David Wendell

Translocation of double-stranded DNA through membrane-adapted phi29 motor protein nanopores.

Biological pores have been used to study the transport of DNA and other molecules but most pores have channels that allow only the movement of small molecules and single-stranded DNA and RNA. The bacteriophage phi29 DNA-packaging motor, which allows double-stranded DNA to enter and exit during a viral infection, contains a connector protein that has a 3.6 – 6.0 nm wide channel. Here we show that a modified version of the connector protein, when reconstituted into liposomes and inserted into planar lipid bilayers, can act as conductive channels to allow the translocation of double-stranded DNA. Single-channel conductance assays and quantitative PCR confirmed the translocation through the pore. The measured conductance of a single connector channel was 4.8 nS in 1 M KCl. This engineered and membrane-adapted phage connector is expected to have interesting applications in nanotechnology and nanomedicine, such as MEMS sensing, microreactors, gene delivery, drug loading, and DNA sequencing.

Artificial Photosynthesis in Ranaspumin-2 Based Foam

We present a cell-free artificial photosynthesis platform that couples the requisite enzymes of the Calvin cycle with a nanoscale photophosphorylation system engineered into a foam architecture using the Tungara frog surfactant protein Ranaspumin-2. This unique protein surfactant allowed lipid vesicles and coupled enzyme activity to be concentrated to the microscale Plateau channels of the foam, directing photoderived chemical energy to the singular purpose of carbon fixation and sugar synthesis, with chemical conversion efficiencies approaching 96%.

Distribution of human-specific bacteroidales and fecal indicator bacteria in an urban watershed impacted by sewage pollution, determined using RNA- and DNA-based quantitative PCR assays.

ABSTRACT The identification of fecal pollution sources is commonly carried out using DNA-based methods. However, there is evidence that DNA can be associated with dead cells or present as “naked DNA” in the environment. Furthermore, it has been shown that rRNA-targeted reverse transcription-quantitative PCR (RT-qPCR) assays can be more sensitive than rRNA gene-based qPCR assays since metabolically active cells usually contain higher numbers of ribosomes than quiescent cells. To this end, we compared the detection frequency of host-specific markers and fecal bacteria using RNA-based RT-qPCR and DNA-based qPCR methods for water samples collected in sites impacted by combined sewer overflows. As a group, fecal bacteria were more frequently detected in most sites using RNA-based methods. Specifically, 8, 87, and 85% of the samples positive for general enterococci, Enterococcus faecalis, and Enterococcus faecium markers, respectively, were detected using RT-qPCR, but not with the qPCR assay counterpart. On average, two human-specific Bacteroidales markers were not detected when using DNA in 12% of the samples, while they were positive for all samples when using RNA (cDNA) as the template. Moreover, signal intensity was up to three orders of magnitude higher in RT-qPCR assays than in qPCR assays. The human-specific Bacteroidales markers exhibited moderate correlation with conventional fecal indicators using RT-qPCR results, suggesting the persistence of nonhuman sources of fecal pollution or the presence of false-positive signals. In general, the results from this study suggest that RNA-based assays can increase the detection sensitivity of fecal bacteria in urban watersheds impacted with human fecal sources.

Correlative Assessment of Fecal Indicators using Human Mitochondrial DNA as a Direct Marker

Identifying the source of surface water fecal contamination is paramount to mitigating pollution and risk to human health. Fecal bacteria such as E. coli have been staple indicator organisms for over a century, however there remains uncertainty with E. coli-based metrics since these bacteria are abundant in the environment. The relationships between the presence of direct indicator of human waste (human mitochondrial DNA), human-specific Bacteroidales, and E. coli were studied for water samples taken from an urban creek system (Duck Creek Watershed, Cincinnati, OH) impacted by combined sewer overflows. Logistic regression analysis shows that human-specific Bacteroidales correlates much more closely to human mitochondrial DNA (R = 0.62) relative to E. coli (R = 0.33). We also examine the speciation of Bacteroidales within the Duck Creek Watershed using next-generation sequencing technology (Ion Torrent) and show the most numerous populations to be associated with sewage. Here we demonstrate that human-specific Bacteroidales closely follow the dynamics of human mitochondrial DNA concentration changes, indicating that these obligate anaerobes are more accurate than E. coli for fecal source tracking, lending further support to risk overestimation using coliforms, especially fecal coliforms and E. coli.

Electrophysiology of single and aggregate Cx43 hemichannels.

Connexin43 (Cx43) is the most ubiquitous gap junction protein in the human body and is essential for cell-to-cell communication in a variety of organs and organ systems. As a result, Cx43 is responsible for mediating both electrical and chemical signals, passing dissolved solutes and small signaling molecules between cells in a coordinated fashion. Here, we explore the electrophysiological properties of hemichannels formed from Cx43 and Cx43 fused to eGFP (Cx43eGFP) and their interactions in a planar lipid membrane (BLM). Unlike in vivo patch clamp experiments, Cx43 was purified and isolated from other membrane constituents allowing elucidation of individual protein responses to various electrical and chemical stimuli. Using this system, we examined hemichannel electrophysiology and the roles of several well-known gap junction blockers, namely: lanthanum, heptanol, carbenoxalone and lindane. We also observed a critical number of hemichannels required for an accelerated conductance increase, an emergent electrical signature indicative of plaque formation.

Treatment of hydrophobic VOCs in trickling bed air biofilter: Emphasis on long-term effect of initial alternate use of hydrophilic VOCs and microbial species evolution

The main research objective of this study is to enhance the removal of recalcitrant compounds that are not readily bioavailable due to limiting mass transfer rate between the liquid and gas phases. Four trickle-bed air biofilters (TBABs), loaded with pelletized diatomaceous earth support media, were run at an empty bed residence time (EBRT) of 120 sec. After an acclimation period at constant loading rate (LR) of n-hexane (13.2 g m−3 hr−1) and intermittent feeding of methanol, n-hexane influent LR was then increased in step-wise fashion to 47.7 g m−3 hr−1 for biofilters receiving acidic nutrients (pH 4), and to 36.3 g m−3 hr−1 for biofilters receiving nutrient at pH 7. The results have shown that for TBABs receiving nutrient at pH 4, greater elimination capacities were obtained as compared to TBABs working at pH 7. n-Hexane removal efficiency of more than 84% at LR up to 47.7 g m−3 hr−1 was obtained for pH 4 nutrient-fed biofilters, while for biofilters with nutrients fed at pH 7, the removal efficiency did not exceed 64% for n-hexane LR of 36.3 g m−3 hr−1. The microbial analysis revealed that no fungal community was detected in TBABs run at neutral pH. The fungi communities that were initially acclimating TBABs run at pH 4, namely, Aspergillus niger and Fusarium solani, were not detected at the end of the experiment, while Gibberella moniliformis (Fusarium verticillioides) genus became the dominant species. Gibberella moniliformis (Fusarium verticillioides) was present along all the biofilter media and sustained very high n-hexane elimination at steady-state condition. Implications: With growing apprehension about sustainability and environmental protection, with limited resources available, and with the passage of the 1990 Amendments to the Clean Air Act, there is more need for using air pollution control techniques that are sound economically and proven environmentally friendly. Biofiltration systems, namely, trickle-bed air biofilters, were for decades recognized as efficient in treating air pollutants. Thus, the application of this technique over a wide industrial spectrum would certainly contribute to reduction of hazardous gas emissions.

Sequencing Human Mitochondrial Hypervariable Region II as a Molecular Fingerprint for Environmental Waters

To protect environmental water from human fecal contamination, authorities must be able to unambiguously identify the source of the contamination. Current identification methods focus on tracking fecal bacteria associated with the human gut, but many of these bacterial indicators also thrive in the environment and in other mammalian hosts. Mitochondrial DNA could solve this problem by serving as a human-specific marker for fecal contamination. Here we show that the human mitochondrial hypervariable region II can function as a molecular fingerprint for human contamination in an urban watershed impacted by combined sewer overflows. We present high-throughput sequencing analysis of hypervariable region II for spatial resolution of the contaminated sites and assessment of the population diversity of the impacting regions. We propose that human mitochondrial DNA from public waste streams may serve as a tool for identifying waste sources definitively, analyzing population diversity, and conducting other anthropological investigations.

Engineering bacterial efflux pumps for solar-powered bioremediation of surface waters.

Antibiotics are difficult to selectively remove from surface waters by present treatment methods. Bacterial efflux pumps have evolved the ability to discriminately expel antibiotics and other noxious agents via proton and ATP driven pathways. Here, we describe light-dependent removal of antibiotics by engineering the bacterial efflux pump AcrB into a proteovesicle system. We have created a chimeric protein with the requisite proton motive force by coupling AcrB to the light-driven proton pump Delta-rhodopsin (dR) via a glycophorin A transmembrane domain. This creates a solar powered protein material capable of selectively capturing antibiotics from bulk solutions. Using environmental water and direct sunlight, our AcrB-dR vesicles removed almost twice as much antibiotic as the treatment standard, activated carbon. Altogether, the AcrB-dR system provides an effective means of extracting antibiotics from surface waters as well as potential antibiotic recovery through vesicle solubilization.

Microsphere dynamics for actin based nanorobotic motility

Actin is the principle building block of the cytoskeleton. It provides eukaryotic cells with their characteristic shape, as well as the means to produce the force required for cell motility. Similarly, actin also provides the motile force behind the movement of prokaryotic organisms such as Listeria monocytogenes. Listerias movement is achieved by hijacking the cytoskeleton proteins of the eukaryotic host through the use of its transmembrane protein, ActA, a nucleation factor which interacts with the host Arp2/3 complex, VASP, and Actin. Previous investigations with ActA have included microbead motility tests in cytoplasmic extract, and a prokaryotic over-expression system in an artificial motility media consisting of several essential actin binding proteins, (ABPs), ATP, and salt. From both of these studies, actin nucleation was observed at the bead surface interface with ActA, in the form of comet tails assembled from crosslinked microfilaments. Here we report comet tail formation using ActA coated microspheres in a motility system consisting of several key ABPs. This movement is being further characterized using a microcantilever-microsphere assembly, which we are using to measure the force output of our actin-based motility system. The ultimate objective of this research is to place the actin-based motility system into polymer vesicles and create an enclosed nanorobotic system, which produces a controllable amoeboid-like movement through an externally applied electric field.

Solar photo-Fenton treatment of microcystin-LR in aqueous environment: Transformation products and toxicity in different water matrices

Transformation products and toxicity patterns of microcystin-LR (MC-LR), a common cyanotoxin in freshwaters, during degradation by solar photo-Fenton process were studied in the absence and presence of two major water components, namely fulvic acid and alkalinity. The transformation products m/z 795, 835, 515/1030 and 532 can be formed through attack of OH on the conjugated carbon double bonds of Adda. Transformation products with m/z 1010, 966 and 513 can be generated through the attack of OH on the methoxy group of Adda. The transformation products m/z 783, 508 and 1012 can be originated from the attack of OH on the cyclic structure of MC-LR. Transformation products (m/z 522, 1028, 1012, 1046 and 514) formed after hydroxylation of the aromatic ring with OH were also identified in this study. The toxicity study revealed that fulvic acid and alkalinity strongly influence the toxicity profiles of solar photo-Fenton treated MC-LR. Fulvic acid enhanced the detoxification whereas low level total alkalinity (1.8 mg L-1 CaCO3) inhibited the detoxification of MC-LR by solar photo-Fenton process as assessed by protein phosphatase-1 (PP-1) inhibition assay. This work provides insights on the utility of solar photo-Fenton destruction of MC-LR in water based on transformation products and toxicity data.