Engineered Escherichia coli Silver-Binding Periplasmic Protein That Promotes Silver Tolerance

This is the published version. Copyright © 2012, American Society for Microbiology. All Rights Reserved.Silver toxicity is a problem that microorganisms face in medical and environmental settings. Through exposure to silver compounds, some bacteria have adapted to growth in high concentrations of silver ions. Such adapted microbes may be dangerous as pathogens but, alternatively, could be potentially useful in nanomaterial-manufacturing applications. While naturally adapted isolates typically utilize efflux pumps to achieve metal resistance, we have engineered a silver-tolerant Escherichia coli strain by the use of a simple silver-binding peptide motif. A silver-binding peptide, AgBP2, was identified from a combinatorial display library and fused to the C terminus of the E. coli maltose-binding protein (MBP) to yield a silver-binding protein exhibiting nanomolar affinity for the metal. Growth experiments performed in the presence of silver nitrate showed that cells secreting MBP-AgBP2 into the periplasm exhibited silver tolerance in a batch culture, while those expressing a cytoplasmic version of the fusion protein or MBP alone did not. Transmission electron microscopy analysis of silver-tolerant cells revealed the presence of electron-dense silver nanoparticles. This is the first report of a specifically engineered metal-binding peptide exhibiting a strong in vivo phenotype, pointing toward a novel ability to manipulate bacterial interactions with heavy metals by the use of short and simple peptide motifs. Engineered metal-ion-tolerant microorganisms such as this E. coli strain could potentially be used in applications ranging from remediation to interrogation of biomolecule-metal interactions in vivo

Toward Identifying the Next Generation of Superfund and Hazardous Waste Site Contaminants

Reproduced with permission from Environmental Health Perspectives."This commentary evolved from a workshop sponsored by the National Institute of Environmental Health Sciences titled "Superfund Contaminants: The Next Generation" held in Tucson, Arizona, in August 2009. All the authors were workshop participants." doi:10.1289/ehp.1002497Our aim was to initiate a dynamic, adaptable process for identifying contaminants of emerging concern (CECs) that are likely to be found in future hazardous waste sites, and to identify the gaps in primary research that cause uncertainty in determining future hazardous waste site contaminants. Superfund-relevant CECs can be characterized by specific attributes: they are persistent, bioaccumulative, toxic, occur in large quantities, and have localized accumulation with a likelihood of exposure. Although still under development and incompletely applied, methods to quantify these attributes can assist in winnowing down the list of candidates from the universe of potential CECs. Unfortunately, significant research gaps exist in detection and quantification, environmental fate and transport, health and risk assessment, and site exploration and remediation for CECs. Addressing these gaps is prerequisite to a preventive approach to generating and managing hazardous waste sites.Support for the workshop, from which this article evolved, was provided by the National Institute of Environmental Health Sciences Superfund Research Program (P42-ES04940)

Targeting Hepatitis B Virus with Zinc Finger Nucleases

Despite an existing effective vaccine, hepatitis B virus (HBV) remains a major public health concern. There are effective suppressive therapies for HBV, but they remain expensive and inaccessible to many, and not all patients respond well. Furthermore, HBV can persist as genomic covalently closed circular DNA (cccDNA) that remains in hepatocytes even during otherwise effective therapy and facilitates rebound in patients after treatment has stopped. Therefore, the need for an effective treatment that targets active and persistent HBV infections remains. As a novel approach to treat HBV, we have targeted the HBV genome for disruption to prevent viral reactivation and replication. We generated 3 zinc finger nucleases (ZFNs) that target sequences within the HBV polymerase, core and X genes. Upon the formation of ZFN-induced DNA double strand breaks (DSB), imprecise repair by non-homologous end joining leads to mutations that inactivate HBV genes. We delivered HBV-specific ZFNs using self-complementary adeno-associated virus (scAAV) vectors and tested their anti-HBV activity in HepAD38 cells. HBV-ZFNs efficiently disrupted HBV target sites by inducing site-specific mutations. Cytotoxicity was seen with one of the ZFNs. scAAV-mediated delivery of a ZFN targeting HBV polymerase resulted in complete inhibition of HBV DNA replication and production of infectious HBV virions in HepAD38 cells. This effect was sustained for at least 2 weeks following only a single treatment. Furthermore, high specificity was observed for all ZFNs, as negligible off-target cleavage was seen via high-throughput sequencing of 7 closely matched potential off-target sites. These results show that HBV-targeted ZFNs can efficiently inhibit active HBV replication and suppress the cellular template for HBV persistence, making them promising candidates for eradication therapy

The percutaneous toxicokinetics of sulphur mustard in a damaged skin porcine model and the evaluation of WoundStat™ as a topical decontaminant

This is the peer reviewed version of the following article: Charlotte A. Hall, et al, 'The percutaneous toxicokinetics of Sulphur mustard in a damaged skin porcine model and the evaluation of WoundStat™ as a topical decontaminant', Journal of Applied Toxicology, July 2017, which has been published in final form at DOI: 10.1002/jat.3453. This article may be used for non-commercial purposes in accordance with Wiley Terms and Conditions for Self-Archiving. Copyright © 2017 John Wiley & Sons, Ltd.This study used a damaged skin, porcine model to evaluate the in vivo efficacy of WoundStat™ for decontamination of superficial (non-haemorrhaging), sulphur mustard-contaminated wounds. The dorsal skin of 12 female pigs was subjected to controlled physical damage and exposed to 10 μL 14C–radiolabelled sulphur mustard (14C–SM). Animals were randomly assigned to either a control or a treatment group. In the latter, WoundStat™ was applied 30 s post exposure and left in situ for 1 h. Skin lesion progression and decontaminant efficacy were quantified over 6 h using a range of biophysical measurements. Skin, blood and organ samples were taken post mortem for histopathological assessment, 14C–SM distribution and toxicokinetic analyses. Application of SM to damaged skin without decontamination was rapidly followed by advanced signs of toxicity, including ulceration and decreased blood flow at the exposure site in all animals. WoundStat™ prevented ulceration and improved blood flow at the exposure site in all decontaminated animals (n = 6). Furthermore, significantly smaller quantities of 14C–SM were detected in the blood (45% reduction), and recovered from skin (70% reduction) and skin surface swabs (99% reduction) at 6 h post-challenge. Overall, the distribution of 14C–SM in the internal organs was similar for both groups, with the greatest concentration in the kidneys, followed by the liver and small intestine. WoundStat™ significantly reduced the amount of 14C–SM recovered from the liver, a key organ for SM metabolism and detoxification. This study demonstrates that WoundStat™ is a suitable product for reducing the ingress and toxicity of a chemical warfare agent.Peer reviewedFinal Accepted Versio

Bacterial laccases: some recent advances and applications

Laccases belong to the large family of multi-copper oxidases (MCOs) that couple the one-electron oxidation of substrates with the four-electron reduction of molecular oxygen to water. Because of their high relative non-specific oxidation capacity particularly on phenols and aromatic amines as well as the lack of requirement for expensive organic cofactors, they have found application in a large number of biotechnological fields. The vast majority of studies and applications were performed using fungal laccases, but bacterial laccases show interesting properties such as optimal temperature above 50 °C, optimal pH at the neutral to alkaline range, thermal and chemical stability and increased salt tolerance. Additionally, bacterial systems benefit from a wide range of molecular biology tools that facilitates their engineering and achievement of high yields of protein production and set-up of cost-effective bioprocesses. In this review we will provide up-to-date information on the distribution and putative physiological role of bacterial laccases and highlight their distinctive structural and biochemical properties, discuss the key role of copper in the biochemical properties, discuss thermostability determinants and, finally, review biotechnological applications with a focus on catalytic mechanisms on phenolics and aromatic amines.info:eu-repo/semantics/publishedVersio

Isolation of homologous arborvirus cultures from heterologous mixtures using limit dilution and virus-specific enzyme imunoassays

Viral cultures were identified recently that contained both Kunjin virus and the closely related flavivirus West Nile. The observation that the KUN virus population grew more efficiently in a mosquito cell line (C6/36) while the WN population replicated more effectively in mammalian cells (Vero) allowed enrichment for either virus by culturing the mixture in the appropriate cell line. Limit dilution of the enriched virus preparations was then performed by infecting microtitre cultures with serial ten fold dilutions. Culture wells that contained a pure population of virus were then identified by immunostaining fixed cell monolayers with virus-specific monoclonal antibodies. Subsequent passage of the 'cloned' viruses in either C6/36 or Vero cells and analysis of the infected cultures by specific monoclonal antibody staining, PCR and nucleotide sequencing confirmed the identity of the virus and that in each case an homogeneous virus population had been obtained. This procedure is particularly useful for isolating virus populations from heterogeneous mixtures that fail to develop discrete plaques in infected cell monolayers