Search

Summary Objective A disintegrin and metalloproteinase with thrombospondin motifs 5 (ADAMTS5) is a key enzyme in degradation of cartilage in osteoarthritis (OA). We report the pharmacological characterization of GLPG1972/S201086, a new, potent and selective small-molecule ADAMTS5 inhibitor. Methods Potency and selectivity of GLPG1972/S201086 for ADAMTS5 were determined using fluorescently-labeled peptide substrates. Inhibitory effects of GLPG1972/S201086 on interleukin-1α-stimulated glycosaminoglycan release in mouse femoral head cartilage explants, and on interleukin-1β-stimulated release of an ADAMTS5-derived aggrecan neoepitope (quantified with ELISA) in human articular cartilage explants were determined. In the destabilization of the medial meniscus (DMM) mouse and menisectomized (MNX) rat models, effects of oral GLPG1972/S201086 on relevant OA histological and histomorphometric parameters were evaluated. Results GLPG1972/S201086 inhibited human and rat ADAMTS5 (IC50 ± SD: 19 ± 2 nM and 5,000-fold selectivity over other related proteases in humans. GLPG1972/S201086 dose-dependently inhibited cytokine-stimulated aggrenolysis in mouse and human cartilage explants (100% at 20 μM and 10 μM, respectively). In DMM mice, GLPG1972/S201086 (30–120 mg/kg b.i.d) versus vehicle reduced femorotibial cartilage proteoglycan loss (23–37%), cartilage structural damage (23–39%) and subchondral bone sclerosis (21–36%). In MNX rats, GLPG1972/S201086 (10–50 mg/kg b.i.d) versus vehicle reduced cartilage damage (OARSI score reduction, 6–23%), and decreased proteoglycan loss (∼27%) and subchondral bone sclerosis (77–110%). Conclusions GLPG1972/S201086 is a potent, selective and orally available ADAMTS5 inhibitor, demonstrating significant protective efficacy on both cartilage and subchondral bone in two relevant in vivo preclinical OA models.

BACKGROUND. Prenatal alcohol exposure (PAE) can produce behavioral deficits in the presence or absence of growth and morphological deficits. Here, we describe a murine PAE model having parallels to the clinical diagnosis of alcohol-related neurodevelopmental deficit (ARND). METHODS. Pregnant C57BL/6J mice were gavaged with alcohol (ALC, 3 g/kg) or maltodextrin daily on embryonic days (E) E8.5 through E17.5. Blood alcohol levels were 211 ± 14 mg/dl at 30 min post-gavage. Offspring behavior was tested at adolescence. RESULTS. ALC dams gained less weight during the alcohol exposure period (p=0.035). ALC male and female pups weighed more than controls at P15 (p≤0.001) and P22 (p≤0.001), but not at P37, perhaps because their dams were pair-housed. During the training session for accelerating rotarod, control offspring trended to stay longer on the rotarod than did ALC (F((1,54)) =2.892, p=0.095). In the Y-maze, ALC offspring had a higher percent alternation than did controls (F((1,54)) =16.577, p

OBJECTIVE The objective of this study was to determine the repellency and efficacy of a 10% imidacloprid/4.5% flumethrin (Seresto® , Elanco) collar over an 8-month period against the eastern paralysis tick (Ixodes holocyclus) on cats. METHODS Two non-blinded, open gender, randomised, placebo-controlled pen studies were conducted, with 26 cats enrolled in each study. Prior to inclusion, cats were immunised with I. holocyclus holocyclotoxin. Cats were treated on Day 0 with either an imidacloprid/flumethrin or placebo collar. Tick infestations with 20 unfed adult female eastern paralysis ticks commenced on Day 7, and were repeated monthly for 8 months. Repellency was determined by comparing the mean number of attached ticks on imidacloprid/flumethrin treated cats, to placebo collar treated cats at 6 and 24 h post infestation. Efficacy was determined by comparing the mean number of live ticks on imidacloprid/flumethrin collar treated cats to placebo collar treated cats at 72 h post infestation. RESULTS Efficacy was 100% (P

Bamboo is a functionally graded material with natural hygroscopicity, and the study of the response of its cell structure to humidity will help expand its use in textiles and engineering structures. For this purpose, in this study, the hygroscopicity of moso bamboo (Phyllostachys edulis) with a gradient fiber structure was assessed by a dynamic vapor sorption (DVS) apparatus and fitted with a Haillwood–Horrobin (H–H) model. The effects of the chemical composition, gradient fiber structure, and mesopore structure of the bamboo were also investigated. The results demonstrate that the hygroscopicity of bamboo gradually increases from the outer to the inner layer along the thickness of the culm wall, which is related to the gradient distribution of the fibers. Based on the H–H model, the hygroscopic mechanism of bamboo was determined as follows. The primary sites derived from amorphous cellulose and hemicellulose act in the low-humidity stage, while the mesopores provide a huge specific surface area (153–132 m2/g), which helps condense water vapor in high-humidity environments. Based on numerical modeling, the hygroscopic ratio of parenchyma cells and fibers was found to be 13:5, indicating that parenchyma cells contribute more to the overall hygroscopicity of moso bamboo. Moreover, the contribution of cellulose to hygroscopicity was found to become gradually higher than that of hemicellulose from the inner to the outer layer due to the increase of the amorphous area (–OH groups). The structure–function relationships between the chemical composition, multi-scale structure, and hygroscopicity identified in this study provide a theoretical basis for bamboo drying and storage technology, as well as the processing and application of bamboo fiber-based composites.

Synucleinopathies are neurodegenerative disorders involving pathological alpha-synuclein (αSyn) protein, including dementia with Lewy bodies, multiple system atrophy and Parkinson's disease (PD). Current in vivo models of synucleinopathy include transgenic mice overexpressing αSyn variants and methods based on administration of aggregated, exogenous αSyn. Combining these techniques offers the ability to study consequences of introducing pathological αSyn into primed neuronal environments likely to develop synucleinopathy. Herein, we characterize the impacts pre-formed fibrils (PFFs) of recombinant, human αSyn have in mice overexpressing human A30P αSyn, a mutation associated with autosomal dominant PD. A30P mouse brain contains detergent insoluble αSyn biochemically similar to PD brain, and these mice develop Lewy-like synucleinopathy with age. Administration of PFFs in A30P mice resulted in regionally-specific accumulations of phosphorylated synuclein, microglial induction and a motor phenotype that differed from PFF-induced effects in wildtype mice. Surprisingly, PFF-induced losses of tyrosine hydroxylase were similar in A30P and wildtype mice. Thus, the PFF-A30P model recapitulates key aspects of synucleinopathy with induction of microglia, creating an appropriate system for evaluating neurodegenerative therapeutics.

Guanosine triphosphate (GTP) cyclohydrolase II (RibA) is one of three enzymes that hydrolytically cleave the C8-N9 bond of the GTP guanine. RibA also catalyzes a subsequent hydrolytic attack at the base liberating formate and in addition cleaves the α-β phosphodiester bond of the triphosphate to form pyrophosphate (PPi). These hydrolytic reactions are promoted by tandem active-site metal ions, zinc and magnesium, that respectively function at the GTP guanine and triphosphate moieties. The RibA reaction is part of riboflavin biosynthesis and forms 2,5-diamino-6-β-pyrimidinone 5'-phosphate, an exocyclic pyrimidine nucleotide that ultimately forms the pyrimidine ring of the isoalloxazine of riboflavin. The stoichiometry of the RibA reaction was defined in the study that first identified this activity in Escherichia coli (Foor, F., Brown, G. M. J. Biol. Chem., 1975, 250, 9, 3545-3551) and has not been quantitatively evaluated in subsequent works. Using primarily transient state approaches we examined the interaction of RibA from E. coli with the GTP, inosine triphosphate, and PPi. Our data indicate that PPi is a slow substrate for RibA that is cleaved to form two phosphate ions (Pi). A combination of real-time enzymatically coupled Pi reporter assays and end-point 31P NMR revealed that Pi is formed at a catalytically relevant rate in the native reaction of RibA with GTP, redefining the reaction stoichiometry. Furthermore, our data indicate that both PPi and GTP stimulate conformational changes prior to hydrolytic chemistry, and we conclude that the cleavage of PPi bound as a substrate or an intermediate state results in conformational relaxation.

Deposition of hyperphosphorylated and aggregated tau protein in the central nervous system is characteristic of Alzheimer disease and other tauopathies. Tau is subject to O-linked N-acetylglucosamine (O-GlcNAc) modification, and O-GlcNAcylation of tau has been shown to influence tau phosphorylation and aggregation. Inhibition of O-GlcNAcase (OGA), the enzyme that removes O-GlcNAc moieties, is a novel strategy to attenuate the formation of pathologic tau. Here we described the in vitro and in vivo pharmacological properties of a novel and selective OGA inhibitor, MK-8719. In vitro, this compound is a potent inhibitor of the human OGA enzyme with comparable activity against the corresponding enzymes from mouse, rat, and dog. In vivo, oral administration of MK-8719 elevates brain and peripheral blood mononuclear cell O-GlcNAc levels in a dose-dependent manner. In addition, positron emission tomography imaging studies demonstrate robust target engagement of MK-8719 in the brains of rats and rTg4510 mice. In the rTg4510 mouse model of human tauopathy, MK-8719 significantly increases brain O-GlcNAc levels and reduces pathologic tau. The reduction in tau pathology in rTg4510 mice is accompanied by attenuation of brain atrophy, including reduction of forebrain volume loss as revealed by volumetric magnetic resonance imaging analysis. These findings suggest that OGA inhibition may reduce tau pathology in tauopathies. However, since hundreds of O-GlcNAcylated proteins may be influenced by OGA inhibition, it will be critical to understand the physiologic and toxicological consequences of chronic O-GlcNAc elevation in vivo. SIGNIFICANCE STATEMENT: MK-8719 is a novel, selective, and potent O-linked N-acetylglucosamine (O-GlcNAc)-ase (OGA) inhibitor that inhibits OGA enzyme activity across multiple species with comparable in vitro potency. In vivo, MK-8719 elevates brain O-GlcNAc levels, reduces pathological tau, and ameliorates brain atrophy in the rTg4510 mouse model of tauopathy. These findings indicate that OGA inhibition may be a promising therapeutic strategy for the treatment of Alzheimer disease and other tauopathies.

Background The essential nutrient choline provides one-carbon units for metabolite synthesis and epigenetic regulation in tissues including brain. Dietary choline intake is often inadequate, and higher intakes are associated with improved cognitive function. Objective Choline supplements confer cognitive improvement for those diagnosed with fetal alcohol spectrum disorder (FASD), a common set of neurodevelopmental impairments; however, the effect sizes have been modest. In this retrospective analysis, we report that genetic polymorphisms affecting choline utilization are associated with cognitive improvement following choline intervention. Methods Fifty-two children from the upper midwestern United States and diagnosed with FASD, ages 2-5 y, were randomly assigned to receive choline (500 mg/d; n = 26) or placebo (n = 26) for 9 mo, and were genotyped for 384 choline-related single nucleotide polymorphisms (SNPs). Memory and cognition were assessed at enrollment, study terminus, and at 4-y follow-up for a subset. Results When stratified by intervention (choline vs. placebo), 14-16 SNPs within the cellular choline transporter gene solute carrier family 44 member 1 (SLC44A1) were significantly associated with performance in an elicited imitation sequential memory task, wherein the effect alleles were associated with the greatest pre-/postintervention improvement. Of these, rs3199966 is a structural variant (S644A) and rs2771040 is a single-nucleotide variant within the 3' untranslated region of the plasma membrane isoform. An additive genetic model best explained the genotype associations. Lesser associations were observed for cognitive outcome and polymorphisms in flavin monooxygenase-3 (FMO3), methylenetetrahydrofolate dehydrogenase-1 (MTHFD1), fatty acid desaturase-2 (FADS2), and adiponectin receptor 1 (ADIPOR1). Conclusions These SLC44A1 variants were previously associated with greater vulnerability to choline deficiency. Our data potentially support the use of choline supplements to improve cognitive function in individuals diagnosed with FASD who carry these effect alleles. Although these findings require replication in both retrospective and prospective confirmatory trials, they emphasize the need to incorporate similar genetic analyses of choline-related polymorphisms in other FASD-choline trials, and to test for similar associations within the general FASD population. This trial was registered at www.clinicaltrials.gov as NCT01149538.

Using optical tweezers, we investigate target search and cleavage by CRISPR–Cas12a on force-stretched λ-DNA. Cas12a uses fast, one-dimensional hopping to locate its target. Binding and cleavage occur rapidly and specifically at low forces (≤5 pN), with a 1.8 nm rate-limiting conformational change. Mechanical distortion slows diffusion, increases off-target binding but hinders cleavage., CRISPR-Cas12a target search and cleavage on force-stretched λ-DNA using optical tweezers.

The effect of frustrated Lewis donors on metal selectivity between actinides and lanthanides was studied using a series of novel organic ligands. Structures and thermodynamic energies were predicted in the gas phase, in water, and in butanol using 9-coordinate, explicitly solvated (H2O) Eu, Gd, Am, and Cm in the +III oxidation state as reactants in the formation of complexes with 2-(6-[1,2,4]-triazin-3-yl-pyridin-2-yl)-1H-indole (Core 1), 3-[6-(2H-pyrazol-3-yl)pyridin-2-yl]-1,2,4-triazine (Core 2), and several derivatives. These complexations were studied using density functional theory (DFT) incorporating scalar relativistic effects on the actinides and lanthanides using a small core pseudopotential and corresponding basis set. A self-consistent reaction field approach was used to model the effect of water and butanol as solvents. Coordination preferences and metal selectivity are predicted for each ligand. Several ligands are predicted to have a high degree of selectivity, particularly when a low ionization potential in the ligand permits charge transfer to Eu(III), reducing it to Eu(II) and creating a half-filled f7 shell. Reasonable separation is predicted between Cm(III) and Gd(III) with Core 1 ligands, possibly due to ligand donor frustration. This separation is largely absent from Core 2 ligands, which are predicted to lose their frustration due to proton transfer from the 2N to the 3N position of the pyrazole component of the ligands via tautomerization.

Background and aims The type of fat consumed in animal-based western diets, typically rich in the saturated fat palmitate, has been implicated in cardiometabolic disease risk. In contrast, the most abundant mono- and polyunsaturated fats, more typical in a vegetarian or plant-based diet, potentiate less deleterious effects. This study determined differences in plasma and urine metabolites when switching from omnivorous to vegetarian diet, including metabolites involved in fatty acid utilization. Methods and results A prospective cohort of 38 European (EA) and African American (AA) omnivorous females were matched by age (25.7 ± 5.3y) and BMI (22.4 ± 1.9 kg/m2). Pre-intervention samples were collected while subjects consumed habitual animal-based diet. Changes in metabolites were assessed by ultra-high-performance liquid chromatography-tandem mass spectroscopy (Metabolon, Inc.) upon completing four days of novel vegetarian diet provided by the Vanderbilt Metabolic Kitchen. Changes in several diet-derived metabolites were observed, including increases in compounds derived from soy food metabolism along with decreases in metabolites of xanthine and histidine. Significant changes occurred in metabolites of saturated, monounsaturated and polyunsaturated fatty acids along with significant differences between EA and AA women in changes in plasma concentrations of acylcarnitines, which reflect the completeness of fatty acid oxidation (versus storage). Conclusion These data suggest improvements in fatty acid metabolism (oxidation vs storage), a key factor in energy homeostasis, may be promoted rapidly by adoption of a vegetarian (plant-based) diet. Mechanistic differences in response to diet interventions must be understood to effectively provide protection against the widespread development of obesity and cardiometabolic disease in population subgroups, such as AA women.

OBJECTIVE To evaluate the efficacy of bovine amniotic membrane homogenate (BAMH) on wounded ex vivo rabbit corneas. PROCEDURE Eighteen corneas obtained from normal rabbit eyes were wounded equally using a 6 mm trephine and cultured into an air-liquid interface model. Corneas were treated with phosphate-buffered saline (PBS) (n = 6, control group), 0.2% ethylenediaminetetraacetic acid (EDTA; n = 6), or BAMH (n = 6). All treatments were applied topically 6 times/day. Each cornea was macrophotographed daily with and without fluorescein stain to assess epithelialization and haziness. After 7 days, corneal transparency was evaluated, and the tissues prepared for histologic analysis of viability, and total and epithelial thickness. RESULTS The mean epithelialization time was 6.2 ± 0.82 days for the control group, 6.2 ± 0.75 days for the EDTA-treated group, and 5.1 ± 0.40 days for the BAMH-treated group, demonstrating a significant difference between the BAMH and the other groups. The corneas that received EDTA had better transparency compared with the other groups. Histologically, all corneas had adequate morphology and architecture after healing. Analysis of corneal and epithelial thickness revealed no significant difference among groups. CONCLUSION Bovine amniotic membrane homogenate is an effective and promising treatment for stromal and epithelial ulcers.

Proton transfer reactions (PTR) have emerged as a powerful tool for the study of intact proteins. When coupled with m/z-selective kinetic excitation, such as parallel ion parking (PIP), one can exert exquisite control over rates of reaction with a high degree of specificity. This allows one to “concentrate”, in the gas-phase, nearly all the signal from an intact protein charge state envelope into a single charge state improving signal-to-noise by 10x or more. While this approach has been previously reported, here we show that implementing these technologies on a 21 T FT-ICR MS provides tremendous advantage for intact protein analysis. Advanced strategies for performing PTR with PIP were developed to complement this unique instrument, including subjecting all analyte ions entering the mass spectrometer to PTR and PIP. This experiment, which we call “PTR-MS(1)-PIP”, generates a pseudo-MS(1) spectrum derived from ions that are exposed to PTR reagent and PIP waveforms, but have not undergone any prior true mass filtering or ion isolation. The result is an extremely rapid and significant improvement in spectral signal-to-noise (S:N) of intact proteins. This permits observation of many more proteoforms and reduces ion injection periods for subsequent tandem mass spectrometry characterization. Additionally, the product ion parking waveform has been optimized to enhance the proton transfer reaction rate without compromise to parking efficiency. We demonstrate that this process, called “rapid park”, can improve reaction rates by 5–10x and explore critical factors discovered to influence this process. Finally, we demonstrate how coupling PTR-MS(1) and rapid park provides a ten-fold reduction in ion injection time, improving the rate of tandem MS sequencing.

The synthesis of a chlorin e6-nevirapine conjugate is reported, in which the nevirapine moiety is attached to the 131-position of chlorin e6 via a diethylene glycol linker. This conjugate was found to be nontoxic in the dark (IC[Formula: see text] > 200 [Formula: see text]M), but highly phototoxic (IC[Formula: see text] = 0.21 [Formula: see text]M at 1.5 J/cm[Formula: see text] toward human HEp2 cells. The chlorin e6-nevirapine conjugate accumulated within cells in multiple organelles, including the Golgi, lysosomes and mitochondria. On the other hand, nevirapine was found to be nontoxic to HEp2 cells.

We expressed a bacterial glucan synthase (Agrobacterium GlgA) in the cytosol of developing endosperm cells in wheat grains, to discover whether it could generate a glucan from cytosolic ADP-glucose. Transgenic lines had high glucan synthase activity during grain filling, but did not accumulate glucan. Instead, grains accumulated very high concentrations of maltose. They had large volumes during development due to high water content, and very shrivelled grains at maturity. Starch synthesis was severely reduced. We propose that cytosolic glucan synthesized by the glucan synthase was immediately hydrolysed to maltose by cytosolic β-amylase(s). Maltose accumulation resulted in a high osmotic potential in developing grain, drawing in excess water that stretched the seed coat and pericarp. Loss of water during grain maturation then led to shrinkage when the grains matured. Maltose accumulation is likely to account for the reduced starch synthesis in transgenic grains, through signalling and toxic effects. Using bioinformatics, we identify an isoform of β-amylase likely to be responsible for maltose accumulation. Removal of this isoform through identification of TILLING mutants or genome editing, combined with co-expression of heterologous glucan synthase and a glucan branching enzyme, may in future enable elevated yields of carbohydrate through simultaneous accumulation of starch and cytosolic glucan.

This work reports on the generation of a graphite-conjugated diimine macrocyclic Co catalyst (GCC-CoDIM) that is assembled at o-quinone edge defects on graphitic carbon electrodes. X-ray photoelectron spectroscopy and X-ray absorption spectroscopy confirm the existence of a new Co surface species with a coordination environment that is the same as that of the molecular analogue, [Co(DIM)Br2]+. GCC-CoDIM selectively reduces nitrite to ammonium with quantitative Faradaic efficiency and at a rate that approaches enzymatic catalysis. Preliminary mechanistic investigations suggest that the increased rate is accompanied by a change in mechanism from the molecular analogue. These results provide a template for creating macrocycle-based electrocatalysts based on first-row transition metals conjugated to an extreme redox-active ligand.

Lung cancer (LC) is the leading cause of cancer‐related mortality. However, the molecular mechanisms associated with the development of metastasis are poorly understood. Understanding the biology of LC metastasis is critical to unveil the molecular mechanisms for designing targeted therapies. We developed two genetically engineered LC mouse models KrasG12D/+; Trp53R172H/+; Ad‐Cre (KPA) and KrasG12D/+; Ad‐Cre (KA). Survival analysis showed significantly (P = 0.0049) shorter survival in KPA tumor‐bearing mice as compared to KA, suggesting the aggressiveness of the model. Our transcriptomic data showed high expression of N‐acetylgalactosaminide alpha‐2, 6‐sialyltransferase 1 (St6galnac‐I) in KPA compared to KA tumors. ST6GalNAc‐I is an O‐glycosyltransferase, which catalyzes the addition of sialic acid to the initiating GalNAc residues forming sialyl Tn (STn) on glycoproteins, such as mucins. Ectopic expression of species‐specific p53 mutants in the syngeneic mouse and human LC cells led to increased cell migration and high expression of ST6GalNAc‐I, STn, and MUC5AC. Immunoprecipitation of MUC5AC in the ectopically expressing p53R175H cells exhibited higher affinity toward STn. In addition, ST6GalNAc‐I knockout (KO) cells also showed decreased migration, possibly due to reduced glycosylation of MUC5AC as observed by low STn on the glycoprotein. Interestingly, ST6GalNAc‐I KO cells injected mice developed less liver metastasis (P = 0.01) compared to controls, while colocalization of MUC5AC and STn was observed in the liver metastatic tissues of control mice. Collectively, our findings support the hypothesis that mutant p53R175H mediates ST6GalNAc‐I expression, leading to the sialyation of MUC5AC, and thus contribute to LC liver metastasis., In the presence of mutant p53R175H, ST6GalNAc‐I mediates MUC5AC sialylation that increases the aggressiveness of lung adenocarcinoma (LUAD). MUC5AC sialylation is required for integrin β4 interaction that induces angiogenesis and liver metastasis. Hence, targeting the ST6GalNAc‐I/MUC5AC could prevent LUAD metastasis.

Diol dehydratase, dependent on coenzyme B12 (B12 -dDDH), displays a peculiar feature of being inactivated by its native substrate glycerol (GOL). Surprisingly, the isofunctional enzyme, B12 -independent glycerol dehydratase (B12 -iGDH), does not undergo suicide inactivation by GOL. Herein we present a series of QM/MM and MD calculations aimed at understanding the mechanisms of substrate-induced suicide inactivation in B12 -dDDH and that of resistance of B12 -iGDH to inactivation. We show that the first step in the enzymatic transformation of GOL, hydrogen abstraction, can occur from both ends of the substrate (either C1 or C3 of GOL). Whereas C1 abstraction in both enzymes leads to product formation, C3 abstraction in B12 -dDDH results in the formation of a low energy radical intermediate, which is effectively trapped within a deep well on the potential energy surface. The long lifetime of this radical intermediate likely enables its side reactions, leading to inactivation. In B12 -iGDH, by comparison, C3 abstraction is an endothermic step; consequently, the resultant radical intermediate is not of low energy, and the reverse process of reforming the reactant is possible.

AIM Diagnosing and monitoring vascular activity in giant cell arteritis (GCA) is difficult due to the paucity of specific serological biomarkers. We assessed the utility of 8 novel biomarkers in an inception cohort of newly suspected GCA patients. METHOD Consecutive patients were enrolled between May 2016 and December 2017. Serum was collected within 72 hours of commencing corticosteroids and at 6 months. It was analyzed for levels of intra-cellular adhesion molecule 1, vascular endothelial growth factor (VEGF), pentraxin 3, von Willebrand factor and procalcitonin (5-plex R&D Systems multiplex assay) and interleukin (IL)6, IL12 and interferon-γ (high-sensitivity 3-plex ProcartaPlex multiplex assay). A GCA specific positron emission tomography / computed tomography (PET/CT) scan was performed at enrolment with uptake in each vascular territory graded and summed to derive a total vascular score (TVS). RESULTS For the 63 patients enrolled, 12 (19%) had a final diagnosis of biopsy-positive GCA and a further 9 had a clinical diagnosis of biopsy-negative GCA. None of the 8 biomarkers was significantly higher in GCA patients compared with those with alternative diagnoses, or demonstrated a positive correlation with the PET/CT TVS. This was in contrast to the C-reactive protein (CRP) and erythrocyte sedimentation rate (ESR) which were higher in the biopsy-positive GCA cohort (P

Introduction Matrix metalloproteinases (MMPs)-2 and -9 are present in corneal ulcers, and an imbalance between MMPs and tissue inhibitors of metalloproteinases (TIMPs) leads to further corneal degradation. Amniotic membrane homogenate (AMH) has proteolytic properties beneficial for corneal healing, but it is unknown whether AMH possesses TIMPs or effectively inhibits MMP-2 and MMP-9 activity. Objective To determine if bovine and equine AMH reduce in vitro MMP-2 and MMP-9 activities associated with the presence of TIMPs. Procedures Undiluted and diluted twofold series (0-fold to 16-fold dilutions) of equine amniotic membrane homogenates (EAMH, n = 8) and bovine amniotic membrane homogenates (BAMH, n = 8) were subjected to fluorescence resonance energy transfer, and the fluorescence emitted was recorded over time. Average fluorescence was calculated versus recombinant concentration. Enzyme-linked immunosorbent assays for TIMPs 1-4 were applied to quantify TIMPs in the samples. Results AMH from both species were able to inhibit MMP-2 and MMP-9 activities in vitro, and the inhibition efficacy decreased gradually with dilution. BAMH was significantly more effective than EAMH at inhibiting MMP-2 and MMP-9 in vitro. TIMPs -2 and -3 were present in EAMH and BAMH. TIMP-1 was detected only in BAMH, and TIMP-4 was not detected in any samples. Conclusion Both EAMH and BAMH directly inhibited MMP-2 and MMP-9 in vitro without dilution, and BAMH showed better inhibition of MMP-2 and MMP-9 before and after dilution compared to EAMH.

Background: Determine the overall, sex-, and racially/ethnically-appropriate population-level cotinine and total nicotine equivalents (TNE-2, the molar sum of the two major nicotine metabolites) cut-points to distinguish tobacco users from nonusers across multiple definitions of use (e.g., exclusive vs. polytobacco, and daily vs. non-daily). Methods: Using Wave 1 (2013–2014) of the U.S. Population Assessment of Tobacco and Health (PATH) Study, we conducted weighted Receiver Operating Characteristic (ROC) analysis to determine the optimal urinary cotinine and TNE-2 cut-points, stratified by sex and race/ethnicity. Results: For past 30-day exclusive cigarette users, the cotinine cut-point that distinguished them from nonusers was 40.5 ng/mL, with considerable variation by sex (male: 22.2 ng/mL; female: 43.1 ng/mL) and between racial/ethnic groups (non-Hispanic other: 5.2 ng/mL; non-Hispanic black: 297.0 ng/mL). A similar, but attenuated, pattern emerged when assessing polytobacco cigarette users (overall cut-point = 39.1 ng/mL, range = 5.5 ng/mL–80.4 ng/mL) and any tobacco users (overall cut-point = 39.1 ng/mL, range = 4.8 ng/mL–40.0 ng/mL). Using TNE-2, which is less impacted by racial differences in nicotine metabolism, produced a comparable pattern of results although reduced the range magnitude. Conclusions: Because of similar frequency of cigarette use among polytobacco users, overall cut-points for exclusive cigarette use were not substantially different from cut-points that included polytobacco cigarette use or any tobacco use. Results revealed important differences in sex and race/ethnicity appropriate cut-points when evaluating tobacco use status and established novel urinary TNE-2 cut-points. Impact: These cut-points may be used for biochemical verification of self-reported tobacco use in epidemiologic studies and clinical trials.

Hydrogen sulfide (H(2)S) is an important biomolecule, and self-immolative thiocarbamates have shown great promise as triggerable H(2)S donors with suitable analogous control compounds, however thiocarbamates with electron-deficient payloads are less efficient H(2)S donors. We report here the synthesis and study of a series of N-methylated esterase-triggered thiocarbamates that block the postulated unproductive deprotonation-based pathway for these compounds. The relative reaction profiles for H(2)S release across a series of electron-rich and electron-poor N-Me aniline payloads are examined experimentally and computationally. We show that thiocarbamate N-methylation does block some side reactivity and increases the H(2)S release profiles for electron-poor donors. Additionally, we show that isothiocyanate release is not a competitive pathway, and rather that the reduced efficiency of electron-poor donors is likely due to other side reactions.

Hexaferrites have a wide range of technological applications as well as a rich and complicated structural chemistry, with iron oxide layers inducing highly anisotropic ferrimagnetism that remains i...

Reduction of the three-coordinate iron(III) imido [Ph2B(tBuIm)2Fe═NDipp] (1) affords [Ph2B(tBuIm)2Fe═NDipp][K(18-C-6)THF2] (2), a rare example of a high-spin (S = 2) iron(II) imido complex. Unusually for a late metal imido complex, the imido ligand in 2 has nucleophilic character, as demonstrated by the reaction with DippNH2, which establishes an equilibrium with the bis(anilido) complex [Ph2B(tBuIm)2Fe(NHDipp)2][K(18-C-6)THF2] (3). In an unusual transformation, formal insertion of iPrN═C═NiPr into the Fe═N(imido) bond yields the guanidinate [Ph2B(tBuIm)2Fe(iPrN)2CNDipp][K(18-C-6)THF2] (4). Reaction of 4 with excess DippNH2 provides 3, along with the guanidine (iPrNH)2C═NDipp. As suggested by these stoichiometric reactions, 2 is an efficient catalyst for the guanylation of carbodiimides, converting a wide range of aniline substrates under mild conditions.

[Image: see text] Hematopoietic progenitor kinase 1 (HPK1), a serine/threonine kinase, is a negative immune regulator of T cell receptor (TCR) and B cell signaling that is primarily expressed in hematopoietic cells. Accordingly, it has been reported that HPK1 loss-of-function in HPK1 kinase-dead syngeneic mouse models shows enhanced T cell signaling and cytokine production as well as tumor growth inhibition in vivo, supporting its value as an immunotherapeutic target. Herein, we present the structurally enabled discovery of novel, potent, and selective diaminopyrimidine carboxamide HPK1 inhibitors. The key discovery of a carboxamide moiety was essential for enhanced enzyme inhibitory potency and kinome selectivity as well as sustained elevation of cellular IL-2 production across a titration range in human peripheral blood mononuclear cells. The elucidation of structure–activity relationships using various pendant amino ring systems allowed for the identification of several small molecule type-I inhibitors with promising in vitro profiles.

OBJECTIVE Describe the secretion and profile of adrenal steroids in patients with adrenal incidentalomas compared to control subjects. DESIGN, SETTING AND PARTICIPANTS A prospective study, 73 patients with adrenal incidentalomas, 21 bilateral and 52 unilateral and 34 matched controls in University Hospital. METHODS Collect fasting blood sample before and 60 min after ACTH test (250 µg IV). One week later, perform overnight 1 mg dexamethasone test. The following steroids were measured by liquid chromatography-mass spectrometry (LC-MS): pregnenolone, 17-OH pregnenolone, 17-OH progesterone, 11-deoxycorticosterone, 11-deoxyortisol, 21-deoxycortisol, corticosterone, cortisol, androstenedione and aldosterone. RESULTS Mean baseline serum cortisol was higher in incidentalomas, bilateral 361 ± 124, (range 143-665) nmol/L,(p

[Image: see text] Hematopoietic progenitor kinase (HPK1), a negative regulator of TCR-mediated T-cell activation, has been recognized as a novel antitumor immunotherapy target. Structural optimization of kinase inhibitor 4 through a systematic two-dimensional diversity screen of pyrazolopyridines led to the identification of potent and selective compounds. Crystallographic studies with HPK1 revealed a favorable water-mediated interaction with Asp155 and a salt bridge to Asp101 with optimized heterocyclic solvent fronts that were critical for enhanced potency and selectivity. Computational studies of model systems revealed differences in torsional profiles that allowed for these beneficial protein–ligand interactions. Further optimization of molecular properties led to identification of potent and selective reverse indazole inhibitor 36 that inhibited phosphorylation of adaptor protein SLP76 in human PBMC and exhibited low clearance with notable bioavailability in in vivo rat studies.

Introduction Concurrent use of tobacco cigarettes and e-cigarettes (“dual use”) is common among tobacco users. Little is known about differences in demographics and toxicant exposure among subsets of dual users. Aims and Methods We analyzed data from adult dual users (current every/some day users of tobacco cigarettes and e-cigarettes, n = 792) included in the PATH Study Wave 1 (2013–2014) and provided urine samples. Samples were analyzed for biomarkers of exposure to nicotine and selected toxicants (tobacco-specific nitrosamine NNK [NNAL], lead, cadmium, naphthalene [2-naphthol], pyrene [1-hydroxypyrene], acrylonitrile [CYMA], acrolein [CEMA], and acrylamide [AAMA]). Subsets of dual users were compared on demographic, behavioral, and biomarker measures to exclusive cigarette smokers (n = 2411) and exclusive e-cigarette users (n = 247). Results Most dual users were predominant cigarette smokers (70%), followed by daily dual users (13%), non-daily concurrent dual users (10%), and predominant vapers (7%). Dual users who smoked daily showed significantly higher biomarker concentrations compared with those who did not smoke daily. Patterns of e-cigarette use had little effect on toxicant exposure. Dual users with high toxicant exposure were generally older, female, and smoked more cigarettes per day. Dual users who had low levels of biomarkers of exposure were generally younger, male, and smoked non-daily. Conclusions In 2013–2014, most dual users smoked cigarettes daily and used e-cigarettes occasionally. Cigarette smoking appears to be the primary driver of toxicant exposure among dual users, with little-to-no effect of e-cigarette use on biomarker levels. Results reinforce the need for dual users to stop smoking tobacco cigarettes to reduce toxicant exposure. Implications With considerable dual use of tobacco cigarettes and e-cigarettes in the United States, it is important to understand differences in toxicant exposure among subsets of dual users, and how these differences align with user demographics. Findings suggest most dual users smoke daily and use e-cigarettes intermittently. Low exposure to toxicants was most common among younger users, males, and intermittent smokers; high exposure to toxicants was most common among older users, females, and heavier cigarette smokers. Results underscore the heterogeneity occurring within dual users, and the need to quit smoking cigarettes completely in order to reduce toxicant exposure.

A new facility has been developed which allows for a stable and reproducible production of ambient-like model aerosols (PALMA) in the laboratory. The set-up consists of multiple aerosol generators, a custom-made flow tube homogeniser, isokinetic sampling probes, and a system to control aerosol temperature and humidity. Model aerosols containing elemental carbon, secondary organic matter from the ozonolysis of α-pinene, inorganic salts such as ammonium sulfate and ammonium nitrate, mineral dust particles, and water were generated under different environmental conditions and at different number and mass concentrations. The aerosol physical and chemical properties were characterised with an array of experimental methods, including scanning mobility particle sizing, ion chromatography, total reflection X-ray fluorescence spectroscopy and thermo-optical analysis. The facility is very versatile and can find applications in the calibration and performance characterisation of aerosol instruments monitoring ambient air. In this study, we performed, as proof of concept, an intercomparison of three different commercial PM (particulate matter) monitors (TEOM 1405, DustTrak DRX 8533 and Fidas Frog) with the gravimetric reference method under three simulated environmental scenarios. The results are presented and compared to previous field studies. We believe that the laboratory-based method for simulating ambient aerosols presented here could provide in the future a useful alternative to time-consuming and expensive field campaigns, which are often required for instrument certification and calibration.

A series of 1,2,3-triazolium-functionalized cellulose derivatives were prepared using an azide–alkyne “click” cyclization strategy, followed by quaternization. As cellulose represents an inexpensiv...

The use of manganese in homogeneous hydrogenation catalysis has been a recent focus in the pursuit of more environmentally benign base metal catalysts. It has great promise with its unique reactivity when coupled with metal–ligand cooperation of aminophosphine pincer ligands. Here, a manganese precatalyst Mn(P–N–P′)(CO)2, where P–N–P′ is the amido form of the ligand (S,S)-PPh2CHPhCHPhNHCH2CH2PiPr2, has been synthesized and used for base-free ketone hydrogenation. This catalyst shows exceptionally high enantioselectivity and good activity, with tolerance for base-sensitive substrates. NMR structural analysis of intermediates formed by the reaction of the amido complex with hydrogen under pressure identified a reactive hydride with an NOE contact with the syn amine proton. Computational analysis of the catalytic cycle reveals that the heterolytic splitting of dihydrogen across the MnN bond in the amido complex has a low barrier while the hydride transfer to the ketone is the turnover-limiting step. The pro-S transition state is found to be usually much lower in energy than the pro-R transition state depending on the ketone structure, consistent with the high (S) enantiomeric excess in the alcohol products. The energy to reach the transition state is higher for the distortion of the in-coming ketone than that of the hydride complex. In a one-to-one comparison with the similar iron catalyst FeH2(CO)(P–NH–P′), the manganese catalyst is found to have higher enantioselectivity, often over 95% ee, while the iron catalyst has higher activity and productivity. An explanation of these differences is provided on the basis of the more deformable iron hydride complex due to the smaller hydride ligands.

Disclosed in this communication is a thorough study on the dearomative addition of organomagnesium nucleophiles to N-alkyl pyridinium electrophiles. The regiochemical outcomes have observable and predictable trends associated with the substituent patterns on the pyridinium electrophile. Often, the substituent effects can be either additive, giving high selectivities, or ablative, giving competing outcomes. Additionally, the nature of the organometallic nucleophilic component was also investigated for its role in the regioselective outcome. The effects of either reactive component are important to both the overall reactivity and site of nucleophilic addition. The utility of these observed trends is demonstrated in a concise, dearomative synthesis of a tricyclic compound shown to have insecticidal activity.

Ligand-based mixed valent (MV) complexes of Al(iii) incorporating electron donating (ED) and electron withdrawing (EW) substituents on bis(imino)pyridine ligands (I2P) have been prepared. The MV states containing EW groups are both assigned as Class II/III, and those with ED functional groups are Class III and Class II/III in the (I2P-)(I2P2-)Al and [(I2P2-)(I2P3-)Al]2- charge states, respectively. No abrupt changes in delocalization are observed with ED and EW groups and from this we infer that ligand and metal valence p-orbitals are well-matched in energy and the absence of LMCT and MLCT bands supports the delocalized electronic structures. The MV ligand charge states (I2P-)(I2P2-)Al and [(I2P2-)(I2P3-)Al]2- show intervalence charge transfer (IVCT) transitions in the regions 6850-7740 and 7410-9780 cm-1, respectively. Alkali metal cations in solution had no effect on the IVCT bands of [(I2P2-)(I2P3-)Al]2- complexes containing -PhNMe2 or -PhF5 substituents. Minor localization of charge in [(I2P2-)(I2P3-)Al]2- was observed when -PhOMe substituents are included.

A reduction in cytosolic invertase in Arabidopsis roots changes the sucrose to hexose ratio, resulting in profound alterations of metabolism, growth, development, and patterns of gene expression., Plant roots depend on sucrose imported from leaves as the substrate for metabolism and growth. Sucrose and hexoses derived from it are also signalling molecules that modulate growth and development, but the importance for signalling of endogenous changes in sugar levels is poorly understood. We report that reduced activity of cytosolic invertase, which converts sucrose to hexoses, leads to pronounced metabolic, growth, and developmental defects in roots of Arabidopsis (Arabidopsis thaliana) seedlings. In addition to altered sugar and downstream metabolite levels, roots of cinv1 cinv2 mutants have reduced elongation rates, cell and meristem size, abnormal meristematic cell division patterns, and altered expression of thousands of genes of diverse functions. Provision of exogenous glucose to mutant roots repairs relatively few of the defects. The extensive transcriptional differences between mutant and wild-type roots have hallmarks of both high sucrose and low hexose signalling. We conclude that the mutant phenotype reflects both low carbon availability for metabolism and growth and complex sugar signals derived from elevated sucrose and depressed hexose levels in the cytosol of mutant roots. Such reciprocal changes in endogenous sucrose and hexose levels potentially provide rich information about sugar status that translates into flexible adjustments of growth and development.

The objective of this study is to convert bamboo into a transparent material with great optical transmittance and good strength. Bamboo has a much faster regeneration rate than wood, but its high density and high extractive content make it challenging to produce transparent products. This study presents a simple and effective approach that could address this challenge. Pretreatment of bamboo with low concentration sodium hydroxide greatly improved the preparation efficiency of transparent bamboo. The transparent bamboo with a thickness of 1 mm and cellulose volume fraction of 22% made from the pretreated bamboo exhibited an improved total optical transmissivity up to 80%, which was 60% higher than that of untreated bamboo. Compared to transparent wood (TW), although the transmissivity of transparent bamboo was slightly lower, its mechanical strength was almost doubled. Besides, the developed transparent bamboo exhibited a low heat conductivity of 0.203 W m-1 K-1, being about 10% lower than that of TW (0.225 W m-1 K-1) and approximately 80% lower than that of common glass material (0.974 W m-1 K-1). The transparent bamboo would significantly enhance energy-saving performance, being a promising alternative to traditional glass.

In 2013 we presented a model for identifying nursing behavior from primate teeth based on rapid postnatal concentration changes in the non-essential trace element barium. Here we leverage the permanent neonatal (birth) line in the enamel of several dozen primate M1 cusps to compare pre- and postnatal trends in barium, zinc, strontium, and oxygen, as each element is believed to evince developmental patterning. Barium and zinc are the most consistent biomarkers of nursing initiation; a majority of M1 cusps shows concentration increases from prenatal to postnatal enamel, whereas strontium shows decreases or no change with similar frequency. Exceptions to the pattern of barium increase occurred in cusps that had been mineralizing for less than three weeks, suggesting that subsequent enamel maturation has only a minor impact on detecting real time events. Oxygen isotope compositions (δ18O) show rapid and marked fluctuations (∼1–2‰) within two weeks of birth in 93% of M1 cusps (n = 27/29). This is likely due to measurements of hypomineralized perinatal enamel and physiological changes in the body water of newborn infants. Ongoing work integrating elemental concentration gradients with isotopic variation will help establish the degree to which milk intake may cause elevated δ18O in teeth. We show that chemical identification of pre-to postnatal transitions may be robust to slight planar deviations that commonly obscure growth increments under light microscopy, and could help validate the identification of potential neonatal lines, making this approach a useful complement to bioarchaeological studies and public health investigations.

Context and purpose There is an urgent need to develop vitamin D dietary recommendations for dark-skinned populations resident at high latitude. Using data from randomised controlled trials (RCTs) with vitamin D3-supplements/fortified foods, we undertook an individual participant data-level meta-regression (IPD) analysis of the response of wintertime serum 25-hydroxyvitamin (25(OH)D) to total vitamin D intake among dark-skinned children and adults residing at ≥ 40° N and derived dietary requirement values for vitamin D. Methods IPD analysis using data from 677 dark-skinned participants (of Black or South Asian descent; ages 5-86 years) in 10 RCTs with vitamin D supplements/fortified foods identified via a systematic review and predefined eligibility criteria. Outcome measures were vitamin D intake estimates across a range of 25(OH)D thresholds. Results To maintain serum 25(OH)D concentrations ≥ 25 and 30 nmol/L in 97.5% of individuals, 23.9 and 27.3 µg/day of vitamin D, respectively, were required among South Asian and 24.1 and 33.2 µg/day, respectively, among Black participants. Overall, our age-stratified intake estimates did not exceed age-specific Tolerable Upper Intake Levels for vitamin D. The vitamin D intake required by dark-skinned individuals to maintain 97.5% of winter 25(OH)D concentrations ≥ 50 nmol/L was 66.8 µg/day. This intake predicted that the upper 2.5% of individuals could potentially achieve serum 25(OH)D concentrations ≥ 158 nmol/L, which has been linked to potential adverse effects in older adults in supplementation studies. Conclusions Our IPD-derived vitamin D intakes required to maintain 97.5% of winter 25(OH)D concentrations ≥ 25, 30 and 50 nmol/L are substantially higher than the equivalent estimates for White individuals. These requirement estimates are also higher than those currently recommended internationally by several agencies, which are based predominantly on data from Whites and derived from standard meta-regression based on aggregate data. Much more work is needed in dark-skinned populations both in the dose-response relationship and risk characterisation for health outcomes. Trail registration PROSPERO International Prospective Register of Systematic Reviews (Registration Number: CRD42018097260).

PurposeGlaucoma patients with peripheral vision loss have in the past subjectively described their field loss as ‘blurred’ or ‘no vision compromise’. We developed an iPad app for patients to self-characterise perception within areas of glaucomatous visual field loss.MethodsTwelve glaucoma patients with visual acuity ≥20/40 in each eye, stable and reliable Humphrey Visual Field (HVF) over 2 years were enrolled. An iPad app (held at 33 cm) allowed subjects to modify ‘blur’ or ‘dimness’ to match their perception of a 2×2 m wall-mounted poster at 1 m distance. Subjects fixated at the centre of the poster (spanning 45° of field from centre). The output was degree of blur/dim: normal, mild and severe noted on the iPad image at the 54 retinal loci tested by the HVF 24-2 and was compared to threshold sensitivity values at these loci. Monocular (Right eye (OD), left eye (OS)) HVF responses were used to calculate an integrated binocular (OU) visual field index (VFI). All three data sets were analysed separately.Results36 HVF and iPad responses from 12 subjects (mean age 71±8.2y) were analysed. The mean VFI was 77% OD, 76% OS, 83% OU. The most common iPad response reported was normal followed by blur. No subject reported dim response. The mean HVF sensitivity threshold was significantly associated with the iPad response at the corresponding retinal loci (For OD, OS and OU, respectively (dB): normal: 23, 25, 27; mild blur: 18, 16, 22; severe blur: 9, 9, 11). On receiver operative characteristic (ROC) curve analysis, the HVF retinal sensitivity cut-off at which subjects reported blur was 23.4 OD, 23 OS and 23.3 OU (dB).ConclusionsGlaucoma subjects self-pictorialised their field defects as blur; never dim or black. Our innovation allows translation of HVF data to quantitatively characterise visual perception in patients with glaucomatous field defects.