In JoVE (1)

Other Publications (27)

Articles by Edwin Garcia in JoVE

 JoVE Biology

Open Source High Content Analysis Utilizing Automated Fluorescence Lifetime Imaging Microscopy

1Photonics Group, Department of Physics, Imperial College London, 2Institute for Chemical Biology, Department of Chemistry, Imperial College London, 3MRC Clinical Sciences Centre, Hammersmith Hospital, 4Chemical Biology Section, Department of Chemistry, Imperial College London, 5Retroscreen Virology Ltd, 6Pfizer Global Research and Development, Pfizer Limited, Sandwich, Kent, UK, 7Centre for Histopathology, Imperial College London


JoVE 55119

Other articles by Edwin Garcia on PubMed

Molecular Cloning and Characterization of Two Hsp 70 Homologous Genes from the Dimorphic Fungus Paracoccidioides Brasiliensis

Biomedica : Revista Del Instituto Nacional De Salud. Dec, 2003  |  Pubmed ID: 14968921

Paracoccidioides brasiliensis, a dimorphic fungus, is the etiologic agent of Paracoccidioidomycosis (PCM), one of the most important systemic mycosis in Latin America. Two genes (2.2 and 1DB5) were cloned, characterized and sequenced; they showed homology with members of hsp70 gene family. By using several probe fragments derived from these genes, levels of expression for each gene were determined by Northern blot during transition to the yeast phase. The highest level of hsp70 transcript occurred between 30 min to 6 hours after temperature shift, with significant reduction after 36-48 hours. However, after 72 hours, the level of the transcription increased until yeast phase was reached. As a response to temperature increase, hsp 70 genes are expressed during the transition phase and possibly play a role in the differentiation process.

Haplotypes of the Beta2-adrenergic Receptor Gene Are Associated with Essential Hypertension in a Singaporean Chinese Population

Journal of Hypertension. Nov, 2004  |  Pubmed ID: 15480094

To investigate the relation between the gene encoding the beta2-adrenergic receptor (B2AR) and essential hypertension in a Singaporean Chinese cohort.

Cannabinoids and Ghrelin Have Both Central and Peripheral Metabolic and Cardiac Effects Via AMP-activated Protein Kinase

The Journal of Biological Chemistry. Jul, 2005  |  Pubmed ID: 15899896

Endocannabinoids and ghrelin are potent appetite stimulators and are known to interact at a hypothalamic level. However, both also have important peripheral actions, including beneficial effects on the ischemic heart and increasing adipose tissue deposition, while ghrelin has direct effects on carbohydrate metabolism. The AMP-activated protein kinase (AMPK) is a heterotrimeric enzyme that functions as a fuel sensor to regulate energy balance at both cellular and whole body levels, and it may mediate the action of anti-diabetic drugs such as metformin and peroxisome proliferator-activated receptor gamma agonists. Here we show that both cannabinoids and ghrelin stimulate AMPK activity in the hypothalamus and the heart, while inhibiting AMPK in liver and adipose tissue. These novel effects of cannabinoids on AMPK provide a mechanism for a number of their known actions, such as the reduction in infarct size in the myocardium, an increase in adipose tissue, and stimulation of appetite. The beneficial effects of ghrelin on heart function, including reduction of myocyte apoptosis, and its effects on lipogenesis and carbohydrate metabolism, can also be explained by its ability to activate AMPK. Our data demonstrate that AMPK not only links the orexigenic effects of endocannabinoids and ghrelin in the hypothalamus but also their effects on the metabolism of peripheral tissues.

Haplotypes of the Beta-2 Adrenergic Receptor Associate with High Diastolic Blood Pressure in the Caerphilly Prospective Study

Journal of Hypertension. Mar, 2006  |  Pubmed ID: 16467650

Current evidence demonstrates that both genetic and environmental factors influence blood pressure. The sympathetic nervous system is a key player in blood pressure control and functional genetic variants of the beta-2 adrenergic receptor (B2AR) have been identified and implicated in the pathogenesis of hypertension. The present study aimed to determine the effects of common haplotypes of the B2AR gene upon blood pressure in the Caerphilly Prospective Study.

Ghrelin and Cardiovascular Health

Current Opinion in Pharmacology. Apr, 2006  |  Pubmed ID: 16483844

Ghrelin and its receptor are widely distributed in cardiovascular tissues, and there is no doubt that the effects of ghrelin in the cardiovascular system are mediated not only via its growth-hormone-releasing effect but also by direct effects on the heart. Indeed, new pharmacological approaches with animal and cell models using elegant study designs have described new functions of ghrelin, providing new potential therapeutic opportunities for ghrelin in cardiovascular medicine.

Ionic Colloidal Crystals: Ordered, Multicomponent Structures Via Controlled Heterocoagulation

Physical Review. E, Statistical, Nonlinear, and Soft Matter Physics. Jan, 2006  |  Pubmed ID: 16486135

We propose a new type of ordered colloid, the "ionic colloidal crystal" (ICC), which is stabilized by attractive electrostatic interactions analogous to those in atomic ionic materials. The rapid self-organization of colloids via this method should result in a diversity of orderings that are analogous to ionic compounds. Most of these complex structures would be difficult to produce by other methods. We use a Madelung summation approach to evaluate the conditions where ICC's are thermodynamically stable. Using this model, we compare the relative electrostatic energies of various structures showing that the regions of ICC stability are determined by two dimensionless parameters representing charge balance and the spatial extent of the electrostatic interactions. Parallels and distinctions between ICC's and classical ionic crystals are discussed. Monte Carlo simulations are utilized to examine the glass transition and melting temperatures, between which crystallization can occur, of a model system having the rocksalt structure. These tools allow us to make a first-order prediction of the experimentally accessible regions of surface charge, particle size, ionic strength, and temperature where ICC formation is probable.

Residual Brain Infection in Relapsing-fever Borreliosis

The Journal of Infectious Diseases. May, 2006  |  Pubmed ID: 16619194

Neurological involvement is common in the spirochetal infection relapsing fever (RF) in both humans and experimental animals. RF is best known for antigenic variation caused by the sequential expression of variable outer membrane lipoproteins of 2 sizes, variable small (Vsp) and variable large (Vlp) proteins. Less understood is the persistence of RF borreliae in the brain after they are cleared from the blood, referred to as residual brain infection (RBI). Our goal was to investigate the phenomenon of RBI in RF.

A Mutation and Expression Analysis of the Oncogene BRAF in Pituitary Adenomas

Clinical Endocrinology. Mar, 2007  |  Pubmed ID: 17302867

BRAF is an oncogene that is commonly mutated in both melanomas and papillary thyroid carcinomas, usually at position V600E that leads to constitutive activity in the Ras-mitogen-activated protein kinase (MAPK) pathway. We speculated that this same gene may be either mutated at this site, or overexpressed, in pituitary adenomas.

Examining the Candidacy of Ghrelin As a Gene Responsible for Variation in Adult Stature in a United Kingdom Population with Type 2 Diabetes

The Journal of Clinical Endocrinology and Metabolism. Jun, 2007  |  Pubmed ID: 17389697

Recently, a quantitative trait locus for stature was reported on chromosome 3p26 in patients with type 2 diabetes.

Coinfection with Borrelia Turicatae Serotype 2 Prevents the Severe Vestibular Dysfunction and Earlier Mortality Caused by Serotype 1

The Journal of Infectious Diseases. Jun, 2007  |  Pubmed ID: 17471439

Relapsing fever (RF) is a multisystemic spirochetal infection caused by different Borrelia species. Studies in our laboratory have shown that disease severity varies depending on the infecting serotype. However, the relative contribution of each serotype to pathogenesis during mixed infections is not known. To investigate this, we compared the outcome of infection with isogenic serotypes 1 (Bt1) or 2 (Bt2) of the RF agent B. turicatae alone or in combination.

The Molecular Phenotype of Human Cardiac Myosin Associated with Hypertrophic Obstructive Cardiomyopathy

Cardiovascular Research. Aug, 2008  |  Pubmed ID: 18411228

The aim of the study was to compare the functional and structural properties of the motor protein, myosin, and isolated myocyte contractility in heart muscle excised from hypertrophic cardiomyopathy patients by surgical myectomy with explanted failing heart and non-failing donor heart muscle.

Ghrelin Receptor Gene Polymorphisms and Body Size in Children and Adults

The Journal of Clinical Endocrinology and Metabolism. Oct, 2008  |  Pubmed ID: 18647811

The GH secretagogue receptor type 1a gene (GHSR) encodes the cognate receptor of ghrelin, a gut hormone that regulates food intake and pituitary GH secretion. Previous studies in U.S. families and a German population suggested GHSR to be a candidate quantitative locus for association with human obesity and growth.

Application of a High Throughput Bioluminescence-based Method and Mathematical Model for the Quantitative Comparison of Polymer Microbicide Efficiency

Biomacromolecules. May, 2009  |  Pubmed ID: 19338347

Quaternized poly(4-vinyl pyridine)-based copolymers are known to be effective against a wide range of bacteria and possess biocompatible properties. Extensive testing of a wide range of copolymers is necessary to further explore and enhance the biocidal properties. However, testing is hampered by labor-intensive bacteria testing techniques. The present paper presents a new testing method, based on bioluminescent reporter strains to enable fast evaluation of bactericidal properties. The reported method enables us to create real-time characterization of bacteria behavior with far less labor than required through traditional testing methods. A mathematical model was also developed to characterize the change in bacteria populations exposed to biocides and to enable the quantitative comparison of minimum bactericidal concentrations.

Loss of T-tubules and Other Changes to Surface Topography in Ventricular Myocytes from Failing Human and Rat Heart

Proceedings of the National Academy of Sciences of the United States of America. Apr, 2009  |  Pubmed ID: 19342485

T-tubular invaginations of the sarcolemma of ventricular cardiomyocytes contain junctional structures functionally coupling L-type calcium channels to the sarcoplasmic reticulum calcium-release channels (the ryanodine receptors), and therefore their configuration controls the gain of calcium-induced calcium release (CICR). Studies primarily in rodent myocardium have shown the importance of T-tubular structures for calcium transient kinetics and have linked T-tubule disruption to delayed CICR. However, there is disagreement as to the nature of T-tubule changes in human heart failure. We studied isolated ventricular myocytes from patients with ischemic heart disease, idiopathic dilated cardiomyopathy, and hypertrophic obstructive cardiomyopathy and determined T-tubule structure with either the fluorescent membrane dye di-8-ANNEPs or the scanning ion conductance microscope (SICM). The SICM uses a scanning pipette to produce a topographic representation of the surface of the live cell by a non-optical method. We have also compared ventricular myocytes from a rat model of chronic heart failure after myocardial infarction. T-tubule loss, shown by both ANNEPs staining and SICM imaging, was pronounced in human myocytes from all etiologies of disease. SICM imaging showed additional changes in surface structure, with flattening and loss of Z-groove definition common to all etiologies. Rat myocytes from the chronic heart failure model also showed both T-tubule and Z-groove loss, as well as increased spark frequency and greater spark amplitude. This study confirms the loss of T-tubules as part of the phenotypic change in the failing human myocyte, but it also shows that this is part of a wider spectrum of alterations in surface morphology.

The Role of Ghrelin and Ghrelin-receptor Gene Variants and Promoter Activity in Type 2 Diabetes

European Journal of Endocrinology. Aug, 2009  |  Pubmed ID: 19460888

Ghrelin and its receptor play an important role in glucose metabolism and energy homeostasis, and therefore they are functional candidates for genes carrying susceptibility alleles for type 2 diabetes.

Matrix Metalloproteinase-9 Delays Wound Healing in a Murine Wound Model

Surgery. Feb, 2010  |  Pubmed ID: 20004432

Metalloproteinase-9 (MMP-9) is a type IV collagenase found at elevated levels in chronic wounds. As wounds heal, MMP-9 diminishes. In this study, we investigated whether MMP-9 directly contributes to chronic wound pathogenesis.

Dislocation Filtering in GaN Nanostructures

Nano Letters. May, 2010  |  Pubmed ID: 20397703

Dislocation filtering in GaN by selective area growth through a nanoporous template is examined both by transmission electron microscopy and numerical modeling. These nanorods grow epitaxially from the (0001)-oriented GaN underlayer through the approximately 100 nm thick template and naturally terminate with hexagonal pyramid-shaped caps. It is demonstrated that for a certain window of geometric parameters a threading dislocation growing within a GaN nanorod is likely to be excluded by the strong image forces of the nearby free surfaces. Approximately 3000 nanorods were examined in cross-section, including growth through 50 and 80 nm diameter pores. The very few threading dislocations not filtered by the template turn toward a free surface within the nanorod, exiting less than 50 nm past the base of the template. The potential active region for light-emitting diode devices based on these nanorods would have been entirely free of threading dislocations for all samples examined. A greater than 2 orders of magnitude reduction in threading dislocation density can be surmised from a data set of this size. A finite element-based implementation of the eigenstrain model was employed to corroborate the experimentally observed data and examine a larger range of potential nanorod geometries, providing a simple map of the different regimes of dislocation filtering for this class of GaN nanorods. These results indicate that nanostructured semiconductor materials are effective at eliminating deleterious extended defects, as necessary to enhance the optoelectronic performance and device lifetimes compared to conventional planar heterostructures.

SERCA2a Gene Transfer Decreases Sarcoplasmic Reticulum Calcium Leak and Reduces Ventricular Arrhythmias in a Model of Chronic Heart Failure

Circulation. Arrhythmia and Electrophysiology. Jun, 2011  |  Pubmed ID: 21406682

Sarcoplasmic reticulum calcium ATPase 2a (SERCA2a) gene therapy improves mechanical function in heart failure and is under evaluation in a clinical trial. A critical question is whether SERCA2a gene therapy predisposes to increased sarcoplasmic reticulum calcium (SR Ca(2+)) leak, cellular triggered activity, and ventricular arrhythmias in the failing heart.

Genetic Studies on the Ghrelin, Growth Hormone Secretagogue Receptor (GHSR) and Ghrelin O-acyl Transferase (GOAT) Genes

Peptides. Nov, 2011  |  Pubmed ID: 21930173

Ghrelin is a 28 amino acid peptide hormone that is produced both centrally and peripherally. Regulated by the ghrelin O-acyl transferase enzyme, ghrelin exerts its action through the growth hormone secretagogue receptor, and is implicated in a diverse range of physiological processes. These implications have placed the ghrelin signaling pathway at the center of a large number of candidate gene and genome-wide studies which aim to identify the genetic basis of human heterogeneity. In this review we summarize the available data on the genetic variability of ghrelin, its receptor and its regulatory enzyme, and their association with obesity, stature, type 2 diabetes, cardiovascular disease, eating disorders, and reward seeking behavior.

Built-in Electric Field Minimization in (In, Ga)N Nanoheterostructures

Nano Letters. Nov, 2011  |  Pubmed ID: 21942457

(In, Ga)N nanostructures show great promise as the basis for next generation LED lighting technology, for they offer the possibility of directly converting electrical energy into light of any visible wavelength without the use of down-converting phosphors. In this paper, three-dimensional computation of the spatial distribution of the mechanical and electrical equilibrium in nanoheterostructures of arbitrary topologies is used to elucidate the complex interactions between geometry, epitaxial strain, remnant polarization, and piezoelectric and dielectric contributions to the self-induced internal electric fields. For a specific geometry-nanorods with pyramidal caps-we demonstrate that by tuning the quantum well to cladding layer thickness ratio, h(w)/h(c), a minimal built-in electric field can be experimentally realized and canceled, in the limit of h(w)/h(c) = 1.28, for large h(c) values.

Combined Blockade of Signalling Pathways Shows Marked Anti-tumour Potential in Phaeochromocytoma Cell Lines

Journal of Molecular Endocrinology. Oct, 2012  |  Pubmed ID: 22715163

Currently, there is no completely effective therapy available for metastatic phaeochromocytomas (PCCs) and paragangliomas. In this study, we explore new molecular targeted therapies for these tumours, using one more benign (mouse phaeochromocytoma cell (MPC)) and one more malignant (mouse tumour tissue (MTT)) mouse PCC cell line - both generated from heterozygous neurofibromin 1 knockout mice. Several PCC-promoting gene mutations have been associated with aberrant activation of PI3K/AKT, mTORC1 and RAS/RAF/ERK signalling. We therefore investigated different agents that interfere specifically with these pathways, including antagonism of the IGF1 receptor by NVP-AEW541. We found that NVP-AEW541 significantly reduced MPC and MTT cell viability at relatively high doses but led to a compensatory up-regulation of ERK and mTORC1 signalling at suboptimal doses while PI3K/AKT inhibition remained stable. We subsequently investigated the effect of the dual PI3K/mTORC1/2 inhibitor NVP-BEZ235, which led to a significant decrease of MPC and MTT cell viability at doses below 50 nM but again increased ERK signalling. Accordingly, we next examined the combination of NVP-BEZ235 with the established agent lovastatin, as this has been described to inhibit ERK signalling. Lovastatin alone significantly reduced MPC and MTT cell viability at therapeutically relevant doses and inhibited both ERK and AKT signalling, but increased mTORC1/p70S6K signalling. Combination treatment with NVP-BEZ235 and lovastatin showed a significant additive effect in MPC and MTT cells and resulted in inhibition of both AKT and mTORC1/p70S6K signalling without ERK up-regulation. Simultaneous inhibition of PI3K/AKT, mTORC1/2 and ERK signalling suggests a novel therapeutic approach for malignant PCCs.

Characterization of SNARE Proteins in Human Pituitary Adenomas: Targeted Secretion Inhibitors As a New Strategy for the Treatment of Acromegaly?

The Journal of Clinical Endocrinology and Metabolism. Dec, 2013  |  Pubmed ID: 24152687

Targeted secretion inhibitors (TSIs), a new class of recombinant biotherapeutic proteins engineered from botulinum toxin, represent a novel approach for treating diseases with excess secretion. They inhibit hormone secretion from targeted cell types through cleavage of SNARE (soluble N-ethylmaleimide-sensitive factor-activating protein receptor) proteins. qGHRH-LH(N)/D is a TSI targeting pituitary somatotroph through binding to the GHRH-receptor and cleavage of the vesicle-associated membrane protein (VAMP) family of SNARE proteins.

Donor-site Preferences in Women During Autologous Skin Grafting

Plastic and Reconstructive Surgery. Mar, 2014  |  Pubmed ID: 24572883

Autologous split-thickness skin grafting has been proven to provide the best cosmetic and functional outcome after cutaneous burn injuries and thus is the standard of care. Clinical observations have shown that female burn patients frequently have greater difficulty choosing a donor site than do male burn patients. However, there is a lack of data characterizing donor-site preferences among women with burns.

Spheronization Process Particle Kinematics Determined by Discrete Element Simulations and Particle Image Velocimentry Measurements

International Journal of Pharmaceutics. Dec, 2014  |  Pubmed ID: 25304094

Spheronization is an important pharmaceutical manufacturing technique to produce spherical agglomerates of 0.5-2mm diameter. These pellets have a narrow size distribution and a spherical shape. During the spheronization process, the extruded cylindrical strands break in short cylinders and evolve from a cylindrical to a spherical state by deformation and attrition/agglomeration mechanisms. Using the discrete element method, an integrated modeling-experimental framework is presented, that captures the particle motion during the spheronization process. Simulations were directly compared and validated against particle image velocimetry (PIV) experiments with monodisperse spherical and dry γ-Al2O3 particles.

Cancer-associated Fibroblasts Predict Poor Outcome and Promote Periostin-dependent Invasion in Oesophageal Adenocarcinoma

The Journal of Pathology. Feb, 2015  |  Pubmed ID: 25345775

Interactions between cancer cells and cancer-associated fibroblasts (CAFs) play an important role in tumour development and progression. In this study we investigated the functional role of CAFs in oesophageal adenocarcinoma (EAC). We used immunochemistry to analyse a cohort of 183 EAC patients for CAF markers related to disease mortality. We characterized CAFs and normal oesophageal fibroblasts (NOFs) using western blotting, immunofluorescence and gel contraction. Transwell assays, 3D organotypic culture and xenograft models were used to examine the effects on EAC cell function and to dissect molecular mechanisms regulating invasion. Most EACs (93%) contained CAFs with a myofibroblastic (α-SMA-positive) phenotype, which correlated significantly with poor survival [p = 0.016; HR 7. 1 (1.7-29.4)]. Primary CAFs isolated from EACs have a contractile, myofibroblastic phenotype and promote EAC cell invasion in vitro (Transwell assays, p ≤ 0.05; organotypic culture, p < 0.001) and in vivo (p ≤ 0.05). In vitro, this pro-invasive effect is modulated through the matricellular protein periostin. Periostin is secreted by CAFs and acts as a ligand for EAC cell integrins αvβ3 and αvβ5, promoting activation of the PI3kinase-Akt pathway. In patient samples, periostin expression at the tumour cell-stromal interface correlates with poor overall and disease-free survival. Our study highlights the importance of the tumour stroma in EAC progression. Paracrine interaction between CAF-secreted periostin and EAC-expressed integrins results in PI3 kinase-Akt activation and increased tumour cell invasion. Most EACs contain a myofibroblastic CAF-rich stroma; this may explain the aggressive, highly infiltrative nature of the disease, and suggests that stromal targeting may produce therapeutic benefit in EAC patients.

Authentication and Characterisation of a New Oesophageal Adenocarcinoma Cell Line: MFD-1

Scientific Reports. Sep, 2016  |  Pubmed ID: 27600491

New biological tools are required to understand the functional significance of genetic events revealed by whole genome sequencing (WGS) studies in oesophageal adenocarcinoma (OAC). The MFD-1 cell line was isolated from a 55-year-old male with OAC without recombinant-DNA transformation. Somatic genetic variations from MFD-1, tumour, normal oesophagus, and leucocytes were analysed with SNP6. WGS was performed in tumour and leucocytes. RNAseq was performed in MFD-1, and two classic OAC cell lines FLO1 and OE33. Transposase-accessible chromatin sequencing (ATAC-seq) was performed in MFD-1, OE33, and non-neoplastic HET1A cells. Functional studies were performed. MFD-1 had a high SNP genotype concordance with matched germline/tumour. Parental tumour and MFD-1 carried four somatically acquired mutations in three recurrent mutated genes in OAC: TP53, ABCB1 and SEMA5A, not present in FLO-1 or OE33. MFD-1 displayed high expression of epithelial and glandular markers and a unique fingerprint of open chromatin. MFD-1 was tumorigenic in SCID mouse and proliferative and invasive in 3D cultures. The clinical utility of whole genome sequencing projects will be delivered using accurate model systems to develop molecular-phenotype therapeutics. We have described the first such system to arise from the oesophageal International Cancer Genome Consortium project.

Induction of Fibroblast Senescence Generates a Non-fibrogenic Myofibroblast Phenotype That Differentially Impacts on Cancer Prognosis

Aging. Dec, 2016  |  Pubmed ID: 27992856

Cancer-associated fibroblasts (CAF) remain a poorly characterized, heterogeneous cell population. Here we characterized two previously described tumor-promoting CAF sub-types, smooth muscle actin (SMA)-positive myofibroblasts and senescent fibroblasts, identifying a novel link between the two. Analysis of CAF cultured ex vivo, showed that senescent CAF are predominantly SMA-positive; this was confirmed by immunochemistry in head & neck (HNSCC) and esophageal (EAC) cancers. In vitro, we found that fibroblasts induced to senesce develop molecular, ultrastructural and contractile features typical of myofibroblasts and this is dependent on canonical TGF-β signaling. Similar to TGF-β1-generated myofibroblasts, these cells secrete soluble factors that promote tumor cell motility. However, RNA-sequencing revealed significant transcriptomic differences between the two SMA-positive CAF groups, particularly in genes associated with extracellular matrix (ECM) deposition and organization, which differentially promote tumor cell invasion. Notably, second harmonic generation imaging and bioinformatic analysis of SMA-positive human HNSCC and EAC showed that collagen fiber organization correlates with poor prognosis, indicating that heterogeneity within the SMA-positive CAF population differentially impacts on survival. These results show that non-fibrogenic, SMA-positive myofibroblasts can be directly generated through induction of fibroblast senescence and suggest that senescence and myofibroblast differentiation are closely linked processes.

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