Publications by authors named "David J Evans"

200 Publications

Nerve-associated transient receptor potential ion channels can contribute to intrinsic resistance to bacterial adhesion in vivo.

FASEB J 2021 10;35(10):e21899

Vision Science Program, University of California, Berkeley, California, USA.

The cornea of the eye differs from other mucosal surfaces in that it lacks a viable bacterial microbiome and by its unusually high density of sensory nerve endings. Here, we explored the role of corneal nerves in preventing bacterial adhesion. Pharmacological and genetic methods were used to inhibit the function of corneal sensory nerves or their associated transient receptor potential cation channels TRPA1 and TRPV1. Impacts on bacterial adhesion, resident immune cells, and epithelial integrity were examined using fluorescent labeling and quantitative confocal imaging. TRPA1/TRPV1 double gene-knockout mice were more susceptible to adhesion of environmental bacteria and to that of deliberately-inoculated Pseudomonas aeruginosa. Supporting the involvement of TRPA1/TRPV1-expressing corneal nerves, P. aeruginosa adhesion was also promoted by treatment with bupivacaine, or ablation of TRPA1/TRPV1-expressing nerves using RTX. Moreover, TRPA1/TRPV1-dependent defense was abolished by enucleation which severs corneal nerves. High-resolution imaging showed normal corneal ultrastructure and surface-labeling by wheat-germ agglutinin for TRPA1/TRPV1 knockout murine corneas, and intact barrier function by absence of fluorescein staining. P. aeruginosa adhering to corneas after perturbation of nerve or TRPA1/TRPV1 function failed to penetrate the surface. Single gene-knockout mice showed roles for both TRPA1 and TRPV1, with TRPA1 more susceptible to P. aeruginosa adhesion while TRPV1 corneas instead accumulated environmental bacteria. Corneal CD45+/CD11c+ cell responses to P. aeruginosa challenge, previously shown to counter bacterial adhesion, also depended on TRPA1/TRPV1 and sensory nerves. Together, these results demonstrate roles for corneal nerves and TRPA1/TRPV1 in corneal resistance to bacterial adhesion in vivo and suggest that the mechanisms involve resident immune cell populations.
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http://dx.doi.org/10.1096/fj.202100874RDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8486357PMC
October 2021

Imprecise recombinant viruses evolve via a fitness-driven, iterative process of polymerase template-switching events.

PLoS Pathog 2021 Aug 20;17(8):e1009676. Epub 2021 Aug 20.

BSRC and School of Biology, University of St Andrews, St Andrews, United Kingdom.

Recombination is a common feature of many positive-strand RNA viruses, playing an important role in virus evolution. However, to date, there is limited understanding of the mechanisms behind the process. Utilising in vitro assays, we have previously shown that the template-switching event of recombination is a random and ubiquitous process that often leads to recombinant viruses with imprecise genomes containing sequence duplications. Subsequently, a process termed resolution, that has yet to be mechanistically studied, removes these duplicated sequences resulting in a virus population of wild type length genomes. Using defined imprecise recombinant viruses together with Oxford Nanopore and Illumina high throughput next generation sequencing technologies we have investigated the process of resolution. We show that genome resolution involves subsequent rounds of template-switching recombination with viral fitness resulting in the survival of a small subset of recombinant genomes. This alters our previously held understanding that recombination and resolution are independent steps of the process, and instead demonstrates that viruses undergo frequent and continuous recombination events over a prolonged period until the fittest viruses, predominantly those with wild type length genomes, dominate the population.
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http://dx.doi.org/10.1371/journal.ppat.1009676DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8409635PMC
August 2021

Human tear fluid modulates the Pseudomonas aeruginosa transcriptome to alter antibiotic susceptibility.

Ocul Surf 2021 Jul 28;22:94-102. Epub 2021 Jul 28.

School of Optometry, University of California, Berkeley, CA, USA; Graduate Groups in Vision Science, Microbiology, and Infectious Diseases & Immunity, University of California, Berkeley, CA, USA. Electronic address:

Purpose: Previously, we showed that tear fluid protects corneal epithelial cells against Pseudomonas aeruginosa without suppressing bacterial viability. Here, we studied how tear fluid affects bacterial gene expression.

Methods: RNA-sequencing was used to study the P. aeruginosa transcriptome after tear fluid exposure (5 h, 37 C). Outcomes were further investigated by biochemical and physiological perturbations to tear fluid and tear-like fluid (TLF) and assessment of bacterial viability following tear/TLF pretreatment and antibiotic exposure.

Results: Tear fluid deregulated ~180 P. aeruginosa genes ≥8 fold versus PBS including downregulating lasI, rhlI, qscR (quorum sensing/virulence), oprH, phoP, phoQ (antimicrobial resistance) and arnBCADTEF (polymyxin B resistance). Upregulated genes included algF (biofilm formation) and hemO (iron acquisition). qPCR confirmed tear down-regulation of oprH, phoP and phoQ. Tear fluid pre-treatment increased P. aeruginosa resistance to meropenem ~5-fold (4 μg/ml), but enhanced polymyxin B susceptibility ~180-fold (1 μg/ml), the latter activity reduced by dilution in PBS. Media containing a subset of tear components (TLF) also sensitized bacteria to polymyxin B, but only ~22.5-fold, correlating with TLF/tear fluid Ca and Mg concentrations. Accordingly, phoQ mutants were not sensitized by TLF or tear fluid. Superior activity of tear fluid versus TLF against wild-type P. aeruginosa was heat resistant but proteinase K sensitive.

Conclusion: P. aeruginosa responds to human tear fluid by upregulating genes associated with bacterial survival and adaptation. Meanwhile, tear fluid down-regulates multiple virulence-associated genes. Tears also utilize divalent cations and heat resistant/proteinase K sensitive component(s) to enhance P. aeruginosa sensitivity to polymyxin B.
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http://dx.doi.org/10.1016/j.jtos.2021.07.004DOI Listing
July 2021

First come, first served: superinfection exclusion in Deformed wing virus is dependent upon sequence identity and not the order of virus acquisition.

ISME J 2021 Jun 30. Epub 2021 Jun 30.

Biomedical Sciences Research Complex, University of St. Andrews, North Haugh, St. Andrews, UK.

Deformed wing virus (DWV) is the most important globally distributed pathogen of honey bees and, when vectored by the ectoparasite Varroa destructor, is associated with high levels of colony losses. Divergent DWV types may differ in their pathogenicity and are reported to exhibit superinfection exclusion upon sequential infections, an inevitability in a Varroa-infested colony. We used a reverse genetic approach to investigate competition and interactions between genetically distinct or related virus strains, analysing viral load over time, tissue distribution with reporter gene-expressing viruses and recombination between virus variants. Transient competition occurred irrespective of the order of virus acquisition, indicating no directionality or dominance. Over longer periods, the ability to compete with a pre-existing infection correlated with the genetic divergence of the inoculae. Genetic recombination was observed throughout the DWV genome with recombinants accounting for ~2% of the population as determined by deep sequencing. We propose that superinfection exclusion, if it occurs at all, is a consequence of a cross-reactive RNAi response to the viruses involved, explaining the lack of dominance of one virus type over another. A better understanding of the consequences of dual- and superinfection will inform development of cross-protective honey bee vaccines and landscape-scale DWV transmission and evolution.
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http://dx.doi.org/10.1038/s41396-021-01043-4DOI Listing
June 2021

Final Exon Frameshift Biallelic Variants Are Associated with Microcephalic Complex Hereditary Spastic Paraplegia.

Brain Sci 2021 May 11;11(5). Epub 2021 May 11.

College of Medicine and Health, RILD Wellcome Wolfson Centre, University of Exeter, Royal Devon & Exeter NHS Foundation Trust, Barrack Road, Exeter EX2 5DW, UK.

The hereditary spastic paraplegias (HSPs) are a large clinically heterogeneous group of genetic disorders classified as 'pure' when the cardinal feature of progressive lower limb spasticity and weakness occurs in isolation and 'complex' when associated with other clinical signs. Here, we identify a homozygous frameshift alteration occurring in the last coding exon of the protein tyrosine phosphatase type 23 () gene in an extended Palestinian family associated with autosomal recessive complex HSP. encodes a catalytically inert non-receptor protein tyrosine phosphatase that has been proposed to interact with the endosomal sorting complex required for transport (ESCRT) complex, involved in the sorting of ubiquitinated cargos for fusion with lysosomes. In view of our data, we reviewed previously published candidate pathogenic variants to clarify clinical outcomes associated with pathogenic gene variants. This determined that a number of previously proposed candidate alterations are likely benign and revealed that pathogenic biallelic alterations cause a varied clinical spectrum comprising of complex HSP associated with microcephaly, which may occur without intellectual impairment or involve more severe neurological disease. Together, these findings highlight the importance of the inclusion of the gene on HSP gene testing panels globally.
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http://dx.doi.org/10.3390/brainsci11050614DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8151426PMC
May 2021

Deformed wing virus: using reverse genetics to tackle unanswered questions about the most important viral pathogen of honey bees.

FEMS Microbiol Rev 2021 Aug;45(4)

Biomedical Sciences Research Complex, University of St Andrews, St Andrews, KY16 9ST, UK.

Deformed wing virus (DWV) is the most important viral pathogen of honey bees. It usually causes asymptomatic infections but, when vectored by the ectoparasitic mite Varroa destructor, it is responsible for the majority of overwintering colony losses globally. Although DWV was discovered four decades ago, research has been hampered by the absence of an in vitro cell culture system or the ability to culture pure stocks of the virus. The recent developments of reverse genetic systems for DWV go some way to addressing these limitations. They will allow the investigation of specific questions about strain variation, host tropism and pathogenesis to be answered, and are already being exploited to study tissue tropism and replication in Varroa and non-Apis pollinators. Three areas neatly illustrate the advances possible with reverse genetic approaches: (i) strain variation and recombination, in which reverse genetics has highlighted similarities rather than differences between virus strains; (ii) analysis of replication kinetics in both honey bees and Varroa, in studies that likely explain the near clonality of virus populations often reported; and (iii) pathogen spillover to non-Apis pollinators, using genetically tagged viruses to accurately monitor replication and infection.
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http://dx.doi.org/10.1093/femsre/fuaa070DOI Listing
August 2021

Tungsten Ligand-Based Sulfur-Atom-Transfer Catalysts: Synthesis, Characterization, Sustained Anaerobic Catalysis, and Mode of Aerial Deactivation.

Inorg Chem 2020 Dec 17;59(23):16824-16828. Epub 2020 Nov 17.

Department of Chemistry and Physics, La Trobe Institute of Molecular Sciences, La Trobe University, Melbourne, Victoria 3086, Australia.

The synthesis, properties, X-ray structures, and catalytic sulfur-atom-transfer (SAT) reactions of W(μ-S)(μ-S)(dtc)(dped) [; dtc = SCNR, where R = Me, Et, Bu, and Bn; dped = SCPh] and W(μ-S)(dtc)(dped) () are reported. These complexes represent the oxidized () and reduced () forms of anaerobic SAT catalysts operating through the bidirectional, ligand-based half-reaction (μ-S)(μ-S) ↔ (μ-S) + S. The catalysts are deactivated in air through the formation of catalytically inactive oxo complexes, (dtc)WO(μ-S)(μ-dped)W(dtc)(dped) (), prompting us to recommend that group 6 SAT activity be assessed under strictly anaerobic conditions.
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http://dx.doi.org/10.1021/acs.inorgchem.0c02915DOI Listing
December 2020

Evidence for and against deformed wing virus spillover from honey bees to bumble bees: a reverse genetic analysis.

Sci Rep 2020 10 8;10(1):16847. Epub 2020 Oct 8.

Biomedical Sciences Research Complex, University of St. Andrews, North Haugh, St. Andrews, KY16 9ST, UK.

Deformed wing virus (DWV) is a persistent pathogen of European honey bees and the major contributor to overwintering colony losses. The prevalence of DWV in honey bees has led to significant concerns about spillover of the virus to other pollinating species. Bumble bees are both a major group of wild and commercially-reared pollinators. Several studies have reported pathogen spillover of DWV from honey bees to bumble bees, but evidence of a sustained viral infection characterized by virus replication and accumulation has yet to be demonstrated. Here we investigate the infectivity and transmission of DWV in bumble bees using the buff-tailed bumble bee Bombus terrestris as a model. We apply a reverse genetics approach combined with controlled laboratory conditions to detect and monitor DWV infection. A novel reverse genetics system for three representative DWV variants, including the two master variants of DWV-type A and B-was used. Our results directly confirm DWV replication in bumble bees but also demonstrate striking resistance to infection by certain transmission routes. Bumble bees may support DWV replication but it is not clear how infection could occur under natural environmental conditions.
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http://dx.doi.org/10.1038/s41598-020-73809-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7546617PMC
October 2020

A Potential MRI Agent and an Anticancer Drug Encapsulated within CPMV Virus-Like Particles.

Comb Chem High Throughput Screen 2021 ;24(10):1557-1571

John Innes Centre, Norwich Research Park, Norwich, NR4 7UH, United Kingdom.

Background: Virus nanoparticles have been extensively studied over the past decades for theranostics applications. Viruses are well-characterized, naturally occurring nanoparticles that can be produced in high quantity with a high degree of similarity in both structure and composition.

Objectives: The plant virus Cowpea Mosaic Virus (CPMV) has been innovatively used as a nanoscaffold. Utilization of the internal cavity of empty Virus-Like Particles (VLPs) for the inclusion of therapeutics within the capsid has opened many opportunities in drug delivery and imaging applications.

Methods: The encapsidation of magnetic materials and anticancer drugs was achieved. CPMV denotes molecules attached to the external surface of CPMV and CPMV denotes molecules within the interior of the capsid.

Results: Here, the generation of novel VLPs incorporating iron-platinum nanoparticles CPMV and cisplatin (Cis) (CPMV) is reported. CPMV exhibited a cytotoxic IC of CPMV on both A549 and MDA-MB-231 cell lines of 1.8 μM and 3.9 μM, respectively after 72 hours of incubation. The CPMV were prepared as potential MRI contrast agents.

Conclusion: Cisplatin loaded VLP (CPMV) is shown to enhance cisplatin cytotoxicity in cancer cell lines with its potency increased by 2.3-folds.
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http://dx.doi.org/10.2174/1386207323666200914110012DOI Listing
January 2021

Resistance of the murine cornea to bacterial colonization during experimental dry eye.

PLoS One 2020 29;15(5):e0234013. Epub 2020 May 29.

Vision Science Program, University of California, Berkeley, CA, United States of America.

The healthy cornea is remarkably resistant to infection, quickly clearing deliberately inoculated bacteria such as Pseudomonas aeruginosa and Staphylococcus aureus. Contrasting with the adjacent conjunctiva and other body surfaces, it also lacks a resident viable bacterial microbiome. Corneal resistance to microbes depends on intrinsic defenses involving tear fluid and the corneal epithelium. Dry eye, an ocular surface disease associated with discomfort and inflammation, can alter tear fluid composition and volume, and impact epithelial integrity. We previously showed that experimentally-induced dry eye (EDE) in mice does not increase corneal susceptibility to P. aeruginosa infection. Here, we explored if EDE alters corneal resistance to bacterial colonization. EDE was established in mice using scopolamine injections and dehumidified air-flow, and verified by phenol-red thread testing after 5 and 10 days. As expected, EDE corneas showed increased fluorescein staining versus controls consistent with compromised epithelial barrier function. Confocal imaging using mT/mG knock-in mice with red-fluorescent membranes revealed no other obvious morphological differences between EDE corneas and controls for epithelium, stroma, and endothelium. EDE corneas were imaged ex vivo and compared to controls after alkyne-functionalized D-alanine labeling of metabolically-active colonizing bacteria, or by FISH using a universal 16S rRNA gene probe. Both methods revealed very few viable bacteria on EDE corneas after 5 or 10 days (median of 0, upper quartile of ≤ 1 bacteria per field of view for each group [9-12 eyes per group]) similar to control corneas. Furthermore, there was no obvious difference in abundance of conjunctival bacteria, which included previously reported filamentous forms. Thus, despite reduced tear flow and apparent compromise to corneal barrier function (fluorescein staining), EDE murine corneas continue to resist bacterial colonization and maintain the absence of a resident viable bacterial microbiome.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0234013PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7259750PMC
August 2020

Green Bees: Reverse Genetic Analysis of Deformed Wing Virus Transmission, Replication, and Tropism.

Viruses 2020 05 12;12(5). Epub 2020 May 12.

Biomedical Sciences Research Complex, University of St. Andrews, St. Andrews KY16 9ST, UK.

Environmental and agricultural pollination services by honey bees, , and honey production are compromised by high levels of annual colony losses globally. The majority are associated with disease caused by deformed wing virus (DWV), a positive-strand RNA virus, exacerbated by the ectoparasitic mite . To improve honey bee health, a better understanding of virus transmission and pathogenesis is needed which requires the development of tools to study virus replication, transmission, and localisation. We report the use of reverse genetic (RG) systems for the predominant genetically distinct variants of DWV to address these questions. All RG-recovered viruses replicate within 24 h post-inoculation of pupae and could recapitulate the characteristic symptoms of DWV disease upon eclosion. Larvae were significantly less susceptible but could be infected orally and subsequently developed disease. Using genetically tagged RG DWV and an feeding system, we demonstrate virus replication in the mite by accumulation of tagged negative-strand viral replication intermediates. We additionally apply a modified DWV genome expressing a fluorescent reporter protein for direct observation of virus distribution in injected pupae or fed larvae. Using this, we demonstrate extensive sites of virus replication in a range of pupal tissues and organs and in the nascent wing buds in larvae fed high levels of virus, indicative of a direct association between virus replication and pathogenesis. These studies provide insights into virus replication kinetics, tropism, transmission, and pathogenesis, and produce new tools to help develop the understanding needed to control DWV-mediated colony losses.
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http://dx.doi.org/10.3390/v12050532DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7291132PMC
May 2020

Chronic bee paralysis as a serious emerging threat to honey bees.

Nat Commun 2020 05 1;11(1):2164. Epub 2020 May 1.

School of Natural and Environmental Sciences, Newcastle University, Newcastle upon Tyne, Tyne and Wear, NE1 7RU, UK.

Chronic bee paralysis is a well-defined viral disease of honey bees with a global distribution that until recently caused rare but severe symptomatology including colony loss. Anecdotal evidence indicates a recent increase in virus incidence in several countries, but no mention of concomitant disease. We use government honey bee health inspection records from England and Wales to test whether chronic bee paralysis is an emerging infectious disease and investigate the spatiotemporal patterns of disease. The number of chronic bee paralysis cases increased exponentially between 2007 and 2017, demonstrating chronic bee paralysis as an emergent disease. Disease is highly clustered spatially within most years, suggesting local spread, but not between years, suggesting disease burnt out with periodic reintroduction. Apiary and county level risk factors are confirmed to include scale of beekeeping operation and the history of honey bee imports. Our findings offer epidemiological insight into this damaging emerging disease.
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http://dx.doi.org/10.1038/s41467-020-15919-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7195492PMC
May 2020

Diffuse lamellar keratitis associated with tabletop autoclave biofilms: case series and review.

J Cataract Refract Surg 2020 Mar;46(3):340-349

From the Department of Ophthalmology (Sorenson), Alta Bates Summit Medical Center, and School of Optometry (Sorenson, Tran, Evans, Lin), University of California, Berkeley, California, USA; Department of Ophthalmology (Holland), University of British Columbia, British Columbia, Vancouver, Canada; College of Pharmacy (Evans), Touro University California, Vallejo, Vision Science Graduate Group (Lin), University of California, Berkeley, California, and Moran Eye Center (Mamalis), University of Utah Medical Center, Salt Lake City, Utah, and Altos Eye Physicians (Chang), Los Altos, California, USA.

Purpose: To report a diffuse lamellar keratitis (DLK) cluster attributed to autoclave reservoir biofilm and to review the risk and prevention of DLK and toxic anterior segment syndrome (TASS) caused by such biofilms.

Setting: Refractive Surgery Center, University of California, Berkeley.

Design: Observational case-control study and review of literature.

Methods: Eyes were evaluated for DLK following laser in situ keratomileusis (LASIK) over a 5-year period. Multiple changes in surgical and operating room protocols were prompted by a cluster of DLK cases. The autoclave reservoir chamber wall was cultured for microbial contamination. The MEDLINE database was used to identify relevant past publications.

Results: From January 7, 2010, to December 18, 2014, 1115 eyes received LASIK. Between September 2, 2010, and June 11, 2012, 147 eyes of 395 LASIK cases developed DLK (37.2%). Systematic modifications in surgical protocols were unsuccessful in ending the prolonged cluster of DLK cases until the STATIM 2000 autoclave was replaced with a new STATIM autoclave and a reservoir sterilization and surveillance protocol implemented. Over the subsequent 30 months, DLK incidence was reduced to 2.2% (14 DLK cases from 632 total LASIK cases, P < .0001). The retired autoclave reservoir chamber wall cultures grew Pseudomonas aeruginosa and the Burkholderia cepacia complex.

Conclusions: Fluid reservoirs of tabletop steam autoclaves can readily develop polymicrobial biofilms harboring microbial pathogens, whose inert molecular byproducts can cause DLK and TASS when introduced to the eye by surgical instruments. Stringent reservoir cleaning and maintenance may significantly reduce this risk by preventing and removing these biofilms.
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http://dx.doi.org/10.1097/j.jcrs.0000000000000070DOI Listing
March 2020

Contact lens-related corneal infection: Intrinsic resistance and its compromise.

Prog Retin Eye Res 2020 05 20;76:100804. Epub 2019 Nov 20.

School of Optometry, University of California, Berkeley, CA, USA; College of Pharmacy, Touro University California, Vallejo, CA, USA.

Contact lenses represent a widely utilized form of vision correction with more than 140 million wearers worldwide. Although generally well-tolerated, contact lenses can cause corneal infection (microbial keratitis), with an approximate annualized incidence ranging from ~2 to ~20 cases per 10,000 wearers, and sometimes resulting in permanent vision loss. Research suggests that the pathogenesis of contact lens-associated microbial keratitis is complex and multifactorial, likely requiring multiple conspiring factors that compromise the intrinsic resistance of a healthy cornea to infection. Here, we outline our perspective of the mechanisms by which contact lens wear sometimes renders the cornea susceptible to infection, focusing primarily on our own research efforts during the past three decades. This has included studies of host factors underlying the constitutive barrier function of the healthy cornea, its response to bacterial challenge when intrinsic resistance is not compromised, pathogen virulence mechanisms, and the effects of contact lens wear that alter the outcome of host-microbe interactions. For almost all of this work, we have utilized the bacterium Pseudomonas aeruginosa because it is the leading cause of lens-related microbial keratitis. While not yet common among corneal isolates, clinical isolates of P. aeruginosa have emerged that are resistant to virtually all currently available antibiotics, leading the United States CDC (Centers for Disease Control) to add P. aeruginosa to its list of most serious threats. Compounding this concern, the development of advanced contact lenses for biosensing and augmented reality, together with the escalating incidence of myopia, could portent an epidemic of vision-threatening corneal infections in the future. Thankfully, technological advances in genomics, proteomics, metabolomics and imaging combined with emerging models of contact lens-associated P. aeruginosa infection hold promise for solving the problem - and possibly life-threatening infections impacting other tissues.
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http://dx.doi.org/10.1016/j.preteyeres.2019.100804DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7237316PMC
May 2020

Characterization of Historical Methane Occurrence Frequencies from U.S. Underground Natural Gas Storage Facilities with Implications for Risk Management, Operations, and Regulatory Policy.

Risk Anal 2020 03 5;40(3):588-607. Epub 2019 Nov 5.

ProbabilityManagement.org, Stanford, CA, USA.

Defining a baseline for the frequency of occurrences at underground natural gas storage facilities is critical to maintaining safe operation and to the development of appropriate risk management plans and regulatory approaches. Currently used frequency-estimation methods are reviewed and broadened in this article to include critical factors of cause, severity, and uncertainty that contribute to risk. A Bayesian probabilistic analysis characterizes the aleatoric historical occurrence frequencies given imperfect sampling. Frequencies for the three main storage facility types in the United States (depleted oil-and-gas field storage, aquifer storage, solution-mined salt cavern storage) are generally on the order of 3 to 9 × 10 occurrences, of all causes (surface, well integrity, subsurface integrity) and severities (nuisance, serious, catastrophic), per facility-year. Loss of well integrity is associated with many, but not all, occurrences either within the subsurface or from there up to the surface. The probability of one serious or catastrophic leakage occurrence to the ground surface within the next 10 years, assuming constant number of facilities, is approximately 0.1-0.3% for any facility type. Storage operators and industry regulators can use occurrence frequencies, their associated probabilities and uncertainties, and forecasts of severity magnitudes to better prioritize resources, establish a baseline against which progress toward achieving a reduction target could be measured, and develop more effective mitigation/monitoring/reduction programs in a risk management plan.
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http://dx.doi.org/10.1111/risa.13417DOI Listing
March 2020

Gold-coated plant virus as computed tomography imaging contrast agent.

Beilstein J Nanotechnol 2019 7;10:1983-1993. Epub 2019 Oct 7.

John Innes Centre, Norwich Research Park, Norwich, UK.

Chemical modification of the surface of viruses, both the interior and the exterior, imparts new functionalities, that have potential applications in nanomedicine. In this study, we developed novel virus-based nanomaterials as a contrast agent for computed tomography (CT) imaging in vitro. The gold-coated cowpea mosaic virus (Au-CPMV) particles were generated by the electrostatic adsorption of positively charged electrolyte on the virus capsid with the subsequent incubation and reduction of anionic gold complexes. Au-CPMV particles as a CT contrast agent offer a fast scan time (less than 2 min), low cost, and biocompatibility and allow for high-resolution imaging with ca. 150 Hounsfield units (HU). The Au-CPMV surface was further modified allowing for the incorporation of targeting molecules of specific cell types.
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http://dx.doi.org/10.3762/bjnano.10.195DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6808194PMC
October 2019

DMBT1 inhibition of Pseudomonas aeruginosa twitching motility involves its N-glycosylation and cannot be conferred by the Scavenger Receptor Cysteine-Rich bacteria-binding peptide domain.

Sci Rep 2019 09 11;9(1):13146. Epub 2019 Sep 11.

School of Optometry, University of California, Berkeley, CA, USA.

The scavenging capacity of glycoprotein DMBT1 helps defend mucosal epithelia against microbes. DMBT1 binding to multiple bacterial species involves its conserved Scavenger Receptor Cysteine-Rich (SRCR) domains, localized to a 16-mer consensus sequence peptide, SRCRP2. Previously, we showed that DMBT1 bound Pseudomonas aeruginosa pili, and inhibited twitching motility, a pilus-mediated movement important for virulence. Here, we determined molecular characteristics required for twitching motility inhibition. Heat-denatured DMBT1 lost capacity to inhibit twitching motility and showed reduced pili binding (~40%). Size-exclusion chromatography of Lys-C-digested native DMBT1 showed that only high-Mw fractions retained activity, suggesting involvement of the N-terminal containing repeated SRCR domains with glycosylated SRCR-Interspersed Domains (SIDs). However, individual or pooled consensus sequence peptides (SRCRPs 1 to 7) showed no activity and did not bind P. aeruginosa pili; nor did recombinant DMBT1 (aa 1-220) or another SRCR-rich glycoprotein, CD163. Enzymatic de-N-glycosylation of DMBT1, but not de-O-glycosylation, reduced its capacity to inhibit twitching motility (~57%), without reducing pili binding. Therefore, DMBT1 inhibition of P. aeruginosa twitching motility involves its N-glycosylation, its pili-binding capacity is insufficient, and it cannot be conferred by the SRCR bacteria-binding peptide domain, either alone or mixed with other unlinked SRCRPs, suggesting an additional mechanism for DMBT1-mediated mucosal defense.
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http://dx.doi.org/10.1038/s41598-019-49543-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6739395PMC
September 2019

Type IV Pili Can Mediate Bacterial Motility within Epithelial Cells.

mBio 2019 08 20;10(4). Epub 2019 Aug 20.

School of Optometry, University of California, Berkeley, California, USA

is among bacterial pathogens capable of twitching motility, a form of surface-associated movement dependent on type IV pili (T4P). Previously, we showed that T4P and twitching were required for to cause disease in a murine model of corneal infection, to traverse human corneal epithelial multilayers, and to efficiently exit invaded epithelial cells. Here, we used live wide-field fluorescent imaging combined with quantitative image analysis to explore how twitching contributes to epithelial cell egress. Results using time-lapse imaging of cells infected with wild-type PAO1 showed that cytoplasmic bacteria slowly disseminated throughout the cytosol at a median speed of >0.05 μm s while dividing intracellularly. Similar results were obtained with flagellin () and flagellum assembly () mutants, thereby excluding swimming, swarming, and sliding as mechanisms. In contrast, mutants (lacking T4P) and mutants (twitching motility defective) appeared stationary and accumulated in expanding aggregates during intracellular division. Transmission electron microscopy confirmed that these mutants were not trapped within membrane-bound cytosolic compartments. For the wild type, dissemination in the cytosol was not prevented by the depolymerization of actin filaments using latrunculin A and/or the disruption of microtubules using nocodazole. Together, these findings illustrate a novel form of intracellular bacterial motility differing from previously described mechanisms in being directly driven by bacterial motility appendages (T4P) and not depending on polymerized host actin or microtubules. Host cell invasion can contribute to disease pathogenesis by the opportunistic pathogen Previously, we showed that the type III secretion system (T3SS) of invasive strains modulates cell entry and subsequent escape from vacuolar trafficking to host lysosomes. However, we also showed that mutants lacking either type IV pili (T4P) or T4P-dependent twitching motility (i) were defective in traversing cell multilayers, (ii) caused less pathology , and (iii) had a reduced capacity to exit invaded cells. Here, we report that after vacuolar escape, intracellular can use T4P-dependent twitching motility to disseminate throughout the host cell cytoplasm. We further show that this strategy for intracellular dissemination does not depend on flagellin and resists both host actin and host microtubule disruption. This differs from mechanisms used by previously studied pathogens that utilize either host actin or microtubules for intracellular dissemination independently of microbe motility appendages.
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http://dx.doi.org/10.1128/mBio.02880-18DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6703432PMC
August 2019

Seneca Valley Virus Exploits TEM8, a Collagen Receptor Implicated in Tumor Growth.

Front Oncol 2018 6;8:506. Epub 2018 Nov 6.

Department of Chemistry, Wichita State University, Wichita, KS, United States.

Recent studies reveal that Seneca Valley Virus (SVV) exploits tumor endothelial marker 8 (TEM8) for cellular entry, the same surface receptor pirated by bacterial-derived anthrax toxin. This observation is particularly significant as SVV is a known oncolytic virus which selectively infects and kills tumor cells, particularly those of neuroendocrine origin. TEM8 is a transmembrane glycoprotein that is preferentially upregulated in some tumor cell and tumor-associated stromal cell populations. Both TEM8 and SVV have been evaluated for targeting of tumors of multiple origins, but the connection between the two was previously unknown. Here, we review currently understood interactions between TEM8 and SVV, anthrax protective antigen (PA), and collagen VI, a native binding partner of TEM8, with an emphasis on potential therapeutic directions moving forward.
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http://dx.doi.org/10.3389/fonc.2018.00506DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6232524PMC
November 2018

A novel murine model for contact lens wear reveals clandestine IL-1R dependent corneal parainflammation and susceptibility to microbial keratitis upon inoculation with Pseudomonas aeruginosa.

Ocul Surf 2019 01 12;17(1):119-133. Epub 2018 Nov 12.

School of Optometry, University of California, Berkeley, CA, 94720, USA; Graduate Group in Vision Science, University of California, Berkeley, CA, 94720, USA; Graduate Groups in Microbiology, Infectious Diseases & Immunity, University of California, Berkeley, CA, 94720, USA. Electronic address:

Purpose: Contact lens wear carries a risk of complications, including corneal infection. Solving these complications has been hindered by limitations of existing animal models. Here, we report development of a new murine model of contact lens wear.

Methods: C57BL/6 mice were fitted with custom-made silicone-hydrogel contact lenses with or without prior inoculation with Pseudomonas aeruginosa (PAO1-GFP). Contralateral eyes served as controls. Corneas were monitored for pathology, and examined ex vivo using high-magnification, time-lapse imaging. Fluorescent reporter mice allowed visualization of host cell membranes and immune cells. Lens-colonizing bacteria were detected by viable counts and FISH. Direct-colony PCR was used for bacterial identification.

Results: Without deliberate inoculation, lens-wearing corneas remained free of visible pathology, and retained a clarity similar to non-lens wearing controls. CD11c-YFP reporter mice revealed altered numbers, and distribution, of CD11c-positive cells in lens-wearing corneas after 24 h. Worn lenses showed bacterial colonization, primarily by known conjunctival or skin commensals. Corneal epithelial cells showed vacuolization during lens wear, and after 5 days, cells with phagocyte morphology appeared in the stroma that actively migrated over resident keratocytes that showed altered morphology. Immunofluorescence confirmed stromal Ly6G-positive cells after 5 days of lens wear, but not in MyD88 or IL-1R gene-knockout mice. P. aeruginosa-contaminated lenses caused infectious pathology in most mice from 1 to 13 days.

Conclusions: This murine model of contact lens wear appears to faithfully mimic events occurring during human lens wear, and could be valuable for experiments, not possible in humans, that help solve the pathogenesis of lens-related complications.
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http://dx.doi.org/10.1016/j.jtos.2018.11.006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6365008PMC
January 2019

Structural and functional analysis of the roles of the HCV 5' NCR miR122-dependent long-range association and SLVI in genome translation and replication.

PeerJ 2018 6;6:e5870. Epub 2018 Nov 6.

BSRC and School of Biology, University of St Andrews, St Andrews, UK.

The hepatitis C virus RNA genome possesses a variety of conserved structural elements, in both coding and non-coding regions, that are important for viral replication. These elements are known or predicted to modulate key life cycle events, such as translation and genome replication, some involving conformational changes induced by long-range RNA-RNA interactions. One such element is SLVI, a stem-loop (SL) structure located towards the 5' end of the core protein-coding region. This element forms an alternative RNA-RNA interaction with complementary sequences in the 5' untranslated regions that are independently involved in the binding of the cellular microRNA 122 (miR122). The switch between 'open' and 'closed' structures involving SLVI has previously been proposed to modulate translation, with lower translation efficiency associated with the 'closed' conformation. In the current study, we have used selective 2'-hydroxyl acylation analysed by primer extension to validate this RNA-RNA interaction in the absence and presence of miR122. We show that the long-range association (LRA) only forms in the absence of miR122, or otherwise requires the blocking of miR122 binding combined with substantial disruption of SLVI. Using site-directed mutations introduced to promote open or closed conformations of the LRA we demonstrate no correlation between the conformation and the translation phenotype. In addition, we observed no influence on virus replication compared to unmodified genomes. The presence of SLVI is well-documented to suppress translation, but these studies demonstrate that this is not due to its contribution to the LRA. We conclude that, although there are roles for SLVI in translation, the LRA is not a riboswitch regulating the translation and replication phenotypes of the virus.
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http://dx.doi.org/10.7717/peerj.5870DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6225842PMC
November 2018

Impact of topical corticosteroid pretreatment on susceptibility of the injured murine cornea to Pseudomonas aeruginosa colonization and infection.

Exp Eye Res 2019 02 19;179:1-7. Epub 2018 Oct 19.

School of Optometry, University of California, Berkeley, CA, USA; Vision Science Program, University of California, Berkeley, CA, USA; Graduate Groups in Microbiology, And Infectious Diseases & Immunity, University of California, Berkeley, CA, USA. Electronic address:

Research with animal models of Pseudomonas aeruginosa keratitis has shown that use of a topical corticosteroid alone against an established infection can significantly increase the number of colonizing bacteria or worsen clinical disease. Moreover, retrospective analysis has suggested that corticosteroid use in humans is associated with an increased risk of keratitis in eyes with pre-existing disease. Thus, while corticosteroids are often used to reduce ocular inflammation in the absence of infection, the risk of opportunistic infection remains a concern. However, the effect of corticosteroids on the intrinsic barrier function of uninfected corneas is unknown. Here, we tested if short-term topical corticosteroid treatment of an uninfected murine cornea would increase susceptibility to P. aeruginosa colonization or infection after epithelial injury. Topical prednisolone acetate (1%) was administered to one eye of C57BL/6 mice three times a day for 3 days; control eyes were treated with sterile PBS. Prior to inoculation with a cytotoxic P. aeruginosa corneal isolate strain 6206, corneas were subject to superficial-injury by tissue paper blotting, or scratch-injured followed by 12 h of healing. Previously we have shown that blotting renders mouse corneas susceptible to P. aeruginosa adhesion, but not infection, while 12 h healing reduces susceptibility to infection after scratching. Corneas were evaluated at 48 h for bacterial colonization and microbial keratitis (MK). To monitor impact on wound healing, corneal integrity was examined by fluorescein staining immediately after scarification and after 12 h healing. For both the tissue paper blotting and scratch-injury models, there was no significant difference in P. aeruginosa colonization at 48 h between corticosteroid-pretreated eyes and controls. With the blotting model, one case of MK was observed in a control (PBS-pretreated) cornea; none in corticosteroid-pretreated corneas. With the 12 h healing model, MK occurred in 6 of 17 corticosteroid-pretreated eyes versus 2 of 17 controls, a difference not statistically significant. Corticosteroid-pretreated eyes showed greater fluorescein staining 12 h after scarification injury, but this did not coincide with increased colonization or MK. Together, these data show that short-term topical corticosteroid therapy on an uninfected murine cornea does not necessarily enhance its susceptibility to P. aeruginosa colonization or infection after injury, even when it induces fluorescein staining.
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http://dx.doi.org/10.1016/j.exer.2018.10.010DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6360113PMC
February 2019

Mechanisms and consequences of positive-strand RNA virus recombination.

J Gen Virol 2018 10 29;99(10):1345-1356. Epub 2018 Aug 29.

Biomedical Sciences Research Complex and School of Biology, University of St Andrews, St Andrews, UK.

Genetic recombination in positive-strand RNA viruses is a significant evolutionary mechanism that drives the creation of viral diversity by the formation of novel chimaeric genomes. The process and its consequences, for example the generation of viruses with novel phenotypes, has historically been studied by analysis of the end products. More recently, with an appreciation that there are both replicative and non-replicative mechanisms at work, and with new approaches and techniques to analyse intermediate products, the viral and cellular factors that influence the process are becoming understood. The major influence on replicative recombination is the fidelity of viral polymerase, although RNA structures and sequences may also have an impact. In replicative recombination the viral polymerase is necessary and sufficient, although roles for other viral or cellular proteins may exist. In contrast, non-replicative recombination appears to be mediated solely by cellular components. Despite these insights, the relative importance of replicative and non-replicative mechanisms is not clear. Using single-stranded positive-sense RNA viruses as exemplars, we review the current state of understanding of the processes and consequences of recombination.
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http://dx.doi.org/10.1099/jgv.0.001142DOI Listing
October 2018

IL-1R and MyD88 Contribute to the Absence of a Bacterial Microbiome on the Healthy Murine Cornea.

Front Microbiol 2018 29;9:1117. Epub 2018 May 29.

School of Optometry, University of California, Berkeley, Berkeley, CA, United States.

Microbial communities are important for the health of mucosal tissues. Traditional culture and gene sequencing have demonstrated bacterial populations on the conjunctiva. However, it remains unclear if the cornea, a transparent tissue critical for vision, also hosts a microbiome. Corneas of wild-type, IL-1R (-/-) and MyD88 (-/-) C57BL/6 mice were imaged after labeling with alkyne-functionalized D-alanine (alkDala), a probe that only incorporates into the peptidoglycan of metabolically active bacteria. Fluorescence hybridization (FISH) was also used to detect viable bacteria. AlkDala labeling was rarely observed on healthy corneas. In contrast, adjacent conjunctivae harbored filamentous alkDala-positive forms, that also labeled with DMN-Tre, a Corynebacterineae-specific probe. FISH confirmed the absence of viable bacteria on healthy corneas, which also cleared deliberately inoculated bacteria within 24 h. Differing from wild-type, both IL-1R (-/-) and MyD88 (-/-) corneas harbored numerous alkDala-labeled bacteria, a result abrogated by topical antibiotics. IL-1R (-/-) corneas were impermeable to fluorescein suggesting that bacterial colonization did not reflect decreased epithelial integrity. Thus, in contrast to the conjunctiva and other mucosal surfaces, healthy murine corneas host very few viable bacteria, and this constitutive state requires the IL-1R and MyD88. While this study cannot exclude the presence of fungi, viruses, or non-viable or dormant bacteria, the data suggest that healthy murine corneas do not host a resident viable bacterial community, or microbiome, the absence of which could have important implications for understanding the homeostasis of this tissue.
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http://dx.doi.org/10.3389/fmicb.2018.01117DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5986933PMC
May 2018

Internal Deposition of Cobalt Metal and Iron Oxide Within CPMV eVLPs.

Methods Mol Biol 2018 ;1776:189-201

John Innes Centre, Norwich Research Park, Norwich, UK.

Empty (containing no genomic material) CPMV virus-like particles are loaded within the virus capsid with metal or metal oxide. Metal ions are allowed to diffuse through pores in the capsid surface and are reduced or hydrolyzed to metallic nanoparticles. The external surface of the virus-like particles remains amenable to further chemical modification.
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http://dx.doi.org/10.1007/978-1-4939-7808-3_12DOI Listing
February 2019

The Impact of ExoS on Internalization by Epithelial Cells Is Independent of and Correlates with Bistability of Type Three Secretion System Gene Expression.

mBio 2018 05 1;9(3). Epub 2018 May 1.

School of Optometry, University of California, Berkeley, Berkeley, California, USA

is internalized into multiple types of epithelial cell and and yet is often regarded as an exclusively extracellular pathogen. Paradoxically, ExoS, a type three secretion system (T3SS) effector, has antiphagocytic activities but is required for intracellular survival of and its occupation of bleb niches in epithelial cells. Here, we addressed mechanisms for this dichotomy using invasive (ExoS-expressing) and corresponding effector-null isogenic T3SS mutants, effector-null mutants of cytotoxic with and without ExoS transformation, antibiotic exclusion assays, and imaging using a T3SS-GFP reporter. Except for effector-null PA103, all strains were internalized while encoding ExoS. Intracellular bacteria showed T3SS activation that continued in replicating daughter cells. Correcting the mutation in effector-null PA103 promoted internalization by >10-fold with or without ExoS. Conversely, mutating in PAO1 reduced internalization by >10-fold, also with or without ExoS. Effector-null PA103 remained less well internalized than PAO1 matched for status, but only with ExoS expression, suggesting additional differences between these strains. Quantifying T3SS activation using GFP fluorescence and quantitative reverse transcription-PCR (qRT-PCR) showed that T3SS expression was hyperinducible for strain PA103Δ versus other isolates and was unrelated to status. These findings support the principle that is not exclusively an extracellular pathogen, with internalization influenced by the relative proportions of T3SS-positive and T3SS-negative bacteria in the population during host cell interaction. These data also challenge current thinking about T3SS effector delivery into host cells and suggest that T3SS bistability is an important consideration in studying pathogenesis. is often referred to as an extracellular pathogen, despite its demonstrated capacity to invade and survive within host cells. Fueling the confusion, encodes T3SS effectors with anti-internalization activity that, paradoxically, play critical roles in intracellular survival. Here, we sought to address why ExoS does not prevent internalization of the strains that natively encode it. Results showed that ExoS exerted unusually strong anti-internalization activity under conditions of expression in the effector-null background of strain PA103, often used to study T3SS effector activity. Inhibition of internalization was associated with T3SS hyperinducibility and ExoS delivery. PA103 mutation, preventing flagellar assembly, further reduced internalization but did so independently of ExoS. The results revealed intracellular T3SS expression by all strains and suggested that T3SS bistability influences internalization. These findings reconcile controversies in the literature surrounding internalization and support the principle that is not exclusively an extracellular pathogen.
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http://dx.doi.org/10.1128/mBio.00668-18DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5930308PMC
May 2018

Models for aerobic carbon monoxide dehydrogenase: synthesis, characterization and reactivity of paramagnetic MoO(μ-S)Cu complexes.

Chem Sci 2018 Jan 20;9(4):876-888. Epub 2017 Nov 20.

Department of Chemistry and Physics , La Trobe Institute for Molecular Science , La Trobe University , Melbourne , Victoria 3086 , Australia . Email:

Reaction of [CoCp][TpMoOS(OAr)] [Cp = η-cyclopentadienyl; Tp = hydrotris(3-isopropylpyrazol-1-yl)borate; OAr = phenolate or derivative thereof] with [Cu(NCMe)(Metcn)]BF (Metcn = 1,4,7-trimethyl-1,4,7-triazacyclononane) in MeCN at -30 °C results in the formation of red-brown/black, paramagnetic, μ-sulfido-Mo(v)/Cu(i) complexes, TpMoO(OAr)(μ-S)Cu(Metcn). The complexes possess the MoO(μ-S)Cu core found in aerobic carbon monoxide dehydrogenases (CODHs) and exhibit X-band EPR spectra closely related to those of semi-reduced CODH, with ∼ 1.937, hyperfine coupling to Mo ( = 39-42 × 10 cm) and strong superhyperfine coupling to Cu ( = 34-63 × 10 cm). Anisotropic spectra exhibit monoclinic symmetry with ∼ 1.996, ∼ 1.944 and ∼ 1.882, and nearly isotropic values (75-90 × 10 cm). The X-ray structures of four derivatives (Ar = Ph, CH Bu-2, CHBu-2, CHPh-4) are reported and discussed along with that of the Ar = CH Bu-3,5 derivative (communicated in C. Gourlay, D. J. Nielsen, J. M. White, S. Z. Knottenbelt, M. L. Kirk and C. G. Young, , 2006, , 2164). The complexes exhibit distorted octahedral oxo-Mo(v) and distorted tetrahedral Cu(i) centres bridged by a single bent μ-sulfido ligand, with Mo-S and Cu-S distances and Mo-S-Cu angles in the ranges 2.262-2.300 Å, 2.111-2.134 Å and 115.87-134.27°, respectively. The 2 -butyl derivative adopts a unique phenolate conformation with O 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 Mo-O-Cα and O 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 Mo-S-Cu torsion angles of 92.7 and 21.1°, respectively, very different from those of the other structurally characterized derivatives (31-47 and 33-45°, respectively) and exhibits a relatively short Mo···Cu distance [3.752(2) Å 3.806(7)-4.040(2) Å]. As well, the value of this complex (34.3 × 10 cm) is much lower than the values observed for other members of the series (55-63 × 10 cm), supporting the hypothesis that the electronic structure of the MoO(μ-S)Cu core unit and the degree of intermetallic communication are strongly dependent on the geometry of the MoO(OR)(μ-S)Cu unit. The complexes participate in an electrochemically reversible Mo(vi)/Mo(v) redox couple and react with cyanide undergoing decupration and desulfurization reactions of the type observed for CODH.
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http://dx.doi.org/10.1039/c7sc04239fDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5873225PMC
January 2018

Synthesis of Gold Nanoparticles Using Leaf Extract of Ziziphus zizyphus and their Antimicrobial Activity.

Nanomaterials (Basel) 2018 Mar 19;8(3). Epub 2018 Mar 19.

John Innes Centre, Norwich Research Park, Norwich NR4 7UH, UK.

(1) Background: There is a growing need for the development of new methods for the synthesis of nanoparticles. The interest in such particles has raised concerns about the environmental safety of their production methods; (2) Objectives: The current methods of nanoparticle production are often expensive and employ chemicals that are potentially harmful to the environment, which calls for the development of "greener" protocols. Herein we describe the synthesis of gold nanoparticles (AuNPs) using plant extracts, which offers an alternative, efficient, inexpensive, and environmentally friendly method to produce well-defined geometries of nanoparticles; (3) Methods: The phytochemicals present in the aqueous leaf extract acted as an effective reducing agent. The generated AuNPs were characterized by Transmission electron microscopy (TEM), Scanning electron microscope (SEM), and Atomic Force microscopy (AFM), X-ray diffraction (XRD), UV-visible spectroscopy, energy dispersive X-ray (EDX), and thermogravimetric analyses (TGA); (4) Results and Conclusions: The prepared nanoparticles were found to be biocompatible and exhibited no antimicrobial or antifungal effect, deeming the particles safe for various applications in nanomedicine. TGA analysis revealed that biomolecules, which were present in the plant extract, capped the nanoparticles and acted as stabilizing agents.
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http://dx.doi.org/10.3390/nano8030174DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5869665PMC
March 2018

Macrolide antibiotics for bronchiectasis.

Cochrane Database Syst Rev 2018 03 15;3:CD012406. Epub 2018 Mar 15.

Faculty of Health and Social Care, Edge Hill University, Ormskirk, UK.

Background: Bronchiectasis is a chronic respiratory disease characterised by abnormal and irreversible dilatation and distortion of the smaller airways. Bacterial colonisation of the damaged airways leads to chronic cough and sputum production, often with breathlessness and further structural damage to the airways. Long-term macrolide antibiotic therapy may suppress bacterial infection and reduce inflammation, leading to fewer exacerbations, fewer symptoms, improved lung function, and improved quality of life. Further evidence is required on the efficacy of macrolides in terms of specific bacterial eradication and the extent of antibiotic resistance.

Objectives: To determine the impact of macrolide antibiotics in the treatment of adults and children with bronchiectasis.

Search Methods: We identified trials from the Cochrane Airways Trials Register, which contains studies identified through multiple electronic searches and handsearches of other sources. We also searched trial registries and reference lists of primary studies. We conducted all searches on 18 January 2018.

Selection Criteria: We included randomised controlled trials (RCTs) of at least four weeks' duration that compared macrolide antibiotics with placebo or no intervention for the long-term management of stable bronchiectasis in adults or children with a diagnosis of bronchiectasis by bronchography, plain film chest radiograph, or high-resolution computed tomography. We excluded studies in which participants had received continuous or high-dose antibiotics immediately before enrolment or before a diagnosis of cystic fibrosis, sarcoidosis, or allergic bronchopulmonary aspergillosis. Our primary outcomes were exacerbation, hospitalisation, and serious adverse events.

Data Collection And Analysis: Two review authors independently screened the titles and abstracts of 103 records. We independently screened the full text of 40 study reports and included 15 trials from 30 reports. Two review authors independently extracted outcome data and assessed risk of bias for each study. We analysed dichotomous data as odds ratios (ORs) and continuous data as mean differences (MDs) or standardised mean differences (SMDs). We used standard methodological procedures as expected by Cochrane.

Main Results: We included 14 parallel-group RCTs and one cross-over RCT with interventions lasting from 8 weeks to 24 months. Of 11 adult studies with 690 participants, six used azithromycin, four roxithromycin, and one erythromycin. Four studies with 190 children used either azithromycin, clarithromycin, erythromycin, or roxithromycin.We included nine adult studies in our comparison between macrolides and placebo and two in our comparison with no intervention. We included one study with children in our comparison between macrolides and placebo and one in our comparison with no intervention.In adults, macrolides reduced exacerbation frequency to a greater extent than placebo (OR 0.34, 95% confidence interval (CI) 0.22 to 0.54; 341 participants; three studies; I = 65%; moderate-quality evidence). This translates to a number needed to treat for an additional beneficial outcome of 4 (95% CI 3 to 8). Data show no differences in exacerbation frequency between use of macrolides (OR 0.31, 95% CI 0.08 to 1.15; 43 participants; one study; moderate-quality evidence) and no intervention. Macrolides were also associated with a significantly better quality of life compared with placebo (MD -8.90, 95% CI -13.13 to -4.67; 68 participants; one study; moderate-quality evidence). We found no evidence of a reduction in hospitalisations (OR 0.56, 95% CI 0.19 to 1.62; 151 participants; two studies; I = 0%; low-quality evidence), in the number of participants with serious adverse events, including pneumonia, respiratory and non-respiratory infections, haemoptysis, and gastroenteritis (OR 0.49, 95% CI 0.20 to 1.23; 326 participants; three studies; I = 0%; low-quality evidence), or in the number experiencing adverse events (OR 0.83, 95% CI 0.51 to 1.35; 435 participants; five studies; I = 28%) in adults with macrolides compared with placebo.In children, there were no differences in exacerbation frequency (OR 0.40, 95% CI 0.11 to 1.41; 89 children; one study; low-quality evidence); hospitalisations (OR 0.28, 95% CI 0.07 to 1.11; 89 children; one study; low-quality evidence), serious adverse events, defined within the study as exacerbations of bronchiectasis or investigations related to bronchiectasis (OR 0.43, 95% CI 0.17 to 1.05; 89 children; one study; low-quality evidence), or adverse events (OR 0.78, 95% CI 0.33 to 1.83; 89 children; one study), in those receiving macrolides compared to placebo. The same study reported an increase in macrolide-resistant bacteria (OR 7.13, 95% CI 2.13 to 23.79; 89 children; one study), an increase in resistance to Streptococcus pneumoniae (OR 13.20, 95% CI 1.61 to 108.19; 89 children; one study), and an increase in resistance to Staphylococcus aureus (OR 4.16, 95% CI 1.06 to 16.32; 89 children; one study) with macrolides compared with placebo. Quality of life was not reported in the studies with children.

Authors' Conclusions: Long-term macrolide therapy may reduce the frequency of exacerbations and improve quality of life, although supporting evidence is derived mainly from studies of azithromycin, rather than other macrolides, and predominantly among adults rather than children. However, macrolides should be used with caution, as limited data indicate an associated increase in microbial resistance. Macrolides are associated with increased risk of cardiovascular death and other serious adverse events in other populations, and available data cannot exclude a similar risk among patients with bronchiectasis.
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http://dx.doi.org/10.1002/14651858.CD012406.pub2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6494352PMC
March 2018
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