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Mouse Tmem135 mutation reveals a mechanism involving mitochondrial dynamics that leads to age-dependent retinal pathologies.

Sun, 2017-11-05 06:18
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Mouse Tmem135 mutation reveals a mechanism involving mitochondrial dynamics that leads to age-dependent retinal pathologies.

Elife. 2016 Nov 15;5:

Authors: Lee WH, Higuchi H, Ikeda S, Macke EL, Takimoto T, Pattnaik BR, Liu C, Chu LF, Siepka SM, Krentz KJ, Rubinstein CD, Kalejta RF, Thomson JA, Mullins RF, Takahashi JS, Pinto LH, Ikeda A

Abstract
While the aging process is central to the pathogenesis of age-dependent diseases, it is poorly understood at the molecular level. We identified a mouse mutant with accelerated aging in the retina as well as pathologies observed in age-dependent retinal diseases, suggesting that the responsible gene regulates retinal aging, and its impairment results in age-dependent disease. We determined that a mutation in the transmembrane 135 (Tmem135) is responsible for these phenotypes. We observed localization of TMEM135 on mitochondria, and imbalance of mitochondrial fission and fusion in mutant Tmem135 as well as Tmem135 overexpressing cells, indicating that TMEM135 is involved in the regulation of mitochondrial dynamics. Additionally, mutant retina showed higher sensitivity to oxidative stress. These results suggest that the regulation of mitochondrial dynamics through TMEM135 is critical for protection from environmental stress and controlling the progression of retinal aging. Our study identified TMEM135 as a critical link between aging and age-dependent diseases.

PMID: 27863209 [PubMed - indexed for MEDLINE]

What Are the Costs of Trauma Center Readiness? Defining and Standardizing Readiness Costs for Trauma Centers Statewide.

Sun, 2017-10-01 01:44
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What Are the Costs of Trauma Center Readiness? Defining and Standardizing Readiness Costs for Trauma Centers Statewide.

Am Surg. 2017 Sep 01;83(9):979-990

Authors: Ashley DW, Mullins RF, Dente CJ, Garlow L, Medeiros RS, Atkins EV, Solomon G, Abston D, Ferdinand CH

Abstract
Trauma center readiness costs are incurred to maintain essential infrastructure and capacity to provide emergent services on a 24/7 basis. These costs are not captured by traditional hospital cost accounting, and no national consensus exists on appropriate definitions for each cost. Therefore, in 2010, stakeholders from all Level I and II trauma centers developed a survey tool standardizing and defining trauma center readiness costs. The survey tool underwent minor revisions to provide further clarity, and the survey was repeated in 2013. The purpose of this study was to provide a follow-up analysis of readiness costs for Georgia's Level I and Level II trauma centers. Using the American College of Surgeons Resources for Optimal Care of the Injured Patient guidelines, four readiness cost categories were identified: Administrative, Clinical Medical Staff, Operating Room, and Education/Outreach. Through conference calls, webinars and face-to-face meetings with financial officers, trauma medical directors, and program managers from all trauma centers, standardized definitions for reporting readiness costs within each category were developed. This resulted in a survey tool for centers to report their individual readiness costs for one year. The total readiness cost for all Level I trauma centers was $34,105,318 (avg $6,821,064) and all Level II trauma centers was $20,998,019 (avg $2,333,113). Methodology to standardize and define readiness costs for all trauma centers within the state was developed. Average costs for Level I and Level II trauma centers were identified. This model may be used to help other states define and standardize their trauma readiness costs.

PMID: 28958278 [PubMed - in process]

Evaluation of sFLT1 protein levels in human eyes with the FLT1 rs9943922 polymorphism.

Sun, 2017-10-01 01:44
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Evaluation of sFLT1 protein levels in human eyes with the FLT1 rs9943922 polymorphism.

Ophthalmic Genet. 2017 Sep 26;:1-5

Authors: Chirco KR, Lewis CJ, Scheetz TE, Johnston RM, Tucker BA, Stone EM, Fingert JH, Mullins RF

Abstract
PURPOSE: Age-related macular degeneration (AMD) is a devastating disease characterized by central vision impairment in individuals with advanced age. Neovascular AMD is a form of end-stage disease in which choroidal vessel outgrowth occurs beneath the retina. While many hypotheses have been raised as to what triggers the formation of pathological choroidal neovascular membranes, the exact mechanism for their initiation remains unresolved. Polymorphisms in the FLT1 gene have previously been associated with neovascular AMD risk, including the rs9943922 single nucleotide polymorphism (SNP). Here, we aimed to determine the association between the high-risk FLT1 genotype and FLT1 protein levels in human retina or retinal pigment epithelium (RPE)/choroid tissue.
METHODS: Retina and RPE/choroid tissue from 10 human donor eyes was selected from a collection of eyes genotyped for the rs9943922 SNP. Differences in soluble and membrane bound FLT1 protein levels were assessed for retina versus RPE/choroid donor tissue using ELISA and Western blotting analyses. Genotype-associated changes in FLT1 protein levels were also evaluated.
RESULTS: We found soluble FLT1 levels in the RPE/choroid tissue to be approximately three times higher than that of the retina (p < 0.001), while both samples have similar levels of the membrane bound form. When tissue with the rs9943922 SNP was compared with controls, no significant genotypic differences in FLT1 protein levels were observed.
CONCLUSIONS: Based on these data, we conclude that the rs9943922 SNP in the FLT1 gene does not result in a large difference in FLT1 protein levels, regardless of whether it is the soluble or the membrane bound form.

PMID: 28949775 [PubMed - as supplied by publisher]

Transgenic TBK1 mice have features of normal tension glaucoma.

Sun, 2017-10-01 01:44
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Transgenic TBK1 mice have features of normal tension glaucoma.

Hum Mol Genet. 2017 Jan 01;26(1):124-132

Authors: Fingert JH, Miller K, Hedberg-Buenz A, Roos BR, Lewis CJ, Mullins RF, Anderson MG

Abstract
Duplication of the TBK1 gene is associated with 1-2% of normal tension glaucoma, a common cause of vision loss and blindness that occurs without grossly abnormal intraocular pressure. We generated a transgenic mouse that has one copy of the human TBK1 gene (native promoter and gene structure) incorporated into the mouse genome (Tg-TBK1). Expression of the TBK1 transgene in the retinae of these mice was demonstrated by real-time PCR. Using immunohistochemistry TBK1 protein was predominantly localized to the ganglion cell layer of the retina, the cell type most affected by glaucoma. More intense TBK1 labelling was detected in the retinal ganglion cells (RGCs) of Tg-TBK1 mice than in wild-type littermates. Tg-TBK1 mice exhibit the cardinal sign of glaucoma, a progressive loss of RGCs. Hemizygous Tg-TBK1 mice (with one TBK1 transgene per genome) had a 13% loss of RGCs by 18 months of age (P = 1.5 × 10-8). Homozygous Tg-TBK1 mice had 7.6% fewer RGCs than hemizygous Tg-TBK1 mice and 20% fewer RGCs than wild-type mice (P = 1.9 × 10-5) at 6 months of age. No difference in intraocular pressures was detected between Tg-TBK1 mice and wild-type littermates as they aged (P > 0.05). Tg-TBK1 mice with extra doses of the TBK1 gene recapitulate the phenotype of normal tension glaucoma in human patients with a TBK1 gene duplication. Together, these studies confirm the pathogenicity of the TBK1 gene duplication in human glaucoma and suggest that excess production of TBK1 kinase may have a role in the pathology of glaucoma.

PMID: 28025332 [PubMed - indexed for MEDLINE]

A Method for Sectioning and Immunohistochemical Analysis of Stem Cell-Derived 3-D Organoids.

Sat, 2017-09-02 18:41
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A Method for Sectioning and Immunohistochemical Analysis of Stem Cell-Derived 3-D Organoids.

Curr Protoc Stem Cell Biol. 2016 May 12;37:1C.19.1-1C.19.11

Authors: Wiley LA, Beebe DC, Mullins RF, Stone EM, Tucker BA

Abstract
This unit describes a protocol for embedding, sectioning, and immunocytochemical analysis of pluripotent stem cell-derived 3-D organoids. Specifically, we describe a method to embed iPSC-derived retinal cups in low-melt agarose, acquire thick sections using a vibratome tissue slicer, and perform immunohistochemical analysis. This method includes an approach for antibody labeling that minimizes the amount of antibody needed for individual experiments and that utilizes large-volume washing to increase the signal-to-noise ratio, allowing for clean, high-resolution imaging of developing cell types. The universal methods described can be employed regardless of the type of pluripotent stem cell used and 3-D organoid generated. © 2016 by John Wiley & Sons, Inc.

PMID: 27171793 [PubMed - indexed for MEDLINE]

Generation of Xeno-Free, cGMP-Compliant Patient-Specific iPSCs from Skin Biopsy.

Sat, 2017-08-19 13:09
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Generation of Xeno-Free, cGMP-Compliant Patient-Specific iPSCs from Skin Biopsy.

Curr Protoc Stem Cell Biol. 2017 Aug 14;42:4A.12.1-4A.12.14

Authors: Wiley LA, Anfinson KR, Cranston CM, Kaalberg EE, Collins MM, Mullins RF, Stone EM, Tucker BA

Abstract
This unit describes protocols for the generation of clinical-grade patient-specific induced pluripotent stem cell (iPSC)-derived retinal cells from patients with inherited retinal degenerative blindness. Specifically, we describe how, using xeno-free reagents in an ISO class 5 environment, one can isolate and culture dermal fibroblasts, generate iPSCs, and derive autologous retinal cells via 3-D differentiation. The universal methods described herein for the isolation of dermal fibroblasts and generation of iPSCs can be employed regardless of disease, tissue, or cell type of interest. © 2017 by John Wiley & Sons, Inc.

PMID: 28806854 [PubMed - in process]