High performance computing enabling exhaustive analysis of higher order single nucleotide polymorphism interaction in Genome Wide Association Studies.

Genome-wide association studies (GWAS) are a common approach for systematic discovery of single nucleotide polymorphisms (SNPs) which are associated with a given disease. Univariate analysis approaches commonly employed may miss important SNP associations that only appear through multivariate analysis in complex diseases. However, multivariate SNP analysis is currently limited by its inherent computational complexity. In this work, we present a computational framework that harnesses supercomputers. Based on our results, we estimate a three-way interaction analysis on 1.1 million SNP GWAS data requiring over 5.8 years on the full "Avoca" IBM Blue Gene/Q installation at the Victorian Life Sciences Computation Initiative. This is hundreds of times faster than estimates for other CPU based methods and four times faster than runtimes estimated for GPU methods, indicating how the improvement in the level of hardware applied to interaction analysis may alter the types of analysis that can be performed. Furthermore, the same analysis would take under 3 months on the currently largest IBM Blue Gene/Q supercomputer "Sequoia" at the Lawrence Livermore National Laboratory assuming linear scaling is maintained as our results suggest. Given that the implementation used in this study can be further optimised, this runtime means it is becoming feasible to carry out exhaustive analysis of higher order interaction studies on large modern GWAS.This research was partially funded by NHMRC grant 1033452 and was supported by a Victorian Life Sciences Computation Initiative (VLSCI) grant number 0126 on its Peak Computing Facility at the University of Melbourne, an initiative of the Victorian Government, Australia

Systematic Y2H screening reveals extensive effector-complex formation

During infection pathogens secrete small molecules, termed effectors, to manipulate and control the interaction with their specific hosts. Both the pathogen and the plant are under high selective pressure to rapidly adapt and co-evolve in what is usually referred to as molecular arms race. Components of the host’s immune system form a network that processes information about molecules with a foreign origin and damage-associated signals, integrating them with developmental and abiotic cues to adapt the plant’s responses. Both in the case of nucleotide-binding leucine-rich repeat receptors and leucine-rich repeat receptor kinases interaction networks have been extensively characterized. However, little is known on whether pathogenic effectors form complexes to overcome plant immunity and promote disease. Ustilago maydis, a biotrophic fungal pathogen that infects maize plants, produces effectors that target hubs in the immune network of the host cell. Here we assess the capability of U. maydis effector candidates to interact with each other, which may play a crucial role during the infection process. Using a systematic yeast-two-hybrid approach and based on a preliminary pooled screen, we selected 63 putative effectors for one-on-one matings with a library of nearly 300 effector candidates. We found that 126 of these effector candidates interacted either with themselves or other predicted effectors. Although the functional relevance of the observed interactions remains elusive, we propose that the observed abundance in complex formation between effectors adds an additional level of complexity to effector research and should be taken into consideration when studying effector evolution and function. Based on this fundamental finding, we suggest various scenarios which could evolutionarily drive the formation and stabilization of an effector interactome

Use of a Novel Nonparametric Version of DEPTH to Identify Genomic Regions Associated with Prostate Cancer Risk.

BACKGROUND: We have developed a genome-wide association study analysis method called DEPTH (DEPendency of association on the number of Top Hits) to identify genomic regions potentially associated with disease by considering overlapping groups of contiguous markers (e.g., SNPs) across the genome. DEPTH is a machine learning algorithm for feature ranking of ultra-high dimensional datasets, built from well-established statistical tools such as bootstrapping, penalized regression, and decision trees. Unlike marginal regression, which considers each SNP individually, the key idea behind DEPTH is to rank groups of SNPs in terms of their joint strength of association with the outcome. Our aim was to compare the performance of DEPTH with that of standard logistic regression analysis. METHODS: We selected 1,854 prostate cancer cases and 1,894 controls from the UK for whom 541,129 SNPs were measured using the Illumina Infinium HumanHap550 array. Confirmation was sought using 4,152 cases and 2,874 controls, ascertained from the UK and Australia, for whom 211,155 SNPs were measured using the iCOGS Illumina Infinium array. RESULTS: From the DEPTH analysis, we identified 14 regions associated with prostate cancer risk that had been reported previously, five of which would not have been identified by conventional logistic regression. We also identified 112 novel putative susceptibility regions. CONCLUSIONS: DEPTH can reveal new risk-associated regions that would not have been identified using a conventional logistic regression analysis of individual SNPs. IMPACT: This study demonstrates that the DEPTH algorithm could identify additional genetic susceptibility regions that merit further investigation. Cancer Epidemiol Biomarkers Prev; 25(12); 1619-24. ©2016 AACR.National Health and Medical Research Council Australia (Grant ID: 1033452, Senior Principal Research Fellowship, Senior Research Fellowship), Cancer Research UK (Grant IDs: C5047/A7357, C1287/A10118, C1287/A5260, C5047/A3354, C5047/A10692, C16913/A6135 and C16913/A6835), Prostate Research Campaign UK (now Prostate Cancer UK), The Institute of Cancer Research and The Everyman Campaign, The National Cancer Research Network UK, The National Cancer Research Institute (NCRI) UK, National Institute for Health Research funding to the NIHR Biomedical Research Centre at The Institute of Cancer Research and The Royal Marsden NHS Foundation Trust, Prostate Cancer Research Program of Cancer Council Victoria from The National Health and Medical Research Council, Australia (Grant IDs: 126402, 209057, 251533, 396414, 450104, 504700, 504702, 504715, 623204, 940394, 614296), VicHealth, Cancer Council Victoria, The Prostate Cancer Foundation of Australia, The Whitten Foundation, PricewaterhouseCoopers, Tattersall’sThis is the author accepted manuscript. The final version is available from the American Association for Cancer Research via http://dx.doi.org/10.1158/1055-9965.EPI-16-030

Disease modeling in cerebral organoids

Viele neurologische Erkrankungen können mit aktuellen in vitro und in vivo Modellen nicht zufriedenstellend beschrieben werden, da entweder die komplexe 3D-Struktur eines Organs, oder der menschliche genetische Hintergrund nicht vorhanden sind. In dieser Arbeit werden krankheitsrelevante Mechanismen in zerebralen Organoiden beschrieben, dreidimensionale gehirnähnliche in vitro-Gewebe, die in zukünftigen Studien bei der Forschung an neurologischen Erkrankungen helfen können. Wir fokussieren uns auf die entwicklungsbiologischen Aspekte der Migration von Interneuronen und der Neuralrohrfaltung, bei denen zerebrale Organoide einen Vorteil gegenüber anderen Modellsystemen haben können. Außerdem wird die Entwicklung einer skalierbaren Plattform für das Wachstum von zerebralen Organoiden präsentiert. Neocorticale Interneurone haben eine wichtige regulatorische Funktion, da sie das inhibitorische Potential im Gehirn modellieren. Kommt es zu einer Beeinträchtigung von bestimmten Interneuronen- Subtypen, kann dies zu der Entwicklung von neurologischen Erkrankungen wie Schizophrenie und Epilepsie führen. Während der Entwicklung des Neocortex haben Interneurone eine komplexe Entwicklung, da sie von ihrem Entstehungsort im ventralen Prosencephalon tangential über lange Strecken migrieren, bevor sie sich im dorsalen Prosencephalon in neuronale Schaltkreise integrieren. Durch die Fusion von zwei unterschiedlich differenzierten zerebralen Organoiden, die das ventrale und das dorsale Prosencephalon präsentieren, konnten wir eine ventral-dorsale Achse in zerebralen Organoiden darstellen, an der Interneuronen von dem ventralen in den dorsalen Bereich migrieren. Mithilfe von Immunfärbungen konnten verschiedene Interneuron-Subtypen nachgewiesen werden, welche typische Migrationseigenschaften zeigten. Dieses Interneuronen-Modell kann dazu benutzt werden, krankheitsrelevante Aspekte bei der Entstehung, der Migration und der Integration von Interneuronen zu untersuchen. Außerdem können Drug Screens für potentielle therapeutische Substanzen ausgeführt werden. In einem zweiten Projekt wurden die Elemente der Neurulation und des Neuralrohrs in neuronalen Rosetten von zerebralen Organoiden untersucht. Fehlbildungen des Neuralrohrs können zu tödlichen oder stark beeinträchtigenden Effekten auf den entstehenden Embryo führen. Allerdings gibt es signifikante Unterschiede in der Faltung des Neuralrohrs in menschlichen Embryos im Vergleich zu den meisten eingesetzten Modellsystemen. Darum wäre ein Modellsystem mit menschlichem genetischem Hintergrund von Interesse. Wir analysieren Ähnlichkeiten der Entwicklung des Neuronalrohrs zwischen zerebralen Organoiden und der Neuralrohrentwicklung. Wir konnten zeigen, dass die Zellen der frühen Entwicklung von neuronalen Rosetten in zerebralen von ähnlicher Identität sind wie das Neuroepithelium. Außerdem präsentieren wir zwei modifizierte zerebrale Organoid- Protokolle, die die Beobachtung von a) früh entwickelnden, struktur-stabilen neuronalen Rosetten, und 5 b) faltungsähnlichen Strukturen erlaubt. Wir schlussfolgern aus diesen Ergebnissen, dass man in zerebralen Organoiden einige, aber nicht alle Elemente eines Neuralrohrs beobachten kann. Zuletzt werden die ersten Entwicklungsschritte eines Projektes diskutiert, welches Upscaling des zerebralen Organoid-Protokolls erlauben soll. Das aktuelle Protokoll erfordert einen hohen Arbeitsaufwand, weswegen ein vereinfachtes Protokoll auf einer all-in-one Plattform von großem Nutzen wäre. In dieser Arbeit präsentieren wir Ergebnisse die zeigen, dass das Wachstum der initialen Entwicklungsschritte von zerebralen Organoiden auf diesen Plattformen bereits möglich ist. Wir hoffen, dass diese Plattform in späteren Entwicklungsschritten zu einem reduzierten Arbeitsaufwand und zur automatisierten Produktion von zerebralen Organoiden führen wird.Using current in vitro 2D and in vivo systems, many human neurological diseases cannot be investigated in a satisfactory level, as either the complex 3D structure or the human genetic background are missing. In this work, we study two disease-relevant developmental aspects in cerebral organoids which could address some of these limitations. Additionally, we present an approach to upscale cerebral organoid growth, which could help in large scale-screens. Forebrain interneurons have a major function in the human brain as the main inhibitory source for excitatory neurons and impairments of interneuron function are strongly associated with psychiatric diseases such as Epilepsy and Schizophrenia. One aspect of forebrain interneuron impairment is their complex emergence during human brain development and their long-distance migration from ventral into dorsal forebrain, which may be impaired in the named neurological disorders. Using cerebral organoids, we tried a novel approach and fused 2 distinct brain region organoids, depicting the ventral and the dorsal forebrain, together in one organoid “fusion” to generate a ventral-dorsal axis. We could observe robust and targeted migration of interneurons from the ventral into the dorsal cerebral tissue and further characterized multiple different interneuron subtypes in our model. With this interneuron migration assay, disease relevant studies of the emergence, migration and integration of interneurons in early human brain development could be addressed in future studies, and drug screens could be applied for testing potential therapeutic compounds against neurological diseases. We also investigated whether cerebral organoids can be used to study the neural tube and neural tube closure. Neural tube closure defects can have severe to even fatal consequences on developing embryos and neural tube closure in humans and mice differs significantly, thus a human model system could be useful in understanding neural tube closure defects. We investigated several components of neural tube, from cell identity to morphological characterizations, and describe modified cerebral organoid protocols where we could observe a) the emergence of temporally stable neural rosettes in 6 early embryoid body (EB) formation and b) could observe a folding event of neural rosettes in cerebral organoids. We conclude that these experiments show the usability of cerebral organoids for the analysis of neural tube elements in cerebral organoids. We also discuss the initial steps of a project which addresses the upscaling of the cerebral organoid method. The organoid method is labor intensive, so a simplified protocol with the potential of automatization would help tremendously in using the cerebral organoids in large scales. We currently engineer an all-in-one platform for cerebral organoid growth which targets the initial development of cerebral organoids. As intermediate results, we present a simplified generation step of EB for cerebral organoid growth. We hope that the design of this platform will help in the future to make the cerebral organoid protocol less time-consuming and allow upscaling as well as automatization of the growth of cerebral organoids

Etstablishment of a facility for stability studies

Diese Arbeit befasst sich mit der Erstellung eines Lastenheftes für ein GMP-konformes Stabilitätslager, sowie mit der Erstellung eines Qualifizierungsplans. Das Stabilitätslager ist ein Teil des neu in das Unternehmen CELLsius eingegliederten Stabilitätslabors. Bei der Erstellung beider Dokumente wird insbesondere auf die AMBO 2009, die EU-GMP-Guidelines, das aktuell gültige AMG und die ICH-Guidelines mit einem Hauptaugenmerk auf ICH-Q1 (stability) eingegangen. Den wichtigsten Punkt des Lastenheftes stellen die Konstantklimakammern dar, die die zentrale Rolle der Probenlagerung für das Stabilitätslabor übernehmen. Eine ausreichende Qualifizierung des Lagers durch DQ, IQ, OQ und PQ sorgt für eine risikominimierte Durchführung der Errichtung und einen problemlosen Betrieb.This paper describes the creation of a specification sheet and a qualification plan for a GMP-compliant facility for stability studies. The facility for stability studies is part of the newly incorporated stability laboratory of the company CELLsius. For the creation of the documents, particularly the legislative texts AMBO 2009, AMG, the EU-GMP-Guidelines and the ICH-Guidelines, with a main focus on ICH-Q1 (stability) were important. The most important components of the specification sheet are the constant climate chambers, which are the central part in the stability laboratory. Through an adequate qualification with DQ, IQ, OQ and PQ, the realisation and operation will run under low risk

Fused dorsal-ventral cerebral organoids model human cortical interneuron migration

AbstractDevelopment of the forebrain involves the migration of GABAergic interneurons over long distances from ventral into dorsal regions. Although defects in interneuron migration are implicated in neuropsychiatric diseases such as Epilepsy, Autism, and Schizophrenia, model systems to study this process in humans are currently lacking. Here, we describe a method for analyzing human interneuron migration using 3D organoid culture. By fusing cerebral organoids specified toward dorsal and ventral forebrain, we generate a continuous dorsal-ventral axis. Using fluorescent reporters, we demonstrate robust directional GABAergic interneuron migration from ventral into dorsal forebrain. We describe methodology for time lapse imaging of human interneuron migration that is inhibited by the CXCR4 antagonist AMD3100. Our results demonstrate that cerebral organoid fusion cultures can model complex interactions between different brain regions. Combined with reprogramming technology, fusions offer a possibility to analyze complex neurodevelopmental defects using cells from neuropsychiatric disease patients, and to test potential therapeutic compounds.</jats:p

Thesium Anatomicarum ex publicis Lectionibus, de Circulatione Sanguinis depromtarum Fasciculus II. quem placidae cum Eruditis Disputationi, in Auditorio mai. d. 9. Maii habendae sistunt Ioh. Daniel Maior, D. & P.P. & sub Praesidio eius Respondens Michael Meinhard Werner, Regio monte Prussus. Philosoph. Cultor

THESIUM ANATOMICARUM EX PUBLICIS LECTIONIBUS, DE CIRCULATIONE SANGUINIS DEPROMTARUM FASCICULUS II. QUEM PLACIDAE CUM ERUDITIS DISPUTATIONI, IN AUDITORIO MAI. D. 9. MAII HABENDAE SISTUNT IOH. DANIEL MAIOR, D. & P.P. & SUB PRAESIDIO EIUS RESPONDENS MICHAEL MEINHARD WERNER, REGIO MONTE PRUSSUS. PHILOSOPH. CULTOR Thesium Anatomicarum ex publicis Lectionibus, de Circulatione Sanguinis depromtarum Fasciculus II. quem placidae cum Eruditis Disputationi, in Auditorio mai. d. 9. Maii habendae sistunt Ioh. Daniel Maior, D. & P.P. & sub Praesidio eius Respondens Michael Meinhard Werner, Regio monte Prussus. Philosoph. Cultor ( - ) Title page ( - ) Caput III. De Vibratoriâ Sanguinis Agitatione. ( - ) Cap. IV. De Concussivo hinc inde Motu Sanguinis. ( - ) Cap. V. De ulteriore Progressu Sanguinis. ( - ) Section ( -