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Objectives This study sought to assess the relationship between mean vs peak calcified plaque density and their impact on calculating coronary artery calcium (CAC) scores and to compare the corresponding differential prediction of atherosclerotic cardiovascular disease (ASCVD) and coronary heart disease (CHD) mortality. Background The Agatston CAC score is quantified per lesion as the product of plaque area and a 4-level categorical peak calcium density factor. However, mean calcium density may more accurately measure the heterogenous mixture of lipid-rich, fibrous, and calcified plaque reflective of ASCVD risk. Methods We included 10,373 individuals from the CAC Consortium who had CAC >0 and per-vessel measurements of peak calcium density factor and mean calcium density. Area under the curve and continuous net reclassification improvement analyses were performed for CHD and ASCVD mortality to compare the predictive abilities of mean calcium density vs peak calcium density factor when calculating the Agatston CAC score. Results Participants were on average 53.4 years of age, 24.4% were women, and the median CAC score was 68 Agatston units. The average values for mean calcium density and peak calcium density factor were 210 ± 50 Hounsfield units and 3.1 ± 0.5, respectively. Individuals younger than 50 years of age and/or those with a total plaque area 100. Conclusion Mean and peak calcium density may differentially describe plaque composition early in the atherosclerotic process. Mean calcium density performs better than peak calcium density factor when combined with plaque area for ASCVD mortality prediction among persons with Agatston CAC 1-99.

The modulator 2-fluorobenzoic acid (2-fba) is widely used to prepare RE clusters in metal-organic frameworks (MOFs). In contrast to known RE MOF structures containing hydroxide bridging groups, we report for the first time the possible presence of fluoro bridging groups in RE MOFs. In this report we discuss the synthesis of a holmium-UiO-66 analogue as well as a novel holmium MOF, where evidence of fluorinated clusters is observed. The mechanism of fluorine extraction from 2-fba is discussed as well as the implications that these results have for previously reported RE MOF structures.

Introduction: Enzymatic cross-linking of the collagens within the extracellular matrix (ECM) catalyzed by enzymes such as lysyl oxidase (LOX) and lysyl oxidase like-enzymes 1–4 (LOXL), transglutaminase 2 (TG2), and peroxidasin (PXDN) contribute to fibrosis progression through extensive collagen cross-linking. Studies in recent years have begun elucidating the important role of collagen cross-linking in perpetuating progression of organ fibrosis independently of inflammation through an increasingly stiff and noncompliant ECM. Therefore, collagen cross-linking and the cross-linking enzymes have become new targets in anti-fibrotic therapy as well as targets of novel biomarkers to properly assess resolution of the fibrotic ECM. Areas covered: The enzymatic actions of enzymes catalyzing collagen cross-linking and their relevance in organ fibrosis. Potential biomarkers specifically quantifying proteolytic fragments of collagen cross-linking is discussed based on Pubmed search done in November 2020 as well as the authors knowledge. Expert opinion: Current methods for the assessment of fibrosis involve the use of invasive and/or cumbersome and expensive methods such as tissue biopsies. Thus, an unmet need exists for the development and validation of minimally invasive biomarkers of proteolytic fragments of cross-linked collagens. These biomarkers may aid in the development and proper assessment of fibrosis resolution in coming years.

The PKC-θ isoform of protein kinase C is selectively expressed in T lymphocytes and plays an important role in the T cell antigen receptor (TCR)-triggered activation of mature T cells, T cell proliferation, and the subsequent release of cytokines such as interleukin-2 (IL-2). Herein, we report the synthesis and structure-activity relationship (SAR) of a novel series of PKC-θ inhibitors. Through a combination of structure-guided design and exploratory SAR, suitable replacements for the basic C4 amine of the original lead (3) were identified. Property-guided design enabled the identification of appropriately substituted C2 groups to afford potent analogs with metabolic stability and permeability to support in vivo testing. With exquisite general kinase selectivity, cellular inhibition of T cell activation as assessed by IL-2 expression, a favorable safety profile, and demonstrated in vivo efficacy in models of acute and chronic T cell activation with oral dosing, CC-90005 (57) was selected for clinical development.

The anionic surfactant sodium dodecyl sulfate (SDS) interacts strongly with most globular proteins and denatures and unfolds them. While scattering studies using X-rays and neutrons have shown that this denaturation generally leads to protein-decorated SDS micelles, a different SDS-decorated polypeptide model has recently been suggested for complexes between SDS and Ubiquitin (UBI), in which individual SDS molecules are distributed on a partially stretched protein. To resolve this apparent discrepancy, we have investigated the SDS-UBI system by a number of complementary techniques. Small-angle X-ray scattering (SAXS) provides the overall structure of the SDS-UBI complexes, Tyr fluorescence and circular dichroism follow changes in tertiary and secondary structure, and isothermal titration calorimetry determines the stoichiometries of complexes and the amount of free SDS as a function of [SDS]. At low [SDS], UBI preserves its folded structure but dimerizes to a small extent. At 4 SDS per UBI, a complex is formed with two UBI and a small shared SDS cluster with 8 SDS molecules. In these complexes UBI preserves most of its native fold. At 10-12 SDS per UBI, which remains below the critical micelle concentration under our conditions, UBI-covered SDS micelles form with four UBIs around a core of 40 SDSs. This implies a protein-assisted micellization and an associated unfolding of UBI involving a change from mainly β-strands to mainly α-helical secondary structure. As [SDS] is increased, the complex gradually changes so that finally only one UBI covers one micelle with a similar number of SDS molecules at SDS saturation. Thus, we conclude that SDS unfolds UBI by mechanisms very similar to those observed for other globular proteins, leading to a protein-decorated SDS micelle rather than an SDS-decorated unfolded polypeptide chain.

Aim: To investigate the effect of femoral head size on blood metal-ion levels caused by taper corrosion in metal-on-polyethylene total hip arthroplasty, comparing 36- to 44-mm heads with 32-mm heads. Methods: In a randomised, controlled, single-blinded trial, 96 patients were allocated to receive either a 32-mm metal head or the largest possible metal head (36–44 mm) that could be accommodated in the thinnest available vitamin E, cross-linked polyethylene insert. Blood metal ion levels were collected at 1- and 2-year follow-ups. Results: At 1-year, metal-ion levels did not differ between the groups. The median (interquartile range) blood-ion levels for the 32-mm versus the 36- to 44-mm group were 0.11 µg/L (0.08–0.15) versus 0.12 µg/L (0.08–0.22), p = 0.546, for cobalt, 0.50 µg/L (0.50–0.59) versus 0.50 µg/L (0.50–1.20), p = 0.059, for chromium and 1.58 µg/L (1.38–2.05) versus 1.48 µg/L (1.14–1.87), p = 0.385, for titanium. At 2 years, there was no difference either and the corresponding values were 0.15 µg/L (0.12–0.24) versus 0.18 µg/L (0.12–0.28), p = 0.682 for cobalt, 0.50 µg/L (0.50–0.50) versus 0.50 µg/L (0.50–0.57), p = 0.554, for chromium and 1.54 µg/L (1.16–1.87) versus 1.42 µg/L (1.01–1.72), p = 0.207 for titanium. Conclusions: The use of the largest possible metal head (36–44 mm) compared to a 32-mm head in metal–on-polyethylene bearings does not appear to elevate blood metal-ion levels up to 2 years postoperatively. As taper corrosion is probably time-dependent, longer-term reports are needed to evaluate the association between large metal heads and blood metal ion levels. Trial registration: ClinicalTrials.gov (reg. ID NCT0231 6704)

Feasible process conditions for reforming of gas mixtures of CH4 and CO2 into CO-rich synthesis gas are dictated by the risk of carbon formation. To overcome this risk, a minimum steam requirement exists for the process as determined by thermodynamic considerations. This work describes an approach for using a well-known nickel-based catalyst system to enable production of CO-rich synthesis gas at low steam-to-hydrocarbon-carbon (S/C) ratios. The concept utilizes a two-reactor approach where traditional steam reforming is done initially, followed by CO2 addition to the hot synthesis gas and subsequent conversion in an adiabatic reactor, a so-called Adiabatic POst Converter (APOC). This allows for operating at overall S/C ratios of below 1.5; which is much lower than what is possible in a conventional fired reformer. The central element of the APOC is that the temperature is kept higher than 700–800 °C to circumvent the carbon limits. The process elements were demonstrated by a combination of thermodynamic calculations, process calculations, and laboratory experiments. The concept allows for design of significantly more compact reformers (up to 30 % reduced size), which in turn also reduces firing and thereby CO2 emissions. The concept can also be used in existing plants where the capacity can be boosted by adding an APOC.

In recent years, there has been an increasing demand for the replacement of synthetic food colorants with naturally derived alternatives. Filamentous fungi are prolific producers of secondary metabolites including polyketide-derived pigments, many of which have not been fully characterized yet. During our ongoing investigations of black aspergilli, we noticed that Aspergillus homomorphus turned yellow when cultivated on malt extract agar plates. Chemical discovery guided by UV and MS led to the isolation of two novel yellow natural products, and their structures were elucidated as aromatic α-pyrones homopyrones A (1) and B (2) by HRMS and NMR. Combined investigations including retro-biosynthesis, genome mining, and gene deletions successfully linked both compounds to their related biosynthetic gene clusters. This demonstrated that homopyrones are biosynthesized by using cinnamoyl-CoA as the starter unit, followed by extension with three malonyl-CoA units, and lactonization to give the core hybrid backbone structure. The polyketide synthase AhpA includes a C-methylation domain, which however seems to be promiscuous since only 2 is C-methylated. Altogether, the homopyrones represent a rare case of hybrid phenylpropanoid- and polyketide-derived natural products in filamentous fungi. KEY POINTS: • Homopyrones represent a rare type of fungal polyketides synthesized from cinnamic-CoA. • CRISPR/Cas9 technology has been firstly applied in Aspergillus homomorphus.

DNA-encoded chemical libraries are typically screened against purified protein targets. Recently, cell-based selections with encoded chemical libraries have been described, commonly revealing suboptimal performance due to insufficient recovery of binding molecules. We used carbonic anhydrase IX (CAIX)-expressing tumor cells as a model system to optimize selection procedures with code-specific quantitative polymerase chain reaction (qPCR) as selection readout. Salt concentration and performing PCR on cell suspension had the biggest impact on selection performance, leading to 15-fold enrichment factors for high-affinity monovalent CAIX binders (acetazolamide; KD=8.7 nM). Surprisingly, the homobivalent display of acetazolamide at the extremities of both complementary DNA strands led to a substantial improvement of both ligand recovery and enrichment factors (above 100-fold). The optimized procedures were used for selections with a DNA-encoded chemical library comprising 1 million members against tumor cell lines expressing CAIX, leading to a preferential recovery of known and new ligands against this validated tumor-associated target. This work may facilitate future affinity selections on cells against target proteins which might be difficult to express otherwise. © 2021 Wiley-VCH GmbH ISSN:0947-6539 ISSN:1521-3765

Sufficient nitrogen (N) supply is decisive to achieve high grass seed yields while overfertilization will lead to negative environmental impact. From the literature, estimation of N rates taking into account the crop’s N status and its yield potential, seems promising for attaining high yields and averting adverse environmental impacts. This study aimed at an evaluation of remote sensing to predict final seed yield, N traits of the grass seed crop and the usability of nitrogen nutrition index (NNI) to measure additional N requirement. It included four years’ data and eight N application rates and strategies. Several reflectance measurements were made and used for the calculation of 18 vegetation indices. The predictions were made using partial least square regression and support vector machine. Three different yield responses to N fertilization were noted; one with linear response, one with optimum economic nitrogen (EON) at ~188 kg N ha−1, and one with EON at ~138 kg N ha−1. We conclude that although it is possible to make in-season predictions of NNI, it does not always portray the differences in yield potential; thus, it is challenging to utilize it to optimize N application.

The present opinion deals with the re‐evaluation of thaumatin (E 957) when used as a food additive. Thaumatin is a natural plant protein, consisting of thaumatin I and thaumatin II proteins together with minor amounts of plant constituents, obtained by acidic aqueous extraction of the arils of the fruit of Thaumatococcus daniellii plant. The Panel followed the conceptual framework for the risk assessment of certain food additives and considered that thaumatin is a digestible protein; adequate exposure estimates were available; there was no concern with respect to the genotoxicity; no conclusion on oral allergenicity could be drawn from the available human data; no adverse effects were observed in sub‐chronic toxicity studies in rats and dogs at the highest dose tested of up 5,200 and 1,476 mg/kg bodyweight (bw) per day, respectively, and in a prenatal developmental toxicity study up to 2,000 mg/kg bw per day; moderate confidence in the body of evidence supported the absence of association between exposure to thaumatin and adverse health outcomes. Therefore, the Panel concluded that there is no need for a numerical acceptable daily intake (ADI) for thaumatin (E 957) and, based on a margin of safety (MOS) of 5,417, considered to be an underestimate and derived using the highest 95th percentile (P95) exposure of 0.48 mg/kg bw per day in consumers only, there is no safety concern for thaumatin (E 957) at the regulatory maximum level exposure assessment scenario, which was considered the most appropriate. The Panel recommended that European Commission considers introducing in the EU specifications for thaumatin (E 957) a new specification limit for the minimum combined content of thaumatin I and II proteins in E 957, a specification limit for yeast, mould counts and Salmonella spp and lowering the existing maximum limit for arsenic along with the inclusion of maximum limits for mercury and cadmium., This publication is linked to the following EFSA Supporting Publications article: http://onlinelibrary.wiley.com/doi/10.2903/sp.efsa.2021.EN-6918/full

Mono- and diglycerides of fatty acids (E 471) was re-evaluated in 2017 by the former EFSA Panel on Food Additives and Nutrient sources added to Food (ANS). As a follow-up to this assessment, the Panel on Food Additives and Flavouring was requested to assess mono- and diglycerides of fatty acids (E 471) for its use as food additive in food for infants below 16 weeks of age belonging to food categories 13.1.1 (Infant formulae) and 13.1.5.1 (Dietary foods for infants for special medical purposes and special formulae for infants). In addition, the FAF Panel was requested to address the issues already identified during the re-evaluation of the food additive in 2017 when used in food for the general population. The Panel considered that there is no indication of adverse effects from the available animal studies at the highest dose tested and from the post marketing data. A comparison was made between the daily exposure to the sum of mono- and di-acylglycerols from breast milk and that resulting from the use of E 471 in the infant formula. The Panel noted that the resulting exposures are in the same order of magnitude. Overall, the Panel concluded that there is no reason for a safety concern when E 471 used as food additive in FC 13.1.1 and 13.1.5.1 and according to the Annex III to Regulation (EC) No 1333/2008. The risk assessment for toxic elements and impurities clearly indicated the need to lower the current maximum limits for arsenic, lead, cadmium and mercury and to include limits for glycidyl esters, 3-monochloropropane diol and erucic acid in the EU specifications of E 471. © 2021 European Food Safety Authority. EFSA Journal published by John Wiley and Sons Ltd on behalf of European Food Safety Authority

Cross-linked Tetronic star block copolymer gels, based on poly(ethylene oxide) and poly(propylene oxide), behave quite regular with respect to mechanical properties, but exhibits unusual absence of structural response to strain. The elastic response is linear up to more than 100% strain, with a steady-state modulus of the order of 0.01 MPa after an initial stress relaxation. Neutron and X-ray scattering experiments show a consistent but unexpected response to uniaxial strain, with no changes in characteristic molecular dimensions. Upon strain beyond about 100%, that is, when the stress-strain curve is no longer linear, structural texture appears and becomes even more pronounced upon further strain, thus, indicating alignment of the self-assembled hexagonally ordered cylindrical micelles with the cylinder-axis perpendicular to the strain. It is proposed that the main structural response to large-amplitude strain is related to a layer-dominated structure of cross-linked star molecules.

Presence of an ultra high molecular weight (UHMw) fraction in flowing polymer melts is known to facilitate formation of oriented crystalline structures significantly. The UHMw fraction manifests itself as a minor tail in the molar mass distribution and is hardly detectable in the canonical characterization methods. In this study, alternatively, we demonstrate how the nonlinear extensional rheology reveals to be a very sensitive characterization tool for investigating the effect of the UHMw-tail on the structural ordering mechanism. Samples containing a UHMw-tail relative to samples without, exhibit a clear increase in extensional stress that is directly correlated with the crystalline orientation of the quenched samples. Extensional rheology, particularly, in combination with linear creep measurements, thus, enables the conformational evolution of the UHMw-tail to be studied and linked to the enhanced formation of oriented structures.

Adding small amounts of ring polymers to a matrix of their linear counterparts is known to increase the zero-shear-rate viscosity because of linear-ring threading. Uniaxial extensional rheology measurements show that, unlike its pure linear and ring constituents, the blend exhibits an overshoot in the stress growth coefficient. By combining these measurements with ex-situ small-angle neutron scattering and nonequilibrium molecular dynamics simulations, this overshoot is shown to be driven by a transient threading–unthreading transition of rings embedded within the linear entanglement network. Prior to unthreading, embedded rings deform affinely with the linear entanglement network and produce a measurably stronger elongation of the linear chains in the blend compared to the pure linear melt. Thus, rings uniquely alter the mechanisms of transient elongation in linear polymers.

Background: Electronic cigarettes (e-cigarettes) generally have a more favorable toxicant profile than conventional cigarettes; however, limited information exists for women of reproductive age (WRA). Our aim was to compare biomarkers of toxicant exposure, inflammation, and oxidative stress among WRA who self-report exclusive e-cigarette use, exclusive cigarette smoking, or never tobacco use (controls). Methods: Multivariable linear regression models were used to compare the geometric means of urinary biomarkers of toxicant exposure and their metabolites, serum markers of inflammation [highly sensitive C-reactive protein, soluble intercellular adhesion molecule (sICAM), interleukin 6, fibrinogen], and a measurement of oxidative stress [prostaglandin F2a-8-isoprostane (F2PG2a)] among WRA from the Population Assessment of Tobacco and Health survey. Results: E-cigarette users had higher levels of lead, tobacco-specific nitrosamines, nicotine metabolites, and some volatile organic compounds (VOCs) than controls. Except for cadmium and lead, e-cigarette users had lower levels of the analyzed urinary toxicant biomarkers compared with cigarette smokers. Cigarette smokers had higher levels of all the biomarkers of toxicant exposure than controls. There were no significant differences in the levels of markers of inflammation and oxidative stress between e-cigarette users and controls. E-cigarette users and controls had lower levels of sICAM and F2PG2a than cigarette smokers. Conclusion: WRA who use e-cigarettes had lower levels of some of the evaluated urinary biomarkers of toxicant exposure and serum biomarkers of inflammation and oxidative stress than those who smoke cigarettes, but higher lead, nicotine metabolites, and some VOCs than controls, which can increase health risks.

PET/computed tomography (CT) imaging increasingly is used in neuroendocrine neoplasms (NENs) for diagnosis, staging, monitoring, prognostication, and choosing treatment. Somatostatin PET analog tracers have added to the specificity by obtaining higher affinity to somatostatin receptors with 68Ga-labeled or 64Cu-labeled DOTA peptides compared with single-photon emission CT imaging isotopes. PET uptake correlates to tumor grade and is an essential part of theranostics with peptide receptor radionuclide treatment. This article focuses on the literature on head-to-head studies and meta-analyses of different combinations of peptide agonists and a few antagonists. Overall, the published data support the diagnostic capability of PET/CT imaging in NENs.

Synthetic mimics of antimicrobial peptides (AMPs) is a promising class of molecules for a variety of antimicrobial applications. Several hurdles must be passed before effective systemic infection therapies with AMPs can be achieved, but the path to effective topical treatment of skin, nail, and soft tissue infections appears less challenging to navigate. Skin and soft tissue infection is closely coupled to the emergence of antibiotic resistance and represents a major burden to the healthcare system. The present study evaluates the promising synthetic cationic AMP mimic, AMC-109, for treatment of skin infections in vivo. The compound is evaluated both in impregnated cotton wound dressings and in a gel formulation against skin infections caused by Staphylococcus aureus and methicillin resistant S. aureus. Both the ability to prevent colonization and formation of an infection, as well as eradicate an ongoing infection in vivo with a high bacterial load, were evaluated. The present work demonstrates that AMC-109 displays a significantly higher antibacterial activity with up to a seven-log reduction in bacterial loads compared to current clinical standard therapy; Altargo cream (1% retapamulin) and Fucidin cream (2% fusidic acid) in the in vivo wound models. It is thus concluded that AMC-109 represents a promising entry in the development of new and effective remedies for various skin infections.

Context The calcium-sensing receptor (CaSR) is essential to maintain a stable calcium concentration in serum. Spermatozoa are exposed to immense changes in concentrations of CaSR ligands such as calcium, magnesium, and spermine during epididymal maturation, in the ejaculate, and in the female reproductive environment. However, the role of CaSR in human spermatozoa is unknown. Objective This work aimed to investigate the role of CaSR in human spermatozoa. Methods We identified CaSR in human spermatozoa and characterized the response to CaSR agonists on intracellular calcium, acrosome reaction, and 3′,5′-cyclic adenosine 5′-monophosphate (cAMP) in spermatozoa from men with either loss-of-function or gain-of-function mutations in CASR and healthy donors. Results CaSR is expressed in human spermatozoa and is essential for sensing extracellular free ionized calcium (Ca2+) and Mg2+. Activators of CaSR augmented the effect of sperm-activating signals such as the response to HCO3– and the acrosome reaction, whereas spermatozoa from men with a loss-of-function mutation in CASR had a diminished response to HCO3–, lower progesterone-mediated calcium influx, and were less likely to undergo the acrosome reaction in response to progesterone or Ca2+. CaSR activation increased cAMP through soluble adenylyl cyclase (sAC) activity and increased calcium influx through CatSper. Moreover, external Ca2+ or Mg2+ was indispensable for HCO3– activation of sAC. Two male patients with a CASR loss-of-function mutation in exon 3 presented with normal sperm counts and motility, whereas a patient with a loss-of-function mutation in exon 7 had low sperm count, motility, and morphology. Conclusion CaSR is important for the sensing of Ca2+, Mg2+, and HCO3– in spermatozoa, and loss-of-function may impair male sperm function.

The single nucleotide polymorphism rs7804356 located in the Src kinase-associated phosphoprotein 2 (SKAP2) gene is associated with type 1 diabetes (T1D), suggesting SKAP2 as a causal candidate gene. The objective of the study was to investigate if SKAP2 has a functional role in the b-cells in relation to T1D. In a cohort of children with newly diagnosed T1D, rs7804356 predicted glycemic control and residual b-cell function during the 1st year after diagnosis. In INS-1E cells and rat and human islets, proinflammatory cytokines reduced the content of SKAP2. Functional studies revealed that knockdown of SKAP2 aggravated cytokine-induced apoptosis in INS-1E cells and primary rat b-cells, suggesting an antiapoptotic function of SKAP2. In support of this, overexpression of SKAP2 afforded protection against cytokine-induced ap-optosis, which correlated with reduced nuclear content of S536-phosphorylated nuclear factor-kB (NF-kB) subunit p65, lower nitric oxide production, and diminished CHOP expression indicative of decreased endoplasmic reticu-lum stress. Knockdown of CHOP partially counteracted the increase in cytokine-induced apoptosis caused by SKAP2 knockdown. In conclusion, our results suggest that SKAP2 controls b-cell sensitivity to cytokines possibly by affecting the NF-kB–inducible nitric oxide synthase– endoplasmic reticulum stress pathway., SCOPUS: ar.j, info:eu-repo/semantics/published

There is a growing interest in the antibody-based delivery of cytokines to the tumor environment as a means to boost the anti-cancer activity of tumor-resident T cells and NK cells. Here, we describe the expression and characterization of fusion proteins, featuring the L19 antibody (specific to the alternatively-spliced EDB domain of fibronectin) and an engineered cytokine with interleukin-2 and interleukin-15 properties. The cytokine moiety was fused either at the N-terminal or at the C-terminal extremity and both fusion proteins showed a selective tumor accumulation in a quantitative biodistribution experiment. The N-terminal fusion inhibited tumor growth in immunocompetent mice bearing F9 carcinomas or WEHI-164 sarcomas when used as single agent. The anticancer activity was compared to the one of the same cytokine payload used as recombinant protein or fused to an anti-hen egg lysozyme antibody, serving as negative control of irrelevant specificity in the mouse. These results indicate that the antibody-based delivery of engineered cytokines to the tumor neovasculature may mediate a potent anticancer activity.

BACKGROUND: Findings of historical studies suggest that elevated LDL cholesterol is not associated with increased risk of myocardial infarction and atherosclerotic cardiovascular disease in patients older than 70 years. We aimed to test this hypothesis in a contemporary population of individuals aged 70-100 years.METHODS: We included in our analysis individuals (aged 20-100 years) from the Copenhagen General Population Study (CGPS) who did not have atherosclerotic cardiovascular disease or diabetes at baseline and who were not taking statins. Standard hospital assays were used to measure LDL cholesterol. We calculated hazard ratios (HRs) and absolute event rates for myocardial infarction and atherosclerotic cardiovascular disease, and we estimated the number needed to treat (NNT) in 5 years to prevent one event.FINDINGS: Between Nov 25, 2003, and Feb 17, 2015, 91 131 individuals were enrolled in CGPS. During mean 7·7 (SD 3·2) years of follow-up (to Dec 7, 2018), 1515 individuals had a first myocardial infarction and 3389 had atherosclerotic cardiovascular disease. Risk of myocardial infarction per 1·0 mmol/L increase in LDL cholesterol was augmented for the overall population (HR 1·34, 95% CI 1·27-1·41) and was amplified for all age groups, particularly those aged 70-100 years. Risk of atherosclerotic cardiovascular disease was also raised per 1·0 mmol/L increase in LDL cholesterol overall (HR 1·16, 95% CI 1·12-1·21) and in all age groups, particularly those aged 70-100 years. Risk of myocardial infarction was also increased with a 5·0 mmol/L or higher LDL cholesterol (ie, possible familial hypercholesterolaemia) versus less than 3·0 mmol/L in individuals aged 80-100 years (HR 2·99, 95% CI 1·71-5·23) and in those aged 70-79 years (1·82, 1·20-2·77). Myocardial infarction and atherosclerotic cardiovascular disease events per 1000 person-years for every 1·0 mmol/L increase in LDL cholesterol were highest in individuals aged 70-100 years, with number of events lower with younger age. The NNT in 5 years to prevent one myocardial infarction or atherosclerotic cardiovascular disease event if all people were given a moderate-intensity statin was lowest for individuals aged 70-100 years, with the NNT increasing with younger age.INTERPRETATION: In a contemporary primary prevention cohort, people aged 70-100 years with elevated LDL cholesterol had the highest absolute risk of myocardial infarction and atherosclerotic cardiovascular disease and the lowest estimated NNT in 5 years to prevent one event. Our data are important for preventive strategies aimed at reducing the burden of myocardial infarction and atherosclerotic cardiovascular disease in the growing population aged 70-100 years.FUNDING: None.

The present opinion deals with an updated safety assessment of the food additive titanium dioxide(E 171) based on new relevant scientific evidence considered by the Panel to be reliable, includingdata obtained with TiO2nanoparticles (NPs) and data from an extended one-generation reproductivetoxicity (EOGRT) study. Less than 50% of constituent particles by number in E 171 have a minimumexternal dimension30 nm) up to the highest dose tested of100 mg/kg bw per day. No effects on reproductive and developmental toxicity were observed up to adose of 1,000 mg E 171/kg bw per day, the highest dose tested in the EOGRT study. However,observations of potential immunotoxicity and inflammation with E 171 and potential neurotoxicity withTiO2NPs, together with the potential induction of aberrant crypt foci with E 171, may indicate adverseeffects. With respect to genotoxicity, the Panel concluded that TiO2particles have the potential toinduce DNA strand breaks and chromosomal damage, but not gene mutations. No clear correlationwas observed between the physico-chemical properties of TiO2particles and the outcome of eitherin vitroorin vivogenotoxicity assays. A concern for genotoxicity of TiO2particles that may be presentin E 171 could therefore not be ruled out. Several modes of action for the genotoxicity may operate inparallel and the relative contributions of different molecular mechanisms elicited by TiO2particles arenot known. There was uncertainty as to whether a threshold mode of action could be assumed. Inaddition, a cut-off value for TiO2particle size with respect to genotoxicity could not be identified. Noappropriately designed study was available to investigate the potential carcinogenic effects of TiO2NPs. Based on all the evidence available, a concern for genotoxicity could not be ruled out, and giventhe many uncertainties, the Panel concluded that E 171 can no longer be considered as safe whenused as a food additive.©2021 European Food Safety Authority. EFSA Journalpublished by John Wiley and Sons Ltd on behalfof European Food Safety Authority.Keywords:Titanium dioxide, E 171, CAS No 13463-67-7EFSA Journal 2021;19(5):6585www.efsa.europa.eu/efsajournal

The reinforcing efficacy of cocaine is largely determined by its capacity to inhibit the dopamine transporter (DAT), and emerging evidence suggests that differences in cocaine potency are linked to several symptoms of cocaine use disorder. Despite this evidence, the neural processes that govern cocaine potencyin vivoremain unclear. In male rats, we used chemogenetics with intra-VTA microinfusions of the agonist clozapine-n-oxide to bidirectionally modulate dopamine neurons. Usingex vivofast scan cyclic voltammetry, pharmacological probes of the DAT, biochemical assessments of DAT membrane availability and phosphorylation, and cocaine self-administration, we tested the effects of chemogenetic manipulations on cocaine potency at distal DATs in the nucleus accumbens as well as the behavioral economics of cocaine self-administration. We discovered that chemogenetic manipulation of dopamine neurons produced rapid, bidirectional modulation of cocaine potency at DATs in the nucleus accumbens. We then provided evidence that changes in cocaine potency are associated with alterations in DAT affinity for cocaine and demonstrated that this change in affinity coincides with DAT conformation biases and changes in DAT phosphorylation state. Finally, we showed that chemogenetic manipulation of dopamine neurons alters cocaine consumption in a manner consistent with changes in cocaine potency at distal DATs. Based on the spatial and temporal constraints inherent to our experimental design, we posit that changes in cocaine potency are driven by alterations in dopamine neuron activity. When considered together, these observations provide a novel mechanism through which GPCRs regulate cocaine's pharmacological and behavioral effects.SIGNIFICANCE STATEMENTDifferences in the pharmacological effects of cocaine are believed to influence the development and progression of cocaine use disorder. However, the biological and physiological processes that determine sensitivity to cocaine remain unclear. In this work, we use a combination of chemogenetics, fast scan cyclic voltammetry, pharmacology, biochemistry, and cocaine self-administration with economic demand analysis to demonstrate a novel mechanism by which cocaine potency is determinedin vivo. These studies identify a novel process by which the pharmacodynamics of cocaine are derivedin vivo, and thus this work has widespread implications for understanding the mechanisms that regulate cocaine consumption across stages of addiction.

Metal organic frameworks (MOFs) have garnered increased attention over the past 20 years. Due to their porosity, high surface area, and nearly limitless customization and tunability MOFs have been designed for applications ranging from gas storage and separation to catalysis to sensing to biomedical engineering. Within the latter category, MOFs offer an appealing function for drug delivery as they can be loaded with multiple therapeutic moieties tailored to target specific disorders with triggered and controlled release characteristics. However, there is an unmet need to assess their viability for pulmonary treatment via inhalation. Targeting pulmonary disorders including infectious diseases by delivering medication directly to the lungs attacks the primary site of infection rather than relying on systemic distribution. The inherent advantage of this strategy is maximizing local lung concentrations of the drug. An introduction to inhaled therapies is provided here as a preamble to a brief summary of the current development state of MOF drug delivery systems. This review is intended to highlight the relative disparity between research toward MOFs as pulmonary drug delivery vehicles compared to other delivery platforms. Prospective biomedical applications for inhalable MOFs are also discussed.

Fluorescence imaging of immunolabeled brain slices is a key tool in neuroscience that enable mapping of proteins or DNA/RNA at resolutions not possible with non-invasive techniques, including magnetic resonance or nuclear imaging. The signal in specific regions is usually quantified after manually drawing regions of interest, risking operator-bias. Automated segmentation methods avoid this risk but require multi-sample average atlases with similar image contrast as the images to be analyzed. We here present the first population-based average atlas of the C57BL/6 mouse brain constructed from brain sections labeled with the fluorescence nuclear stain DAPI. The data set constitutes a rich three-dimensional representation of the average mouse brain in the DAPI staining modality reconstructed from coronal slices and includes an automatic segmentation/spatial normalization pipeline for novel coronal slices. It constitutes the final population-based average template, individual reconstructed brain volumes, and native coronal slices. The comprehensive data set and accompanying spatial normalization/segmentation software are provided. We encourage the community to utilize it to improve and validate methods for automated brain slice analysis., Measurement(s) brain measurement Technology Type(s) epifluorescence microscopy • magnetic resonance imaging (MRI) Factor Type(s) brain region Sample Characteristic - Organism Mus musculus • C57BL/6 mouse strain Machine-accessible metadata file describing the reported data: 10.6084/m9.figshare.12554666

Surface modification by different quaternary ammonium compounds (QAC) makes nanoclays more compatible with various polymeric matrices, thereby expanding their potential applications. The growing industrial use of nanoclays could potentially pose a health risk for workers. Here, we assessed how surface modification of nanoclays modulates their pulmonary toxicity. An in vitro screening of the unmodified nanoclay Bentonite (montmorillonite) and four organomodified nanoclays (ONC); coated with various QAC, including benzalkonium chloride (BAC), guided the selection of the materials for the in vivo study. Mice were exposed via a single intratracheal instillation to 18, 54, and 162 µg of unmodified Bentonite or dialkyldimethyl-ammonium-coated ONC (NanofilSE3000), or to 6, 18, and 54 µg of a BAC-coated ONC (Nanofil9), and followed for one, 3, or 28 days. All materials induced dose- and time-dependent responses in the exposed mice. However, all doses of Bentonite induced larger, but reversible, inflammation (BAL neutrophils) and acute phase response (Saa3 gene expression in lung) than the two ONC. Similarly, highest levels of DNA strand breaks were found in BAL cells of mice exposed to Bentonite 1 day post-exposure. A significant increase of DNA strand breaks was detected also for NanofilSE3000, 3 days post-exposure. Only mice exposed to Bentonite showed increased Tgf-β gene expression in lung, biomarker of pro-fibrotic processes and hepatic extravasation, 3 days post-exposure. This study indicates that Bentonite treatment with some QAC changes main physical-chemical properties, including shape and surface area, and may decrease their pulmonary toxicity in exposed mice.

We explore the influence (i) of the interaction between aluminium and alumina, and (ii) of sodium impurities present in Bayer alumina, on the pressure infiltration of alumina particle preforms with molten aluminium. At 1000°C or above, although the aluminium/alumina system is non-reactive, capillarity-driven solution-reprecipitation processes cause the liquid-solid interface to become mobile. Data show that this can result in infiltration kinetics that resemble those observed with reaction-driven pressure infiltration, namely a continuously increasing melt saturation under fixed infiltration pressure. Resulting isobaric saturation velocities are measured at 1000°C, 1050°C and 1100°C. The role of alumina particle shape and of Na-containing inclusions is investigated. It is found that the main factors affecting the rate of high-temperature isobaric infiltration in this system is the particle geometry. Measured steady infiltration rates give an activation volume on the order of ≈ 200 nm3 and an activation energy in the range of 300-500 k J m o l , suggesting that isobaric infiltration kinetics are governed by diffusion through the solid alumina. Sodium impurities of Bayer alumina are present within β″-Al2O3. They do not influence steady pressure infiltration but ease initial melt penetration into the preform, possibly because evaporated Na2O alters the oxide skin layer that lines the surface of molten aluminium.

We characterized the global transcriptome of Escherichia coli MG1655:: tetA grown in the presence of ½ MIC (14 mg/L) of OTC, and for comparison WT MG1655 strain grown with 1//2 MIC of OTC (0.25 mg/L OTC). 1646 genes changed expression significantly (FDR > 0.05) in the resistant strain, the majority of which (1246) were also regulated in WT strain. Genes involved in purine synthesis and ribosome structure and function were top-enriched among up-regulated genes, and anaerobic respiration, nitrate metabolism and aromatic amino acid biosynthesis genes among down-regulated genes. Blocking of the purine-synthesis- did not affect resistance phenotypes (MIC and growth rate with OTC), while blocking of protein synthesis using low concentrations of chloramphenicol or gentamicin, lowered MIC towards OTC. Metabolic-modeling, using a novel model for MG1655 and continuous weighing factor that reflected the degree of up or down regulation of genes encoding a reaction, identified 102 metabolic reactions with significant change in flux in MG1655:: tetA when grown in the presence of OTC compared to growth without OTC. These pathways could not have been predicted by simply analyzing functions of the up and down regulated genes, and thus this work has provided a novel method for identification of reactions which are essential in the adaptation to growth in the presence of antimicrobials.

Shewanella woodyi cultures were used to correlate bioluminescence intensity with changes in the electrochemical potential of a saltwater medium using soluble electron acceptors. A relationship between the concentration of NaNO3 or CoCl2 to bioluminescence intensity was confirmed using aerobic cultures of S. woodyi at 20°C with glucose as the sole carbon source. In general, increasing the concentration of nitrate or Co(II) reduced the bioluminescence per cell, with complete luminescence being repressed at ≥5 mM nitrate and ≥0.5 mM Co(II). Results from cell viability fluorescent staining concluded that increasing the concentration of Co(II) or nitrate did not affect the overall viability of the cells when compared with cultures lacking Co(II) or nitrate. These data show that potentials of

Marine n-3 PUFA exert beneficial effects that might inhibit atherosclerosis and reduce vascular disease. Previous studies have, however, reported conflicting results and here we have summarised the early history and the most recent findings from follow-up studies and randomised clinical trials investigating marine n-3 PUFA in relation to the risk of atherosclerotic CVD. Most follow-up studies have suggested that the intake of marine n-3 PUFA may be associated with a lower risk of CVD. Recent studies have also shown that it is important to focus on substitution issues and dietary patterns. Further, the use of gold standard biomarkers of fatty acid exposure such as adipose tissue should be encouraged. Findings from clinical supplemental trials have shown conflicting results and findings from previous meta-analyses and guidelines have generally not supported the use of fish oil supplements for the prevention of CVD. However, a recent meta-analysis including three recent large clinical trials with fish oil supplements reported a moderate beneficial effect on cardiovascular endpoints. Interestingly, results from a large clinical trial (REDUCE-IT) have suggested that supplementation with a high dose of purified EPA ethyl ester for 4⋅9 years significantly and markedly reduced the risk of cardiovascular events in patients with CVD and mild hypertriglyceridaemia; findings that need to be confirmed. While it seems appropriate to recommend consumption of fish, particular fatty fish for prevention of CVD, an effect of fish oil supplements is probably at best marginal perhaps apart from patients with hypertriglyceridaemia.

Oxygen consumption measurement was used to study potential oxidative degradation reactions occurring in wooden artefacts from the Viking age Oseberg collection in Norway. Model samples of fresh birch were impregnated with iron, calcium and alum salts to mimic concentrations of such compounds found in Oseberg artefacts and to assess their effect on oxygen consumption rates. The results showed that heated impregnation with alum salt (KAl(SO4)2·12H2O) significantly increased the rate of oxygen consumption, confirming a previously observed link between alum-treatment and wood oxidation. The presence of iron salts in alum-treated wood specimens, even at low concentrations, also substantially increased the oxidation rate. However, the mechanism by which this occurred appeared to be influenced by the alum-treatment. Samples treated with both iron and calcium salts were also studied, in order to investigate a proposed inhibition of iron-induced oxidation by calcium ions. However, these did not appear to consume oxygen at significantly different rates. In Oseberg samples, a large variation in oxygen consumption rates from 0.48 to an apparent 8.2 μg O2 (g wood)−1 day−1 was observed, and these values were consistently higher than those for reference fresh wood. The results demonstrated that oxygen consumption measurement is a viable method of evaluating chemical stability in this case, but is best applied to model samples with limited compositional variability., Heritage Science, 8 (1), ISSN:2050-7445