Mass spectrometry-based thermal shift assay for protein-ligand binding analysis.

Described here is a mass spectrometry-based screening assay for the detection of protein-ligand binding interactions in multicomponent protein mixtures. The assay utilizes an oxidation labeling protocol that involves using hydrogen peroxide to selectively oxidize methionine residues in proteins in order to probe the solvent accessibility of these residues as a function of temperature. The extent to which methionine residues in a protein are oxidized after specified reaction times at a range of temperatures is determined in a MALDI analysis of the intact proteins and/or an LC-MS analysis of tryptic peptide fragments generated after the oxidation reaction is quenched. Ultimately, the mass spectral data is used to construct thermal denaturation curves for the detected proteins. In this proof-of-principle work, the protocol is applied to a four-protein model mixture comprised of ubiquitin, ribonuclease A (RNaseA), cyclophilin A (CypA), and bovine carbonic anhydrase II (BCAII). The new protocol's ability to detect protein-ligand binding interactions by comparing thermal denaturation data obtained in the absence and in the presence of ligand is demonstrated using cyclosporin A (CsA) as a test ligand. The known binding interaction between CsA and CypA was detected using both the MALDI- and LC-MS-based readouts described here.

Microbial inactivation of Pseudomonas putida and Pichia pastoris using gene silencing.

Antisense deoxyoligonucleotide (ASO) gene silencing was investigated as a potential disinfection tool for industrial and drinking water treatment application. ASOs bind with their reverse complementary mRNA transcripts thereby blocking protein translation. While ASO silencing has mainly been studied in medicine, it may be useful for modulating gene expression and inactivating microorganisms in environmental applications. In this proof of concept work, gene targets were sh ble (zeocin resistance) and todE (catechol-2,3-dioxygenase) in Pichia pastoris and npt (kanamycin resistance) in Pseudomonas putida. A maximum 0.5-fold decrease in P. pastoris cell numbers was obtained following a 120 min incubation with single-stranded DNA (ssDNA) concentrations ranging from 0.2 to 200 nM as compared to the no ssDNA control. In P. putida, a maximum 5.2-fold decrease was obtained after 90 min with 400 nM ssDNA. While the silencing efficiencies varied for the 25 targets tested, these results suggest that protein activity as well as microbial growth can be altered using ASO gene silencing-based tools. If successful, this technology has the potential to eliminate some of the environmental and health issues associated with the use of strong chemical biocides. However, prior to its dissemination, more research is needed to increase silencing efficiency and develop effective delivery methods.

Large-area nanopatterning of self-assembled monolayers of alkanethiolates by interferometric lithography.

We demonstrate that interferometric lithography provides a fast, simple approach to the production of patterns in self-assembled monolayers (SAMs) with high resolution over square centimeter areas. As a proof of principle, two-beam interference patterns, formed using light from a frequency-doubled argon ion laser (244 nm), were used to pattern methyl-terminated SAMs on gold, facilitating the introduction of hydroxyl-terminated adsorbates and yielding patterns of surface free energy with a pitch of ca. 200 nm. The photopatterning of SAMs on Pd has been demonstrated for the first time, with interferometric exposure yielding patterns of surface free energy with similar features sizes to those obtained on gold. Gold nanostructures were formed by exposing SAMs to UV interference patterns and then immersing the samples in an ethanolic solution of mercaptoethylamine, which etched the metal substrate in exposed areas while unoxidized thiols acted as a resist and protected the metal from dissolution. Macroscopically extended gold nanowires were fabricated using single exposures and arrays of 66 nm gold dots at 180 nm centers were formed using orthogonal exposures in a fast, simple process. Exposure of oligo(ethylene glycol)-terminated SAMs to UV light caused photodegradation of the protein-resistant tail groups in a substrate-independent process. In contrast to many protein patterning methods, which utilize multiple steps to control surface binding, this single step process introduced aldehyde functional groups to the SAM surface at exposures as low as 0.3 J cm(-2), significantly less than the exposure required for oxidation of the thiol headgroup. Although interferometric methods rely upon a continuous gradient of exposure, it was possible to fabricate well-defined protein nanostructures by the introduction of aldehyde groups and removal of protein resistance in nanoscopic regions. Macroscopically extended, nanostructured assemblies of streptavidin were formed. Retention of functionality in the patterned materials was demonstrated by binding of biotinylated proteins.

Noninvasive monitoring of tissue hemoglobin using UV-VIS diffuse reflectance spectroscopy: a pilot study.

We conducted a pilot study on 10 patients undergoing general surgery to test the feasibility of diffuse reflectance spectroscopy in the visible wavelength range as a noninvasive monitoring tool for blood loss during surgery. Ratios of raw diffuse reflectance at wavelength pairs were tested as a first-pass for estimating hemoglobin concentration. Ratios can be calculated easily and rapidly with limited post-processing, and so this can be considered a near real-time monitoring device. We found the best hemoglobin correlations were when ratios at isosbestic points of oxy- and deoxyhemoglobin were used, specifically 529/500 nm. Baseline subtraction improved correlations, specifically at 520/509 nm. These results demonstrate proof-of-concept for the ability of this noninvasive device to monitor hemoglobin concentration changes due to surgical blood loss. The 529/500 nm ratio also appears to account for variations in probe pressure, as determined from measurements on two volunteers.

The characterization of twenty sequenced human genomes.

We present the analysis of twenty human genomes to evaluate the prospects for identifying rare functional variants that contribute to a phenotype of interest. We sequenced at high coverage ten "case" genomes from individuals with severe hemophilia A and ten "control" genomes. We summarize the number of genetic variants emerging from a study of this magnitude, and provide a proof of concept for the identification of rare and highly-penetrant functional variants by confirming that the cause of hemophilia A is easily recognizable in this data set. We also show that the number of novel single nucleotide variants (SNVs) discovered per genome seems to stabilize at about 144,000 new variants per genome, after the first 15 individuals have been sequenced. Finally, we find that, on average, each genome carries 165 homozygous protein-truncating or stop loss variants in genes representing a diverse set of pathways.

High-throughput identification of chemical inhibitors of E. coli Group 2 capsule biogenesis as anti-virulence agents.

Rising antibiotic resistance among Escherichia coli, the leading cause of urinary tract infections (UTIs), has placed a new focus on molecular pathogenesis studies, aiming to identify new therapeutic targets. Anti-virulence agents are attractive as chemotherapeutics to attenuate an organism during disease but not necessarily during benign commensalism, thus decreasing the stress on beneficial microbial communities and lessening the emergence of resistance. We and others have demonstrated that the K antigen capsule of E. coli is a preeminent virulence determinant during UTI and more invasive diseases. Components of assembly and export are highly conserved among the major K antigen capsular types associated with UTI-causing E. coli and are distinct from the capsule biogenesis machinery of many commensal E. coli, making these attractive therapeutic targets. We conducted a screen for anti-capsular small molecules and identified an agent designated "C7" that blocks the production of K1 and K5 capsules, unrelated polysaccharide types among the Group 2-3 capsules. Herein lies proof-of-concept that this screen may be implemented with larger chemical libraries to identify second-generation small-molecule inhibitors of capsule biogenesis. These inhibitors will lead to a better understanding of capsule biogenesis and may represent a new class of therapeutics.

Pharmacogenomics in early-phase clinical development.

Pharmacogenomics (PGx) offers the promise of utilizing genetic fingerprints to predict individual responses to drugs in terms of safety, efficacy and pharmacokinetics. Early-phase clinical trial PGx applications can identify human genome variations that are meaningful to study design, selection of participants, allocation of resources and clinical research ethics. Results can inform later-phase study design and pipeline developmental decisions. Nevertheless, our review of the clinicaltrials.gov database demonstrates that PGx is rarely used by drug developers. Of the total 323 trials that included PGx as an outcome, 80% have been conducted by academic institutions after initial regulatory approval. Barriers for the application of PGx are discussed. We propose a framework for the role of PGx in early-phase drug development and recommend PGx be universally considered in study design, result interpretation and hypothesis generation for later-phase studies, but PGx results from underpowered studies should not be used by themselves to terminate drug-development programs.

Phylotastic! Making tree-of-life knowledge accessible, reusable and convenient.

BACKGROUND: Scientists rarely reuse expert knowledge of phylogeny, in spite of years of effort to assemble a great "Tree of Life" (ToL). A notable exception involves the use of Phylomatic, which provides tools to generate custom phylogenies from a large, pre-computed, expert phylogeny of plant taxa. This suggests great potential for a more generalized system that, starting with a query consisting of a list of any known species, would rectify non-standard names, identify expert phylogenies containing the implicated taxa, prune away unneeded parts, and supply branch lengths and annotations, resulting in a custom phylogeny suited to the user's needs. Such a system could become a sustainable community resource if implemented as a distributed system of loosely coupled parts that interact through clearly defined interfaces. RESULTS: With the aim of building such a "phylotastic" system, the NESCent Hackathons, Interoperability, Phylogenies (HIP) working group recruited 2 dozen scientist-programmers to a weeklong programming hackathon in June 2012. During the hackathon (and a three-month follow-up period), 5 teams produced designs, implementations, documentation, presentations, and tests including: (1) a generalized scheme for integrating components; (2) proof-of-concept pruners and controllers; (3) a meta-API for taxonomic name resolution services; (4) a system for storing, finding, and retrieving phylogenies using semantic web technologies for data exchange, storage, and querying; (5) an innovative new service, DateLife.org, which synthesizes pre-computed, time-calibrated phylogenies to assign ages to nodes; and (6) demonstration projects. These outcomes are accessible via a public code repository (GitHub.com), a website (http://www.phylotastic.org), and a server image. CONCLUSIONS: Approximately 9 person-months of effort (centered on a software development hackathon) resulted in the design and implementation of proof-of-concept software for 4 core phylotastic components, 3 controllers, and 3 end-user demonstration tools. While these products have substantial limitations, they suggest considerable potential for a distributed system that makes phylogenetic knowledge readily accessible in computable form. Widespread use of phylotastic systems will create an electronic marketplace for sharing phylogenetic knowledge that will spur innovation in other areas of the ToL enterprise, such as annotation of sources and methods and third-party methods of quality assessment.

Atherosclerosis: Viewing the problem from a different perspective including possible treatment options

This paper proposes that atherosclerosis is initiated by a signaling event that deposits calcium hydroxyapatite (Ca-HAP). This event is preceded by a loss of mechanical structure in the arterial wall. After Ca-HAP has been deposited, it is unlikely that it will be reabsorbed because the solubility product constant (K sp) is very small, and the large stores of Ca +2 and PO 4-3 in the bones oppose any attempts to dissolve Ca-HAP by decreasing the common ions. The hydroxide ion (OH -) of Ca-HAP can be displaced in nature by fluoride (F -) and carbonate (CO 3-2) ions, and it is proposed that anions associated with cholesterol ester hydrolysis and, in very small quantities, the enolate of 7-ketocholesterol could also displace the OH -of Ca-HAP, forming an ionic bond. The free energy of hydration of Ca-HAP at 310 K is most likely negative, and the ionic radii of the anions associated with the hydrolysis of cholesterol ester are compatible with the substitution. Furthermore, examination of the pathology of atherosclerotic lesions by Raman and NMR spectroscopy and confocal microscopy supports deposition of Ca-HAP associated with cholesterol. Investigating the affinity of intermediates of cholesterol hydrolysis for Ca-HAP compared to lipoproteins such as HDL, LDL, and VLDL using isothermic titration calorimetry could add proof of this concept and may lead to the development of a new class of medications targeted at the deposition of cholesterol within Ca-HAP. Treatment of acute ischemic events as a consequence of atherosclerosis with denitrogenation and oxygenation is discussed. © the author(s), publisher and licensee Libertas Academica Ltd.

Chromatin accessibility mapping identifies mediators of basal transcription and retinoid-induced repression of OTX2 in medulloblastoma.

Despite an emerging understanding of the genetic alterations giving rise to various tumors, the mechanisms whereby most oncogenes are overexpressed remain unclear. Here we have utilized an integrated approach of genomewide regulatory element mapping via DNase-seq followed by conventional reporter assays and transcription factor binding site discovery to characterize the transcriptional regulation of the medulloblastoma oncogene Orthodenticle Homeobox 2 (OTX2). Through these studies we have revealed that OTX2 is differentially regulated in medulloblastoma at the level of chromatin accessibility, which is in part mediated by DNA methylation. In cell lines exhibiting chromatin accessibility of OTX2 regulatory regions, we found that autoregulation maintains OTX2 expression. Comparison of medulloblastoma regulatory elements with those of the developing brain reveals that these tumors engage a developmental regulatory program to drive OTX2 transcription. Finally, we have identified a transcriptional regulatory element mediating retinoid-induced OTX2 repression in these tumors. This work characterizes for the first time the mechanisms of OTX2 overexpression in medulloblastoma. Furthermore, this study establishes proof of principle for applying ENCODE datasets towards the characterization of upstream trans-acting factors mediating expression of individual genes.

A Peptide Uncoupling BDNF Receptor TrkB from Phospholipase Cγ1 Prevents Epilepsy Induced by Status Epilepticus.

The BDNF receptor tyrosine kinase, TrkB, underlies nervous system function in both health and disease. Excessive activation of TrkB caused by status epilepticus promotes development of temporal lobe epilepsy (TLE), revealing TrkB as a therapeutic target for prevention of TLE. To circumvent undesirable consequences of global inhibition of TrkB signaling, we implemented a novel strategy aimed at selective inhibition of the TrkB-activated signaling pathway responsible for TLE. Our studies of a mouse model reveal that phospholipase Cγ1 (PLCγ1) is the dominant signaling effector by which excessive activation of TrkB promotes epilepsy. We designed a novel peptide (pY816) that uncouples TrkB from PLCγ1. Treatment with pY816 following status epilepticus inhibited TLE and prevented anxiety-like disorder yet preserved neuroprotective effects of endogenous TrkB signaling. We provide proof-of-concept evidence for a novel strategy targeting receptor tyrosine signaling and identify a therapeutic with promise for prevention of TLE caused by status epilepticus in humans.