Publications by authors named "Sanjoy K Bhattacharya"

142 Publications

Parallel Multi-Omics in High-Risk Subjects for the Identification of Integrated Biomarker Signatures of Type 1 Diabetes.

Biomolecules 2021 Mar 4;11(3). Epub 2021 Mar 4.

Diabetes Research Institute, University of Miami Miller School of Medicine, Miami, FL 33136, USA.

Background: Biomarkers are crucial for detecting early type-1 diabetes (T1D) and preventing significant β-cell loss before the onset of clinical symptoms. Here, we present proof-of-concept studies to demonstrate the potential for identifying integrated biomarker signature(s) of T1D using parallel multi-omics.

Methods: Blood from human subjects at high risk for T1D (and healthy controls; = 4 + 4) was subjected to parallel unlabeled proteomics, metabolomics, lipidomics, and transcriptomics. The integrated dataset was analyzed using Ingenuity Pathway Analysis (IPA) software for disturbances in the at-risk subjects compared to controls.

Results: The final quadra-omics dataset contained 2292 proteins, 328 miRNAs, 75 metabolites, and 41 lipids that were detected in all samples without exception. Disease/function enrichment analyses consistently indicated increased activation, proliferation, and migration of CD4 T-lymphocytes and macrophages. Integrated molecular network predictions highlighted central involvement and activation of NF-κB, TGF-β, VEGF, arachidonic acid, and arginase, and inhibition of miRNA Let-7a-5p. IPA-predicted candidate biomarkers were used to construct a putative integrated signature containing several miRNAs and metabolite/lipid features in the at-risk subjects.

Conclusions: Preliminary parallel quadra-omics provided a comprehensive picture of disturbances in high-risk T1D subjects and highlighted the potential for identifying associated integrated biomarker signatures. With further development and validation in larger cohorts, parallel multi-omics could ultimately facilitate the classification of T1D progressors from non-progressors.
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http://dx.doi.org/10.3390/biom11030383DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7999903PMC
March 2021

Lipidomics dataset of PTEN deletion-induced optic nerve regeneration mouse model.

Data Brief 2021 Feb 26;34:106699. Epub 2020 Dec 26.

Bascom Palmer Eye Institute, Miller School of Medicine at University of Miami, Miami, FL, 33136, USA.

The optic nerve is part of the mammalian adult central nervous system (CNS) and has limited capability to regenerate after injury. Deletion of phosphatase and tensin homolog (PTEN), a negative regulator of the PI3 kinase/Akt pathway, has been shown to promote regeneration in retinal ganglion cells (RGCs) after optic nerve injury [1]. We present the lipidome of adult PTEN mice subjected to intravitreal injection of adeno-associated viruses expressing Cre (AAV-Cre) as a model of CNS neuroregeneration. At 4 weeks old, PTEN mice were intravitreally-injected with 2-3 μl of either AAV-Cre (KO) or AAV-PLAP (control), and two weeks later optic nerve crush was performed. At indicated time-points after crush (0 days, 7 days, 14 days), mice were euthanized and optic nerves were immediately dissected out, and then flash frozen on dry ice. A modified Bligh and Dyer [2] method was used for lipid extraction from the optic nerves, followed by liquid chromatography-mass spectrometry (LC MS-MS) lipid profiling using a Q-Exactive Orbitrap instrument coupled with Accela 600 HPLC. The raw scans were analysed with LipidSearch 4.2 and the statistical analysis was conducted through Metaboanalyst 4.0. This data is available at Metabolomics Workbench, study ID ST001477.
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http://dx.doi.org/10.1016/j.dib.2020.106699DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7797512PMC
February 2021

Capillary Electrophoresis Assessment of Plasma Protein Changes in an African Penguin () With Aspergillosis.

ACS Omega 2020 Dec 15;5(51):33280-33289. Epub 2020 Dec 15.

Miami Integrative Metabolomics Research Center, University of Miami, Miami, Florida 33136, United States.

A decrease of avian biodiversity in the African continent has been the result of anthropogenic pressure in the region. This has resulted in the African penguin () being placed on the endangered species list and requires conservation efforts to maintain its free-ranging population and placement under managed care. In the latter environment, infection by can be common. The diagnosis and treatment of this fungal disease in birds has presented with many difficulties, largely due to the diversity and limited knowledge that exists about this species. In this study, we implement a high-resolution capillary electrophoresis system for the fractionation of African penguin plasma, followed by mass spectrometry analysis for the identification of proteins associated with aspergillosis. Several protein differences were revealed, including changes in acute phase proteins and lipid metabolism. In addition, our results demonstrated that fibrinogen β chain is a protein largely present during the inflammatory process in an African penguin infected with . These findings present a new avenue for the measurement of plasma proteins as a potential method for identifying important biomarkers to aid in monitoring African penguin health.
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http://dx.doi.org/10.1021/acsomega.0c04983DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7774288PMC
December 2020

Multi-omics insights into neuronal regeneration and re-innervation.

Neural Regen Res 2021 Feb;16(2):296-297

Miami Integrative Metabolomics Research Center, Bascom Palmer Eye Institute; Program In Biomedical Sciences and Neuroscience Graduate Program, University of Miami, Miami, FL, USA.

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http://dx.doi.org/10.4103/1673-5374.289434DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7896231PMC
February 2021

Lipid profile dataset of optogenetics induced optic nerve regeneration.

Data Brief 2020 Aug 5;31:106001. Epub 2020 Jul 5.

Bascom Palmer Eye Institute, Miller School of Medicine at University of Miami, Miami, FL 33136, United States.

The optic nerve transfers visual information from the retina to the brain through the axons of retinal ganglion cells (RGCs). In adult mammals, optic nerve injuries and progressive degenerative diseases lead to the irreversible loss of RGCs, resulting in vision loss and blindness. Optogenetic models have proved useful in manipulating the growth of RGCs through expression and stimulation of channelrhodopsins (Chr2) in RGCs using the RGC-specific thy-1 promoter. Using transgenic Chr2 mouse (Thy1-ChR2-EYFP) as a model of regeneration, we profile the lipid changes which occur after traumatic optic nerve crush, light stimulation and forced RGC axonal growth. Thy1-ChR2-EYFP and control (C57BL/6) mice were divided in four groups each - 1) no crush and no stimulation, 2) no crush with stimulation, 3) crush and without stimulation, and 4) crush with stimulation. After euthanasia, the optic nerves were collected for lipidomic analysis. The Bligh and Dyer method was used for lipid extraction, followed by mass spectrometry lipid profiling with a Q-Exactive Orbitrap Liquid Chromatography-Mass Spectrometer (LC MS-MS). The raw scans were analysed with LipidSearch 4.1.3 and the statistical analysis was conducted through Metaboanalyst 4.0. This data is available at Metabolomics Workbench, study ID ST001381: [https://www.metabolomicsworkbench.org/data/DRCCMetadata.php?Mode=Study&StudyID=ST001381&StudyType=MS&ResultType=5].
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http://dx.doi.org/10.1016/j.dib.2020.106001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7358746PMC
August 2020

Myelin Basic Protein Phospholipid Complexation Likely Competes with Deimination in Experimental Autoimmune Encephalomyelitis Mouse Model.

ACS Omega 2020 Jun 16;5(25):15454-15467. Epub 2020 Jun 16.

Department of Ophthalmology, Bascom Palmer Eye Institute, University of Miami, Miami, Florida 33136, United States.

Multiple sclerosis has complex pathogenesis encompassing a variety of components (immunologic, genetic, and environmental). The autoimmunogenicity against the host's myelin basic protein is a major contributor. An increase in myelin basic protein deimination (a post-translational modification) and a change in phospholipid composition have been associated with multiple sclerosis. The interaction of myelin basic protein with phospholipids in the myelin membrane is an important contributor to the stability and maintenance of proper myelin sheath function. The study of this aspect of multiple sclerosis is an area that has yet to be fully explored and that the present study seeks to understand. Several biochemical methods, a capillary electrophoresis coupled system and mass spectrometry, were used in this study. These methods identified four specific phospholipids complexing with myelin basic protein. We show that lysophosphatidylcholine 18:1 provides a robust competitive effect against hyper-deimination. Our data suggest that lysophosphatidylcholine 18:1 has a different biochemical behavior when compared to other phospholipids and lysophosphatidylcholines 14:0, 16:0, and 18:0.
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http://dx.doi.org/10.1021/acsomega.0c01590DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7331039PMC
June 2020

Alteration in Lysophospholipids and Converting Enzymes in Glaucomatous Optic Nerves.

Invest Ophthalmol Vis Sci 2020 06;61(6):60

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Purpose: To determine whether lysophospholipid (LPL) profiles and corresponding conversion enzymes in the LPL pathways are altered in the optic nerve (ON) between human control and glaucoma samples.

Methods: Lipids extracted from control (n = 11) and glaucomatous (n = 12) ON samples using the Bligh and Dyer method were subjected to high-resolution mass spectrometry on a Q-exactive mass spectrometer coupled with a high-performance liquid chromatography (Accela 600) system. Analysis was performed for LPLs (lysophosphatidylcholines, lysophosphatidylserines, lysophosphatidylethanolamines, lysophosphatidylinositols, and lysosphingomyelines) using LipidSearch v.4.1, MZmine v.2.0, and MetaboAnalyst v.4.0. LPL synthesis and degradation pathway maps, utilizing UniProt and BRENDA database entries as needed, were created using Kyoto Encyclopedia of Genes and Genomes (KEGG)-based tools. The mRNA expression level in normal and glaucomatous human ON were analyzed using Gene Expression Omnibus (GEO) entry GSE45570. Protein amounts were determined using PHAST gel and dot blot and were used for normalization of protein amounts across samples. Western blot, ELISA, and protein quantification were performed using established protocols.

Results: Principal component analysis of ON LPL profile placed control and glaucomatous ONs in two distinct separate groups. Mass spectrometric analysis of ON revealed decrease in lysophosphatidic acid, lysophosphatidylethanolamine, lysophosphatidylcholine, and significant increase in diacylglycerol in glaucomatous ON. Statistical analysis of LPL conversion enzymes revealed significant overexpression of phosphatidate phosphatase LPIN2, phospholipid phosphatase 3, phosphatidylcholine-sterol acyltransferase, and calcium-dependent phospholipase 2, and significant downregulation of glycerol-3-phosphate acyltransferase 4 at mRNA level in glaucomatous ON. Western blot and ELISA confirmed proteomic differences between normal and diseased ON.

Conclusions: Our analysis revealed alterations in specific LPL levels and corresponding select enzyme-level changes in glaucomatous ON.
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http://dx.doi.org/10.1167/iovs.61.6.60DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7415893PMC
June 2020

Mitochondrial lipid profiling data of a traumatic optic neuropathy model.

Data Brief 2020 Jun 30;30:105649. Epub 2020 Apr 30.

Department of Ophthalmology, Bascom Palmer Eye Institute, University of Miami Miller School of Medicine, Miami, FL 33136, USA.

Traumatic optic neuropathy (TON) is a degenerative process that occurs in a subset of patients following blunt force trauma to the head. This condition is characterized by retinal ganglion cell (RGC) death and axon degeneration within the optic nerve [1]. At the cellular level, mitochondrial changes are associated with many optic neuropathies [2, 3]. Here, we provide a dataset demonstrating changes in the optic nerve mitochondrial lipid profile of a sonication-induced traumatic optic neuropathy (SI-TON) mouse model at 1, 7, and 14 days after injury. 32 C57BL/6J mice were separated into 4 groups (control, 1, 7, and 14 days) of 8, with 4 males and 4 females in each. Mice were exposed to sonication-induced trauma as described previously (by Tao et al) and optic nerves were harvested at 1, 7, or 14 days following injury [4]. Mitochondria were isolated from homogenized optic nerves and lipids were extracted. Extracted mitochondrial lipids were analysed with a Q-Exactive Orbitrap Liquid Chromatography-Mass Spectrometer (LC MS-MS). Further analysis of raw data was conducted with LipidSearch 4.1.3 and Metaboanalyst 4.0. This data is publicly available at the Metabolomics Workbench, http://www.metabolomicsworkbench.org (Project ID: PR000905).
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http://dx.doi.org/10.1016/j.dib.2020.105649DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7221166PMC
June 2020

The effect of extrinsic Wnt/β-catenin signaling in Muller glia on retinal ganglion cell neurite growth.

Dev Neurobiol 2020 03 17;80(3-4):98-110. Epub 2020 Apr 17.

Bascom Palmer Eye Institute, University of Miami Miller School of Medicine, Miami, FL, USA.

Muller glia are the predominant glial cell type in the retina, and they structurally and metabolically support retinal neurons. Wnt/β-catenin signaling pathways play essential roles in the central nervous system, including glial and neuronal differentiation, axonal growth, and neuronal regeneration. We previously demonstrated that Wnt signaling activation in retinal ganglion cells (RGC) induces axonal regeneration after injury. However, whether Wnt signaling within the adjacent Muller glia plays an axongenic role is not known. In this study, we characterized the effect of Wnt signaling in Muller glia on RGC neurite growth. Primary Muller glia and RGC cells were grown in transwell co-cultures and adenoviral constructs driving Wnt regulatory genes were used to activate and inhibit Wnt signaling specifically in primary Muller glia. Our results demonstrated that activation of Wnt signaling in Muller glia significantly increased RGC average neurite length and branch site number. In addition, the secretome of Muller glia after induction or inhibition of Wnt signaling was characterized using protein profiling of conditioned media by Q Exactive mass spectrometry. The Muller glia secretome after activation of Wnt signaling had distinct and more numerous proteins involved in regulation of axon extension, axon projection and cell adhesion. Furthermore, we showed highly redundant expression of Wnt signaling ligands in Muller glia and Frizzled receptors in RGCs and Muller glia. Therefore, this study provides new information about potential neurite growth promoting molecules in the Muller glia secretome, and identified Wnt-dependent target proteins that may mediate the axonal growth.
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http://dx.doi.org/10.1002/dneu.22741DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7377969PMC
March 2020

Differentiation of soluble aqueous humor metabolites in primary open angle glaucoma and controls.

Exp Eye Res 2020 05 1;194:108024. Epub 2020 Apr 1.

Bascom Palmer Eye Institute, University of Miami, Miami, FL, USA; Miami Integrative Metabolomics Research Center, University of Miami, Miami, FL, USA. Electronic address:

We report an analysis of the aqueous humor (AH) metabolome of primary open angle glaucoma (POAG) in comparison to normal controls. The AH samples were obtained from human donors [control (n = 35), POAG (n = 23)]. The AH samples were subjected to one-dimensional H nuclear magnetic resonance (NMR) analyses on a Bruker Avance 600 MHz instrument with a 1.7 mM NMR probe. The same samples were then subjected to isotopic ratio outlier analysis (IROA) using a Q Exactive orbitrap mass spectrometer after chromatography on an Accela 600 HPLC. Clusterfinder Build 3.1.10 was used for identification and quantification based on long-term metabolite matrix standards. In total, 278 metabolites were identified in control samples and 273 in POAG AH. The metabolites identified were fed into previously reported proteome and genome information and the OmicsNet interaction network generator to construct a protein-metabolite interactions network with an embedded protein-protein network. Significant differences in metabolite composition in POAG compared to controls were identified indicating potential protein/gene pathways associated with these metabolites. These results will expand our previous understanding of the impeded AH metabolite composition, provide new insight into the regulation of AH outflow, and likely aid in future AH and trabecular meshwork multi-omics network analyses.
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http://dx.doi.org/10.1016/j.exer.2020.108024DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7229990PMC
May 2020

Aqueous humor metabolite profile of pseudoexfoliation glaucoma is distinctive.

Mol Omics 2020 10;16(5):425-435

Bascom Palmer Eye Institute, University of Miami, Miami, Florida, USA. and Miami Integrative Metabolomics Research Center, University of Miami, Miami, Florida, USA.

Pseudoexfoliation (PEX) is a known cause of secondary open angle glaucoma. PEX glaucoma is associated with structural and metabolic changes in the eye. Despite similarities, PEX and primary open angle glaucoma (POAG) may have differences in the composition of metabolites. We analyzed the metabolites of the aqueous humor (AH) of PEX subjects sequentially first using nuclear magnetic resonance (1H NMR: HSQC and TOCSY), and subsequently with liquid chromatography tandem mass spectrometry (LC-MS/MS) implementing isotopic ratio outlier analysis (IROA) quantification. The findings were compared with previous results for POAG and control subjects analyzed using identical sequential steps. We found significant differences in metabolites between the three conditions. Principle component analysis (PCA) and partial least squares discriminant analysis (PLS-DA) indicated clear grouping based on the metabolomes of the three conditions. We used machine learning algorithms and a percentage set of the data to train, and utilized a different or larger dataset to test whether a trained model can correctly classify the test dataset as PEX, POAG or control. Three different algorithms: linear support vector machines (SVM), deep learning, and a neural network were used for prediction. They all accurately classified the test datasets based on the AH metabolome of the sample. We next compared the AH metabolome with known AH and TM proteomes and genomes in order to understand metabolic pathways that may contribute to alterations in the AH metabolome in PEX. We found potential protein/gene pathways associated with observed significant metabolite changes in PEX.
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http://dx.doi.org/10.1039/c9mo00192aDOI Listing
October 2020

Endogenous ocular lipids as potential modulators of intraocular pressure.

J Cell Mol Med 2020 04 23;24(7):3856-3900. Epub 2020 Feb 23.

Department of Ophthalmology, Bascom Palmer Eye Institute, University of Miami, Miami, FL, USA.

Elevated intraocular pressure (IOP) is a risk factor in glaucoma, a group of irreversible blinding diseases. Endogenous lipids may be involved in regulation of IOP homeostasis. We present comparative fold analysis of phospholipids and sphingolipids of aqueous humour and trabecular meshwork from human control vs primary open-angle glaucoma and mouse control (normotensive) vs ocular hypertensive state. The fold analysis in control vs disease state was based on ratiometric mass spectrometric data for above classes of lipids. We standardized in vitro assays for rapid characterization of lipids undergoing significant diminishment in disease state. Evaluation of lipids using in vitro assays helped select a finite number of lipids that may potentially expand cellular interstitial space embedded in an artificial matrix or increase fluid flow across a layer of cells. These assays reduced a number of lipids for initial evaluation using a mouse model, DBA/2J with spontaneous IOP elevation. These lipids were then used in other mouse models for confirmation of IOP lowering potential of a few lipids that were found promising in previous assessments. Our results provide selected lipid molecules that can be pursued for further evaluation and studies that may provide insight into their function.
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http://dx.doi.org/10.1111/jcmm.14975DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7171415PMC
April 2020

Multi-Omic Analyses of Growth Cones at Different Developmental Stages Provides Insight into Pathways in Adult Neuroregeneration.

iScience 2020 Feb 14;23(2):100836. Epub 2020 Jan 14.

Bascom Palmer Eye Institute, University of Miami Miller School of Medicine, Miami, FL 33136, USA; Miami Integrative Metabolomics Research Center, University of Miami Miller School of Medicine, Miami, FL 33136, USA; Program in Biomedical Sciences & Neuroscience Graduate Program, University of Miami Miller School of Medicine, Miami, FL 33136, USA. Electronic address:

Growth cones (GCs) are structures associated with growing neurons. GC membrane expansion, which necessitates protein-lipid interactions, is critical to axonal elongation in development and in adult neuritogenesis. We present a multi-omic analysis that integrates proteomics and lipidomics data for the identification of GC pathways, cell phenotypes, and lipid-protein interactions, with an analytic platform to facilitate the visualization of these data. We combine lipidomic data from GC and adult axonal regeneration following optic nerve crush. Our results reveal significant molecular variability in GCs across developmental ages that aligns with the upregulation and downregulation of lipid metabolic processes and correlates with distinct changes in the lipid composition of GC plasmalemma. We find that these processes also define the transition into a growth-permissive state in the adult central nervous system. The insight derived from these analyses will aid in promoting adult regeneration and functional innervation in devastating neurodegenerative diseases.
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http://dx.doi.org/10.1016/j.isci.2020.100836DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6997871PMC
February 2020

Lipidomics dataset of sonication-induced traumatic optic neuropathy in mice.

Data Brief 2020 Apr 16;29:105147. Epub 2020 Jan 16.

Bascom Palmer Eye Institute, Department of Ophthalmology, University of Miami, Miami, FL, 33136, USA.

Traumatic optic neuropathy (TON) is the loss of vision secondary to trauma. Approximately two weeks after traumatic damage, diffuse retinal ganglion cell loss and axon degeneration of the optic nerve are exhibited [1]. Here we present the changes that occur in the optic nerve lipidome of two-month-old C57BL/6J mice following sonication-induced TON (SI-TON), which closely models the indirect clinical mechanism in TON. Optic nerves were harvested at three time points following injury: 1-day, 7-days, and 14-days for comparison with the control group (uninjured optic nerves from 2-month-old mice). The optic nerves were subjected to mass spectrometry and bioinformatic analysis using LipidSearch 4.1.3 and Metaboanalyst 4.0. This data pertains to the lipidome at each time point following indirect trauma to the optic nerve. The data presented here will augment investigation into the neurodegenerative process. The data is available at Metabolomics Workbench [http://www.metabolomicsworkbench.org (Project ID: PR000859)].
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http://dx.doi.org/10.1016/j.dib.2020.105147DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6994518PMC
April 2020

Nuclear prelamin a recognition factor and iron dysregulation in multiple sclerosis.

Metab Brain Dis 2020 02 10;35(2):275-282. Epub 2019 Dec 10.

Department of Ophthalmology & Bascom Palmer Eye Institute, University of Miami, 1638 NW 10th Avenue, Miami, Florida, USA.

Dysregulation of iron metabolism and aberrant iron deposition has been associated with multiple sclerosis. However, the factors that contribute to this pathological state remain to be understood. In this study, human multiple sclerosis and mice brain samples were analyzed through mass spectrometry as well as histological and immunoblot techniques, which demonstrated that iron deposition is associated with increased levels of nuclear prelamin A recognition factor (NARF). NARF is a protein associated with the mitochondria which has also been linked to mitochondrial defects in multiple sclerosis. We report NARF to be associated in multiple sclerosis pathology and aberrant iron deposition.
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http://dx.doi.org/10.1007/s11011-019-00515-zDOI Listing
February 2020

Significant upregulation of small heat shock protein αA-crystallin in retinal detachment.

Exp Eye Res 2019 12 21;189:107811. Epub 2019 Sep 21.

The Ohio State University Department of Ophthalmology and Visual Science, Havener Eye Institute, The Ohio State University Wexner Medical Center, 915 Olentangy River Road, Columbus, OH, 43212, USA. Electronic address:

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http://dx.doi.org/10.1016/j.exer.2019.107811DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6886572PMC
December 2019

Lipid profiling dataset of the Wnt3a-induced optic nerve regeneration.

Data Brief 2019 Aug 24;25:103966. Epub 2019 May 24.

Bascom Palmer Eye Institute, Miller School of Medicine at University of Miami, Miami, FL, 33136, USA.

We present lipid profiling data from mouse retina and optic nerve after optic nerve crush and during Wnt3a-induced axonal regeneration at 7 and 15 days post-crush. This data is available at the Metabolomics Workbench, http://www.metabolomicsworkbench.org (Project ID: PR000718).
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http://dx.doi.org/10.1016/j.dib.2019.103966DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6723204PMC
August 2019

Comparative lipid profiling dataset of the inflammation-induced optic nerve regeneration.

Data Brief 2019 Jun 25;24:103950. Epub 2019 Apr 25.

Stein Eye Institute, David Geffen School of Medicine at UCLA, Los Angeles, CA 90095, USA.

In adult mammals, retinal ganglion cells (RGCs) fail to regenerate following damage. As a result, RGCs die after acute injury and in progressive degenerative diseases such as glaucoma; this can lead to permanent vision loss and, eventually, blindness. Lipids are crucial for the development and maintenance of cell membranes, myelin sheaths, and cellular signaling pathways, however, little is known about their role in axon injury and repair. Studies examining changes to the lipidome during optic nerve (ON) regeneration could greatly inform treatment strategies, yet these are largely lacking. Experimental animal models of ON regeneration have facilitated the exploration of the molecular determinants that affect RGC axon regeneration. Here, we analyzed lipid profiles of the ON and retina in an ON crush rat model using liquid chromatography-mass spectrometry. Furthermore, we investigated lipidome changes after ON crush followed by intravitreal treatment with Zymosan, a yeast cell wall derivative known to enhance RGC regeneration. This data is available at the NIH Common Fund's Metabolomics Data Repository and Coordinating Center (supported by NIH grant, U01-DK097430) website, the Metabolomics Workbench, http://www.metabolomicsworkbench.org, where it has been assigned Project ID: PR000661. The data can be accessed directly via it's Project DOI: doi: 10.21,228/M87D53.
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http://dx.doi.org/10.1016/j.dib.2019.103950DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6517575PMC
June 2019

Assessment of Transport of Lipid Metabolites Within Trabecular Meshwork Cells.

Methods Mol Biol 2019 ;1996:187-197

Bascom Palmer Eye Institute, University of Miami Miller School of Medicine, Miami, FL, USA.

Lipids from trabecular meshwork (TM) cells are of particular interest to ophthalmological researchers as a therapeutic target for lowering intraocular pressure (IOP) in glaucomatous eyes. Fluorescence-based lipid transport assays (FBLTA) and immunocytochemistry (ICC) are dynamic fluorescence analysis techniques that allow for quantitative and qualitative comparisons, respectively, between multiple samples. Here we describe methods for FBLTA, ICC, and mass spectroscopy designed to measure the kinetics and localization of lipid metabolites within the trabecular meshwork.
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http://dx.doi.org/10.1007/978-1-4939-9488-5_16DOI Listing
April 2020

Isotopic Ratio Outlier Analysis (IROA) of Aqueous Humor for Metabolites.

Methods Mol Biol 2019 ;1996:179-185

Bascom Palmer Eye Institute, University of Miami Miller School of Medicine, Miami, FL, USA.

"Omics" revolution during the last few decades has vastly increased the understanding of the biological processes that remained obscure in part due to the lack of quality data. Genomics, transcriptomics, epigenomics, proteomics, lipidomics, metabolomics, and microbiomics form an invaluable array of fields that contribute holistically to a global understanding of the intertwined roles of the basic constituents of life and how they comprehensively paint a picture of disease. Metabolomics, in particular, is the study of metabolites, which are the products of cellular metabolic processes (small molecules, i.e., fatty acids, amino acids, or carbohydrates, are part of metabolomics). In this chapter, it is explained how to achieve a metabolomics profiling of aqueous humor using a special isotopic carbon labeling, a novel technique that combines a tremendous effectiveness with simplicity. The very nature of this technique guarantees the exclusion of false positives, since the algorithms are based on the recognition of unique isotopic patterns.
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http://dx.doi.org/10.1007/978-1-4939-9488-5_15DOI Listing
April 2020

HSD18B7 Enzyme Assay Technique Using a Triple Quadrupole Mass Spectrometer.

Methods Mol Biol 2019 ;1996:155-159

Bascom Palmer Eye Institute, University of Miami Miller School of Medicine, Miami, FL, USA.

Enzymes exist in all biological systems to catalyze vital biochemical reactions. The reactivity of an enzyme and the extent of its influence on product formation can give insight to understanding the physiological changes that can take place. The enzyme HSD17B7, involved in the cholesterol biosynthesis pathway, may play a role in influencing underlying changes during transition of disease, specifically in eyes at normal state to eyes that have glaucoma. In this work, we present a method to test the enzymatic activity level of HSD17B7 between normal and glaucomatous optic nerves to assess whether enzymatic upregulation of cholesterol biosynthesis may play a role in glaucoma.
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http://dx.doi.org/10.1007/978-1-4939-9488-5_13DOI Listing
April 2020

Immature and Mature Collagen Crosslinks Quantification Using High-Performance Liquid Chromatography and High-Resolution Mass Spectrometry in Orbitrap™.

Methods Mol Biol 2019 ;1996:101-111

Bascom Palmer Eye Institute, University of Miami Miller School of Medicine, Miami, FL, USA.

Different methodologies for collagen quantification have been described in the past. Introduction of mass spectrometry combined with high-performance liquid chromatography (HPLC) is a high-resolution tool, which has generated novel applications in biomedical research. In this study, HPLC coupled to electrospray ionization (ESI) tandem mass spectrometry (HPLC-ESI-MS/MS) was used to characterize tissue samples from AVFs done in rats. These findings helped create a protocol for identifying and quantifying components of immature and mature collagen crosslink moieties. Two different internal standards were used: epinephrine and pyridoxine. Quantification curves were drawn by means of these standards. The goal of the experiment was to achieve accurate quantification with the minimum amount of sample. Time and cost of experiment were considerably minimized. Up to date, this method has not been tested for crosslinking quantification.
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http://dx.doi.org/10.1007/978-1-4939-9488-5_10DOI Listing
April 2020

Protein-Lipid Complex Separation Utilizing a Capillary Electrophoresis System.

Methods Mol Biol 2019 ;1996:95-100

Bascom Palmer Eye Institute, University of Miami Miller School of Medicine, Miami, FL, USA.

The separation and analysis of protein-lipid complexes has proven to be challenging due to the harsh conditions required by conventional methods of protein or lipid isolation, which disrupt the fine forces that govern the interactions between lipid head groups and protein side chains. The method described in this publication presents an alternative for the separation of protein-lipid complexes while maintaining the integrity of their interactions. The method exploits the specific electrophoretic forces that are unique to the geometry of the capillary system and allows purification of intact complexes and the systematic analysis of its constituents. This technique is specifically applied for the separation of native protein-lipid complexes found in the central nervous system.
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http://dx.doi.org/10.1007/978-1-4939-9488-5_9DOI Listing
April 2020

Analyses of Cholesterol and Derivatives in Ocular Tissues Using LC-MS/MS Methods.

Methods Mol Biol 2019 ;1996:53-59

Bascom Palmer Eye Institute, University of Miami Miller School of Medicine, Miami, FL, USA.

Cholesterol is considered one of the most abundant sterols present in mammals, amphipathic in nature, and a key constituent of the cell membrane. Its unique chemical structure consisting of four linked hydrocarbon rings, with an aliphatic chain on one end and a hydroxyl group on the other, confers it the ability to form hydrogen bonds with other lipid classes, like phospholipids or sphingolipids head groups. However, due to its hydrophobic moiety, the fatty acid chain, free cholesterol (FC) it is difficult to be analyzed via electrospray ionization (ESI). The method presented in this chapter to identify and quantify free cholesterol and cholesteryl esters (CE) is based on the chemical derivatization of the sample, strategy devised to avoid the problematic of ESI. However, relevant mention should be made to an alternative separation protocol, which uses ultrahigh performance liquid chromatography and in-source collision-induced dissociation to achieve a simultaneous quantification of FC, CE, and triglycerides (TG).
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http://dx.doi.org/10.1007/978-1-4939-9488-5_6DOI Listing
April 2020

Analyses of Cholesterol Metabolites of Optic Nerve Using GC-MS Methods.

Methods Mol Biol 2019 ;1996:47-51

Bascom Palmer Eye Institute, University of Miami Miller School of Medicine, Miami, FL, USA.

Gas chromatography-mass spectrometry (GC-MS) is considered the gold standard for analyzing and quantifying the presence of biological compounds in tissue samples due to its high sensitivity, peak resolution, and reproducibility. In this chapter, we describe a step-by-step modified Bligh and Dyer protocol for lipid extraction from the optic nerve tissue and a procedure for GC-MS analyses of the lipid extract. These protocols are based on our experience and can be modified depending on samples and compounds of interest.
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http://dx.doi.org/10.1007/978-1-4939-9488-5_5DOI Listing
April 2020

Quantitative Metabolomics Using Isotope Residue Outlier Analysis (IROA) with Internal Standards.

Methods Mol Biol 2019 ;1996:41-46

Department of Surgery, Miller School of Medicine, University of Miami, Miami, FL, USA.

Various research strategies involving biomarker discovery and mechanistic studies in system biology depend on reproducible and reliable quantification of all metabolites from tissue(s) of interest. Contemporary analytical methods rely on mass spectrometry-based targeted and/or untargeted metabolomics platforms. The robustness of these analyses depends on the cleanliness of the samples, accuracy of the database, resolution of the instrument, and, the most variable of the list, the personal preferences of the researcher and the instrument operator. In this chapter, we introduce a simple method to prepare murine liver samples and carry it through the Isotope Ratio Outlier Analysis (IROA) pipeline. This pipeline encompasses sample preparation, LC-MS-based peak acquisition, proprietary software-based library creation, normalization, and quantification of metabolites. IROA offers a unique platform to create and normalize a local library and account for run-to-run variability over years of acquisition using the internal standards (IROA-IS) and long-term reference standards (IROA-LTRS).
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http://dx.doi.org/10.1007/978-1-4939-9488-5_4DOI Listing
April 2020

Labeled quantitative mass spectrometry to study the host response during aspergillosis in the common bottlenose dolphin (Tursiops truncatus).

Vet Microbiol 2019 May 30;232:42-49. Epub 2019 Mar 30.

University of Miami, Division of Comparative Pathology, Department of Pathology & Laboratory Medicine, Miller School of Medicine, Miami, FL, 33136, USA. Electronic address:

Aspergillosis is a fungal infection caused by Aspergillus molds that can affect both humans and animals. Despite advances in diagnostics and therapy, medical management of this disease remains difficult. Expansion of the basic knowledge regarding its pathophysiology in animals is critical to aid in the identification of new biomarkers of infection for diagnosis and therapeutic targets. For such a purpose, proteomics can be used by addressing protein changes during various disease processes. In the present study, a mass spectrometry analysis based on isobaric tagging for relative and absolute quantitation (iTRAQ) was applied for direct identification and relative quantitation of proteins in blood collected from 32 Aspergillus-diseased common bottlenose dolphins (Tursiops truncatus, 32 samples) in comparison with blood from 55 other dolphins (55 samples from 41 clinically-normal controls and from 14 cetaceans with miscellaneous non-Aspergillus inflammation diseases) and ten convalescent dolphins (28 samples). Sixty-six and 40 proteins were found to be ≥2.0-fold over- and underrepresented versus miscellaneous non-Aspergillus inflammatory dolphins, respectively, and most were confirmed vs. clinically-normal controls and convalescents. Many proteins which play a role in the adaptive immune response were identified, including MHC proteins and others involved in catalytic activity like the NADPH-ubiquinone oxido-reductases. Overall, iTRAQ appears to be a convenient proteomic tool greatly suited for exploratory ex vivo studies focusing on pathophysiology. This technique should be considered as a preliminary step before validation of new diagnostic markers.
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http://dx.doi.org/10.1016/j.vetmic.2019.03.030DOI Listing
May 2019

Optic Nerve Lipidomics Reveal Impaired Glucosylsphingosine Lipids Pathway in Glaucoma.

Invest Ophthalmol Vis Sci 2019 04;60(5):1789-1798

Department of Ophthalmology & Bascom Palmer Eye Institute, University of Miami, Miami, Florida, United States.

Purpose: To determine major differences in lipid profile between human control and glaucomatous optic nerve. To assess major enzymes in lipid pathway if aberration is revealed for a lipid class by profiling.

Methods: Optic nerve (ON) samples were obtained from human cadaveric donors [control (n = 11) and primary open-angle glaucoma (POAG; n = 12)]; the lipids were extracted using Bligh and Dyer methods. Control and glaucoma donors were all Caucasians age 72.3 ± 5.9 and 70.3 ± 10.5 (inclusive of both sexes), respectively. Lipids were extracted after weighing the tissue; the protein amounts in the corresponding aqueous phase of organic solvent extraction were recorded. High-resolution mass spectrometry was performed using a Q-exactive mass spectrometer coupled with an EASY-nLC 1000 liquid chromatograph instrument. Bioinformatics and statistical analysis were performed using LipidSearch v.4.1 and MetaboAnalyst 4.0/STATA 14.2. Protein amounts were determined using Bradford's method. Western blot, ELISA, and immunohistochemistry utilized established protocols and were performed for protein quantification and localization, respectively. Additional donor tissues were utilized for Western blot, ELISA, and immunohistochemistry.

Results: Principal component analysis (PCA) placed control and glaucomatous ONs in two distinct groups based on analysis of lipid profiles. Total lipid, total phospholipids, total ceramide, and total sphingolipids were similar (without significant difference) between control and glaucoma. However, we found a significant increase in glucosylsphingosine in glaucoma compared to control samples. We found similar levels of glucocerebrosidase (GBA), ceramide glucosyltransferase (UGCG), decreased nonlysosomal glucocerebrosidase (GBA2), and increased lysosomal and nonlysosomal acylsphingosine amidohydrolase (ASAH1 and ASAH2) levels in glaucomatous ON compared to control.

Conclusions: We found significant differences in glucosylsphingosine lipids, consistent with decreased GBA and GBA2 and increased ASAH1 and ASAH2 immunoreactivity in glaucoma, suggesting the potential impairment of sphingolipid enzymatic pathways in lysosomal and nonlysosomal cellular compartments.
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http://dx.doi.org/10.1167/iovs.18-25802DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6485987PMC
April 2019

Dataset of growth cone-enriched lipidome and proteome of embryonic to early postnatal mouse brain.

Data Brief 2019 Jun 26;24:103865. Epub 2019 Mar 26.

Bascom Palmer Eye Institute, Department of Ophthalmology, University of Miami, Miami, FL 33136, USA.

A growth cone is a part of a neuron considered as a hub for axon growth, motility and guidance functions. Growth cones are thought to play a critical role during development of neurons. Growth cones also play a significant role in adult regeneration. Here, we present a dataset on the lipid and protein profiling of the growth cone-enriched fractions derived from C57BL/6J mice forebrains of developmental stage: E18, P0, P3, P6, and P9. For comparison, we analyzed non-growth cone membranes from the same samples. Lipid data is available at the Metabolomics Workbench [http://www.metabolomicsworkbench.org (Project ID: PR000746)]. Protein data is available at Proteomics Identifications (PRIDE) partner repository (PRIDE identifier PXD012134).
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http://dx.doi.org/10.1016/j.dib.2019.103865DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6468181PMC
June 2019

A novel myelin basic protein transcript variant in the murine central nervous system.

Mol Biol Rep 2019 Apr 12;46(2):2547-2553. Epub 2019 Feb 12.

Bascom Palmer Eye Institute, University of Miami, Miami, FL, USA.

Myelin basic protein is a multifunctional protein whose primary role is to adhere membranes of the myelin sheath. There are various isoforms that have been identified, 6 distinct isoforms in human and 13 distinct isoforms in mice. These distinct isoforms are the product of alternative splicing of a single gene. The present study sought out to identify the different isoforms found in the murine central nervous system. Neuronal tissue (brain) from five different C57BL6/J mice at 2 months of age was harvested and used for mRNA extraction. mRNA was reversed transcribed to cDNA and transcripts were detected through PCR amplification and DNA agarose gel separation. Primers for exon 1, exon 5b and exon 11 of the myelin basic protein gene were used to capture all the possible transcripts that are naturally found in the murine central nervous system. Unknown transcript was sequenced at Genewiz facilities (South Plainfield, NJ) and mass spectrometry protein sequence analysis demonstrated the presence of a novel myelin basic protein transcript variant. We identified a novel transcript variant of myelin basic protein. This novel transcript variant corresponds to a myelin basic protein of 32.5 kDa which has not been previously reported. This novel transcript variant presents relevant clinical significance to various demyelinating diseases due to its contribution to the understanding of the natural state of the murine central nervous system.
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http://dx.doi.org/10.1007/s11033-019-04635-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6472899PMC
April 2019