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"Anderson, Michael G."
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Anterior chamber depth in mice is controlled by several quantitative trait loci
Anterior chamber depth (ACD) is a quantitative trait associated with primary angle closure glaucoma (PACG). Although ACD is highly heritable, known genetic variations explain a small fraction of the phenotypic variability. The purpose of this study was to identify additional ACD-influencing loci using strains of mice. Cohorts of 86 N2 and 111 F2 mice were generated from crosses between recombinant inbred BXD24/TyJ and wild-derived CAST/EiJ mice. Using anterior chamber optical coherence tomography, mice were phenotyped at 10–12 weeks of age, genotyped based on 93 genome-wide SNPs, and subjected to quantitative trait locus (QTL) analysis. In an analysis of ACD among all mice, six loci passed the significance threshold of p = 0.05 and persisted after multiple regression analysis. These were on chromosomes 6, 7, 11, 12, 15 and 17 (named Acdq6 , Acdq7 , Acdq11 , Acdq12 , Acdq15 , and Acdq17 , respectively). Our findings demonstrate a quantitative multi-genic pattern of ACD inheritance in mice and identify six previously unrecognized ACD-influencing loci. We have taken a unique approach to studying the anterior chamber depth phenotype by using mice as genetic tool to examine this continuously distributed trait.
Journal Article
Reduction of ER stress via a chemical chaperone prevents disease phenotypes in a mouse model of primary open angle glaucoma
Mutations in myocilin (MYOC) are the most common genetic cause of primary open angle glaucoma (POAG), but the mechanisms underlying MYOC-associated glaucoma are not fully understood. Here, we report the development of a transgenic mouse model of POAG caused by the Y437H MYOC mutation; the mice are referred to herein as Tg-MYOC(Y437H) mice. Analysis of adult Tg-MYOC(Y437H) mice, which we showed express human MYOC containing the Y437H mutation within relevant eye tissues, revealed that they display glaucoma phenotypes (i.e., elevated intraocular pressure [IOP], retinal ganglion cell death, and axonal degeneration) closely resembling those seen in patients with POAG caused by the Y437H MYOC mutation. Mutant myocilin was not secreted into the aqueous humor but accumulated in the ER of the trabecular meshwork (TM), thereby inducing ER stress in the TM of Tg-MYOC(Y437H) mice. Furthermore, chronic and persistent ER stress was found to be associated with TM cell death and elevation of IOP in Tg-MYOC(Y437H) mice. Reduction of ER stress with a chemical chaperone, phenylbutyric acid (PBA), prevented glaucoma phenotypes in Tg-MYOC(Y437H) mice by promoting the secretion of mutant myocilin in the aqueous humor and by decreasing intracellular accumulation of myocilin in the ER, thus preventing TM cell death. These results demonstrate that ER stress is linked to the pathogenesis of POAG and may be a target for treatment in human patients.
Journal Article
Ketamine/Xylazine-Induced Corneal Damage in Mice
We have observed that the commonly used ketamine/xylazine anesthesia mix can induce a focally severe and permanent corneal opacity. The purpose of this study was to establish the clinical and histological features of this deleterious side effect, its sensitivity with respect to age and anesthesia protocol, and approaches for avoiding it.
Young C57BL/6J, C57BLKS/J, and SJL/J mice were treated with permutations of anesthesia protocols and compared using slit-lamp exams, optical coherence tomography, histologic analyses, and telemetric measurements of body temperature.
Ketamine/xylazine induces corneal damage in mice with a variable frequency. Among 12 experimental cohorts, corneal damage associated with ketamine/xylazine was observed in 9 of them. Despite various treatments to avoid corneal dehydration during anesthesia, the frequency of corneas experiencing damage among responding cohorts was 42% (26% inclusive of all cohorts), which is significantly greater than the natural prevalence (5%). The damage was consistent with band keratopathy. It appeared as a white or gray horizontal band located proximal to the pupil and was positive for subepithelial calcium deposition with von Kossa stain.
The sum of our clinical and histological observations is consistent with ketamine/xylazine-induced band keratopathy in mice. This finding is relevant for mouse studies involving the eye and/or vision-dependent behavioral assays, which would both be prone to artifact without appreciation of the damage caused by ketamine/xylazine anesthesia. Use of yohimbine is suggested as a practical means of avoiding this complication.
Journal Article
Axonopathy precedes cell death in ocular damage mediated by blast exposure
Traumatic brain injuries (TBI) of varied types are common across all populations and can cause visual problems. For military personnel in combat settings, injuries from blast exposures (bTBI) are prevalent and arise from a myriad of different situations. To model these diverse conditions, we are one of several groups modeling bTBI using mice in varying ways. Here, we report a refined analysis of retinal ganglion cell (RGC) damage in male C57BL/6J mice exposed to a blast-wave in an enclosed chamber. Ganglion cell layer thickness, RGC density (BRN3A and RBPMS immunoreactivity), cellular density of ganglion cell layer (hematoxylin and eosin staining), and axon numbers (paraphenylenediamine staining) were quantified at timepoints ranging from 1 to 17-weeks. RNA sequencing was performed at 1-week and 5-weeks post-injury. Earliest indices of damage, evident by 1-week post-injury, are a loss of RGC marker expression, damage to RGC axons, and increase in glial markers expression. Blast exposure caused a loss of RGC somas and axons—with greatest loss occurring by 5-weeks post-injury. While indices of glial involvement are prominent early, they quickly subside as RGCs are lost. The finding that axonopathy precedes soma loss resembles pathology observed in mouse models of glaucoma, suggesting similar mechanisms.
Journal Article
Whole-exome sequencing prioritizes candidate genes for hereditary cataract in the Emory mouse mutant
The Emory cataract (Em) mouse mutant has long been proposed as an animal model for age-related or senile cataract in humans—a leading cause of visual impairment. However, the genetic defect(s) underlying the autosomal dominant Em phenotype remains elusive. Here, we confirmed development of the cataract phenotype in commercially available Em/J mice [but not ancestral Carworth Farms White (CFW) mice] at 6–8 months of age and undertook whole-exome sequencing of candidate genes for Em. Analysis of coding and splice-site variants did not identify any disease-causing/associated mutations in over 450 genes known to underlie inherited and age-related forms of cataract and other lens disorders in humans and mice, including genes for lens crystallins, membrane/cytoskeleton proteins, DNA/RNA-binding proteins, and those associated with syndromic/systemic forms of cataract. However, we identified three cataract/lens-associated genes each with one novel homozygous variant including predicted missense substitutions in Prx (p.R167C) and Adamts10 (p.P761L) and a disruptive in-frame deletion variant (predicted missense) in Abhd12 (p.L30_A32delinsS) that were absent in CFW and over 35 other mouse strains. In silico analysis predicted that the missense substitutions in Prx and Adamts10 were borderline neutral/damaging and neutral, respectively, at the protein function level, whereas, that in Abhd12 was functionally damaging. Both the human counterparts of Adamts10 and Abhd12 are clinically associated with syndromic forms of cataract known as Weil-Marchesani syndrome 1 and polyneuropathy, hearing loss, ataxia, retinitis pigmentosa, and cataract syndrome, respectively. Overall, while we cannot exclude Prx and Adamts10, our data suggest that Abhd12 is a promising candidate gene for cataract in the Em/J mouse.
Journal Article
Making room for zoom in focus group methods: opportunities and challenges for novice researchers (during and beyond COVID-19)
Als die COVID-19-Pandemie über die Welt hereinbrach, waren viele Menschen gezwungen, sich auf online-basierte Routinen einzustellen, darunter auch qualitative Forscher*innen, die nach alternativen Möglichkeiten zur Erhebung aussagekräftiger Daten suchten. Während Fokusgruppen traditionell Face to Face durchgeführt werden, bieten Fortschritte bei Online-Videokonferenzanwendungen neue Methoden zur Datenerhebung, die jedoch bisher nur selten untersucht wurden. In diesem Artikel berichten wir über die Erfahrungen von 12 Doktorand*innen mit der Durchführung von Fokusgruppen unter Verwendung von Zoom im Rahmen eines Kurses zu qualitativen Interviewmethoden. Wir reflektieren Chancen und Herausforderungen, die wir als Moderator*innen und Teilnehmer*innen bei der Nutzung von Zoom erlebten z.B. bei der Vorbereitung oder in Bezug auf Rapport, die Einbindung anderer digitaler Tools und von Internetverbindungen. Zusammenfassend lässt sich sagen, dass die Durchführung von Online-Fokusgruppen unter Verwendung von Zoom insgesamt eine positive Erfahrung war und mit Face-to-Face-Fokusgruppen vergleichbar ist. Möglichkeiten der Teilnehmer*innenrekrutierung, die Sicherheitsmerkmale von Zoom und die Nutzung von Zoom und allgemeiner neuen Technologien sollten auch jenseits der Pandemie weiter erforscht werden.
As the COVID-19 pandemic swept through the world, it forced many people to adapt to an online-based routine, including qualitative researchers looking for alternative ways to collect meaningful data. While focus groups are traditionally conducted in-person, advances with online videoconferencing applications present a new method to collect data, however, few studies have explored this. In this article we present 12 doctoral students' experiences with conducting focus groups using the videoconferencing application Zoom during a qualitative methods course on interviewing methods. Through this self-study qualitative analysis, participants reflected on the opportunities and challenges experienced as both moderators and participants using Zoom including: preparation, rapport, incorporating other digital tools, and internet connectivity. In conclusion, doing focus groups online using Zoom was a positive experience overall and comparable to in-person focus groups for collecting qualitative data, despite the introduction of technology. More research on participant recruitment, new technology, Zoom's security features, and Zoom's use outside of a pandemic should be further explored.
Journal Article
Exome-based investigation of the genetic basis of human pigmentary glaucoma
Background
Glaucoma is a leading cause of visual disability and blindness. Release of iris pigment within the eye, pigment dispersion syndrome (PDS), can lead to one type of glaucoma known as pigmentary glaucoma. PDS has a genetic component, however, the genes involved with this condition are largely unknown. We sought to discover genes that cause PDS by testing cohorts of patients and controls for mutations using a tiered analysis of exome data.
Results
Our primary analysis evaluated melanosome-related genes that cause dispersion of iris pigment in mice (
TYRP1
,
GPNMB
,
LYST
,
DCT
, and
MITF
). We identified rare mutations, but they were not statistically enriched in PDS patients. Our secondary analyses examined
PMEL
(previously linked with PDS),
MRAP
, and 19 other genes. Four
MRAP
mutations were identified in PDS cases but not in controls (
p
= 0.016). Immunohistochemical analysis of human donor eyes revealed abundant MRAP protein in the iris, the source of pigment in PDS. However, analysis of
MRAP
in additional cohorts (415 cases and 1645 controls) did not support an association with PDS. We also did not confirm a link between
PMEL
and PDS in our cohorts due to lack of reported mutations and similar frequency of the variants in PDS patients as in control subjects.
Conclusions
We did not detect a statistical enrichment of mutations in melanosome-related genes in human PDS patients and we found conflicting data about the likely pathogenicity of
MRAP
mutations. PDS may have a complex genetic basis that is not easily unraveled with exome analyses.
Journal Article
Ensemble emission of isolated organic chromophores incorporated into an organometallic single crystal
Molecular quantum emitters are becoming increasingly important in quantum information and communication. As a stepping stone towards a single-molecule quantum system, the collective emission from the ensemble of isolated organic chromophores, randomly and sparsely incorporated into an organometallic host crystal, is characterized by Raman and temperature-dependent photoluminescence spectroscopies. The tetracene or rubrene guest chromophores are deposited at very low densities when the ferrocene host is grown in a crystalline form, so that each of the chromophores is well isolated by its organometallic molecular neighbors. The ensemble emission of the chromophores is compared to that of the crystalline or dissolved forms to identify its unique spectral features. The enhanced quantum yield and reduced spectral linewidth with a significant blue-shift in photoluminescence suggest that ferrocene is a novel type of host matrix, maximizing the ability of the tetracene guest to act as a well-isolated quantum entity, while suppressing unwanted environmental decoherence by confining it within the ferromagnetic (organometallic) host material.
Journal Article
Inherited glaucoma in DBA/2J mice: Pertinent disease features for studying the neurodegeneration
The glaucomas are neurodegenerative diseases involving death of
retinal ganglion cells and optic nerve head excavation. A major risk
factor for this neurodegeneration is a harmfully elevated intraocular
pressure (IOP). Human glaucomas are typically complex, progressive
diseases that are prevalent in the elderly. Family history and genetic
factors are clearly important in human glaucoma. Mouse studies have proven
helpful for investigating the genetic and mechanistic basis of complex
diseases. We previously reported inherited, age-related progressive
glaucoma in DBA/2J mice. Here, we report our updated findings from
studying the disease in a large number of DBA/2J mice. The period when
mice have elevated IOP extends from 6 months to 16 months, with 8–9
months representing an important transition to high IOP for many mice.
Optic nerve degeneration follows IOP elevation, with the majority of optic
nerves being severely damaged by 12 months of age. This information should
help with the design of experiments, and we present the data in a manner
that will be useful for future studies of retinal ganglion cell
degeneration and optic neuropathy.
Journal Article