Publications by authors named "Giovanni Cuda"

110 Publications

Mass Spectrometry-Based Glycoproteomics and Prostate Cancer.

Int J Mol Sci 2021 May 14;22(10). Epub 2021 May 14.

Research Centre for Advanced Biochemistry and Molecular Biology, Department of Experimental and Clinical Medicine, Magna Graecia University of Catanzaro, 88100 Catanzaro, Italy.

Aberrant glycosylation has long been known to be associated with cancer, since it is involved in key mechanisms such as tumour onset, development and progression. This review will focus on protein glycosylation studies in cells, tissue, urine and serum in the context of prostate cancer. A dedicated section will cover the glycoforms of prostate specific antigen, the molecule that, despite some important limitations, is routinely tested for helping prostate cancer diagnosis. Our aim is to provide readers with an overview of mass spectrometry-based glycoproteomics of prostate cancer. From this perspective, the first part of this review will illustrate the main strategies for glycopeptide enrichment and mass spectrometric analysis. The molecular information obtained by glycoproteomic analysis performed by mass spectrometry has led to new insights into the mechanism linking aberrant glycosylation to cancer cell proliferation, migration and immunoescape.
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http://dx.doi.org/10.3390/ijms22105222DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8156230PMC
May 2021

Proteomic Profile of EPS-Urine through FASP Digestion and Data-Independent Analysis.

J Vis Exp 2021 May 8(171). Epub 2021 May 8.

Research Centre for Advanced Biochemistry and Molecular Biology, Department of Experimental and Clinical Medicine, Magna Graecia University of Catanzaro.

Filter-aided sample protocol (FASP) is widely used for proteomics sample preparation because it allows to concentrate diluted samples and it is compatible with a wide variety of detergents. Bottom-up proteomics workflows like FASP increasingly rely on LC-MS/MS methods performed in data-independent analysis (DIA) mode, a scanning method that allows deep proteome coverage and low incidence of missing values. In this report, we will provide the details of a workflow that combines a FASP protocol, a double StageTip purification step and LC-MS/MS in DIA mode for urinary proteome mapping. As a model sample, we analyzed expressed prostatic secretions (EPS)-urine, a sample collected after a digital rectal exam (DRE), which is of interest in prostate cancer biomarker discovery studies.
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http://dx.doi.org/10.3791/62512DOI Listing
May 2021

Generation of human induced pluripotent stem cell lines (UNIMGi003-A and UNIMGi004-A) from two Italian siblings affected by Unverricht-Lundborg disease.

Stem Cell Res 2021 May 9;53:102329. Epub 2021 Apr 9.

Department of Clinical and Experimental Medicine, Magna Græcia University, 88100 Catanzaro, Italy. Electronic address:

Unverricht-Lundborg disease (ULD) is an inherited form of progressive myoclonus epilepsy caused by mutations in the gene encoding Cystatin B (CSTB), an inhibitor of lysosomal proteases. The most common mutation described in ULD patients is an unstable expansion of a dodecamer sequence located in the CSTB gene promoter. This expansion is causative of the downregulation of CSTB gene expression and, consequently, of its inhibitory activity. Here we report the generation of induced pluripotent stem cell (iPSC) lines from two Italian siblings having a family history of ULD and affected by different clinical and pathological phenotypes of the disease.
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http://dx.doi.org/10.1016/j.scr.2021.102329DOI Listing
May 2021

Deciphering the Role of Wnt and Rho Signaling Pathway in iPSC-Derived ARVC Cardiomyocytes by In Silico Mathematical Modeling.

Int J Mol Sci 2021 Feb 18;22(4). Epub 2021 Feb 18.

Department of Clinical and Experimental Medicine, Magna Græcia University, 88100 Catanzaro, Italy.

Arrhythmogenic Right Ventricular cardiomyopathy (ARVC) is an inherited cardiac muscle disease linked to genetic deficiency in components of the desmosomes. The disease is characterized by progressive fibro-fatty replacement of the right ventricle, which acts as a substrate for arrhythmias and sudden cardiac death. The molecular mechanisms underpinning ARVC are largely unknown. Here we propose a mathematical model for investigating the molecular dynamics underlying heart remodeling and the loss of cardiac myocytes identity during ARVC. Our methodology is based on three computational models: firstly, in the context of the Wnt pathway, we examined two different competition mechanisms between β-catenin and Plakoglobin (PG) and their role in the expression of adipogenic program. Secondly, we investigated the role of RhoA-ROCK pathway in ARVC pathogenesis, and thirdly we analyzed the interplay between Wnt and RhoA-ROCK pathways in the context of the ARVC phenotype. We conclude with the following remark: both Wnt/β-catenin and RhoA-ROCK pathways must be inactive for a significant increase of expression, suggesting that a crosstalk mechanism might be responsible for mediating ARVC pathogenesis.
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http://dx.doi.org/10.3390/ijms22042004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7923182PMC
February 2021

Cytoplasmic cleavage of IMPA1 3' UTR is necessary for maintaining axon integrity.

Cell Rep 2021 Feb;34(8):108778

MRC Laboratory for Molecular Cell Biology, University College London, London WC1E 6BT, UK. Electronic address:

The 3' untranslated regions (3' UTRs) of messenger RNAs (mRNAs) are non-coding sequences involved in many aspects of mRNA metabolism, including intracellular localization and translation. Incorrect processing and delivery of mRNA cause severe developmental defects and have been implicated in many neurological disorders. Here, we use deep sequencing to show that in sympathetic neuron axons, the 3' UTRs of many transcripts undergo cleavage, generating isoforms that express the coding sequence with a short 3' UTR and stable 3' UTR-derived fragments of unknown function. Cleavage of the long 3' UTR of Inositol Monophosphatase 1 (IMPA1) mediated by a protein complex containing the endonuclease argonaute 2 (Ago2) generates a translatable isoform that is necessary for maintaining the integrity of sympathetic neuron axons. Thus, our study provides a mechanism of mRNA metabolism that simultaneously regulates local protein synthesis and generates an additional class of 3' UTR-derived RNAs.
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http://dx.doi.org/10.1016/j.celrep.2021.108778DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7918530PMC
February 2021

Daidzein Pro-cognitive Effects Coincided with Changes of Brain Neurotensin1 Receptor and Interleukin-10 Expression Levels in Obese Hamsters.

Neurotox Res 2021 Jun 11;39(3):645-657. Epub 2021 Jan 11.

Comparative Neuroanatomy Laboratory of Biology, Ecology and Earth Science Department (DiBEST), University of Calabria, Arcavacata Di Rende, Ponte P. Bucci 4B, 87036, Cosenza, Italy.

At present, concerns are pointing to "tasteful" high-fat diets as a cause of conditioning physical-social states that through alterations of some key emotional- and nutritional-related limbic circuits such as hypothalamic and amygdalar areas lead to obesity states. Feeding and energetic homeostatic molecular mechanisms are part of a complex neuronal circuit accounting for this metabolic disorder. In an attempt to exclude conventional drugs for treating obesity, daidzein, a natural glycosidic isoflavone, which mimics estrogenic neuroprotective properties against increased body weight, is beginning to be preferred. In this study, evident anxiolytic-like behaviors were detected following treatment of high-fat diet hamsters with daidzein as shown by extremely evident (p < 0.001) exploration tendencies in novel object recognition test and a notably greater amount of time spent (p < 0.01) in open arms of elevated plus maze. Moreover, the isoflavone promoted a protective role against neurodegeneration processes as shown by few, if any, amino cupric silver granules in amygdalar, hypothalamic and hippocampal neuronal fields when compared with obese hamsters. Interestingly, elevated expression levels of the anorexic neuropeptide receptor neurotensin1 in the above limbic areas of obese hamsters were extremely reduced by daidzein, especially during recovery of cognitive events. Contextually, such effects were strongly paralleled by increased levels of the anti-neuroinflammatory cytokine, interleukin-10. Our results corroborate a neuroprotective ability of this natural glycosidic isoflavone, which through its interaction with the receptor neurotensin1 and interleukin-10 pathways is correlated not only to improved feeding states, and subsequently obesity conditions, but above all to cognitive performances.
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http://dx.doi.org/10.1007/s12640-020-00328-4DOI Listing
June 2021

Generation of iPSC lines from two patients affected by febrile seizure due to inherited missense mutation in SCN1A gene.

Stem Cell Res 2020 12 7;49:102083. Epub 2020 Nov 7.

Department of Experimental and Clinical Medicine, Research Center for Advanced Biochemistry and Molecular Biology, University "Magna Graecia" of Catanzaro, Italy.

Here, we described the generation of human induced pluripotent stem cell lines (hiPSCs) from fibroblasts isolated by punch biopsies of two siblings carrying inherited mutation (c.434 T > C) in the SCN1A gene, encoding for the neuronal voltage gated sodium channel Na1.1. The mutation leads to the substitution of a highly conserved methionine with a threonine (M145T) in the protein sequence, leading to infant febrile seizures (FS). The older brother, affected by complex FS, also developed temporal lobe epilepsy (TLE) during adolescence.
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http://dx.doi.org/10.1016/j.scr.2020.102083DOI Listing
December 2020

Statins Stimulate New Myocyte Formation After Myocardial Infarction by Activating Growth and Differentiation of the Endogenous Cardiac Stem Cells.

Int J Mol Sci 2020 Oct 26;21(21). Epub 2020 Oct 26.

Department of Experimental and Clinical Medicine, Magna Graecia University, 88100 Catanzaro, Italy.

The 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors (statins) exert pleiotropic effects on cardiac cell biology which are not yet fully understood. Here we tested whether statin treatment affects resident endogenous cardiac stem/progenitor cell (CSC) activation in vitro and in vivo after myocardial infarction (MI). Statins (Rosuvastatin, Simvastatin and Pravastatin) significantly increased CSC expansion in vitro as measured by both BrdU incorporation and cell growth curve. Additionally, statins increased CSC clonal expansion and cardiosphere formation. The effects of statins on CSC growth and differentiation depended on Akt phosphorylation. Twenty-eight days after myocardial infarction by permanent coronary ligation in rats, the number of endogenous CSCs in the infarct border zone was significantly increased by Rosuvastatin-treatment as compared to untreated controls. Additionally, commitment of the activated CSCs into the myogenic lineage (c-kit/Gata4 CSCs) was increased by Rosuvastatin administration. Accordingly, Rosuvastatin fostered new cardiomyocyte formation after MI. Finally, Rosuvastatin treatment reversed the cardiomyogenic defects of CSCs in c-kit haploinsufficient mice, increasing new cardiomyocyte formation by endogenous CSCs in these mice after myocardial infarction. In summary, statins, by sustaining Akt activation, foster CSC growth and differentiation in vitro and in vivo. The activation and differentiation of the endogenous CSC pool and consequent new myocyte formation by statins improve myocardial remodeling after coronary occlusion in rodents. Similar effects might contribute to the beneficial effects of statins on human cardiovascular diseases.
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http://dx.doi.org/10.3390/ijms21217927DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7663580PMC
October 2020

DJ-1 Proteoforms in Breast Cancer Cells: The Escape of Metabolic Epigenetic Misregulation.

Cells 2020 08 26;9(9). Epub 2020 Aug 26.

Laboratory of Proteomics, Research Center on Advanced Biochemistry and Molecular Biology, Department of Experimental and Clinical Medicine, Magna Græcia Universityof Catanzaro, S Venuta University Campus, 88100 Catanzaro, Italy.

Enhanced glycolysis is a hallmark of breast cancer. In cancer cells, the high glycolytic flux induces carbonyl stress, a damaging condition in which the increase of reactive carbonyl species makes DNA, proteins, and lipids more susceptible to glycation. Together with glucose, methylglyoxal (MGO), a byproduct of glycolysis, is considered the main glycating agent. MGO is highly diffusible, enters the nucleus, and can react with easily accessible lysine- and arginine-rich tails of histones. Glycation adducts on histones undergo oxidization and further rearrange to form stable species known as advanced glycation end-products (AGEs). This modification alters nucleosomes stability and chromatin architecture deconstructing the histone code. Formation of AGEs has been associated with cancer, diabetes, and several age-related diseases. Recently, DJ-1, a cancer-associated protein that protects cells from oxidative stress, has been described as a deglycase enzyme. Although its role in cell survival results still controversial, in several human tumors, its expression, localization, oxidation, and phosphorylation were found altered. This work aimed to explore the molecular mechanism that triggers the peculiar cellular compartmentalization and the specific post-translational modifications (PTM) that, occurring in breast cancer cells, influences the DJ-1 dual role. Using a proteomic approach, we identified on DJ-1 a novel threonine phosphorylation (T125) that was found, by the in-silico tool scansite 4, as part of a putative Akt consensus. Notably, this threonine is in addition to histidine 126, a key residue involved in the formation of catalytic triade (glu18-Cys106-His126) inside the glioxalase active site of DJ. Interestingly, we found that pharmacological modulation of Akt pathway induces a functional tuning of DJ-1 proteoforms, as well as their shuttle from cytosol to nucleus, pointing out that pathway as critical in the development of DJ-1 pro-tumorigenic abilities. Deglycase activity of DJ-1 on histones proteins, investigated by coupling 2D tau gel with LC-MS/MS and 2D-TAU (Triton-Acid-Urea)-Western blot, was found correlated with its phosphorylation status that, in turn, depends from Akt activation. In normal conditions, DJ-1 acts as a redox-sensitive chaperone and as an oxidative stress sensor. In cancer cells, glycolytic rewiring, inducing increased reactive oxygen species (ROS) levels, enhances AGEs products. Alongside, the moderate increase of ROS enhances Akt signaling that induces DJ-1-phosphorylation. When phosphorylated DJ-1 increases its glyoxalase activity, the level of AGEs on histones decreases. Therefore, phospho-DJ-1 prevents glycation-induced histones misregulation and its Akt-related hyperactivity represents a way to preserve the epigenome landscape sustaining proliferation of cancer cells. Together, these results shed light on an interesting mechanism that cancer cells might execute to escape the metabolic induced epigenetic misregulation that otherwise could impair their malignant proliferative potential.
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http://dx.doi.org/10.3390/cells9091968DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7563694PMC
August 2020

Modeling Cardiac Disease Mechanisms Using Induced Pluripotent Stem Cell-Derived Cardiomyocytes: Progress, Promises and Challenges.

Int J Mol Sci 2020 Jun 19;21(12). Epub 2020 Jun 19.

Department of Experimental and Clinical Medicine, Research Center for Advanced Biochemistry and Molecular Biology, University "Magna Graecia" of Catanzaro, 88100 Loc. Germaneto, Catanzaro, Italy.

Cardiovascular diseases (CVDs) are a class of disorders affecting the heart or blood vessels. Despite progress in clinical research and therapy, CVDs still represent the leading cause of mortality and morbidity worldwide. The hallmarks of cardiac diseases include heart dysfunction and cardiomyocyte death, inflammation, fibrosis, scar tissue, hyperplasia, hypertrophy, and abnormal ventricular remodeling. The loss of cardiomyocytes is an irreversible process that leads to fibrosis and scar formation, which, in turn, induce heart failure with progressive and dramatic consequences. Both genetic and environmental factors pathologically contribute to the development of CVDs, but the precise causes that trigger cardiac diseases and their progression are still largely unknown. The lack of reliable human model systems for such diseases has hampered the unraveling of the underlying molecular mechanisms and cellular processes involved in heart diseases at their initial stage and during their progression. Over the past decade, significant scientific advances in the field of stem cell biology have literally revolutionized the study of human disease in vitro. Remarkably, the possibility to generate disease-relevant cell types from induced pluripotent stem cells (iPSCs) has developed into an unprecedented and powerful opportunity to achieve the long-standing ambition to investigate human diseases at a cellular level, uncovering their molecular mechanisms, and finally to translate bench discoveries into potential new therapeutic strategies. This review provides an update on previous and current research in the field of iPSC-driven cardiovascular disease modeling, with the aim of underlining the potential of stem-cell biology-based approaches in the elucidation of the pathophysiology of these life-threatening diseases.
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http://dx.doi.org/10.3390/ijms21124354DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7352327PMC
June 2020

A Disposable Passive Microfluidic Device for Cell Culturing.

Biosensors (Basel) 2020 Feb 29;10(3). Epub 2020 Feb 29.

Department of Experimental and Clinical Medicine, Univ. of Catanzaro, Germaneto, 88100 Catanzaro, Italy.

In this work, a disposable passive microfluidic device for cell culturing that does not require any additional/external pressure sources is introduced. By regulating the height of fluidic columns and the aperture and closure of the source wells, the device can provide different media and/or drug flows, thereby allowing different flow patterns with respect to time. The device is made of two Polymethylmethacrylate (PMMA) layers fabricated by micro-milling and solvent assisted bonding and allows us to ensure a flow rate of 18.6 μl/ℎ - 7%/day, due to a decrease of the fluid height while the liquid is driven from the reservoirs into the channels. Simulations and experiments were conducted to characterize flows and diffusion in the culture chamber. Melanoma tumor cells were used to test the device and carry out cell culturing experiments for 48 hours. Moreover, HeLa, Jurkat, A549 and HEK293T cell lines were cultivated successfully inside the microfluidic device for 72 hours.
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http://dx.doi.org/10.3390/bios10030018DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7146476PMC
February 2020

miR-128a Acts as a Regulator in Cardiac Development by Modulating Differentiation of Cardiac Progenitor Cell Populations.

Int J Mol Sci 2020 Feb 10;21(3). Epub 2020 Feb 10.

Department of Cardiovascular Surgery, German Heart Center Munich at the Technische Universität München, 80636 Munich, Germany.

MicroRNAs (miRs) appear to be major, yet poorly understood players in regulatory networks guiding cardiogenesis. We sought to identify miRs with unknown functions during cardiogenesis analyzing the miR-profile of multipotent enhancer cardiac progenitor cells (NkxCE-CPCs). Besides well-known candidates such as miR-1, we found about 40 miRs that were highly enriched in NkxCE-CPCs, four of which were chosen for further analysis. Knockdown in zebrafish revealed that only miR-128a affected cardiac development and function robustly. For a detailed analysis, loss-of-function and gain-of-function experiments were performed during in vitro differentiations of transgenic murine pluripotent stem cells. MiR-128a knockdown (1) increased , , and (cardiac transcription factors) but reduced at the onset of cardiogenesis, (2) upregulated -positive CPCs, whereas NkxCE-positive CPCs were downregulated, and (3) increased the expression of the ventricular cardiomyocyte marker accompanied by a reduced beating frequency of early cardiomyocytes. Overexpression of miR-128a (4) diminished the expression of , , , and , but increased , (5) enhanced NkxCE-positive CPCs, and (6) favored nodal-like cardiomyocytes (, , ) accompanied by increased beating frequencies. In summary, we demonstrated that miR-128a plays a so-far unknown role in early heart development by affecting the timing of CPC differentiation into various cardiomyocyte subtypes.
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http://dx.doi.org/10.3390/ijms21031158DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7038042PMC
February 2020

Histone proteomics reveals novel post-translational modifications in breast cancer.

Aging (Albany NY) 2019 12 8;11(23):11722-11755. Epub 2019 Dec 8.

Laboratory of Proteomics, Research Center on Advanced Biochemistry and Molecular Biology, Department of Experimental and Clinical Medicine, Magna Græcia University of Catanzaro, Salvatore Venuta University Campus, Catanzaro, Italy.

Histones and their variants are subjected to several post-translational modifications (PTMs). Histones PTMs play an important role in the regulation of gene expression and are critical for the development and progression of many types of cancer, including breast cancer. In this study, we used two-dimensional TAU/SDS electrophoresis, coupled with mass spectrometry for a comprehensive profiling of histone PTMs in breast cancer cell lines.Proteomic approach allowed us to identify 85 histone PTMs, seventeen of which are not reported in the UniProt database. Western blot analysis was performed to confirm a peculiar pattern of PTMs in the sporadic and hereditary breast cancer cell lines compared to normal cells. Overlapping mass spectrometry data with western blotting results, we identified, for the first time to our knowledge, a tyrosine phosphorylation on histone H1, which is significantly higher in breast cancer cells. Additionally, by inhibiting specific signaling paths, such as PI3K, PPARγ and FAK pathways, we established a correlation between their regulation and the presence of new histone PTMs. Our results may provide new insight on the possible implication of these modifications in breast cancer and may offer new perspectives for future clinical applications.
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http://dx.doi.org/10.18632/aging.102577DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6932915PMC
December 2019

Stem Cells: The Game Changers of Human Cardiac Disease Modelling and Regenerative Medicine.

Int J Mol Sci 2019 Nov 16;20(22). Epub 2019 Nov 16.

Department of Experimental and Clinical Medicine, Research Center for Advanced Biochemistry and Molecular Biology, University "Magna Graecia" of Catanzaro, 88100 Loc., Germaneto, Catanzaro, Italy.

A comprehensive understanding of the molecular basis and mechanisms underlying cardiac diseases is mandatory for the development of new and effective therapeutic strategies. The lack of appropriate cell models that faithfully mirror the human disease phenotypes has hampered the understanding of molecular insights responsible of heart injury and disease development. Over the past decade, important scientific advances have revolutionized the field of stem cell biology through the remarkable discovery of reprogramming somatic cells into induced pluripotent stem cells (iPSCs). These advances allowed to achieve the long-standing ambition of modelling human disease in a dish and, more interestingly, paved the way for unprecedented opportunities to translate bench discoveries into new therapies and to come closer to a real and effective stem cell-based medicine. The possibility to generate patient-specific iPSCs, together with the new advances in stem cell differentiation procedures and the availability of novel gene editing approaches and tissue engineering, has proven to be a powerful combination for the generation of phenotypically complex, pluripotent stem cell-based cellular disease models with potential use for early diagnosis, drug screening, and personalized therapy. This review will focus on recent progress and future outcome of iPSCs technology toward a customized medicine and new therapeutic options.
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http://dx.doi.org/10.3390/ijms20225760DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6888119PMC
November 2019

Similar miRNomic signatures characterize the follicular fluids collected after follicular and luteal phase stimulations in the same ovarian cycle.

J Assist Reprod Genet 2020 Jan 7;37(1):149-158. Epub 2019 Nov 7.

Università degli Studi Magna Graecia di Catanzaro, Catanzaro, Italy.

Purpose: To detect putative differences in the miRNomic profile of follicular fluids collected after follicular-phase-stimulation (FPS-FFs) and paired luteal-phase-stimulation (LPS-FFs) in the same ovarian cycles (DuoStim).

Methods: Exploratory study at a private IVF center and University involving FPS-FFs and paired-LPS-FFs collected from 15 reduced ovarian reserve and advanced maternal age women undergoing DuoStim (n = 30 paired samples). The samples were combined in 6 paired pools (5 samples each) and balanced according to maternal age and number of cumulus-oocyte-complexes. Micro-RNAs were isolated and sequenced. Four miRNAs were then selected for further validation on 6 single pairs of FPS-FFs and LPS-FFs by qPCR.

Results: Forty-three miRNAs were detected in both FPS-FFs and paired-LPS-FFs after sequencing and no statistically significant differences were reported. Thirty-three KEGG pathways were identified as regulated from the detected miRNAs. Four miRNAs (miR-146b, miR-191, miR-320a, and miR-483) were selected for qPCR validation since consistently expressed in our samples and possibly involved in the regulation/establishment of a healthy follicular environment. Again, no significant differences were reported between FPS-FFs and paired-LPS-FFs, also when the analysis was corrected for maternal age and number of cumulus-oocyte-complexes in generalized linear models.

Conclusions: These data complement the embryological, chromosomal, and clinical evidence of equivalence between FPS and LPS published to date.
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http://dx.doi.org/10.1007/s10815-019-01607-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7000610PMC
January 2020

Establishment and characterization of induced pluripotent stem cells (iPSCs) from central nervous system lupus erythematosus.

J Cell Mol Med 2019 11 19;23(11):7382-7394. Epub 2019 Sep 19.

Department of Experimental and Clinical Medicine, Stem Cell Laboratory, Research Center for Advanced Biochemistry and Molecular Biology, "Magna Graecia" University, Catanzaro, Italy.

Involvement of the central nervous system (CNS) is an uncommon feature in systemic lupus erythematosus (SLE), making diagnosis rather difficult and challenging due to the poor specificity of neuropathic symptoms and neurological symptoms. In this work, we used human-induced pluripotent stem cells (hiPSCs) derived from CNS-SLE patient, with the aim to dissect the molecular insights underlying the disease by gene expression analysis and modulation of implicated pathways. CNS-SLE-derived hiPSCs allowed us to provide evidence of Erk and Akt pathways involvement and to identify a novel cohort of potential biomarkers, namely CHCHD2, IDO1, S100A10, EPHA4 and LEFTY1, never reported so far. We further extended the study analysing a panel of oxidative stress-related miRNAs and demonstrated, under normal or stress conditions, a strong dysregulation of several miRNAs in CNS-SLE-derived compared to control hiPSCs. In conclusion, we provide evidence that iPSCs reprogrammed from CNS-SLE patient are a powerful useful tool to investigate the molecular mechanisms underlying the disease and to eventually develop innovative therapeutic approaches.
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http://dx.doi.org/10.1111/jcmm.14598DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6815917PMC
November 2019

A Passive Microfluidic Device for Chemotaxis Studies.

Micromachines (Basel) 2019 Aug 20;10(8). Epub 2019 Aug 20.

Department of Experimental and Clinical Medicine, University "Magna Graecia" of Catanzaro, 88100 Catanzaro, Italy.

This work presents a disposable passive microfluidic system, allowing chemotaxis studies, through the generation of a concentration gradient. The device can handle liquid flows without an external supply of pressure or electric gradients, but simply using gravity force. It is able to ensure flow rates of 10 µL/h decreasing linearly with 2.5% in 24 h. The device is made of poly(methylmethacrylate) (PMMA), a biocompatible material, and it is fabricated by micro-milling and solvent assisted bonding. It is assembled into a mini incubator, designed properly for cell biology studies in passive microfluidic devices, which provides control of temperature and humidity levels, a contamination-free environment for cells with air and 5% of CO. Furthermore, the mini incubator can be mounted on standard inverted optical microscopes. By using our microfluidic device integrated into the mini incubator, we are able to evaluate and follow in real-time the migration of any cell line to a chemotactic agent. The device is validated by showing cell migration at a rate of 0.36 µm/min, comparable with the rates present in scientific literature.
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http://dx.doi.org/10.3390/mi10080551DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6722731PMC
August 2019

Comprehensive proteogenomic analysis of human embryonic and induced pluripotent stem cells.

J Cell Mol Med 2019 08 25;23(8):5440-5453. Epub 2019 Jun 25.

Research Center for Advanced Biochemistry and Molecular Biology, Department of Experimental and Clinical Medicine, University "Magna Graecia" of Catanzaro, Catanzaro, Italy.

Although the concepts of somatic cell reprogramming and human-induced pluripotent stem cells (hiPSCs) generation have undergone several analyses to validate the usefulness of these cells in research and clinic, it remains still controversial whether the hiPSCs are equivalent to human embryonic stem cells (hESCs), pointing to the need of further characterization for a more comprehensive understanding of pluripotency. Most of the experimental evidence comes from the transcriptome analysis, while a little is available on protein data, and even less is known about the post-translational modifications. Here, we report a combined strategy of mass spectrometry and gene expression profiling for proteogenomic analysis of reprogrammed and embryonic stem cells. The data obtained through this integrated, multi-"omics" approach indicate that a small, but still significant, number of distinct pathways is enriched in reprogrammed versus embryonic stem cells, supporting the view that pluripotency is an extremely complex, multifaceted phenomenon, with peculiarities that are characteristic of each cell type.
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http://dx.doi.org/10.1111/jcmm.14426DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6653499PMC
August 2019

In Preclinical Model of Ovarian Cancer, the SGK1 Inhibitor SI113 Counteracts the Development of Paclitaxel Resistance and Restores Drug Sensitivity.

Transl Oncol 2019 Aug 1;12(8):1045-1055. Epub 2019 Jun 1.

Department of "Scienze della Salute", University "Magna Graecia" of Catanzaro, Viale Europa, Catanzaro. Electronic address:

Ovarian cancer is the second most common gynecological malignancy worldwide. Paclitaxel is particularly important in the therapy of ovarian carcinomas, but the treatment efficacy is counteracted by the development of resistance to chemotherapy. The identification of target molecules that can prevent or control the development of chemoresistance might provide important tools for the management of patients affected by ovarian cancer. Serum- and glucocorticoid-regulated kinase 1 (SGK1) appears to be a key determinant of resistance to chemo- and radiotherapy. Specifically, SGK1 affects paclitaxel sensitivity in RKO colon carcinoma cells by modulating the specificity protein 1 (SP1)-dependent expression of Ran-specific GTPase-activating protein (RANBP1), a member of the GTP-binding nuclear protein Ran (RAN) network that is required for the organization and function of the mitotic spindle. SGK1 inhibition might thus be useful for counteracting the development of paclitaxel resistance. Here, we present in vitro data obtained using ovarian carcinoma cell lines that indicate that the SGK1 inhibitor SI113 inhibits cancer cell proliferation, potentiates the effects of paclitaxel-based chemotherapy, counteracts the development of paclitaxel resistance, and restores paclitaxel sensitivity in paclitaxel-resistant A2780 ovarian cancer cells. The results were corroborated by preclinical studies of xenografts generated in nude mice through the implantation of paclitaxel-resistant human ovarian cancer cells. The SGK1 inhibitor SI113 synergizes with paclitaxel in the treatment of xenografted ovarian cancer cells. Taken together, these data suggest that SGK1 inhibition should be investigated in clinical trials for the treatment of paclitaxel-resistant ovarian cancer.
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http://dx.doi.org/10.1016/j.tranon.2019.05.008DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6545392PMC
August 2019

High-throughput detection of low abundance sialylated glycoproteins in human serum by TiO enrichment and targeted LC-MS/MS analysis: application to a prostate cancer sample set.

Anal Bioanal Chem 2019 Jan 28;411(3):755-763. Epub 2018 Nov 28.

Department of Experimental and Clinical Medicine, Magna Graecia University of Catanzaro, Campus "S. Venuta", Viale Europa, Loc. Germaneto, 88100, Catanzaro, Italy.

Glycopeptide enrichment can be a strategy to allow the detection of peptides belonging to low abundance proteins in complex matrixes such as blood serum or plasma. Though several glycopeptide enrichment protocols have shown excellent sensitivities in this respect, few reports have demonstrated the applicability of these methods to relatively large sample cohorts. In this work, a fast protocol based on TiO enrichment and highly sensitive mass spectrometric analysis by Selected Reaction Monitoring (SRM) has been applied to a cohort of serum samples from prostate cancer and benign prostatic hyperplasia patients in order to detect low abundance proteins in a single LC-MS/MS analysis in nanoscale format, without immunodepletion or peptide fractionation. A peptide library of over 700 formerly N-glycosylated peptides was created by data dependent analysis. Then, 16 medium to low abundance proteins were selected for detection by single injection LC-MS/MS based on selected-reaction monitoring. Results demonstrated the consistent detection of the low-level proteins under investigation. Following label-free quantification, four proteins (Adipocyte plasma membrane-associated protein, Periostin, Cathepsin D and Lysosome-associated membrane glycoprotein 2) were found significantly increased in prostate cancer sera compared to the control group. Graphical abstract ᅟ.
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http://dx.doi.org/10.1007/s00216-018-1497-5DOI Listing
January 2019

On-Tissue Hydrogel-Mediated Nondestructive Proteomic Characterization: Application to fr/fr and FFPE Tissues and Insights for Quantitative Proteomics Using a Case of Cardiac Myxoma.

Proteomics Clin Appl 2019 01 12;13(1):e1700167. Epub 2018 Nov 12.

Research Center for Advanced Biochemistry and Molecular Biology, Department of Experimental and Clinical Medicine, Magna Graecia University of Catanzaro, Campus "S. Venuta,", Viale Europa, Loc. Germaneto, 88100 Catanzaro, Italy.

Purpose: The application of a methodology for quantitative protein analysis from formalin-fixed and paraffin-embedded (FFPE) tissue by using hydrogels. Miniaturized polymeric gels are placed onto histologically defined tissue regions in order to perform localized digestion for bottom-up proteomics. Hydrogel-extracted peptides are then labeled with tandem mass tags (TMT) reagents for relative protein quantification. A cardiac myxoma biopsy is used.

Experimental Design: Multiple hydrogels, incorporating the proteolytic enzyme trypsin, are placed on serial tissue sections, and processed for digestion and TMT derivatization. SCX fractionation before LC-MS/MS analysis and bioinformatics analysis are carried out.

Results: Two histologically different areas on both FFPE and frozen sections of the same cardiac myxoma biopsy are compared. In total, 1949 (FFPE) and 2491 (frozen) proteins are identified, with a total overlap of 56%. The quantitative comparison highlighted 15 (FFPE) and 138 (frozen) differentially expressed proteins between myxoma regions.

Conclusion: The methodology successfully detects numerous protein signals from FFPE and frozen specimens and is able to differentiate between tissue regions. A fast and reliable tissue preparation for quantitative protein analysis by minimum sample manipulation is developed. This offers an option for on-tissue proteomics analysis while preserving the inherent spatial information on the tissue.
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http://dx.doi.org/10.1002/prca.201700167DOI Listing
January 2019

Proteomic analysis of S-nitrosylated nuclear proteins in rat cortical neurons.

Sci Signal 2018 07 3;11(537). Epub 2018 Jul 3.

Medical Research Council Laboratory for Molecular Cell Biology, University College London, WC1E 6BT London, UK.

Neurons modulate gene expression in response to extrinsic signals to enable brain development, cognition, and learning and to process stimuli that regulate systemic physiological functions. This signal-to-gene communication is facilitated by posttranslational modifications such as S-nitrosylation, the covalent attachment of a nitric oxide (NO) moiety to cysteine thiols. In the cerebral cortex, S-nitrosylation of histone deacetylase 2 (HDAC2) is required for gene transcription during neuronal development, but few other nuclear targets of S-nitrosylation have been identified to date. We used S-nitrosothiol resin-assisted capture on NO donor-treated nuclear extracts from rat cortical neurons and identified 614 S-nitrosylated nuclear proteins. Of these, 131 proteins have not previously been shown to be S-nitrosylated in any system, and 555 are previously unidentified targets of S-nitrosylation in neurons. The sites of S-nitrosylation were identified for 59% of the targets, and motifs containing single lysines were found at 33% of these sites. In addition, lysine motifs were necessary for promoting the S-nitrosylation of HDAC2 and methyl-CpG binding protein 3 (MBD3). Moreover, S-nitrosylation of the histone-binding protein RBBP7 was necessary for dendritogenesis of cortical neurons in culture. Together, our findings characterize S-nitrosylated nuclear proteins in neurons and identify S-nitrosylation motifs that may be shared with other targets of NO signaling.
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http://dx.doi.org/10.1126/scisignal.aar3396DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6726469PMC
July 2018

Integration of "Omics" Strategies for Biomarkers Discovery and for the Elucidation of Molecular Mechanisms Underlying Brugada Syndrome.

Proteomics Clin Appl 2018 11 20;12(6):e1800065. Epub 2018 Jul 20.

Laboratory of Proteomics, Research Center of Advanced Biochemistry and Molecular Biology, Department of Experimental and Clinical Medicine, Magna Graecia University of Catanzaro, 88100, Catanzaro, Italy.

Purpose: The Brugada syndrome (BrS) is a severe inherited cardiac disorder. Given the high genetic and phenotypic heterogeneity of this disease, three different "omics" approaches are integrated in a synergic way to elucidate the molecular mechanisms underlying the pathophysiology of BrS as well as for identifying reliable diagnostic/prognostic markers.

Experimental Design: The profiling of plasma Proteome and MiRNome is perfomed in a cohort of Brugada patients that were preliminary subjected to genomic analysis to assess a peculiar gene mutation profile.

Results: The integrated analysis of "omics" data unveiled a cooperative activity of mutated genes, deregulated miRNAs and proteins in orchestrating transcriptional and post-translational events that are critical determining factors for the development of the Brugada pattern.

Conclusions And Clinical Relevance: This study provides the basis to shed light on the specific molecular fingerprints underlying BrS development and to gain further insights on the pathogenesis of this life-threatening cardiac disease.
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http://dx.doi.org/10.1002/prca.201800065DOI Listing
November 2018

Interplay of cell-cell contacts and RhoA/MRTF-A signaling regulates cardiomyocyte identity.

EMBO J 2018 06 15;37(12). Epub 2018 May 15.

Klinik und Poliklinik Innere Medizin I, Klinikum rechts der Isar - Technical University of Munich, Munich, Germany

Cell-cell and cell-matrix interactions guide organ development and homeostasis by controlling lineage specification and maintenance, but the underlying molecular principles are largely unknown. Here, we show that in human developing cardiomyocytes cell-cell contacts at the intercalated disk connect to remodeling of the actin cytoskeleton by regulating the RhoA-ROCK signaling to maintain an active MRTF/SRF transcriptional program essential for cardiomyocyte identity. Genetic perturbation of this mechanosensory pathway activates an ectopic fat gene program during cardiomyocyte differentiation, which ultimately primes the cells to switch to the brown/beige adipocyte lineage in response to adipogenesis-inducing signals. We also demonstrate by fate mapping and clonal analysis of cardiac progenitors that cardiac fat and a subset of cardiac muscle arise from a common precursor expressing Isl1 and Wt1 during heart development, suggesting related mechanisms of determination between the two lineages.
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http://dx.doi.org/10.15252/embj.201798133DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6003642PMC
June 2018

Proteomics Analysis to Assess the Role of Mitochondria in BRCA1-Mediated Breast Tumorigenesis.

Proteomes 2018 Mar 27;6(2). Epub 2018 Mar 27.

Laboratory of Proteomics, Research Center of Advanced Biochemistry and Molecular Biology, Department of Experimental and Clinical Medicine, Magna Græcia University of Catanzaro, Catanzaro 88100, Italy.

Mitochondria are the organelles deputed to energy production, but they are also involved in carcinogenesis, cancer progression, and metastasis, playing a role in altered energy metabolism in cancer cells. Mitochondrial metabolism is connected with several mitochondrial pathways such as ROS signaling, Ca homeostasis, mitophagy, and mitochondrial biogenesis. These pathways are merged in an interactive super-network that seems to play a crucial role in cancer. Germline mutations of the gene account for 5-10% of breast cancers and confer a risk of developing the disease 10- to 20-fold much higher than in non-carriers. By considering metabolic networks that could reconcile both genetic and non-genetic causal mechanisms in driven tumorigenesis, we herein based our study on the hypothesis that haploinsufficiency might drive metabolic rewiring in breast epithelial cells, acting as a push toward malignant transformation. Using 2D-DIGE we analyzed and compared the mitochondrial proteomic profile of sporadic breast cancer cell line (MCF7) and mutated breast cancer cell line (HCC1937). Image analysis was carried out with Decider Software, and proteins differentially expressed were identified by LC-MS/MS on a quadrupole-orbitrap mass spectrometer Q-Exactive. Ingenuity pathways analysis software was used to analyze the fifty-three mitochondrial proteins whose expression resulted significantly altered in response to mutation status. Mitochondrial Dysfunction and oxidative phosphorylation, and energy production and nucleic acid metabolism were, respectively, the canonical pathway and the molecular function mainly affected. Western blotting analysis was done to validate the expression and the peculiar mitochondrial compartmentalization of specific proteins such us HSP60 and HIF-1α. Particularly intriguing is the correlation between mutation status and HIF-1α localization into the mitochondria in a dependent manner. Data obtained led us to hypothesize an interesting connection between and mitochondria pathways, capable to trigger metabolic changes, which, in turn, sustain the high energetic and anabolic requirements of the malignant phenotype.
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http://dx.doi.org/10.3390/proteomes6020016DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6027205PMC
March 2018

Reactivation of the Nkx2.5 cardiac enhancer after myocardial infarction does not presage myogenesis.

Cardiovasc Res 2018 07;114(8):1098-1114

Department of Cardiovascular Surgery, German Heart Center Munich at the Technische Universität München, Lazarettstraße 36, 80636 Munich, Germany.

Aims: The contribution of resident stem or progenitor cells to cardiomyocyte renewal after injury in adult mammalian hearts remains a matter of considerable debate. We evaluated a cell population in the adult mouse heart induced by myocardial infarction (MI) and characterized by an activated Nkx2.5 enhancer element that is specific for multipotent cardiac progenitor cells (CPCs) during embryonic development. We hypothesized that these MI-induced cells (MICs) harbour cardiomyogenic properties similar to their embryonic counterparts.

Methods And Results: MICs reside in the heart and mainly localize to the infarction area and border zone. Interestingly, gene expression profiling of purified MICs 1 week after infarction revealed increased expression of stem cell markers and embryonic cardiac transcription factors (TFs) in these cells as compared to the non-mycoyte cell fraction of adult hearts. A subsequent global transcriptome comparison with embryonic CPCs and fibroblasts and in vitro culture of MICs unveiled that (myo-)fibroblastic features predominated and that cardiac TFs were only expressed at background levels.

Conclusions: Adult injury-induced reactivation of a cardiac-specific Nkx2.5 enhancer element known to specifically mark myocardial progenitor cells during embryonic development does not reflect hypothesized embryonic cardiomyogenic properties. Our data suggest a decreasing plasticity of cardiac progenitor (-like) cell populations with increasing age. A re-expression of embryonic, stem or progenitor cell features in the adult heart must be interpreted very carefully with respect to the definition of cardiac resident progenitor cells. Albeit, the abundance of scar formation after cardiac injury suggests a potential to target predestinated activated profibrotic cells to push them towards cardiomyogenic differentiation to improve regeneration.
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http://dx.doi.org/10.1093/cvr/cvy069DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6279078PMC
July 2018

Secretome Analysis of Hypoxia-Induced 3T3-L1 Adipocytes Uncovers Novel Proteins Potentially Involved in Obesity.

Proteomics 2018 04;18(7):e1700260

Department of Health Sciences, University "Magna Graecia" of Catanzaro, Catanzaro, Italy.

In the obese state, as adipose tissue expands, adipocytes become hypoxic and dysfunctional, leading to changes in the pattern of adipocyte-secreted proteins. To better understand the role of hypoxia in the mechanisms linked to obesity, we comparatively analyzed the secretome of murine differentiated 3T3-L1 adipocytes exposed to normoxia or hypoxia for 24 h. Proteins secreted into the culture media were precipitated by trichloroacetic acid and then digested with trypsin. The peptides were labeled with dimethyl labeling and analyzed by reversed phase nanoscale liquid chromatography coupled to a quadrupole Orbitrap mass spectrometer. From a total of 1508 identified proteins, 109 were differentially regulated, of which 108 were genuinely secreted. Factors significantly downregulated in hypoxic conditions included adiponectin, a known adipokine implicated in metabolic processes, as well as thrombospondin-1 and -2, and matrix metalloproteinase-11, all multifunctional proteins involved in extracellular matrix (ECM) homeostasis. Findings were validated by Western blot analysis. Expression studies of the relative genes were performed in parallel experiments in vitro, in differentiated 3T3-L1 adipocytes, and in vivo, in fat tissues from obese versus lean mice. Our observations are compatible with the concept that hypoxia may be an early trigger for both adipose cell dysfunction and ECM remodeling.
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http://dx.doi.org/10.1002/pmic.201700260DOI Listing
April 2018

HMGA1 and MMP-11 Are Overexpressed in Human Non-melanoma Skin Cancer.

Anticancer Res 2018 02;38(2):771-778

Department of Health Sciences, University "Magna Græcia" of Catanzaro, Catanzaro, Italy

Background/aim: The High-Mobility Group A1 (HMGA1) protein has been implicated in human malignancies, playing an important role in cancer proliferation, angiogenesis and metastasis. Increased HMGA1 expression has been found in skin mouse tumors, whereas Hmga1-null mice were protected against skin carcinogenesis. Here, we examined the expression of HMGA1 in human skin tumors, squamous cell carcinoma and basal cell carcinoma.

Materials And Methods: Tumor and normal skin tissues from 15 affected patients were surgically excised, and mRNA and protein extraction was performed. mRNA and protein content for both HMGA1 and MMP-11, a proteinase enzyme that plays a role in tumor development and progression, was measured by real-time PCR and western blotting, respectively. Data were analyzed by the SPSS software.

Results: HMGA1 mRNA and protein expression patterns were higher in neoplastic skin lesions, compared to normal skin (p<0.001). Similar results were observed for MMP-11.

Conclusion: Our data confirm previous observations in mice studies, and suggest that HMGA1 and MMP-11 may play a key role in the proliferation and progression of skin tumors in humans.
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http://dx.doi.org/10.21873/anticanres.12283DOI Listing
February 2018

Short-term retinoic acid treatment sustains pluripotency and suppresses differentiation of human induced pluripotent stem cells.

Cell Death Dis 2018 01 5;9(1). Epub 2018 Jan 5.

Department of Experimental and Clinical Medicine, Stem Cell Laboratory, Research Center of Advanced Biochemistry and Molecular Biology, University "Magna Graecia" of Catanzaro, Viale Europa, 88100, Catanzaro, Italy.

Human pluripotent stem cells (hPSCs), including human embryonic stem cells (hESCs) derived from blastocyst and human induced pluripotent stem cells (hiPSCs) generated from somatic cells by ectopic expression of defined transcriptional factors, have both the ability to self-renew and to differentiate into all cell types. Here we explored the two antagonistic effects of retinoic acid (RA) on hiPSCs. Although RA has been widely described as a pharmacological agent with a critical role in initiating differentiation of pluripotent stem cells, we demonstrate that short-term RA exposure not only antagonizes cell differentiation and sustains pluripotency of hiPSCs, but it also boosts and improves their properties and characteristics. To shed light on the mechanistic insights involved in the resistance to differentiation of hiPSCs cultured in RA conditions, as well as their improved pluripotency state, we focused our attention on the Wnt pathway. Our findings show that RA inhibits the Wnt canonical pathway and positively modulates the Akt/mTOR signaling, explaining why such perturbations, under our experimental conditions, do not lead to hiPSCs differentiation. Altogether, these data uncover a novel role for RA in favouring the maintenance of ground-state pluripotency, supporting its bivalent role, dose- and time-dependent, for hiPSCs differentiation and self-renewal processes.
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http://dx.doi.org/10.1038/s41419-017-0028-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5849042PMC
January 2018