Biology subjects

Moore, J.

Publications and source records attributed to Moore, J..

11 recordsLinked to original sources

bioRxiv2018

A unified model for microtubule rescue

How microtubules transition from depolymerization to polymerization, known as rescue, is poorly understood. Here we examine two models for rescue: 1) an end-driven model in which the depolymerizing end stochastically switches to a stable state; and 2) a lattice-driven model in which rescue-sites are integrated into the microtubule prior to depolymerization. We test these models using a combination of computational simulations and in vitro experiments with purified tubulin. Our findings support the lattice-driven model by identifying repeated rescue sites in microtubules. In addition, we discover an important role for divalent cations in determining the frequency and location of rescue sites. We use wash-in experiments to show that divalent cations inhibit rescue during depolymerization, but not during the polymerization. We propose a unified model in which rescues are driven by embedded rescue sites in microtubules, but the activity of these sites is influenced by changes in the depolymerizing ends.

cell biology↗

bioRxiv2018

TUBA1A mutations identified in lissencephaly patients dominantly disrupt neuronal migration and impair dynein activity

Tubulinopathies are severe human brain malformations associated with mutations in tubulin genes. Despite the identification of many tubulin mutations in patients, we do not understand how these mutations impact the microtubule cytoskeleton, how the changes to microtubule function lead to brain malformations, or how different tubulin isotypes regulate microtubules to support normal neurodevelopment. TUBA1A -tubulin is the most commonly affected tubulin isotype in tubulinopathy patients. Heterozygous mutations in TUBA1A have been identified in patients with diverse cortical malformations including microlissencephaly, lissencephaly, pachygyria, and polymicrogyria. Here we focus on mutations affecting the conserved arginine at position 402 (R402), which account for 30% of all reported TUBA1A mutations in patients. We demonstrate that exogenous expression of TUBA1A-R402C and TUBA1A-R402H patient alleles is sufficient to dominantly disrupt cortical neuron migration in the developing mouse brain, recapitulating the human lissencephaly phenotype. Intriguingly, ectopic expression of TUBA1A-R402C/H alleles does not alter morphology, axonal trafficking, or microtubule polymerization rates in cultured neurons, but does lead to subtle changes in axonal microtubule orientation. Further, we find that budding yeast -tubulin with analogous R402C and R402H mutations assembles into microtubules but disrupts the activity of the microtubule motor dynein. The level of dynein impairment scales with abundance of R402 mutant -tubulin in the cell. Together, our results support a model in which tubulinopathy mutations at R402 poison the microtubule network in young neurons by creating defective binding sites for dynein at the microtubule surface.

developmental biology↗

bioRxiv2018

A Fixed Moderate-dose Combination of Tiletamine+Zolazepam Outperforms Midazolam in Induction of Short-term Immobilization of Ball Pythons (Python regius)

Laboratory animals are commonly anesthetized to prevent pain and distress and to provide safe handling. Anesthesia procedures are well-developed for common laboratory mammals, but not as well established in reptiles. We assessed the performance of intramuscularly injected tiletamine (dissociative anesthetic) and zolazepam (benzodiazepine sedative) in fixed combination (2 mg/kg and 3 mg/kg) in comparison to 2 mg/kg of midazolam (benzodiazepine sedative) in ball pythons (Python regius). We measured heart and respiratory rates and quantified induction parameters (i.e., time to loss of righting reflex, time to loss of withdrawal reflex) and recovery parameters (i.e., time to regain righting reflex, withdrawal reflex, normal behavior). Mild decreases in heart and respiratory rates (median decrease of <10 beats per minute and <5 breaths per minute) were observed for most time points among all three anesthetic dose groups. No statistically significant difference between the median time to loss of righting reflex was observed among animals of any group (p = 0.783). However, the withdrawal reflex was lost in all snakes receiving 3mg/kg of tiletamine+zolazepam but not in all animals of the other two groups (p = 0.0004). In addition, the time for animals to regain the righting reflex and resume normal behavior was longer in the drug combination dose groups compared to the midazolam group (p = 0.0055). Our results indicate that midazolam is an adequate sedative for ball pythons but does not suffice to achieve reliable immobilization or anesthesia, whereas tiletamine+zolazepam achieves short-term anesthesia in a dose-dependent manner.

zoology↗

bioRxiv2018

Validation of a library of cGMP-compliant human pluripotent stem cell lines for use in liver therapy

Recent advancements in the production of hepatocytes from human pluripotent stem cells (hPSC-Heps) afford tremendous possibilities for treatment of patients with liver disease. Validated current good manufacturing practice (cGMP) lines are an essential prerequisite for such applications but have only recently been established. Whether such cGMP lines are capable of hepatic differentiation is not known. To address this knowledge gap, we examined the proficiency of three recently derived cGMP lines (two hiPSC and one hESC) to differentiate into hepatocytes and their suitability for therapy. hPSC-Heps generated using a chemically defined four-step hepatic differentiation protocol uniformly demonstrated highly reproducible phenotypes and functionality. Seeding into a 3D PEG-DA fabricated inverted colloid crystal (ICC) scaffold converted these immature progenitors into more advanced hepatic tissue structures. Hepatic constructs could also be successfully encapsulated into the immune-privileged material alginate. This is the first report we are aware of demonstrating cGMP-compliant hPSCs can generate cells with advanced hepatic function potentially suitable for future therapeutic applications.

cell biology↗

bioRxiv2018

Endogenous protection from ischemic brain injury by preconditioned monocytes

Exposure to low dose lipopolysaccharide prior to cerebral ischemia is neuroprotective in stroke models, a phenomenon termed preconditioning. While it is well established that lipopolysaccharide-preconditioning induces central and peripheral immune responses, the cellular mechanisms modulating ischemic injury remain unclear. Here, we investigated the role of immune cells in the brain protection afforded by preconditioning and we tested whether monocytes may be reprogrammed by ex vivo lipopolysaccharide exposure thus modulating the inflammatory injury after cerebral ischemia in male mice. We found that systemic injection of low-dose lipopolysaccharide induces a distinct subclass of CD115+Ly6Chi monocytes that protect the brain after transient middle cerebral artery occlusion in mice. Remarkably, adoptive transfer of monocytes isolated from preconditioned mice into naive mice 7 hours after transient middle cerebral artery occlusion reduced brain injury. Gene expression and functional studies showed that IL-10, iNOS and CCR2 in monocytes are essential for the neuroprotection. This protective activity was elicited even if mouse or human monocytes were exposed ex vivo to lipopolysaccharide and then injected into male mice after stroke. Cell tracking studies showed that protective monocytes are mobilized from the spleen and reach brain and meninges, wherein they suppressed post-ischemic inflammation and neutrophils influx into the brain parenchyma. Our findings unveil a previously unrecognized subpopulation of splenic monocytes capable to protect the brain with an extended therapeutic window, and provide the rationale for cell therapies based on the delivery of autologous or allogeneic protective monocytes into patients with ischemic stroke.\n\nSignificance StatementInflammation is a key component of the pathophysiology of the brain in stroke, a leading cause of death and disability with limited therapeutic options. Here, we investigate endogenous mechanisms of protection against cerebral ischemia. Using LPS preconditioning as an approach to induce ischemic tolerance in mice, we found the generation of neuroprotective monocytes within the spleen from where they traffic to the brain and meninges suppressing post-ischemic inflammation. Importantly, systemic LPS preconditioning can be mimicked by adoptive transfer of in vitro-preconditioned mouse or human monocytes at translational relevant time points after stroke. This model of neuroprotection may facilitate clinical efforts to increase the efficacy of bone marrow mononuclear cell treatments in acute neurological diseases such as cerebral ischemia.

neuroscience↗

bioRxiv2017

Transcriptome-wide analysis of the functional intronome using spliceosome profiling

Full understanding of eukaryotic transcriptomes and how they respond to different conditions requires deep knowledge of all sites of intron excision. Although RNA-Seq provides much of this information, the low abundance of many spliced transcripts (often due to their rapid cytoplasmic decay) limits the ability of RNA-Seq alone to reveal the full repertoire of spliced species. Here we present \"spliceosome profiling\", a strategy based on deep sequencing of RNAs co-purifying with late stage spliceosomes. Spliceosome profiling allows for unambiguous mapping of intron ends to single nucleotide resolution and branchpoint identification at unprecedented depths. Our data reveal hundreds of new introns in S. pombe and numerous others that were previously misannotated. By providing a means to directly interrogate sites of spliceosome assembly and catalysis genome-wide, spliceosome profiling promises to transform our understanding of RNA processing in the nucleus much like ribosome profiling has transformed our understanding mRNA translation in the cytoplasm.

genomics↗

bioRxiv2017

A Feed-forward Relay between Bicoid and Orthodenticle Regulates the Timing ofEmbryonic Patterning in Drosophila

The K50 homeodomain (K50HD) protein Orthodenticle (Otd) is critical for anterior patterning and brain and eye development in most metazoans. In Drosophila melanogaster, another K50HD protein, Bicoid (Bcd), has evolved to replace Otds ancestral function in embryo patterning. Bcd is distributed as a long-range maternal gradient and activates transcription of a large number of target genes including otd. Otd and Bcd bind similar DNA sequences in vitro, but how their transcriptional activities are integrated to pattern anterior regions of the embryo is unknown. Here we define three major classes of enhancers that are differentially sensitive to binding and transcriptional activation by Bcd and Otd. Class 1 enhancers are initially activated by Bcd, and activation is transferred to Otd via a feed-forward relay (FFR) that involves sequential binding of the two proteins to the same DNA motif. Class 2 enhancers are activated by Bcd, and maintained by an Otd-independent mechanism. Class 3 enhancers are never bound by Bcd, but Otd binds and activates them in a second wave of zygotic transcription. The specific activities of enhancers in each class are mediated by DNA motif variants preferentially bound by Bcd or Otd, and the presence or absence of sites for cofactors that interact with these proteins. Our results define specific patterning roles for Bcd and Otd, and provide mechanisms for coordinating the precise timing of gene expression patterns during embryonic development.

molecular biology↗

bioRxiv2017

The structured demography of open populations in fluctuating environments

At the spatial scale relevant to many field studies and management policies, populations may experience more external recruitment than internal recruitment. These sources of recruitment, as well as local demography, are often subject to stochastic fluctuations in environmental conditions. Here, we introduce a class of stochastic models accounting for these complexities and provide analytic methods for understanding their long-term behavior. The population state n(x) of these stochastic models is a function or vector keeping track of densities of individuals with continuous (e.g. size) or discrete (e.g. age) traits x taking values in a compact metric space. This state variable is updated by a stochastic affine equation nt+1 = At+1nt + bt+1 where At+1 is a time varying operator (e.g. an integral operator or a matrix) that updates the local demography, and bt+1 is a time varying function or vector representing external recruitment. When the realized per-capita growth rate of the local demography is negative, we show that all initial conditions converge to the same time-varying trajectory. Furthermore, when A1, A2... and b1, b2, ... are stationary sequences, this limiting behavior is determined by a unique stationary distribution. When the stationary sequences are periodic, uncorrelated, or a mixture of these two types of stationarity, we derive explicit formulas for the mean, within-year covariance, and auto-covariance of the stationary distribution. Sensitivity formulas for these statistical features are also given. The analtyic methods are illustrated with applications to discrete size-structured models of space-limited coral populations, and continuously size-structured models of giant clam populations.

ecology↗

bioRxiv2017

Restoration of Eastern oyster populations with positive density dependence

AO_SCPCAPBSTRACTC_SCPCAPPositive density dependence can create a threshold of population states below which extinction of the population occurs. The existence of this threshold, which can often be a complex, multi-dimensional surface, rather than a single point, is of particular importance in degraded populations for which there is a desire for successful restoration. Here, we incorporated positive density dependence into a closed, size- and age-structured integral projection model parameterized with empirical data from an Eastern oyster, Crassostrea virginica, population in Pamlico Sound, North Carolina. To understand the properties of the threshold surface, and implications for restoration, we introduced a general method based on a linearization of the threshold surface at its unique, unstable equilibrium. We estimated the number of oysters of a particular age (i.e. stock enhancement), or the surface area of hard substrate required (i.e. habitat enhancement), to move a population from an extinction trajectory to a persistent trajectory. The location of the threshold surface was strongly affected by changes in the amount of local larval retention. Traditional stock enhancement with oysters less than a year old (i.e. spat) required three times as many oysters relative to stock enhancement with oysters between ages three and seven, while the success of habitat enhancement depended upon the initial size distribution of the population. The methodology described here demonstrates the importance of considering positive density dependence in oyster populations, and also provides insights into effective management and restoration strategies when dealing with a high dimensional threshold separating extinction and persistence.

ecology↗

bioRxiv2016

The Image Data Resource: A Scalable Platform for Biological Image Data Access, Integration, and Dissemination

Access to primary research data is vital for the advancement of science. To extend the data types supported by community repositories, we built a prototype Image Data Resource (IDR) that collects and integrates imaging data acquired across many different imaging modalities. IDR links high-content screening, super-resolution microscopy, time-lapse and digital pathology imaging experiments to public genetic or chemical databases, and to cell and tissue phenotypes expressed using controlled ontologies. Using this integration, IDR facilitates the analysis of gene networks and reveals functional interactions that are inaccessible to individual studies. To enable re-analysis, we also established a computational resource based on IPython notebooks that allows remote access to the entire IDR. IDR is also an open source platform that others can use to publish their own image data. Thus IDR provides both a novel on-line resource and a software infrastructure that promotes and extends publication and re-analysis of scientific image data.

bioinformatics↗

bioRxiv2016

OME Files - An open source reference library for the OME-XML metadata model and the OME-TIFF file format

Digital imaging is now used throughout biological and biomedical research to measure the architecture, composition and dynamics of cells, tissues and organisms. The many different imaging technologies create data in many different formats. This diversity of data formats arises because the creation of common, cross-domain data standards is difficult and the pace of innovation challenges the durability of any defined standard and the implementation of data stewardship principles such as FAIR1. The Open Microscopy Environment (OME; http://openmicroscopy.org) has therefore developed the OME Data Model, an open, extensible specification for imaging metadata, that supports metadata related to an imaging experiment, data acquisition and any derived analytic results2-4. The OME Data Model has been implemented in OME-XML so that metadata can be stored and accessed by any sof ...

bioinformatics↗