Dissecting the Enterococcal Polysaccharide Antigen (EPA) structure to explore innate immune evasion and phage specificity.

Streptococci, Lactococci and Enterococci all produce L-rhamnose-containing cell wall polysaccharides which define Lancefield serotypes and represent promising candidates for the design of glycoconjugate vaccines. The L-rhamnose containing Enterococcal Polysaccharide Antigen (EPA), produced by the opportunistic pathogen Enterococcus faecalis, plays a critical role in normal growth, division, biofilm formation, antimicrobial resistance, phage susceptibility, and innate immune evasion. Despite the critical role of this polymer in E.

Acquired resistance to oxaliplatin is not directly associated with increased resistance to DNA damage in SK-N-ASrOXALI4000, a newly established oxaliplatin-resistant sub-line of the neuroblastoma cell line SK-N-AS.

The formation of acquired drug resistance is a major reason for the failure of anti-cancer therapies after initial response. Here, we introduce a novel model of acquired oxaliplatin resistance, a sub-line of the non-MYCN-amplified neuroblastoma cell line SK-N-AS that was adapted to growth in the presence of 4000 ng/mL oxaliplatin (SK-N-ASrOXALI4000). SK-N-ASrOXALI4000 cells displayed enhanced chromosomal aberrations compared to SK-N-AS, as indicated by 24-chromosome fluorescence in situ hybridisation.

Inappropriate expression of the translation elongation factor 1A disrupts genome stability and metabolism.

The translation elongation factor eEF1A is one of the most abundant proteins found within cells, and its role within protein synthesis is well documented. Levels of eEF1A are tightly controlled, with inappropriate expression linked to oncogenesis. However, the mechanisms by which increased eEF1A expression alters cell behaviour are unknown.

New links between SOD1 and metabolic dysfunction from a yeast model of amyotrophic lateral sclerosis.

A number of genes have been linked to familial forms of the fatal motor neuron disease amyotrophic lateral sclerosis (ALS). Over 150 mutations within the gene encoding superoxide dismutase 1 (SOD1) have been implicated in ALS, but why such mutations lead to ALS-associated cellular dysfunction is unclear. In this study, we identify how ALS-linked SOD1 mutations lead to changes in the cellular health of the yeast Saccharomyces cerevisiae We find that it is not the accumulation of aggregates but the loss of Sod1 protein stability that drives cellular dysfunction.

Combined ligand-observe (19)F and protein-observe (15)N,(1)H-HSQC NMR suggests phenylalanine as the key Δ-somatostatin residue recognized by human protein disulfide isomerase.

Human protein disulphide isomerase (hPDI) is an endoplasmic reticulum (ER) based isomerase and folding chaperone. Molecular detail of ligand recognition and specificity of hPDI are poorly understood despite the importance of the hPDI for folding secreted proteins and its implication in diseases including cancer and lateral sclerosis. We report a detailed study of specificity, interaction and dissociation constants (Kd) of the peptide-ligand Δ-somatostatin (AGSKNFFWKTFTSS) binding to hPDI using (19)F ligand-observe and (15)N,(1)H-HSQC protein-observe NMR methods.

CydDC-mediated reductant export in Escherichia coli controls the transcriptional wiring of energy metabolism and combats nitrosative stress.

The glutathione/cysteine exporter CydDC maintains redox balance in Escherichia coli. A cydD mutant strain was used to probe the influence of CydDC upon reduced thiol export, gene expression, metabolic perturbations, intracellular pH homoeostasis and tolerance to nitric oxide (NO). Loss of CydDC was found to decrease extracytoplasmic thiol levels, whereas overexpression diminished the cytoplasmic thiol content.

The Fanconi Anemia DNA Repair Pathway Is Regulated by an Interaction between Ubiquitin and the E2-like Fold Domain of FANCL.

The Fanconi Anemia (FA) DNA repair pathway is essential for the recognition and repair of DNA interstrand crosslinks (ICL). Inefficient repair of these ICL can lead to leukemia and bone marrow failure. A critical step in the pathway is the monoubiquitination of FANCD2 by the RING E3 ligase FANCL.

Q2DSTD NMR deciphers epitope-mapping variability for peptide recognition of integrin αvβ6.

Integrin αvβ6 is a cell surface arginine-glycine-aspartic acid (RGD)-specific heterodimeric glycoprotein that is only expressed on epithelia during processes of tissue remodelling, including cancer. The specificity and molecular nature of interactions toward this integrin are poorly understood and new insights into such processes are important to cell biologists and pharmaceutical drug discovery. This study demonstrates the application of quantitative two-dimensional saturation transfer (Q2DSTD) NMR to obtain precise details of peptide interactions with integrin αvβ6 and their correlation to specificity for the integrin.

New insights into stop codon recognition by eRF1.

In eukaryotes, translation termination is performed by eRF1, which recognizes stop codons via its N-terminal domain. Many previous studies based on point mutagenesis, cross-linking experiments or eRF1 chimeras have investigated the mechanism by which the stop signal is decoded by eRF1. Conserved motifs, such as GTS and YxCxxxF, were found to be important for termination efficiency, but the recognition mechanism remains unclear.

Solution structure of a bacterial microcompartment targeting peptide and its application in the construction of an ethanol bioreactor.

Targeting of proteins to bacterial microcompartments (BMCs) is mediated by an 18-amino-acid peptide sequence. Herein, we report the solution structure of the N-terminal targeting peptide (P18) of PduP, the aldehyde dehydrogenase associated with the 1,2-propanediol utilization metabolosome from Citrobacter freundii. The solution structure reveals the peptide to have a well-defined helical conformation along its whole length.

FAD binding, cobinamide binding and active site communication in the corrin reductase (CobR).

Adenosylcobalamin, the coenzyme form of vitamin B12, is one Nature's most complex coenzyme whose de novo biogenesis proceeds along either an anaerobic or aerobic metabolic pathway. The aerobic synthesis involves reduction of the centrally chelated cobalt metal ion of the corrin ring from Co(II) to Co(I) before adenosylation can take place. A corrin reductase (CobR) enzyme has been identified as the likely agent to catalyse this reduction of the metal ion.

19F NMR spectroscopy monitors ligand binding to recombinantly fluorine-labelled b'x from human protein disulphide isomerase (hPDI).

We report a protein-observe (19)F NMR-based ligand titration binding study of human PDI b'x with Δ-somatostatin that also emphasises the need to optimise recombinant protein fluorination when using 5- or 6-fluoroindole. This study highlights a recombinant preference for 5-fluoroindole over 6-fluoroindole; most likely due to the influence of fluorine atomic packing within the folded protein structure. Fluorination affords a single (19)F resonance probe to follow displacement of the protein x-linker as ligand is titrated and provides a dissociation constant of 23 ± 4 μM.

Protein disulfide-isomerase interacts with a substrate protein at all stages along its folding pathway.

In contrast to molecular chaperones that couple protein folding to ATP hydrolysis, protein disulfide-isomerase (PDI) catalyzes protein folding coupled to formation of disulfide bonds (oxidative folding). However, we do not know how PDI distinguishes folded, partly-folded and unfolded protein substrates. As a model intermediate in an oxidative folding pathway, we prepared a two-disulfide mutant of basic pancreatic trypsin inhibitor (BPTI) and showed by NMR that it is partly-folded and highly dynamic.

Optimising selective excitation pulses to maximise saturation transfer difference NMR spectroscopy.

A simple method is presented that optimizes the STD NMR Gaussian pulse to deliver significant increases in STD amplification factors with minimal perturbation of the ligand. This approach is practically demonstrated using the wheat-germ agglutinin/N-acetyl-D-glucosamine protein-ligand system.

Structural guided scaffold phage display libraries as a source of bio-therapeutics.

We have developed a structurally-guided scaffold phage display strategy for identification of ligand mimetic bio-therapeutics. As a proof of concept we used the ligand of integrin αvβ6, a tumour cell surface receptor and a major new target for imaging and therapy of many types of solid cancer. NMR structure analysis showed that RGD-helix structures are optimal for αvβ6 ligand-interaction, so we designed novel algorithms to generate human single chain fragment variable (scFv) libraries with synthetic VH-CDR3 encoding RGD-helix hairpins with helices of differing pitch, length and amino acid composition.

Structural definition is important for the propagation of the yeast [PSI+] prion.

Prions are propagated in Saccharomyces cerevisiae with remarkable efficiency, yet we know little about the structural basis of sequence variations in the prion protein that support or prohibit propagation of the prion conformation. We show that certain single-amino-acid substitutions in the prion protein Sup35 impact negatively on the maintenance of the associated prion-based [PSI(+)] trait by combining in vivo phenotypic analysis with solution NMR structural studies. A clear correlation is observed between mutationally induced conformational differences in one of the oligopeptide repeats (R2) in the N terminus of Sup35 and the relative ability to propagate [PSI(+)].

Measuring protein reduction potentials using 15N HSQC NMR spectroscopy.

NMR spectroscopy was used to measure reduction potentials of four redox proteins by following multiple (15)N HSQC protein resonances across a titration series using mixtures of oxidised and reduced glutathione. Results for PDI a, PDI ab and DsbA agree with the literature and our result for ERp18 confirms this protein as an oxidoreductase of comparable or greater reducing strength than PDI a.

High-resolution NMR studies of structure and dynamics of human ERp27 indicate extensive interdomain flexibility.

ERp27 (endoplasmic reticulum protein 27.7 kDa) is a homologue of PDI (protein disulfide-isomerase) localized to the endoplasmic reticulum. ERp27 is predicted to consist of two thioredoxin-fold domains homologous with the non-catalytic b and b' domains of PDI.

NMR relaxation and structural elucidation of peptides in the presence and absence of trifluoroethanol illuminates the critical molecular nature of integrin αvβ6 ligand specificity.

Integrin αvβ6 is an important emerging target for both imaging and therapy of cancer that requires specific ligands based on Arg-Gly-Asp (RGD) peptides. There remains little correlation between integrin-RGD ligand specificity despite studies suggesting an RGD-turn-helix ligand motif is required. Here, we describe the application of N NMR relaxation analyses and structure determination of αvβ6 peptide ligands in the presence and absence of trifluoroethanol (TFE) to identify their critical molecular nature that influences specificity, interaction and function.

One-bond and two-bond J couplings help annotate protein secondary-structure motifs: J-coupling indexing applied to human endoplasmic reticulum protein ERp18.

NMR coupling constants, both direct one-bond ((1)J) and geminal two-bond ((2)J), are employed to analyze the protein secondary structure of human oxidized ERp18. Coupling constants collected and evaluated for the 18 kDa protein comprise 1268 values of (1)J(CαHα), (1)J(CαCβ), (1)J(CαC'), (1)J(C'N'), (1)J(N'Cα), (1)J(N') (HN), (2)J(CαN'), (2)J(HNCα), (2)J(C'HN), and (2)J(HαC'). Comparison with (1)J and (2)J data from reference proteins and pattern analysis on a per-residue basis permitted main-chain ϕ,ψ torsion-angle combinations of many of the 149 amino-acid residues in ERp18 to be narrowed to particular secondary-structure motifs.

Solution structure and dynamics of ERp18, a small endoplasmic reticulum resident oxidoreductase .

Here we report the solution structure of oxidized ERp18 as determined using NMR spectroscopy. ERp18 is the smallest member of the protein disulfide isomerase (PDI) family of proteins to contain a Cys-Xxx-Xxx-Cys active site motif. It is an 18 kDa endoplasmic reticulum resident protein with unknown function although sequence similarity to individual domains of the thiol-disulfide oxidoreductase PDI suggests ERp18 may have a similar structure and function.

Four-year longitudinal study of behavioral changes in coping with stress.

Objective: To examine the relationship between stress and coping.

Methods: Short- and long-term approaches to behavioral change were evaluated in 3 groups (short-term, long-term, control).

Results: Subjects participating in a 6-week stress management program designed to develop stress management skills reported temporary decreases in burnout, while subjects receiving additional "refresher" sessions showed decreases in burnout throughout a 4-year period.

Stress and verbal abuse in nursing: do burned out nurses eat their young?

Aim: The purpose of this study was to explore the types and frequency of verbal abuse of nurses by other nurses. Further, this study explored the components, characteristics, consequences and effects of abuse in an effort to better understand the dynamics of verbal abuse of nurses in the workplace. Nurses who experience occupational burnout are more likely to abuse other nurses.