Methods For Inhibiting Clc-2 Channel With Gatx2

Compositions and methods of using scorpion venom peptide that is a ligand for ClC channels are provided. One aspect provides a pharmaceutical composition containing an amount of GaTx2 effective to inhibit ClC activity. Methods of treating a disorder or symptom of a disorder related to aberrant ClC channel activity are also provided.Georgia Tech Research Corporatio

Integrative high-throughput study of arsenic hyper-accumulation in Pteris vittata

Arsenic is a natural contaminant in the soil and ground water, which raises considerable concerns in food safety and human health worldwide. The fernPteris vittata (Chinese brake fern) is the first identified arsenic hyperaccumulator[1]. It and its close relatives have un-paralleled ability to tolerant arsenic and feature unique arsenic metabolisms. The focus of the research presented in this thesis is to elucidate the fundamentals of arsenic tolerance and hyper-accumulation in Pteris vittata through high throughput technology and bioinformatics tools. The transcriptome of the P. vittatagametophyte under arsenate stress was obtained using RNA-Seq technology and Trinity de novo assembly. Functional annotation of the transcriptome was performed in terms of blast search, Gene Ontology term assignment, Eukaryotic Orthologous Groups (KOG) classification, and pathway analysis. Differentially expressed genes induced by arsenic stress were identified, which revealed several key players in arsenic hyper-accumulation. As part of the efforts to annotate differentially expressed genes, literature of plant arsenic tolerance was collected and built into a searchable database using the Textpresso text-mining tool, which greatly facilitates the retrieval of biological facts involving arsenic related gene. In addition, an SVM-based named-entity recognition system was constructed to identify new references to genes in literature. The results provide excellent sequence resources for arsenic tolerance study in P.vittata, and establish a platform for integrative study using data of multiple types

Mineral binding peptides by phage display: experimental and bioinformatics studies

Phage display has attracted a great deal of interest in the identification of peptides specific to nanomaterials revealing distinctive binding behaviour. Though significant progress has been made in selecting and screening of biomolecule binding peptides, the accuracy of molecular recognition for inorganic materials is still challenging due to the limitations of phage display libraries and biopanning process. The study presented in this thesis is aimed at isolating mineral binding peptides by phage display and verifying them experimentally and/ or bioinformatically; exploring the role of electrostatic/ non-electrostatic interactions in the aqueous phase and the factors responsible for the adsorption or desorption of peptide or phage from the mineral surface. Firstly, silica binding peptides LPVRLDW, NDLMNRA, GQSEKHL and GASESYL have been identified using the phage display technique by varying experimental conditions including pH, detergent, washing and elution buffers to remove unique 7-mer peptide binding phages from amorphous hydrophilic silica nanoparticles via disruption of the molecular interactions between the phage attached peptides and the nanoparticles. A repanning method reported here, has experimentally reproduced the majority of the initially discovered silica binders; alongside identifying/ recovering additional peptide sequences HYIDFRW, KIAVIST and YSLKQYQ that may have been overlooked in the routine approach to biopanning. Secondly, an alternative three step elution method reported here, has eluted and recovered most target silica binders including ADIRHIK in the early panning rounds and removed the phage clones that are bound to silica by hydrophobic, hydrogen bonding and electrostatic attractions or repulsions; as opposed to one specific buffer being used for all panning rounds including elution steps in traditional biopanning experiments. Also, the phage clones that resist detection to single elution step have been eluted in the other successive elution steps, thereby recovering and improving the elution procedure for silica surfaces. In addition, these three different elution buffers have eluted phage clones that are interaction or charge specific subject to change in the elution buffer pH condition. The experimental results demonstrate that this sequential three step elution process was able to isolate tightly bound target silica binders in one or two biopanning rounds than the more typical four to five; thereby reducing biopanning rounds, cost and effort. Moreover, the bioinformatic analysis to cross check the authenticity/ quality of target binders has been reported. Furthermore, selected silica binding peptides isolated from phage display experiments were synthesized by a solid phase peptide synthesis approach and peptide-silica interactions explored in vitro, using quantitative and qualitative techniques. The fluorometric analysis of these peptides revealed that the peptide adsorption to silica surfaces would have more than one type of interactions (i.e. electrostatic/ hydrophobic/ H-bonding and Van der Waals) and could be influenced by the experimental conditions. More significantly, an increase in binding activity to negatively charged silica nanoparticles was noticed for the peptides (HYIDFRW, KIAVIST and YSLKQYQ) modified with an amide (NH2) group as opposed to a carboxyl group at the C-terminal end; driving an increase in overall charge or pI of the peptides. Insights from the studies presented may provide valuable information for designing and engineering of silica directed constructs for a range of biomedical and nanotechnological applications

Bioinformatics Techniques for Studying Drug Resistance In HIV and Staphylococcus Aureus

The worldwide HIV/AIDS pandemic has been partly controlled and treated by antivirals targeting HIV protease, integrase and reverse transcriptase, however, drug resistance has become a serious problem. HIV-1 drug resistance to protease inhibitors evolves by mutations in the PR gene. The resistance mutations can alter protease catalytic activity, inhibitor binding, and stability. Different machine learning algorithms (restricted boltzmann machines, clustering, etc.) have been shown to be effective machine learning tools for classification of genomic and resistance data. Application of restricted boltzmann machine produced highly accurate and robust classification of HIV protease resistance. They can also be used to compare resistance profiles of different protease inhibitors. HIV drug resistance has also been studied by enzyme kinetics and X-ray crystallography. Triple mutant HIV-1 protease with resistance mutations V32I, I47V and V82I has been used as a model for the active site of HIV-2 protease. The effects of four investigational antiviral inhibitors was measured for Triple mutant. The tested compounds had significantly worse inhibition of triple mutant with Ki values of 17-40 nM compared to 2-10 pM for wild type protease. The crystal structure of triple mutant in complex with GRL01111 was solved and showed few changes in protease interactions with inhibitor. These new inhibitors are not expected to be effective for HIV-2 protease or HIV-1 protease with changes V32I, I47V and V82I. Methicillin-resistant Staphylococcus aureus (MRSA) is an opportunistic pathogen that causes hospital and community-acquired infections. Antibiotic resistance occurs because of newly acquired low-affinity penicillin-binding protein (PBP2a). Transcriptome analysis was performed to determine how MuM (mutated PBP2 gene) responds to spermine and how Mu50 (wild type) responds to spermine and spermine–β-lactam synergy. Exogenous spermine and oxacillin were found to alter some significant gene expression patterns with major biochemical pathways (iron, sigB regulon) in MRSA with mutant PBP2 protein

The Mechanism for RNA Recognition by ANTAR Regulators of Gene Expression

ANTAR proteins are widespread bacterial regulatory proteins that have RNA–binding output domains and utilize antitermination to control gene expression at the post-initiation level. An ANTAR protein, EutV, regulates the ethanolamine-utilization genes (eut) in Enterococcus faecalis. Using this system, we present genetic and biochemical evidence of a general mechanism of antitermination used by ANTARs, including details of the antiterminator structure. The novel antiterminator structure consists of two small hairpins with highly conserved terminal loop residues, both features being essential for successful antitermination. The ANTAR protein dimerizes and associates with its substrate RNA in response to signal-induced phosphorylation. Furthermore, bioinformatic searches using this conserved antiterminator motif identified many new ANTAR target RNAs in phylogenetically diverse bacterial species, some comprising complex regulons. Despite the unrelatedness of the species in which they are found, the majority of the ANTAR–associated genes are thematically related to nitrogen management. These data suggest that the central tenets for gene regulation by ANTAR antitermination occur widely in nature to specifically control nitrogen metabolism

Minimalistic Peptide-Based Supramolecular Systems Relevant to the Chemical Origin of Life

All forms of life are based on biopolymers, which are made up of a selection of simple building blocks, such as amino acids, nucleotides, fatty acids and sugars. Their individual properties govern their interactions, giving rise to complex supramolecular structures with highly specialized functionality, including ligand recognition, catalysis and compartmentalization. In this thesis, we aim to answer the question whether short peptides could have acted as precursors of modern proteins during prebiotic evolution. Using a combination of experimental and computational techniques, we screened a large molecular search space for peptide sequences that are capable of forming supramolecular complexes with adenosine triphosphate (ATP), life’s ubiquitous energy currency, and uridine triphosphate (UTP). Our results demonstrate that peptides as short as heptamers can form dynamic supramolecular complexes, adapt their structure to a ligand upon binding, undergo phase-separation into spatially confined compartments and catalyze nucleotide-hydrolysis

Mitochondrial Functions are Major Targets of Isocyanide Activity in \u3ci\u3eSaccharomyces cerevisiae\u3c/i\u3e

The isocyanide functional group is important in the synthesis of many organic compounds and is found in natural products produced by plants, bacteria, marine invertebrates, and fungi. The antimicrobial activities of isocyanide compounds have been documented for almost 70 years, however, the biochemical targets and mechanisms of action remain poorly defined. We report antimicrobial activity of 4-para-nitrophenyl-isocyanide (p-NPIC) against a model fungus, Saccharomyces cerevisiae, and the human fungal pathogen Candida albicans. To identify the cellular and molecular targets of p-NPIC, we screened the non-essential single gene-deletion collection of S. cerevisiae. We aimed to identify genes which, when absent, rendered the resulting strain incapable of growth on solid media containing 1.5 µM p-NPIC. We identified 167 strains that were hypersensitive to p-NPIC and determined the minimum inhibitory concentration of p-NPIC for each of these mutant strains. The most sensitive deletion-strains (MIC \u3c 3.0 µM in liquid media) were enriched in mitochondrial functions including the mitochondrial type II fatty acid synthase, lipoic acid biosynthesis and protein lipoylation, synthesis and assembly of iron-sulfur clusters, and assembly and maintenance of cytochrome c oxidase. The identification of essentially all components of the proton-pumping vacuolar membrane ATPase, as well as some pH sensitive components of membrane lipid biosynthesis, also suggest a role for regulation of cytoplasmic pH as a key determinant of p-NPIC tolerance. Taken together, these results suggest that mitochondrial metal homeostasis and reactive oxygen scavenging are disrupted by p-NPIC treatment and provide new information about the potential mechanisms of action of isocyanide natural products. Advisor: Wayne R. Riekho

Microfluidic Selection of Aptamers towards Applications in Precision Medicine

Precision medicine represents a shift in medicine where large datasets are gathered for massive patient groups to draw correlations between disease cohorts. An individual patient can then be compared to these large datasets to determine the best treatment strategy. While electronic health records and next generation sequencing techniques have enabled much of the early applications for precision medicine, the human genome only represents a fraction of the information present and important to a person’s health. A person’s proteome (peptides and proteins) and glycome (glycans and glycosylation patterns) contain biomarkers that indicate health and disease; however, tools to detect and analyze such biomarkers remain scarce. Thus, precision medicine databases are lacking a major source of phenotypic data due to the absence of available methods to explore these domains, despite the potential of such data to allow further stratification of patients and individualized therapeutic strategies. Available methods to detect non-nucleic acid biomarkers are currently not well suited to address the needs of precision medicine. Mass spectrometry techniques, while capable of generating high throughput data, lack standardization, require extensive preparative steps, and have many sources of errors. Immunoassays rely on antibodies which are time consuming and expensive to produce for newly discovered biomarkers. Aptamers, analogous to antibodies but composed of nucleotides and isolated through in vitro methods, have potential to identify non-nucleic acid biomarkers but methods to isolate aptamers remain labor and resource intensive and time consuming. Recently, microfluidic technology has been applied to the aptamer discovery process to reduce the aptamer development time, while consuming smaller amounts of reagents. Methods have been demonstrated that employ capillary electrophoresis, magnetic mixers, and integrated functional chambers to select aptamers. However, these methods are not yet able to fully integrate the entire aptamer discovery process on a single chip and must rely on off-chip processes to identify aptamers. In this thesis, new approaches for aptamer selection are developed that aim to integrate the entire process for aptamer discovery on a single chip. These approaches are capable of performing efficient aptamer selection and polymerase chain reaction based amplification while utilizing highly efficient bead-based reactions. The approaches use pressure driven flow, electrokinetic flow or a combination of both to transfer aptamer candidates through multiple rounds of affinity selection and PCR amplification within a single microfluidic device. As such, the approaches are capable of isolating aptamer candidates within a day while consuming <500 µg of a target molecule. The utility of the aptamer discovery approach is then demonstrated with examples in precision medicine over a broad spectrum (small molecule to protein) of molecular targets. Seeking to demonstrate the potential of the device to generate probes capable of accessing the human glycome (an emerging source of precision medicine biomarkers), aptamers are isolated against gangliosides GM1, GM3, and GD3, and a glycosylated peptide. Finally, personalized, patient specific aptamers are isolated against a multiple myeloma patient serum sample. The aptamers have high affinity only for the patient derived antibody