Quorum sensing and DNA release in bacterial biofilms

The multicellular behavior of bacteria has been the subject of much recent interest. This behavior includes coordinated control of virulence, luminescence, competence and biofilm formation; each of these appears to be regulated or influenced by quorum sensing. An understanding of what biofilms constitute, and how they develop, is emerging. It is clear that biofilm formation is a carefully orchestrated process that is dependent on quorum sensing. Somewhat surprisingly, several independent observations have noted an important role for DNA in the structure of biofilms. Recent studies describe a mechanism for linking DNA release to quorum sensing, providing a possible mechanism for the coordinated release of DNA, and its integration into a biofilm. A review of the literature reveals that similar observations have been made for biofilms of both Gram-positive and Gram-negative organisms. Further study will determine whether this is a general trend, however.     

Conformation and assembly of polypeptide scaffolds in templating the synthesis of silica: an example of a polylysine macromolecular "switch"

Although the role of polycationic macromolecules in catalyzing the synthesis of silica structures is well established, detailed understanding of the mechanisms behind the production of silica structures of controlled morphologies remains unclear. In this study, we have used both poly-L-lysine (PLL) and/or poly-D-lysine (PDL) for silica synthesis to investigate mechanisms controlling inorganic morphologies. The formation of both spherical silica particles and hexagonal plates was observed. The formation of hexagonal plates was suggested, via circular dichroic spectroscopy (CD), to result from the assembly of helical polylysine molecules. We confirm that the formation of PLL helices is a prerequisite to the hexagonal silica synthesis. In addition, we present for the first time that the handedness of the helicity of the macromolecule does not affect the formation of hexagonal silica. We also show, by using two different silica precursors, that the precursor does not have a direct effect on the formation of hexagonal silica plates. Furthermore, when polylysine helices were converted to beta-sheet structure, only silica particles were obtained, thus suggesting that the adoption of a helical conformation by PLL is required for the formation of hexagonally organized silica. These results demonstrate that the change in polylysine conformation can act as a "switch" in silica structure formation and suggest the potential for controlling morphologies and structures of inorganic materials via control of the conformation of soft macromolecular templates.     

Cortical development: receiving reelin

Recent genetic and biochemical studies indicate that lipoprotein receptors are components of the neuronal receptor for Reelin, mediating the glycoprotein's essential function in cortical development. At least eight cadherin-related neuronal receptors may also play a part in this signalling system.     

Substrate analogues to study cell-wall biosynthesis and its inhibition

It has been known for more than 30 years that Lipid II is an intermediate in peptidoglycan synthesis. Recently, it has become apparent that it is also an important target of numerous antibiotics, including the glycopeptides, the lantibiotics and ramoplanin. It is also utilized by sortases in the construction of Gram-positive cell walls. Recent progress has been made in the synthesis of peptidoglycan intermediates that can be used to study enzymes which make peptidoglycan. These intermediates also enable studies to probe the mechanism of action of a variety of substrate-binding antibiotics.     

Structure and reorientational dynamics of angiotensin I and II: a microscopic physical insight

We present a study of structural analysis and reorientational dynamics of Angiotensin I (AngI) and Angiotensin II (AngII) in aqueous solution. AngI is a decapeptide that acts as a precursor to the octapeptide AngII in the Renin-Angiotensin-Aldosterone system for blood pressure regulation. Experimental structural characterization of these peptides, carried out with circular dichroism and infrared spectroscopy, showed that the angiotensins are mostly disordered but exhibit a measurable population of ordered structures at room temperature. Interestingly, these change from the unordered polyproline-like conformation for AngI to a more compact and ordered conformation for AngII as the length of the peptide is decreased. Anisotropy decay measurements with picosecond time resolution indicate slower overall tumbling and a greater amplitude of internal motion in AngI compared to AngII, consistent with more compact and less flexible structure of the active form of the peptide. To model the microscopic behavior of the peptides, 2-μs molecular dynamics simulation trajectories were generated for AngI and AngII, at 300?K using the OPLS-AA potential and SPC water. The structures sampled in the simulations mostly agree with the experimental results, showing the prevalence of disordered structures, turns, and polyproline helices. Additionally, the computational results predict fewer sampled conformations, tighter side-chain packing and marked increase of Phe8 solvent accessibility upon AngI truncation to AngII. Our combined approach of experiment and extensive computer simulation thus yields new information on the conformational dynamics of the angiotensins, helping provide insight into the structural basis for the potency of AngI relative to AngII.     

Elucidation and chemical modulation of sulfolipid-1 biosynthesis in Mycobacterium tuberculosis

Mycobacterium tuberculosis possesses unique cell-surface lipids that have been implicated in virulence. One of the most abundant is sulfolipid-1 (SL-1), a tetraacyl-sulfotrehalose glycolipid. Although the early steps in SL-1 biosynthesis are known, the machinery underlying the final acylation reactions is not understood. We provide genetic and biochemical evidence for the activities of two proteins, Chp1 and Sap (corresponding to gene loci rv3822 and rv3821), that complete this pathway. The membrane-associated acyltransferase Chp1 accepts a synthetic diacyl sulfolipid and transfers an acyl group regioselectively from one donor substrate molecule to a second acceptor molecule in two successive reactions to yield a tetraacylated product. Chp1 is fully active in vitro, but in M. tuberculosis, its function is potentiated by the previously identified sulfolipid transporter MmpL8. We also show that the integral membrane protein Sap and MmpL8 are both essential for sulfolipid transport. Finally, the lipase inhibitor tetrahydrolipstatin disrupts Chp1 activity in M. tuberculosis, suggesting an avenue for perturbing SL-1 biosynthesis in vivo. These data complete the SL-1 biosynthetic pathway and corroborate a model in which lipid biosynthesis and transmembrane transport are coupled at the membrane-cytosol interface through the activity of multiple proteins, possibly as a macromolecular complex.     

Time-gated luminescence assay using nonmetal probes for determination of protein kinase activity-based disease markers

A novel nonmetal optical probe ARC-1063 whose long-lifetime luminescence is induced by association with the target protein kinase is used for the measurement of the concentration of catalytic subunit of protein kinase A (PKAc) in complicated biological solutions. High affinity (K(D) = 10 pM toward PKAc) and unique optical properties of the probe enable its application for the measurement of picomolar concentrations of PKAc in the presence of high concentrations of other proteins. The described assay is applicable in the high-throughput format with the instrument setups designed for lanthanide-based time-gated (time-resolved) luminescence methods. The assay is used for demonstration that extracellular PKAc (ECPKA) is present in plasma samples of all healthy persons and cancer patients but great care must be taken for procedures of treatment of blood samples to avoid disruption, damage, or activation of platelets in the course of plasma (or serum) preparation and conservation.     

Quantifying the reduction in potential health risks by determining the sensitivity of poliovirus type 1 chat strain and rotavirus SA-11 to electron beam irradiation of iceberg lettuce and spinach

Fresh produce, such as lettuce and spinach, serves as a route of food-borne illnesses. The U.S. FDA has approved the use of ionizing irradiation up to 4 kGy as a pathogen kill step for fresh-cut lettuce and spinach. The focus of this study was to determine the inactivation of poliovirus and rotavirus on lettuce and spinach when exposed to various doses of high-energy electron beam (E-beam) irradiation and to calculate the theoretical reduction in infection risks that can be achieved under different contamination scenarios and E-beam dose applications. The D(10) value (dose required to reduce virus titers by 90%) (standard error) of rotavirus on spinach and lettuce was 1.29 (± 0.64) kGy and 1.03 (± 0.05) kGy, respectively. The D(10) value (standard error) of poliovirus on spinach and lettuce was 2.35 (± 0.20) kGy and 2.32 (± 0.08) kGy, respectively. Risk assessment of data showed that if a serving (～14 g) of lettuce was contaminated with 10 PFU/g of poliovirus, E-beam irradiation at 3 kGy will reduce the risk of infection from >2 in 10 persons to approximately 6 in 100 persons. Similarly, if a serving size (～0.8 g) of spinach is contaminated with 10 PFU/g of rotavirus, E-beam irradiation at 3 kGy will reduce infection risks from >3 in 10 persons to approximately 5 in 100 persons. The results highlight the value of employing E-beam irradiation to reduce public health risks but also the critical importance of adhering to good agricultural practices that limit enteric virus contamination at the farm and in packing houses.     

Circulating and synovial antibody profiling of juvenile arthritis patients by nucleic acid programmable protein arrays

Introduction

Juvenile idiopathic arthritis (JIA) is a heterogeneous disease characterized by chronic joint inflammation of unknown cause in children. JIA is an autoimmune disease and small numbers of autoantibodies have been reported in JIA patients. The identification of antibody markers could improve the existing clinical management of patients.

Methods

A pilot study was performed on the application of a high-throughput platform, the nucleic acid programmable protein array (NAPPA), to assess the levels of antibodies present in the systemic circulation and synovial joint of a small cohort of juvenile arthritis patients. Plasma and synovial fluid from 10 JIA patients was screened for antibodies against 768 proteins on NAPPAs.

Results

Quantitative reproducibility of NAPPAs was demonstrated with > 0.95 intra-array and inter-array correlations. A strong correlation was also observed for the levels of antibodies between plasma and synovial fluid across the study cohort (r = 0.96). Differences in the levels of 18 antibodies were revealed between sample types across all patients. Patients were segregated into two clinical subtypes with distinct antibody signatures by unsupervised hierarchical cluster analysis.

Conclusion

The NAPPAs provide a high-throughput quantitatively reproducible platform to screen for disease-specific autoantibodies at the proteome level on a microscope slide. The strong correlation between the circulating antibody levels and those of the inflamed joint represents a novel finding and provides confidence to use plasma for discovery of autoantibodies in JIA, thus circumventing the challenges associated with joint aspiration. We expect that autoantibody profiling of JIA patients on NAPPAs could yield antibody markers that can act as criteria to stratify patients, predict outcomes and understand disease etiology at the molecular level.