Mullins Lab Publications

Subscribe to Mullins Lab Publications feed Mullins Lab Publications
Updated: 5 days 38 min ago

Bulk and single-cell gene expression analyses reveal aging human choriocapillaris has pro-inflammatory phenotype.

Wed, 2020-06-17 13:20
Related Articles

Bulk and single-cell gene expression analyses reveal aging human choriocapillaris has pro-inflammatory phenotype.

Microvasc Res. 2020 Jun 09;:104031

Authors: Voigt AP, Whitmore SS, Mulfaul K, Chirco KR, Giacalone JC, Flamme-Wiese MJ, Stockman A, Stone EM, Tucker BA, Scheetz TE, Mullins RF

Abstract
The human choroidal vasculature is subject to age-related structural and gene expression changes implicated in age-related macular degeneration (AMD). In this study, we performed both bulk and single-cell RNA sequencing on infant (n = 4 for bulk experiments, n = 2 for single-cell experiments) and adult (n = 13 for bulk experiments, n = 6 for single-cell experiments) human donors to characterize how choroidal gene expression changes with age. Differential expression analysis revealed that aged choroidal samples were enriched in genes encoding pro-inflammatory transcription factors and leukocyte transendothelial cell migration adhesion proteins. Such genes were observed to be differentially expressed specifically within choroidal endothelial cells at the single-cell level. Immunohistochemistry experiments support transcriptional findings that CD34 is elevated in infant choriocapillaris endothelial cells while ICAM-1 is enriched in adults. These results suggest several potential drivers of the pro-inflammatory vascular phenotype observed with advancing age.

PMID: 32531351 [PubMed - as supplied by publisher]

Visualization of Mouse Choroidal and Retinal Vasculature Using Fluorescent Tomato Lectin Perfusion.

Wed, 2020-06-10 10:56
Related Articles

Visualization of Mouse Choroidal and Retinal Vasculature Using Fluorescent Tomato Lectin Perfusion.

Transl Vis Sci Technol. 2020 Jan;9(1):1

Authors: Jiao C, Adler K, Liu X, Sun W, Mullins RF, Sohn EH

Abstract
Purpose: To develop a reliable and simplified method to assess choroid and retinal vasculature on whole mount and cross sections in mice using tomato lectin (TL; Lycopersicon esculentum).
Methods: Albino mice (n = 27) received 1 mg/mL of TL (conjugated to Dylight-594) intravascularly through the tail vein, jugular vein, or cardiac left ventricle. Whole mounts of the retina and choroid were evaluated using fluorescence microscopy. Perfusion with GSL-IB4 conjugated to Dylight-594 and fluorescein isothiocyanate was performed to compare against labeling with TL. Co-labeling of choroidal endothelial cells with perfused TL on cross-sections with antibodies directed against the choriocapillaris-restricted endothelial cell marker CA4 was performed. The percentage of perfused choroidal and retinal vessels was assessed semiquantitatively. One mouse was subjected to thermal laser damage before perfusion to cause retinal and choroidal vasculature ablation.
Results: Intravascular injection of TL led to consistent, robust labeling of retinal and choroidal vascular walls. On cross-sections, choriocapillaris was co-labeled with CA4 and TL. On flat mount, TL perfusion resulted in better labeling of choroidal vessels using tail/jugular vein injection compared with cardiac perfusion (P < .01). More consistent labeling of the choroidal and retinal vascular trees was observed with TL than with GSL-IB4. Vascular damage caused by laser ablation was detected readily using this method.
Conclusions: TL injection intravascularly can reliably label normal and ablated choroid and retinal vasculature in mouse in a quick, simple manner.
Translational Relevance: These data will help to facilitate modeling in rodents for diseases such as age-related macular degeneration, diabetes, and other ischemic/angiogenic processes that can also be used for treatment evaluation.

PMID: 32509436 [PubMed]

Optimizing Donor Cellular Dissociation and Subretinal Injection Parameters for Stem Cell-Based Treatments.

Wed, 2020-06-10 10:56
Related Articles

Optimizing Donor Cellular Dissociation and Subretinal Injection Parameters for Stem Cell-Based Treatments.

Stem Cells Transl Med. 2019 08;8(8):797-809

Authors: Scruggs BA, Jiao C, Cranston CM, Kaalberg E, Wang K, Russell SR, Wiley LA, Mullins RF, Stone EM, Tucker BA, Sohn EH

Abstract
Subretinal delivery of stem cell-derived retinal cells as a strategy to treat retinal degenerative blindness holds great promise. Currently, two clinical trials are underway in which human fetal retinal progenitor cells (RPCs) are being delivered to patients by intravitreal or subretinal injection to preserve or restore vision, respectively. With the advent of the induced pluripotent stem cell (iPSC), and in turn three-dimensional derivation of retinal tissue, it is now possible to generate autologous RPCs for cell replacement. The purpose of this study was to evaluate the effect of commonly used cell isolation and surgical manipulation strategies on donor cell viability. iPSC-RPCs were subjected to various conditions, including different dissociation and isolation methods, injection cannula sizes, and preinjection storage temperatures and times. The effects of commonly used surgical techniques on both host and donor cell viability were evaluated in Yucatan mini-pigs (n = 61 eyes). We found a significant increase in cell viability when papain was used for RPC isolation. In addition, a significant decrease in cell viability was detected when using the 41G cannula compared with 31G and at storage times of 4 hours compared with 30 minutes. Although 96.4% of all eyes demonstrated spontaneous retinal reattachment following injection, retinal pigment epithelium (RPE) abnormalities were seen more frequently in eyes receiving injections via a 31G cannula; interestingly, eyes that received cell suspensions were relatively protected against such RPE changes. These findings indicate that optimization of donor cell isolation and delivery parameters should be considered when developing a subretinal cell replacement strategy. Stem Cells Translational Medicine 2019;8:797&809.

PMID: 31004408 [PubMed - indexed for MEDLINE]

How much green does it take to be orange? Determining the cost associated with trauma center readiness.

Wed, 2020-06-03 09:12
Related Articles

How much green does it take to be orange? Determining the cost associated with trauma center readiness.

J Trauma Acute Care Surg. 2019 05;86(5):765-773

Authors: Ashley DW, Mullins RF, Dente CJ, Johns TJ, Garlow LE, Medeiros RS, Atkins EV, Solomon G, Abston D, Ferdinand CH, Georgia Research Institute for Trauma Study Group

Abstract
BACKGROUND: Readiness costs are real expenses incurred by trauma centers to maintain essential infrastructure to provide emergent services on a 24/7 basis. Although the components for readiness are well described in the American College of Surgeons' Resources for Optimal Care of the Injured Patient, the cost associated with each component is not well defined. We hypothesized that meeting the requirements of the 2014 Resources for Optimal Care of the Injured Patient would result in significant costs for trauma centers.
METHODS: The state trauma commission in conjunction with trauma medical directors, program managers, and financial officers of each trauma center standardized definitions for each component of trauma center readiness cost and developed a survey tool for reporting. Readiness costs were grouped into four categories: administrative/program support staff, clinical medical staff, in-house operating room, and education/outreach. To verify consistent cost reporting, a financial auditor analyzed all data. Trauma center outliers were further evaluated to validate variances. All level I/level II trauma centers (n = 16) completed the survey on 2016 data.
RESULTS: Average annual readiness cost is US $10,078,506 for a level I trauma center and US $4,925,103 for level IIs. Clinical medical staff was the costliest component representing 55% of costs for level Is and 64% for level IIs. Although education/outreach is mandated, levels I and II trauma centers only spend approximately US $100,000 annually on this category (1%-2%), demonstrating a lack of resources.
CONCLUSION: This study defines the cost associated with each component of readiness as defined in the Resources for Optimal Care of the Injured Patient manual. Average readiness cost for a level I trauma center is US $10,078,506 and US $4,925,103 for a level II. The significant cost of trauma center readiness highlights the need for additional trauma center funding to meet the requirements set forth by the American College of Surgeons.
LEVEL OF EVIDENCE: Economic and value-based evaluations, level III.

PMID: 30768564 [PubMed - indexed for MEDLINE]

Subretinal pseudocyst: A novel optical coherence tomography finding in age-related macular degeneration.

Wed, 2020-05-13 02:47
Related Articles

Subretinal pseudocyst: A novel optical coherence tomography finding in age-related macular degeneration.

Eur J Ophthalmol. 2020 May;30(3):NP24-NP26

Authors: Sacconi R, Mullins RF, Lutty GA, Borrelli E, Bandello F, Querques G

Abstract
PURPOSE: To report the presence of a new structural optical coherence tomography finding, namely, subretinal pseudocysts, in a patient affected by age-related macular degeneration.
METHODS: Case report including multimodal imaging discussion.
CASE REPORT: We report a case of a 77-year-old woman affected by age-related macular degeneration from 7 years. Best corrected visual acuity was counting fingers and 20/40 in the right and left eye, respectively. The left eye was affected by type 1 macular neovascularization treated by 34 intravitreal injections of anti-vascular endothelial growth factor (22 ranibizumab and 12 aflibercept injections). Interestingly, structural optical coherence tomography showed the persistence of a subretinal cystoid space (i.e. 'subretinal pseudocyst') after the last anti-vascular endothelial growth factor treatment, even in absence of other signs of exudation.
CONCLUSIONS: Subretinal pseudocysts are a new structural optical coherence tomography entity. We reported for the first time the evidence that pseudocysts may develop in the subretinal space in a case of age-related macular degeneration.

PMID: 31018677 [PubMed - indexed for MEDLINE]

Single-cell transcriptomics of the human retinal pigment epithelium and choroid in health and macular degeneration.

Tue, 2020-04-28 21:13
Related Articles

Single-cell transcriptomics of the human retinal pigment epithelium and choroid in health and macular degeneration.

Proc Natl Acad Sci U S A. 2019 11 26;116(48):24100-24107

Authors: Voigt AP, Mulfaul K, Mullin NK, Flamme-Wiese MJ, Giacalone JC, Stone EM, Tucker BA, Scheetz TE, Mullins RF

Abstract
The human retinal pigment epithelium (RPE) and choroid are complex tissues that provide crucial support to the retina. Disease affecting either of these supportive tissues can lead to irreversible blindness in the setting of age-related macular degeneration. In this study, single-cell RNA sequencing was performed on macular and peripheral regions of RPE-choroid from 7 human donor eyes in 2 independent experiments. In the first experiment, total RPE/choroid preparations were evaluated and expression profiles specific to RPE and major choroidal cell populations were identified. As choroidal endothelial cells represent a minority of the total RPE/choroidal cell population but are strongly implicated in age-related macular degeneration (AMD) pathogenesis, a second single-cell RNA-sequencing experiment was performed using endothelial cells enriched by magnetic separation. In this second study, we identified gene expression signatures along the choroidal vascular tree, classifying the transcriptome of human choriocapillaris, arterial, and venous endothelial cells. We found that the choriocapillaris highly and specifically expresses the regulator of cell cycle gene (RGCC), a gene that responds to complement activation and induces apoptosis in endothelial cells. In addition, RGCC was the most up-regulated choriocapillaris gene in a donor diagnosed with AMD. These results provide a characterization of the human RPE and choriocapillaris transcriptome, offering potential insight into the mechanisms of choriocapillaris response to complement injury and choroidal vascular disease in age-related macular degeneration.

PMID: 31712411 [PubMed - indexed for MEDLINE]

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

Tue, 2020-04-28 21:13
Related Articles

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

Cell Rep. 2019 02 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 - indexed for MEDLINE]