Publications by authors named "Alfonso Blanco Fernández"

16 Publications

  • Page 1 of 1

Cyclophilin A regulates secretion of tumour-derived extracellular vesicles.

Transl Oncol 2021 May 10;14(8):101112. Epub 2021 May 10.

UCD School of Biomolecular & Biomedical Science, Conway Institute, University College Dublin (UCD), Belfield, Dublin 4, Ireland. Electronic address:

Extracellular Vesicles (EVs) are a heterogenous population of particles that play an important role in cell-cell communication in physiological and pathophysiological situations. In this study we reveal that the peptidyl prolyl isomerase Cyclophilin A (CypA) is enriched in cancer-derived EVs from a range of haematopoietic malignancies. CypA-enriched blood cancer EVs were taken up by normal monocytes independent of EV surface trypsin-sensitive proteins and potently stimulated pro-inflammatory MMP9 and IL-6 secretion. Further characterisation revealed that CypA is intravesicular, however, it is not present in all EVs derived from the haematopoietic cells, instead, it is predominantly located in high density EVs with a range of 1.15-1.18 g/ml. Furthermore, loss of CypA expression in haematological cancer cells attenuates high density EV-induced pro-inflammatory MMP9 and IL-6 secretion from monocytes. Mechanistically, we reveal that homozygous loss or siRNA knockdown of CypA expression significantly reduced the secretion of EVs in the range of 100-200 nm from blood cancer cells under normal and hypoxic conditions. Overall, this work reveals a novel role for CypA in cancer cell EV biogenesis.
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http://dx.doi.org/10.1016/j.tranon.2021.101112DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8131927PMC
May 2021

Tetrameric and polymeric silver complexes of the omeprazole scaffold; synthesis, structure, in vitro and in vivo antimicrobial activities and DNA interaction.

J Inorg Biochem 2018 09 31;186:317-328. Epub 2018 May 31.

Department of Physics, Chemistry and Pharmacy, University of Southern Denmark, Campusvej 55, 5230 Odense M, Denmark. Electronic address:

Two complexes [AgI(pmtbH)] (1) and {[Ag(pmtbH)(NO)·2X} (2) (where pmtbH is 2-[(2-pyridinylmethyl)thio]-1H-benzimidazole and X is HO or MeOH) were synthesised and structurally characterised. Complex 2 showed therapeutic potential against Candida albicans, Escherichia coli, Staphylococcus aureus and Pseudomonas aeruginosa but complex 1 did not show significant activity in vitro. Further in vivo studies using larvae of the insect Galleria mellonella indicated that complex 2 significantly stimulates the immune system and that pre-treatment with the complex offers appreciable protection against all three bacteria. Real-time flow cytometry data support the observed antimicrobial profile of complex 2 and suggest the antimicrobial response may be linked to a form of bacterial programmed cell death (PCD). Complex 2 was found to interact with DNA in the bacterial and fungal cells but it did not cleave plasmid DNA isolated from the three bacteria.
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http://dx.doi.org/10.1016/j.jinorgbio.2018.05.018DOI Listing
September 2018

A Novel Retinal Ganglion Cell Promoter for Utility in AAV Vectors.

Front Neurosci 2017 21;11:521. Epub 2017 Sep 21.

School of Genetics and Microbiology, Smurfit Institute of Genetics, Trinity College DublinDublin, Ireland.

Significant advances in gene therapy have enabled exploration of therapies for inherited retinal disorders, many of which are in preclinical development or clinical evaluation. Gene therapy for retinal conditions has led the way in this growing field. The loss of retinal ganglion cells (RGCs) is a hallmark of a number of retinal disorders. As the field matures innovations that aid in refining therapies and optimizing efficacy are in demand. Gene therapies under development for RGC-related disorders, when delivered with recombinant adeno associated vectors (AAV), have typically been expressed from ubiquitous promoter sequences. Here we describe how a novel promoter from the murine gene was selected to drive transgene expression in RGCs. The promoter, in an AAV2/2 vector, was shown to drive preferential EGFP expression in murine RGCs following intravitreal injection. In contrast, EGFP expression from a promoter was observed not only in RGCs, but throughout the inner nuclear layer and in amacrine cells located within the ganglion cell layer (GCL). Of note, the promoter sequence is sufficiently compact to be readily accommodated in AAV vectors, where transgene size represents a significant constraint. Moreover, this promoter should in principle provide a more targeted and potentially safer alternative for RGC-directed gene therapies.
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http://dx.doi.org/10.3389/fnins.2017.00521DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5613148PMC
September 2017

Subfertility in bulls carrying a nonsense mutation in transmembrane protein 95 is due to failure to interact with the oocyte vestments.

Biol Reprod 2017 Jul;97(1):50-60

School of Agriculture and Food Science, University College Dublin, Belfield, Dublin 4, Ireland.

In a recent genome-wide association study, 40 Fleckvieh bulls with exceptionally poor fertility were found to be homozygous for a nonsense mutation in the transmembrane protein 95 (TMEM95) encoding gene. Ejaculates from these individuals exhibited normal sperm concentration, morphology, viability, and motility. However, only 1.7% of inseminations resulted in pregnancies. The aim of this study was to examine the effect of this mutation in TMEM95 on bovine sperm function in vitro. Sperm from homozygous (mt/mt) males had lower in vitro fertility than sperm from wild-type (wt/wt) or heterozygous (wt/mt) bulls (P < 0.01). In addition, early embryo division was affected in the mt/mt group (P < 0.01). This translated into a lower (P < 0.01) blastocyst rate at day 8. Fluorescent staining revealed that TMEM95 is lost after the acrosome reaction. This led us to hypothesize that TMEM95 might be involved in events that lead to sperm-oocyte interaction. After fertilization, a lower number (P < 0.01) of sperm from mt/mt bulls bound to the zona pellucida (ZP). Sperm from mt/mt bulls were also less able to penetrate oocytes with no ZP (P< 0.01). However, when sperm from these animals were injected into mouse oocytes, they could decondense as successfully as sperm from wt/wt bulls. No differences between genotypes were observed in the ability of sperm to retain motility in an ex vivo oviduct, or in the percentage of sperm exhibiting markers for capacitation and acrosomal reaction. These results suggest that fertilization failure in mt/mt bulls is due to the inability of their sperm to interact with the oocyte vestments.
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http://dx.doi.org/10.1093/biolre/iox065DOI Listing
July 2017

Real time kinetic flow cytometry measurements of cellular parameter changes evoked by nanosecond pulsed electric field.

Cytometry A 2016 05 15;89(5):472-9. Epub 2016 Mar 15.

Conway Institute of Biomolecular and Biomedical Research, University College Dublin, Belfield, Dublin 4, Ireland.

Nanosecond pulsed electric field (nsPEF) is a novel method to increase cell proliferation rate. The phenomenon is based on the microporation of cellular organelles and membranes. However, we have limited information on the effects of nsPEF on cell physiology. Several studies have attempted to describe the effects of this process, however no real time measurements have been conducted to date. In this study we designed a model system which allows the measurement of cellular processes before, during and after nsPEF treatment in real time. The system employs a Vabrema Mitoplicator(TM) nsPEF field generating instrument connected to a BD Accuri C6 cytometer with a silicon tube led through a peristaltic pump. This model system was applied to observe the effects of nsPEF in mammalian C6 glioblastoma (C6 glioma) and HEK-293 cell lines. Viability (using DRAQ7 dye), intracellular calcium levels (using Fluo-4 dye) and scatter characteristics were measured in a kinetic manner. Data were analyzed using the FACSKin software. The viability and morphology of the investigated cells was not altered upon nsPEF treatment. The response of HEK-293 cells to ionomycin as positive control was significantly lower in the nsPEF treated samples compared to non-treated cells. This difference was not observed in C6 cells. FSC and SSC values were not altered significantly by the nsPEF treatment. Our results indicate that this model system is capable of reliably investigating the effects of nsPEF on cellular processes in real time. © 2016 International Society for Advancement of Cytometry.
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http://dx.doi.org/10.1002/cyto.a.22838DOI Listing
May 2016

Nonlinear signalling networks and cell-to-cell variability transform external signals into broadly distributed or bimodal responses.

J R Soc Interface 2014 Sep;11(98):20140383

Systems Biology Ireland, University College Dublin, Belfield, Dublin 4, Ireland Conway Institute of Biomolecular and Biomedical Research, University College Dublin, Belfield, Dublin 4, Ireland School of Medicine and Medical Science, University College Dublin, Belfield, Dublin 4, Ireland

We show theoretically and experimentally a mechanism behind the emergence of wide or bimodal protein distributions in biochemical networks with nonlinear input-output characteristics (the dose-response curve) and variability in protein abundance. Large cell-to-cell variation in the nonlinear dose-response characteristics can be beneficial to facilitate two distinct groups of response levels as opposed to a graded response. Under the circumstances that we quantify mathematically, the two distinct responses can coexist within a cellular population, leading to the emergence of a bimodal protein distribution. Using flow cytometry, we demonstrate the appearance of wide distributions in the hypoxia-inducible factor-mediated response network in HCT116 cells. With help of our theoretical framework, we perform a novel calculation of the magnitude of cell-to-cell heterogeneity in the dose-response obtained experimentally.
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http://dx.doi.org/10.1098/rsif.2014.0383DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4233687PMC
September 2014

Remote access technology offers tremendous possibilities to the cytometry community.

Cytometry A 2013 Dec 8;83(12):1062-5. Epub 2013 Nov 8.

Flow Cytometry Facility, School of Biochemistry and Immunology, Trinity Biomedical Sciences Institute, Trinity College, Dublin, Ireland.

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http://dx.doi.org/10.1002/cyto.a.22409DOI Listing
December 2013

Monitoring of dynamic microbiological processes using real-time flow cytometry.

PLoS One 2013 14;8(11):e80117. Epub 2013 Nov 14.

Department of Environmental Microbiology, Eawag - Swiss Federal Institute for Aquatic Science and Technology, Dübendorf, Switzerland ; Department of Environmental Systems Science, ETH Zürich, Zürich, Switzerland.

We describe a straightforward approach to continuously monitor a variety of highly dynamic microbiological processes in millisecond resolution with flow cytometry, using standard bench-top instrumentation. Four main experimental examples are provided, namely: (1) green fluorescent protein expression by antibiotic-stressed Escherichia coli, (2) fluorescent labeling of heat-induced membrane damage in an autochthonous freshwater bacterial community, (3) the initial growth response of late stationary E. coli cells inoculated into fresh growth media, and (4) oxidative disinfection of a mixed culture of auto-fluorescent microorganisms. These examples demonstrate the broad applicability of the method to diverse biological experiments, showing that it allows the collection of detailed, time-resolved information on complex processes.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0080117PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3828236PMC
August 2014

Efficacy of codelivery of dual AAV2/5 vectors in the murine retina and hippocampus.

Hum Gene Ther 2012 Aug 20;23(8):847-58. Epub 2012 Jul 20.

Department of Genetics, Trinity College Dublin, Dublin 2, Ireland.

Recombinant adeno-associated virus (AAV) represents an efficient system for neuronal transduction. However, a potential drawback of AAV is its restricted packaging capacity of approximately 5 kb. To bypass this limitation, a number of dual- and triple-vector strategies divide the transgene(s) between two or three AAVs. The success of these approaches relies directly on efficient cotransduction of the component AAVs. Although proof of concept for these stratagems has been demonstrated, the underlying cotransduction rate has not been analyzed quantitatively. In this study, cotransduction efficiencies in both retina and hippocampus have been investigated, using two reporter AAVs expressing either a green (GFP) or red (DsR) fluorescent protein. Transduction efficiencies were monitored via microscopy, flow cytometry, and quantitative PCR. After viral transduction with 1.5×10(9) viral particles of each of the reporter AAVs, approximately one-third of the retinal cells expressed one or both transgenes at levels detectable by native fluorescence. Notably, the majority of the remaining retinal cells were also transduced and expressed the reporters at lower levels, which were detectable only by immunolabeling. Flow cytometric analysis demonstrated cotransduction rates of up to 55% with the two reporter AAVs in retinal cells. Modifying the ratio of the two coadministered AAVs resulted in altered mRNA expression levels of the two reporter genes in cotransduced cell populations. The study suggests that codelivery of AAV is an efficient means of expanding the therapeutic application of AAV in neurons.
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http://dx.doi.org/10.1089/hum.2011.142DOI Listing
August 2012

Enhancement of BODIPY505/515 lipid fluorescence method for applications in biofuel-directed microalgae production.

J Microbiol Methods 2012 Aug 14;90(2):137-43. Epub 2012 Apr 14.

Charles Parsons Energy Research Programme, Bioresources Research Centre, School of Biosystems Engineering, University College Dublin, Belfield, Dublin 4, Ireland.

This paper describes a microalgal cell lipid fluorescence enhancement method using BODIPY(505/515), which can be used to screen for lipids in wild-type microalgae and to monitor lipid content within microalgae production processes to determine optimal harvesting time. The study was based on four microalgae species (Dunaliella teteriolecta, Tetraselmis suecica, Nannochloropsis oculata, and Nannochloris atomus) selected because of their inherent high lipid content. An extended analysis was carried out with N. oculata due to the depressed fluorescence observed when compared with the other experimental strains. BODIPY(505/515) lipid fluorescence was determined for two solvent pre-treatment methods (DMSO and glycerol) and four staining condition parameters (analysis time, staining temperature, dye concentration, and algal cell concentration). It was found that lipid fluorescence of thick cell-walled microalgae, such as N. oculata, is significantly enhanced by both the pre-treatment methods and staining condition parameters, thereby significantly enhancing lipid fluorescence by ca. 800 times the base autofluorescence. The lipid fluorescence enhancement method provides a quick and simple index for in vivo Flow Cytometry quantification of total lipid contents for purposes of species screening or whole culture monitoring in biofuel-directed microalgae production.
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http://dx.doi.org/10.1016/j.mimet.2012.03.020DOI Listing
August 2012

Regulator of G-protein signaling 18 integrates activating and inhibitory signaling in platelets.

Blood 2012 Apr 10;119(16):3799-807. Epub 2012 Jan 10.

Conway Institute, University College Dublin, Ireland.

Regulator of G-protein signaling 18 (RGS18) is a GTPase-activating protein for the G-α-q and G-α-i subunits of heterotrimeric G-proteins that turns off signaling by G-protein coupled receptors. RGS18 is highly expressed in platelets. In the present study, we show that the 14-3-3γ protein binds to phosphorylated serines 49 and 218 of RGS18. Platelet activation by thrombin, thromboxane A2, or ADP stimulates the association of 14-3-3 and RGS18, probably by increasing the phosphorylation of serine 49. In contrast, treatment of platelets with prostacyclin and nitric oxide, which trigger inhibitory cyclic nucleotide signaling involving cyclic AMP-dependent protein kinase A (PKA) and cyclic GMP-dependent protein kinase I (PKGI), induces the phosphorylation of serine 216 of RGS18 and the detachment of 14-3-3. Serine 216 phosphorylation is able to block 14-3-3 binding to RGS18 even in the presence of thrombin, thromboxane A2, or ADP. 14-3-3-deficient RGS18 is more active compared with 14-3-3-bound RGS18, leading to a more pronounced inhibition of thrombin-induced release of calcium ions from intracellular stores. Therefore, PKA- and PKGI-mediated detachment of 14-3-3 activates RGS18 to block Gq-dependent calcium signaling. These findings indicate cross-talk between platelet activation and inhibition pathways at the level of RGS18 and Gq.
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http://dx.doi.org/10.1182/blood-2011-11-390369DOI Listing
April 2012

5-AZA-2'-deoxycytidine induced demethylation influences N-glycosylation of secreted glycoproteins in ovarian cancer.

Epigenetics 2011 Nov 1;6(11):1362-72. Epub 2011 Nov 1.

Dublin-Oxford NIBRT Glycobiology Laboratory, NIBRT, Conway Institute, University College Dublin, Belfield, Dublin, Ireland.

Glycosylation is the most common posttranslational modification of proteins and is highly reflective of changes in the environment of a cell. Epigenetic modifications to the genome are stably transmitted to daughter cells without the requirement for genetic sequence alterations. Aberrant regulation of both epigenetic programming and glycosylation patterning are integral aspects of carcinogenesis. The objective of this study was to determine the interplay between these two complex cellular processes. We demonstrate that global DNA methylation changes in ovarian cancer epithelial cells (OVCAR3) resulted in significant alterations in the glycosylation of secreted glycoproteins. These changes included a reduction in core fucosylation, increased branching and increased sialylation. We further show that the change in core fucose levels was mirrored by altered expression of GMDS and FX, key enzymes in fucose biosynthesis. Alterations in the expression of key glycosyltransferase enzymes such as MGAT5 reflect the changes seen in the branching and sialylation of secreted glycans. Overall, our results highlight that modifications to the epigenetic machinery have a profound effect on the glycan structures generated by cells, which may be a key step in understanding metastasis and drug resistance during cancer progression.
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http://dx.doi.org/10.4161/epi.6.11.17977DOI Listing
November 2011

A flow-cytometric method for continuous measurement of intracellular Ca(2+) concentration.

Cytometry A 2010 Nov;77(11):1091-7

UCD School of Biomolecular and Biomedical Science, UCD-Conway Institute, University College Dublin, Belfield, Dublin 4, Ireland.

Alterations in intracellular Ca(2+) concentration are amongst the most rapid responses to a variety of stimuli in mammalian cells. In the nervous system in particular, responses occur within nanoseconds. A major challenge in intracellular Ca(2+) analysis is to achieve measurements within this very fast time frame. To date, the dynamic intracellular Ca(2+) concentration has been monitored by confocal microscopy, plate-based assays, spectrofluorometry, and flow cytometry, although there are issues with the number of cells analyzed or gaps in recording due to the addition of compounds, with significant loss of detail of a rapid Ca(2+) response. The new generation of flow cytometers (such as Accuri C6) resolves this problem by allowing the addition of test compounds with continuous monitoring of thousands of cells, providing a method for dynamic Ca(2+) measurements. This system was tested with commonly used Ca(2+) modulating agents in C6 glioma cells. Thapsigargin (TG), a blocker of Ca(2+) uptake into the endoplasmic reticulum (ER), causes a significant increase in the intracellular calcium concentration via ER emptying followed by Ca(2+) entry via store-operated Ca(2+) channels (SOCC). This well-established pathway can be partially inhibited by 2-aminoethoxydiphenyl borate (2-APB), a blocker of SOCC. Both the increase with TG alone and the partial increase when coincubated with 2-APB were observed with continuous recording along with calibration curves using an Accuri C6 flow cytometer. With these new cytometers, dynamic Ca(2+) concentration measurement becomes extremely accessible and accurate, while also providing extensive and valuable data regarding population health and responsiveness.
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http://dx.doi.org/10.1002/cyto.a.20974DOI Listing
November 2010

AAV-mediated chronic over-expression of SNAP-25 in adult rat dorsal hippocampus impairs memory-associated synaptic plasticity.

J Neurochem 2010 Feb 30;112(4):991-1004. Epub 2009 Nov 30.

Applied Neurotherapeutics Research Group, Smurfit Institute of Genetics, Trinity College Dublin, Dublin, Ireland.

Long-term memory is formed by alterations in glutamate-dependent excitatory synaptic transmission, which is in turn regulated by synaptosomal protein of 25 kDa (SNAP-25), a key component of the soluble N-ethylmaleimide-sensitive factor attachment protein receptor complex essential for exocytosis of neurotransmitter-filled synaptic vesicles. Both reduced and excessive SNAP-25 activity has been implicated in various disease states that involve cognitive dysfunctions such as attention deficit hyperactivity disorder, schizophrenia and Alzheimer's disease. Here, we over-express SNAP-25 in the adult rat dorsal hippocampus by infusion of a recombinant adeno-associated virus vector, to evaluate the consequence of late adolescent-adult dysfunction of the soluble N-ethylmaleimide-sensitive factor attachment protein receptor protein in the absence of developmental disruption. We report a specific and significant increase in the levels of extracellular glutamate detectable by microdialysis and a reduction in paired-pulse facilitation in the hippocampus. In addition, SNAP-25 over-expression produced cognitive deficits, delaying acquisition of a spatial map in the water maze and impairing contextual fear conditioning, both tasks known to be dorsal hippocampal dependent. The high background transmission state and pre-synaptic dysfunction likely result in interference with requisite synapse selection during spatial and fear memory consolidation. Together these studies provide the first evidence that excess SNAP-25 activity, restricted to the adult period, is sufficient to mediate significant deficits in the memory formation process.
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http://dx.doi.org/10.1111/j.1471-4159.2009.06516.xDOI Listing
February 2010

The effect of retinoic acid and deoxycholic acid on the differentiation of primary human esophageal keratinocytes.

Dig Dis Sci 2008 Nov 27;53(11):2851-7. Epub 2008 Mar 27.

UCD School of Medicine, UCD Conway Institute of Biomolecular and Biomedical Research, Dublin, Ireland.

The mechanism linking gastroduodenal reflux disease to intestinal metaplasia in the esophagus (Barrett's esophagus) has not been determined. Active conjugate metabolites of retinoic acid, in addition to bile acids, undergo an enterohepatic circulation in bile. Retinoic acid and bile acids are candidate mediators of keratinocyte transdifferentiation in Barrett's esophagus. We studied the effects of retinoic acid on the differentiation of primary human esophageal keratinocytes cultured in vitro. Retinoic acid induces expression of a marker of intestinal differentiation, MUC2, in these cells. However, retinoic acid, alone or in combination with the hydrophobic bile acid, deoxycholic acid, does not affect esophageal keratinocyte squamous differentiation as assessed by involucrin expression and cellular morphology. The ability of retinoic acid to induce MUC2 expression may be relevant to the pathogenesis of Barrett's esophagus. However, this does not result in suppression of squamous differentiation.
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http://dx.doi.org/10.1007/s10620-008-0240-zDOI Listing
November 2008