In situ STM study of homoepitaxial electrodeposition on Au(1 0 0)

Peer reviewedPostprin

Familial hypercholesterolaemia in children and adolescents from 48 countries: a cross-sectional study

Background: Approximately 450 000 children are born with familial hypercholesterolaemia worldwide every year, yet only 2·1% of adults with familial hypercholesterolaemia were diagnosed before age 18 years via current diagnostic approaches, which are derived from observations in adults. We aimed to characterise children and adolescents with heterozygous familial hypercholesterolaemia (HeFH) and understand current approaches to the identification and management of familial hypercholesterolaemia to inform future public health strategies. Methods: For this cross-sectional study, we assessed children and adolescents younger than 18 years with a clinical or genetic diagnosis of HeFH at the time of entry into the Familial Hypercholesterolaemia Studies Collaboration (FHSC) registry between Oct 1, 2015, and Jan 31, 2021. Data in the registry were collected from 55 regional or national registries in 48 countries. Diagnoses relying on self-reported history of familial hypercholesterolaemia and suspected secondary hypercholesterolaemia were excluded from the registry; people with untreated LDL cholesterol (LDL-C) of at least 13·0 mmol/L were excluded from this study. Data were assessed overall and by WHO region, World Bank country income status, age, diagnostic criteria, and index-case status. The main outcome of this study was to assess current identification and management of children and adolescents with familial hypercholesterolaemia. Findings: Of 63 093 individuals in the FHSC registry, 11 848 (18·8%) were children or adolescents younger than 18 years with HeFH and were included in this study; 5756 (50·2%) of 11 476 included individuals were female and 5720 (49·8%) were male. Sex data were missing for 372 (3·1%) of 11 848 individuals. Median age at registry entry was 9·6 years (IQR 5·8-13·2). 10 099 (89·9%) of 11 235 included individuals had a final genetically confirmed diagnosis of familial hypercholesterolaemia and 1136 (10·1%) had a clinical diagnosis. Genetically confirmed diagnosis data or clinical diagnosis data were missing for 613 (5·2%) of 11 848 individuals. Genetic diagnosis was more common in children and adolescents from high-income countries (9427 [92·4%] of 10 202) than in children and adolescents from non-high-income countries (199 [48·0%] of 415). 3414 (31·6%) of 10 804 children or adolescents were index cases. Familial-hypercholesterolaemia-related physical signs, cardiovascular risk factors, and cardiovascular disease were uncommon, but were more common in non-high-income countries. 7557 (72·4%) of 10 428 included children or adolescents were not taking lipid-lowering medication (LLM) and had a median LDL-C of 5·00 mmol/L (IQR 4·05-6·08). Compared with genetic diagnosis, the use of unadapted clinical criteria intended for use in adults and reliant on more extreme phenotypes could result in 50-75% of children and adolescents with familial hypercholesterolaemia not being identified. Interpretation: Clinical characteristics observed in adults with familial hypercholesterolaemia are uncommon in children and adolescents with familial hypercholesterolaemia, hence detection in this age group relies on measurement of LDL-C and genetic confirmation. Where genetic testing is unavailable, increased availability and use of LDL-C measurements in the first few years of life could help reduce the current gap between prevalence and detection, enabling increased use of combination LLM to reach recommended LDL-C targets early in life

Robust estimation of bacterial cell count from optical density

Optical density (OD) is widely used to estimate the density of cells in liquid culture, but cannot be compared between instruments without a standardized calibration protocol and is challenging to relate to actual cell count. We address this with an interlaboratory study comparing three simple, low-cost, and highly accessible OD calibration protocols across 244 laboratories, applied to eight strains of constitutive GFP-expressing E. coli. Based on our results, we recommend calibrating OD to estimated cell count using serial dilution of silica microspheres, which produces highly precise calibration (95.5% of residuals <1.2-fold), is easily assessed for quality control, also assesses instrument effective linear range, and can be combined with fluorescence calibration to obtain units of Molecules of Equivalent Fluorescein (MEFL) per cell, allowing direct comparison and data fusion with flow cytometry measurements: in our study, fluorescence per cell measurements showed only a 1.07-fold mean difference between plate reader and flow cytometry data

Gold nanoparticles as electronic bridges for laccase-based biocathodes

Direct electron transfer (DET) reactions between redox enzymes and electrodes can be maximized by oriented immobilization of the enzyme mols. onto an electroactive surface modified with functionalized Au nanoparticles (AuNPs). Here, the authors present such strategy for obtaining a DET-based laccase (Lc) cathode for O2 electroredn. at low overpotentials. The stable nanostructured enzymic electrode is based on the step-by-step covalent attachment of AuNPs and Lc mols. to porous graphite electrodes using the diazonium salt redn. strategy. Oriented immobilization of the enzyme mols. on adequately functionalized AuNPs allows establishing very fast DET with the electrode via their Cu T1 site. The measured electrocatalytic waves of O2 redn. can be deconvoluted into two contributions. The one at lower overpotentials corresponds to immobilized Lc mols. that are efficiently wired by the AuNPs with a heterogeneous electron transfer rate const. k0 » 400 s-1

Combined ATR-SEIRAS and EC-STM study of the immobilization of laccase on chemically modified au electrodes

The successive steps of laccase immobilization on chem. modified Au electrodes were monitored using ATR-SEIRAS and in situ STM. Successful covalent immobilization of the enzyme on Au electrodes modified by a mixed aminophenyl-mercaptohexanol adlayer and on Au electrodes modified by a 4-aminothiophenyl SAM via a Schiff base reaction followed by the formation of amide bonds is revealed by the emergence of the corresponding bands in the ATR-SEIRA spectra, and an enzyme coverage on aminophenyl-mercaptohexanol-modified Au electrodes of about (7.27 ± 1.93) × 1011 laccase units per cm2 was calcd. from STM images. The small differences between the ATR-SEIRA spectra of the enzyme immobilized on aminophenyl-mercaptohexanol-modified Au electrodes and the ATR-SEIRA spectra of the enzyme immobilized on 4-aminothiophenyl-modified Au electrodes are attributed to a different orientation of the immobilized enzyme due to the presence on the surface of aminophenyl-mercaptohexanol-modified Au electrodes of OH functional groups that favor an orientation of laccase with the Cu T1 center of the enzyme facing the electrode surface, thus, allowing a high activity for direct electrocatalysis of the ORR at low overpotentials

Direct electron transfer reactions between human ceruloplasmin and electrodes.

In an effort to find conditions favouring bioelectrocatalytic reduction of oxygen by surface-immobilised human ceruloplasmin (Cp), direct electron transfer (DET) reactions between Cp and an extended range of surfaces were considered. Exploiting advances in surface nanotechnology, bare and carbon-nanotube-modified spectrographic graphite electrodes as well as bare, thiol- and gold-nanoparticle-modified gold electrodes were considered, and ellipsometry provided clues as to the amount and form of adsorbed Cp. DET was studied under different conditions by cyclic voltammetry and chronoamperometry. Two Faradaic processes with midpoint potentials of about 400 mV and 700 mV vs. NHE, corresponding to the redox transformation of copper sites of Cp, were clearly observed. In spite of the significant amount of Cp adsorbed on the electrode surfaces, as well as the quite fast DET reactions between the redox enzyme and electrodes, bioelectrocatalytic reduction of oxygen by immobilised Cp was never registered. The bioelectrocatalytic inertness of this complex multi-functional redox enzyme interacting with a variety of surfaces might be associated with a very complex mechanism of intramolecular electron transfer involving a kinetic trapping behaviour

Specific and oriented immobilization of proteins on gold nanoparticles

Over the past years, there has been a noticeable interest on the coverage of gold surfaces with monolayers of proteins based on the molecular recognition properties of biological systems T. In this sense, the immobilization of proteins on surfaces retaining their full activity and stability constitutes a challenging goal. Most of the common methods are difficult to control and usually yield randomly bound proteins. On the contrary, an ideal immobilization would produce saturation coverage of specifically bound proteins. The formation of protein layers is induced by anchoring them to gold surfaces functionalized with active molecules, such as transition metal complexes with affinity to repetitive histidine sequences. A feasible method to uniformly cover gold surfaces consists on the self-assembly of thiols by oxidative-chemisorption over the gold. Reversible monolayers of histidine-tagged proteins have been produced using a gold layer covered with a chelator thioalkane monolayer. In order to avoid a highly complex organic synthetic work, step-by-step construction of the functional monolayer over a template of thiocarboxylic acid chemisorbed onto gold has been developed. Because of its simplicity, both, from a conceptual as well as from a practical point of view, the step-by-step synthesis of a functional self assembled monolayer is accessible to most of the laboratories working on enzyme technology in spite of having limited facilities for organic synthesis. This synthetic strategy allows, by a judicious design of the synthetic route, the development of a multiplicity of architectures on SAMs. Different SAM strategies have been developed in our group for controlled and oriented immobilization of enzymes onto gold surfaces, using them as amperometric electrodes in the characterization of the enzymatic catalytic performance. We present a next step of these SAM strategies towards functionalization of gold nanoparticles’ surface for oriented immobilization of model proteins. The SAM provides the ability to discriminate between specific and non-specific proteins attachment

Bioelectrochemical studies of azurin and laccase confined in three-dimensional chips based on gold-modified nano-/microstructured silicon

Double-sided three-dimensional porous silicon chips, 6 mm x 6 mm, covered with a 40 nm gold (nano)layer, were fabricated from a porous silicon wafer. Scanning electron microscopy along with electrochemical characterisation showed sample conductivity, mechanical stability, and high surface area of the thus fabricated devices, viz. 10 times higher electrochemically active surface area compared to the geometric area. The three-dimensional gold coated silicon chips were further modified with thiol layers, followed by immobilisation of a simple copper-containing redox protein, azurin, or a complex multicopper redox enzyme, laccase. The bioelectrochemical studies showed very high surface concentrations of azurin and laccase, i.e. close to the theoretical monolayer coverage. However, direct electron transfer reactions between the biomolecules and gold surfaces were observed only for a small percentage of the immobilised redox protein and enzyme, respectively. Thus, highly efficient oxygen-bioelectroreduction on laccase-modified 3D thiol-gold-porous silicon chips (as compared to planar laccase-modified gold electrodes, 42 mu A/cm(2) vs. 7 mu A/cm(2), respectively) was obtained only in the presence of an efficient soluble redox mediator. (C) 2009 Elsevier B.V. All rights reserved

Laccase electrode for direct electrocatalytic reduction of O2 to H2O with high-operational stability and resistance to chloride inhibition

Laccase from Trametes hirsuta basidiomycete has been covalently bound to graphite electrodes electrochem. modified with Ph derivs. as a way to attach the enzyme mols. with an adequate orientation for direct electron transfer (DET). Current densities up to 0.5 mA/cm2 of electrocatalytic redn. of O2 to H2O were obtained in absence of redox mediators, suggesting preferential orientation of the T1 Cu center of the laccase towards the electrode. The covalent attachment of the laccase mols. to the functionalized electrodes permitted remarkable operational stability. Moreover, O2 bioelectroredn. based on DET between the laccase and the electrode was not inhibited by chloride ions, whereas mediated bioelectrocatalysis was. In contrast, fluoride ions inhibited both direct and mediated electron transfers-based bioelectrocatalytic redn. of O2. Thus, two different modes of laccase inhibition by halides are discussed

Combinatorial saturation mutagenesis of the Myceliophthora thermophila laccase T2 mutant : the connection between the C-terminal plug and the conserved 509VSG511 tripeptide

A mutant laccase from the Ascomycete Myceliophthora thermophila has been submitted to iterative cycles of combinatorial satn. mutagenesis through in vivo overlap extension in Saccharomyces cerevisiae. Over 180,000 clones were explored, among which the S510G mutant revealed a direct interaction between the conserved 509VSG511 tripeptide, located in the neighborhood of the T1 site, and the C-terminal plug. The KmO2 value of the mutant increased 1.5-fold, and the electron transfer pathway between the reducing substrate and the T1 copper ion was altered, improving the catalytic efficiency towards non-phenolic and phenolic substrates by about 3- and 8-fold. Although the geometry at the T1 site was perturbed by the mutation, paradoxically the laccase redox potential was not significantly altered. Together, the results obtained in this study suggest that the 509VSG511 tripeptide may play a hitherto unrecognized role in regulating the traffic of oxygen through the C-terminal plug, the latter blocking access to the T2/T3 copper cluster in the native enzyme