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Background This analysis of the IMPACT study assessed the cardiovascular (CV) safety of single-inhaler triple therapy with fluticasone furoate/umeclidinium/vilanterol (FF/UMEC/VI) versus FF/VI and UMEC/VI dual therapy. Methods IMPACT was a 52-week, randomized, double-blind, multicenter Phase III study comparing the efficacy and safety of FF/UMEC/VI 100/62.5/25 mcg with FF/VI 100/25 mcg or UMEC/VI 62.5/25 mcg in patients ≥40 years of age with symptomatic chronic obstructive pulmonary disease (COPD) and ≥1 moderate/severe exacerbation in the previous year. The inclusion criteria for the study were intentionally designed to permit the enrollment of patients with significant concurrent CV disease/risk. CV safety assessments included proportion of patients with and exposure-adjusted rates of on-treatment CV adverse events of special interest (CVAESI) and major adverse cardiac events (MACE), as well as time-to-first (TTF) CVAESI, and TTF CVAESI resulting in hospitalization/prolonged hospitalization or death. Results Baseline CV risk factors were similar across treatment groups. Overall, 68% of patients (n = 7012) had ≥1 CV risk factor and 40% (n = 4127) had ≥2. At baseline, 29% of patients reported a current/past cardiac disorder and 58% reported a current/past vascular disorder. The proportion of patients with on-treatment CVAESI was 11% for both FF/UMEC/VI and UMEC/VI, and 10% for FF/VI. There was no statistical difference for FF/UMEC/VI versus FF/VI or UMEC/VI in TTF CVAESI (hazard ratio [HR]: 0.98, 95% confidence interval [CI]: 0.85, 1.11; p = 0.711 and HR: 0.92, 95% CI: 0.78, 1.08; p = 0.317, respectively) nor TTF CVAESI leading to hospitalization/prolonged hospitalization or death (HR: 1.19, 95% CI: 0.93, 1.51; p = 0.167 and HR: 0.96, 95% CI: 0.72, 1.27; p = 0.760, respectively). On-treatment MACE occurred in ≤3% of patients across treatment groups, with similar prevalence and rates between treatments. Conclusions In a symptomatic COPD population with a history of exacerbations and a high rate of CV disease/risk, the proportion of patients with CVAESI and MACE was 10–11% and 1–3%, respectively, across treatment arms, and the risk of CVAESI was low and similar across treatment arms. There was no statistically significant increased CV risk associated with the use of FF/UMEC/VI versus FF/VI or UMEC/VI, and UMEC/VI versus FF/VI. Trial registration NCT02164513 (GSK study number CTT116855).

Here we report electron-microprobe data and unit-cell parameters for jinshajiangite, ideally NaBaFe2+4Ti2(Si2O7)2O2(OH)2F, from a new locality: the Oktyabr'skii massif in the coastal area of the Azov Sea, Donetsk region, Ukraine. Chemical analysis by electron microprobe gave Nb2O5 1.59, ZrO2 0.61, TiO2 17.07, SiO2 27.60, Al2O3 0.08, Fe2O3 2.04, FeO 16.42, BaO 9.81, ZnO 0.76, MnO 12.97, CaO 1.82, MgO 0.07, K2O 2.05, Na2O 2.51, F 2.48, H2O 1.92, O = F –1.04, sum 98.76 wt.%; H2O was determined in accord with the required number of monovalent anions for the Ti-dominant perraultite-type minerals: OH + F = 3 pfu; the Fe3+/Fe2+ ratio was assigned in accord with Mössbauer-spectroscopy results for jinshajiangite from a different locality. The empirical formula calculated on the basis of 19 (O + F) is (Na0.71Ca0.28□0.01)Σ1(Ba0.56K0.38□0.06)Σ1(Fe2+1.99Mn1.59Fe3+0.22Zn0.08Mg0.02Al0.01□0.09)Σ4 (Ti1.86Nb0.10Zr0.04)Σ2(Si4.00O14)O2[(OH)1.86F0.14]Σ2F1.00, Z = 4. Unit-cell parameters from the single-crystal data were determined by least-squares refinement of 9807 reflections with I > 10σI and are as follows: a = 10.726(8), b = 13.834(10), c = 11.065(8) Å, α = 108.172(5), β = 99.251(7), γ = 90.00(1)°, V = 1537.5(3.4) Å3, space group C .

A structure hierarchy is developed for chain-, ribbon- and tube-silicate based on the connectedness of one-dimensional polymerisations of (TO4)n−tetrahedra, where T = Si4+plus P5+, V5+, As5+, Al3+, Fe3+, B3+, Be2+, Zn2+and Mg2+. Such polymerisations are described by ageometrical repeat unit(with ngtetrahedra) and atopological repeat unit(or graph) (with ntvertices). The connectivity of the tetrahedra (vertices) in the geometrical (topological) repeat units is denoted by the expressioncTr(cVr) wherecis the connectivity (degree) of the tetrahedron (vertex) andris the number of tetrahedra (vertices) of connectivity (degree)cin the repeat unit. ThuscTr=1Tr12Tr23Tr34Tr4(cVr=1Vr12Vr23Vr34Vr4) represents all possible connectivities (degrees) of tetrahedra (vertices) in the geometrical (topological) repeat units of such one-dimensional polymerisations. We may generate all possiblecTr(cVr) expressions for chains (graphs) with tetrahedron (vertex) connectivities (degrees)c= 1 to 4 wherer= 1 tonby sequentially increasing the values ofcandr, and by ranking them accordingly. The silicate (sensu lato) units of chain-, ribbon- and tube-silicate minerals are identified and associated with the relevantcTr(cVr) symbols. Following description and association with the relevantcTr(cVr) symbols of the silicate units in all chain-, ribbon- and tube-silicate minerals, the minerals are arranged into decreasing O:T ratio from 3.0 to 2.5, an arrangement that reflects their increasing structural connectivity. Considering only the silicate component, the compositional range of the chain-, ribbon- and tube-silicate minerals strongly overlaps that of the sheet-silicate minerals. Of the chain-, ribbon- and tube-silicates and sheet silicates with the same O:T ratio, some have the samecVrsymbols (vertex connectivities) but the tetrahedra link to each other in different ways and are topologically different. The abundance of chain-, ribbon- and tube-silicate minerals decreases as O:T decreases from 3.0 to 2.5 whereas the abundance of sheet-silicate minerals increases from O:T = 3.0 to 2.5 and decreases again to O:T = 2.0. Some of the chain-, ribbon- and tube-silicate minerals have more than one distinct silicate unit: (1) vinogradovite, revdite, lintisite (punkaruaivite) and charoite have mixed chains, ribbons and/or tubes; (2) veblenite, yuksporite, miserite and okenite have clusters or sheets in addition to chains, ribbons and tubes. It is apparent that some chain-ribbon-tube topologies are favoured over others as of the ~450 inosilicate minerals, ~375 correspond to only four topologically unique graphs, the other ~75 minerals correspond to ~46 topologically unique graphs.

To inform cost-effective monitoring of offshore geological storage of carbon dioxide (CO 2), a unique field experiment, designed to simulate leakage of CO 2 from a sub-seafloor storage reservoir, was carried out in the central North Sea. A total of 675 kg of CO 2 were released into the shallow sediments (∼3 m below seafloor) for 11 days at flow rates between 6 and 143 kg d -1. A set of natural, inherent tracers ( 13C, 18O) of injected CO 2 and added, non-toxic tracer gases (octafluoropropane, sulfur hexafluoride, krypton, methane) were used to test their applicability for CO 2 leakage attribution and quantification in the marine environment. All tracers except 18O were capable of attributing the CO 2 source. Tracer analyses indicate that CO 2 dissolution in sediment pore waters ranged from 35 % at the lowest injection rate to 41% at the highest injection rate. Direct measurements of gas released from the sediment into the water column suggest that 22 % to 48 % of the injected CO 2 exited the seafloor at, respectively, the lowest and the highest injection rate. The remainder of injected CO 2 accumulated in gas pockets in the sediment. The methodologies can be used to rapidly confirm the source of leaking CO 2 once seabed samples are retrieved.

The germline is the only cellular lineage capable of transferring genetic information from one generation to the next. Intergenerational transmission of epigenetic memory through the germline, in the form of DNA methylation, has been proposed; however, in mammals this is largely prevented by extensive epigenetic erasure during germline definition. Here we report that, unlike mammals, the continuously-defined ‘preformed’ germline of zebrafish does not undergo genome-wide erasure of DNA methylation during development. Our analysis also uncovers oocyte-specific germline amplification and demethylation of an 11.5-kb repeat region encoding 45S ribosomal RNA (fem-rDNA). The peak of fem-rDNA amplification coincides with the initial expansion of stage IB oocytes, the poly-nucleolar cell type responsible for zebrafish feminisation. Given that fem-rDNA overlaps with the only zebrafish locus identified thus far as sex-linked, we hypothesise fem-rDNA expansion could be intrinsic to sex determination in this species., Germline cells transfer genetic information to offspring, and in zebrafish, drive sex determination. Here the authors report that, unlike mammals, the germline of zebrafish does not undergo genome-wide DNA methylation erasure, while amplifying and demethylating sex-linked rDNA during feminisation.

Rationale: In the IMPACT (Informing the Pathway of COPD Treatment) trial, single-inhaler fluticasone furoate/umeclidinium/vilanterol (FF/UMEC/VI) triple therapy reduced exacerbation risk versus FF/VI and UMEC/VI and mortality risk versus UMEC/VI. However, pneumonia incidence was higher in the inhaled corticosteroid (FF)containing arms, raising questions about the relative benefit of exacerbation reduction compared with the increased risk of pneumonia.Objectives: Determine benefit-risk of the three treatments by evaluating time-to-first and rates of composite exacerbation or pneumonia outcomes.Methods: We evaluated time-to-first (prespecified) and rates (post hoc) of investigator-reported pneumonia, serious pneumonia leading to hospitalization or death, and the composite endpoints of 1) moderate (required antibiotics/corticosteroids)/severe (hospitalized) exacerbation or pneumonia and 2) severe exacerbation or serious (hospitalized) pneumonia. Analyses were repeated for radiographically confirmed pneumonia (post hoc).Results: Moderate/severe exacerbations occurred in 47%, 49%, and 50% of patients randomized to FF/UMEC/VI, FF/VI andUMEC/VI, and pneumonias in 8%, 7%, and 5%, respectively. FF/UMEC/VI reduced the risk of combined moderate/severe exacerbation or pneumonia (time-to-first) versus FF/VI (hazard ratio, 0.87 [95% confidence interval (CI), 0.82-0.92]) and UMEC/VI (0.87 [0.81-0.94]), as well as the risk of combined severe exacerbation or serious pneumonia versus UMEC/VI (0.83 [0.72-0.96]). FF/UMEC/VI reduced the rate of combined moderate/severe exacerbation or pneumonia (rate ratio, 0.78 [0.72-0.84]) and combined severe exacerbation or serious pneumonia (rate ratio, 0.76 [0.65-0.89]) versus UMEC/VI. Results were similar for radiographically confirmed pneumonia endpoints.Conclusions: Despite higher incidence of pneumonia in FF-containing arms, these composite exacerbation/pneumonia outcomes support a favorable benefit-risk profile of FF/UMEC/VI versus FF/VI and UMEC/VI in patients with symptomatic chronic obstructive pulmonary disease and a history of exacerbations.

Although the mechanism of DNA demethylating drugs has been understood for many years, the direct effect of these drugs on methylation of the complementary strands of DNA has not been formally demonstrated. By using hairpin-bisulphite sequencing, we describe the kinetics and pattern of DNA methylation following treatment of cells by the DNA methyltransferase 1 (DNMT1) inhibitor, decitabine. As expected, we demonstrate complete loss of methylation on the daughter strand following S-phase in selected densely methylated genes in synchronized Jurkat cells. Thereafter, cells showed a heterogeneous pattern of methylation reflecting replication of the unmethylated strand and restoration of methylation.

Intermediate waters (500 - 2000 m) from the equatorial- to North Pacific are currently hypoxic (oxygen concentrations below 120 µmol/kg), while deeper waters are well oxygenated. For the last ice-age, some proxy records suggested that this trend was reversed, with well-oxygenated Pacific intermediate waters, and lower oxygenated deeper waters associated with an increased deep carbon reservoir. Recent work however suggests that there was an overall expansion of oxygen depleted water in the eastern tropical North Pacific during the last glacial period (Hoogakker et al., 2018). To further assess the natural variability in intermediate water dissolved oxygen concentrations over longer time-scales we extend the bottom water oxygen record of ODP Site 1242 (1360 m depth located in the eastern tropical north Pacific), to 140,000 years, using the benthic foraminifera carbon isotope gradient approach of Hoogakker et al. (2015). Our reconstructions suggest that oxygen concentrations varied with an approximate 40 kyr period; with lowest concentration during cool periods of the penultimate glacial, MIS 5b, 4 and 2.

CaveCalc is a PHREEQC-based numerical model for cave dripwater and speleothem chemistry, designed to aid in the interpretation of speleothem palaeoclimate records and cave monitoring datasets. CaveCalc forward-models dripwater and carbonate chemistry and isotopes through a variety of soil, karst and cave processes. Such processes include soil water & gas equilibration, carbonate bedrock dissolution, secondary carbonate precipitation and CO2 degassing. CaveCalc is able to quantitatively model bedrock dissolution under semi-open conditions — a feature necessary to accurately simulate the gas chemistry occurring in real cave environments. The model allows coupled modelling of multiple proxy systems, including δ18O, δ13C, a14C, δ44/40Ca and trace-elements (Mg/Ca, Sr/Ca, Ba/Ca), within a single framework. Additional proxy systems, chemical processes and calibration data may be added to the model as required.

In this work we report on a complete crystal-chemical characterization of a near end-member riebeckite from Malawi, and use the available data to critically compare information obtained from different analytical methods. The sample occurs as well-formed and very large single crystals in pegmatitic rocks. Accurate site-populations were determined by combining single-crystal structure refinement and electron microprobe analysis (EMPA). The Fe3+/Fe2+ ratio was obtained from Mössbauer spectroscopy. Lithium was quantified by Laser Ablation Inductively Coupled Plasma Mass Spectroscopy (LA-ICP-MS).Fourier-Transform Infrared (FTIR) spectra, collected both on powders and single crystals, are presented and discussed. FTIR spectra in the NIR region are also presented for the first time for this amphibole. The FTIR data are compatible with complete local ordering of A cations close to F, and complete Fe2+/Mg disorder at M(1,3). Polarized Raman-scattering data collected from single crystals confirm this conclusion. In addition, it was found that FTIR data collected on powders provide the best agreement with the site occupancies derived from chemical (EMPA and LA-ICP-MS) and crystal-chemical data, possibly because they do not depend on experimental issues such as orientation and polarization.

Background: The HER family of proteins (EGFR, HER2, HER3 and HER4) have long been thought to be therapeutic targets for bladder cancer, but previous clinical trials targeting these proteins have been disappointing. Second generation agents may be more effective. Objective: The aim of this study was to evaluate responses to two second-generation irreversible tyrosine kinase inhibitors, dacomitinib and afatinib, in bladder cancer cell lines. Methods: Cell lines were characterized by targeted next generation DNA sequencing, RNA sequencing, western blotting and flow cytometry. Cell survival responses to dacomitinib or afatinib were determined using (3-[4,5-dimethylthioazol-2-yl]-2,5-diphenyl tetrazolium bromide) (MTT) or [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) and phenazine methosylfate (PMS) cell survival assays. Results: Only two cell lines of 12 tested were sensitive to afatinib. Sensitivity to afatinib was significantly associated with mutation in either HER2 or HER3 (p

The functional membrane proteins on tumor-cell-derived EVs contain a large amount of biomolecular information, and can serve as a comprehensive marker to delineate the molecular nature of cancer. However, due to low secretion rates, it is difficult to perform accurate quantification and biomolecular analysis with conventional EV analysis technologies such as the Western blots. Here, we introduce a multifunctional EV analysis technology based on an automated microfluidic chemiluminescent ELISA (Enzyme-Linked ImmunoSorbent Assay) platform. With this system, we were able to achieve rapid EV quantification (

Mutations in the subunits of the cohesin complex, particularly in the STAG2 subunit, have been identified in a range of myeloid malignancies, but it is unclear how these mutations progress leukaemia. Here, we created isogenic K562 erythromyeloid leukaemia cells with and without the known leukemic STAG2 null mutation, R614*. STAG2 null cells acquired stem cell and extracellular matrix gene expression signatures that accompanied an adherent phenotype. Chromatin accessibility was dramatically altered in STAG2 null K562 cells, consistent with gene expression changes. Enhanced chromatin accessibility was observed at genes encoding hematopoietic transcription factors, ERG and RUNX1. Upon phorbol 12-myristate 13-acetate (PMA)-induced megakaryocytic differentiation, STAG2-null cells showed precocious spike in RUNX1 transcription from its P2 promoter. A similar precocious spike was observed in transcription of ERG. Interestingly, spikes in RUNX1-P2 and ERG only occurred as immediate early response to differentiation induction. Treatment of STAG2 null cells with enhancer-blocking BET inhibitor, JQ1, dampened precocious RUNX1 P2 expression and led to a complete loss of RUNX1 P1 and ERG transcription during PMA stimulation in both parental and STAG2 null K562 cells. These results suggest that precocious RUNX1 and ERG expression in STAG2 null cells is enhancer-driven. Furthermore, JQ1 treatment reduced stem cell-associated KIT expression in STAG2 null cells. We conclude that STAG2 depletion in leukemic cells amplifies an enhancer-driven transcriptional response to differentiation signals, and this characteristic is dampened by BET inhibition. The results have relevance to the development of therapeutic strategies for myeloid leukaemia

Oxygen, hydrogen, sulfur, and carbon isotopes have been analyzed in the Pea Ridge magnetite-apatite deposit, the largest historic producer among the known iron deposits in the southeast Missouri portion of the 1.5 to 1.3 Ga eastern granite-rhyolite province. The data were collected to investigate the sources of ore fluids, conditions of ore formation, and provenance of sulfur, and to improve the general understanding of the copper, gold, and rare earth element potential of iron deposits regionally. The δ 18 O values of Pea Ridge magnetite are 1.9 to 4.0‰, consistent with a model in which some magnetite crystallized from a melt and other magnetite—perhaps the majority—precipitated from an aqueous fluid of magmatic origin. The δ 18 O values of quartz, apatite, actinolite, K-feldspar, sulfates, and calcite are significantly higher, enough so as to indicate growth or equilibration under cooler conditions than magnetite and/or in the presence of a fluid that was not entirely magmatic. A variety of observations, including stable isotope observations, implicate a second fluid that may ultimately have been meteoric in origin and may have been modified by isotopic exchange with rocks or by evaporation during storage in lakes. Sulfur isotope analyses of sulfides from Pea Ridge and seven other mineral deposits in the region reveal two distinct populations that average 3 and 13‰. Two sulfur sources are implied. One was probably igneous melts or rocks belonging to the mafic- to intermediate-composition volcanic suite that is present at or near most of the iron deposits; the other was either melts or volcanic rocks that had degassed very extensively, or else volcanic lakes that had trapped rising magmatic gases. The higher δ 34 S values correspond to deposits or prospects where copper is noteworthy—the Central Dome portion of the Boss deposit, the Bourbon deposit, and the Vilander prospective area. The correspondence suggests that (1) sulfur either limited the deposition of copper or was cotransported with copper, and (2) sulfur isotope analysis may be useful in evaluating southeast Missouri iron deposits for copper and possibly for gold.

New O and Fe stable isotope ratios are reported for magnetite samples from high-grade massive magnetite of the Mesoproterozoic Pea Ridge and Pilot Knob magnetite-apatite ore deposits and these results are compared with data for other iron oxide-apatite deposits to shed light on the origin of the southeast Missouri deposits. The δ 18 O values of magnetite from Pea Ridge ( n = 12) and Pilot Knob ( n = 3) range from 1.0 to 7.0 and 3.3 to 6.7‰, respectively. The δ 56 Fe values of magnetite from Pea Ridge ( n = 10) and Pilot Knob ( n = 6) are 0.03 to 0.35 and 0.06 to 0.27‰, respectively. These δ 18 O and the δ 56 Fe values suggest that magnetite crystallized from a silicate melt (typical igneous δ 56 Fe ranges 0.06–0.49‰) and grew in equilibrium with a magmatic-hydrothermal aqueous fluid. We propose that the δ 18 O and δ 56 Fe data for the Pea Ridge and Pilot Knob magnetite-apatite deposits are consistent with the flotation model recently proposed by Knipping et al. (2015a), which invokes flotation of a magmatic magnetite-fluid suspension and offers a plausible explanation for the igneous (i.e., up to ~15.9 wt % TiO 2 in magnetite) and hydrothermal features of the deposits.

Purpose: A post-hoc analysis of the IMPACT study in the subgroup of patients taking triple therapy at screening. The effect of ICS and LAMA step down was analyzed by comparing triple therapy vs dual therapy for rate of exacerbations (exb), change in FEV1 and SGRQ. Methods: IMPACT was a 52-week randomized, double-blind study in patients ≥40 yrs with symptomatic COPD and ≥1 exb in the prior 12 months. The primary endpoint was annual rate of on-treatment moderate/severe (mod/sev) exb comparing fluticasone furoate/umeclidinium/vilanterol (FF/UMEC/VI) 100/62.5/25µg vs FF/VI and UMEC/VI. Data were analysed based on the medications they were taking at screening. Results: 10,355 patients were in the ITT population (FF/UMEC/VI, n=4151; FF/VI, n=4134; UMEC/VI, n=2070). At baseline, 40% of each treatment group were taking triple therapy. Data for the comparison of FF/UMEC/VI vs FF/VI and vs UMEC/VI for the endpoints mod/sev exb, severe exb, change from baseline in trough FEV1, and change from baseline in SGRQ are presented in table 1. Conclusion: In patients maintained on ICS or LAMA therapy the magnitude of reduction in exacerbations, hospitalizations, and improvement in lung function, quality of life was greater than in those who had withdrawal of these therapies.

BACKGROUND The benefits of triple therapy for chronic obstructive pulmonary disease (COPD) with an inhaled glucocorticoid, a long-acting muscarinic antagonist (LAMA), and a long-acting β2-agonist (LABA), as compared with dual therapy (either inhaled glucocorticoid–LABA or LAMA–LABA), are uncertain. METHODS In this randomized trial involving 10,355 patients with COPD, we compared 52 weeks of a once-daily combination of fluticasone furoate (an inhaled glucocorticoid) at a dose of 100 μg, umeclidinium (a LAMA) at a dose of 62.5 μg, and vilanterol (a LABA) at a dose of 25 μg (triple therapy) with fluticasone furoate–vilanterol (at doses of 100 μg and 25 μg, respectively) and umeclidinium–vilanterol (at doses of 62.5 μg and 25 μg, respectively). Each regimen was administered in a single Ellipta inhaler. The primary outcome was the annual rate of moderate or severe COPD exacerbations during treatment. RESULTS The rate of moderate or severe exacerbations in the triple-therapy group was 0.91 per year, as compared with 1.07 per year in the fluticasone furoate–vilanterol group (rate ratio with triple therapy, 0.85; 95% confidence interval [CI], 0.80 to 0.90; 15% difference; P

© 2015 Journal of NeuroVirology, Inc. Cell-free mitochondiral DNA (mtDNA) is an immunogenic molecule associated with many inflammatory conditions. We evaluated the relationship between cell-free mtDNA in cerebrospinal fluid (CSF) and neurocognitive performance and inflammation during HIV infection. In a cross-sectional analysis, we evaluated the association of mtDNA levels with clinical assessments, inflammatory markers, and neurocognitive performance in 28 HIV-infected individuals. In CSF, we measured mtDNA levels by droplet digital PCR, and soluble CD14 and CD163, neurofilament light, and neopterin by ELISA. In blood and CSF, we measured soluble IP-10, MCP-1, TNF-α, and IL-6 by ELISA, and intracellular expression of IL-2, IFN-γ, and TNF-α in CD4+ and CD8+ T cells by flow cytometry. We also evaluated the relationship between CSF pleocytosis and mtDNA longitudinally in another set of five individuals participating in an antiretroviral treatment (ART) interruption study. Cell-free CSF mtDNA levels strongly correlated with neurocognitive performance among individuals with neurocognitive impairment (NCI) (r = 0.77, p = 0.001). CSF mtDNA also correlated with levels of IP-10 in CSF (r = 0.70, p = 0.007) and MCP-1 in blood plasma (r = 0.66, p = 0.01) in individuals with NCI. There were no significant associations between inflammatory markers and mtDNA in subjects without NCI, and levels of mtDNA did not differ between subjects with and without NCI. MtDNA levels preceded pleocytosis and HIV RNA following ART interruption. Cell-free mtDNA in CSF was strongly associated with the severity of neurocognitive dysfunction and inflammation only in individuals with NCI. Our findings suggest that within a subset of subjects cell-free CSF mtDNA is associated with inflammation and degree of NCI.

The elemental carbon (EC) tracer method has often been used to estimate the primary and secondary organic aerosol (OA) fractions using field measurements of organic carbon (OC) and EC. In this observation-based approach, EC is used as a tracer for primary OC (POC), which allows for the estimation of secondary OC (SOC). The accuracy of this approach is evaluated using concentrations generated by PMCAMx, a three-dimensional chemical transport model that simulates the complex processes leading to SOC formation (including evaporation and chemical processing of POC and chemical aging of semivolatile and intermediate volatility organics). The ratio of primary organic to elemental carbon [OC/EC]p is estimated in various locations in the Eastern United States, and is then used to calculate the primary and secondary OC concentrations. To estimate the [OC/EC]p from simulated concentrations, we use both a traditional approach and the high EC edge method, in which only values with the highest EC/OC ratio are used. Both methods perform best on a daily-averaged basis, because of the variability of the [OC/EC]p ratio during the day. The SOC estimated by the EC tracer methods corresponds to the biogenic and anthropogenic SOC formed during the oxidation of volatile organic compounds. On the other hand, the estimated POC corresponds to the sum of the fresh POC, the SOC from oxidation of the evaporated POC and the intermediate volatility organic compounds, and the OC from long-distance transport. With this correspondence, the traditional EC tracer method tends to overpredict primary OC and underpredict secondary OC for the selected urban areas in the eastern United States. The high EC edge method performs better, especially in areas where the primary contribution to OC is smaller. Despite the weaknesses of models like the one used here, the conclusions about the accuracy of observation-based methods like the EC-tracer approach should be relatively robust due to the internal consistency of the data and the approach.

The chemical transport model PMCAMx is used to examine the effect of climate change on fine (under 2.5 µms) particulate matter (PM2.5) during the summer in the eastern United States. Meteorology from 10 years in the 1990s (present) and 10 years in the 2050s (future) based on the Intergovernmental Panel on Climate Change A2 scenario is used. Anthropogenic pollutant emissions are assumed to remain constant, while biogenic emissions are climate sensitive and, depending on species, increase between 15 and 27% on average. The predicted changes of PM2.5 are modest (increases of less than 10% on average across the domain) and quite variable in space, ranging from +13% in the Plains to −7% in the Northeast. Variability is driven concurrently by changes in temperature, wind speed, rainfall, and relative humidity, with no single dominant meteorological factor. Sulfate and organic aerosol are responsible for most of the PM2.5 change. The improved treatment of organic aerosol using the volatility basis set does not increase significantly its sensitivity to climate change compared to traditional treatments that neglect the volatility of primary particles and do not simulate the chemical aging processes. Future organic aerosol is predicted to be more oxidized due to increases of its secondary biogenic and anthropogenic components. These results suggest that the effects of planned and expected emission anthropogenic emission controls will be more important than those of climate change for PM2.5 concentrations in 2050. Maximum daily 8 h average ozone increases by 5% on average are predicted, with a marked increase in the Northeast, Southeast, and Midwest.

Widespread and global efforts to improve degraded coastal ecosystems, especially those experiencing hypoxia, warrant a renewed focus on understanding and quantifying restoration trajectories. We describe a whole-ecosystem experiment to manipulate dissolved oxygen concentrations using large-scale destratification aeration that was used to document the biogeochemical response of a small estuary to changes in oxygen availability. The experiment was successful in creating oxic and anoxic bottom water conditions at a spatial scale much larger than that encompassed by the aerators. After a period of anoxic conditions, the return of oxygenated bottom water by destratification resulted in rapid decreases in sediment phosphate fluxes, uptake of nitrate and nitrite, and an increase in simulated denitrification rates. Bottom water nutrient concentrations responded near-simultaneously to these changes to benthic–pelagic fluxes. The rapidity with which the ecosystem responded to increases in bottom water oxygen confirms the critical role of dissolved oxygen concentrations in modulating nutrient cycling. This result also provides insight into the likely response of hypoxic and anoxic estuaries to remediation of oxygen deficits at the sediment–water interface.

The muscleblind-like (MBNL) family of proteins are key developmental regulators of alternative splicing. Sequestration of MBNL proteins by expanded CUG/CCUG repeat RNA transcripts is a major pathogenic mechanism in the neuromuscular disorder myotonic dystrophy (DM). MBNL1 contains four zinc finger (ZF) motifs that form two tandem RNA binding domains (ZF1–2 and ZF3–4) which each bind YGCY RNA motifs. In an effort to determine the differences in function between these domains, we designed and characterized synthetic MBNL proteins with duplicate ZF1–2 or ZF3–4 domains, referred to as MBNL-AA and MBNL-BB, respectively. Analysis of splicing regulation revealed that MBNL-AA had up to 5-fold increased splicing activity while MBNL-BB had 4-fold decreased activity compared to a MBNL protein with the canonical arrangement of zinc finger domains. RNA binding analysis revealed that the variations in splicing activity are due to differences in RNA binding specificities between the two ZF domains rather than binding affinity. Our findings indicate that ZF1–2 drives splicing regulation via recognition of YGCY RNA motifs while ZF3–4 acts as a general RNA binding domain. Our studies suggest that synthetic MBNL proteins with improved or altered splicing activity have the potential to be used as both tools for investigating splicing regulation and protein therapeutics for DM and other microsatellite diseases.

Background Mitochondria are abundant organelles critical for energy metabolism and brain function. Mitochondrial DNA (mtDNA), released during cellular injury and as part of the innate immune response to viral pathogens, contains CpG motifs that act as TLR-9 ligands. We investigated relationships between cerebrospinal fluid (CSF) cell-free mtDNA levels and HIV viral load (VL), biomarkers of inflammation and iron transport, and neurocognitive (NC) function in the CNS HIV Antiretroviral Therapy Effects Research (CHARTER) cohort. Methods We quantified cell-free mtDNA in CSF by droplet digital PCR in 332 CHARTER participants who underwent comprehensive neuropsychiatric evaluation. NC performance was assessed using the global deficit score (GDS) as either a continuous or a binary measure (GDS ≥ 0.5, impaired vs. GDS

Nucleos(t)ide reverse transcriptase inhibitor (NRTI)-sparing regimens may potentially minimize antiretroviral (ART) toxicities, but demonstrate mixed efficacy and toxicity results. The impact of an integrase strand transfer inhibitor (INSTI) and protease inhibitor (PI) regimen on HIV viral dynamics and T cell kinetics remains underdescribed.To compare the effect of raltegravir + ritonavir boosted lopinavir (RAL + LPV/r) to efavirenz/tenofovir disoproxil fumarate/emtricitabine (EFV/TDF/FTC) on HIV kinetics and T cell dynamics.Fifty participants naïve to ART underwent HIV viral kinetic sampling evaluated using biexponential mixed effects modeling. A subset of 28 subjects (with complete viral suppression) underwent flow cytometry and evaluation of soluble markers of inflammation at weeks 0, 4, and 48 of ART.RAL + LPV/r compared to EFV/TDF/FTC resulted in a prolonged first phase viral decay rate (18 vs. 13 days p 0.01). From weeks 0 to 4, RAL + LPV/r was associated with a trend toward greater decreases in activated CD4The prolonged first phase viral decay observed with RAL + LPV/r in persons starting ART did not result in differences in viral suppression at week 48. We also observed trends in declines in certain cellular markers of immune activation but it remains unclear if this could translate to long-term immunologic benefits in persons on an INSTI + PI.

Significance Mass spectrometry experiments with a nickel-containing respiratory hydrogenase from Escherichia coli provide conclusive proof that it catalyzes the four-electron reduction of oxygen by hydrogen, a reaction analogous to combustion. Hydrogenase-1 is a membrane-bound enzyme that oxidizes hydrogen in the periplasm to reduce respiratory-chain quinones. If oxygen is present and enters the buried active site, a package of electrons, derived from hydrogen oxidation and held in the iron–sulfur cluster relay system, transfers back to convert it cleanly to water. The enzyme thus avoids production of reactive oxygen intermediates that would otherwise cause inactivation. This study establishes the basis of the oxygen tolerance in respiratory nickel–iron hydrogenases and demonstrates biology’s unexpected use of nickel in the active site of a four-electron oxidase.

Objectives: To compare the Roche Cell-Free DNA Collection Tubes® against the Streck Cell-Free DNA BCT®s for sample stability using Cell Free DNA (cfDNA) from healthy volunteers (n = 20). Design & methods: Whole blood was drawn into five Roche and five Streck tubes per volunteer, stored at room temperature and processed at five different time points (Days 0, 4, 7, 10 and 14). One volunteer had blood drawn into ×10 K3EDTA tubes to observe the effect of no preservation buffer on White Blood Cell (WBC) lysis. DNA was extracted from the plasma and the concentration (ng/μL) measured using the Qubit Fluorometer® at each time point. The eluted DNA was further analysed by capillary electrophoresis to determine the proportion of cfDNA and gDNA contamination in the samples over the 14 days. Results: There was no difference in individual (p = 0.097) and median paired (p = 0.26) DNA concentration across the five time points between the two tubes. However, a difference was observed for samples in the Roche tubes for pair days 0–7 (p = 0.01), 0 to 10 (p = 0.046) and 0 to 14 (p = 0.0016) in contrast to the Streck tubes after adjustment for multiple testing. Conclusion: The findings of this study indicate that the Roche Cell-Free DNA Collection Tubes® are a suitable alternative for sample collection and storage at room temperature, albeit for a duration of less than 7 days. Keywords: Cell free DNA, Roche cell-free DNA collection tubes, Plasma DNA, Blood-based biomarker, Genomic DNA contamination

Purpose: To describe small hyperreflective areas using spectral-domain optical coherence tomography (SD-OCT) imaging in eyes that have had silicone oil tamponade. Methods: Retrospective case series of 11 eyes of 11 patients. The authors retrospectively identified patients who underwent vitrectomy and silicone oil tamponade secondary to a rhegmatogenous retinal detachment (nine patients), panuveitis with retinal necrosis (one patient), or recurrent full-thickness macular hole surgery (one patient) who had manifestations of silicone oil emulsion on SD-OCT imaging. Patients were monitored during the postoperative period by clinical examination and using SD-OCT. A model eye in which emulsified silicone oil had been injected in the anterior chamber was used to obtain anterior segment SD-OCT images for comparison. Results: The mean age of our patients was 50 years (range, 39–76 years). In eight eyes, the SD-OCT examination was carried out after silicone oil removal, and in three eyes, the SD-OCT examination was carried out with the oil in situ. Of the nine eyes treated for rhegmatogenous retinal detachment, five had a relieving retinectomy for advanced anterior proliferative vitreoretinopathy or for traumatic retinal incarceration (one eye). The eye treated for full-thickness macular hole had a vitrectomy, internal limiting membrane peel, and silicone oil injection for recurrent macular hole. Ten eyes showed hyperreflective, spherical, tiny droplets using SD-OCT imaging. These were thought to represent silicone oil droplets intraretinally or underneath epiretinal membranes, and one eye showed hyperreflective areas subretinally (retina detached). One additional patient was found to have tiny intravitreal silicone oil droplets after silicone oil removal. Similarly, the silicone oil appeared as multiple hyperreflective spherical droplets as detected by SD-OCT. Anterior segment studies of silicone oil emulsification in the experimental model revealed a similar appearance to that seen with in vivo SD-OCT imaging. Conclusion: The authors have found small hyperreflective areas intraretinally, subretinally, and underneath epiretinal membranes on SD-OCT in eyes that have had silicone oil tamponade for a variety of indications. The authors have seen a similar appearance when silicone oil emulsification is examined in vivo. The authors conclude that the hyperreflective areas are likely (but not certain) to be very small bubbles of emulsified silicone. Further studies are required to determine the incidence, clinicopathologic, and functional significance of probable silicone oil emulsification and deposition within the retinal layers.

A new analytical method was developed to quantify 26 newly-identified and 21 legacy (e.g. perfluoroalkyl carboxylates, perfluoroalkyl sulfonates, and fluorotelomer sulfonates) per and polyfluorinated alkyl substances (PFAS) in groundwater and aqueous film forming foam (AFFF) formulations. Prior to analysis, AFFF formulations were diluted into methanol and PFAS in groundwater were micro liquid-liquid extracted. Methanolic dilutions of AFFF formulations and groundwater extracts were analyzed by large-volume injection (900 μL) high-performance liquid chromatography tandem mass spectrometry. Orthogonal chromatography was performed using cation exchange (silica) and anion exchange (propylamine) guard columns connected in series to a reverse-phase (C18) analytical column. Method detection limits for PFAS in groundwater ranged from 0.71 ng/L to 67 ng/L, and whole-method accuracy ranged from 96% to 106% for analytes for which matched authentic analytical standards were available. For analytes without authentic analytical standards, whole-method accuracy ranged from 78 % to 144 %, and whole-method precision was less than 15 % relative standard deviation for all analytes. A demonstration of the method on groundwater samples from five military bases revealed eight of the 26 newly-identified PFAS present at concentrations up to 6900 ng/L. The newly-identified PFAS represent a minor fraction of the fluorinated chemicals in groundwater relative to legacy PFAS. The profiles of PFAS in groundwater differ from those found in fluorotelomer- and electrofluorination-based AFFF formulations, which potentially indicates environmental transformation of PFAS.

Members of the human epidermal growth factor receptor (HER) family play a significant role in bladder cancer progression and may underlie the development of chemotherapy resistance. Dacomitinib is an irreversible tyrosine kinase inhibitor with structural specificity for the catalytic domains of epidermal growth factor receptor (EGFR), HER2 and HER4 that has exhibited vigorous efficacy against other solid tumors. We evaluated the antitumor activity of dacomitinib in human bladder cancer cell lines expressing varying levels of HER family receptors. These cell lines also were established as bladder cancer xenografts in nonobese diabetic/severe combined immunodeficiency (NOD/SCID) mice to assess dacomitinib activity in vivo. Significant cytotoxic and cytostatic effects were noted in cells expressing elevated levels of the dacomitinib target receptors with apoptosis and cell cycle arrest being the predominant mechanisms of antitumor activity. Cells expressing lower levels of HER receptors were much less sensitive to dacomitinib. Interestingly, dacomitinib was more active than either trastuzumab or cetuximab in vitro, and exhibited increased growth inhibition of bladder tumor xenografts compared with lapatinib. Pharmacodynamic effects of dacomitinib included decreased E-cadherin (E-cad) expression, reduction of EGFR and extracellular signal-regulated kinase (ERK) phosphorylation and reduced mitotic count. Dacomitinib also inhibited tumor growth in a chemotherapy-resistant xenograft and, when combined with chemotherapy in a sensitive xenograft, exhibited superior antitumor effects compared with individual treatments. Evaluation in xenograft-bearing mice revealed that this combination was broadly feasible and well tolerated. In conclusion, dacomitinib exhibited pronounced activity both as a single agent and when combined with chemotherapy in human bladder cancer models. Further investigation of dacomitinib in the preclinical and clinical trial settings is being pursued.

Speleothem oxygen isotopes and growth rates are valuable proxies for reconstructing climate history. There is debate, however, about the conditions that allow speleothems to grow in oxygen isotope equilibrium, and about the correct equilibrium fractionation factors. We report results from a series of carbonate growth experiments in karst-analogue conditions in the laboratory. The setup closely mimics natural processes (e.g. precipitation driven by CO₂-degassing, low ionic strength solution, thin solution film) but with a tight control on growth conditions (temperature, pCO₂, drip rate, calcite saturation index and the composition of the intial solution). Calcite is dissolved in water in a 20,000 ppmV pCO₂ environment. This solution is dripped onto glass plates (coated with seed-carbonate) in a lower pCO₂ environment (0.00679T +(e0.00248T-2)*(-0.779d_r² + 10.05d_r + 11.69). This relationship indicates a substantial increase of growth mass with temperature, a smaller influence of drip rate on growth mass at low temperature and a non-linear relationship between drip rate and growth mass at higher temperatures. Low temperature, fast dripping conditions are found to be most favourable for reducing effects associated with evaporation and rapid depletion of the dissolved inorganic carbon reservoir (rapid DIC-depletion). The impact of evaporation can be so large so caves with high relative humidity are also preferable for palaeoclimate reconstruction. Even allowing for the maximum offsets that may have been induced by evaporation and rapid DIC-depletion, δ18O measured in some of our experiments remained higher than those predicted by Kim and O'Neil (1997). Our new results are well explained by equilibrium at a significantly higher αcalcite-water, with a kinetic-isotope effect that favours 16O incorporation as growth rate increases. This scenario agrees with recent studies by Coplen (2007) and Dietzel et al. (2009). Overall, our results suggest that three separate processes cause δ18O to deviate from true isotope equilibrium in the cave environment. Two of these drive δ18O to higher values (evaporation and rapid DIC-depletion) while one drives δ18O to lower values (preferential incorporation of 16O in the solid carbonate at faster growth rates). While evaporation and DIC-depletion can be avoided in some settings, the third may be inescapable in the cave environment and means that any temperature to δ18O relationship is an approximation. The controlled conditions of the present experiments also display limitations in the use of the Hendy test to identifying equilibrium growth.

Cell-free mitochondrial DNA (mtDNA) is a highly immunogenic molecule that is associated with several inflammatory conditions and with neurocognitive impairment during untreated HIV infection. Here, we investigate how cell-free mtDNA in cerebrospinal fluid (CSF) is associated with inflammation, neuronal damage, and neurocognitive functioning in the context of long-term suppressive antiretroviral therapy (ART). We quantified the levels of cell-free mtDNA in the CSF from 41 HIV-infected individuals with completely suppressed HIV RNA levels in blood plasma (

ObjectiveIn this work, we evaluated the association of human immunodeficiency virus (HIV) infection and methamphetamine (METH) use with mitochondrial injury in the brain and its implication on neurocognitive impairment.DesignMitochondria carry their genome (mtDNA) and play a critical role in cellular processes in the central nervous system. METH is commonly used in HIV-infected populations. HIV infection and METH use can cause damage to mtDNA and lead to neurocognitive morbidity. We evaluated HIV infection and METH use with mitochondrial injury in the brain.MethodsWe obtained white and gray matter from Brodmann areas 7, 8, 9, 46 of the following: HIV-infected individuals with history of past METH use (HIV+METH+, n = 16), HIV-infected individuals with no history of past METH use (HIV+METH-, n = 11), and HIV-negative controls (HIV-METH-, n = 30). We used the 'common deletion', a 4977 bp mutation, as a measurement of mitochondrial injury, and quantified levels of mtDNA and 'common deletion' by droplet digital PCR, and evaluated in relation to neurocognitive functioning [Global Deficit Score (GDS)].ResultsLevels of mtDNA and mitochondrial injury were highest in white matter of Brodmann area 46. A higher relative proportion of mtDNA carrying the 'common deletion' was associated with lower GDS (P

We present the first study of Ca isotope cycling in a natural cave system, with measurements of bedrock, dripwater and recently formed carbonate, coupled to a first stalagmite time-series spanning the 8.2 kyr event. Dripwaters at Heshang Cave (Central China; 30°27′N, 110°25′E) are isotopically heavy relative to the dolomite bedrock, the result of prior calcite precipitation (PCP) occurring earlier in the drip flow path. A simple Rayleigh fractionation model quantifies the extent of PCP in the modern environment at 36% Ca removal. The observed in situ calcium isotope fractionation factor between dripwater and carbonate is Δ 44 / 42 Ca = − 0.63 ± 0.03 ‰ and does not vary during the annual cycle. Measurements of speleothem carbonate spanning the 8.2 kyr event show the response of Ca isotopes to changing climate. δ44/42Ca increases by 0.35‰ at the onset of the event, coeval with changes in δ18O and Mg/Ca, and remains high for 80 yr. This change is explained by decreased rainfall leading to increased PCP; an interpretation supported by established PCP proxies (Mg/Ca, Sr/Ca and Ba/Ca). Ca isotopes indicate that PCP increased to 60% Ca removal during the event, which, from application of a simple box model, suggests mean annual rainfall decreased by approximately a third in Central China during the 8.2 kyr event. The response of Ca isotopes across this event demonstrates their potential for the assessment of past conditions, including past dripwater flow rates and rainfall.

Changes in summertime organic aerosol (OA) concentrations in the Eastern U.S. are investigated for different temperature change scenarios using the chemical transport model PMCAMx-2008. OA is simulated using the volatility basis set approach, assuming that the primary emissions are semi-volatile and that the intermediate volatile and semi-volatile organic compounds are oxidized in the gas phase, resulting in products with lower volatility. For the basic temperature change scenario where biogenic emissions are kept constant, ground-level OA decreases by −0.3% K−1 on average. Increases in the north (+0.1% K−1) and decreases in the south (−0.5% K−1) are predicted. The effect of the uncertain temperature dependence of the aging rate constant is modest, changing the OA by only 0.1% K−1 over the temperature-independent case. For the more realistic scenario in which biogenic OA precursor emissions are allowed to increase with temperature (up to 10% K−1), however, average OA increases by 4.1% K−1, with even higher increases in southern regions. These results suggest that as temperature increases, complicated changes in production, partitioning and chemical aging will take place. Nevertheless, the change in biogenic emissions and subsequent production of biogenic OA is more than an order of magnitude more important than the changes in the rates of chemical and physical atmospheric processes.

We have measured δ44/42Ca of laboratory-precipitated calcite grown in an experimental setup that closely replicates stalagmite formation. Calcium solutions were dripped onto two different substrates in tightly-controlled conditions and calcite precipitated due to rapid CO2 degassing. With seeded glass slides as the substrate, we observe a Ca isotope ratio in the calcite which is ∼0.5‰ per amu lower than that in the growth solution. This fractionation is generally almost twice that observed in previously published calcite growth experiments and indicates a large kinetic effect on Ca isotopes in the stalagmite growth environment. The precipitate forming near the spot where the drip lands shows slightly greater solution-to-precipitate fractionation than calcite further from the drip reflecting a decrease in this kinetic fractionation as precipitation continues. We interpret these results in the context of the model of Fantle and DePaolo (2007) which involves surface entrapment of light Ca isotopes to decrease calcite δ44/42Ca, and depletion of Ca from the solution in the direct vicinity of the growing calcite to increase calcite δ44/42Ca. In the stalagmite setting, the second of these effects is minimized so that calcite Ca isotope ratios are unusually light. This interpretation suggests that stalagmite Ca isotope ratios should decrease with the saturation state of the drip water (i.e. with the growth rate of calcite). Ca isotopes might therefore allow reconstruction of surface entrapment of trace metals and isotopes more generally and might, for instance, allow an assessment of the appropriate relationship between oxygen isotope fractionation and temperature for periods of past growth in stalagmites.

A nondestructive, chemical-free method is presented for the extraction of DNA from small insects. Blackflies were submerged in sterile, distilled water and sonicated for varying lengths of time to provide DNA which was assessed in terms of quantity, purity and amplification efficiency. A verified DNA barcode was produced from DNA extracted from blackfly larvae, pupae and adult specimens. A 60-second sonication period was found to release the highest quality and quantity of DNA although the amplification efficiency was found to be similar regardless of sonication time. Overall, a 66% amplification efficiency was observed. Examination of post-sonicated material confirmed retention of morphological characters. Sonication was found to be a reliable DNA extraction approach for barcoding, providing sufficient quality template for polymerase chain reaction amplification as well as retaining the voucher specimen for post-barcoding morphological evaluation.

This paper describes the design, fabrication and characterization of a microfluidic gas centrifuge for separating dilute gas mixtures based on the molecular weights of their constituents. The principal advantage of this approach is its fast response time compared to other methods that are based on permeation or adsorption/desorption. This would allow it to serve as a real-time preconcentrator for improving the sensitivity of miniature chemical sensors. Devices with nozzle throat widths as small as 3.6 μm have been fabricated using photolithography, deep reactive ion etching (DRIE) and silicon-glass anodic bonding. Measurements of the device's performance show that a single stage can achieve a two-fold enrichment of an initially 1% mixture of SF 6 in N 2 in 0.01 ms. These experimental findings are consistent with the results of two-dimensional numerical simulations of the flow through the device. The simulations suggest that the performance of a single stage could be improved significantly by changing the geometry of the entrance flow. Further improvements in performance could be achieved by cascading the devices.

Morphological changes in the retinal vascular network are associated with future risk of many systemic and vascular diseases. However, uncertainty over the presence and nature of some of these associations exists. Analysis of data from large population based studies will help to resolve these uncertainties. The QUARTZ (QUantitative Analysis of Retinal vessel Topology and siZe) retinal image analysis system allows automated processing of large numbers of retinal images. However, an image quality assessment module is needed to achieve full automation. In this paper, we propose such an algorithm, which uses the segmented vessel map to determine the suitability of retinal images for use in the creation of vessel morphometric data suitable for epidemiological studies. This includes an effective 3-dimensional feature set and support vector machine classification. A random subset of 800 retinal images from UK Biobank (a large prospective study of 500,000 middle aged adults; where 68,151 underwent retinal imaging) was used to examine the performance of the image quality algorithm. The algorithm achieved a sensitivity of 95.33% and a specificity of 91.13% for the detection of inadequate images. The strong performance of this image quality algorithm will make rapid automated analysis of vascular morphometry feasible on the entire UK Biobank dataset (and other large retinal datasets), with minimal operator involvement, and at low cost. Changes in retinal vasculature prospectively associated with disease outcomes.Large population based studies help to resolve uncertainties in these associations.QUARTZ software extracts morphometric data from large numbers of retinal images.Automated image quality assessment is required to achieve full automation.This addition into QUARTZ makes processing the entire UK Biobank dataset feasible.

International controlled trials have demonstrated increasing sustained virological response (SVR) rates to interferon-based therapies in hepatitis-C-treated patients. Response rates of 6-20% in the era of interferon monotherapy are compared with 42-82% with pegylated interferon plus ribavirin. The virological durability of the SVR is unknown and the optimal follow-up for these patients is unclear. The aim of our study was to determine SVR rates and the durability of the response to interferon-based therapies in the clinical setting. From our database of 1540 hepatitis C patients, 344 treatment courses of at least 12 weeks duration were identified, including interferon monotherapy (175 patients), interferon plus ribavirin (96 patients) and peginterferon plus ribavirin (73 patients). Interferon monotherapy was associated with an SVR rate of 5% in 103 genotype 1 patients and 25% in 72 genotype 2/3 patients. Response rates were higher (P0.001) with interferon plus ribavirin-41% in 34 genotype 1 patients and 73% in 62 genotype 2/3 patients-and with peginterferon plus ribavirin-47% in 47 genotype 1 patients and 79% in 26 genotype 2/3 patients. Of 147 patients with an SVR, 146 (99%) remained hepatitis C virus PCR negative during a mean 2.3 years (range 0.3-10.3) of follow-up. In conclusion, with advances in therapies, we are achieving higher response rates in hepatitis C patients treated in the clinical setting. We can now expect an SVR in over half of the treated patients. Importantly, the response is durable and medium and long-term follow-up of these patients are of low yield and largely unnecessary.

We have studied the effect of key neurotrophic factors (NTFs) on morphology, levels of the vanilloid receptor-1 (TRPV1) and responses to capsaicin in adult human sensory neurons in vitro. Avulsed dorsal root ganglia (DRG, n = 5) were cultured with or without a combination of nerve growth factor (NGF), glial cell (line)-derived growth factor (GDNF) and neurotrophin3 (NT3) for 5 days. In the absence of NTFs, the diameter of neurons ranged from 20 to 100 microm (mean 42 +/- 4 microm). Adding NTFs caused a significant increase in neuronal sizes, up to 120 microm (mean diameter 62 +/- 5 microm, P < 0.01, t-test), an overall 35% increase of TRPV1-positive neurons (P < 0.003), and notably of large TRPV1-positive neurons > 80 microm (P < 0.05). Responses to capsaicin were significantly enhanced with calcium ratiometry (P < 0.0001). Short duration (1h) exposure of dissociated sensory neurons to NTFs increased numbers of TRPV1-positive neurons, but not of TRPV3, Nav 1.8 and IK1 and the morphological size-distribution remained similar to intact post-mortem DRG neurons. NTFs thus increase size, elevate TRPV1 levels and enhance capsaicin responses in cultured human DRG neurons; these changes may relate to pathophysiology in disease states, and provide an in vitro model to study novel analgesics.

We report here a preliminary study in which dynamic secondary ion mass spectrometry (SIMS) has provided images of boron-10 (10B) in biological tissue as used in research into boron neutron capture therapy. Cultured tumour cells incubated in media containing known concentrations of a 10B-containing compound, p-boronophenylalanine (BPA), and intracranial tumour tissue from animals previously injected with BPA were analysed by an in-house constructed SIMS. Investigations were conducted in positive secondary ion detection mode using a 25-keV, 5-nA gallium primary ion source. For calibration purposes, tissue standards were also analysed and their boron-to-carbon signal ratios correlated to bulk boron concentrations measured by inductively coupled plasma atomic emission spectroscopy (ICP-AES). Ion maps of 10B, 12C, 23Na and 39K showing gross tissue and cell features were acquired. SIMS and ICP-AES standard measurements were in good agreement. Tissue regions with high or low 10B concentrations were identified along with 10B hotspots in normal brain areas. Cultured cells revealed the intracellular localization of 10B. SIMS is capable of producing images showing the distribution of 10B at p.p.m. levels in cells and in normal and tumour-bearing brain tissue.

Bioluminescence, the conversion of chemical energy into light in living organisms, is dependent on two principal components, an enzyme luciferase and the substrate luciferin. In beetles, the enzyme luciferase has been extensively studied, with significant enzymological, sequence and structural data now available. Furthermore, the enzyme has been employed in a remarkable number of important applications, from microbial detection and medical imaging to GM gene expression studies. However, there is little information regarding the biosynthesis of beetle luciferin, and here we review the literature and speculate as to its evolutionary origins. Luciferin consists of a benzothiazole moiety attached to a thiazole carboxylic acid moiety, the former being rarely observed in nature but the latter being observed in a broad range of biologically derived molecules. Benzothiazoles are, however, observed in melanogenesis and we speculate as to whether this may be relevant to the understanding of luciferin biosynthesis in beetles. This review examines recent novel insights into beetle luciferin recycling and we assess a range of possible biosynthetic mechanisms.

The microstructural change, crack initiation and spallation of a vacuum plasma sprayed (VPS) thermal barrier coating on an INCONEL-738 superalloy substrate were investigated after successive 300 h thermal cycles at 1050 °C. The coating was characterised using Raman spectroscopy, scanning electron microscopy (SEM) energy dispersive X-ray analysis (EDX) and Auger electron spectroscopy (AES). Localised micro-cracks at the yttrium (III) oxide stabilised zirconium (IV) oxide (YSZ) ceramic coating/thermally grown oxide (TGO) interface were observed after 8 cycles. Spallation of the YSZ coating occurred after approximately 21 cycles. Significant amounts of the elements titanium, tantalum and chromium were found within the TGO together with the formation of nickel, cobalt and chromium-rich oxides at this TGO/YSZ interface.

The E-cadherin protein mediates Ca(2+)-dependent interepithelial adhesion. Association of E-cadherin with the catenin family of proteins is critical for the maintenance of a functional adhesive complex. We have identified a novel truncated E-cadherin species of 100-kDa (E-cad(100)) in prostate and mammary epithelial cells. E-cad(100) was generated by treatment of cells with ionomycin or TPA. Cell-permeable calpain inhibitors prevented E-cad(100) induction by ionomycin. Immunoblotting for spectrin and mu-calpain confirmed calpain activation in response to ionomycin treatment. Both the mu- and m-isoforms of calpain efficiently generated E-cad(100) in vitro. The E-cad(100) fragment was unable to bind to beta-catenin, gamma-catenin, and p120, suggesting that this cleavage event would disrupt the E-cadherin adhesion complex. Mutational analysis localized the calpain cleavage site to the cytosolic domain upstream of the beta- and gamma-catenin binding motifs of E-cadherin. Because E-cadherin is inactivated in many adenocarcinomas we hypothesized that calpain may play a role in prostate tumorigenesis. A prostate cDNA microarray data base was analyzed for calpain expression in which it was found that m-calpain was up-regulated in localized prostate cancer, and to an even higher degree in metastatic prostate cancer compared with normal prostate tissue. Furthermore, we examined the cleavage of E-cadherin in prostate cancer specimens and found that E-cad(100) accumulated in both localized and metastatic prostate tumors, supporting the cDNA microarray data. These findings demonstrate a novel mechanism by which E-cadherin is functionally inactivated through calpain-mediated proteolysis and suggests that E-cadherin is targeted by calpain during the tumorigenic progression of prostate cancer.