Draft genome sequence of potentially dikaryotic black fungus Aureobasidium melanogenum isolated from aircraft.

The Ascomycota yeast Aureobasidium melanogenum strain W12 was isolated from an aircraft polymer-coated surface. The genome size is 53,160,883 bp with a G + C content of 50.13%. The genome contains fatty acid transporters, cutinases, hydroxylases, and lipases potentially used for survival on polymer coatings on aircraft.

Host Cell Transcriptional Tuning with CRISPR/dCas9 to Mitigate the Effects of Toxin Exposure.

Anthrax infection is caused byBacillus anthracis, a bacterium that once established within the host releases lethal toxin (LeTx). Anthrax LeTx is internalized by the capillary morphogenesis protein 2/anthrax toxin receptor 2 (CMG2/ANTXR2) cell surface receptor on mammalian cells. Once inside the cell, LeTx cleaves mitogen-activated protein kinases (MAPKs), ultimately leading to cell death. Previous reports have shown that decreased expression of ANTXR2 reduces cell susceptibility to LeTx. By ablating the ANTXR2 gene in cells in vitro, we observed complete resistance to LeTx-induced cell death. Here, we directed CRISPR/dCas9-based tools to the ANTXR2 promoter to modulate ANTXR2 expression without altering the underlying gene sequence in human cell lines that express the receptor at high levels. We hypothesized that downregulating the expression of the ANTXR2 gene at the genomic level would mitigate the impact of toxin exposure. In one epigenetic editing approach, we employed the fusion of DNMT3A DNA methyltransferase and dCas9 (dCas9-DNMT3A) to methylate CpGs within the CpG island of the ANTXR2 promoter and found this repressed ANTXR2 gene expression resulting in significant resistance to LeTx-induced cell death. Furthermore, by multiplexing gRNAs to direct dCas9-DNMT3A to multiple sites in the ANTXR2 promoter, we applied a broader distribution of CpG methylation along the gene promoter resulting in enhanced repression and resistance to LeTx. In parallel, we directed the dCas9-KRAB-MeCP2 transcriptional repressor to the ANTXR2 promoter to quickly and robustly repress ANTXR2 expression. With this approach, in as little as two weeks, we created resistance to LeTx at a similar level to ANTXR2 gene-ablated cells. Overall, we present a transcriptional tuning approach to inhibit the effects of LeTx and provide a framework to repress toxin-binding cell surface receptors.

Draft Genome Sequence of the Nonmotile Tremellomycetes Yeast Naganishia albida, Isolated from Aircraft.

The Basidiomycota yeast Naganishia albida strain 5307AI was isolated from an aircraft polymer-coated surface. The genome size is 20,642,279 bp, with a G+C content of 53.99%. The genome contains fatty acid transporters, cutinases, hydroxylases, and lipases that are likely used for survival on polymer coatings on aircraft.

In vitro fermentation test bed for evaluation of engineered probiotics in polymicrobial communities.

In vitro fermentation systems offer significant opportunity for deconvoluting complex metabolic dynamics within polymicrobial communities, particularly those associated with the human gut microbiome. In vitro gut models have broad experimental capacity allowing rapid evaluation of multiple parameters, generating knowledge to inform design of subsequent in vivo studies. Here, our method describes an in vitro fermentation test bed to provide a physiologically-relevant assessment of engineered probiotics circuit design functions. Typically, engineered probiotics are evaluated under pristine, mono- or co-culture conditions and transitioned directly into animal or human studies, commonly resulting in a loss of desired function when introduced to complex gut communities. Our method encompasses a systematic workflow entailing fermentation, molecular and functional characterization, and statistical analyses to validate an engineered probiotic's persistence, plasmid stability and reporter response. To demonstrate the workflow, simplified polymicrobial communities of human gut microbial commensals were utilized to investigate the probiotic Escherichia coli Nissle 1917 engineered to produce a fluorescent reporter protein. Commensals were assembled with increasing complexity to produce a mock community based on nutrient utilization. The method assesses engineered probiotic persistence in a competitive growth environment, reporter production and function, effect of engineering on organism growth and influence on commensal composition. The in vitro test bed represents a new element within the Design-Build-Test-Learn paradigm, providing physiologically-relevant feedback for circuit re-design and experimental validation for transition of engineered probiotics to higher fidelity animal or human studies.

A Pilot Study of the Effect of Deployment on the Gut Microbiome and Traveler's Diarrhea Susceptibility.

Traveler's diarrhea (TD) is a recurrent and significant issue for many travelers including the military. While many known enteric pathogens exist that are causative agents of diarrhea, our gut microbiome may also play a role in TD susceptibility. To this end, we conducted a pilot study of the microbiome of warfighters prior to- and after deployment overseas to identify marker taxa relevant to TD. This initial study utilized full-length 16S rRNA gene sequencing to provide additional taxonomic resolution toward identifying predictive taxa.16S rRNA analyses of pre- and post-deployment fecal samples identified multiple marker taxa as significantly differentially abundant in subjects that reported diarrhea, including Weissella, Butyrivibrio, Corynebacterium, uncultivated Erysipelotrichaceae, Jeotgallibaca, unclassified Ktedonobacteriaceae, Leptolinea, and uncultivated Ruminiococcaceae. The ability to identify TD risk prior to travel will inform prevention and mitigation strategies to influence diarrhea susceptibility while traveling.

Draft Genome Sequence of Filamentous Fungus Phialemoniopsis curvata, Isolated from Diesel Fuel.

Phialemoniopsis curvata D216 is a filamentous fungus isolated from contaminated diesel fuel. The genome size is 40.3 Mbp with a G+C content of 54.81%. Its genome encodes enzymes and pathways likely involved in the degradation of and survival in fuel, including lipases, fatty acid transporters, and beta oxidation.

Genome Sequence of a Byssochlamys sp. Strain Isolated from Fouled B20 Biodiesel.

Byssochlamys sp. strain AF001 is a filamentous fungus isolated from fouled B20 biodiesel. Its growth on B20 biodiesel results in the degradation and fouling of the fuel and higher rates of corrosion in affected storage tanks. The genome of Byssochlamys sp. AF001 is 35.9 Mbp and is composed of 10 scaffolds, with a G+C content of 45.89%.

A plasmid containing the human metallothionein II gene can function as an antibody-assisted electrophoretic biosensor for heavy metals.

Different forms of heavy metals affect biochemical systems in characteristic ways that cannot be detected with typical metal analysis methods like atomic absorption spectrometry. Further, using living systems to analyze interaction of heavy metals with biochemical systems can be laborious and unreliable. To generate a reliable easy-to-use biologically-based biosensor system, the entire human metallothionein-II (MT-II) gene was incorporated into a plasmid (pUC57-MT) easily replicated in Escherichia coli. In this system, a commercial polyclonal antibody raised against human metal-responsive transcription factor-1 protein (MTF-1 protein) could modify the electrophoretic migration patterns (i.e. cause specific decreases in agarose gel electrophoretic mobility) of the plasmid in the presence or absence of heavy metals other than zinc (Zn). In the study here, heavy metals, MTF-1 protein, and polyclonal anti-MTF-1 antibody were used to assess pUC57-MT plasmid antibody-assisted electrophoretic mobility. Anti-MTF-1 antibody bound both MTF-1 protein and pUC57-MT plasmid in a non-competitive fashion such that it could be used to differentiate specific heavy metal binding. The results showed that antibody-inhibited plasmid migration was heavy metal level-dependent. Zinc caused a unique mobility shift pattern opposite to that of other metals tested, i.e. Zn blocked the antibody ability to inhibit plasmid migration, despite a greatly increased affinity for DNA by the antibody when Zn was present. The Zn effect was reversed/modified by adding MTF-1 protein. Additionally, antibody inhibition of plasmid mobility was resistant to heat pre-treatment and trypsinization, indicating absence of residual DNA extraction-resistant bacterial DNA binding proteins. DNA binding by anti-DNA antibodies may be commonly enhanced by xenobiotic heavy metals and elevated levels of Zn, thus making them potentially effective tools for assessment of heavy metal bioavailability in aqueous solutions and fluid obtained from metal implant sites.

Detection of orexin A neuropeptide in biological fluids using a zinc oxide field effect transistor.

Biomarkers which are indicative of acute physiological and emotional states are studied in a number of different areas in cognitive neuroscience. Currently, many cognitive studies are conducted based on programmed tasks followed by timed biofluid sampling, central laboratory processing, and followed by data analysis. In this work, we present a sensor platform capable of rapid biomarker detection specific for detecting neuropeptide orexin A, found in blood and saliva and known as an indicator of fatigue and cognitive performance. A peptide recognition element that selectively binds to orexin A was designed, characterized, and functionalized onto a zinc oxide field effect transistor to enable rapid detection. The detection limit using the sensor platform was sub-picomolar in water, and picomolar to nanomolar levels in saliva and serum. The transistor and recognition element sensor platform can be easily expanded, allowing for multiple biomarkers to be detected simultaneously, lending itself to complex biomarker analysis applicable to rapid feedback for neuroscience research and physiological monitoring.

Keratin-based antimicrobial textiles, films, and nanofibers.

The combination of appealing structural properties, biocompatibility, and the availability of renewable and inexpensive raw materials, make keratin-based materials attractive for a variety of applications. In this paper, we report on the antimicrobial functionalization of keratin-based materials, including wool cloth and regenerated cellulose/keratin composite films and nanofibers. The functionalization of these materials was accomplished utilizing a facile chlorination reaction that converts the nitrogen-bearing moieties of keratin into halamine compounds. Halamine-charged wool cloth exhibited rapid and potent bactericidal activity against several species of bacteria and induced up to a 5.3 log (i.e., 99.9995%) reduction in the colony forming units of Bacillus thuringiensis spores within 10 min. Keratin-containing composites were prepared by the spin coating and coaxial electrospinning of extracted/oxidized alpha-keratin and cellulose acetate (CA) solubilized in formic acid, followed by CA deacetylation. Regenerated cellulose/keratin materials chlorinated to display halamines were also effective in killing Escherichia coli and Staphylococcus aureus bacteria. Electrospun core/shell nanofibers engineered to maximize keratin-Cl surface area displayed higher activity against S. aureus than films composed of the same materials. The halamine-based antimicrobial functionalization methods demonstrated for keratin-based materials in this paper are anticipated to translate to other protein biopolymers of interest to the biomaterials community.

Microelectrode array biosensor for studying carbohydrate-mediated interactions.

Carbohydrate-mediated host-pathogen interactions are essential to bacterial and viral pathogenesis, and represent an attractive target for the development of antiadhesives to prevent infection. We present a versatile microelectrode array-based platform to investigate carbohydrate-mediated protein and bacterial binding, with the objective of developing a generalizable method for screening inhibitors of host-microbe interactions. Microelectrode arrays are well suited for interrogating biological binding events, including proteins and whole-cells, and are amenable to electrochemical derivitization, facilitating rapid deposition of biomolecules. In this study, we achieve microelectrode functionalization with carbohydrates via controlled polymerization of pyrrole to individual microelectrodes, followed by physisorption of neoglycoconjugates to the polypyrrole-coated electrodes. Bioactivity of the immobilized carbohydrates was confirmed with carbohydrate-binding proteins (lectins) detected by both fluorescent and electrochemical means. The platform's ability to analyze whole-cell binding was demonstrated using strains of Escherichia coli and Salmonella enterica, and the dose-dependent inhibition of S. enterica by a soluble carbohydrate antiadhesive.

Sporicidal/bactericidal textiles via the chlorination of silk.

Bacterial spores, such as those of the Bacillus genus, are extremely resilient, being able to germinate into metabolically active cells after withstanding harsh environmental conditions or aggressive chemical treatments. The toughness of the bacterial spore in combination with the use of spores, such as those of Bacillus anthracis, as a biological warfare agent necessitates the development of new antimicrobial textiles. In this work, a route to the production of fabrics that kill bacterial spores and cells within minutes of exposure is described. Utilizing this facile process, unmodified silk cloth is reacted with a diluted bleach solution, rinsed with water, and dried. The chlorination of silk was explored under basic (pH 11) and slightly acidic (pH 5) conditions. Chloramine-silk textiles prepared in acidified bleach solutions were found to have superior breaking strength and higher oxidative Cl contents than those prepared under caustic conditions. Silk cloth chlorinated for ≥1 h at pH 5 was determined to induce >99.99996% reduction in the colony forming units of Escherichia coli, as well as Bacillus thuringiensis Al Hakam (B. anthracis simulant) spores and cells within 10 min of contact. The processing conditions presented for silk fabric in this study are highly expeditionary, allowing for the on-site production of protein-based antimicrobial materials from a variety of agriculturally produced feed-stocks.

Theophylline detection using an aptamer and DNA-gold nanoparticle conjugates.

A detection system for theophylline that combined the recognition properties of an aptamer and the plasmonic response of gold nanoparticles (AuNPs) is presented. The aptamer was used as a linker for AuNPs functionalized with complementary sequences to the aptamer (DNA-AuNPs), producing supramolecular complexes that disassemble when exposed to theophylline due to aptamer binding. The detection event was reported as a change in the AuNPs plasmonic peak and intensity. Addition of a spacer on the DNA immobilized on the AuNPs facing the aptamer binding site improved the aggregates' response, doubling the detection range of system response to theophylline. Modification of the oligonucleotides immobilized on the AuNPs that reduced the interparticle distance in the aggregated state suppressed their response to theophylline and addition of the spacer recovered it. This work demonstrated that the design of oligonucleotides immobilized on the AuNPs could be used to improve their plasmonic response without affecting aptamer performance.

Targeted deposition of antibodies on a multiplex CMOS microarray and optimization of a sensitive immunoassay using electrochemical detection.

BACKGROUND: The CombiMatrix ElectraSense microarray is a highly multiplex, complementary metal oxide semiconductor with 12,544 electrodes that are individually addressable. This platform is commercially available as a custom DNA microarray; and, in this configuration, it has also been used to tether antibodies (Abs) specifically on electrodes using complementary DNA sequences conjugated to the Abs. METHODOLOGY/PRINCIPAL FINDINGS: An empirical method is described for developing and optimizing immunoassays on the CombiMatrix ElectraSense microarray based upon targeted deposition of polypyrrole (Ppy) and capture Ab. This process was automated using instrumentation that can selectively apply a potential or current to individual electrodes and also measure current generated at the electrodes by an enzyme-enhanced electrochemical (ECD) reaction. By designating groups of electrodes on the array for different Ppy deposition conditions, we determined that the sensitivity and specificity of a sandwich immunoassay for staphylococcal enterotoxin B (SEB) is influenced by the application of different voltages or currents and the application time. The sandwich immunoassay used a capture Ab adsorbed to the Ppy and a reporter Ab labeled for fluorescence detection or ECD, and results from these methods of detection were different. CONCLUSIONS/SIGNIFICANCE: Using Ppy deposition conditions for optimum results, the lower limit of detection for SEB using the ECD assay was between 0.003 and 0.01 pg/ml, which represents an order of magnitude improvement over a conventional enzyme-linked immunosorbant assay. In the absence of understanding the variables and complexities that affect assay performance, this highly multiplexed electrode array provided a rapid, high throughput, and empirical approach for developing a sensitive immunoassay.

Use of a multiplexed CMOS microarray to optimize and compare oligonucleotide binding to DNA probes synthesized or immobilized on individual electrodes.

The CombiMatrix microarray with 12,544 electrodes supports in situ electrochemical synthesis of user-defined DNA probes. As an alternative, we immobilized commercially synthesized DNA probes on individual electrodes coated with electropolymerized polypyrrole (Ppy). Hybridization was measured using a biotinylated target oligonucleotide and either Cy5-streptavidin and fluorescence detection or horseradish peroxidase-streptavidin and enzyme-enhanced electrochemical detection. Detection efficiencies were optimized by varying the deposition of the Ppy, the terminal groups on the DNA probes, and other factors that impacted fluorescence quenching and electrical conductivity. Optimized results were compared against those obtained using a microarray with the same DNA sequences synthesized in situ. Immobilized probes produced higher fluorescence signals, possibly by providing a greater stand off between the Cy5 on the target oligonucleotide and the quenching effects of the Ppy and the platinum electrode.

Identification of upper respiratory tract pathogens using electrochemical detection on an oligonucleotide microarray.

Bacterial and viral upper respiratory infections (URI) produce highly variable clinical symptoms that cannot be used to identify the etiologic agent. Proper treatment, however, depends on correct identification of the pathogen involved as antibiotics provide little or no benefit with viral infections. Here we describe a rapid and sensitive genotyping assay and microarray for URI identification using standard amplification and hybridization techniques, with electrochemical detection (ECD) on a semiconductor-based oligonucleotide microarray. The assay was developed to detect four bacterial pathogens (Bordetella pertussis, Streptococcus pyogenes, Chlamydia pneumoniae and Mycoplasma pneumoniae) and 9 viral pathogens (adenovirus 4, coronavirus OC43, 229E and HK, influenza A and B, parainfluenza types 1, 2, and 3 and respiratory syncytial virus. This new platform forms the basis for a fully automated diagnostics system that is very flexible and can be customized to suit different or additional pathogens. Multiple probes on a flexible platform allow one to test probes empirically and then select highly reactive probes for further iterative evaluation. Because ECD uses an enzymatic reaction to create electrical signals that can be read directly from the array, there is no need for image analysis or for expensive and delicate optical scanning equipment. We show assay sensitivity and specificity that are excellent for a multiplexed format.

Use of semiconductor-based oligonucleotide microarrays for influenza a virus subtype identification and sequencing.

In the face of concerns over an influenza pandemic, identification of virulent influenza A virus isolates must be obtained quickly for effective responses. Rapid subtype identification, however, is difficult even in well-equipped virology laboratories or is unobtainable in the field under more austere conditions. Here we describe a genome assay and microarray design that can be used to rapidly identify influenza A virus hemagglutinin subtypes 1 through 15 and neuraminidase subtypes 1 through 9. Also described is an array-based enzymatic assay that can be used to sequence portions of both genes or any other sequence of interest.

Isolation and Purification of Colicin ECL 12, a Bacteriocin That Inhibits Strains of Escherichia coli O157:H7 †.

A swine fecal isolate, identified as Escherichia coli ECL12, was found to produce an antimicrobial substance designated as colicin ECL12. Colicin ECL12 was inhibitory against 20 strains of E. coli O157:H7 previously isolated from both human and bovine feces. Identification of the producer strain was determined phenotypically by biochemical and morphological tests. Colicin ECL12 was sensitive to several proteolytic enzymes. Adsorption of colicin ECL12 to sensitive cells of E. coli O157:H7 was bactericidal, resulting in a 2 log reduction in viable cell counts. Colicin ECL12 was purified from strain ECL12 by cell extraction and ion-exchange chromatography. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of colicin ECL12 resolved a single protein with a molecular weight of approximately 65,000.