Biocompatibility of Antifogging SiO-doped Diamond-Like Carbon Laparoscope Coatings.

Laparoscopes can suffer from fogging and contamination difficulties, resulting in a reduced field of view during surgery. A series of diamond-like carbon films, doped with SiO, were produced by pulsed laser deposition for evaluation as biocompatible, antifogging coatings. DLC films doped with SiO demonstrated hydrophilic properties with water contact angles under 40°. Samples subjected to plasma cleaning had improved contact angle results, with values under 5°. Doping the DLC films with SiO led to an average 40% decrease in modulus and 60% decrease in hardness. Hardness of the doped films, 12.0 - 13.2 GPa, was greater than that of the uncoated fused silica substrate, 9.2 GPa. The biocompatibility was assessed through CellTiter-Glo assays, with the films demonstrating statistically similar levels of cell viability when compared to the control media. The absence of ATP released by blood platelets in contact with the DLC coatings suggests in vivo hemocompatibility. The SiO doped films displayed improved transparency levels in comparison to undoped films, achieving up to an average of 80% transmission over the visible spectrum and an attenuation coefficient of 1.1 × 104 cm-1 at the 450 nm wavelength. The SiO doped DLC films show promise as a method of fog prevention for laparoscopes.

Shift-field refinement of macromolecular atomic models.

The aim of crystallographic structure solution is typically to determine an atomic model which accurately accounts for an observed diffraction pattern. A key step in this process is the refinement of the parameters of an initial model, which is most often determined by molecular replacement using another structure which is broadly similar to the structure of interest. In macromolecular crystallography, the resolution of the data is typically insufficient to determine the positional and uncertainty parameters for each individual atom, and so stereochemical information is used to supplement the observational data. Here, a new approach to refinement is evaluated in which a `shift field' is determined which describes changes to model parameters affecting whole regions of the model rather than individual atoms only, with the size of the affected region being a key parameter of the calculation which can be changed in accordance with the resolution of the data. It is demonstrated that this approach can improve the radius of convergence of the refinement calculation while also dramatically reducing the calculation time.

The metabolic impact of extracellular nitrite on aerobic metabolism of Paracoccus denitrificans.

Nitrite, in equilibrium with free nitrous acid (FNA), can inhibit both aerobic and anaerobic growth of microbial communities through bactericidal activities that have considerable potential for control of microbial growth in a range of water systems. There has been much focus on the effect of nitrite/FNA on anaerobic metabolism and so, to enhance understanding of the metabolic impact of nitrite/FNA on aerobic metabolism, a study was undertaken with a model denitrifying bacterium Paracoccus denitrificans PD1222. Extracellular nitrite inhibits aerobic growth of P. denitrificans in a pH-dependent manner that is likely to be a result of both nitrite and free nitrous acid (pKa = 3.25) and subsequent reactive nitrogen oxides generated from the intracellular passage of FNA into P. denitrificans. Increased expression of a gene encoding a flavohemoglobin protein (Fhp) (Pden_1689) was observed in response to extracellular nitrite. Construction and analysis of a deletion mutant established Fhp to be involved in endowing nitrite/FNA resistance at high extracellular nitrite concentrations. Global transcriptional analysis confirmed nitrite-dependent expression of fhp and indicated that P. denitrificans expressed a number of stress response systems associated with protein, DNA and lipid repair. It is therefore suggested that nitrite causes a pH-dependent stress response that is due to the production of associated reactive nitrogen species, such as nitric oxide from the internalisation of FNA.

DNA barcoding of freshwater fishes and the development of a quantitative qPCR assay for the species-specific detection and quantification of fish larvae from plankton samples.

The barcoding of mitochondrial cytochrome c oxidase subunit 1 (coI) gene was amplified and sequenced from 16 species of freshwater fishes found in Lake Wivenhoe (south-eastern Queensland, Australia) to support monitoring of reservoir fish populations, ecosystem function and water health. In this study, 630-650 bp sequences of the coI barcoding gene from 100 specimens representing 15 genera, 13 families and two subclasses of fishes allowed 14 of the 16 species to be identified and differentiated. The mean ± s.e. Kimura 2 parameter divergence within and between species was 0.52 ± 0.10 and 23.8 ± 2.20% respectively, indicating that barcodes can be used to discriminate most of the fish species accurately. The two terapontids, Amniataba percoides and Leiopotherapon unicolor, however, shared coI DNA sequences and could not be differentiated using this gene. A barcoding database was established and a qPCR assay was developed using coI sequences to identify and quantify proportional abundances of fish species in ichthyoplankton samples from Lake Wivenhoe. These methods provide a viable alternative to the time-consuming process of manually enumerating and identifying ichthyoplankton samples.

An in vitro study assessing the effect of mesh morphology and suture fixation on bacterial adherence.

PURPOSE: Prosthetic infections, although relatively uncommon in hernia surgery, are a source of considerable morbidity and cost. The aims of this experimental study were to assess the influence of the morphological properties of the mesh on bacterial adherence in vitro. The morphological properties assessed were the polymer type, filament type, filament diameter, mesh weight, mean pore size, and the addition of silver chlorhexidine and titanium coatings. In addition, the study assessed the effect on bacterial adherence of adding a commonly used suture to the mesh and compared adherence rates to self-gripping mesh that does not require suture fixation. METHODS: Eight commercially sourced flat hernia meshes with different material characteristics were included in the study. These were Prolene(®) (Ethicon(®)), DualMesh(®) (Gore(®)), DualMesh(®) Plus (Gore(®)), Parietex™ ProGrip (Covidien™), TiMesh(®) Light (GfE Medical), Bard(®) Soft Mesh (Bard(®)), Vypro(®) (Ethicon(®)), and Omyra(®) (Braun(®)). Individual meshes were inoculated with Staphylococcus epidermidis and Staphylococcus aureus with a bacterial inoculum of 10(2) bacteria. To assess the effect of suture material on bacterial adhesion, a sterile piece of commonly used monofilament suture material (2.0 Prolene(®), ZB370 Ethicon(®)) was sutured to selected meshes (chosen to represent different commonly used polymers and/or the presence of an antibacterial coating). Inoculated meshes were incubated for 18 h in tryptone soy broth and then analysed using scanning electron microscopy. A previously validated method for enumeration of bacteria using automated stage movement electron microscopy was used for direct bacterial counting. The final fraction of the bacteria adherent to the mesh was compared between the meshes and for each morphological variable. One-way analysis of variance (ANOVA) was performed on the bacterial counts. Tukey's test was used to determine the difference between the different biomaterials in the event the ANOVA was significant. RESULTS: Properties that significantly increased the mean bacterial adherence were the expanded polytetrafluoroethylene polymer (P < 0.001); multifilament meshes (P < 0.001); increased filament diameter (P < 0.001); increased mesh weight (P < 0.001); and smaller mean pore size (P < 0.001). In contrast, mesh coating with antibacterial silver chlorhexidine significantly reduced bacterial adhesion (S. epidermidis mean bacterial count 140.7 ± 19.1 SE with DualMesh(®) vs. 2.3 ± 1.2 SE with DualMesh(®) Plus, P < 0.001; S. aureus mean bacterial count 371.7 ± 22.7 SE with DualMesh(®) vs. 19.3 ± 4.7 SE with DualMesh(®) Plus, P = 0.002). The addition of 2.0 Prolene suture material significantly increased the mean number of adherent bacteria independent of the mesh polymer or mesh coating (P = 0.04 to <0.001). CONCLUSION: The present study demonstrates the significant influence of the prosthetic load on bacterial adherence. In patients at increased risk of infection, low prosthetic load materials, i.e., lightweight meshes with large pores, may be beneficial. Furthermore self-fixing meshes, which avoid increasing the prosthetic load and antibacterial impregnated meshes, may have an advantage in this setting.

Determining the mechanisms for aerobic granulation from mixed seed of floccular and crushed granules in activated sludge wastewater treatment.

Aerobic granulation is a novel and promising technology for wastewater treatment. However, long start-up periods required for the development of granules from floccular sludge, and the loss of biomass in this period leading to poor nutrient removal performance are key challenges. In a recent study the addition of crushed granules to a floccular sludge significantly reduced the start-up period, and also maintained the nutrient removal performance during granulation. In this study, we examined the mechanisms responsible for the fast granulation from a mixture of floccular and granular sludges. Fluorescent microbead particles (4 µm diameter) were successfully applied to differentially label the surfaces of floccular and crushed granular aggregates. Labelled flocs and crushed granules were added to a laboratory scale wastewater treatment reactor, and the granule formation process was monitored using confocal laser scanning microscopy over an 80 day period. Flocs were observed to attach to the surface of the seeding granules, resulting in reduced biomass washout during granulation. This mechanism not only reduces the granulation period, but also maintains the nutrient removal performance of the reactor. The results indicate that the granules acted as nuclei for floccular particle attachment, which accelerated granule formation.

Microbial community analysis during continuous fermentation of thermally hydrolysed waste activated sludge.

Acidogenic fermentation of thermally hydrolysed waste activated sludge was carried out at laboratory scale in two reactors operated under different hydraulic retention times (HRT). Process performance was assessed in terms of volatile fatty acid (VFA) composition and yield. The diversity of the microbial population was investigated by constructing a 16S rRNA gene library and subsequent phylogenetic analysis of clones. Fluorescence in situ hybridization (FISH) was used to assess the relative abundance of different bacterial groups. Bacteroidetes and Firmicutes were the dominant taxonomic groups representing 93% of the total sequences obtained in the reactor with 4 d HRT. A similar VFA yield (0.4-0.5 g VFA(COD) g SCOD(-1)) was obtained for the HRTs tested (1-4 d), indicating that extended retention times were not useful. Within Firmicutes, Clostridia was the major group detected in the clone sequences. These had close affiliation to Sporanaerobacter acetigenes, suggesting organisms of this group were important for hydrolysis of the protein fraction of the substrate. However, FISH analysis failed to detect the major portion of the bacteria, and this is most likely due to the lack of appropriate probes. This work emphasizes the diversity of fermentative communities, and indicates that more work is needed to identify and detect the important members.

Enhancing aerobic granulation for biological nutrient removal from domestic wastewater.

This study focuses on the enhancement of aerobic granulation and biological nutrient removal maintenance treating domestic wastewater. Two sequencing batch reactors (SBRs) were inoculated with either only floccular sludge (100%-floc SBR) or supplemented with 10% crushed granules (90%-floc SBR). Granules developed in both reactors. The 100%-floc SBR achieved 75% of nitrogen and 93% of phosphorus removal at the end of the performance, but some floccular sludge remained in the system. The 90%-floc SBR became fully granulated and finished with 84% and 99% of nitrogen and phosphorus removal, respectively. Regarding biological phosphorus removal, nitrite was identified as an inhibitor of the process. Nitrite levels lower than 5 mg N-NO2-L(-1) were used for anoxic phosphate uptake while higher concentrations inhibited the process.

Evidence of compositional differences between the extracellular and intracellular DNA of a granular sludge biofilm.

AIMS: Extracellular polymeric substances (EPS) are an important component of microbial biofilms, and it is becoming increasingly apparent that extracellular DNA (eDNA) has a functional role in EPS. This study characterizes the eDNA extracted from the novel activated sludge biofilm process of aerobic granules. METHODS AND RESULTS: Exposing the sludge to cation exchange resin (CER) was used for the extraction of eDNA and intracellular DNA (iDNA) from aerobic granules. This was optimized for eDNA yield while causing minimal cell lysis. We then compared the DNA composition of these extractions using randomly amplified polymorphic DNA (RAPD) fingerprinting and PCR-based denaturing gradient-gel electrophoresis (DGGE). Upon the analysis of the genomic DNA and the 16S rRNA genes, differences were detected between the sludge biofilm eDNA and iDNA. CONCLUSIONS: Different bacteria within the biofilm disproportionally release DNA into the EPS matrix of the biofilm. SIGNIFICANCE AND IMPACT OF THE STUDY: The findings further the idea that eDNA has a functional role in the biofilm state, which is an important conceptual information for industrial application of biofilms.

Monitoring associations between clade-level variation, overall community structure and ecosystem function in enhanced biological phosphorus removal (EBPR) systems using terminal-restriction fragment length polymorphism (T-RFLP).

The role of Candidatus "Accumulibacter phosphatis" (Accumulibacter) in enhanced biological phosphorus removal (EBPR) is well established but the relevance of different Accumulibacter clades to the performance of EBPR systems is unknown. We developed a terminal-restriction fragment length polymorphism (T-RFLP) technique to monitor changes in the relative abundance of key members of the bacterial community, including Accumulibacter clades, in four replicate mini-sequencing batch reactors (mSBRs) operated for EBPR over a 35-day period. The ability of the T-RFLP technique to detect trends was confirmed using fluorescence in situ hybridisation (FISH). EBPR performance varied between reactors and over time; by day 35, performance was maintained in mSBR2 whilst it had deteriorated in mSBR1. However, reproducible trends in structure-function relationships were detected in the mSBRs. EBPR performance was strongly associated with the relative abundance of total Accumulibacter. A shift in the ratio of the dominant Accumulibacter clades was also detected, with Type IA associated with good EBPR performance and Type IIC associated with poor EBPR performance. Changes in ecosystem function of the mSBRs in the early stages of the experiment were more closely associated with changes in the abundance of (unknown) members of the flanking community than of either Accumulibacter or Candidatus "Competibacter phosphatis". This study therefore reveals a hitherto unrecorded and complex relationship between Accumulibacter clades, the flanking community and ecosystem function of laboratory-scale EBPR systems.

Towards exposure of elusive metabolic mixed-culture processes: the application of metaproteomic analyses to activated sludge.

Protein expression is a direct reflection of specific microbial activities in any ecosystem. In order to assess protein expression in mixed microbial communities, the feasibility of applying proteomic techniques to activated sludge samples has recently been demonstrated. We report the application of metaproteomics to two activated sludges from a laboratory-scale sequencing batch reactor with dissimilar phosphorus removal performances. Fluorescence in situ hybridization (FISH) revealed that the sludge with good enhanced biological phosphorus removal performance (EBPR) was dominated by Betaproteobacteria (65% of EUBMIX binding cells) and gave positive signals for the Rhodocyclus-type PAO specific probe (59%). The non-EBPR sludge was dominated by tetrad-forming Alphaproteobacteria (75%). With regard to the proteomic investigation, 630 individual protein spots were matched across the replicate groups of the anaerobic and aerobic phases of the EBPR sludge with 9.4% of all spots being statistically different between the two phases. The non-EBPR metaproteomic maps exhibited 590 matched spots with 14.7% statistical differences between the two phases. Overall, the non-EBPR sludge expressed around 30% more significant differences than the EBPR sludge. The comparison of protein expression in the two sludges showed that their metaproteomes were substantially different and this was reflected in their microbial community structures and metabolic transformations.

Simulation studies of the interactions between membrane proteins and detergents.

Interactions between membrane proteins and detergents are important in biophysical and structural studies and are also biologically relevant in the context of folding and transport. Despite a paucity of high-resolution data on protein-detergent interactions, novel methods and increased computational power enable simulations to provide a means of understanding such interactions in detail. Simulations have been used to compare the effect of lipid or detergent on the structure and dynamics of membrane proteins. Moreover, some of the longest and most complex simulations to date have been used to observe the spontaneous formation of membrane protein-detergent micelles. Common mechanistic steps in the micelle self-assembly process were identified for both alpha-helical and beta-barrel membrane proteins, and a simple kinetic mechanism was proposed. Recently, simplified (i.e. coarse-grained) models have been utilized to follow long timescale transitions in membrane protein-detergent assemblies.

Molecular simulations and lipid-protein interactions: potassium channels and other membrane proteins.

Molecular dynamics simulations may be used to probe the interactions of membrane proteins with lipids and with detergents at atomic resolution. Examples of such simulations for ion channels and for bacterial outer membrane proteins are described. Comparison of simulations of KcsA (an alpha-helical bundle) and OmpA (a beta-barrel) reveals the importance of two classes of side chains in stabilizing interactions with the head groups of lipid molecules: (i) tryptophan and tyrosine; and (ii) arginine and lysine. Arginine residues interacting with lipid phosphate groups play an important role in stabilizing the voltage-sensor domain of the KvAP channel within a bilayer. Simulations of the bacterial potassium channel KcsA reveal specific interactions of phosphatidylglycerol with an acidic lipid-binding site at the interface between adjacent protein monomers. A combination of molecular modelling and simulation reveals a potential phosphatidylinositol 4,5-bisphosphate-binding site on the surface of Kir6.2.

Residential radon exposure and lung cancer risk: commentary on Cohen's county-based study.

The large United States county-based study () in which an inverse relationship has been suggested between residential low-dose radon levels and lung cancer mortality has been reviewed. While this study has been used to evaluate the validity of the linear nonthreshold theory, the grouped nature of its data limits the usefulness of this application. Our assessment of the study's approach, including a reanalysis of its data, also indicates that the likelihood of strong, undetected confounding effects by cigarette smoking, coupled with approximations of data values and uncertainties in accuracy of data sources regarding levels of radon exposure and intensity of smoking, compromises the study's analytic power. The most clear data for estimating lung cancer risk from low levels of radon exposure continue to rest with higher-dose studies of miner populations in which projections to zero dose are consistent with estimates arising from most case-control studies regarding residential exposure.

The tumescent technique to significantly reduce blood loss during burn surgery.

INTRODUCTION: Burn surgery is complicated by blood loss. The tumescent technique of subdermal injection of epinephrine has been utilized to decrease intraoperative blood loss. We hypothesized that this would safely decrease blood loss during burn surgery. METHODS: Twenty patients utilized the tumescent technique. The tumescent group had subdermal injections of epinephrine beneath the excision and donor site plus thrombin spray and warm saline soaked laparotomy pads. Ten patients grafted prior to adopting the tumescent technique utilized thrombin spray and warm saline soaked laparotomy pads for hemostasis. Blood loss was determined by operative estimation and calculation. Data were analyzed by Student's t-test and paired t-test. RESULTS: The two groups were demographically similar. The tumescent group had significantly less total blood loss and blood loss per unit area excised. There were no clinically detectable arrythmias, changes in heart rate or blood pressure noted. CONCLUSIONS: The tumescent technique significantly reduced intraoperative blood loss. It is safe, inexpensive and easy to use. The subdermal epinephrine/saline injection creates a smooth, tense surface which assists with debridement and donor harvest.

The R71G BRCA1 is a founder Spanish mutation and leads to aberrant splicing of the transcript.

In a BRCA1 screening in familial breast cancer carried out in different centres in Spain, France, and United Kingdom, a missense mutation 330A>G which results in a Arg to Gly change at codon 71 (R71G) was independently identified in 6 families, all of them with Spanish ancestors. This residue coincides with the -2 position of the exon 5 donor splice site. We further investigated the effect of this base substitution on the splicing of BRCA1 mRNA. The sequence analysis of the cDNA indicated that 22 bp of exon 5 were deleted, creating with the first bases of exon 6 a termination codon at position 64, which results in a truncated protein. The BRCA1 haplotype of the R71G carrier patients and Spanish controls was analysed by use of six microsatellites located within or near BRCA1. Our results are consistent with the possibility that these families shared a common ancestry with BRCA1 R71G being a founder mutation of Spanish origin.