Benjamin D. Reeves

Global Analysis of Viral Infection in an Archaeal Model System

The origin and evolutionary relationship of viruses is poorly understood. This makes archaeal virus-host systems of particular interest because the hosts generally root near the base of phylogenetic trees, while some of the viruses have clear structural similarities to those that infect prokaryotic and eukaryotic cells. Despite the advantageous position for use in evolutionary studies, little is known about archaeal viruses or how they interact with their hosts, compared to viruses of bacteria and eukaryotes. In addition, many archaeal viruses have been isolated from extreme environments and present a unique opportunity for elucidating factors that are important for existence at the extremes. In this article we focus on virus-host interactions using a proteomics approach to study Sulfolobus Turreted Icosahedral Virus (STIV) infection of Sulfolobus solfataricus P2. Using cultures grown from the ATCC cell stock, a single cycle of STIV infection was sampled six times over a 72 h period. More than 700 proteins were identified throughout the course of the experiments. Seventy one host proteins were found to change their concentration by nearly twofold (p < 0.05) with 40 becoming more abundant and 31 less abundant. The modulated proteins represent 30 different cell pathways and 14 clusters of orthologous groups. 2D gel analysis showed that changes in post-translational modifications were a common feature of the affected proteins. The results from these studies showed that the prokaryotic antiviral adaptive immune system CRISPR-associated proteins (CAS proteins) were regulated in response to the virus infection. It was found that regulated proteins come from mRNAs with a shorter than average half-life. In addition, activity-based protein profiling (ABPP) profiling on 2D-gels showed caspase, hydrolase, and tyrosine phosphatase enzyme activity labeling at the protein isoform level. Together, this data provides a more detailed global view of archaeal cellular responses to viral infection, demonstrates the power of quantitative two-dimensional differential gel electrophoresis and ABPP using 2D gel compatible fluorescent dyes.

Expanding the paradigm of thiol redox in the thermophilic root of life.

BACKGROUND The current paradigm of intracellular redox chemistry maintains that cells establish a reducing environment maintained by a pool of small molecule and protein thiol to protect against oxidative damage. This strategy is conserved in mesophilic organisms from all domains of life, but has been confounded in thermophilic organisms where evidence suggests that intracellular proteins have abundant disulfides. METHODS Chemical labeling and 2-dimensional gel electrophoresis were used to capture disulfide bonding in the proteome of the model thermophile Sulfolobus solfataricus. The redox poise of the metabolome was characterized using both chemical labeling and untargeted liquid chromatography mass spectrometry. Gene annotation was undertaken using support vector machine based pattern recognition. RESULTS Proteomic analysis indicated the intracellular protein thiol of S. solfataricus was primarily in the disulfide form. Metabolic characterization revealed a lack of reduced small molecule thiol. Glutathione was found primarily in the oxidized state (GSSG), at relatively low concentration. Combined with genetic analysis, this evidence shows that pathways for synthesis of glutathione do exist in the archaeal domain. CONCLUSIONS In observed thermophilic organisms, thiol abundance and redox poise suggest that this system is not directly utilized for protection against oxidative damage. Instead, a more oxidized intracellular environment promotes disulfide bonding, a critical adaptation for protein thermostability. GENERAL SIGNIFICANCE Based on the placement of thermophilic archaea close to the last universal common ancestor in rRNA phylogenies, we hypothesize that thiol-based redox systems are derived from metabolic pathways originally tasked with promoting protein stability.

Selective trapping of SNO-BSA and GSNO by benzenesulfinic acid sodium salt: mechanistic study of thiosulfonate formation and feasibility as a protein S-nitrosothiol detection strategy

The conversion of S-nitrosothiols to thiosulphonates by reaction with the sodium salt of benzenesulfinic acid (PhSO2Na) has been examined in detail with the exemplary substrates S-nitrosoglutathione (GSNO) and S-nitrosylated bovine serum albumin (SNO-BSA). The reaction stoichiometry (2:1, PhSO2Na:RSNO) and the rate law (first order in both PhSO2Na and RSNO) have been determined under mild acidic conditions (pH 4.0). The products have been identified as the corresponding thiosulphonates (GSSO2Ph and BSA-SSO2Ph) along with PhSO2NHOH obtained in a 1:1 ratio. GSH, GSSG, and BSA were unreactive to PhSO2Na.

Enhanced sensitivity employing zwitterionic and pI balancing dyes (Z-CyDyes) optimized for 2D-gel electrophoresis based on side chain modifications of CyDye fluorophores. New tools for use in proteomics and diagnostics.

The CyDye family of fluorescent dyes is currently the overwhelming choice for applications in proteomic analysis, using two-dimensional difference gel electrophoresis (2D-DIGE). Protein labeling with CyDyes is hampered by protein precipitation and gel smearing when used above minimal labeling. The solubility of labeled protein may be improved by introducing water solubilizing groups on the dye such as cysteic acids. However, addition of a negatively charged functionality will have the undesired effect of shifting the pI in relation to the unlabeled protein. These limitations have been addressed through the synthesis of highly water-soluble and pI balancing zwitterionic CyDye fluorophores (Z-CyDyes). The new dyes feature a cysteic acid motif, a titratable amine functionality and a NHS activated ester group. In side by side 2D-DIGE comparisons of Z-CyDyes and CyDyes, the new dyes significantly enhanced protein spot volume and the number of spots that were detected. Z-CyDyes have the potential to enhance the depth of proteome coverage and provide a general strategy for improving the performance of protein tagging reagents.

Aggregatibacter actinomycetemcomitans biofilm killing by a targeted ciprofloxacin prodrug

A pH-sensitive ciprofloxacin prodrug was synthesized and targeted against biofilms of the periodontal pathogen Aggregatibacter actinomycetemcomitans (Aa). The dose required to reduce the viability of a mature biofilm of Aa by ∼80% was in the range of ng cm−2 of colonized area (mean biofilm density 2.33 × 109 cells cm−2). A mathematical model was formulated that predicts the temporal change in the concentration of ciprofloxacin in the Aa biofilm as the drug is released and diffuses into the bulk medium. The predictions of the model were consistent with the extent of killing obtained. The results demonstrate the feasibility of the strategy to induce mortality, and together with the mathematical model, provide the basis for design of targeted antimicrobial prodrugs for the topical treatment of oral infections such as periodontitis. The targeted prodrug approach offers the possibility of optimizing the dose of available antimicrobials in order to kill a chosen pathogen while leaving the commensal microbiota relatively undisturbed.

New Dendritic Materials as Potential OLED Transport and Emitter Moeities

Traditionally, organic light-emitting devices (OLEDs) are prepared with discrete layers for hole and electron transport. Different materials must be used for these layers because most materials will preferentially transport one charge carrier more efficiently than the other. In most cases, the emitter material serves a dual purpose as both the emitter and the hole or electron transporter. One of the major failure modes of OLEDs results from thermal instabilities of the insulating organic layers caused by joule heating during device operation. The problem is most pronounced for the hole transporting layer (HTL) material which are usually tertiary aromatic amines (i.e., TPD and NPD). This has been attributed to the relatively lower glass transition temperatures (Tg) and resulting inferior thermal stabilities compared to the other materials making up the device. Many researchers have produced HTL materials with higher Tgs based on tertiary aromatic amine oligomers and starburst compounds. Starburst or model dendritic materials offer the advantages of high thermal stabilities and multi-functionality.

Spectroscopy and nonlinear optical absorption of bis(diphenylamino) diphenyl polyenes

The spectroscopy and nonlinear absorption of bis(diphenylamino) diphenyl polyenes have been studied in octane and dichloromethane solvents. The amines exhibit high fluorescence quantum yield and two photon excited emission. Two photon absorption cross section, (sigma) 2, was measured by Z-scan experiments. Strong two photon absorption is indicated by high values for (sigma) 2. Solvent has strong influence in the measurement of (sigma) 2 values.

Design, Synthesis and Characterization of New Bimechanistic Optical Power Limiters Based on Reverse Saturable and Two-photon Absorption

During the past five years there has been considerable progress in the design of organic materials for optical power limiting (OPL) applications. One of the more promising and widely studied material approaches involves reverse saturable absorption (RSA) from various excited states. We have recently been examining the efficacy of utilizing highly absorbing photogenerated charge states for RSA. Both polaronic radical cations and bipolaronic dications are possible candidates for this mode of optical limiting. Equally intriguing are new approaches to designing chromophores with large two-photon absorption (TPA) cross-sections. For some pulse durations the effective two-photon cross-section for bis-(diphenylamino)diphenylpolyenes, and dendrimers based on these repeat units, are extraordinaryly large, indicative of excited state absorption. In this presentation we will discuss the possibility of combining these two optical limiting mechanistic paradigms in single structures, which may then be considered as bimechanistic optical power limiters.

Model dendrons and dendrimers incorporating diphenylamino-substituted diphenylpolyene and PPV-oligomer moieties for NLO applications

Bis-(diphenylamino)diphenylpolyenes have been shown to form exceptionally stable, highly absorbing bipolaronic dications in solution and thin film. Replacement of one diphenylamino substituent with a N-(hydroxyethyl), N-ethylaminophenyl moiety yields a polyene series that also form stable bipolarons, and are intensely fluorescent. These new chromophores are also easily attached to either a PMMA backbone or to 3,5-dihydroxybenzyl alcohol to yield functionalized dendrons capable of attachment to various core molecules to yield functionalized dendrimers. Diphenylamino-substituted PPV oligomers can also be obtained with similar functionality. These new materials all possess large two-photon cross-sections and display optical limiting for nanosecond pulses with peak activity extending into the visible portion of the spectrum. In this presentation we will discuss the synthesis of these new materials and preliminary characterization as two-photon absorbers, photoluminescent materials suitable for organic light- emitting diodes, and as dendrimers capable of 3D charge delocalization and exceptionally large third order hyperpolarizabilities.

Photonic Applications of Bipolaron Formation in Bis-(Diphenylamino)Diphenylpolyene

Bis-(dialkylamino) and bis-(diphenylamino) substituted diphenylpolyenes have recently been shown to have exceptionally large two-photon cross-sections, and have also been used in optical power limiting (OPL) applications. Another potential mechanism for OPL in these materials involves the photogeneration of highly absorbing polaronic radical-cations or bipolaronic dications which can then function as highly absorbing reverse saturable absorbers (RSAs). The synthesis of series of these unique polyenes has been carried out, as well as studies of chemically induced bipolaron formation in solution. In order to actually be used in OPL devices, it is probable that these chromophores will need to be incorporated into polymer formulations, In this paper we describe three distinct ways that these chromophores can be incorporated in polymer structures: (1) as pendant groups attached to a PMMA backbone, (2) as formal repeat units in a copolymer, and (3) perhaps the most interesting, as surface attached functionalities in dendrimer formulations. The formation of bipolaron charge species in these polymers will be addressed.