Gary J. Vora

Combining chemoselective ligation with polyhistidine-driven self-assembly for the modular display of biomolecules on quantum dots.

One of the principle hurdles to wider incorporation of semiconductor quantum dots (QDs) in biology is the lack of facile linkage chemistries to create different types of functional QD--bioconjugates. A two-step modular strategy for the presentation of biomolecules on CdSe/ZnS core/shell QDs is described here which utilizes a chemoselective, aniline-catalyzed hydrazone coupling chemistry to append hexahistidine sequences onto peptides and DNA. This specifically provides them the ability to ratiometrically self-assemble to hydrophilic QDs. The versatility of this labeling approach was highlighted by ligating proteolytic substrate peptides, an oligoarginine cell-penetrating peptide, or a DNA-probe to cognate hexahistidine peptidyl sequences. The modularity allowed subsequently self-assembled QD constructs to engage in different types of targeted bioassays. The self-assembly and photophysical properties of individual QD conjugates were first confirmed by gel electrophoresis and Forster resonance energy transfer analysis. QD-dye-labeled peptide conjugates were then used as biosensors to quantitatively monitor the proteolytic activity of caspase-3 or elastase enzymes from different species. These sensors allowed the determination of the corresponding kinetic parameters, including the Michaelis constant (K(M)) and the maximum proteolytic activity (V(max)). QDs decorated with cell-penetrating peptides were shown to be successfully internalized by HEK 293T/17 cells, while nanocrystals displaying peptide--DNA conjugates were utilized as fluorescent probes in hybridization microarray assays. This modular approach for displaying peptides or DNA on QDs may be extended to other more complex biomolecules such as proteins or utilized with different types of nanoparticle materials.

Nucleic Acid Amplification Strategies for DNA Microarray-Based Pathogen Detection

ABSTRACT DNA microarray-based screening and diagnostic technologies have long promised comprehensive testing capabilities. However, the potential of these powerful tools has been limited by front-end target-specific nucleic acid amplification. Despite the sensitivity and specificity associated with PCR amplification, the inherent bias and limited throughput of this approach constrain the principal benefits of downstream microarray-based applications, especially for pathogen detection. To begin addressing alternative approaches, we investigated four front-end amplification strategies: random primed, isothermal Klenow fragment-based, φ29 DNA polymerase-based, and multiplex PCR. The utility of each amplification strategy was assessed by hybridizing amplicons to microarrays consisting of 70-mer oligonucleotide probes specific for enterohemorrhagic Escherichia coli O157:H7 and by quantitating their sensitivities for the detection of O157:H7 in laboratory and environmental samples. Although nearly identical levels of hybridization specificity were achieved for each method, multiplex PCR was at least 3 orders of magnitude more sensitive than any individual random amplification approach. However, the use of Klenow-plus-Klenow and φ29 polymerase-plus-Klenow tandem random amplification strategies provided better sensitivities than multiplex PCR. In addition, amplification biases among the five genetic loci tested were 2- to 20-fold for the random approaches, in contrast to >4 orders of magnitude for multiplex PCR. The same random amplification strategies were also able to detect all five diagnostic targets in a spiked environmental water sample that contained a 63-fold excess of contaminating DNA. The results presented here underscore the feasibility of using random amplification approaches and begin to systematically address the versatility of these approaches for unbiased pathogen detection from environmental sources.

Use of Oligonucleotide Microarrays for Rapid Detection and Serotyping of Acute Respiratory Disease-Associated Adenoviruses

ABSTRACT The cessation of the adenovirus vaccination program for military trainees has resulted in several recent acute respiratory disease (ARD) outbreaks. In the absence of vaccination, rapid detection methods are necessary for the timely implementation of measures to prevent adenovirus transmission within military training facilities. To this end, we have combined a fluorogenic real-time multiplex PCR assay with four sets of degenerate PCR primers that target the E1A, fiber, and hexon genes with a long oligonucleotide microarray capable of identifying the most common adenovirus serotypes associated with adult respiratory tract infections (serotypes 3, 4, 7, 16, and 21) and a representative member of adenovirus subgroup C (serotype 6) that is a common cause of childhood ARD and that often persists into adulthood. Analyses with prototype strains demonstrated unique hybridization patterns for representative members of adenovirus subgroups B1, B2, C, and E, thus allowing serotype determination. Microarray-based sensitivity assessments revealed lower detection limits (between 1 and 100 genomic copies) for adenovirus serotype 4 (Ad4) and Ad7 cell culture lysates, clinical nasal washes, and throat swabs and purified DNA from clinical samples. When adenovirus was detected from coded clinical samples, the results obtained by this approach demonstrated an excellent concordance with those obtained by the more established method of adenovirus identification as well as by cell culture with fluorescent-antibody staining. Finally, the utility of this method was further supported by its ability to detect adenoviral coinfections, contamination, and, potentially, recombination events. Taken together, the results demonstrate the usefulness of the simple and rapid diagnostic method developed for the unequivocal identification of ARD-associated adenoviral serotypes from laboratory or clinical samples that can be completed in 1.5 to 4.0 h.

Genomic and proteomic analyses of the coral pathogen Vibrio coralliilyticus reveal a diverse virulence repertoire.

Vibrio coralliilyticus has been implicated as an important pathogen of coral species worldwide. In this study, the nearly complete genome of Vibrio coralliilyticus strain P1 (LMG23696) was sequenced and proteases implicated in virulence of the strain were specifically investigated. The genome sequence of P1 (5 513 256 bp in size) consisted of 5222 coding sequences and 58 RNA genes (53 tRNAs and at least 5 rRNAs). Seventeen metalloprotease and effector (vgrG, hlyA and hcp) genes were identified in the genome and expressed proteases were also detected in the secretome of P1. As the VcpA zinc-metalloprotease has been considered an important virulence factor of V. coralliilyticus, a vcpA deletion mutant was constructed to evaluate the effect of this gene in animal pathogenesis. Both wild-type and mutant (ΔvcpA) strains exhibited similar virulence characteristics that resulted in high mortality in Artemia and Drosophila pathogenicity bioassays and strong photosystem II inactivation of the coral dinoflagellate endosymbiont (Symbiodinium). In contrast, the ΔvcpA mutant demonstrated higher hemolytic activity and secreted 18 proteins not secreted by the wild type. These proteins included four types of metalloproteases, a chitinase, a hemolysin-related protein RbmC, the Hcp protein and 12 hypothetical proteins. Overall, the results of this study indicate that V. coralliilyticus strain P1 has a diverse virulence repertoire that possibly enables this bacterium to be an efficient animal pathogen.

Virulence Gene- and Pandemic Group-Specific Marker Profiling of Clinical Vibrio parahaemolyticus Isolates

ABSTRACT Vibrio parahaemolyticus is a halophilic bacterium capable of causing food- and waterborne gastroenteritis, wound infections, and septicemia in humans. The organism has recently received increasing attention, as the emergence of a new clone, V. parahaemolyticus O3:K6, has resulted in the first documented pandemic spread of V. parahaemolyticus. We used microarray analyses to explore the presence of known virulence factors and genetic markers thought to be specific for V. parahaemolyticus O3:K6 and its clonal derivatives. Analyses of 48 human clinical isolates collected between 1997 and 2005 revealed that the V. parahaemolyticus chromosome 2 type III secretion system is not specifically associated with pandemic strains and can be found in tdh-negative (i.e., Kanagawa-negative) clinical isolates. These results highlight the genetic dynamism of V. parahaemolyticus and aid in refining the genetic definition of the pandemic group members.

Detection and Genotyping of Entamoeba histolytica, Entamoeba dispar, Giardia lamblia, and Cryptosporidium parvum by Oligonucleotide Microarray

ABSTRACT Entamoeba histolytica, Giardia lamblia, and Cryptosporidium parvum are the most frequently identified protozoan parasites causing waterborne disease outbreaks. The morbidity and mortality associated with these intestinal parasitic infections warrant the development of rapid and accurate detection and genotyping methods to aid public health efforts aimed at preventing and controlling outbreaks. In this study, we describe the development of an oligonucleotide microarray capable of detecting and discriminating between E. histolytica, Entamoeba dispar, G. lamblia assemblages A and B, and C. parvum types 1 and 2 in a single assay. Unique hybridization patterns for each selected protozoan were generated by amplifying six to eight diagnostic sequences/organism by multiplex PCR; fluorescent labeling of the amplicons via primer extension; and subsequent hybridization to a set of genus-, species-, and subtype-specific covalently immobilized oligonucleotide probes. The profile-based specificity of this methodology not only permitted for the unequivocal identification of the six targeted species and subtypes, but also demonstrated its potential in identifying related species such as Cryptosporidium meleagridis and Cryptosporidium muris. In addition, sensitivity assays demonstrated lower detection limits of five trophozoites of G. lamblia. Taken together, the specificity and sensitivity of the microarray-based approach suggest that this methodology may provide a promising tool to detect and genotype protozoa from clinical and environmental samples.

Electrochemical Investigation of a Microbial Solar Cell Reveals a Nonphotosynthetic Biocathode Catalyst

ABSTRACT Microbial solar cells (MSCs) are microbial fuel cells (MFCs) that generate their own oxidant and/or fuel through photosynthetic reactions. Here, we present electrochemical analyses and biofilm 16S rRNA gene profiling of biocathodes of sediment/seawater-based MSCs inoculated from the biocathode of a previously described sediment/seawater-based MSC. Electrochemical analyses indicate that for these second-generation MSC biocathodes, catalytic activity diminishes over time if illumination is provided during growth, whereas it remains relatively stable if growth occurs in the dark. For both illuminated and dark MSC biocathodes, cyclic voltammetry reveals a catalytic-current–potential dependency consistent with heterogeneous electron transfer mediated by an insoluble microbial redox cofactor, which was conserved following enrichment of the dark MSC biocathode using a three-electrode configuration. 16S rRNA gene profiling showed Gammaproteobacteria, most closely related to Marinobacter spp., predominated in the enriched biocathode. The enriched biocathode biofilm is easily cultured on graphite cathodes, forms a multimicrobe-thick biofilm (up to 8.2 μm), and does not lose catalytic activity after exchanges of the reactor medium. Moreover, the consortium can be grown on cathodes with only inorganic carbon provided as the carbon source, which may be exploited for proposed bioelectrochemical systems for electrosynthesis of organic carbon from carbon dioxide. These results support a scheme where two distinct communities of organisms develop within MSC biocathodes: one that is photosynthetically active and one that catalyzes reduction of O2 by the cathode, where the former partially inhibits the latter. The relationship between the two communities must be further explored to fully realize the potential for MSC applications.

Co-infections of Adenovirus Species in Previously Vaccinated Patients

Adenoviral infections associated with respiratory illness in military trainees involve multiple co-infecting species and serotypes.

A Previously Uncharacterized, Nonphotosynthetic Member of the Chromatiaceae Is the Primary CO2-Fixing Constituent in a Self-Regenerating Biocathode

ABSTRACT Biocathode extracellular electron transfer (EET) may be exploited for biotechnology applications, including microbially mediated O2 reduction in microbial fuel cells and microbial electrosynthesis. However, biocathode mechanistic studies needed to improve or engineer functionality have been limited to a few select species that form sparse, homogeneous biofilms characterized by little or no growth. Attempts to cultivate isolates from biocathode environmental enrichments often fail due to a lack of some advantage provided by life in a consortium, highlighting the need to study and understand biocathode consortia in situ. Here, we present metagenomic and metaproteomic characterization of a previously described biocathode biofilm (+310 mV versus a standard hydrogen electrode [SHE]) enriched from seawater, reducing O2, and presumably fixing CO2 for biomass generation. Metagenomics identified 16 distinct cluster genomes, 15 of which could be assigned at the family or genus level and whose abundance was roughly divided between Alpha- and Gammaproteobacteria. A total of 644 proteins were identified from shotgun metaproteomics and have been deposited in the the ProteomeXchange with identifier PXD001045. Cluster genomes were used to assign the taxonomic identities of 599 proteins, with Marinobacter, Chromatiaceae, and Labrenzia the most represented. RubisCO and phosphoribulokinase, along with 9 other Calvin-Benson-Bassham cycle proteins, were identified from Chromatiaceae. In addition, proteins similar to those predicted for iron oxidation pathways of known iron-oxidizing bacteria were observed for Chromatiaceae. These findings represent the first description of putative EET and CO2 fixation mechanisms for a self-regenerating, self-sustaining multispecies biocathode, providing potential targets for functional engineering, as well as new insights into biocathode EET pathways using proteomics.

Surveillance of transcriptomes in basic military trainees with normal, febrile respiratory illness, and convalescent phenotypes

Gene expression profiles permit analysis of host immune response at the transcriptome level. We used the Pax gene Blood RNA (PAX) System and Affymetrix microarrays (HG-U133A&B) to survey profiles in basic military trainees and to classify them as healthy, febrile respiratory illness (FRI) without adenovirus, FRI with adenovirus, and convalescent from FRI with adenovirus. We assessed quality metrics of RNA processing for microarrays. Class prediction analysis discovered nested sets of transcripts that could categorize the phenotypes with optimized accuracy of 99% (nonfebrile vs febrile, P<0.0005), 87% (healthy vs convalescent, P=0.001), and 91% (febrile without vs with adenovirus, P<0.0005). The discovered set for classification of nonfebrile vs febrile patients consisted of 40 transcripts with functions related to interferon induced genes, complement cascades, and TNF and IL1 signaling. The set of seven transcripts for distinguishing healthy vs convalescent individuals included those associated with ribosomal structure, humoral immunity, and cell adhesion. The set of 10 transcripts for distinguishing FRI without vs with adenovirus had functions related to interferon induced genes, IL1 receptor accessory protein, and cell interactions. These results are the first in vivo demonstration of classification of infectious diseases via host signature transcripts and move us towards using the transcriptome in biosurveillance.