Biology subjects

King, R.

Publications and source records attributed to King, R..

5 recordsLinked to original sources

bioRxiv2018

FLASH: A next-generation CRISPR diagnostic for multiplexed detection of antimicrobial resistance sequences

The growing prevalence of deadly microbes with resistance to previously life-saving drug therapies is a dire threat to human health. Detection of low abundance pathogen sequences remains a challenge for metagenomic Next Generation Sequencing (NGS). We introduce FLASH (Finding Low Abundance Sequences by Hybridization), a next-generation CRISPR/Cas9 diagnostic method that takes advantage of the efficiency, specificity and flexibility of Cas9 to enrich for a programmed set of sequences. FLASH-NGS achieves up to 5 orders of magnitude of enrichment and sub-attomolar gene detection with minimal background. We provide an open-source software tool (FLASHit) for guide RNA design. Here we applied it to detection of antimicrobial resistance genes in respiratory fluid and dried blood spots, but FLASH-NGS is applicable to all areas that rely on multiplex PCR.

microbiology↗

bioRxiv2017

Genomic Locus Modulating Corneal Thickness in the Mouse Identifies POU6F2 as a Potential Risk of Developing Glaucoma

Purpose: Central corneal thickness (CCT) is one of the most heritable ocular traits and it is also a phenotypic risk factor for primary open angle glaucoma (POAG). The present study uses the BXD Recombinant Inbred (RI) strains to identify novel quantitative trait loci (QTLs) modulating CCT in the mouse with the potential of identifying a molecular link between CCT and risk of developing POAG.\n\nMethods: The BXD RI strain set was used to define mammalian genomic loci modulating CCT, with a total of 818 corneas measured from 61 BXD RI strains (between 60-100 days of age). The mice were anesthetized and the eyes were positioned in front of the lens of the Phoenix Micron IV Image-Guided OCT system or the Bioptigen OCT system. CCT data for each strain was averaged and used to identify quantitative trait loci (QTLs) modulating this phenotype using the bioinformatics tools on GeneNetwork (www.genenetwork.org). The candidate genes and genomic loci identified in the mouse were then directly compared with the summary data from a human primary open-angle glaucoma (POGA) genome wide association study (NEIGHBORHOOD) to determine if any genomic elements modulating mouse CCT are also risk factors for POAG.\n\nResults: This analysis revealed one significant QTL on Chr 13 and a suggestive QTL on Chr 7. The significant locus on Chr 13 (13 to 19 Mb) was examined further to define candidate genes modulating this eye phenotype. For the Chr 13 QTL in the mouse, only one gene in the region (Pou6f2) contained nonsynonymous SNPs. Of these five nonsynonymous SNPs in Pou6f2, two resulted in changes in the amino acid proline which could result in altered secondary structure affecting protein function. The 7 Mb region under the mouse Chr 13 peak distributes over 2 chromosomes in the human: Chr 1 and Chr 7. These genomic loci were examined in the NEIGHBORHOOD database to determine if they are potential risk factors for human glaucoma identified using meta-data from human GWAS. The top 50 hits all resided within one gene (POU6F2), with the highest significance level of p = 10-6 for SNP rs76319873. POU6F2 is found in retinal ganglion cells and in corneal limbal stem cells. To test the effect of POU6F2 on CCT we examined the corneas of a Pou6f2-null mice and the corneas were thinner than those of wild-type littermates. In addition, these POU6F2 RGCs die early in the DBA/2J model of glaucoma than most RGCs.\n\nConclusions: Using a mouse genetic reference panel, we identified a transcription factor, Pou6f2, that modulates CCT in the mouse. POU6F2 is also found in a subset of retinal ganglion cells and these RGCs are sensitive to injury.\n\nAuthors SummaryGlaucoma is a complex group of diseases with several known causal mutations and many known risk factors. One well-known risk factor for developing primary open angle glaucoma is the thickness of the central cornea. The present study leverages a unique blend of systems biology methods using BXD recombinant inbred mice and genome-wide association studies from humans to define a putative molecular link between a phenotypic risk factor (central corneal thickness) and glaucoma. We identified a transcription factor, POU6F2, that is found in the developing retinal ganglion cells and cornea. POU6F2 is also present in a subpopulation of retinal ganglion cells and in stem cells of the cornea. Functional studies reveal that POU6F2 is associated the central corneal thickness and with susceptibility of retinal ganglion cells to injury.

genetics↗

bioRxiv2017

Genomic Loci Modulating Retinal Ganglion Cell Death Following Elevated IOP in the Mouse

The present study was designed to identify genomic loci modulating the susceptibility of retinal ganglion cells (RGC) to elevated intraocular pressure (IOP) in the BXD recombinant inbred mouse strain set. IOP was elevated by injecting magnetic microspheres into the anterior chamber and blocking the trabecular meshwork using a handheld magnet to impede drainage. The IOP was then measured over the next 21 days. Only animals with IOP greater than 25 mmHg for two consecutive days or an IOP above 30 mmHg on a single day after microsphere-injection were used in this study. On day 21, mice were sacrificed and the optic nerve was processed for histology. Axons were counted for both the injected and the control eye in 49 BXD strains, totaling 181 normal counts and 191 counts associated with elevated IOP. The axon loss for each strain was calculated and the data were entered into genenetwork.org. The average number of normal axons in the optic nerve across all strains was 54,788 {+/-} 16% (SD), which dropped to 49,545 {+/-} 20% in animals with artificially elevated IOP. Interval mapping demonstrated a relatively similar genome-wide map for both conditions with a suggestive Quantitative Trait Locus (QTL) on proximal Chromosome 3. When the relative axon loss was used to generate a genome-wide interval map, we identified one significant QTL (p<0.05) on Chromosome 18 between 53.6 and 57 Mb. Within this region, the best candidate gene for modulating axon loss was Aldh7a1. Immunohistochemistry demonstrated ALDH7A1 expression in mouse RGCs. ALDH7A1 variants were not significantly associated with glaucoma in the NEIGHBORHOOD GWAS dataset, but this enzyme was identified as part of the butanoate pathway previously associated with glaucoma risk. Our results suggest that genomic background influences susceptibility to RGC degeneration and death in an inducible glaucoma model.

genetics↗

bioRxiv2017

Genetic Locus Modulating IOP

PurposeIntraocular pressure (IOP) is the primary risk factor for developing glaucoma. The present study examines genomic contribution to the normal regulation of IOP in the mouse.\n\nMethodsThe BXD recombinant inbred (RI) strain set was used to identify genomic loci modulating IOP. We measured the IOP from 532 eyes from 34 different strains. The IOP data will be subjected to conventional quantitative trait analysis using simple and composite interval mapping along with epistatic interactions to define genomic loci modulating normal IOP.\n\nResultsThe analysis defined one significant quantitative trait locus (QTL) on Chr.8 (100 to 106 Mb). The significant locus was further examined to define candidate genes that modulate normal IOP. There are only two good candidate genes within the 6 Mb over the peak, Cdh8 (Cadherin 8) and Cdh11 (Cadherin 11). Expression analysis on gene expression and immunohistochemistry indicate that Cdh11 is the best candidate for modulating the normal levels of IOP.\n\nConclusionsWe have examined the genomic regulation of IOP in the BXD RI strain set and found one significant QTL on Chr. 8. Within this QTL that are two potential candidates for modulating IOP with the most likely gene being Cdh11.

bioinformatics↗

bioRxiv2017

The BXD Mouse Strains are a Model System for Studying Optic Nerve Regeneration

The present study is designed to identify the influences of genetic background to optic nerve regeneration using the two parental strains C57BL/6J and DBA/2J and 7 BXD recombinant inbred strains. To study regeneration in the optic nerve, Pten was knocked down in the retinal ganglion cells using AAV, and a mild inflammatory response was induced by an intravitreal injection of zymosan with CPT-cAMP, and the axons were damaged by optic nerve crush (ONC). Regenerating axons were labeled by Cholera Toxin B and quantified 14 days after ONC. The number of axons at 0.5 mm and 1 mm from the crush site were counted. In addition, we measured the distance that 5 axons had grown down the nerve and the longest distance a single axon reached. Results showed a considerable amount of differential axonal growth across all 9 BXD strains. There was a significant difference (P=0.014 Mann-Whitney U test) in the regenerative capacity in the number of axons reaching 0.5 mm from a low of 1487.6 {+/-} 264.9 axons in BXD102 to a high of 4175.8 {+/-} 648.6 axons in BXD29. There were also significant differences (P=0.014 Mann-Whitney U test) in the distance axons traveled, looking at a minimum of 5 axons with the shortest distance was 787.2 {+/-} 46.5{micro}m in BXD102 to a maximum distance of 2025.5 {+/-} 223.3{micro}m in BXD29. These results reveal that genetic background can modulate axonal regeneration and that the BXD strains are a particularly well-suited model system.

neuroscience↗