Deciphering the Acylation Pattern of Yersinia enterocolitica Lipid A

Pathogenic bacteria may modify their surface to evade the host innate immune response. Yersinia enterocolitica modulates its lipopolysaccharide (LPS) lipid A structure, and the key regulatory signal is temperature. At 21°C, lipid A is hexa-acylated and may be modified with aminoarabinose or palmitate. At 37°C, Y. enterocolitica expresses a tetra-acylated lipid A consistent with the 3'-O-deacylation of the molecule. In this work, by combining genetic and mass spectrometric analysis, we establish that Y. enterocolitica encodes a lipid A deacylase, LpxR, responsible for the lipid A structure observed at 37°C. Western blot analyses indicate that LpxR exhibits latency at 21°C, deacylation of lipid A is not observed despite the expression of LpxR in the membrane. Aminoarabinose-modified lipid A is involved in the latency. 3-D modelling, docking and site-directed mutagenesis experiments showed that LpxR D31 reduces the active site cavity volume so that aminoarabinose containing Kdo(2)-lipid A cannot be accommodated and, therefore, not deacylated. Our data revealed that the expression of lpxR is negatively controlled by RovA and PhoPQ which are necessary for the lipid A modification with aminoarabinose. Next, we investigated the role of lipid A structural plasticity conferred by LpxR on the expression/function of Y. enterocolitica virulence factors. We present evidence that motility and invasion of eukaryotic cells were reduced in the lpxR mutant grown at 21°C. Mechanistically, our data revealed that the expressions of flhDC and rovA, regulators controlling the flagellar regulon and invasin respectively, were down-regulated in the mutant. In contrast, the levels of the virulence plasmid (pYV)-encoded virulence factors Yops and YadA were not affected in the lpxR mutant. Finally, we establish that the low inflammatory response associated to Y. enterocolitica infections is the sum of the anti-inflammatory action exerted by pYV-encoded YopP and the reduced activation of the LPS receptor by a LpxR-dependent deacylated LPS.

Defining the molecular basis for the first potent and selective orthosteric agonists of the FFA2 free fatty acid receptor

FFA2 is a G protein-coupled receptor that responds to short chain fatty acids (SCFAs) and has generated interest as a therapeutic target for metabolic and inflammatory conditions. However, definition of its functions has been slowed by a dearth of selective ligands that can distinguish it from the closely related FFA3. At present, the only selective ligands described for FFA2 suffer from either poor potency, altered signaling due to allosteric modes of action, or a lack of function at non-human orthologs of the receptor. To address the need for novel selective ligands, we synthesized two compounds potentially having FFA2 activity and examined the molecular basis of their function. These compounds were confirmed to be potent and selective FFA2 agonists that interact with the orthosteric binding site. A combination of ligand structure-activity relationship, pharmacological analysis, homology modeling, species ortholog comparisons and mutagenesis studies were then employed to define the molecular basis of selectivity and function of these ligands. From this, we identified key residues within both extracellular loop 2 (ECL2) and the transmembrane domain (TM) regions of FFA2 critical for ligand function. One of these ligands was active with reasonable potency at rodent orthologs of FFA2 and demonstrated the role of FFA2 in the regulation of lipolysis in murine 3T3-L1 adipocytes. Together, these findings describe the first potent and selective FFA2 orthosteric agonists and demonstrate key aspects of ligand interaction within the orthosteric binding site of FFA2 that will be invaluable in future ligand development at this receptor.

Molecular and clinicopathological markers of prognosis in breast cancer

A vast body of research in breast cancer prognostication has accumulated. Yet despite this, patients within current prognostic categories may have significantly different outcomes. There is a need to more accurately divide those cancer types associated with an excellent prognosis from those requiring more aggressive therapy. Gene expression array studies have revealed the numerous molecular breast cancer subtypes that are associated with differing outcomes. Furthermore, as next generation technologies evolve and further reveal the complexities of breast cancer, it is likely that existing prognostic approaches will become progressively refined. Future prognostication in breast cancer requires a morphomolecular, multifaceted approach involving the assessment of anatomical disease extent and levels of protein, DNA and RNA expression. One of the major challenges in prognostication will be the integration of potential assays into existing clinical systems and identification of appropriate patient subgroups for analysis.

Are there species smaller than 1 mm?

The rapid advance in genetic sequencing technologies has provided an unprecedented amount of data on the biodiversity of meiofauna. It was hoped that these data would allow the identification and counting of species, distinguished as tight clusters of similar genomes. Surprisingly, this appears not to be the case. Here, we begin a theoretical discussion of this phenomenon, drawing on an individual-based ecological model to inform our arguments. The determining factor in the emergence (or not) of distinguishable genetic clusters in the model is the product of population size with mutation rate—a measure of the adaptability of the population as a whole. This result suggests that indeed one should not expect to observe clearly distinguishable species groupings in data gathered from ultrasequencing of meiofauna.

Water-hydrophobic compound interactions with the microbial cell

The structural interactions of biological macromolecules, their biochemical activities and, ultimately, the metabolic function of cellular systems are dependent upon weak inter- and intra-molecular forces such as hydrogen bonds, Van der Waals forces, and the hydrophobic effect. Water molecules, and those of hydrophobic substances such as hydrocarbons, can take part in and/or modify these interactions and thereby determine the operational and structural stability of the microbial cell and its macromolecular systems. We explain how the cytosol, plasma membrane and the extracellular solution form a material and energetic continuum; and discuss the behavior of hydrophobic substances of extracellular origin as they migrate into the plasma membrane and into the cell's interior. The adverse effects of substances with a log P octanol-water =2, that partition into the hydrophobic domains of biological macromolecules, are discussed in relation to microbial cell function; and we speculate whether the cellular stress that they induce is symmetrical or asymmetrical in nature. In the context of the microbial environment, we take a situational-functional approach to consider how hydrophobic stressors interact with the microbial cell, and what types of evasion tactics microbes can employ to minimize their inhibitory activities. Finally, we discuss the ecological implications of hydrocarbon-induced cellular stress for microbial systems.

Triose phosphate isomerase from the blood fluke Schistosoma mansoni:Biochemical characterisation of a potential drug and vaccine target

The glycolytic enzyme triose phosphate isomerase from Schistosoma mansoni is a potential target for drugs and vaccines. Molecular modelling of the enzyme predicted that a Ser-Ala-Asp motif which is believed to be a helminth-specific epitope is exposed. The enzyme is dimeric (as judged by gel filtration and cross-linking), resistant to proteolysis and highly stable to thermal denaturation (melting temperature of 82.0°C). The steady-state kinetic parameters are high (Km for dihydroxyacetone phosphate is 0.51mM; Km for glyceraldehyde 3-phosphate is 1.1mM; kcat for dihydroxyacetone phosphate is 7800s(-1) and kcat for glyceraldehyde 3-phosphate is 6.9s(-1)).

The Continuing Debate on Deep Molluscan Phylogeny: Evidence for Serialia (Mollusca, Monoplacophora + Polyplacophora)

Molluscs are a diverse animal phylum with a formidable fossil record. Although there is little doubt about the monophyly of the eight extant classes, relationships between these groups are controversial.We analysed a comprehensive multilocus molecular data set for molluscs, the first to include multiple species from all classes, including five monoplacophorans in both extant families. Our analyses of fivemarkers resolve two major clades: the first includes gastropods and bivalves sister to Serialia (monoplacophorans and chitons), and the second comprises scaphopods sister to aplacophorans and cephalopods. Traditional groupings such as Testaria, Aculifera, and Conchifera are rejected by our data with significant Approximately Unbiased (AU) test values. A new molecular clock indicates that molluscs had a terminal Precambrian origin with rapid divergence of all eight extant classes in the Cambrian. Therecovery of Serialia as a derived, Late Cambrian clade is potentially in line with the stratigraphic chronology of morphologically heterogeneous early mollusc fossils. Serialia is in conflict with traditional molluscan classifications and recent phylogenomic data. Yet our hypothesis, as others from molecular data, implies frequent molluscan shell and body transformations by heterochronic shifts in development and multiple convergent adaptations, leading to the variable shells and body plans in extant lineages.

A generalized functional response for predators that switch between multiple prey species

We develop a theory for the food intake of a predator that can switch between multiple prey species. The theory addresses empirical observations of prey switching and is based on the behavioural assumption that a predator tends to continue feeding on prey that are similar to the prey it has consumed last, in terms of, e.g., their morphology, defences, location, habitat choice, or behaviour. From a predator's dietary history and the assumed similarity relationship among prey species, we derive a general closed-form multi-species functional response for describing predators switching between multiple prey species. Our theory includes the Holling type II functional response as a special case and makes consistent predictions when populations of equivalent prey are aggregated or split. An analysis of the derived functional response enables us to highlight the following five main findings. (1) Prey switching leads to an approximate power-law relationship between ratios of prey abundance and prey intake, consistent with experimental data. (2) In agreement with empirical observations, the theory predicts an upper limit of 2 for the exponent of such power laws. (3) Our theory predicts deviations from power-law switching at very low and very high prey-abundance ratios. (4) The theory can predict the diet composition of a predator feeding on multiple prey species from diet observations for predators feeding only on pairs of prey species. (5) Predators foraging on more prey species will show less pronounced prey switching than predators foraging on fewer prey species, thus providing a natural explanation for the known difficulties of observing prey switching in the field. (C) 2013 Elsevier Ltd. All rights reserved.

Identifying cell class specific losses from serially generated electroretinogram components

Purpose. Processing of information through the cellular layers of the retina occurs in a serial manner. In the electroretinogram (ERG), this complicates interpretation of inner retinal changes as dysfunction may arise from "upstream" neurons or may indicate a direct loss to that neural generator. We propose an approach that addresses this issue by defining ERG gain relationships.

Methods. Regression analyses between two serial ERG parameters in a control cohort of rats are used to define gain relationships. These gains are then applied to two models of retinal disease.

Results. The PIIIamp to PIIamp gain is unity whereas the PIIamp to pSTRamp and PIIamp to nSTRamp gains are greater than unity, indicating "amplification" (P <0.05). Timing relationships show amplification between PIIIit to PIIit and compression for PIIit to pSTRit and PIIit to nSTRit, (P <0.05). Application of these gains to ?-3-deficiency indicates that all timing changes are downstream of photoreceptor changes, but a direct pSTR amplitude loss occurs (P <0.05). Application to diabetes indicates widespread inner retinal dysfunction which cannot be attributed to outer retinal changes (P <0.05).

Conclusions. This simple approach aids in the interpretation of inner retinal ERG changes by taking into account gain characteristics found between successive ERG components of normal animals.

Evaluation of a worksite cervical screening initiative to increase Pap smear uptake in Malaysia:a cluster randomized controlled trial

Despite the significant burden of cervical cancer, Malaysia like many middle-income countries relies on opportunistic cervical screening as opposed to a more organized population-based program. The aim of this study was to ascertain the effectiveness of a worksite screening initiative upon Papanicolaou smear test (Pap test) uptake among educated working women in Malaysia.

The Saccharomyces cerevisiae quinone oxidoreductase Lot6p: stability, inhibition and cooperativity

Lot6p (EC 1.5.1.39; Ylr011wp) is the sole quinone oxidoreductase in the budding yeast, Saccharomyces cerevisiae. Using hexahistidine tagged, recombinant Lot6p, we determined the steady-state enzyme kinetic parameters with both NADH and NADPH as electron donors; no cooperativity was observed with these substrates. The NQO1 inhibitor curcumin, the NQO2 inhibitor resveratrol, the bacterial nitroreductase inhibitor nicotinamide and the phosphate mimic vanadate all stabilise the enzyme towards thermal denaturation as judged by differential scanning fluorimetry. All except vanadate have no observable effect on the chemical cross-linking of the two subunits of the Lot6p dimer. These compounds all inhibit Lot6p's oxidoreductase activity, and all except nicotinamide exhibit negative cooperativity. Molecular modelling suggests that curcumin, resveratrol and nicotinamide all bind over the isoalloxazine ring of the FMN cofactor in Lot6p. Resveratrol was predicted to contact an α-helix that links the two active sites. Mutation of Gly-142 (which forms part of this helix) to serine does not greatly affect the thermal stability of the enzyme. However, this variant shows less cooperativity towards resveratrol than the wild type. This suggests a plausible hypothesis for the transmission of information between the subunits and, thus, the molecular mechanism of negative cooperativity in Lot6p.

A conserved spider silk domain acts as a molecular switch that controls fibre assembly

A huge variety of proteins are able to form fibrillar structures(1), especially at high protein concentrations. Hence, it is surprising that spider silk proteins can be stored in a soluble form at high concentrations and transformed into extremely stable fibres on demand(2,3). Silk proteins are reminiscent of amphiphilic block copolymers containing stretches of polyalanine and glycine-rich polar elements forming a repetitive core flanked by highly conserved non-repetitive amino-terminal(4,5) and carboxy-terminal(6) domains. The N-terminal domain comprises a secretion signal, but further functions remain unassigned. The C-terminal domain was implicated in the control of solubility and fibre formation(7) initiated by changes in ionic composition(8,9) and mechanical stimuli known to align the repetitive sequence elements and promote beta-sheet formation(10-14). However, despite recent structural data(15), little is known about this remarkable behaviour in molecular detail. Here we present the solution structure of the C-terminal domain of a spider dragline silk protein and provide evidence that the structural state of this domain is essential for controlled switching between the storage and assembly forms of silk proteins. In addition, the C-terminal domain also has a role in the alignment of secondary structural features formed by the repetitive elements in the backbone of spider silk proteins, which is known to be important for the mechanical properties of the fibre.

Epstein-Barr Virus Large Tegument Protein BPLF1 Contributes to Innate Immune Evasion through Interference with Toll-Like Receptor Signaling

Viral infection triggers an early host response through activation of pattern recognition receptors, including Toll-like receptors (TLR). TLR signaling cascades induce production of type I interferons and proinflammatory cytokines involved in establishing an anti-viral state as well as in orchestrating ensuing adaptive immunity. To allow infection, replication, and persistence, (herpes)viruses employ ingenious strategies to evade host immunity. The human gamma-herpesvirus Epstein-Barr virus (EBV) is a large, enveloped DNA virus persistently carried by more than 90% of adults worldwide. It is the causative agent of infectious mononucleosis and is associated with several malignant tumors. EBV activates TLRs, including TLR2, TLR3, and TLR9. Interestingly, both the expression of and signaling by TLRs is attenuated during productive EBV infection. Ubiquitination plays an important role in regulating TLR signaling and is controlled by ubiquitin ligases and deubiquitinases (DUBs). The EBV genome encodes three proteins reported to exert in vitro deubiquitinase activity. Using active site-directed probes, we show that one of these putative DUBs, the conserved herpesvirus large tegument protein BPLF1, acts as a functional DUB in EBV-producing B cells. The BPLF1 enzyme is expressed during the late phase of lytic EBV infection and is incorporated into viral particles. The N-terminal part of the large BPLF1 protein contains the catalytic site for DUB activity and suppresses TLR-mediated activation of NF-κB at, or downstream of, the TRAF6 signaling intermediate. A catalytically inactive mutant of this EBV protein did not reduce NF-κB activation, indicating that DUB activity is essential for attenuating TLR signal transduction. Our combined results show that EBV employs deubiquitination of signaling intermediates in the TLR cascade as a mechanism to counteract innate anti-viral immunity of infected hosts.

Saccharomyces uvarum, a distinct group within Saccharomyces sensu stricto

A natural subgroup (that we refer to as Saccharomyces uvarum) was identified, within the heterogeneous species Saccharomyces bayanus. The typical electrophoretic karyotype, interfertility of hybrids between strains, distinctive sugar fermentation pattern, and uniform fermentation characteristics in must, indicated that this subgroup was not only highly homogeneous, but also clearly distinguishable from other species within the Saccharomyces sensu stricto group. Investigation of the S. bayanus type strain and other strains that have been classified as S. bayanus, confirmed the apparent lack of homogeneity and, in some cases, supported the hypothesis that they are natural hybrids. Copyright (C) 1999 Federation of European Microbiological Societies.

Integrating molecular diagnostics into histopathology training: the Belfast model

Molecular medicine is transforming modern clinical practice, from diagnostics to therapeutics. Discoveries in research are being incorporated into the clinical setting with increasing rapidity. This transformation is also deeply changing the way we practise pathology. The great advances in cell and molecular biology which have accelerated our understanding of the pathogenesis of solid tumours have been embraced with variable degrees of enthusiasm by diverse medical professional specialties. While histopathologists have not been prompt to adopt molecular diagnostics to date, the need to incorporate molecular pathology into the training of future histopathologists is imperative. Our goal is to create, within an existing 5-year histopathology training curriculum, the structure for formal substantial teaching of molecular diagnostics. This specialist training has two main goals: (1) to equip future practising histopathologists with basic knowledge of molecular diagnostics and (2) to create the option for those interested in a subspecialty experience in tissue molecular diagnostics to pursue this training. It is our belief that this training will help to maintain in future the role of the pathologist at the centre of patient care as the integrator of clinical, morphological and molecular information.