by major and minor glands, as well as by-products of host oral cells, oral bacteria, gingival crevicular fluid, and exogenous compounds. Major salivary glands include the paired parotid, submandibular, and sublingual glands. The secreted fluids of the salivary glands vary in composition, flow rate, site of release, and clearance suggesting that different types of saliva fulfill different functions and therefore can provide unique biological information. Consequently, for the comprehension of the functionality of the salivary components, spatially resolved investigations are warranted. To understand and comprehensively map the highly heterogeneous environment of the oral cavity, advanced spatial sampling techniques for metabolomics analysis are needed. Here, we present a systematic evaluation of collection devices for spatially resolved sampling aimed at untargeted metabolomics and propose a comprehensive and reproducible collection and analysis protocol for the spatially resolved analysis of the human oral metabolome.Proteomic

The Human Fungal Pathogen Cryptococcus neoformans Escapes Macrophages by a Phagosome Emptying Mechanism That Is Inhibited by Arp2/3 Complex-Mediated Actin Polymerisation

The lysis of infected cells by disease-causing microorganisms is an efficient but risky strategy for disseminated infection, as it exposes the pathogen to the full repertoire of the host's immune system. Cryptococcus neoformans is a widespread fungal pathogen that causes a fatal meningitis in HIV and other immunocompromised patients. Following intracellular growth, cryptococci are able to escape their host cells by a non-lytic expulsive mechanism that may contribute to the invasion of the central nervous system. Non-lytic escape is also exhibited by some bacterial pathogens and is likely to facilitate long-term avoidance of the host immune system during latency. Here we show that phagosomes containing intracellular cryptococci undergo repeated cycles of actin polymerisation. These actin ‘flashes’ occur in both murine and human macrophages and are dependent on classical WASP-Arp2/3 complex mediated actin filament nucleation. Three dimensional confocal imaging time lapse revealed that such flashes are highly dynamic actin cages that form around the phagosome. Using fluorescent dextran as a phagosome membrane integrity probe, we find that the non-lytic expulsion of Cryptococcus occurs through fusion of the phagosome and plasma membranes and that, prior to expulsion, 95% of phagosomes become permeabilised, an event that is immediately followed by an actin flash. By using pharmacological agents to modulate both actin dynamics and upstream signalling events, we show that flash occurrence is inversely related to cryptococcal expulsion, suggesting that flashes may act to temporarily inhibit expulsion from infected phagocytes. In conclusion, our data reveal the existence of a novel actin-dependent process on phagosomes containing cryptococci that acts as a potential block to expulsion of Cryptococcus and may have significant implications for the dissemination of, and CNS invasion by, this organism.\ud \u

Genomic analysis reveals Lactobacillus sanfranciscensis as stable element in traditional sourdoughs

Sourdough has played a significant role in human nutrition and culture for thousands of years and is still of eminent importance for human diet and the bakery industry. Lactobacillus sanfranciscensis is the predominant key bacterium in traditionally fermented sourdoughs

Thermodynamics of Black Holes in Two (and Higher) Dimensions

A comprehensive treatment of black hole thermodynamics in two-dimensional dilaton gravity is presented. We derive an improved action for these theories and construct the Euclidean path integral. An essentially unique boundary counterterm renders the improved action finite on-shell, and its variational properties guarantee that the path integral has a well-defined semi-classical limit. We give a detailed discussion of the canonical ensemble described by the Euclidean partition function, and examine various issues related to stability. Numerous examples are provided, including black hole backgrounds that appear in two dimensional solutions of string theory. We show that the Exact String Black Hole is one of the rare cases that admits a consistent thermodynamics without the need for an external thermal reservoir. Our approach can also be applied to certain higher-dimensional black holes, such as Schwarzschild-AdS, Reissner-Nordstrom, and BTZ.Comment: 63 pages, 3 pdf figures, v2: added reference