Differential reactivity of 9-NH2-ellipticine on apurinic and apyrimidinic sites in circular DNA

Endonucleases for apurinic sites as well as chemical compounds reacting with aldehydes do not generally differentiate between apurinic and apyrimidinic sites. We have studied the effect of the apurinic site reagent, 9-NH2-ellipticine, on apyrimidinic sites enzymatically generated on PBR322 DNA and compared it to its' action on apurinic PM2 and PBR322 DNAs. In conditions where this compound induces breakage of apurinic sites, it does not display any action on apyrimidinic sites.     

Spotiton: a prototype for an integrated inkjet dispense and vitrification system for cryo-TEM

Over the last three decades, Cryo-TEM has developed into a powerful technique for high-resolution imaging of biological macromolecules in their native vitrified state. However, the method for vitrifying specimens onto EM grids is essentially unchanged - application of ～3 μL sample to a grid, followed by blotting and rapid plunge freezing into liquid ethane. Several trials are often required to obtain suitable thin (few hundred nanometers or less) vitrified layers amenable for cryo-TEM imaging, which results in waste of precious sample and resources. While commercially available instruments provide some level of automation to control the vitrification process in an effort to increase quality and reproducibility, obtaining satisfactory vitrified specimens remains a bottleneck in the Cryo-TEM pipeline. We describe here a completely novel method for EM specimen preparation based on small volume (picoliter to nanoliter) dispensing using inkjet technology. A first prototype system (Spotiton v0.5) demonstrates feasibility of this new approach for specimen vitrification. A piezo-electric inkjet dispenser is integrated with optical real-time cameras (100 Hz frame rate) to analyze picoliter to nanoliter droplet profiles in-flight and spreading dynamics on the grid, and thus provides a method to optimize timing of the process. Using TEM imaging and biochemical assays we demonstrate that the piezo-electric inkjet mechanism does not disrupt the structural or functional integrity of macromolecules. These preliminary studies provide insight into the factors and components that will need further development to enable a robust and repeatable technique for specimen vitrification using this novel approach.     

Serendipitous syntheses of Rh(H)2Cl(PRPh2)3 complexes, and their crystal structures, where R = Me, Cy (cyclohexyl)

Reactions of RhCl(cod)(THP) (cod = 1,5-cyclooctadiene; THP = P(CH₂OH)₃) with PMePh₂ or PCyPh₂ (Cy = cyclohexyl) in acetone/MeOH solution under H₂ surprisingly form the complexes cis, mer-Rh(H)₂Cl(PRPh₂)₃ (R = Me or Cy); both complexes are characterized by crystallography (the first structures in which the hydride ligands of such dihydrido-chloro-trisphosphine complexes have been located), and by detailed ¹H and ³¹P NMR spectroscopy. The key role of the THP in the observed chemistry is discussed.     

Dendritic cells and cytokines in human inflammatory and autoimmune diseases

Dendritic cells (DCs) produce cytokines and are susceptible to cytokine-mediated activation. Thus, interaction of resting immature DCs with TLR ligands, for example nucleic acids, or with microbes leads to a cascade of pro-inflammatory cytokines and skewing of T cell responses. Conversely, several cytokines are able to trigger DC activation (maturation) via autocrine, for example TNF and plasmacytoid DCs, and paracrine, for example type I IFN and myeloid DCs, pathways. By controlling DC activation, cytokines regulate immune homeostasis and the balance between tolerance and immunity. The increased production and/or bioavailability of cytokines and associated alterations in DC homeostasis have been implicated in various human inflammatory and autoimmune diseases. Targeting these cytokines with biological agents as already is the case with TNF and IL-1 represents a success of immunology and the coming years will expand the range of cytokines as therapeutic targets in autoinflammatory and autoimmune pathology.     

Pharmacological rescue of the mutant cystic fibrosis transmembrane conductance regulator (CFTR) detected by use of a novel fluorescence platform

Numerous human diseases arise because of defects in protein folding, leading to their degradation in the endoplasmic reticulum. Among them is cystic fibrosis (CF), caused by mutations in the gene encoding the CF transmembrane conductance regulator (CFTR ), an epithelial anion channel. The most common mutation, F508del, disrupts CFTR folding, which blocks its trafficking to the plasma membrane. We developed a fluorescence detection platform using fluorogen-activating proteins (FAPs) to directly detect FAP-CFTR trafficking to the cell surface using a cell-impermeant probe. By using this approach, we determined the efficacy of new corrector compounds, both alone and in combination, to rescue F508del-CFTR to the plasma membrane. Combinations of correctors produced additive or synergistic effects, improving the density of mutant CFTR at the cell surface up to ninefold over a single-compound treatment. The results correlated closely with assays of stimulated anion transport performed in polarized human bronchial epithelia that endogenously express F508del-CFTR. These findings indicate that the FAP-tagged constructs faithfully report mutant CFTR correction activity and that this approach should be useful as a screening assay in diseases that impair protein trafficking to the cell surface.     

Large-scale production of a therapeutic protein in transgenic tobacco plants: effect of subcellular targeting on quality of a recombinant dog gastric lipase

A recombinant dog gastric lipase with therapeutic potential for the treatment of exocrine pancreatic insufficiency was expressed in transgenic tobacco plants. We targeted the protein using two different signal sequences for either vacuolar retention or secretion. In both cases, an active glycosylated recombinant protein was obtained. The recombinant enzymes and the native enzyme displayed similar properties including acid resistance and acidic optimum pH. The proteolytic maturation and the specific activity of the recombinant proteins, however, were found to be dependent on subcellular compartmentalization. Expression levels of recombinant dog gastric lipase were about 5% and 7% of acid extractable plant proteins for vacuolar retention and secretion respectively. This expression system already has allowed the production of tens of grams of purified lipase through open-field culture of transgenic tobacco plants.     

An eco-informatics tool for microbial community studies: supervised classification of Amplicon Length Heterogeneity (ALH) profiles of 16S rRNA

Support vector machines (SVM) and K-nearest neighbors (KNN) are two computational machine learning tools that perform supervised classification. This paper presents a novel application of such supervised analytical tools for microbial community profiling and to distinguish patterning among ecosystems. Amplicon length heterogeneity (ALH) profiles from several hypervariable regions of 16S rRNA gene of eubacterial communities from Idaho agricultural soil samples and from Chesapeake Bay marsh sediments were separately analyzed. The profiles from all available hypervariable regions were concatenated to obtain a combined profile, which was then provided to the SVM and KNN classifiers. Each profile was labeled with information about the location or time of its sampling. We hypothesized that after a learning phase using feature vectors from labeled ALH profiles, both these classifiers would have the capacity to predict the labels of previously unseen samples. The resulting classifiers were able to predict the labels of the Idaho soil samples with high accuracy. The classifiers were less accurate for the classification of the Chesapeake Bay sediments suggesting greater similarity within the Bay's microbial community patterns in the sampled sites. The profiles obtained from the V1+V2 region were more informative than that obtained from any other single region. However, combining them with profiles from the V1 region (with or without the profiles from the V3 region) resulted in the most accurate classification of the samples. The addition of profiles from the V 9 region appeared to confound the classifiers. Our results show that SVM and KNN classifiers can be effectively applied to distinguish between eubacterial community patterns from different ecosystems based only on their ALH profiles.