An approach to quantifying 3D responses of cells to extreme strain

The tissues of hollow organs can routinely stretch up to 2.5 times their length. Although significant pathology can arise if relatively large stretches are sustained, the responses of cells are not known at these levels of sustained strain. A key challenge is presenting cells with a realistic and well-defined three-dimensional (3D) culture environment that can sustain such strains. Here, we describe an in vitro system called microscale, magnetically-actuated synthetic tissues (micro-MASTs) to quantify these responses for cells within a 3D hydrogel matrix. Cellular strain-threshold and saturation behaviors were observed in hydrogel matrix, including strain-dependent proliferation, spreading, polarization, and differentiation, and matrix adhesion retained at strains sufficient for apoptosis. More broadly, the system shows promise for defining and controlling the effects of mechanical environment upon a broad range of cells

Confidence Intervals for Concentration and Brightness from Fluorescence Fluctuation Measurements

AbstractThe theory of photon count histogram (PCH) analysis describes the distribution of fluorescence fluctuation amplitudes due to populations of fluorophores diffusing through a focused laser beam and provides a rigorous framework through which the brightnesses and concentrations of the fluorophores can be determined. In practice, however, the brightnesses and concentrations of only a few components can be identified. Brightnesses and concentrations are determined by a nonlinear least-squares fit of a theoretical model to the experimental PCH derived from a record of fluorescence intensity fluctuations. The χ2 hypersurface in the neighborhood of the optimum parameter set can have varying degrees of curvature, due to the intrinsic curvature of the model, the specific parameter values of the system under study, and the relative noise in the data. Because of this varying curvature, parameters estimated from the least-squares analysis have varying degrees of uncertainty associated with them. There are several methods for assigning confidence intervals to the parameters, but these methods have different efficacies for PCH data. Here, we evaluate several approaches to confidence interval estimation for PCH data, including asymptotic standard error, likelihood joint-confidence region, likelihood confidence intervals, skew-corrected and accelerated bootstrap (BCa), and Monte Carlo residual resampling methods. We study these with a model two-dimensional membrane system for simplicity, but the principles are applicable as well to fluorophores diffusing in three-dimensional solution. Using simulated fluorescence fluctuation data, we find the BCa method to be particularly well-suited for estimating confidence intervals in PCH analysis, and several other methods to be less so. Using the BCa method and additional simulated fluctuation data, we find that confidence intervals can be reduced dramatically for a specific non-Gaussian beam profile

Report of the Regional Coordination Meeting for the North Atlantic (RCM NA) 2015

The 12th RCM North Atlantic was held in Hamburg (Germany) 14-18 September 2015. The main purpose of the RCM is to coordinate the National Programmes (NP) of the Member States (MS) in the North Atlantic region. National Programmes for 2011-2013 have been rolled over for the period 2014-2016. Therefore, the main focus at this year was to improve regional data collection, analysis and storage and the evolution towards Regional Coordination Groups (RCG).The impact of the introduction of the landing obligation and preparations for its implementation was also discussed taking into account possible changes in scientific sampling schemes. The participation of four National Correspondents make possible to address National administration issues related to the oncoming EU MAP. A data call was launched by the chairs of the RCM NA, RCM Baltic and RCM NS&EA where MS were requested to upload data for 2014 in the regional database (RDB Fishframe) hosted by ICES. All MS except France and Northern Ireland complied with this request on landings and effort data. All MS except France uploaded sample data for 2014. French data were available for the meeting using a web base interface. Evaluation of the data call for submission data to the RDB revealed the numbers of species in landings and sample data and the numbers of metiers in effort data are in general data stable. RCM NA see big improvements in the work MS are doing regarding data calls coming from a situation where some countries didn´t provide any data to a new scenario where everyone is providing data; at the same time the overall quality has significantly improved, which is a large step forward. Regional data collection, analysis, storage and the evolution towards Regional Coordination Groups (RCG). Optimizing and harmonizing fisheries management across MS is dependent on improving regional coordination. The group discussed various needs and aspects relevant for facilitating future work of the RCM. Future tasks for the RCM don’t differ much from the current tasks. The discussion was focused on the structure of the RCGs, funding and short term needs to address tasks in an efficient way in the future. Regional coordination encompasses many different aspects, ranging from regional cooperation, sampling design, quality control procedures, data storage and analysis to the actual coordination, reporting and accountancy. Current task sharing and coordination procedures as well as future mechanisms are partially covered under the current MARE study 2014/19 (FISHPI). The project and its progress were presented to the group. The outcomes of this study will demonstrate future procedures based on case studies. As substantial effort and costs are involved to facilitate the process of regional coordination, the group highlighted the importance to access to budgets to cover these costs. Development of the RDB is also crucial for future work of the RCGs; funds are needed for the development. Additionally, RCM NA identified 4 supra regional topics where work can be done intersesionally in cooperation with the rest of RCMs: (1) Cost sharing of funding surveys; (2) Impact of landing obligation; (3) reviewing the ICES list of data needs ; and (4) review and follow up on RDB upload logs. Due to the importance to moving to a regional catch sampling scheme, an exercise was realised using the distribution of landings by harbour and fleet segment as a proxy of sampling frames that could hypothetically operate in a regional probability based design. The exercise was based on landing weight, for the simple reason that this was the only complete variable that was available for all the various national data sets. A regional sampling design can however be optimized in any number of ways (e.g. by landings value, by métier diversity, by species diversity, by number of fishing trips). The aims and aspirations of the end users need to be defined to ascertain which is most appropriate. It is one of the overriding advantages of a regional sampling design (as opposed to the aggregation of national designs) that the overall coverage can be set out to achieve regional goals. The RCM NA analyzed and discussed the main achievements of WKISCON2. It was clear that concurrent sampling at-sea is a long-established practice in most MS and that, where it was applied, concurrent sampling of fishing trips on-shore resulted in substantial increases in species collected without jeopardizing the main uses of data. Stock assessment and discard estimation and management are the major current uses of concurrent sampling data. Concurrent sampling has also been providing other benefits than its initial reason, such as advice to local, national and international authorities, research on MSFD descriptors, mixed fisheries and gear interactions and on mortality of rare species, data-poor stocks and PETS. It was clear that concurrent sampling being a statistically valid method for species selection which has proven to fulfil different end-users needs, implementation constraints hinder concurrent sampling on-shore. Thus, in order to meet end-users needs and to overcome the constraints that may arise from the implementation of concurrent sampling in some countries, particularly on-shore, RCM NA considers that different statistically sound approaches other than concurrent sampling must be developed to be tested in the field, so they may provide useful alternatives. Introduction of the landing obligation and its impact in the implementation in scientific sampling schemes. In terms of evaluating the impact of the introduction of the Landing Obligation (LO) regulation on data collection, there is only limited experience as the current implementation only covers Pelagic and Industrial fisheries in this region but MS have or are preparing for the implementation where they can. It is currently perceived that this year is a transition period for the pelagic fisheries and that these fisheries and control agencies are not fully implementing the LO (managing but not enforcing). As a result MS did not have a lot of comments on the current year and are in general preparing for next year. During the meeting it was decided to gather further information to address this issue by getting member states who were present to fill in a table on “Monitoring the impact of the landing obligation on data collection in the North Atlantic region” outlining the current state of play. This table could be considered as a live document which should be filled in year by year as the Landing Obligation is phased in. This table will then serve to provide an historical record as countries can document the changes year by year and will also provide guidance and act as a learning tool to all member states on how other countries are implementing the LO. National administrations The group discussed the proposal for task sharing and criteria for joint surveys. RCM NS&EA and RCM NA 2014 discussed a cost model for the present joint MS financed surveys and for future joint surveys. In addition to this model, the RCM NA 2015 highlighted that four categories of surveys should be considered in relation to task sharing and criteria for joint surveys. In the light of cost sharing, the group commented that the current DCF recast proposal refers to ‘exploitation of stocks’ rather than EU TAC or landings. Given the relative stability, EU TAC shares are the preferred basis for sharing costs. The exploitation of stocks shall be interpreted as EU-TAC share as a default. In specific cases, RCGs can in the future agree on different interpretation where needed and feasible. Fully agreement among the group was concerning to the engagement and participation of National Correspondent (NC) in this meeting. The future role of the NCs in the RCG context was discussed, indicating a formal role for the NCs in the RCG process to approve and agree on regional arrangements. However, the current recast of the DCF doesn’t include the formal involvement of the NCs in the coordination procedures and meetings. RCM NA highlights this as potentially problematic for the foreseen formal role of the NCs. Other items on the agenda were the consideration of the follow up of relevant recommendations made last years by Liaison Meeting and presentations and relevant development from ICES, EC and SC-RDB

Cellular and Matrix Mechanics of Bioartificial Tissues During Continuous Cyclic Stretch

Bioartificial tissues are useful model systems for studying cell and extra-cellular matrix mechanics. These tissues provide a 3D environment for cells and allow tissue components to be easily modified and quantified. In this study, we fabricated bioartificial tissue rings from a 1 ml solution containing one million cardiac fibroblasts and 1 mg collagen. After 8 days, rings compacted to <1% of original volume and cell number increased 2.4 fold. We initiated continuous cyclic stretching of the rings after 2, 4, or 8 days of incubation, while monitoring the tissue forces. Peak tissue force during each cycle decreased rapidly after initiating stretch, followed by further slow decline. We added 2 μM Cytochalasin-D to some rings prior to initiation of stretch to determine the force contributed by the matrix. Cell force was estimated by subtracting matrix force from tissue force. After 12 h, matrix force-strain curves were highly nonlinear. Cell force-strain curves were linear during loading and showed hysteresis indicating viscoelastic behavior. Cell stiffness increased with stretching frequency from 0.001–0.25 Hz. Cell stiffness decreased with stretch amplitude (5–25%) at 0.1 Hz. The trends in cell stiffness do not fit simple viscoelastic models previously proposed, and suggest possible strain-amplitude related changes during cyclic stretch

Temporal-spatial profiling of pedunculopontine galanin-cholinergic neurons in the lactacystin rat model of Parkinson’s disease

Parkinson’s disease (PD) is conventionally seen as resulting from single-system neurodegeneration affecting nigrostriatal dopaminergic neurons. However, accumulating evidence indicates a multi-system degeneration and neurotransmitter deficiencies, including cholinergic neurons which degenerate in a brainstem nucleus, the pedunculopontine nucleus (PPN), resulting in motor- and cognitive impairments. The neuropeptide galanin can inhibit cholinergic transmission, whilst being upregulated in degenerating brain regions associated with cognitive decline. Here we determined the temporal-spatial profile of progressive expression of endogenous galanin within degenerating cholinergic neurons, across the rostro-caudal axis of the PPN, by utilising the lactacystin-induced rat model of PD. First, we show progressive neuronal death affecting nigral dopaminergic and PPN cholinergic neurons, reflecting that seen in PD patients, to facilitate use of this model for assessing the therapeutic potential of bioactive peptides. Next, stereological analyses of the lesioned brain hemisphere found that the number of PPN cholinergic neurons expressing galanin increased by 11%, compared to sham-lesioned controls, increasing by a further 5% as the neurodegenerative process evolved. Galanin upregulation within cholinergic PPN neurons was most prevalent closest to the intra-nigral lesion site, suggesting that galanin upregulation in such neurons adapt intrinsically to neurodegeneration, to possibly neuroprotect. This is the first report on the extent and pattern of galanin expression in cholinergic neurons across distinct PPN subregions in both the intact rat CNS and lactacystin lesioned rats. The findings pave the way for future work to target galanin signaling in the PPN, to determine the extent to which upregulated galanin expression could offer a viable treatment strategy for ameliorating PD symptoms associated with cholinergic degeneration

A large-scale genome-wide association study meta-analysis of cannabis use disorder

Summary Background Variation in liability to cannabis use disorder has a strong genetic component (estimated twin and family heritability about 50–70%) and is associated with negative outcomes, including increased risk of psychopathology. The aim of the study was to conduct a large genome-wide association study (GWAS) to identify novel genetic variants associated with cannabis use disorder. Methods To conduct this GWAS meta-analysis of cannabis use disorder and identify associations with genetic loci, we used samples from the Psychiatric Genomics Consortium Substance Use Disorders working group, iPSYCH, and deCODE (20 916 case samples, 363 116 control samples in total), contrasting cannabis use disorder cases with controls. To examine the genetic overlap between cannabis use disorder and 22 traits of interest (chosen because of previously published phenotypic correlations [eg, psychiatric disorders] or hypothesised associations [eg, chronotype] with cannabis use disorder), we used linkage disequilibrium score regression to calculate genetic correlations. Findings We identified two genome-wide significant loci: a novel chromosome 7 locus (FOXP2, lead single-nucleotide polymorphism [SNP] rs7783012; odds ratio [OR] 1·11, 95% CI 1·07–1·15, p=1·84 × 10−9) and the previously identified chromosome 8 locus (near CHRNA2 and EPHX2, lead SNP rs4732724; OR 0·89, 95% CI 0·86–0·93, p=6·46 × 10−9). Cannabis use disorder and cannabis use were genetically correlated (rg 0·50, p=1·50 × 10−21), but they showed significantly different genetic correlations with 12 of the 22 traits we tested, suggesting at least partially different genetic underpinnings of cannabis use and cannabis use disorder. Cannabis use disorder was positively genetically correlated with other psychopathology, including ADHD, major depression, and schizophrenia. Interpretation These findings support the theory that cannabis use disorder has shared genetic liability with other psychopathology, and there is a distinction between genetic liability to cannabis use and cannabis use disorder. Funding National Institute of Mental Health; National Institute on Alcohol Abuse and Alcoholism; National Institute on Drug Abuse; Center for Genomics and Personalized Medicine and the Centre for Integrative Sequencing; The European Commission, Horizon 2020; National Institute of Child Health and Human Development; Health Research Council of New Zealand; National Institute on Aging; Wellcome Trust Case Control Consortium; UK Research and Innovation Medical Research Council (UKRI MRC); The Brain & Behavior Research Foundation; National Institute on Deafness and Other Communication Disorders; Substance Abuse and Mental Health Services Administration (SAMHSA); National Institute of Biomedical Imaging and Bioengineering; National Health and Medical Research Council (NHMRC) Australia; Tobacco-Related Disease Research Program of the University of California; Families for Borderline Personality Disorder Research (Beth and Rob Elliott) 2018 NARSAD Young Investigator Grant; The National Child Health Research Foundation (Cure Kids); The Canterbury Medical Research Foundation; The New Zealand Lottery Grants Board; The University of Otago; The Carney Centre for Pharmacogenomics; The James Hume Bequest Fund; National Institutes of Health: Genes, Environment and Health Initiative; National Institutes of Health; National Cancer Institute; The William T Grant Foundation; Australian Research Council; The Virginia Tobacco Settlement Foundation; The VISN 1 and VISN 4 Mental Illness Research, Education, and Clinical Centers of the US Department of Veterans Affairs; The 5th Framework Programme (FP-5) GenomEUtwin Project; The Lundbeck Foundation; NIH-funded Shared Instrumentation Grant S10RR025141; Clinical Translational Sciences Award grants; National Institute of Neurological Disorders and Stroke; National Heart, Lung, and Blood Institute; National Institute of General Medical Sciences.Peer reviewe

Transancestral GWAS of alcohol dependence reveals common genetic underpinnings with psychiatric disorders

Liability to alcohol dependence (AD) is heritable, but little is known about its complex polygenic architecture or its genetic relationship with other disorders. To discover loci associated with AD and characterize the relationship between AD and other psychiatric and behavioral outcomes, we carried out the largest genome-wide association study to date of DSM-IV-diagnosed AD. Genome-wide data on 14,904 individuals with AD and 37,944 controls from 28 case-control and family-based studies were meta-analyzed, stratified by genetic ancestry (European, n = 46,568; African, n = 6,280). Independent, genome-wide significant effects of different ADH1B variants were identified in European (rs1229984; P = 9.8 x 10(-13)) and African ancestries (rs2066702; P = 2.2 x 10(-9)). Significant genetic correlations were observed with 17 phenotypes, including schizophrenia, attention deficit-hyperactivity disorder, depression, and use of cigarettes and cannabis. The genetic underpinnings of AD only partially overlap with those for alcohol consumption, underscoring the genetic distinction between pathological and nonpathological drinking behaviors.Peer reviewe