Molecular typing of enteroviruses: comparing 5'UTR, VP1 and whole genome sequencing methods.

Enteroviruses (EV) commonly cause hand, foot and mouth disease (HFMD), and can also cause potentially fatal neurological and systemic complications. In our laboratory, sequencing 5' untranslated region (UTR) of the viral genome has been the routine method of genotyping EVs. During a recent localised outbreak of aseptic meningitis, sequencing the 5'UTR identified the causative virus as EV-A71, which did not fit with the clinical syndrome or illness severity. When genotyped using a different target gene, VP1, the result was different. This led us to evaluate the accuracy of the two different target genome regions and compare them against whole genome sequencing (WGS). We aimed to optimise the algorithm for detection and characterisation of EVs in the diagnostic laboratory. We hypothesised that VP1 and WGS genotyping would provide different results than 5'UTR in a subset of samples. Clinical samples from around New South Wales which were positive for EV by commercial polymerase chain reaction (PCR) assays were genotyped by targeting three different viral genome regions: the 5'UTR, VP1 and WGS. Sequencing was performed by Sanger and next generation sequencing. The subtyping results were compared. Of the 74/118 (63%) samples that were successfully typed using both the 5'UTR and the VP1 method, the EV typing result was identical for 46/74 (62%) samples compared to WGS as the gold standard. The same EV group but different EV types were found in 22/74 (30%) samples, and 6/74 (8%) samples belonged to different EV groups depending on typing method used. Genotyping with WGS and VP1 is more accurate than 5'UTR. Genotyping by the 5'UTR method is very sensitive, but less specific.

RAD18, WRNIP1 and ATMIN promote ATM signalling in response to replication stress.

The DNA replication machinery invariably encounters obstacles that slow replication fork progression, and threaten to prevent complete replication and faithful segregation of sister chromatids. The resulting replication stress activates ATR, the major kinase involved in resolving impaired DNA replication. In addition, replication stress also activates the related kinase ATM, which is required to prevent mitotic segregation errors. However, the molecular mechanism of ATM activation by replication stress is not defined. Here, we show that monoubiquitinated Proliferating Cell Nuclear Antigen (PCNA), a marker of stalled replication forks, interacts with the ATM cofactor ATMIN via WRN-interacting protein 1 (WRNIP1). ATMIN, WRNIP1 and RAD18, the E3 ligase responsible for PCNA monoubiquitination, are specifically required for ATM signalling and 53BP1 focus formation induced by replication stress, not ionising radiation. Thus, WRNIP1 connects PCNA monoubiquitination with ATMIN/ATM to activate ATM signalling in response to replication stress and contribute to the maintenance of genomic stability.

SETD2 loss-of-function promotes renal cancer branched evolution through replication stress and impaired DNA repair.

Defining mechanisms that generate intratumour heterogeneity and branched evolution may inspire novel therapeutic approaches to limit tumour diversity and adaptation. SETD2 (Su(var), Enhancer of zeste, Trithorax-domain containing 2) trimethylates histone-3 lysine-36 (H3K36me3) at sites of active transcription and is mutated in diverse tumour types, including clear cell renal carcinomas (ccRCCs). Distinct SETD2 mutations have been identified in spatially separated regions in ccRCC, indicative of intratumour heterogeneity. In this study, we have addressed the consequences of SETD2 loss-of-function through an integrated bioinformatics and functional genomics approach. We find that bi-allelic SETD2 aberrations are not associated with microsatellite instability in ccRCC. SETD2 depletion in ccRCC cells revealed aberrant and reduced nucleosome compaction and chromatin association of the key replication proteins minichromosome maintenance complex component (MCM7) and DNA polymerase δ hindering replication fork progression, and failure to load lens epithelium-derived growth factor and the Rad51 homologous recombination repair factor at DNA breaks. Consistent with these data, we observe chromosomal breakpoint locations are biased away from H3K36me3 sites in SETD2 wild-type ccRCCs relative to tumours with bi-allelic SETD2 aberrations and that H3K36me3-negative ccRCCs display elevated DNA damage in vivo. These data suggest a role for SETD2 in maintaining genome integrity through nucleosome stabilization, suppression of replication stress and the coordination of DNA repair.

Optimization of microalgal productivity using an adaptive, non-linear model based strategy.

The optimization of biomass and oil productivities in heterotrophic cultures of Auxenochlorella protothecoides was achieved using a non-linear model-based approach. A 10-fold increase in the average biomass productivity, and a 16-fold increase in the maximum productivity, was observed with respect to batch cultures as a result of the proposed optimization strategy. Final cell density in the optimized culture was 144 g/L (dry weight), with 49.4%w/w oil content. Maximum lipid productivity was 20.16 g/L d, achieved during the exponential growth phase at an average cell density of 86 g/L. Lipid productivity in the optimized microalgal culture was higher than previously reported values for other oleaginous microorganisms. Oil composition analysis showed that the oil has a high quality as biodiesel precursor. The higher productivity and excellent lipid profile of the optimized microalgal culture make A. protothecoides an exceptional source for biodiesel production and a potential source of single cell oil for other applications.

Studies to further investigate the inhibition of human liver microsomal CYP2C8 by the acyl-ß-glucuronide of gemfibrozil.

In previous studies, gemfibrozil acyl-ß-glucuronide, but not gemfibrozil, was found to be a mechanism-based inhibitor of cytochrome P450 2C8. To better understand whether this inhibition is specific for gemfibrozil acyl-ß-glucuronide or whether other glucuronide conjugates are potential substrates for inhibition of this enzyme, we evaluated several pharmaceutical compounds (as their acyl glucuronides) as direct-acting and metabolism-dependent inhibitors of CYP2C8 in human liver microsomes. Of 11 compounds that were evaluated as their acyl glucuronide conjugates, only gemfibrozil acyl-ß-glucuronide exhibited mechanism-based inhibition, indicating that CYP2C8 mechanism-based inhibition is very specific to certain glucuronide conjugates. Structural analogs of gemfibrozil were synthesized, and their glucuronide conjugates were prepared to further examine the mechanism of inhibition. When the aromatic methyl groups on the gemfibrozil moiety were substituted with trifluoromethyls, the resulting glucuronide conjugate was a weaker inhibitor of CYP2C8 and mechanism-based inhibition was abolished. However, the glucuronide conjugates of monomethyl gemfibrozil analogs were mechanism-based inhibitors of CYP2C8, although not as potent as gemfibrozil acyl-ß-glucuronide itself. The ortho-monomethyl analog was a more potent inhibitor than the meta-monomethyl analog, indicating that CYP2C8 favors the ortho position for oxidation and potential inhibition. Molecular modeling of gemfibrozil acyl-ß-glucuronide in the CYP2C8 active site is consistent with the ortho-methyl position being the favored site of covalent attachment to the heme. Moreover, hydrogen bonding to four residues (Ser100, Ser103, Gln214, and Asn217) is implicated.

The dynamics of heterotrophic algal cultures.

In this work, the time varying characteristics of microalgal cultures are investigated. Microalgae are a promising source of biofuels and other valuable chemicals; a better understanding of their dynamic behavior is, however, required to facilitate process scale-up, optimization and control. Growth and oil production rates are evaluated as a function of carbon and nitrogen sources concentration. It is found that nitrogen has a major role in controlling the productivity of microalgae. Moreover, it is shown that there exists a nitrogen source concentration at which biomass and oil production can be maximized. A mathematical model that describes the effect of nitrogen and carbon source on growth and oil production is proposed. The model considers the uncoupling between nutrient uptake and growth, a characteristic of algal cells. Validity of the proposed model is tested on fed-batch cultures.

Nanostructure, dissolution and morphology characteristics of microcidal silver films deposited by magnetron sputtering.

Novel microcidal silver films for burn dressings have been produced by magnetron sputtering. The nanostructure and dissolution characteristics of these films exhibiting antimicrobial behavior were studied as a function of the process conditions, namely, gas composition, gas pressure and input power, using transmission electron microscopy (TEM), high-resolution scanning electron microscopy, X-ray photoelectron spectroscopy (XPS) and resistivity. TEM revealed that bioactive films were nanocrystalline, with a grain size of the order of 15nm and the presence of twins. Surface morphology studies before and after dissolution suggested that bioactive films released silver at therapeutic levels in the form of nanoparticles or grains. Chemical species identification with XPS showed that the biologically active films were metallic in nature. The importance of oxygen in the sputtering environment, the resultant nanostructure and presence of twins are discussed to explain the unique antimicrobial properties of these silver films.

Impact of heat on nanocrystalline silver dressings. Part I: Chemical and biological properties.

Thermal stability of heat-treated nanocrystalline silver dressings was investigated using chemical techniques and biological assays. Dressings were heat-treated for 24h at temperatures from 23 to 110 degrees C. Bactericidal efficacy of heat-treated dressings was measured using a log reduction assay, while antibacterial longevity was determined via plate-to-plate transfer corrected zone of inhibition assays. Over the temperature range tested, biological activity dropped from excellent to negligible. Biological longevity results showed that controlled release properties of the dressings were significantly reduced by heat treatments above 75 degrees C. These data illustrate nanocrystalline silver sensitivity to heat. Further, it was clear that dressing efficacy is determined by total available soluble silver, not total silver in the dressing. It was determined that the quantity of soluble silver decreased significantly with increased heat treatment temperatures. These results should be considered in developing new nanocrystalline drug delivery systems.

Impact of heat on nanocrystalline silver dressings. Part II: Physical properties.

This work explores the effects of elevated temperature on the physical and chemical properties of nanocrystalline silver, and relates it to previously observed thermally induced changes in biological activity [Taylor PL et al. Biomaterials, in press, doi:10.1016/j.biomaterials.2005.05.040]. Microstructural evolution of nanocrystalline silver dressings, heat-treated for 24 h at temperatures from 23 to 110 degrees C, was studied in detail using X-ray diffraction (XRD), scanning electron microscopy (SEM), and X-ray photoelectron spectroscopy (XPS). These analyses indicated that silver nanocrystalline coatings undergo significant changes in structure when exposed to elevated temperature. XRD analysis showed a rapid increase in crystallite size above 75 degrees C along with decomposition of crystalline silver oxide (Ag2O) at the onset of crystallite growth. SEM imaging showed a loss of fine features and sintering of the structure at elevated temperatures. The XPS data indicated that silver-oxygen bonds disappeared completely, with the initial decomposition occurring between 23 and 37 degrees C, and total oxygen in the coating decreased from 16-17% to 6.5% over the temperature range of 75-110 degrees C. A comparison of these results to the data of Taylor et al. [Biomaterials, in press, doi:10.1016/j.biomaterials.2005.05.040] indicates that the unique biological properties of nanocrystalline silver are related to its nanostructure. This should guide future development of therapeutic nanocrystalline silver delivery systems.

Healing of porcine donor sites covered with silver-coated dressings.

OBJECTIVE: To compare rates of healing of donor sites in pigs between those dressed with silver-coated dressings and those dressed with petrolatum-impregnated absorbent gauze. DESIGN: Open study with each animal acting as its own control. SETTING: University research facility, Canada. ANIMALS: 6 young specific-pathogen-free domestic pigs. INTERVENTIONS: A total of 72 wounds about 1 cm x 2 cm x 0.4 mm were made in rows of eight on each pig with a dermatome. They were divided into three groups of 24, and dressed with petrolatum gauze, or silver-coated dressings moistened with sterile water either once only or daily for 10 days. All dressings were secured in place with an elastic bandage. MAIN OUTCOME MEASURES: Erythema, infection, epidermal migration, and healing. RESULTS: Wounds dressed with moistened silver-coated dressings re-epithelialised significantly more quickly. This resulted in complete re-epithelialisation within 70% of the time taken by those wounds dressed with petrolatum gauze. CONCLUSION: Silver-coated dressings provide a moist environment for the healing wound combined with an effective antimicrobial agent, and this significantly accelerates healing compared with wounds dressed with traditional petrolatum gauze dressings.

Efficacy of topical silver against fungal burn wound pathogens.

BACKGROUND: Fungal infections of burn wounds have become an important cause of burn-associated morbidity and mortality. The nature of fungal infections dictates aggressive treatment to minimize the morbidity associated with these infections. Persons with large total body surface area burns are particularly susceptible to fungal infections and are treated in such a manner as to minimize their risk of infection. METHODS: This study examined the in vitro fungicidal efficacy of a variety of different topical agents. By placing fungal inocula in contact with mafenide acetate, silver nitrate, silver sulfadiazine, and a nanocrystalline silver-coated dressing, we determined the kill kinetics of these topical agents against a spectrum of common burn wound fungal pathogens. RESULTS: The topical antimicrobials that were tested demonstrated varying degrees of efficacy against these pathogens. CONCLUSION: The nanocrystalline silver-based dressing provided the fastest and broadest-spectrum fungicidal activity and may make it a good candidate for use to minimize the potential of fungal infection, thereby reducing complications that delay wound healing.

Comparative evaluation of the antimicrobial activity of ACTICOAT antimicrobial barrier dressing.

This study evaluated the antimicrobial activity of ACTICOAT Antimicrobial Barrier Dressing (Westaim Biomedical Corp, Fort Saskatchewan, Alberta, Canada), a silver-coated wound dressing, and compared it with silver nitrate, silver sulfadiazine, and mafenide acetate. The minimum inhibitory concentrations (MIC), minimum bactericidal concentrations (MBC), zone of inhibition, and killing curves were determined with 5 clinically relevant bacteria. The data indicate that ACTICOAT silver had the lowest MIC and MBC and generated similar zones of inhibition to silver nitrate and silver sulfadiazine. Viable bacteria were undetectable 30 minutes after inoculation with the dressing, whereas it took 2 to 4 hours for silver nitrate and silver sulfadazine to achieve the same result. Mafenide acetate generated the biggest zones of inhibition, but it had the highest MICs and MBCs, and a significant number of bacteria still survived after 6 hours of treatment. The results suggest that ACTICOAT Antimicrobial Barrier Dressing has better antimicrobial performance than either of the existing silver-based products. ACTICOAT dressing killed the bacteria that were tested much faster, which is a very important characteristic for a wound dressing acting as a barrier to invasive infection to have. The study also suggests that a single susceptibility test such as a MIC or zone of inhibition test does not provide a comprehensive profile of antimicrobial activity of a topical antimicrobial agent or dressing. A combination of tests is desirable.

Wound management in an era of increasing bacterial antibiotic resistance: a role for topical silver treatment.

BACKGROUND: Antibiotic-resistant bacteria represent an increasing concern in wound infections. Wound colonization with these organisms normally results in aggressive management of the wound complicated by a greatly limited choice of therapeutic antibiotics. Silver and other noble metals are recognized as potential allies in combating these organisms in wounds. METHODS: Three types of topical silver applications were tested to determine their bactericidal efficacies against clinical isolates of antibiotic-resistant organisms. The silver-based applications represent 3 methods of applying silver to wounds: as a liquid (silver nitrate), incorporated in a cream (silver sulfadiazine) and as a dressing coating (silver-coated dressings). The reduction in the viable bacterial population recovered from test articles after exposure to silver provided a comparative measure of the bactericidal efficacies of these silver applications. RESULTS: All of the products demonstrated an ability to reduce the number of viable bacteria. However, the methods varied in their efficacy against antibiotic-resistant bacteria, with the silver-coated dressing being the most efficacious and silver nitrate the least efficacious. CONCLUSIONS: Silver was demonstrated to be effective at killing the antibiotic-resistant strains tested. The silver-coated dressing was particularly rapid at killing the tested bacteria and was effective against a broader range of bacteria. Silver may be a useful prophylactic or therapeutic agent for the prevention of wound colonization by organisms that impede healing, including antibiotic-resistant bacteria.

A matched-pair, randomized study evaluating the efficacy and safety of Acticoat silver-coated dressing for the treatment of burn wounds.

A new silver-coating technology was developed to prevent wound adhesion, limit nosocomial infection, control bacterial growth, and facilitate burn wound care through a silver-coated dressing material. For the purposes of this article, Acticoat (Westaim Biomedical Inc, Fort Saskatchawan, Alberta, Canada) silver-coated dressing was used. After in vitro and in vivo studies, a randomized, prospective clinical study was performed to assess the efficacy and ease of use of Acticoat dressing as compared with the efficacy and ease of our institution's standard burn wound care. Thirty burn patients with symmetric wounds were randomized to be treated with either 0.5% silver nitrate solution or Acticoat silver-coated dressing. The dressing was evaluated on the basis of overall patient comfort, ease of use for the wound care provider, and level of antimicrobial effectiveness. Wound pain was rated by the patient using a visual analog scale during dressing removal, application, and 2 hours after application. Ease of use was rated by the nurse providing wound care. Antimicrobial effectiveness was evaluated by quantitative burn wound biopsies performed before and at the end of treatment. Patients found dressing removal less painful with Acticoat than with silver nitrate, but they found the pain to be comparable during application and 2 hours after application. According to the nurses, there was no statistically significant difference in the ease of use. The frequency of burn wound sepsis (> 10(5) organisms per gram of tissue) was less in Acticoat-treated wounds than in those treated with silver nitrate (5 vs 16). Secondary bacteremias arising from infected burn wounds were also less frequent with Acticoat than with silver nitrate-treated wounds (1 vs 5). Acticoat dressing offers a new form of dressing for the burn wound, but it requires further investigation with greater numbers of patients in a larger number of centers and in different phases of burn wound care.

Visual detection of protein adsorption onto electrochemically oxidized aluminum surfaces.

Adsorption of proteins onto electrochemically oxidized aluminum surfaces by a simple visual observation was investigated. For this purpose, Ta and then A1 were sputtered onto glass slides (Al/Ta/glass slides). Al/Ta/glass slides were electrochemically oxidized in 0.4 M H3PO4 under the potentiostatic conditions. After the application of aqueous solutions of bovine, rabbit or human immunoglobulin onto the solid Al2O3 surfaces, a change in colour was monitored visually. It was found that all investigated proteins could successfully be adsorbed onto oxidized Al surfaces. This was manifested by a change in colour of the surface from tan to a purple or blue, depending on the concentration of proteins, coating time and degree of oxidation of the Al layer. Most importantly, when an aqueous solution of human IgG was applied on an anti-human IgG coated surface, a change in colour was also observed indicating that the adsorption process did not denature the molecular recognition sites. This type of antibody-antigen reaction was confirmed on the example of anti-human prothrombin--human prothrombin. It is believed that this technology may be useful in developing immunosensors for a variety of applications.

Human responses to bacterial endotoxin.

The literature concerning human responses to bacterial endotoxin, in particular to the purified lipopolysaccharide derivative, has been critically reviewed and summarized. Papers selected for review are those that reported studies of human subjects in a normal state of health administered defined doses of either intravenous or aerosol inhaled material. Emphasis was placed on cardiovascular, pulmonary, metabolic, and inflammatory parameters. The information detailed here can be applied to understanding the pathophysiology of human consequences to spontaneous, clinical diseases induced by this highly inflammatory and ubiquitous substance.