A Potent Peptidomimetic Inhibitor of Botulinum Neurotoxin Serotype A Has a Very Different Conformation than SNAP-25 Substrate

SummaryBotulinum neurotoxin serotype A is the most lethal of all known toxins. Here, we report the crystal structure, along with SAR data, of the zinc metalloprotease domain of BoNT/A bound to a potent peptidomimetic inhibitor (Ki = 41 nM) that resembles the local sequence of the SNAP-25 substrate. Surprisingly, the inhibitor adopts a helical conformation around the cleavage site, in contrast to the extended conformation of the native substrate. The backbone of the inhibitor's P1 residue displaces the putative catalytic water molecule and concomitantly interacts with the “proton shuttle” E224. This mechanism of inhibition is aided by residue contacts in the conserved S1′ pocket of the substrate binding cleft and by the induction of new hydrophobic pockets, which are not present in the apo form, especially for the P2′ residue of the inhibitor. Our inhibitor is specific for BoNT/A as it does not inhibit other BoNT serotypes or thermolysin

Iterative Structure-Based Peptide-Like Inhibitor Design against the Botulinum Neurotoxin Serotype A

The botulinum neurotoxin serotype A light chain (BoNT/A LC) protease is the catalytic component responsible for the neuroparalysis that is characteristic of the disease state botulism. Three related peptide-like molecules (PLMs) were designed using previous information from co-crystal structures, synthesized, and assayed for in vitro inhibition against BoNT/A LC. Our results indicate these PLMS are competitive inhibitors of the BoNT/A LC protease and their Ki values are in the nM-range. A co-crystal structure for one of these inhibitors was determined and reveals that the PLM, in accord with the goals of our design strategy, simultaneously involves both ionic interactions via its P1 residue and hydrophobic contacts by means of an aromatic group in the P2′ position. The PLM adopts a helical conformation similar to previously determined co-crystal structures of PLMs, although there are also major differences to these other structures such as contacts with specific BoNT/A LC residues. Our structure further demonstrates the remarkable plasticity of the substrate binding cleft of the BoNT/A LC protease and provides a paradigm for iterative structure-based design and development of BoNT/A LC inhibitors

A modular approach toward producing nanotherapeutics targeting the innate immune system.

Immunotherapies controlling the adaptive immune system are firmly established, but regulating the innate immune system remains much less explored. The intrinsic interactions between nanoparticles and phagocytic myeloid cells make these materials especially suited for engaging the innate immune system. However, developing nanotherapeutics is an elaborate process. Here, we demonstrate a modular approach that facilitates efficiently incorporating a broad variety of drugs in a nanobiologic platform. Using a microfluidic formulation strategy, we produced apolipoprotein A1-based nanobiologics with favorable innate immune system-engaging properties as evaluated by in vivo screening. Subsequently, rapamycin and three small-molecule inhibitors were derivatized with lipophilic promoieties, ensuring their seamless incorporation and efficient retention in nanobiologics. A short regimen of intravenously administered rapamycin-loaded nanobiologics (mTORi-NBs) significantly prolonged allograft survival in a heart transplantation mouse model. Last, we studied mTORi-NB biodistribution in nonhuman primates by PET/MR imaging and evaluated its safety, paving the way for clinical translation.This work was supported by NIH grants R01 CA220234, R01 HL144072, P01 HL131478, and NWO/ZonMW Vici 91818622 (to W.J.M.M.); R01 HL143814 and P01HL131478 (to Z.A.F.); R01 AI139623 (to J.O.); and P30 CA008748 (to T.R.). M.M.T.v.L. was supported by the American Heart Association (grant 19PRE34380423). M.G.N. was supported by a Spinoza grant from the Netherlands Organization for Scientific Research and an ERC Advanced Grant (no. 833247); L.A.B.J. was supported by a Competitiveness Operational Programme grant of the Romanian Ministry of European Funds (P_37_762, MySMIS 103587).S

Spatial analysis of air pollution and childhood asthma in Hamilton, Canada: comparing exposure methods in sensitive subgroups

<p>Abstract</p> <p>Background</p> <p>Variations in air pollution exposure within a community may be associated with asthma prevalence. However, studies conducted to date have produced inconsistent results, possibly due to errors in measurement of the exposures.</p> <p>Methods</p> <p>A standardized asthma survey was administered to children in grades one and eight in Hamilton, Canada, in 1994–95 (N ~1467). Exposure to air pollution was estimated in four ways: (1) distance from roadways; (2) interpolated surfaces for ozone, sulfur dioxide, particulate matter and nitrous oxides from seven to nine governmental monitoring stations; (3) a kriged nitrogen dioxide (NO₂) surface based on a network of 100 passive NO₂monitors; and (4) a land use regression (LUR) model derived from the same monitoring network. Logistic regressions were used to test associations between asthma and air pollution, controlling for variables including neighbourhood income, dwelling value, state of housing, a deprivation index and smoking.</p> <p>Results</p> <p>There were no significant associations between any of the exposure estimates and asthma in the whole population, but large effects were detected the subgroup of children without hayfever (predominately in girls). The most robust effects were observed for the association of asthma without hayfever and NO₂LUR OR = 1.86 (95%CI, 1.59–2.16) in all girls and OR = 2.98 (95%CI, 0.98–9.06) for older girls, over an interquartile range increase and controlling for confounders.</p> <p>Conclusion</p> <p>Our findings indicate that traffic-related pollutants, such as NO₂, are associated with asthma without overt evidence of other atopic disorders among female children living in a medium-sized Canadian city. The effects were sensitive to the method of exposure estimation. More refined exposure models produced the most robust associations.</p

Altered gene expression and DNA damage in peripheral blood cells from Friedreich's ataxia patients: Cellular model of pathology

The neurodegenerative disease Friedreich's ataxia (FRDA) is the most common autosomal-recessively inherited ataxia and is caused by a GAA triplet repeat expansion in the first intron of the frataxin gene. In this disease, transcription of frataxin, a mitochondrial protein involved in iron homeostasis, is impaired, resulting in a significant reduction in mRNA and protein levels. Global gene expression analysis was performed in peripheral blood samples from FRDA patients as compared to controls, which suggested altered expression patterns pertaining to genotoxic stress. We then confirmed the presence of genotoxic DNA damage by using a gene-specific quantitative PCR assay and discovered an increase in both mitochondrial and nuclear DNA damage in the blood of these patients (p<0.0001, respectively). Additionally, frataxin mRNA levels correlated with age of onset of disease and displayed unique sets of gene alterations involved in immune response, oxidative phosphorylation, and protein synthesis. Many of the key pathways observed by transcription profiling were downregulated, and we believe these data suggest that patients with prolonged frataxin deficiency undergo a systemic survival response to chronic genotoxic stress and consequent DNA damage detectable in blood. In conclusion, our results yield insight into the nature and progression of FRDA, as well as possible therapeutic approaches. Furthermore, the identification of potential biomarkers, including the DNA damage found in peripheral blood, may have predictive value in future clinical trials

Genetic variants in novel pathways influence blood pressure and cardiovascular disease risk.

Blood pressure is a heritable trait influenced by several biological pathways and responsive to environmental stimuli. Over one billion people worldwide have hypertension (≥140 mm Hg systolic blood pressure or  ≥90 mm Hg diastolic blood pressure). Even small increments in blood pressure are associated with an increased risk of cardiovascular events. This genome-wide association study of systolic and diastolic blood pressure, which used a multi-stage design in 200,000 individuals of European descent, identified sixteen novel loci: six of these loci contain genes previously known or suspected to regulate blood pressure (GUCY1A3-GUCY1B3, NPR3-C5orf23, ADM, FURIN-FES, GOSR2, GNAS-EDN3); the other ten provide new clues to blood pressure physiology. A genetic risk score based on 29 genome-wide significant variants was associated with hypertension, left ventricular wall thickness, stroke and coronary artery disease, but not kidney disease or kidney function. We also observed associations with blood pressure in East Asian, South Asian and African ancestry individuals. Our findings provide new insights into the genetics and biology of blood pressure, and suggest potential novel therapeutic pathways for cardiovascular disease prevention

The Canadian Urban Environmental Health Research Consortium - A protocol for building a national environmental exposure data platform for integrated analyses of urban form and health

Background: Multiple external environmental exposures related to residential location and urban form including, air pollutants, noise, greenness, and walkability have been linked to health impacts or benefits. The Canadian Urban Environmental Health Research Consortium (CANUE) was established to facilitate the linkage of extensive geospatial exposure data to existing Canadian cohorts and administrative health data holdings. We hypothesize that this linkage will enable investigators to test a variety of their own hypotheses related to the interdependent associations of built environment features with diverse health outcomes encompassed by the cohorts and administrative data. Methods: We developed a protocol for compiling measures of built environment features that quantify exposure; vary spatially on the urban and suburban scale; and can be modified through changes in policy or individual behaviour to benefit health. These measures fall into six domains: air quality, noise, greenness, weather/climate, and transportation and neighbourhood factors; and will be indexed to six-digit postal codes to facilitate merging with health databases. Initial efforts focus on existing data and include estimates of air pollutants, greenness, temperature extremes, and neighbourhood walkability and socioeconomic characteristics. Key gaps will be addressed for noise exposure, with a new national model being developed, and for transportation-related exposures, with detailed estimates of truck volumes and diesel emissions now underway in selected cities. Improvements to existing exposure estimates are planned, primarily by increasing temporal and/or spatial resolution given new satellite-based sensors and more detailed national air quality modelling. Novel metrics are also planned for walkability and food environments, green space access and function and life-long climate-related exposures based on local climate zones. Critical challenges exist, for example, the quantity and quality of input data to many of the models and metrics has changed over time, making it difficult to develop and validate historical exposures. Discussion: CANUE represents a unique effort to coordinate and leverage substantial research investments and will enable a more focused effort on filling gaps in exposure information, improving the range of exposures quantified, their precision and mechanistic relevance to health. Epidemiological studies may be better able to explore the common theme of urban form and health in an integrated manner, ultimately contributing new knowledge informing policies that enhance healthy urban living

New genetic loci link adipose and insulin biology to body fat distribution.

Body fat distribution is a heritable trait and a well-established predictor of adverse metabolic outcomes, independent of overall adiposity. To increase our understanding of the genetic basis of body fat distribution and its molecular links to cardiometabolic traits, here we conduct genome-wide association meta-analyses of traits related to waist and hip circumferences in up to 224,459 individuals. We identify 49 loci (33 new) associated with waist-to-hip ratio adjusted for body mass index (BMI), and an additional 19 loci newly associated with related waist and hip circumference measures (P < 5 × 10(-8)). In total, 20 of the 49 waist-to-hip ratio adjusted for BMI loci show significant sexual dimorphism, 19 of which display a stronger effect in women. The identified loci were enriched for genes expressed in adipose tissue and for putative regulatory elements in adipocytes. Pathway analyses implicated adipogenesis, angiogenesis, transcriptional regulation and insulin resistance as processes affecting fat distribution, providing insight into potential pathophysiological mechanisms

Ambient PM2.5 and risk of emergency room visits for myocardial infarction: impact of regional PM2.5 oxidative potential: a case-crossover study

Abstract Background Regional differences in the oxidative potential of fine particulate air pollution (PM2.5) may modify its impact on the risk of myocardial infarction. Methods A case-crossover study was conducted in 16 cities in Ontario, Canada to evaluate the impact of regional PM2.5 oxidative potential on the relationship between PM2.5 and emergency room visits for myocardial infarction. Daily air pollution and meteorological data were collected between 2004 and 2011 from provincial monitoring sites and regional estimates of glutathione (OPGSH) and ascorbate-related (OPAA) oxidative potential were determined using an acellular assay based on a synthetic respiratory tract lining fluid. Exposure variables for the combined oxidant capacity of NO2 and O3 were also examined using their sum (Ox) and a weighted average (Ox wt) based on their redox potentials. Results In total, 30,101 cases of myocardial infarction were included in the analysis. For regions above the 90th percentile of OPGSH each 5 μg/m3 increase in same-day PM2.5 was associated with a 7.9 % (95 % CI: 4.1, 12) increased risk of myocardial infarction whereas a 4.1 % (95 % CI: 0.26, 8.0) increase was observed in regions above the 75th percentile and no association was observed below the 50th percentile (p-interaction = 0.026). A significant 3-way interaction was detected with the strongest associations between PM2.5 and myocardial infarction occurring in areas with high regional OPGSH and high Ox wt (p-interaction < 0.001). Conclusions Regional PM2.5 oxidative potential may modify the impact of PM2.5 on the risk of myocardial infarction. The combined oxidant capacity of NO2 and O3 may magnify this effect