The structure of haemoglobin bound to the haemoglobin receptor IsdH from Staphylococcus aureus shows disruption of the native α-globin haem pocket

© 2015 International Union of Crystallography. Staphylococcus aureus is a common and serious cause of infection in humans. The bacterium expresses a cell-surface receptor that binds to, and strips haem from, human haemoglobin (Hb). The binding interface has previously been identified; however, the structural changes that promote haem release from haemoglobin were unknown. Here, the structure of the receptor-Hb complex is reported at 2.6 Å resolution, which reveals a conformational change in the α-globin F helix that disrupts the haem-pocket structure and alters the Hb quaternary interactions. These features suggest potential mechanisms by which the S. aureus Hb receptor induces haem release from Hb

Characterizing Width Uniformity by Wave Propagation

This work describes a novel image analysis approach to characterize the uniformity of objects in agglomerates by using the propagation of normal wavefronts. The problem of width uniformity is discussed and its importance for the characterization of composite structures normally found in physics and biology highlighted. The methodology involves identifying each cluster (i.e. connected component) of interest, which can correspond to objects or voids, and estimating the respective medial axes by using a recently proposed wavefront propagation approach, which is briefly reviewed. The distance values along such axes are identified and their mean and standard deviation values obtained. As illustrated with respect to synthetic and real objects (in vitro cultures of neuronal cells), the combined use of these two features provide a powerful description of the uniformity of the separation between the objects, presenting potential for several applications in material sciences and biology.Comment: 14 pages, 23 figures, 1 table, 1 referenc

Pre-registration of CT pulmonary volumetric image data

Bakalárska práca sa zaoberá predregistráciou pľúcnych objemových CT obrazových dát. Predregistrácia je riešená metódou fázovej korelácie pri rozklade 3D obrazu na 2D rezy usporiadané za sebou. Práca ďalej popisuje geometrické transformácie, interpolácie, výpočet podobnostných kritérií, optimalizáciu registrácie obrazu a proces samotnej registrácie obrazu. Predregistračný softvér je navrhnutý v programovom prostredí MATLAB^®, kde prebieha predregistrácia 3D reálnych CT obrazových dát s dôrazom na rýchlosť procesu.This bachelor thesis is dealing with pre-registration of CT pulmonary volumetric image data. Pre-registration is solved by phase correlation method, which decomposes 3D images into 2D slices arranged in a row. It further describes the geometric transformations, interpolation, calculations of similarity criteria, optimization of registration of images and the image registration process itself. The pre-registration software runs in MATLAB^®, which works with 3D images of real CT image data with an emphasis on process speed.

Human 2'-deoxynucleoside 5'-phosphate N-hydrolase 1 : mechanism of 2'-deoxyuridine 5'-monophosphate hydrolysis

Funding: Industrial Biotechnology Innovation Centre - 2021-01-01.The enzyme 2′-deoxynucleoside 5′-phosphate N-hydrolase 1 (DNPH1) catalyzes the N-ribosidic bond cleavage of 5-hydroxymethyl-2′-deoxyuridine 5′-monophosphate to generate 2-deoxyribose 5-phosphate and 5-hydroxymethyluracil. DNPH1 accepts other 2′-deoxynucleoside 5′-monophosphates as slow-reacting substrates. DNPH1 inhibition is a promising strategy to overcome resistance to and potentiate anticancer poly(ADP-ribose) polymerase inhibitors. We solved the crystal structure of unliganded human DNPH1 and took advantage of the slow reactivity of 2′-deoxyuridine 5′-monophosphate (dUMP) as a substrate to obtain a crystal structure of the DNPH1:dUMP Michaelis complex. In both structures, the carboxylate group of the catalytic Glu residue, proposed to act as a nucleophile in covalent catalysis, forms an apparent low-barrier hydrogen bond with the hydroxyl group of a conserved Tyr residue. The crystal structures are supported by functional data, with liquid chromatography–mass spectrometry analysis showing that DNPH1 incubation with dUMP leads to slow yet complete hydrolysis of the substrate. A direct UV-vis absorbance-based assay allowed characterization of DNPH1 kinetics at low dUMP concentrations. A bell-shaped pH-rate profile indicated that acid–base catalysis is operational and that for maximum kcat/KM, two groups with an average pKa of 6.4 must be deprotonated, while two groups with an average pKa of 8.2 must be protonated. A modestly inverse solvent viscosity effect rules out diffusional processes involved in dUMP binding to and possibly uracil release from the enzyme as rate limiting to kcat/KM. Solvent deuterium isotope effects on kcat/KM and kcat were inverse and unity, respectively. A reaction mechanism for dUMP hydrolysis is proposed.Publisher PDFPeer reviewe

Snapshots of the reaction coordinate of a thermophilic 2'-deoxyribonucleoside/ribonucleoside transferase

Funding: P.T. is funded by IBioIC (IBioIC 2020-2-1), and C.M.C. is funded by the Wellcome Trust (217078/Z/19/Z). C.M.C. and D.H. are funded by research grants from NuCana plc..Nucleosides are ubiquitous to life and are required for the synthesis of DNA, RNA, and other molecules crucial for cell survival. Despite the notoriously difficult organic synthesis of nucleosides, 2′-deoxynucleoside analogues can interfere with natural DNA replication and repair and are successfully employed as anticancer, antiviral, and antimicrobial compounds. Nucleoside 2′-deoxyribosyltransferase (dNDT) enzymes catalyze transglycosylation via a covalent 2′-deoxyribosylated enzyme intermediate with retention of configuration, having applications in the biocatalytic synthesis of 2′-deoxynucleoside analogues in a single step. Here, we characterize the structure and function of a thermophilic dNDT, the protein from Chroococcidiopsis thermalis (CtNDT). We combined enzyme kinetics with structural and biophysical studies to dissect mechanistic features in the reaction coordinate, leading to product formation. Bell-shaped pH-rate profiles demonstrate activity in a broad pH range of 5.5–9.5, with two very distinct pKa values. A pronounced viscosity effect on the turnover rate indicates a diffusional step, likely product (nucleobase1) release, to be rate-limiting. Temperature studies revealed an extremely curved profile, suggesting a large negative activation heat capacity. We trapped a 2′-fluoro-2′-deoxyarabinosyl-enzyme intermediate by mass spectrometry and determined high-resolution structures of the protein in its unliganded, substrate-bound, ribosylated, 2′-difluoro-2′-deoxyribosylated, and in complex with probable transition-state analogues. We reveal key features underlying (2′-deoxy)ribonucleoside selection, as CtNDT can also use ribonucleosides as substrates, albeit with a lower efficiency. Ribonucleosides are the building blocks of RNA and other key intracellular metabolites participating in energy and metabolism, expanding the scope of use of CtNDT in biocatalysis.Peer reviewe

Decreasing intensity of open-ocean convection in the Greenland and Iceland seas

The air–sea transfer of heat and fresh water plays a critical role in the global climate system. This is particularly true for the Greenland and Iceland seas, where these fluxes drive ocean convection that contributes to Denmark Strait overflow water, the densest component of the lower limb of the Atlantic Meridional Overturning Circulation (AMOC). Here we show that the wintertime retreat of sea ice in the region, combined with different rates of warming for the atmosphere and sea surface of the Greenland and Iceland seas, has resulted in statistically significant reductions of approximately 20% in the magnitude of the winter air–sea heat fluxes since 1979. We also show that modes of climate variability other than the North Atlantic Oscillation (NAO) are required to fully characterize the regional air–sea interaction. Mixed-layer model simulations imply that further decreases in atmospheric forcing will exceed a threshold for the Greenland Sea whereby convection will become depth limited, reducing the ventilation of mid-depth waters in the Nordic seas. In the Iceland Sea, further reductions have the potential to decrease the supply of the densest overflow waters to the AMOC

Structural basis for hemoglobin capture by Staphylococcus aureus cell-surface protein, IsdH

Pathogens must steal iron from their hosts to establish infection. In mammals, hemoglobin (Hb) represents the largest reservoir of iron, and pathogens express Hb-binding proteins to access this source. Here, we show how one of the commonest and most significant human pathogens, Staphylococcus aureus, captures Hb as the first step of an iron-scavenging pathway. The x-ray crystal structure of Hb bound to a domain from the Isd (iron-regulated surface determinant) protein, IsdH, is the first structure of a Hb capture complex to be determined. Surface mutations in Hb that reduce binding to the Hb-receptor limit the capacity of S. aureus to utilize Hb as an iron source, suggesting that Hb sequence is a factor in host susceptibility to infection. The demonstration that pathogens make highly specific recognition complexes with Hb raises the possibility of developing inhibitors of Hb binding as antibacterial agents. © 2011 by The American Society for Biochemistry and Molecular Biology, Inc

Exploring the behavioural drivers of veterinary surgeon antibiotic prescribing: a qualitative study of companion animal veterinary surgeons in the UK

Background: Multi-drug resistant bacteria are an increasing concern in both human and veterinary medicine. Inappropriate prescribing and use of antibiotics within veterinary medicine may be a contributory factor to antimicrobial resistance (AMR). The ‘One Health’ Initiative aims to work across species and environments to reduce AMR, however; little is currently known about the factors which influence antibiotic prescribing among veterinary surgeons in companion animal practice. This paper reports on qualitative data analysis of interviews with veterinary surgeons whose practice partially or wholly focuses on companion animals (N = 16). The objective of the research was to explore the drivers of companion animal veterinary surgeons’ antibiotic prescribing behaviours. The veterinary surgeons interviewed were all practising within the UK (England (n = 4), Scotland (n = 11), Northern Ireland (n = 1)). A behavioural thematic analysis of the data was undertaken, which identified barriers and facilitators to specific prescribing-related behaviours. Results: Five components of prescribing behaviours were identified: 1) confirming clinical need for antibiotics; 2) responding to clients; 3) confirming diagnosis; 4) determining dose, duration and type of antibiotic; and 5) preventing infection around surgery (with attendant appropriate and inappropriate antibiotic prescribing behaviours). Barriers to appropriate prescribing identified include: business, diagnostic, fear, habitual practice and pharmaceutical factors. Facilitators include: AMR awareness, infection prevention, professional learning and regulation and government factors. Conclusion: This paper uses a behavioural lens to examine drivers which are an influence on veterinary surgeons’ prescribing behaviours. The paper contributes new understandings about factors which influence antibiotic prescribing behaviours among companion animal veterinary surgeons. This analysis provides evidence to inform future interventions, which are focused on changing prescribing behaviours, in order to address the pressing public health concern of AMR

Chronic inflammatory arthritis drives systemic changes in circadian energy metabolism

SignificanceRheumatoid arthritis (RA) is a debilitating chronic inflammatory disease in which symptoms exhibit a strong time-of-day rhythmicity. RA is commonly associated with metabolic disturbance and increased incidence of diabetes and cardiovascular disease, yet the mechanisms underlying this metabolic dysregulation remain unclear. Here, we demonstrate that rhythmic inflammation drives reorganization of metabolic programs in distal liver and muscle tissues. Chronic inflammation leads to mitochondrial dysfunction and dysregulation of fatty acid metabolism, including accumulation of inflammation-associated ceramide species in a time-of-day-dependent manner. These findings reveal multiple points for therapeutic intervention centered on the circadian clock, metabolic dysregulation, and inflammatory signaling

Microbiome profiling by Illumina sequencing of combinatorial sequence-tagged PCR products

We developed a low-cost, high-throughput microbiome profiling method that uses combinatorial sequence tags attached to PCR primers that amplify the rRNA V6 region. Amplified PCR products are sequenced using an Illumina paired-end protocol to generate millions of overlapping reads. Combinatorial sequence tagging can be used to examine hundreds of samples with far fewer primers than is required when sequence tags are incorporated at only a single end. The number of reads generated permitted saturating or near-saturating analysis of samples of the vaginal microbiome. The large number of reads al- lowed an in-depth analysis of errors, and we found that PCR-induced errors composed the vast majority of non-organism derived species variants, an ob- servation that has significant implications for sequence clustering of similar high-throughput data. We show that the short reads are sufficient to assign organisms to the genus or species level in most cases. We suggest that this method will be useful for the deep sequencing of any short nucleotide region that is taxonomically informative; these include the V3, V5 regions of the bac- terial 16S rRNA genes and the eukaryotic V9 region that is gaining popularity for sampling protist diversity.Comment: 28 pages, 13 figure