A virus-based single-enzyme nanoreactor

Most enzyme studies are carried out in bulk aqueous solution, at the so-called ensemble level, but more recently studies have appeared in which enzyme activity is measured at the level of a single molecule, revealing previously unseen properties. To this end, enzymes have been chemically or physically anchored to a surface, which is often disadvantageous because it may lead to denaturation. In a natural environment, enzymes are present in a confined reaction space, which inspired us to develop a generic method to carry out single-enzyme experiments in the restricted spatial environment of a virus capsid. We report here the incorporation of individual horseradish peroxidase enzymes in the inner cavity of a virus, and describe single-molecule studies on their enzymatic behaviour. These show that the virus capsid is permeable for substrate and product and that this permeability can be altered by changing pH.     

Duodenal Dysbiosis and Relation to the Efficacy of Proton Pump Inhibitors in Functional Dyspepsia

Proton pump inhibitors (PPI) may improve symptoms in functional dyspepsia (FD) through duodenal eosinophil-reducing effects. However, the contribution of the microbiome to FD symptoms and its interaction with PPI remains elusive. Aseptic duodenal brushings and biopsies were performed before and after PPI intake (4 weeks Pantoprazole 40 mg daily, FD-starters and controls) or withdrawal (2 months, FD-stoppers) for 16S-rRNA sequencing. Between- and within-group changes in genera or diversity and associations with symptoms or duodenal factors were analyzed. In total, 30 controls, 28 FD-starters and 19 FD-stoppers were followed. Mucus-associated Porphyromonas was lower in FD-starters vs. controls and correlated with symptoms in FD and duodenal eosinophils in both groups, while Streptococcus correlated with eosinophils in controls. Although clinical and eosinophil-reducing effects of PPI therapy were unrelated to microbiota changes in FD-starters, increased Streptococcus was associated with duodenal PPI effects in controls and remained higher despite withdrawal of long-term PPI therapy in FD-stoppers. Thus, duodenal microbiome analysis demonstrated differential mucus-associated genera, with a potential role of Porphyromonas in FD pathophysiology. While beneficial effects of short-term PPI therapy were not associated with microbial changes in FD-starters, increased Streptococcus and its association with PPIeffects in controls suggest a role for duodenal dysbiosis after long-term PPI therapy.     

Unraveling Protein Interactions between the Temperate Virus Bam35 and Its Bacillus Host Using an Integrative Yeast Two Hybrid–High Throughput Sequencing Approach

Bacillus virus Bam35 is the model Betatectivirus and member of the family Tectiviridae, which is composed of tailless, icosahedral, and membrane-containing bacteriophages. Interest in these viruses has greatly increased in recent years as they are thought to be an evolutionary link between diverse groups of prokaryotic and eukaryotic viruses. Additionally, betatectiviruses infect bacteria of the Bacillus cereus group, which are known for their applications in industry and notorious since it contains many pathogens. Here, we present the first protein–protein interactions (PPIs) network for a tectivirus–host system by studying the Bam35–Bacillus thuringiensis model using a novel approach that integrates the traditional yeast two-hybrid system and high-throughput sequencing (Y2H-HTS). We generated and thoroughly analyzed a genomic library of Bam35′s host B. thuringiensis HER1410 and screened interactions with all the viral proteins using different combinations of bait–prey couples. Initial analysis of the raw data enabled the identification of over 4000 candidate interactions, which were sequentially filtered to produce 182 high-confidence interactions that were defined as part of the core virus–host interactome. Overall, host metabolism proteins and peptidases were particularly enriched within the detected interactions, distinguishing this host–phage system from the other reported host–phage PPIs. Our approach also suggested biological roles for several Bam35 proteins of unknown function, including the membrane structural protein P25, which may be a viral hub with a role in host membrane modification during viral particle morphogenesis. This work resulted in a better understanding of the Bam35–B. thuringiensis interaction at the molecular level and holds great potential for the generalization of the Y2H-HTS approach for other virus–host models.     

The influence of maltodextrins on the structure and properties of compression‐molded starch plastic sheets

Starch plastic sheets were prepared by compression molding of starch‐based plastic granulates. The granulates were prepared by extrusion processing of mixtures of granular potato starch and several maltodextrins (5% w/w) in the presence of glycerol and water as plasticizers and lecithin as melt flow accelerator. The materials were semicrystalline, containing B‐type, V_h‐type, and E_h‐type crystallinity. The properties were dependent on water content. For the materials, a brittle‐to‐ductile transition occurred at a water content in the range of 11–12%, which was in accordance with the observed glass transition temperature. The structural and mechanical properties were a function of starch composition and maltodextrin source as well as molding temperature. The amount of granular remnants and residual B‐type crystallinity decreased with increasing processing temperature. The amount of recrystallized single‐helical amylose increased with increasing temperature. At molding temperatures in the range of 180–200°C, a sharp decrease in starch molecular mass occurred. The influence of molding temperature was reflected in a sharp increase in elongation at molding temperature above 160°C and a gradual decrease in elastic modulus. The tensile strength showed an initial small increase up to 160°C and a sharp decrease at higher molding temperatures. © 1999 John Wiley & Sons, Inc. J Appl Polym Sci 74: 2207–2219, 1999     

A Protein-Adsorbent Hydrogel with Tunable Stiffness for Tissue Culture Demonstrates Matrix-Dependent Stiffness Responses

Although tissue culture plastic has been widely employed for cell culture, the rigidity of plastic is not physiologic. Softer hydrogels used to culture cells have not been widely adopted in part because coupling chemistries are required to covalently capture extracellular matrix (ECM) proteins and support cell adhesion. To create an in vitro system with tunable stiffnesses that readily adsorbs ECM proteins for cell culture, a novel hydrophobic hydrogel system is presented via chemically converting hydroxyl residues on the dextran backbone to methacrylate groups, thereby transforming non-protein adhesive, hydrophilic dextran to highly protein adsorbent substrates. Increasing methacrylate functionality increases the hydrophobicity in the resulting hydrogels and enhances ECM protein adsorption without additional chemical reactions. These hydrophobic hydrogels permit facile and tunable modulation of substrate stiffness independent of hydrophobicity or ECM coatings. Using this approach, it is shown that substrate stiffness and ECM adsorption work together to affect cell morphology and proliferation, but the strengths of these effects vary in different cell types. Furthermore, it is revealed that stiffness-mediated differentiation of dermal fibroblasts into myofibroblasts is modulated by the substrate ECM. The material system demonstrates remarkable simplicity and flexibility to tune ECM coatings and substrate stiffness and study their effects on cell function.     

Domain size-induced heterogeneity as performance limitation of small-domain monolithic columns and other LC support types

We have computed the band broadening and the flow resistance in a series of apparently self-similar porous LC support structures, all having the same mean geometric ratios and external porosity, but with a decreasing scale and disturbed by a scale-independent variance on the size and position of the porous solid zone elements. The study shows in a general and qualitative way that each type of LC support that is produced using a manufacturing process displaying a fixed (i.e., domain-size independent) variance on the size and position of the produced solid zone elements will eventually encounter a limit beyond which a further reduction of the domain size can no longer be expected to yield a significant gain in separation speed. This is currently observed in practice for silica monoliths and could also compromise the performance of photolithographically etched columns.     

An efficient method for the synthesis of peptide aldehyde libraries employed in the discovery of reversible SARS coronavirus main protease (SARS-CoV Mpro) inhibitors

A method for the parallel solid-phase synthesis of peptide aldehydes has been developed. Protected amino acid aldehydes obtained by the racemization-free oxidation of amino alcohols with Dess-Martin periodinane were immobilized on threonyl resins as oxazolidines. Following Boc protection of the ring nitrogen to yield the N-protected oxazolidine linker, peptide synthesis was performed efficiently on this resin. A peptide aldehyde library was designed for targeting the SARS coronavirus main protease, SARS-CoV M(pro)(also known as 3CL(pro)), on the basis of three different reported binding modes and supported by virtual screening. A set of 25 peptide aldehydes was prepared by this method and investigated in inhibition assays against SARS-CoV M(pro). Several potent inhibitors were found with IC(50) values in the low micromolar range. An IC(50) of 7.5 muM was found for AcNSTSQ-H and AcESTLQ-H. Interestingly, the most potent inhibitors seem to bind to SARS-CoV M(pro) in a noncanonical binding mode.     

High-damping carbon nanotube hinged micromirrors

New developments in digital mirror devices (DMDs) require suspension hinges with a good damping and high temperature stability. Carbon nanotubes (CNTs) offer these unique properties. Herein it is shown how CNT hinges can be integrated in micromirrors. The image illustrates a micromirror with a CNT suspension, and a typical overdamped stepresponse (Q-factor < 0.5).