Modeling Binary Time Series Using Gaussian Processes with Application to Predicting Sleep States

Motivated by the problem of predicting sleep states, we develop a mixed effects model for binary time series with a stochastic component represented by a Gaussian process. The fixed component captures the effects of covariates on the binary-valued response. The Gaussian process captures the residual variations in the binary response that are not explained by covariates and past realizations. We develop a frequentist modeling framework that provides efficient inference and more accurate predictions. Results demonstrate the advantages of improved prediction rates over existing approaches such as logistic regression, generalized additive mixed model, models for ordinal data, gradient boosting, decision tree and random forest. Using our proposed model, we show that previous sleep state and heart rates are significant predictors for future sleep states. Simulation studies also show that our proposed method is promising and robust. To handle computational complexity, we utilize Laplace approximation, golden section search and successive parabolic interpolation. With this paper, we also submit an R-package (HIBITS) that implements the proposed procedure.Comment: Journal of Classification (2018

A screening-compatible live cell fluorescence resonance energy transfer-based assay for modulation of Rho GTPase activity

Rho family GTPases are central regulators of cytoskeletal dynamics controlled by guanine nucleotide exchange factors (RhoGEFs) and GTPase-activating proteins (RhoGAPs). This protocol presents a workflow for a robust high-throughput compatible biosensor assay to analyze changes in Rho GTPase activity by these proteins in the native cellular environment. The procedure can be used for semi-quantitative comparison of GEF/GAP function and extended for analysis of additional modulators. The experimental design is applicable also to other monomolecular ratiometric FRET sensors. For complete details on the use and execution of this protocol, please refer to Müller et al. (2020)

RhoGTPase Regulators Orchestrate Distinct Stages of Synaptic Development

Small RhoGTPases regulate changes in post-synaptic spine morphology and density that support learning and memory. They are also major targets of synaptic disorders, including Autism. Here we sought to determine whether upstream RhoGTPase regulators, including GEFs, GAPs, and GDIs, sculpt specific stages of synaptic development. The majority of examined molecules uniquely regulate either early spine precursor formation or later matura- tion. Specifically, an activator of actin polymerization, the Rac1 GEF β-PIX, drives spine pre- cursor formation, whereas both FRABIN, a Cdc42 GEF, and OLIGOPHRENIN-1, a RhoA GAP, regulate spine precursor elongation. However, in later development, a novel Rac1 GAP, ARHGAP23, and RhoGDIs inactivate actomyosin dynamics to stabilize mature synap- ses. Our observations demonstrate that specific combinations of RhoGTPase regulatory pro- teins temporally balance RhoGTPase activity during post-synaptic spine development

Evolutionary dynamics and structural consequences of de novo beneficial mutations and mutant lineages arising in a constant environment

Background: Microbial evolution experiments can be used to study the tempo and dynamics of evolutionary change in asexual populations, founded from single clones and growing into large populations with multiple clonal lineages. High-throughput sequencing can be used to catalog de novo mutations as potential targets of selection, determine in which lineages they arise, and track the fates of those lineages. Here, we describe a long-term experimental evolution study to identify targets of selection and to determine when, where, and how often those targets are hit. Results: We experimentally evolved replicate Escherichia coli populations that originated from a mutator/nonsense suppressor ancestor under glucose limitation for between 300 and 500 generations. Whole-genome, whole-population sequencing enabled us to catalog 3346 de novo mutations that reached \u3e 1% frequency. We sequenced the genomes of 96 clones from each population when allelic diversity was greatest in order to establish whether mutations were in the same or different lineages and to depict lineage dynamics. Operon-specific mutations that enhance glucose uptake were the first to rise to high frequency, followed by global regulatory mutations. Mutations related to energy conservation, membrane biogenesis, and mitigating the impact of nonsense mutations, both ancestral and derived, arose later. New alleles were confined to relatively few loci, with many instances of identical mutations arising independently in multiple lineages, among and within replicate populations. However, most never exceeded 10% in frequency and were at a lower frequency at the end of the experiment than at their maxima, indicating clonal interference. Many alleles mapped to key structures within the proteins that they mutated, providing insight into their functional consequences. Conclusions: Overall, we find that when mutational input is increased by an ancestral defect in DNA repair, the spectrum of high-frequency beneficial mutations in a simple, constant resource-limited environment is narrow, resulting in extreme parallelism where many adaptive mutations arise but few ever go to fixation

Functional Analysis of the Dictyostelium discoideum Rho GTPases RacH and RacA

In this study, we investigated the functions of the two Rho GTPases RacA and RacH in Dictyostelium discoideum. Both genes are constitutively expressed and mRNA and protein are present throughout the complete developmental cycle of Dictyostelium. RacA belongs to the subfamily of Rho BTB proteins that are characterised by a modular organisation, consisting of a GTPase domain, a praline-rich region, a tandem of two BTB domains and a C-terminal region of unknown function. Thus RacA promises to be both, a highly specific adaptor molecule thanks to its BTB domains, and a regulator of signal transduction due to its GTPase domain. The particularity about RacH is that, in contrast to the other characterised Rho GTPases residing at the plasma membrane, RacH was targeted to the nuclear envelope, ER and Golgi apparatus. The pivotal process regulated by Rho GTPases is the rearrangement of the actin cytoskeleton. Therefore we investigated the role of RacA and RacH in cytoskeleton-dependent processes by analysing the performance of the respective knock out mutants in processes depending on the re-organisation of the cortical actin cytoskeleton. RacA-KO cells show conditional defects when cultured in supension: they are severly impaired in growth, cytokinesis and cell shape probably due to lowered cortical tension and F-actin distribution and reveal a slight chemotactical defect as well as reduced rates of pino- and phagocytosis. Considering the correspondence in phenotypes of cells lacking either RacA, PAKa or myosin II and the PAKa-binding by RacA, a model in which RacA acts in concert with Rac1 GTPases as a regulator of PAKa activity working in parallel to the PI3-kinase/PKB pathway that elicits PAKa activity upon chemoattractant stimulation, is proposed. Moreover putative RacA binding partners have been identified. Cells deficient in RacH have a decreased rate of fluid phase endocytosis and exocytosis and a divergent performance in the endocytosis process in comparison to the WT, but no other apparent defects. In a cell-free system RacH stimulated actin polymerisation, suggesting that it might be involved in actin-based trafficking of vesicular compartments, more specifically in the delivery of cargo to early endosomes. The lack of RacH causes defective early endocytosis leading to defects also observed at later stages of this process. By means of chimeric constructs and alanine exchange mutants it was also shown that several regions of the molecule, not only the hypervariable region, determine targeting of RacH and that targeting to the correct membrane compartment probably makes interactions with appropriate regulators and effectors possible, and is therefore essential for function

The role of microtubules in initial neuronal polarization

Neurons are highly polarized cells with two structurally and functionally distinct compartments, axons and dendrites. This dichotomy is the basis for unidirectional signal propagation, the quintessential function of neurons. During neuronal development, the formation of the axon is the initial step in breaking cellular symmetry and the establishment of neuronal polarity. Although a number of polarity regulators involved in this process have been identified, our understanding of the intracellular mechanisms underlying neuronal polarization still remains fragmentary. In my studies, I addressed the role of microtubule dynamics in initial neuronal polarization. To this end I aimed to investigate the following issues: 1) How do microtubule dynamics and stability change during initial neuronal development? 2) Do microtubules play an instructive role in axon formation? 3) What are possible regulators mediating changes in microtubule dynamics during axon formation? Using hippocampal neurons in culture as a model system for neuronal polarization I first addressed the dynamics of microtubules in early developmental stages of neurons. Assessing posttranslational modifications of tubulin which serve as markers of microtubule turnover I found that microtubule stability is increased in a single neurite already before axon formation and in the axon of morphologically polarized cells. This polarized distribution of microtubule stability was confirmed by testing the resistance of neuronal microtubules to pharmacologically induced depolymerization. The axon of polarized neurons and a single neurite in morphologically unpolarized cells showed increased microtubule stability. Thus, I established a correlation between the identity of a process and its microtubule stability. By manipulating specific regulators of neuronal polarity, SAD kinases and GSK-3beta, I analyzed a possible relation between a polarization of microtubule stability and neuronal polarity. I found that a loss of polarity correlated with a loss of polarized microtubule stability in neurons defective for SAD A and SAD B kinases. In marked contrast, the formation of multiple axons, induced by the inhibition of GSK-3beta, was associated with increased microtubule stability in these supernumerary axons. These results suggested that SAD kinases and GSK-3beta regulate neuronal polarization –at least in part– by modulating microtubule dynamics. To establish a possible causal relation between microtubule dynamics and axon formation I assessed the effects of specific pharmacological alterations of microtubule dynamics on neuronal polarization. I found that application of low doses of the microtubule destabilizing drug nocodazole selectively reduced the formation of future dendrites. Conversely, low doses of the microtubule stabilizing drug taxol led to the formation of multiple axons. I also studied microtubule dynamics in living neurons transfected with GFP-tagged EB3, a protein binding specifically to polymerizing microtubule plus ends. In line with my previous observations I found that microtubules are stabilized along the shaft of the growing axons while dynamic microtubules enrich at the tip of the growing process, suggesting that a well- balanced shift of microtubule dynamics towards more stable microtubules is necessary to induce axon formation. By uncaging a photoactivatable analog of taxol I induced a local stabilization of microtubules at the neurite tip of an unpolarized neuron which was sufficient to favor the site of axon formation. This indicates that a transient stabilization of microtubules is sufficient to trigger axon formation. In summary, my data allow the following conclusions: 1) Microtubule stability correlates with the identity of a neuronal process. 2) Microtubule stabilization causes axon formation. 3) Microtubule stabilization precedes axon formation. I therefore deduce that microtubules are actively involved in the process of axon formation and that local microtubule stabilization in one neuronal process is a physiological signal specifying neuronal polarization

De novo identification of universal cell mechanics regulators

Mechanical proprieties determine many cellular functions, such as cell fate specification, migration, or circulation through vasculature. Identifying factors governing cell mechanical phenotype is therefore a subject of great interest. Here we present a mechanomics approach for establishing links between mechanical phenotype changes and the genes involved in driving them. We employ a machine learning-based discriminative network analysis method termed PC-corr to associate cell mechanical states, measured by real-time deformability cytometry (RT-DC), with large-scale transcriptome datasets ranging from stem cell development to cancer progression, and originating from different murine and human tissues. By intersecting the discriminative networks inferred from two selected datasets, we identify a conserved module of five genes with putative roles in the regulation of cell mechanics. We validate the power of the individual genes to discriminate between soft and stiff cell states in silico, and demonstrate experimentally that the top scoring gene, CAV1, changes the mechanical phenotype of cells when silenced or overexpressed. The data-driven approach presented here has the power of de novo identification of genes involved in cell mechanics regulation and paves the way towards engineering cell mechanical properties on demand to explore their impact on physiological and pathological cell functions

The XTT cell proliferation assay applied to cell layers embedded in three-dimensional matrix

Abstract Cell proliferation, a main target in cancer therapy, is influenced by the surrounding three-dimensional (3D) extracellular matrix (ECM). In vitro drug screening is, thus, optimally performed under conditions in which cells are grown (embedded or trapped) in dense 3D matrices, as these most closely mimic the adhesive and mechanical properties of natural ECM. Measuring cell proliferation under these conditions is, however, technically more challenging compared with two-dimensional (2D) culture and other "3D culture conditions," such as growth on top of a matrix (pseudo-3D) or in spongy scaffolds with large pore sizes. Consequently, such measurements are only slowly applied on a wider scale. To advance this, we report on the equal quality (dynamic range, background, linearity) of measuring the proliferation of cell layers embedded in dense 3D matrices (collagen, Matrigel) compared with cells in 2D culture using the easy (one-step) and in 2D well-validated, 2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide (XTT)-assay. The comparison stresses the differences in proliferation kinetics and drug sensitivity of matrix-embedded cells versus 2D culture. Using the specific cell-layer-embedded 3D matrix setup, quantitative measurements of cell proliferation and cell invasion are shown to be possible in similar assay conditions, and cytostatic, cytotoxic, and anti-invasive drug effects can thus be reliably determined and compared in physiologically relevant settings. This approach in the 3D matrix holds promise for improving early-stage, high-throughput drug screening, targeting either highly invasive or highly proliferative subpopulations of cancers or both

Using the RISCI Genetic Screening Platform for Elucidating Apoptosis Signalling Network

Considerable development in the field of nanotechnology is increasingly yielding novel applications of nanoparticles. The unique properties of nanoparticles in particular their high aspect ratio (length : width ratio), however could pose potential risks to the user. A high throughput genetic screening platform, RISCI (robotic single cDNA investigation), was previously established for the systematic evaluation of single gene activities. Here, RISCI was utilised to identify pro-apoptotic genes as well as genes involved in the positive and negative regulation of silica nanoparticle-induced cell death. This project describes the further development of the screening platform by harnessing its capability to screen a cDNA library comprising approximately 30,000 full length, completely annotated, and sequenced human genes for novel regulators of apoptosis. It integrates an extensive skill sets and is broadly organised into three major phases: Setup, Screen and Analysis. The integration of a pro-apoptosis treatment to screen for inhibitors and sensitizers is a novel aspect of the current experimental setup, along with the low redundancy library. The extensive setup phase focused on technical aspects. The cDNA library, acquired as plasmid DNA, was transformed into a bacterial host for replication and subsequent DNA isolation. A new high-throughput process was developed encompassing the production of competent bacteria and a heat shock transformation protocol, which was subsequently transferred onto the robotic platform. In parallel, the software controlling the robots was redeveloped to allow for execution of user-defined protocols while novel transfection protocols were adapted for automation. The screen identified 699 apoptosis inducers, 1,141 inhibitors and 626 sensitizers. Bioinformatics analysis revealed that the inducers were highly enriched for cell death associated terms, while the inhibitors were strongly associated with cancer profiles. Both inducers and sensitizers were predominantly achieving the functional effect on the protein level, but inhibitors were mainly transcription based. Enriched metal response genes also suggest that the silica nanoparticles were causing their toxicity through reactive oxygen species generation. Intriguingly, the screen identified many noncoding sequences as being functionally capable of regulating apoptosis. These noncoding candidates are capable of regulating the protein coding counterparts identified from the screen. The truly interesting part of the project outcome remains those unknown candidates that were implicated in apoptosis regulation for the first time. Dissemination of the consolidated candidate list would help accelerate the experimental validation of these candidates and aid other researchers in deriving novel hypotheses when the candidates are placed in their research context. [For supplementary files please contact author]

Lunar lander and return propulsion system trade study

This trade study was initiated at NASA/JSC in May 1992 to develop and evaluate main propulsion system alternatives to the reference First Lunar Outpost (FLO) lander and return-stage transportation system concept. Thirteen alternative configurations were developed to explore the impacts of various combinations of return stage propellants, using either pressure or pump-fed propulsion systems and various staging options. Besides two-stage vehicle concepts, the merits of single-stage and stage-and-a-half options were also assessed in combination with high-performance liquid oxygen and liquid hydrogen propellants. Configurations using an integrated modular cryogenic engine were developed to assess potential improvements in packaging efficiency, mass performance, and system reliability compared to non-modular cryogenic designs. The selection process to evaluate the various designs was the analytic hierarchy process. The trade study showed that a pressure-fed MMH/N2O4 return stage and RL10-based lander stage is the best option for a 1999 launch. While results of this study are tailored to FLO needs, the design date, criteria, and selection methodology are applicable to the design of other crewed lunar landing and return vehicles