Biology subjects

Grady, S.

Publications and source records attributed to Grady, S..

4 recordsLinked to original sources

bioRxiv2025

A water-based platform test of paired or group social dominance and neural activity in male and female mice

Social hierarchy is an evolutionarily-conserved phenomenon determined by social dominance behavior that has profound influence on health and relevance in neuropsychiatric disorders. Despite this, the neural mechanisms underlying social dominance remain unclear and current behavioral tests are limited. Here, we describe a novel platform test of social dominance where mice compete for space on a small, elevated platform surrounded by cold water and rank is calculated by total time spent on platform. To validate this assay, we conducted tube test followed by platform test in cages of male and female mice. We observed stable rank in both sexes using the platform test and saw notable differences in overall cage hierarchy between tube and platform. Motivated behavior as measured by attempts to get on the platform was reduced over time. Cooperative behavior as measured by shared time on platform was observed across all ranks but predominantly demonstrated by subordinate mice. Corticosterone levels were significantly higher in females but showed no rank-specific differences. Neuronal activity in the prefrontal cortex was not rank-dependent, however activity in the habenula scaled with rank in both sexes and was significantly correlated with dominance in males. We also investigated group social dynamics using the platform test and identified stable hierarchies with more exaggerated dominance compared to paired platform testing. These results introduce the platform test as a novel method for assessing social dominance behavior in male and female mice, which can be used to further our understanding of the neurobiology underlying social hierarchy.

neuroscience↗

bioRxiv2023

Fluorescent human RPA to track assembly dynamics on DNA

DNA metabolic processes including replication, repair, recombination, and telomere maintenance occur on single-stranded DNA (ssDNA). In each of these complex processes, dozens of proteins function together on the ssDNA template. However, when double-stranded DNA is unwound, the transiently open ssDNA is protected and coated by the high affinity heterotrimeric ssDNA binding Replication Protein A (RPA). Almost all downstream DNA processes must first remodel/remove RPA or function alongside to access the ssDNA occluded under RPA. Formation of RPA-ssDNA complexes trigger the DNA damage checkpoint response and is a key step in activating most DNA repair and recombination pathways. Thus, in addition to protecting the exposed ssDNA, RPA functions as a gatekeeper to define functional specificity in DNA maintenance and genomic integrity. RPA achieves functional dexterity through a multi-domain architecture utilizing several DNA binding and protein-interaction domains connected by flexible linkers. This flexible and modular architecture enables RPA to adopt a myriad of configurations tailored for specific DNA metabolic roles. To experimentally capture the dynamics of the domains of RPA upon binding to ssDNA and interacting proteins we here describe the generation of active site-specific fluorescent versions of human RPA (RPA) using 4-azido-L-phenylalanine (4AZP) incorporation and click chemistry. This approach can also be applied to site-specific modifications of other multi-domain proteins. Fluorescence-enhancement through non-canonical amino acids (FEncAA) and Forster Resonance Energy Transfer (FRET) assays for measuring dynamics of RPA on DNA are also described. HighlightsO_LIRPA is an essential protein for most DNA metabolic processes including replication, repair, and recombination. C_LIO_LIRPA is a ssDNA binding protein made of six domains situated across RPA70, RPA32 and RPA14 subunits. Four high affinity DNA binding domains engage the DNA. C_LIO_LISite-specific fluorescent probes were incorporated into two domains of RPA and report on ssDNA binding dynamics. C_LIO_LIBulk-level kinetic and single-molecule assays are described to monitor the binding and remodeling of individual RPA domains on ssDNA. C_LI

biochemistry↗

bioRxiv2021

Voxelated Bioprinting of Mechanically Robust Multiscale Porous Scaffolds for Pancreatic Islets

Three-dimensional (3D) bioprinting additively assembles bio-inks to manufacture tissue-mimicking biological constructs, but with the typical building blocks limited to one-dimensional filaments. Here, we develop a technique for the digital assembly of spherical particles (DASP), which are effectively zero-dimensional voxels - the basic unit of 3D structures. We show that DASP enables on-demand generation, deposition, and assembly of viscoelastic bio-ink droplets. We establish a phase diagram that outlines the viscoelasticity of bio-inks required for printing spherical particles of good fidelity. Moreover, we develop a strategy for engineering bio-inks with independently controllable viscoelasticity and mesh size. Using DASP, we create mechanically robust, multiscale porous scaffolds composed of interconnected yet distinguishable hydrogel particles. Finally, we demonstrate the application of the scaffolds in encapsulating human pancreatic islets for responsive insulin release. Together with the knowledge of bio-ink design, DASP might be used to engineer highly heterogeneous yet tightly organized tissue constructs for therapeutic applications. Table of ContentsA three-dimensional bioprinting technology is developed to enable on-demand generation, deposition, and assembly of viscoelastic bio-ink droplets in a biofriendly environment. A strategy is developed to independently control elasticity, viscosity, and mesh size of bio-inks. The technique allows for easy manufacturing mechanically robust multiscale porous scaffolds, which can be used to encapsulate human pancreatic islets for sustained responsive insulin release. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=182 SRC="FIGDIR/small/449587v1_ufig1.gif" ALT="Figure 1"> View larger version (58K): org.highwire.dtl.DTLVardef@1c0f2eaorg.highwire.dtl.DTLVardef@8bf924org.highwire.dtl.DTLVardef@191bd3aorg.highwire.dtl.DTLVardef@11e5ce1_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioengineering↗

bioRxiv2020

Single amino acid mutations effect Zika virus replication in vitro and virulence in vivo

The 2014-2016 Zika virus (ZIKV) epidemic in the Americas resulted in large deposits of next-generation sequencing data from clinical samples. This resource was mined to identify emerging mutations and trends in mutations as the outbreak progressed over time. Information on transmission dynamics, prevalence and persistence of intra-host mutants, and the position of a mutation on a protein were then used to prioritize 544 reported mutations based on their ability to impact ZIKV phenotype. Using this criteria, six mutants (representing naturally occurring mutations) were generated as synthetic infectious clones using a 2015 Puerto Rican epidemic strain PRVABC59 as the parental backbone. The phenotypes of these naturally occurring variants were examined using both cell culture and murine model systems. Mutants had distinct phenotypes, including changes in replication rate, embryo death, and decreased head size. In particular, a NS2B mutant previously detected during in vivo studies in rhesus macaques was found to cause lethal infections in adult mice, abortions in pregnant females, and increased viral genome copies in both brain tissue and blood of female mice. Additionally, mutants with changes in the region of NS3 that interfaces with NS5 during replication displayed reduced replication in the blood of adult mice. This analytical pathway, integrating both bioinformatic and wet lab experiments, provides a foundation for understanding how naturally occurring single mutations affect disease outcome and can be used to predict the of severity of future ZIKV outbreaks. Author summaryTo determine if naturally occurring individual mutations in the Zika virus epidemic genotype effect viral virulence or replication rate in vitro or in vivo, we generated an infectious clone representing the epidemic genotype of stain Puerto Rico, 2015. Using this clone, six mutants were created by changing nucleotides in the genome to cause one to two amino acid substitutions in the encoded proteins. The six mutants we generated represent mutations that differentiated the early epidemic genotype from genotypes that were either ancestral or that occurred later in the epidemic. We assayed each mutant for changes in growth rate, and for virulence in adult mice and pregnant mice. Three of the mutants caused catastrophic embryo effects including increased embryonic death or significant decrease in head diameter. Three other mutants that had mutations in a genome region associated with replication resulted in changes in in vitro and in vivo replication rates. These results illustrate the potential impact of individual mutations in viral phenotype.

microbiology↗