Genomic Phenotyping by Barcode Sequencing Broadly Distinguishes between Alkylating Agents, Oxidizing Agents, and Non-Genotoxic Agents, and Reveals a Role for Aromatic Amino Acids in Cellular Recovery after Quinone Exposure

Toxicity screening of compounds provides a means to identify compounds harmful for human health and the environment. Here, we further develop the technique of genomic phenotyping to improve throughput while maintaining specificity. We exposed cells to eight different compounds that rely on different modes of action: four genotoxic alkylating (methyl methanesulfonate (MMS), N-Methyl-N-nitrosourea (MNU), N,N′-bis(2-chloroethyl)-N-nitroso-urea (BCNU), N-ethylnitrosourea (ENU)), two oxidizing (2-methylnaphthalene-1,4-dione (menadione, MEN), benzene-1,4-diol (hydroquinone, HYQ)), and two non-genotoxic (methyl carbamate (MC) and dimethyl sulfoxide (DMSO)) compounds. A library of S. cerevisiae 4,852 deletion strains, each identifiable by a unique genetic ‘barcode’, were grown in competition; at different time points the ratio between the strains was assessed by quantitative high throughput ‘barcode’ sequencing. The method was validated by comparison to previous genomic phenotyping studies and 90% of the strains identified as MMS-sensitive here were also identified as MMS-sensitive in a much lower throughput solid agar screen. The data provide profiles of proteins and pathways needed for recovery after both genotoxic and non-genotoxic compounds. In addition, a novel role for aromatic amino acids in the recovery after treatment with oxidizing agents was suggested. The role of aromatic acids was further validated; the quinone subgroup of oxidizing agents were extremely toxic in cells where tryptophan biosynthesis was compromised.Unilever (Firm)National Cancer Institute (U.S.) (R01-CA055042 (now R01-ES022872))Massachusetts Institute of Technology. Center for Environmental Health Sciences (Grant NIEHS P30-ES002109

Restoring brain function after stroke - bridging the gap between animals and humans

Stroke is the leading cause of complex adult disability in the world. Recovery from stroke is often incomplete, which leaves many people dependent on others for their care. The improvement of long-term outcomes should, therefore, be a clinical and research priority. As a result of advances in our understanding of the biological mechanisms involved in recovery and repair after stroke, therapeutic opportunities to promote recovery through manipulation of poststroke plasticity have never been greater. This work has almost exclusively been carried out in preclinical animal models of stroke with little translation into human studies. The challenge ahead is to develop a mechanistic understanding of recovery from stroke in humans. Advances in neuroimaging techniques now enable us to reconcile behavioural accounts of recovery with molecular and cellular changes. Consequently, clinical trials can be designed in a stratified manner that takes into account when an intervention should be delivered and who is most likely to benefit. This approach is expected to lead to a substantial change in how restorative therapeutic strategies are delivered in patients after stroke

Why Are Outcomes Different for Registry Patients Enrolled Prospectively and Retrospectively? Insights from the Global Anticoagulant Registry in the FIELD-Atrial Fibrillation (GARFIELD-AF).

Background: Retrospective and prospective observational studies are designed to reflect real-world evidence on clinical practice, but can yield conflicting results. The GARFIELD-AF Registry includes both methods of enrolment and allows analysis of differences in patient characteristics and outcomes that may result. Methods and Results: Patients with atrial fibrillation (AF) and ≥1 risk factor for stroke at diagnosis of AF were recruited either retrospectively (n = 5069) or prospectively (n = 5501) from 19 countries and then followed prospectively. The retrospectively enrolled cohort comprised patients with established AF (for a least 6, and up to 24 months before enrolment), who were identified retrospectively (and baseline and partial follow-up data were collected from the emedical records) and then followed prospectively between 0-18 months (such that the total time of follow-up was 24 months; data collection Dec-2009 and Oct-2010). In the prospectively enrolled cohort, patients with newly diagnosed AF (≤6 weeks after diagnosis) were recruited between Mar-2010 and Oct-2011 and were followed for 24 months after enrolment. Differences between the cohorts were observed in clinical characteristics, including type of AF, stroke prevention strategies, and event rates. More patients in the retrospectively identified cohort received vitamin K antagonists (62.1% vs. 53.2%) and fewer received non-vitamin K oral anticoagulants (1.8% vs . 4.2%). All-cause mortality rates per 100 person-years during the prospective follow-up (starting the first study visit up to 1 year) were significantly lower in the retrospective than prospectively identified cohort (3.04 [95% CI 2.51 to 3.67] vs . 4.05 [95% CI 3.53 to 4.63]; p = 0.016). Conclusions: Interpretations of data from registries that aim to evaluate the characteristics and outcomes of patients with AF must take account of differences in registry design and the impact of recall bias and survivorship bias that is incurred with retrospective enrolment. Clinical Trial Registration: - URL: http://www.clinicaltrials.gov . Unique identifier for GARFIELD-AF (NCT01090362)

Design development and implementation of an irradiation station at the neutron time-of-flight facility at CERN

A new parasitic, mixed-field, neutron-dominated irradiation station has been recently commissioned at the European Laboratory for Particle Physics (CERN). The station is installed within the neutron time-of-flight (n_TOF) facility, taking advantage of the secondary radiation produced by the neutron spallation target, with neutrons ranging from 0.025 eV to several hundreds of MeV. The new station allows radiation damage studies to be performed in irradiation conditions that are closer to the ones encountered during the operation of particle accelerators; the irradiation tests carried out in the station will be complementary to the standard tests on materials, usually performed with gamma sources. Samples will be exposed to neutron-dominated doses in the MGy range per year, with minimal impact on the n_TOF facility operation. The station has 24 irradiation positions, each hosting up to 100 cm3 of sample material. In view of its proximity to the n_TOF target, inside protective shielding, the irradiation station and its operating procedures have been carefully developed taking into account the safety of personnel and to avoid any unwanted impact on the operation of the n_TOF facility and experiments. Due to the residual radioactivity of the whole area around the n_TOF target and of the irradiated samples, access to the irradiation station is forbidden to human operators even when the n_TOF facility is not in operation. Robots are used for the remote installation and retrieval of the samples, and other optimizations of the handling procedures were developed in compliance with radiation protection regulations and the aim of minimizing doses to personnel. The sample containers were designed to be radiation tolerant, compatible with remote handling, and subject to detailed risk analysis and testing during their development. The whole life cycle of the irradiated materials, including their post-irradiation examinations and final disposal, was considered and optimized

Implications of Placebo and Nocebo Effects for Clinical Practice: Expert Consensus

Background: Placebo and nocebo effects occur in clinical or laboratory medical contexts after administration of an inert treatment or as part of active treatments and are due to psychobiological mechanisms such as expectancies of the patient. Placebo and nocebo studies have evolved from predominantly methodological research into a far-reaching interdisciplinary field that is unravelling the neurobiological, behavioural and clinical underpinnings of these phenomena in a broad variety of medical conditions. As a consequence, there is an increasing demand from health professionals to develop expert recommendations about evidence-based and ethical use of placebo and nocebo effects for clinical practice. Methods: A survey and interdisciplinary expert meeting by invitation was organized as part of the 1st Society for Interdisciplinary Placebo Studies (SIPS) conference in 2017. Twenty-nine internationally recognized placebo researchers participated. Results: There was consensus that maximizing placebo effects and minimizing nocebo effects should lead to better treatment outcomes with fewer side effects. Experts particularly agreed on the importance of informing patients about placebo and nocebo effects and training health professionals in patient-clinician communication to maximize placebo and minimize nocebo effects. Conclusions: The current paper forms a first step towards developing evidence-based and ethical recommendations about the implications of placebo and nocebo research for medical practice, based on the current state of evidence and the consensus of experts. Future research might focus on how to implement these recommendations, including how to optimize conditions for educating patients about placebo and nocebo effects and providing training for the implementation in clinical practice. (C) 2018 S. Karger AG, Base

Disrupted Functional Connectivity with Dopaminergic Midbrain in Cocaine Abusers

Background: Chronic cocaine use is associated with disrupted dopaminergic neurotransmission but how this disruption affects overall brain function (other than reward/motivation) is yet to be fully investigated. Here we test the hypothesis that cocaine addicted subjects will have disrupted functional connectivity between the midbrain (where dopamine neurons are located) and cortical and subcortical brain regions during the performance of a sustained attention task. Methodology/Principal Findings: We measured brain activation and functional connectivity with fMRI in 20 cocaine abusers and 20 matched controls. When compared to controls, cocaine abusers had lower positive functional connectivity of midbrain with thalamus, cerebellum, and rostral cingulate, and this was associated with decreased activation in thalamus and cerebellum and enhanced deactivation in rostral cingulate. Conclusions/Significance: These findings suggest that decreased functional connectivity of the midbrain interferes with the activation and deactivation signals associated with sustained attention in cocaine addicts

Discovery of Novel Hypermethylated Genes in Prostate Cancer Using Genomic CpG Island Microarrays

BACKGROUND: Promoter and 5' end methylation regulation of tumour suppressor genes is a common feature of many cancers. Such occurrences often lead to the silencing of these key genes and thus they may contribute to the development of cancer, including prostate cancer. METHODOLOGY/PRINCIPAL FINDINGS: In order to identify methylation changes in prostate cancer, we performed a genome-wide analysis of DNA methylation using Agilent human CpG island arrays. Using computational and gene-specific validation approaches we have identified a large number of potential epigenetic biomarkers of prostate cancer. Further validation of candidate genes on a separate cohort of low and high grade prostate cancers by quantitative MethyLight analysis has allowed us to confirm DNA hypermethylation of HOXD3 and BMP7, two genes that may play a role in the development of high grade tumours. We also show that promoter hypermethylation is responsible for downregulated expression of these genes in the DU-145 PCa cell line. CONCLUSIONS/SIGNIFICANCE: This study identifies novel epigenetic biomarkers of prostate cancer and prostate cancer progression, and provides a global assessment of DNA methylation in prostate cancer

Differences in Childhood Growth Parameters Between Patients With Somatic and Heritable Retinoblastoma.

PURPOSE: Little is known regarding differences in childhood growth between somatic and heritable retinoblastoma (Rb) populations. We aimed to compare childhood growth parameters between somatic and heritable Rb cohorts at birth and at time of diagnosis with Rb. METHODS: A multinational, longitudinal cohort study was conducted with patients from 11 centers in 10 countries who presented with treatment naïve Rb from January to December 2019. Variables of interest included age, sex, and size characteristics at birth and at time of presentation, as well as germline mutation status. After Bonferroni correction, results were statistically significant if the P value was less than 0.005. RESULTS: We enrolled 696 patients, with 253 analyzed after exclusion criteria applied. Between somatic (n = 39) and heritable (n = 214) Rb cohorts, with males and females analyzed separately, there was no significant difference in birth weight percentile, weight percentile at time of diagnosis, length percentile at time of diagnosis, weight-for-length percentile at time of diagnosis, or change of weight percentile from birth to time of diagnosis. Patients with heritable Rb had a smaller mean weight percentile at birth and smaller mean weight and length percentiles at time of diagnosis with Rb, although this difference was not statistically significant. All cohorts experienced a slight negative change of weight percentile from birth to time of diagnosis. No cohort mean percentiles met criteria for failure to thrive, defined as less than the 5th percentile. CONCLUSIONS: Children with Rb seem to have normal birth and childhood growth patterns. There is no definitive evidence that somatic or heritable Rb has a biological or environmental impact on childhood growth parameters

100% complete assignment of non-labile 1H, 13C, and 15N signals for calcium-loaded calbindin D9k P43G

Here we present the 100% complete assignment chemical shift of non-labile 1H, 15N and 13C nuclei of Calbindin D9k P43G. The assignment includes all non-exchangeable side chain nuclei, including ones that are rarely reported, such as LysNζ as well as the termini. NMR experiments required to achieve truly complete assignments are discussed. To the best of our knowledge our assignments for Calbindin D9k extend beyond previous studies reaching near-completeness (Vis et al. in Biochem 33:14858–14870, 1994; Yamazaki et al. in J Am Chem Soc 116:6464–6465, 1994; Yamazaki et al. in Biochem 32:5656–5669, 1993b)