Biology subjects

Johnson, R. D.

Publications and source records attributed to Johnson, R. D..

8 recordsLinked to original sources

bioRxiv2023

SH2 domain protein E (SHE) and ABL signaling regulate blood vessel size

Blood vessels in different vascular beds vary in lumen diameter, which is essential for their function and fluid flow along the vascular network. Molecular mechanisms involved in the formation of a vascular lumen of appropriate size, or tubulogenesis, are still only partially understood. Src homology 2 domain containing E (She) protein was previously identified in a screen for proteins that interact with Abelson (Abl)-kinase. However, its biological role has remained unknown. Here we demonstrate that She and Abl signaling regulate vascular lumen size in zebrafish embryos and human endothelial cell culture. Zebrafish she mutants displayed increased endothelial cell number and enlarged lumen size of the dorsal aorta (DA) and defects in blood flow. Vascular endothelial specific overexpression of she resulted in a reduced diameter of the DA lumen, which correlated with the reduced arterial cell number and lower endothelial cell proliferation. Chemical inhibition of Abl signaling in zebrafish embryos caused a similar reduction in the DA diameter and alleviated the she mutant phenotype, suggesting that She acts as a negative regulator of Abl signaling. Enlargement of the DA lumen in she mutants correlated with an increased endothelial expression of claudin 5a and 5b (cldn5a / cldn5b), which encode proteins enriched in tight junctions. Inhibition of cldn5a expression partially rescued the enlarged DA in she mutants, suggesting that She regulates DA lumen size, in part, by promoting cldn5a expression. SHE knockdown in human endothelial umbilical vein cells resulted in a similar increase in the diameter of vascular tubes, and also increased phosphorylation of a known ABL downstream effector CRKL. These results argue that SHE functions as an evolutionarily conserved inhibitor of ABL signaling and regulates lumen size during vascular tubulogenesis.

developmental biology↗

bioRxiv2023

Optogenetic urothelial cell stimulation induces bladder contractions and pelvic nerve afferent firing

Urothelial cells, which play an essential role in the barrier function, are also thought to play a sensory role in bladder physiology by releasing signaling molecules in response to sensory stimuli that act upon adjacent sensory neurons. However, it is challenging to study this communication due to the overlap in receptor expression and proximity of urothelial cells to sensory neurons. To overcome this challenge, we have developed a mouse model where we can directly stimulate urothelial cells using optogenetics. We have crossed a uroplakin II-cre mouse (UPK2-Cre) with a mouse that expresses the light-activated cation channel, Channelrhodopsin-2 (ChR2), in the presence of cre-expression. Optogenetic stimulation of urothelial cells cultured from UPK2-ChR2 initiates cellular depolarization and release of adenosine triphosphate. Cystometry recordings demonstrate that optical stimulation of urothelial cells increases bladder pressure and pelvic nerve activity. Increases in bladder pressure persisted, albeit to a lesser extent, when the bladder was excised in an in vitro preparation. The P2X receptor antagonist, PPADS, significantly reduced optically evoked bladder contractions in vivo and ex vivo. Further, corresponding nerve activity was also inhibited with PPADS. Our data suggest that urothelial cells can initiate robust bladder contractions via sensory nerve signaling or contractions through local signaling mechanisms. This data supports a foundation of literature demonstrating communication between sensory neurons and urothelial cells. Importantly, with further use of these optogenetic tools, we hope to scrutinize this signaling mechanism, its importance for normal micturition and nociception, and how it may be altered in pathophysiologic conditions. Animal StudiesAll the procedures involving mice and mouse tissue performed in this study were approved by the University of Florida Institutional Animal Care and Use Committee and in strict accordance with the US National Institute of Health (NIH) Guide for the Care and Use of Laboratory Animals. SignificanceIt has been appreciated for almost two decades that urothelial cells play a sensory role in bladder function. However, it has been particularly challenging to study this communication as both sensory neurons, and urothelial cells express the same sensory receptors. Here we demonstrate an optogenetic technique to specifically stimulate urothelial cells and evaluate the effects on bladder physiology. We used this technique to show that specific urothelial stimulation resulted in bladder contractions. This approach will have a long-lasting impact on how we study urothelial-to-sensory neuron communication and the changes that occur under disease conditions.

physiology↗

bioRxiv2021

Virtual meetings promise to eliminate the geographical and administrative barriers and increase accessibility, diversity, and inclusivity

The COVID-19 pandemic brought a new set of unprecedented challenges not only for healthcare, education, and everyday jobs but also in terms of academic conferences. In this study, we investigate the effect of the broad adoption of virtual platforms for academic conferences as a response to COVID-19 restrictions. We show that virtual platforms enable higher participation from underrepresented minority groups, increased inclusion, and broader geographic distribution. We also discuss emerging challenges associated with the virtual conference format resulting in a decreased engagement of social activities, limited possibilities of cross-fertilization between participants, and reduced peer-to-peer interactions. Lastly, we conclude that a novel comprehensive approach needs to be adopted by the conference organizers to ensure increased accessibility, diversity, and inclusivity of post-pandemic conferences. Our findings provide evidence favoring a hybrid format for future conferences, marrying the strength of both in-person and virtual platforms.

scientific communication and education↗

bioRxiv2021

CRISPR-Cas9 gene editing and rapid detection of gene-edited mutants using high-resolution melting in the apple scab fungus, Venturia inaequalis

BackgroundScab, or black spot, caused by the filamentous fungal pathogen Venturia inaequalis, is the most economically important disease of apple (Malus x domestica) worldwide. To develop durable control strategies against this disease, a better understanding of the genetic mechanisms underlying the growth, reproduction, virulence and pathogenicity of V. inaequalis is required. A major bottleneck for the genetic characterization of V. inaequalis is the inability to easily delete or disrupt genes of interest using homologous recombination. Indeed, no gene deletions or disruptions in V. inaequalis have yet been published. Recently, CRISPR-Cas9 has emerged as an efficient tool for gene editing in filamentous fungi. With this in mind, we set out to establish CRISPR-Cas9 as a gene editing tool in V. inaequalis. ResultsWe showed that CRISPR-Cas9 can be used for gene inactivation in the apple scab fungus. As a proof of concept, we targeted the melanin biosynthesis pathway gene trihydroxynaphthalene reductase (THN), which has previously been shown to result in a light-brown colony phenotype when transcriptionally silenced using RNA interference. Using one of two CRISPR-Cas9 single guide RNAs (sgRNAs) targeted to the THN gene, delivered by a single autonomously replicating Golden Gate-compatible plasmid, we were able to identify six of 36 stable transformants with a light-brown phenotype, indicating an ~16.7% gene inactivation efficiency. Notably, of these six THN mutants, five had an independent mutation. As part of our pipeline, we also report a high-resolution melting (HRM) curve protocol for the rapid detection of CRISPR-Cas9 gene-edited mutants of V. inaequalis. This protocol identified a single base pair deletion mutation in a sample containing only 5% mutant genomic DNA, indicating high sensitivity for mutant screening. ConclusionsIn establishing CRISPR-Cas9 as a tool for gene editing in V. inaequalis, we have provided a strong starting point for studies aiming to decipher the function of genes associated with the growth, reproduction, virulence and pathogenicity of this fungus. The associated HRM curve protocol will enable CRISPR-Cas9 transformants to be screened for gene inactivation in a high-throughput and low-cost manner, which will be particularly powerful in cases where the CRISPR-Cas9-mediated gene inactivation efficiency is low.

microbiology↗

bioRxiv2020

H3k27ac-HiChIP in prostate cell lines identifies risk genes for prostate cancer susceptibility

Genome-wide association studies (GWAS) have identified more than 140 prostate cancer (PrCa) risk regions which provide potential insights into causal mechanisms. Multiple lines of evidence show that a significant proportion of PrCa risk can be explained by germline causal variants that dysregulate nearby target genes in prostate-relevant tissues thus altering disease risk. The traditional approach to explore this hypothesis has been correlating GWAS variants with steady-state transcript levels, referred to as expression quantitative trait loci (eQTLs). In this work, we assess the utility of chromosome conformation capture (3C) coupled with immunoprecipitation (HiChIP) to identify target genes for PrCa GWAS risk loci. We find that interactome data confirms previously reported PrCa target genes identified through GWAS/eQTL overlap (e.g., MLPH). Interestingly, HiChIP identified links between PrCa GWAS variants and genes well-known to play a role in prostate cancer biology (e.g., AR) that are not detected by eQTL-based methods. We validate these findings through CRISPR interference (CRISPRi) perturbation of the variant-containing regulatory elements for NKX3-1 and AR in the LNCaP cell line. Our results demonstrate that looping data harbor additional information beyond eQTLs and expand the number of PrCa GWAS loci that can be linked to candidate susceptibility genes.

genomics↗

bioRxiv2020

Epicardial slices: a 3D organotypic model for the study of epicardial activation and differentiation

The epicardium constitutes an untapped reservoir for cardiac regeneration. Upon myocardial injury, the adult epicardium re-activates the embryonic program, leading to epithelial-to-mesenchymal transition, migration and differentiation. Despite some successes in harnessing the epicardial therapeutic potential by thymosin {beta}4 (T{beta}4) pre-treatment, further translational advancements are hampered by the paucity of representative experimental models. Here we apply innovative protocols to obtain living 3D organotypic slices from porcine hearts, encompassing the epicardial/myocardial interface. In culture, our slices preserve the in vivo architecture and functionality, presenting a continuous epicardium overlaying a healthy and connected myocardium. Upon T{beta}4 treatment of the slices, the epicardial cells become activated upregulating embryonic and EMT genes and invading the myocardium where they differentiate towards the mesenchymal lineage. Our 3D organotypic model enables to investigate the reparative potential of the adult epicardium, offering a new tool to explore ex vivo the complex 3D interactions occurring within the native heart environment.

bioengineering↗

bioRxiv2020

A scalable method for estimating the regional polygenicity of complex traits

A key question in human genetics is understanding the proportion of SNPs modulating a particular phenotype or the proportion of susceptibility SNPs for a disease, termed polygenicity. Previous studies have observed that complex traits tend to be highly polygenic, opposing the previous belief that only a handful of SNPs contribute to a trait. Beyond these genome-wide estimates, the distribution of polygenicity across genomic regions as well as the genomic factors that affect regional polygenicity remain poorly understood. A reason for this gap is that methods for estimating polygenicity utilize SNP effect sizes from GWAS. However, estimating regional polygenicity from GWAS effect sizes involves untangling the correlation between SNPs due to LD, leading to intractable computations for even a small number of SNPs. In this work, we propose a scalable method, BEAVR, to estimate the regional polygenicity of a trait given marginal effect sizes from GWAS and LD information. We implement a Gibbs sampler to estimate the posterior distribution of the regional polygenicity and derive a fast, algorithmic update to circumvent the computational bottlenecks associated with LD. The runtime of our algorithm is [O](MK) for M SNPs and K susceptibility SNPs, where the number of susceptibility SNPs is typically K << M. By modeling the full LD structure, we show that BEAVR provides unbiased estimates of polygenicity compared to previous methods that only partially model LD. Finally, we show how estimates of regional polygenicity for BMI, eczema, and high cholesterol provide insight into the regional genetic architecture of each trait.

genetics↗

bioRxiv2018

A unifying framework for joint trait analysis under a non-infinitesimal model

MotivationA large proportion of risk regions identified by genome-wide association studies (GWAS) are shared across multiple diseases and traits. Understanding whether this clustering is due to sharing of causal variants or chance colocalization can provide insights into shared etiology of complex traits and diseases.\n\nResultsIn this work, we propose a flexible, unifying framework to quantify the overlap between a pair of traits called UNITY (Unifying Non-Infinitesimal Trait analYsis). We formulate a Bayesian generative model that relates the overlap between pairs of traits to GWAS summary statistic data under a non-infinitesimal genetic architecture underlying each trait. We propose a Metropolis-Hastings sampler to compute the posterior density of the genetic overlap parameters in this model. We validate our method through comprehensive simulations and analyze summary statistics from height and BMI GWAS to show that it produces estimates consistent with the known genetic makeup of both traits.\n\nAvailabilityThe UNITY software is made freely available to the research community at: https://github.com/bogdanlab/UNITY\n\nContactruthjohnson@ucla.edu\n\nSupplementary informationSupplementary data are available at Bioinformatics online.

bioinformatics↗