Biology subjects

Bryce, D.

Publications and source records attributed to Bryce, D..

7 recordsLinked to original sources

bioRxiv2022

AutoGater: A Weakly Supervised Neural Network Model to Gate Cells in Flow Cytometric Analyses

Flow cytometry is a useful and efficient method for the rapid characterization of a cell population based on the optical and fluorescence properties of individual cells. Ideally, the cell population would consist of only healthy viable cells as dead cells can confound the analysis. Thus, separating out healthy cells from dying and dead cells, and any potential debris, is an important first step in analysis of flow cytometry data. While gating of debris can be conducted using measured optical properties, identifying dead and dying cells often requires utilizing fluorescent stains (e.g. Sytox, a nucleic acid stain that stains cells with compromised cell membranes) to identify cells that should be excluded from downstream analyses. These stains prolong the experimental preparation process and use a flow cytometers fluorescence channels that could otherwise be used to measure additional fluorescent markers within the cells (e.g. reporter proteins). Here we outline a stain-free method for identifying viable cells for downstream processing by gating cells that are dying or dead. AutoGater is a weakly supervised deep learning model that can separate healthy populations from unhealthy and dead populations using only light-scatter channels. In addition, AutoGater harmonizes different measurements of dead cells such as Sytox and CFUs.

bioinformatics↗

bioRxiv2022

Building an Open Representation for Biological Protocols

Laboratory protocols are critical to biological research and development, yet difficult to communicate and reproduce across projects, investigators, and organizations. While many attempts have been made to address this challenge, there is currently no available protocol representation that is unambiguous enough for precise interpretation and automation, yet simultaneously abstract enough to enable reuse and adaptation. The Protocol Activity Markup Language (PAML) is a free and open protocol representation aiming to address this gap, building on a foundation of UML, Autoprotocol, and SBOL RDF. PAML provides a representation both for protocols and for records of their execution and the resulting data, as well as a framework for exporting from PAML for execution by either humans or laboratory automation. PAML is currently implemented in the form of an RDF knowledge representation, specification document, and Python library, can be exported for execution as either a manual "paper protocol" or Autoprotocol, and is being further developed as an open community effort.

synthetic biology↗

bioRxiv2022

Computational Prediction of Synthetic Circuit Function Across Growth Conditions

A challenge in the design and construction of synthetic genetic circuits is that they will operate within biological systems that have noisy and changing parameter regimes that are largely unmeasurable. The outcome is that these circuits do not operate within design specifications or have a narrow operational envelope in which they can function. This behavior is often observed as a lack of reproducibility in function from day to day or lab to lab. Moreover, this narrow range of operating conditions does not promote reproducible circuit function in deployments where environmental conditions for the chassis are changing, as environmental changes can affect the parameter space in which the circuit is operating. Here we describe a computational method for assessing the robustness of circuit function across broad parameter regions. Previously designed circuits are assessed by this computational method and then circuit performance is measured across multiple growth conditions in budding yeast. The computational predictions are correlated with experimental findings, suggesting that the approach has predictive value for assessing the robustness of a circuit design.

synthetic biology↗

bioRxiv2022

Robustness and reproducibility of simple and complex synthetic logic circuit designs using a DBTL loop

Computational tools addressing various components of design-build-test-learn loops (DBTL) for the construction of synthetic genetic networks exist, but do not generally cover the entire DBTL loop. This manuscript introduces an end-to-end sequence of tools that together form a DBTL loop called DART (Design Assemble Round Trip). DART provides rational selection and refinement of genetic parts to construct and test a circuit. Computational support for experimental process, metadata management, standardized data collection, and reproducible data analysis is provided via the previously published Round Trip (RT) test-learn loop. The primary focus of this work is on the Design Assemble (DA) part of the tool chain, which improves on previous techniques by screening up to thousands of network topologies for robust performance using a novel robustness score derived from dynamical behavior based on circuit topology only. In addition, novel experimental support software is introduced for the assembly of genetic circuits. A complete design-through-analysis sequence is presented using several OR and NOR circuit designs, with and without structural redundancy, that are implemented in budding yeast. The execution of DART tested the predictions of the design tools, specifically with regard to robust and reproducible performance under different experimental conditions. The data analysis depended on a novel application of machine learning techniques to segment bimodal flow cytometry distributions. Evidence is presented that, in some cases, a more complex build may impart more robustness and reproducibility across experimental conditions.

synthetic biology↗

bioRxiv2022

Highly-Automated, High-Throughput Replication of Yeast-based Logic Circuit Design Assessments

We describe an experimental campaign that replicated the performance assessment of logic gates engineered into cells of S. cerevisiae by Gander, et al. Our experimental campaign used a novel high throughput experimentation framework developed under DARPAs Synergistic Discovery and Design (SD2) program: a remote robotic lab at Strateos executed a parameterized experimental protocol. Using this protocol and robotic execution, we generated two orders of magnitude more flow cytometry data than the original experiments. We discuss our results, which largely, but not completely, agree with the original report, and make some remarks about lessons learned.

synthetic biology↗

bioRxiv2020

Glycogen synthase kinase 3 alpha/beta deletion induces precocious growth plate remodeling and cell loss in mice

Glycogen synthase kinase (GSK) 3 acts to negatively regulate multiple signaling pathways, including canonical Wnt signaling. The two mammalian GSK3 proteins (alpha and beta) are at least partially redundant. While Gsk3a KO mice are viable and display a metabolic phenotype, abnormal neuronal development and accelerated aging, Gsk3b KO animals die late in embryogenesis or at birth. Selective Gsk3b KO in bone delayed development of some bones, whereas cartilage-specific Gsk3b KO mice are normal except for elevated levels of GSK3alpha protein. However, the collective role of these two GSK3 proteins in cartilage was not evaluated. To address this, we generated tamoxifen-inducible, cartilage-specific Gsk3a/Gsk3b KO in juvenile mice and investigated their skeletal phenotypes. We found that cartilage-specific Gsk3a/Gsk3b deletion in young, skeletally immature mice causes precocious growth plate remodeling, culminating in shorter long bones and hence, growth retardation. These mice exhibit inefficient breathing patterns at later stages and fail to survive. The disrupted growth plates in KO mice showed progressive loss of cellular and proteoglycan components and Sox9 positive cells, with increased staining for osteocalcin and type II collagen. In addition, an increase in osteoclast recruitment and cell apoptosis was observed in growth plates. Surprisingly, changes in articular cartilage of Gsk3a/Gsk3b KO mice were mild compared to growth plates, signifying differential regulation of articular cartilage vs growth plate tissues. Taken together, these findings emphasize a crucial role of two GSK3 proteins in skeletal development, in particular in the maintenance and function of growth plates. SignificanceGrowth plate cartilage dynamics determine the rate of endochondral bone growth and thus, our final height. These processes are disturbed in many genetic and acquired diseases, but the intracellular mechanisms responsible for normal growth plate function, as well as the cessation of growth plate activity in puberty, are poorly understood. Here, we demonstrate that specific removal of both GSK3 genes (Gsk3a and Gsk3b) in postnatal cartilage of mice leads to a severe reduction of endochondral bone growth, premature remodelling of the growth plate, and early death. In contrast, articular cartilage is only mildly affected by deletion of both genes. These studies identify GSK3 signaling as a key regulator of growth plate dynamics and endochondral bone growth.

developmental biology↗

bioRxiv2018

LRRK2 kinase inhibitors induce a reversible effect in the lungs of non-human primates with no measurable pulmonary deficits

Putative gain-of-function mutations in leucine-rich repeat kinase 2 (LRRK2), resulting in increased kinase activity and cellular toxicity, are a leading genetic cause of Parkinsons disease (PD). Hence, there is strong interest in developing LRRK2 kinase inhibitors as a disease-modifying therapy. Published reports that repeat dosing with two LRRK2 kinase inhibitors (GNE-7915 and GNE-0877) induce histopathological changes in the lung of non-human primates Fuji et al. 2015 (1) raised concerns about potential safety liability of LRRK2 kinase inhibitors. In the present study, we sought to determine whether previously observed effects in the lung: (a) represent on-target pharmacology, but with the potential for margin of safety, (b) are reversible upon drug withdrawal, and (c) are associated with pulmonary function deficits. To this end, we evaluated the histopathological effects, toxicokinetics and target inhibition of three structurally diverse LRRK2 kinase inhibitors, GNE-7915 (30 mg/kg, BID, as a positive control), MLi-2 (15 and 50 mg/kg, QD) and PFE-360 (3 and 6 mg/kg, QD) following 2 weeks of dosing in non-human primates. Subsets of animals dosed with GNE-7915 or MLi-2 were evaluated after 2-week dose-free periods. All three LRRK2 kinase inhibitors induced mild cytoplasmic vacuolation of type II pneumocytes, as reported previously, confirming an on-target effect of these compounds. Interestingly, despite lower doses of both PFE-360 and MLi-2 producing nearly complete inhibition of LRRK2 kinase activity in the brain as assessed by levels of pS935-LRRK2, histopathological changes in lung were absent in animals treated with low-dose PFE-360 and observed only sporadically in the low-dose MLi-2 group. The lung effect was fully reversible at 2 weeks post-dosing of GNE-7915. In a second study of identical dosing with MLi-2 and GNE-7915, no deficits were observed in a battery of translational pulmonary functional tests. In aggregate, these results do not preclude the development of LRRK2 kinase inhibitors for clinical investigation in Parkinsons disease.

pharmacology and toxicology↗