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PyMINEr Finds Gene and Autocrine-Paracrine Networks from Human Islet scRNA-Seq.

Fri, 2019-02-15 11:26
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PyMINEr Finds Gene and Autocrine-Paracrine Networks from Human Islet scRNA-Seq.

Cell Rep. 2019 Feb 12;26(7):1951-1964.e8

Authors: Tyler SR, Rotti PG, Sun X, Yi Y, Xie W, Winter MC, Flamme-Wiese MJ, Tucker BA, Mullins RF, Norris AW, Engelhardt JF

Abstract
Toolsets available for in-depth analysis of scRNA-seq datasets by biologists with little informatics experience is limited. Here, we describe an informatics tool (PyMINEr) that fully automates cell type identification, cell type-specific pathway analyses, graph theory-based analysis of gene regulation, and detection of autocrine-paracrine signaling networks in silico. We applied PyMINEr to interrogate human pancreatic islet scRNA-seq datasets and discovered several features of co-expression graphs, including concordance of scRNA-seq-graph structure with both protein-protein interactions and 3D genomic architecture, association of high-connectivity and low-expression genes with cell type enrichment, and potential for the graph structure to clarify potential etiologies of enigmatic disease-associated variants. We further created a consensus co-expression network and autocrine-paracrine signaling networks within and across islet cell types from seven datasets. PyMINEr correctly identified changes in BMP-WNT signaling associated with cystic fibrosis pancreatic acinar cell loss. This proof-of-principle study demonstrates that the PyMINEr framework will be a valuable resource for scRNA-seq analyses.

PMID: 30759402 [PubMed - in process]

CRISPR-Cas9 genome engineering: Treating inherited retinal degeneration.

Fri, 2019-02-15 11:26
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CRISPR-Cas9 genome engineering: Treating inherited retinal degeneration.

Prog Retin Eye Res. 2018 07;65:28-49

Authors: Burnight ER, Giacalone JC, Cooke JA, Thompson JR, Bohrer LR, Chirco KR, Drack AV, Fingert JH, Worthington KS, Wiley LA, Mullins RF, Stone EM, Tucker BA

Abstract
Gene correction is a valuable strategy for treating inherited retinal degenerative diseases, a major cause of irreversible blindness worldwide. Single gene defects cause the majority of these retinal dystrophies. Gene augmentation holds great promise if delivered early in the course of the disease, however, many patients carry mutations in genes too large to be packaged into adeno-associated viral vectors and some, when overexpressed via heterologous promoters, induce retinal toxicity. In addition to the aforementioned challenges, some patients have sustained significant photoreceptor cell loss at the time of diagnosis, rendering gene replacement therapy insufficient to treat the disease. These patients will require cell replacement to restore useful vision. Fortunately, the advent of induced pluripotent stem cell and CRISPR-Cas9 gene editing technologies affords researchers and clinicians a powerful means by which to develop strategies to treat patients with inherited retinal dystrophies. In this review we will discuss the current developments in CRISPR-Cas9 gene editing in vivo in animal models and in vitro in patient-derived cells to study and treat inherited retinal degenerative diseases.

PMID: 29578069 [PubMed - indexed for MEDLINE]

Generation of an immortalized human choroid endothelial cell line (iChEC-1) using an endothelial cell specific promoter.

Fri, 2018-12-28 02:03
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Generation of an immortalized human choroid endothelial cell line (iChEC-1) using an endothelial cell specific promoter.

Microvasc Res. 2018 Dec 17;:

Authors: Giacalone JC, Miller MJ, Workalemahu G, Reutzel AJ, Ochoa D, Scott Whitmore S, Stone EM, Tucker BA, Mullins RF

Abstract
Age-related macular degeneration (AMD) is a common cause of blindness worldwide. While recent studies have revealed that the loss of choroidal endothelial cells (ChECs) is critical to the disease pathogenesis of dry AMD, in vitro studies are needed to fully elucidate the disease mechanism. However, these studies remain hindered due to the lack of publically available human ChEC lines. To address this need, ChECs were harvested form donor tissue and enriched for by using magnetic cell separation using anti-CD31 conjugated microbeads. Next, lenti-viral vectors with endothelial-specific promoters driving genes necessary for immortalization, CDH5p-hTERT and CDH5p TAg, were generated. Stable integration of both gene cassettes allowed cells to maintain their proliferative state and yielded an immortalized cell line (iChEC-1). Immunocytochemical analysis of iChEC-1 confirmed the expression of important ChEC markers such as CA4, a marker of choriocapillaris endothelial cells, CDH5, and CD34, pan-endothelial cell markers. qRT-PCR analysis of expanded clones from iChEC-1 further showed that the line maintained expression of other important endothelial markers, vWF, PECAM1, and PLVAP, similar to primary cells. Functional responses were characterized by tube-forming assays and repopulation of decellularized choroid with the immortalized cell line. In conclusion, the iChEC-1 line presents a suitable immortalized human ChEC line for future in vitro studies of AMD.

PMID: 30571950 [PubMed - as supplied by publisher]

Correlation of Optical Coherence Tomography and Retinal Histology in Normal and Pro23His Retinal Degeneration Pig.

Thu, 2018-12-13 21:28
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Correlation of Optical Coherence Tomography and Retinal Histology in Normal and Pro23His Retinal Degeneration Pig.

Transl Vis Sci Technol. 2018 Nov;7(6):18

Authors: Cheng J, Sohn EH, Jiao C, Adler KL, Kaalberg EE, Russell SR, Mullins RF, Stone EM, Tucker BA, Han IC

Abstract
Purpose: We correlate optical coherence tomography (OCT) retinal layer thickness measurements with histology in wild-type and retinal degenerative pigs.
Methods: OCT scans were obtained using the Bioptigen Envisu R2200. In normal pigs, three eyes were imaged in vivo, and three eyes were imaged after enucleation. In the Pro23His retinal degeneration pigs (P23H), one eye was imaged in vivo and four eyes were imaged after enucleation. All eyes were fixed in 4% paraformaldehyde and processed for histology. Corresponding retinal locations on OCT and histology were identified using anatomic landmarks (optic nerve, retinal vessels, visual streak). Individual retinal layer thicknesses were measured by two independent, masked graders, and intraclass correlation coefficients were used to determine agreement. OCT and histologic retinal thickness measurements were averaged and compared.
Results: OCT and histologic measurements correlated highly in normal and diseased eyes (R 2 = 0.91 and 0.92, respectively), and scans performed in vivo and ex vivo did not differ significantly. Despite good overall correlation, certain individual retinal layers (e.g., retinal nerve fiber layer [NFL], inner [INL] and outer [ONL] nuclear layers) appeared thicker on OCT compared to histology, while other layers (e.g., retinal pigment epithelium) appeared thinner. No statistically significant difference was found between OCT and histology for any retinal layer thickness measurement.
Conclusions: Retinal layer thickness measurements correlate well with histology in pig eyes, but differences in individual retinal layers may be seen.
Translational Relevance: OCT may be used in pigs to measure retinal thicknesses with good overall correlation to histologic measurements.

PMID: 30519502 [PubMed]

The ARMS2 A69S Polymorphism Is Associated with Delayed Rod-Mediated Dark Adaptation in Eyes at Risk for Incident Age-Related Macular Degeneration.

Thu, 2018-11-08 13:21
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The ARMS2 A69S Polymorphism Is Associated with Delayed Rod-Mediated Dark Adaptation in Eyes at Risk for Incident Age-Related Macular Degeneration.

Ophthalmology. 2018 Oct 30;:

Authors: Mullins RF, McGwin G, Searcey K, Clark ME, Kennedy EL, Curcio CA, Stone EM, Owsley C

Abstract
OBJECTIVE: To examine the association between sequence variants in genetic risk factors for age-related macular degeneration (AMD), and delayed rod-mediated dark adaptation (RMDA), the first functional biomarker for incident AMD, in older adults with normal macular health and early AMD.
DESIGN: Cross-sectional SUBJECTS: Older adults aged ≥60 years in normal macular health (defined as both eyes at step 1 on the Age-Related Eye Disease 9-step AMD classification system) and those with AMD in one or both eyes (defined as steps 2-9).
METHODS: Single nucleotide polymorphisms were genotyped in the CFH and ARMS2 genes using a Taqman assay. RMDA was assessed in one eye after photobleach with targets centered at 5° on the inferior vertical meridian. Rate of dark adaptation was defined by rod intercept time (RIT), duration (minutes) required for sensitivity to reach a criterion sensitivity level in the latter half of the second component of rod recovery. Associations between CFH and ARMS2 polymorphisms and RMDA were adjusted for age and smoking.
MAIN OUTCOME MEASURE: RIT.
RESULTS: The sample consisted of 543 participants having both genotype and RIT determination; 408 were in normal macular health and 135 had AMD, most having early AMD (124 of 135). For the combined sample, higher RIT (slower RMDA) was observed for both the A69S variant in ARMS2 and the Y402H variant in CFH (adjusted p=0.0001 and p=0.0023 respectively). For normal subjects the A69S variant in ARMS2 was associated with higher RIT (adjusted p=0.0011), whereas CFH Y402H was not (adjusted p=0.2175). For AMD cases, the A69S variant of ARMS2 and CFH Y402H were associated with higher RIT (adjusted p=0.0182 and p=0.0222 respectively). Those with a greater number of high-risk ARMS2 and CFH alleles had higher RIT, in both normal and AMD groups (adjusted p=0.0002 and p<0.0001 respectively).
CONCLUSIONS: We report a novel association wherein older adults with high risk ARMS2 and CFH genotypes are more likely to have delayed RMDA, the first functional biomarker for incident early AMD. Before the AMD clinical phenotype is present, those in normal macular health with the ARMS2 A69S allele have delayed RMDA. Understanding ARMS2 function is a research priority.

PMID: 30389424 [PubMed - as supplied by publisher]