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1.
Cereb Cortex ; 34(4)2024 Apr 01.
Article in English | MEDLINE | ID: mdl-38679479

ABSTRACT

Normative ferret brain development was characterized using magnetic resonance imaging. Brain growth was longitudinally monitored in 10 ferrets (equal numbers of males and females) from postnatal day 8 (P8) through P38 in 6-d increments. Template T2-weighted images were constructed at each age, and these were manually segmented into 12 to 14 brain regions. A logistic growth model was used to fit data from whole brain volumes and 8 of the individual regions in both males and females. More protracted growth was found in males, which results in larger brains; however, sex differences were not apparent when results were corrected for body weight. Additionally, surface models of the developing cortical plate were registered to one another using the anatomically-constrained Multimodal Surface Matching algorithm. This, in turn, enabled local logistic growth parameters to be mapped across the cortical surface. A close similarity was observed between surface area expansion timing and previous reports of the transverse neurogenic gradient in ferrets. Regional variation in the extent of surface area expansion and the maximum expansion rate was also revealed. This characterization of normative brain growth over the period of cerebral cortex folding may serve as a reference for ferret studies of brain development.


Subject(s)
Brain , Ferrets , Magnetic Resonance Imaging , Animals , Ferrets/growth & development , Magnetic Resonance Imaging/methods , Male , Female , Brain/growth & development , Brain/diagnostic imaging , Brain/anatomy & histology , Longitudinal Studies , Sex Characteristics
2.
Front Physiol ; 14: 1266444, 2023.
Article in English | MEDLINE | ID: mdl-37942229

ABSTRACT

Developmental programming of chronic adverse cardiovascular health outcomes has been studied both using numerous human populations and an array of animal models. However, the mechanisms that produce transgenerational effects have been difficult to study due to a lack of developmentally relevant models. As such, how increased disease risk is carried to the second generation has been poorly studied. We hypothesized that the endothelium which mediates many acute and chronic vascular inflammatory responses is a key player in these effects, and epidemiological studies implicate transgenerational nutritional effects on endothelial health. To study the mutigenerational effects of maternal undernutrition on offspring endothelial health, we developed a model of transgenerational nutritional stress in guinea pigs, a translationally relevant precocial species with a relatively short lifespan. First- and second-generation offspring were subjected to a high fat diet in adolescence to exacerbate negative cardiovascular health. To assess transcriptional changes, we performed bulk RNA-sequencing in carotid artery endothelial cells, with groups stratified as prenatal control or food restricted, and postnatal control or high fat diet. We detected statistically significant gene alterations for each dietary permutation, some of which were unique to treatments and other transcriptional signatures shared by multiple or all conditions. These findings highlight a core group of genes altered by high fat diet that is shared by all cohorts and a divergence of transgenerational effects between the prenatal ad libitum and dietary restriction groups. This study establishes the groundwork for this model to be used to better understand the interplay of prenatal stress and genetic reprogramming.

3.
J Community Health ; 48(4): 576-584, 2023 Aug.
Article in English | MEDLINE | ID: mdl-36752869

ABSTRACT

BACKGROUND: National quality measures set goals for diabetes management. Hispanic populations are higher risk for diabetes and associated complications, especially low-income communities. Research suggests free clinics provide suboptimal diabetes management. Our quality improvement project aims to improve diabetes management in the Hispanic free clinic population. METHODS: Clínica Latina's volunteer medical students and physicians serve predominantly uninsured Spanish-speaking patients. Established diabetes patients that attended clinic during the study were included. Data was collected regarding patients' diabetes care for two months, then analyzed compared to quality metrics. We implemented paper checklists and electronic medical record (EMR) smart phrases for volunteers to utilize in managing diabetes. RESULTS: 32 patients were included in the study. At baseline, 78% had an A1C check in the past 3 months, 81% were on a statin. In the past year, 81% had a lipid panel, 19% had an eye exam, 63% had a diabetic foot exam, 53% had a urine microalbumin-creatinine screening. After interventions, 97% had an A1C check, 93% were on a statin, 91% had a lipid panel, 31% had an eye exam, 75% had a foot exam, 63% had a urine microalbumin-creatinine. Patients with an LDL < 100 increased from 62 to 66%. The mean A1C did not statistically significantly change. Volunteer smart phrase utilization increased from 37 to 59.1%. CONCLUSION: We implemented a checklist and EMR smart phrase to optimize diabetes management in a student-run Hispanic free clinic, which improved quality metrics. Low-resource clinics serving Spanish-speaking populations may benefit from similar interventions to improve diabetic care.


Subject(s)
Diabetes Mellitus , Hydroxymethylglutaryl-CoA Reductase Inhibitors , Humans , Standard of Care , Glycated Hemoglobin , Creatinine , Diabetes Mellitus/therapy , Diabetes Mellitus/diagnosis , Lipids
4.
Transl Stroke Res ; 14(5): 766-775, 2023 10.
Article in English | MEDLINE | ID: mdl-36181628

ABSTRACT

No current treatments target microvascular reperfusion after stroke, which can contribute to poor outcomes even after successful clot retrieval. The G protein-coupled receptor GPR39 is expressed in brain peri-capillary pericytes, and has been implicated in microvascular regulation, but its role in stroke is unknown. We tested the hypothesis that GPR39 plays a protective role after stroke, in part due to preservation of microvascular perfusion. We generated GPR39 knockout (KO) mice and tested whether GPR39 gene deletion worsens capillary blood flow and exacerbates brain injury and functional deficit after focal cerebral ischemia. Stroke was induced in male and female GPR39 KO and WT littermates by 60-min middle cerebral artery occlusion (MCAO). Microvascular perfusion was assessed via capillary red blood cell (RBC) flux in deep cortical layers in vivo using optical microangiography (OMAG). Brain injury was assessed by measuring infarct size by 2,3,5-triphenyltetrazolium chloride staining at 24 h or brain atrophy at 3 weeks after ischemia. Pole and cylinder behavior tests were conducted to assess neurological function deficit at 1 and 3 weeks post-stroke. Male but not female GPR39 KO mice exhibited larger infarcts and lower capillary RBC flux than WT controls after stroke. Male GPR39 KO mice also exhibited worse neurologic deficit at 1 week post-stroke, though functional deficit disappeared in both groups by 3 weeks. GPR39 deletion worsens brain injury, microvascular perfusion, and neurological function after experimental stroke. Results indicate that GPR39 plays a sex-dependent role in re-establishing microvascular flow and limiting ischemic brain damage after stroke.


Subject(s)
Brain Ischemia , Receptors, G-Protein-Coupled , Stroke , Animals , Male , Mice , Brain Ischemia/genetics , Infarction, Middle Cerebral Artery , Mice, Knockout , Microcirculation , Receptors, G-Protein-Coupled/genetics , Sex Factors , Stroke/genetics
5.
Sci Rep ; 12(1): 8835, 2022 05 25.
Article in English | MEDLINE | ID: mdl-35614104

ABSTRACT

The microcirculation serves crucial functions in adult heart, distinct from those carried out by epicardial vessels. Microvessels are governed by unique regulatory mechanisms, impairment of which leads to microvessel-specific pathology. There are few treatment options for patients with microvascular heart disease, primarily due to limited understanding of underlying pathology. High throughput mRNA sequencing and protein expression profiling in specific cells can improve our understanding of microvessel biology and disease at the molecular level. Understanding responses of individual microvascular cells to the same physiological or pathophysiological stimuli requires the ability to isolate the specific cell types that comprise the functional units of the microcirculation in the heart, preferably from the same heart, to ensure that different cells have been exposed to the same in-vivo conditions. We developed an integrated process for simultaneous isolation and culture of the main cell types comprising the microcirculation in adult mouse heart: endothelial cells, pericytes, and vascular smooth muscle cells. These cell types were characterized with isobaric labeling quantitative proteomics and mRNA sequencing. We defined microvascular cell proteomes, identified novel protein markers, and confirmed established cell-specific markers. Our results allow identification of unique markers and regulatory proteins that govern microvascular physiology and pathology.


Subject(s)
Endothelial Cells , Pericytes , Animals , Endothelial Cells/metabolism , Mice , Microcirculation , Muscle, Smooth, Vascular/metabolism , Pericytes/metabolism , Proteomics , RNA, Messenger/genetics , RNA, Messenger/metabolism
6.
Am J Physiol Cell Physiol ; 322(5): C1011-C1021, 2022 05 01.
Article in English | MEDLINE | ID: mdl-35385329

ABSTRACT

Arachidonic acid metabolites epoxyeicosatrienoates (EETs) and hydroxyeicosatetraenoates (HETEs) are important regulators of myocardial blood flow and coronary vascular resistance (CVR), but their mechanisms of action are not fully understood. We applied a chemoproteomics strategy using a clickable photoaffinity probe to identify G protein-coupled receptor 39 (GPR39) as a microvascular smooth muscle cell (mVSMC) receptor selective for two endogenous eicosanoids, 15-HETE and 14,15-EET, which act on the receptor to oppose each other's activity. The former increases mVSMC intracellular calcium via GPR39 and augments coronary microvascular resistance, and the latter inhibits these actions. Furthermore, we find that the efficacy of both ligands is potentiated by zinc acting as an allosteric modulator. Measurements of coronary perfusion pressure (CPP) in GPR39-null hearts using the Langendorff preparation indicate the receptor senses these eicosanoids to regulate microvascular tone. These results implicate GPR39 as an eicosanoid receptor and key regulator of myocardial tissue perfusion. Our findings will have a major impact on understanding the roles of eicosanoids in cardiovascular physiology and disease and provide an opportunity for the development of novel GPR39-targeting therapies for cardiovascular disease.


Subject(s)
Cytochrome P-450 Enzyme System , Eicosanoids , Arachidonic Acid/metabolism , Coronary Vessels/metabolism , Cytochrome P-450 Enzyme System/metabolism , Eicosanoids/analysis , Eicosanoids/metabolism , Eicosanoids/pharmacology , Vascular Resistance
7.
iScience ; 25(3): 103901, 2022 Mar 18.
Article in English | MEDLINE | ID: mdl-35243258

ABSTRACT

Cells transmit their genomes vertically to daughter cells during cell divisions. Here, we demonstrate the occurrence and extent of horizontal mitochondrial (mt)DNA acquisition between cells that are not in a parent-offspring relationship. Extensive single-cell sequencing from various tissues and organs of adult chimeric mice composed of cells carrying distinct mtDNA haplotypes showed that a substantial fraction of individual cardiomyocytes, neurons, glia, intestinal, and spleen cells captured donor mtDNA at high levels. In addition, chimeras composed of cells with wild-type and mutant mtDNA exhibited increased trafficking of wild-type mtDNA to mutant cells, suggesting that horizontal mtDNA transfer may be a compensatory mechanism to restore compromised mitochondrial function. These findings establish the groundwork for further investigations to identify mtDNA donor cells and mechanisms of transfer that could be critical to the development of novel gene therapies.

8.
Data Brief ; 38: 107343, 2021 Oct.
Article in English | MEDLINE | ID: mdl-34527795

ABSTRACT

Cardiac endothelial cells respond to both ischemia and therapeutic ultrasound; the proteomic changes underlying these responses are unknown. This data article provides raw and processed data resulting from our global, unbiased phosphoproteomics investigation conducted on primary mouse cardiac endothelial cells exposed to ischemia (2-hour oxygen glucose deprivation) and ultrasound (250 kHz, 1.2 MPa) in vitro [1]. Proteins were extracted from cell lysates and enriched phosphopeptides were analyzed with a high mass accuracy liquid chromatrography (LC) - tandem mass spectrometry (MS/MS) proteomic platform, yielding multiple alterations in both total protein levels and phosphorylation events in response to ischemic injury and ultrasound. This dataset can be used as a reference for future studies on the cardiac endothelial response to ischemia and the mechanistic underpinnings of the cellular response to ultrasound, with the potential to yield clinically relevant therapeutic targets.

9.
Int J Mol Sci ; 22(15)2021 Jul 30.
Article in English | MEDLINE | ID: mdl-34360964

ABSTRACT

GPR39, a member of the ghrelin family of G protein-coupled receptors, is zinc-responsive and contributes to the regulation of diverse neurovascular and neurologic functions. Accumulating evidence suggests a role as a homeostatic regulator of neuronal excitability, vascular tone, and the immune response. We review GPR39 structure, function, and signaling, including constitutive activity and biased signaling, and summarize its expression pattern in the central nervous system. We further discuss its recognized role in neurovascular, neurological, and neuropsychiatric disorders.


Subject(s)
Cardiovascular Diseases/genetics , Nervous System Diseases/genetics , Neurovascular Coupling , Receptors, G-Protein-Coupled/metabolism , Animals , Cardiovascular Diseases/metabolism , Cardiovascular Diseases/physiopathology , Homeostasis , Humans , Nervous System Diseases/metabolism , Nervous System Diseases/physiopathology , Receptors, G-Protein-Coupled/genetics , Signal Transduction
10.
J Chem Inf Model ; 61(7): 3442-3452, 2021 07 26.
Article in English | MEDLINE | ID: mdl-34242503

ABSTRACT

G-protein coupled receptors (GPCRs) sense a wide variety of stimuli, including lipids, and transduce signals to the intracellular environment to exert various physiological responses. However, the structural features of GPCRs responsible for detecting and triggering responses to distinct lipid ligands have only recently begun to be revealed. 14,15-epoxyeicosatrienoic acid (14,15-EET) is one such lipid mediator that plays an essential role in the vascular system, displaying both vasodilatory and anti-inflammatory properties. We recently reported multiple low-affinity 14,15-EET-binding GPCRs, but the mechanism by which these receptors sense 14,15-EET remains unclear. Here, we have taken a combined computational and experimental approach to identify and confirm critical residues and properties within the lipid-binding pocket. Furthermore, we generated mutants to engineer selected GPCR-predicted binding sites to either confer or abolish 14,15-EET-induced signaling. Our structure-function analyses indicate that hydrophobic and positively charged residues of the receptor-binding pocket are prerequisites for recognizing lipid ligands such as 14,15-EET and possibly other eicosanoids.


Subject(s)
Lipids , Receptors, G-Protein-Coupled , 8,11,14-Eicosatrienoic Acid/analogs & derivatives , 8,11,14-Eicosatrienoic Acid/metabolism , Binding Sites , Humans , Ligands , Protein Binding , Receptors, G-Protein-Coupled/genetics , Receptors, G-Protein-Coupled/metabolism
11.
Biochim Biophys Acta Proteins Proteom ; 1869(9): 140683, 2021 09.
Article in English | MEDLINE | ID: mdl-34119693

ABSTRACT

Myocardial infarction and subsequent therapeutic interventions activate numerous intracellular cascades in every constituent cell type of the heart. Endothelial cells produce several protective compounds in response to therapeutic ultrasound, under both normoxic and ischemic conditions. How endothelial cells sense ultrasound and convert it to a beneficial biological response is not known. We adopted a global, unbiased phosphoproteomics approach aimed at understanding how endothelial cells respond to ultrasound. Here, we use primary cardiac endothelial cells to explore the cellular signaling events underlying the response to ischemia-like cellular injury and ultrasound exposure in vitro. Enriched phosphopeptides were analyzed with a high mass accuracy liquid chromatrography (LC) - tandem mass spectrometry (MS/MS) proteomic platform, yielding multiple alterations in both total protein levels and phosphorylation events in response to ischemic injury and ultrasound. Application of pathway algorithms reveals numerous protein networks recruited in response to ultrasound including those regulating RNA splicing, cell-cell interactions and cytoskeletal organization. Our dataset also permits the informatic prediction of potential kinases responsible for the modifications detected. Taken together, our findings begin to reveal the endothelial proteomic response to ultrasound and suggest potential targets for future studies of the protective effects of ultrasound in the ischemic heart.


Subject(s)
Endocardium/metabolism , Myocardial Ischemia/physiopathology , Phosphoproteins/metabolism , Amino Acid Sequence , Animals , Chromatography, Liquid/methods , Endocardium/physiology , Endothelial Cells/metabolism , Heart/diagnostic imaging , Ischemia/physiopathology , Male , Mice , Mice, Inbred C57BL , Phosphorylation , Primary Cell Culture , Proteome/metabolism , Proteomics/methods , Signal Transduction , Tandem Mass Spectrometry/methods , Ultrasonic Therapy/methods
12.
Physiol Genomics ; 53(7): 295-308, 2021 07 01.
Article in English | MEDLINE | ID: mdl-34097533

ABSTRACT

Aging is a significant risk factor for cardiovascular disease. Despite the fact that endothelial cells play critical roles in cardiovascular function and disease, the molecular impact of aging on this cell population in many organ systems remains unknown. In this study, we sought to determine age-associated transcriptional alterations in cardiac endothelial cells. Highly enriched populations of endothelial cells (ECs) isolated from the heart, brain, and kidney of young (3 mo) and aged (24 mo) C57/BL6 mice were profiled for RNA expression via bulk RNA sequencing. Approximately 700 cardiac endothelial transcripts significantly differ by age. Gene set enrichment analysis indicated similar patterns for cellular pathway perturbations. Receptor-ligand comparisons indicated parallel alterations in age-affected circulating factors and cardiac endothelial-expressed receptors. Gene and pathway enrichment analyses show that age-related transcriptional response of cardiac endothelial cells is distinct from that of endothelial cells derived from the brain or kidney vascular bed. Furthermore, single-cell analysis identified nine distinct EC subtypes and shows that the Apelin Receptor-enriched subtype is reduced with age in mouse heart. Finally, we identify age-dysregulated genes in specific aged cardiac endothelial subtypes.


Subject(s)
Aging/genetics , Endothelial Cells/physiology , Gene Expression Regulation , Myocardium/cytology , Vascular Cell Adhesion Molecule-1 , Animals , Brain/cytology , Endothelial Cells/classification , Kidney/cytology , Male , Mice, Inbred C57BL , Sequence Analysis, RNA , Single-Cell Analysis , Vascular Cell Adhesion Molecule-1/genetics
13.
J Biomol Struct Dyn ; 38(2): 340-353, 2020 02.
Article in English | MEDLINE | ID: mdl-30706767

ABSTRACT

Neurotensin (NTS) is a 13-amino acid neuropeptide with neuroendocrine and vasoactive functions that is widely expressed in the central nervous system and gastrointestinal tract. NTS is sensed by a multiple cell surface proteins including two G protein-coupling receptors (GPCRs): NTS receptors 1 and 2 (NTSR1 and NTSR2). Crystal structures of NTSR1 have successfully elucidated agonist binding within the orthosteric pocket of receptor but have not revealed the full activation state of the receptor. Recent studies have attempted to address this challenge by improving NTSR1 crystal formation via thermostable mutants; unfortunately, these mutations exhibit functional defects in the G protein coupling of NTSR1. Here, we have used molecular dynamics simulations to gain greater insights into how the amino acid substitutions used in these thermostable mutants (E166A, L310A and F358A) impact receptor activation. Our simulations indicate that wild-type NTSR1 in complex with NTS8-13 shows more active-like features including a 17.7 Å shift in TM6, reflecting a network of polar and aromatic interactions orchestrating agonist-induced receptor conformational changes. We also provide evidence indicating that F358 is a precursor to the rotamer change observed in W321, and our collective analysis also suggests that mutations E166A and F358A are less impactful to G protein coupling than L310A. Furthermore, we believe that our findings can be used to design future NTSR1 mutants that do not interfere with agonist-induced conformational changes and downstream G protein coupling and thus produce structures that will allow visualization of the fully activated receptor conformation.


Subject(s)
Mutation/genetics , Receptors, Neurotensin/genetics , Temperature , Amino Acid Sequence , Molecular Dynamics Simulation , Protein Conformation , Protein Stability , Receptors, Neurotensin/agonists , Receptors, Neurotensin/chemistry , Water/chemistry
14.
Comb Chem High Throughput Screen ; 22(6): 362-369, 2019.
Article in English | MEDLINE | ID: mdl-31272349

ABSTRACT

The glycation of proteins is non-physiological post-translational incorporation of carbohydrates onto the free amines or guanidines of proteins and some lipids. Although the existence of glycated proteins has been known for forty years, a full understanding of their pathogenic nature has been slow in accruing. In recent years, however, glycation has gained widespread acceptance as a contributing factor in numerous metabolic, autoimmune, and neurological disorders, tying together several confounding aspects of disease etiology. From diabetes, arthritis, and lupus, to multiple sclerosis, amyotrophic lateral sclerosis, Alzheimer's, and Parkinson's diseases, an emerging glycation/inflammation paradigm now offers significant new insight into a physiologically important toxicological phenomenon. It exposes novel drug targets and treatment options, and may even lay foundations for long-awaited breakthroughs. This 'current frontier' article briefly profiles current knowledge regarding the underlying causes of glycation, the structural biology implications of such modifications, and their pathological consequences. Although several emerging therapeutic strategies for addressing glycation pathologies are introduced, the primary purpose of this mini-review is to raise awareness of the challenges and opportunities inherent in this emerging new medicinal target area.


Subject(s)
Proteins/metabolism , Glycosylation , Humans , Proteins/chemistry
15.
Neuron ; 100(5): 1007-1009, 2018 12 05.
Article in English | MEDLINE | ID: mdl-30521770

ABSTRACT

In this issue of Neuron, Ambrozkiewicz et al. (2018) define a new molecular circuit controlling neuronal polarization and migration through the transcription factor SOX9 to coordinate the production of regulators of both protein synthesis and degradation.


Subject(s)
MicroRNAs , Ubiquitin-Protein Ligases , Neurons
16.
Physiol Genomics ; 50(2): 104-116, 2018 02 01.
Article in English | MEDLINE | ID: mdl-29212850

ABSTRACT

Hypertension poses a significant challenge to vasculature homeostasis and stands as the most common cardiovascular disease in the world. Its effects are especially profound on endothelial cells that form the inner lining of the vasculature and are directly exposed to the effects of excess pressure. Here, we characterize the in vivo transcriptomic response of cardiac endothelial cells to hypertension by rapidly isolating these cells from the spontaneous hypertension mouse model BPH/2J and its normotensive BPN/3J control strain and performing and RNA sequencing on both. Comparison of transcriptional differences between these groups reveals statistically significant changes in cellular pathways consistent with cardiac fibrosis found in hypertensive animals. Importantly, many of the fibrosis-linked genes identified also differ significantly between juvenile prehypertensive and adult hypertensive BPH/2J mice, suggesting that these transcriptional differences are hypertension related. We examined the dynamic nature of these transcriptional changes by testing whether blood pressure normalization using either a calcium channel blocker (amlodipine) or a angiotensin II receptor blocker (losartan) is able to reverse these expression patterns associated with hypertension. We find that blood pressure reduction is capable of reversing some gene-expression patterns, while other transcripts are recalcitrant to therapeutic intervention. This illuminates the possibility that unmanaged hypertension may irreversibly alter some endothelial transcriptional patterns despite later intervention. This study quantifies how endothelial cells are remodeled at the molecular level in cardiovascular pathology and advances our understanding of the transcriptional events associated with endothelial response to hypertensive challenge.


Subject(s)
Fibrosis/metabolism , Heart Rate/drug effects , Hypertension/metabolism , Amlodipine/therapeutic use , Animals , Blood Pressure/drug effects , Calcium Channel Blockers/therapeutic use , Disease Models, Animal , Fibrosis/genetics , Heart Rate/genetics , Hypertension/drug therapy , Hypertension/genetics , Losartan/therapeutic use , Male , Mice
17.
Am J Surg ; 213(5): 915-920, 2017 May.
Article in English | MEDLINE | ID: mdl-28385379

ABSTRACT

BACKGROUND: Predicting treatment response to chemo-radiotherapy (CRT) in esophageal cancer remains an unrealized goal despite studies linking constellations of genes to prognosis. We aimed to determine if specific expression profiles are associated with pathologic complete response (pCR) after neoadjuvant CRT. METHODS: Eleven genes previously associated with esophageal cancer prognosis were identified. Esophageal adenocarcinoma (EAC) patients treated with neoadjuvant CRT and esophagectomy were included. Patients were classified into two groups: pCR and no-or-incomplete response (NR). Polymerase chain reaction was used to evaluate gene expression. Omnibus testing was applied to overall gene expression differences between groups, and log-rank tests compared individual genes. RESULTS: Eleven pCR and eighteen NR patients were analyzed. Combined expression profiles were significantly different between pCR and NR groups (p < 0.01). The gene CCL28 was over-expressed in pCR patients (Log-HR: 1.53, 95%CI: 0.46-2.59, p = 0.005), and DKK3 was under-expressed in pCR (Log-HR: -1.03 95%CI: -1.97, -0.10, p = 0.031). CONCLUSION: EAC tumors that demonstrated a pCR have genetic profiles that are significantly different from typical NR profiles. The genes CCL28 and DKK3 are potential predictors of treatment response.


Subject(s)
Adenocarcinoma/genetics , Adenocarcinoma/therapy , Biomarkers, Tumor/genetics , Chemoradiotherapy, Adjuvant , Esophageal Neoplasms/genetics , Esophageal Neoplasms/therapy , Gene Expression Regulation, Neoplastic , Neoadjuvant Therapy , Transcriptome , Adenocarcinoma/pathology , Aged , Esophageal Neoplasms/pathology , Esophagectomy , Female , Gene Expression Profiling , Humans , Male , Middle Aged , Prognosis , Registries
18.
Bioinformatics ; 33(3): 447-449, 2017 02 01.
Article in English | MEDLINE | ID: mdl-28171615

ABSTRACT

Summary: Transcriptional profiling using RNA sequencing (RNAseq) has emerged as a powerful methodology to quantify global gene expression patterns in various contexts from single cells to whole tissues. The tremendous amount of data generated by this profiling technology presents a daunting challenge in terms of effectively visualizing and interpreting results. Convenient and intuitive data interfaces are critical for researchers to easily upload, analyze and visualize their RNAseq data. We designed the START (Shiny Transcriptome Analysis Resource Tool) App with these requirements in mind. This application has the power and flexibility to be resident on a local computer or serve as a web-based environment, enabling easy sharing of data between researchers and collaborators. Availability and Implementation: Source Code for the START App is written entirely in R and can be freely available to download at https://github.com/jminnier/STARTapp with the code licensed under GPLv3. It can be launched on any system that has R installed. The START App is also hosted on https://kcvi.shinyapps.io/START for researchers to temporarily upload their data. Contact: minnier@ohsu.edu


Subject(s)
Gene Expression Profiling/methods , High-Throughput Nucleotide Sequencing/methods , Sequence Analysis, RNA/methods , Software , Computational Biology/methods , Internet
19.
Article in English | MEDLINE | ID: mdl-27649858

ABSTRACT

Epoxyeicosatrienoic acids (EETs) are potent vasodilators that play important roles in cardiovascular physiology and disease, yet the molecular mechanisms underlying the biological actions of EETs are not fully understood. Multiple lines of evidence suggest that the actions of EETs are in part mediated via G protein-coupled receptor (GPCR) signaling, but the identity of such a receptor has remained elusive. We sought to identify 14,15-EET-responsive GPCRs. A set of 105 clones were expressed in Xenopus oocyte and screened for their ability to activate cAMP-dependent chloride current. Several receptors responded to micromolar concentrations of 14,15-EET, with the top five being prostaglandin receptor subtypes (PTGER2, PTGER4, PTGFR, PTGDR, PTGER3IV). Overall, our results indicate that multiple low-affinity 14,15-EET GPCRs are capable of increasing cAMP levels following 14,15-EET stimulation, highlighting the potential for cross-talk between prostanoid and other ecosanoid GPCRs. Our data also indicate that none of the 105 GPCRs screened met our criteria for a high-affinity receptor for 14,15-EET.


Subject(s)
8,11,14-Eicosatrienoic Acid/analogs & derivatives , Receptors, G-Protein-Coupled/metabolism , 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid/pharmacology , 8,11,14-Eicosatrienoic Acid/pharmacology , Animals , Cyclic AMP/metabolism , Cystic Fibrosis Transmembrane Conductance Regulator/metabolism , Drug Evaluation, Preclinical , Extracellular Signal-Regulated MAP Kinases/metabolism , HEK293 Cells , Humans , Intracellular Space/drug effects , Intracellular Space/metabolism , Mesenteric Arteries/drug effects , Mesenteric Arteries/physiology , Mice , Oocytes/metabolism , Phosphorylation/drug effects , Protein Transport/drug effects , Receptors, Prostaglandin/metabolism , Receptors, Prostaglandin E, EP2 Subtype/metabolism , Receptors, Prostaglandin E, EP4 Subtype/metabolism , Vasoconstriction/drug effects , Xenopus , beta-Arrestins/metabolism
20.
PLoS One ; 11(5): e0152742, 2016.
Article in English | MEDLINE | ID: mdl-27203283

ABSTRACT

The epoxyeicosatrienoic acid (EET) neutralizing enzyme soluble epoxide hydrolase (sEH) is a neuronal enzyme, which has been localized in both the cytosol and peroxisomes. The molecular basis for its dual localization remains unclear as sEH contains a functional peroxisomal targeting sequence (PTS). Recently, a missense polymorphism was identified in human sEH (R287Q) that enhances its peroxisomal localization. This same polymorphism has also been shown to generate weaker sEH homo-dimers. Taken together, these observations suggest that dimerization may mask the sEH PTS and prevent peroxisome translocation. In the current study, we test the hypothesis that dimerization is a key regulator of sEH subcellular localization. Specifically, we altered the dimerization state of sEH by introducing substitutions in amino acids responsible for the dimer-stabilizing salt-bridge. Green Fluorescent Protein (GFP) fusions of each of mutants were co-transfected into mouse primary cultured cortical neurons together with a PTS-linked red fluorescent protein to constitutively label peroxisomes. Labeled neurons were analyzed using confocal microscopy and co-localization of sEH with peroxisomes was quantified using Pearson's correlation coefficient. We find that dimer-competent sEH constructs preferentially localize to the cytosol, whereas constructs with weakened or disrupted dimerization were preferentially targeted to peroxisomes. We conclude that the sEH dimerization status is a key regulator of its peroxisomal localization.


Subject(s)
Epoxide Hydrolases/metabolism , Peroxisomes/metabolism , Animals , Cells, Cultured , Dimerization , HEK293 Cells , Humans , Mice , Mice, Inbred C57BL , Neurons/metabolism , Protein Transport , Subcellular Fractions/enzymology
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