3D printed microfluidic lab-on-a-chip device for fiber-based dual beam optical manipulation

3D printing of microfluidic lab-on-a-chip devices enables rapid prototyping of robust and complex structures. In this work, we designed and fabricated a 3D printed lab-on-a-chip device for fiber-based dual beam optical manipulation. The final 3D printed chip offers three key features, such as (1) an optimized fiber channel design for precise alignment of optical fibers, (2) an optically clear window to visualize the trapping region, and (3) a sample channel which facilitates hydrodynamic focusing of samples. A square zig–zag structure incorporated in the sample channel increases the number of particles at the trapping site and focuses the cells and particles during experiments when operating the chip at low Reynolds number. To evaluate the performance of the device for optical manipulation, we implemented on-chip, fiber-based optical trapping of different-sized microscopic particles and performed trap stiffness measurements. In addition, optical stretching of MCF-7 cells was successfully accomplished for the purpose of studying the effects of a cytochalasin metabolite, pyrichalasin H, on cell elasticity. We observed distinct changes in the deformability of single cells treated with pyrichalasin H compared to untreated cells. These results demonstrate that 3D printed microfluidic lab-on-a-chip devices offer a cost-effective and customizable platform for applications in optical manipulation

3D printed microfluidic lab-on-a-chip device for fiber-based dual beam optical manipulation

3D printing of microfluidic lab-on-a-chip devices enables rapid prototyping of robust and complex structures. In this work, we designed and fabricated a 3D printed lab-on-a-chip device for fiber-based dual beam optical manipulation. The final 3D printed chip offers three key features, such as (1) an optimized fiber channel design for precise alignment of optical fibers, (2) an optically clear window to visualize the trapping region, and (3) a sample channel which facilitates hydrodynamic focusing of samples. A square zig–zag structure incorporated in the sample channel increases the number of particles at the trapping site and focuses the cells and particles during experiments when operating the chip at low Reynolds number. To evaluate the performance of the device for optical manipulation, we implemented on-chip, fiber-based optical trapping of different-sized microscopic particles and performed trap stiffness measurements. In addition, optical stretching of MCF-7 cells was successfully accomplished for the purpose of studying the effects of a cytochalasin metabolite, pyrichalasin H, on cell elasticity. We observed distinct changes in the deformability of single cells treated with pyrichalasin H compared to untreated cells. These results demonstrate that 3D printed microfluidic lab-on-a-chip devices offer a cost-effective and customizable platform for applications in optical manipulation

A 3D-printed microfluidic gradient generator with integrated photonic silicon sensors for rapid antimicrobial susceptibility testing

With antimicrobial resistance becoming a major threat to healthcare settings around the world, there is a paramount need for rapid point-of-care antimicrobial susceptibility testing (AST) diagnostics. Unfortunately, most currently available clinical AST tools are lengthy, laborious, or are simply inappropriate for point-of-care testing. Herein, we design a 3D-printed microfluidic gradient generator that automatically produces two-fold dilution series of clinically relevant antimicrobials. We first establish the compatibility of these generators for classical AST (i.e., broth microdilution) and then extend their application to include a complete on-chip label-free and phenotypic AST. This is accomplished by the integration of photonic silicon chips, which provide a preferential surface for microbial colonization and allow optical tracking of bacterial behavior and growth at a solid-liquid interface in real-time by phase shift reflectometric interference spectroscopic measurements (PRISM). Using Escherichia coli and ciprofloxacin as a model pathogen-drug combination, we successfully determine the minimum inhibitory concentration within less than 90 minutes. This gradient generator-based PRISM assay provides an integrated AST device that is viable for convenient point-of-care testing and offers a promising and most importantly, rapid alternative to current clinical practices, which extend to 8-24 h

The Impact of Photobleaching on Microarray Analysis

DNA-Microarrays have become a potent technology for high-throughput analysis of genetic regulation. However, the wide dynamic range of signal intensities of fluorophore-based microarrays exceeds the dynamic range of a single array scan by far, thus limiting the key benefit of microarray technology: parallelization. The implementation of multi-scan techniques represents a promising approach to overcome these limitations. These techniques are, in turn, limited by the fluorophores' susceptibility to photobleaching when exposed to the scanner's laser light. In this paper the photobleaching characteristics of cyanine-3 and cyanine-5 as part of solid state DNA microarrays are studied. The effects of initial fluorophore intensity as well as laser scanner dependent variables such as the photomultiplier tube's voltage on bleaching and imaging are investigated. The resulting data is used to develop a model capable of simulating the expected degree of signal intensity reduction caused by photobleaching for each fluorophore individually, allowing for the removal of photobleaching-induced, systematic bias in multi-scan procedures. Single-scan applications also benefit as they rely on pre-scans to determine the optimal scanner settings. These findings constitute a step towards standardization of microarray experiments and analysis and may help to increase the lab-to-lab comparability of microarray experiment results

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

Legitimacy in the Multilevel European Polity

In order to be simultaneously effective and liberal, governments must normally be able to count on voluntary compliance – which, in turn, depends on the support of socially shared legitimacy beliefs. In Western constitutional democracies, such beliefs are derived from the distinct but coexistent traditions of “republican” and “liberal” political philosophy. When judged by these criteria, the European Union – if considered by itself – appears as a thoroughly liberal polity which, however, lacks all republican credentials. But this view (which seems to structure the debates about the “European democratic deficit”) ignores the multilevel nature of the European polity, where the compliance of citizens is requested, and needs to be legitimated by member states – whereas the Union appears as a “government of governments” which is entirely dependent on the voluntary compliance of its member states. What matters primarily, therefore, is the compliance-legitimacy relationship between the Union and its member states – which, however, is normatively constrained by the basic compliance-legitimacy relationship between member governments and their constituents. Given the high consensus requirements of European legislation, member governments could and should be able to assume political responsibility for European policies in which they had a voice, and to justify them in “communicative discourses” in the national public space. This is not necessarily true of “non-political” policy choices imposed by the European Court of Justice. By enforcing its “liberal” program of liberalization and deregulation, the ECJ may presently be undermining the “republican” bases of member-state legitimacy. Where this is the case, open non-compliance is a present danger, and political controls of judicial legislation may be called for.Um gleichzeitig effektiv und liberal sein zu können, ist staatliche Herrschaft auf freiwillige Folgebereitschaft angewiesen – die ihrerseits der Unterstützung durch sozial geteilte Legitimitätsüberzeugungen bedarf. In den demokratischen Verfassungsstaaten des Westens werden solche Überzeugungen aus den unterschiedlichen, aber komplementär zusammenwirkenden Traditionen der „republikanischen“ und der „liberalen“ politischen Philosophie hergeleitet. An diesen Kriterien gemessen erscheint die Europäische Union – wenn man sie für sich betrachtet – als eine „liberale“ politische Ordnung, der jedoch alle „republikanischen“ Legitimitätsmerkmale fehlen. Aber eine solche Sichtweise, die auch die derzeitige Diskussion über ein „europäisches Demokratiedefizit“ bestimmt, verkennt den Mehrebenencharakter des europäischen Gemeinwesens. In ihm sind es die Mitgliedstaaten, die Entscheidungen der Union gegenüber den eigenen Bürgern durchsetzen und auch legitimieren müssen, während es für die Union ihrerseits auf die freiwillige Folgebereitschaft ihrer Mitgliedstaaten ankommt. Dabei werden diese jedoch durch die normativen Grundlagen ihrer eigenen Legitimität begrenzt. Politische Entscheidungen auf europäischer Ebene setzen breiten Konsens voraus, und die Regierungen sollten sie deshalb auch gegenüber den eigenen Bürgern in „kommunikativen Diskursen“ vertreten und dafür die politische Verantwortung übernehmen können. Dies gilt jedoch nicht notwendigerweise auch für Entscheidungen der europäischen Politik, die im nichtpolitischen Modus ohne Beteiligung des Rates und des Parlaments vom Europäischen Gerichtshof bestimmt werden. Mit der gegenwärtigen Radikalisierung seines „liberalen“ Programms der Liberalisierung und Deregulierung des nationalen Rechts könnte der Gerichtshof in der Tat die „republikanischen“ Grundlagen der mitgliedstaatlichen Legitimität unterminieren. In diesem Falle könnte die Union sich nicht länger auf die Folgebereitschaft ihrer Mitgliedstaaten verlassen. Um diese Gefahr für die europäische Integration zu vermeiden, sollte eine stärkere politische Kontrolle der richterlichen Rechtsetzung erwogen werden.1 Legitimacy Republican and liberal legitimating discourses Constitutional democracies – and the EU? 2 Legitimacy in multilevel polities 3 Legitimating member state compliance Political modes of policy making Non-political policy making 4 The need for justification 5 The Court is pushing against the limits of justifiability 6 The liberal undermining of republican legitimacy 7 Needed: A political balance of community and autonomy Reference

Rare Variants in Long Non-Coding RNAs Are Associated With Blood Lipid Levels in the TOPMed Whole-Genome Sequencing Study

Long non-coding RNAs (lncRNAs) are known to perform important regulatory functions in lipid metabolism. Large-scale whole-genome sequencing (WGS) studies and new statistical methods for variant set tests now provide an opportunity to assess more associations between rare variants in lncRNA genes and complex traits across the genome. In this study, we used high-coverage WGS from 66,329 participants of diverse ancestries with measurement of blood lipids and lipoproteins (LDL-C, HDL-C, TC, and TG) in the National Heart, Lung, and Blood Institute (NHLBI) Trans-Omics for Precision Medicine (TOPMed) program to investigate the role of lncRNAs in lipid variability. We aggregated rare variants for 165,375 lncRNA genes based on their genomic locations and conducted rare-variant aggregate association tests using the STAAR (variant-set test for association using annotation information) framework. We performed STAAR conditional analysis adjusting for common variants in known lipid GWAS loci and rare-coding variants in nearby protein-coding genes. Our analyses revealed 83 rare lncRNA variant sets significantly associated with blood lipid levels, all of which were located in known lipid GWAS loci (in a ±500-kb window of a Global Lipids Genetics Consortium index variant). Notably, 61 out of 83 signals (73%) were conditionally independent of common regulatory variation and rare protein-coding variation at the same loci. We replicated 34 out of 61 (56%) conditionally independent associations using the independent UK Biobank WGS data. Our results expand the genetic architecture of blood lipids to rare variants in lncRNAs

Determinants of penetrance and variable expressivity in monogenic metabolic conditions across 77,184 exomes

Penetrance of variants in monogenic disease and clinical utility of common polygenic variation has not been well explored on a large-scale. Here, the authors use exome sequencing data from 77,184 individuals to generate penetrance estimates and assess the utility of polygenic variation in risk prediction of monogenic variants

The Polygenic and Monogenic Basis of Blood Traits and Diseases

Blood cells play essential roles in human health, underpinning physiological processes such as immunity, oxygen transport, and clotting, which when perturbed cause a significant global health burden. Here we integrate data from UK Biobank and a large-scale international collaborative effort, including data for 563,085 European ancestry participants, and discover 5,106 new genetic variants independently associated with 29 blood cell phenotypes covering a range of variation impacting hematopoiesis. We holistically characterize the genetic architecture of hematopoiesis, assess the relevance of the omnigenic model to blood cell phenotypes, delineate relevant hematopoietic cell states influenced by regulatory genetic variants and gene networks, identify novel splice-altering variants mediating the associations, and assess the polygenic prediction potential for blood traits and clinical disorders at the interface of complex and Mendelian genetics. These results show the power of large-scale blood cell trait GWAS to interrogate clinically meaningful variants across a wide allelic spectrum of human variation. Analysis of blood cell traits in the UK Biobank and other cohorts illuminates the full genetic architecture of hematopoietic phenotypes, with evidence supporting the omnigenic model for complex traits and linking polygenic burden with monogenic blood diseases