Publications by authors named "Mauro Costa"

41 Publications

Working With Infertile Couples Seeking Assisted Reproduction: An Interpretative Phenomenological Study With Infertility Care Providers.

Front Psychol 2020 17;11:586873. Epub 2020 Dec 17.

Unit of Clinical Psychology, San Paolo University Hospital, Asst-Santi Paolo e Carlo, Milan, Italy.

Although most studies investigated the impact of infertility and its treatment on the couple, a small body of evidence suggested that infertility care providers may experience different sources of stress related for instance to excessive workload, the complexity of the technique, and relational difficulties with patients. The current study aimed at providing further insight into the understanding of the subjective experience of infertility care providers by highlighting their feelings and emotions, personal meanings, challenges, and opportunities. Following the methodological guidelines of Interpretative Phenomenological Analysis, we conducted individual semi-structured interviews with 23 members of two different fertility units. Interviews were audiotaped and transcribed verbatim. Textual analysis was then conducted to identify emerging dominant themes and subthemes. Three main themes were extracted: (i) , (ii) , (iii) . These themes related to participants experiencing: (i) difficulties in establishing an empathic connection and communicating with couples, such that women were sometimes perceived as "particular patients" and men as poorly involved in the process; (ii) difficulties in dealing with a complex procedure in which errors are not allowed (as reported by embryologists), with a growing number of women aged > 40 seeking assisted reproduction, despite the risks for their health; (iii) being part of a team as a resource, although the huge amount of time spent together can involve conflicts and organizational problems. These findings suggested that patients' overpersistence (rather than just dropout) represents an important source of stress for infertility care providers. At the same time, the concept of particular or difficult patient derives from the combination of multiple factors, including providers' own history and subjective experience. The presence of mental health professionals in fertility units is essential to help providers improve the quality of doctor-patient communication and relieve the stress related to organizational issues and conflicts.
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http://dx.doi.org/10.3389/fpsyg.2020.586873DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7773748PMC
December 2020

Dynamic Interstitial Cell Response during Myocardial Infarction Predicts Resilience to Rupture in Genetically Diverse Mice.

Cell Rep 2020 03;30(9):3149-3163.e6

The Jackson Laboratory, Bar Harbor, ME 04609, USA. Electronic address:

Cardiac ischemia leads to the loss of myocardial tissue and the activation of a repair process that culminates in the formation of a scar whose structural characteristics dictate propensity to favorable healing or detrimental cardiac wall rupture. To elucidate the cellular processes underlying scar formation, here we perform unbiased single-cell mRNA sequencing of interstitial cells isolated from infarcted mouse hearts carrying a genetic tracer that labels epicardial-derived cells. Sixteen interstitial cell clusters are revealed, five of which were of epicardial origin. Focusing on stromal cells, we define 11 sub-clusters, including diverse cell states of epicardial- and endocardial-derived fibroblasts. Comparing transcript profiles from post-infarction hearts in C57BL/6J and 129S1/SvImJ inbred mice, which displays a marked divergence in the frequency of cardiac rupture, uncovers an early increase in activated myofibroblasts, enhanced collagen deposition, and persistent acute phase response in 129S1/SvImJ mouse hearts, defining a crucial time window of pathological remodeling that predicts disease outcome.
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http://dx.doi.org/10.1016/j.celrep.2020.02.008DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7059115PMC
March 2020

Spinal metallosis as a complication of a lodged bullet from a firearm wound: an image-centered case.

Rev Assoc Med Bras (1992) 2018 Aug;64(8):676-679

Department of Radiology and Imaging, DOCUMENTA, São Francisco Hospital, Ribeirão Preto, SP, Brasil.

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http://dx.doi.org/10.1590/1806-9282.64.08.676DOI Listing
August 2018

Metformin intervention prevents cardiac dysfunction in a murine model of adult congenital heart disease.

Mol Metab 2019 02 15;20:102-114. Epub 2018 Nov 15.

The Jackson Laboratory, USA; Australian Regenerative Medicine Institute, Monash University, Australia. Electronic address:

Objective: Congenital heart disease (CHD) is the most frequent birth defect worldwide. The number of adult patients with CHD, now referred to as ACHD, is increasing with improved surgical and treatment interventions. However the mechanisms whereby ACHD predisposes patients to heart dysfunction are still unclear. ACHD is strongly associated with metabolic syndrome, but how ACHD interacts with poor modern lifestyle choices and other comorbidities, such as hypertension, obesity, and diabetes, is mostly unknown.

Methods: We used a newly characterized mouse genetic model of ACHD to investigate the consequences and the mechanisms associated with combined obesity and ACHD predisposition. Metformin intervention was used to further evaluate potential therapeutic amelioration of cardiac dysfunction in this model.

Results: ACHD mice placed under metabolic stress (high fat diet) displayed decreased left ventricular ejection fraction. Comprehensive physiological, biochemical, and molecular analysis showed that ACHD hearts exhibited early changes in energy metabolism with increased glucose dependence as main cardiac energy source. These changes preceded cardiac dysfunction mediated by exposure to high fat diet and were associated with increased disease severity. Restoration of metabolic balance by metformin administration prevented the development of heart dysfunction in ACHD predisposed mice.

Conclusions: This study reveals that early metabolic impairment reinforces heart dysfunction in ACHD predisposed individuals and diet or pharmacological interventions can be used to modulate heart function and attenuate heart failure. Our study suggests that interactions between genetic and metabolic disturbances ultimately lead to the clinical presentation of heart failure in patients with ACHD. Early manipulation of energy metabolism may be an important avenue for intervention in ACHD patients to prevent or delay onset of heart failure and secondary comorbidities. These interactions raise the prospect for a translational reassessment of ACHD presentation in the clinic.
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http://dx.doi.org/10.1016/j.molmet.2018.11.002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6358551PMC
February 2019

Enhancing the efficacy of glycolytic blockade in cancer cells RAD51 inhibition.

Cancer Biol Ther 2019 5;20(2):169-182. Epub 2018 Sep 5.

a Research Department , The Jackson Laboratory , Bar Harbor , Maine , USA.

Targeting the early steps of the glycolysis pathway in cancers is a well-established therapeutic strategy; however, the doses required to elicit a therapeutic effect on the cancer can be toxic to the patient. Consequently, numerous preclinical and clinical studies have combined glycolytic blockade with other therapies. However, most of these other therapies do not specifically target cancer cells, and thus adversely affect normal tissue. Here we first show that a diverse number of cancer models - spontaneous, patient-derived xenografted tumor samples, and xenografted human cancer cells - can be efficiently targeted by 2-deoxy-D-Glucose (2DG), a well-known glycolytic inhibitor. Next, we tested the cancer-cell specificity of a therapeutic compound using the MEC1 cell line, a chronic lymphocytic leukemia (CLL) cell line that expresses activation induced cytidine deaminase (AID). We show that MEC1 cells, are susceptible to 4,4'-Diisothiocyano-2,2'-stilbenedisulfonic acid (DIDS), a specific RAD51 inhibitor. We then combine 2DG and DIDS, each at a lower dose and demonstrate that this combination is more efficacious than fludarabine, the current standard- of- care treatment for CLL. This suggests that the therapeutic blockade of glycolysis together with the therapeutic inhibition of RAD51-dependent homologous recombination can be a potentially beneficial combination for targeting AID positive cancer cells with minimal adverse effects on normal tissue. Implications: Combination therapy targeting glycolysis and specific RAD51 function shows increased efficacy as compared to standard of care treatments in leukemias.
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http://dx.doi.org/10.1080/15384047.2018.1507666DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6343731PMC
April 2020

Deletion of Nkx2-5 in trabecular myocardium reveals the developmental origins of pathological heterogeneity associated with ventricular non-compaction cardiomyopathy.

PLoS Genet 2018 07 6;14(7):e1007502. Epub 2018 Jul 6.

Aix-Marseille Université, CNRS UMR 7288, IBDM, Marseille, France.

Left ventricular non-compaction (LVNC) is a rare cardiomyopathy associated with a hypertrabeculated phenotype and a large spectrum of symptoms. It is still unclear whether LVNC results from a defect of ventricular trabeculae development and the mechanistic basis that underlies the varying severity of this pathology is unknown. To investigate these issues, we inactivated the cardiac transcription factor Nkx2-5 in trabecular myocardium at different stages of trabecular morphogenesis using an inducible Cx40-creERT2 allele. Conditional deletion of Nkx2-5 at embryonic stages, during trabecular formation, provokes a severe hypertrabeculated phenotype associated with subendocardial fibrosis and Purkinje fiber hypoplasia. A milder phenotype was observed after Nkx2-5 deletion at fetal stages, during trabecular compaction. A longitudinal study of cardiac function in adult Nkx2-5 conditional mutant mice demonstrates that excessive trabeculation is associated with complex ventricular conduction defects, progressively leading to strain defects, and, in 50% of mutant mice, to heart failure. Progressive impaired cardiac function correlates with conduction and strain defects independently of the degree of hypertrabeculation. Transcriptomic analysis of molecular pathways reflects myocardial remodeling with a larger number of differentially expressed genes in the severe versus mild phenotype and identifies Six1 as being upregulated in hypertrabeculated hearts. Our results provide insights into the etiology of LVNC and link its pathogenicity with compromised trabecular development including compaction defects and ventricular conduction system hypoplasia.
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http://dx.doi.org/10.1371/journal.pgen.1007502DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6051668PMC
July 2018

NKX2-5 regulates human cardiomyogenesis via a HEY2 dependent transcriptional network.

Nat Commun 2018 04 10;9(1):1373. Epub 2018 Apr 10.

Murdoch Childrens Research Institute, Royal Children's Hospital, Flemington Road, Parkville, VIC, 3052, Australia.

Congenital heart defects can be caused by mutations in genes that guide cardiac lineage formation. Here, we show deletion of NKX2-5, a critical component of the cardiac gene regulatory network, in human embryonic stem cells (hESCs), results in impaired cardiomyogenesis, failure to activate VCAM1 and to downregulate the progenitor marker PDGFRα. Furthermore, NKX2-5 null cardiomyocytes have abnormal physiology, with asynchronous contractions and altered action potentials. Molecular profiling and genetic rescue experiments demonstrate that the bHLH protein HEY2 is a key mediator of NKX2-5 function during human cardiomyogenesis. These findings identify HEY2 as a novel component of the NKX2-5 cardiac transcriptional network, providing tangible evidence that hESC models can decipher the complex pathways that regulate early stage human heart development. These data provide a human context for the evaluation of pathogenic mutations in congenital heart disease.
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http://dx.doi.org/10.1038/s41467-018-03714-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5893543PMC
April 2018

Thrombosed aneurysm of saphenous vein coronary artery bypass grafting.

Rev Assoc Med Bras (1992) 2017 Jun;63(6):488-491

Radiology and Imaging Diagnosis Department, Documenta - Hospital São Francisco, Ribeirão Preto, SP, Brazil.

We describe the case of a male patient, aged 76 years, referred for cardiac investigation due to retrosternal chest pain and dyspnea. He had a history of acute myocardial infarction and angioplasties in the last 30 years, including a saphenous vein coronary artery bypass grafting (SVCABG). Echocardiogram showed hypoechoic oval formation near the right ventricle, suggesting a pericardial cyst. Computed angiotomography revealed a predominantly fusiform and thrombosed aneurysmal dilation of the SVCABG to the right coronary artery. SVCABG aneurysms are very rare and potentially fatal. They usually appear in the late postoperative period, and patients are often asymptomatic. On radiography, it is frequently presented as enlargement of the mediastinum, with echocardiography, computed tomography and magnetic resonance imaging being very useful for diagnosis. Coronary angiography is the gold standard to detect these cases. Our report illustrates a rare situation arising late from a relatively common surgery. Due to its severity, proper recognition in the routine assessment of patients with a similar history is essential.
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http://dx.doi.org/10.1590/1806-9282.63.06.488DOI Listing
June 2017

Breaking bad news in assisted reproductive technology: a proposal for guidelines.

Reprod Health 2017 07 20;14(1):87. Epub 2017 Jul 20.

Department of Health Sciences, Università degli Studi di Milano, San Paolo University Hospital, Via di Rudinì 8, 20142, Milan, Italy.

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http://dx.doi.org/10.1186/s12978-017-0350-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5520370PMC
July 2017

Chronic recurrent multifocal osteomyelitis exhibiting predominance of periosteal reaction.

Rev Assoc Med Bras (1992) 2017 Apr;63(4):303-306

Department of Radiology and Imaging Diagnosis, Documenta, Hospital São Francisco, Ribeirão Preto, SP, Brazil.

Chronic recurrent multifocal osteomyelitis is an idiopathic nonpyogenic autoinflammatory bone disorder involving multiple sites, with clinical progression persisting for more than 6 months and which may have episodes of remission and exacerbation in the long term. It represents up to 2-5% of the cases of osteomyelitis, with an approximate incidence of up to 4/1,000,000 individuals, and average age of disease onset estimated between 8-11 years, predominantly in females. The legs are the most affected, with a predilection for metaphyseal regions along the growth plate. We describe the case of a female patient, aged 2 years and 5 months, with involvement of the left ulna, right jaw and left tibia, showing a predominance of periosteal reaction as main finding.
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http://dx.doi.org/10.1590/1806-9282.63.04.303DOI Listing
April 2017

Point mutations in murine phenocopy human congenital heart disease and induce pathogenic Wnt signaling.

JCI Insight 2017 03 23;2(6):e88271. Epub 2017 Mar 23.

The Jackson Laboratory, Bar Harbor, Maine, USA.

Mutations in the gene are a main cause of congenital heart disease. Several studies have addressed the phenotypic consequences of disrupting the gene locus, although animal models to date failed to recapitulate the full spectrum of the human disease. Here, we describe a new point mutation murine model, akin to its human counterpart disease-generating mutation. Our model fully reproduces the morphological and physiological clinical presentations of the disease and reveals an understudied aspect of -driven pathology, a primary right ventricular dysfunction. We further describe the molecular consequences of disrupting the transcriptional network regulated by in the heart and show that -dependent perturbation of the Wnt signaling pathway promotes heart dysfunction through alteration of cardiomyocyte metabolism. Our data provide mechanistic insights on how regulates heart function and metabolism, a link in the study of congenital heart disease, and confirms that our models are the first murine genetic models to our knowledge to present all spectra of clinically relevant adult congenital heart disease phenotypes generated by mutations in patients.
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http://dx.doi.org/10.1172/jci.insight.88271DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5358496PMC
March 2017

The cardiac fibroblast: Origin, identity and role in homeostasis and disease.

Differentiation 2016 Sep 12;92(3):93-101. Epub 2016 Jul 12.

The Jackson Laboratory, Bar Harbor, ME, USA; Australian Regenerative Medicine Institute, Monash University, Melbourne, Victoria, Australia; National Heart and Lung Institute, Imperial College London, UK.

The mammalian heart is responsible for supplying blood to two separate circulation circuits in a parallel manner. This design provides efficient oxygenation and nutrients to the whole body through the left-sided pump, while the right-sided pump delivers blood to the pulmonary circulation for re-oxygenation. In order to achieve this demanding job, the mammalian heart evolved into a highly specialised organ comprised of working contractile cells or cardiomyocytes, a directional and insulated conduction system, capable of independently generating and conducting electric impulses that synchronises chamber contraction, valves that allow the generation of high pressure and directional blood flow into the circulation, coronary circulation, that supplies oxygenated blood for the heart muscle high metabolically active pumping role and inlet/outlet routes, as the venae cavae and pulmonary veins, aorta and pulmonary trunk. This organization highlights the complexity and compartmentalization of the heart. This review will focus on the cardiac fibroblast, a cell type until recently ignored, but that profoundly influences heart function in its various compartments. We will discuss current advances on definitions, molecular markers and function of cardiac fibroblasts in heart homeostasis and disease.
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http://dx.doi.org/10.1016/j.diff.2016.06.004DOI Listing
September 2016

A novel conditional mouse model for Nkx2-5 reveals transcriptional regulation of cardiac ion channels.

Differentiation 2016 Jan-Mar;91(1-3):29-41. Epub 2016 Feb 17.

Australian Regenerative Medicine Institute, Monash University, Clayton, Vic 3800, Australia; The Jackson Laboratory, ME 04609, United States. Electronic address:

Nkx2-5 is one of the master regulators of cardiac development, homeostasis and disease. This transcription factor has been previously associated with a suite of cardiac congenital malformations and impairment of electrical activity. When disease causative mutations in transcription factors are considered, NKX2-5 gene dysfunction is the most common abnormality found in patients. Here we describe a novel mouse model and subsequent implications of Nkx2-5 loss for aspects of myocardial electrical activity. In this work we have engineered a new Nkx2-5 conditional knockout mouse in which flox sites flank the entire Nkx2-5 locus, and validated this line for the study of heart development, differentiation and disease using a full deletion strategy. While our homozygous knockout mice show typical embryonic malformations previously described for the lack of the Nkx2-5 gene, hearts of heterozygous adult mice show moderate morphological and functional abnormalities that are sufficient to sustain blood supply demands under homeostatic conditions. This study further reveals intriguing aspects of Nkx2-5 function in the control of cardiac electrical activity. Using a combination of mouse genetics, biochemistry, molecular and cell biology, we demonstrate that Nkx2-5 regulates the gene encoding Kcnh2 channel and others, shedding light on potential mechanisms generating electrical abnormalities observed in patients bearing NKX2-5 dysfunction and opening opportunities to the study of novel therapeutic targets for anti-arrhythmogenic therapies.
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http://dx.doi.org/10.1016/j.diff.2015.12.003DOI Listing
December 2016

Cancer and fertility preservation: international recommendations from an expert meeting.

BMC Med 2016 Jan 4;14. Epub 2016 Jan 4.

Physiopathology of Human Reproduction, IRCCS AOU San Martino - IST, Genoa, Italy.

In the last years, thanks to the improvement in the prognosis of cancer patients, a growing attention has been given to the fertility issues. International guidelines on fertility preservation in cancer patients recommend that physicians discuss, as early as possible, with all patients of reproductive age their risk of infertility from the disease and/or treatment and their interest in having children after cancer, and help with informed fertility preservation decisions. As recommended by the American Society of Clinical Oncology and the European Society for Medical Oncology, sperm cryopreservation and embryo/oocyte cryopreservation are standard strategies for fertility preservations in male and female patients, respectively; other strategies (e.g. pharmacological protection of the gonads and gonadal tissue cryopreservation) are considered experimental techniques. However, since then, new data have become available, and several issues in this field are still controversial and should be addressed by both patients and their treating physicians.In April 2015, physicians with expertise in the field of fertility preservation in cancer patients from several European countries were invited in Genova (Italy) to participate in a workshop on the topic of "cancer and fertility preservation". A total of ten controversial issues were discussed at the conference. Experts were asked to present an up-to-date review of the literature published on these topics and the presentation of own unpublished data was encouraged. On the basis of the data presented, as well as the expertise of the invited speakers, a total of ten recommendations were discussed and prepared with the aim to help physicians in counseling their young patients interested in fertility preservation.Although there is a great interest in this field, due to the lack of large prospective cohort studies and randomized trials on these topics, the level of evidence is not higher than 3 for most of the recommendations highlighting the need of further research efforts in many areas of this field. The participation to the ongoing registries and prospective studies is crucial to acquire more robust information in order to provide evidence-based recommendations.
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http://dx.doi.org/10.1186/s12916-015-0545-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4700580PMC
January 2016

CARFMAP: A Curated Pathway Map of Cardiac Fibroblasts.

PLoS One 2015 16;10(12):e0143274. Epub 2015 Dec 16.

Australian Regenerative Medicine Institute, Monash University, Clayton, VIC, 3800, Australia.

The adult mammalian heart contains multiple cell types that work in unison under tightly regulated conditions to maintain homeostasis. Cardiac fibroblasts are a significant and unique population of non-muscle cells in the heart that have recently gained substantial interest in the cardiac biology community. To better understand this renaissance cell, it is essential to systematically survey what has been known in the literature about the cellular and molecular processes involved. We have built CARFMAP (http://visionet.erc.monash.edu.au/CARFMAP), an interactive cardiac fibroblast pathway map derived from the biomedical literature using a software-assisted manual data collection approach. CARFMAP is an information-rich interactive tool that enables cardiac biologists to explore the large body of literature in various creative ways. There is surprisingly little overlap between the cardiac fibroblast pathway map, a foreskin fibroblast pathway map, and a whole mouse organism signalling pathway map from the REACTOME database. Among the use cases of CARFMAP is a common task in our cardiac biology laboratory of identifying new genes that are (1) relevant to cardiac literature, and (2) differentially regulated in high-throughput assays. From the expression profiles of mouse cardiac and tail fibroblasts, we employed CARFMAP to characterise cardiac fibroblast pathways. Using CARFMAP in conjunction with transcriptomic data, we generated a stringent list of six genes that would not have been singled out using bioinformatics analyses alone. Experimental validation showed that five genes (Mmp3, Il6, Edn1, Pdgfc and Fgf10) are differentially regulated in the cardiac fibroblast. CARFMAP is a powerful tool for systems analyses of cardiac fibroblasts, facilitating systems-level cardiovascular research.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0143274PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4684407PMC
June 2016

Microarray profiling to analyse adult cardiac fibroblast identity.

Genom Data 2014 Dec 12;2:345-50. Epub 2014 Oct 12.

Australian Regenerative Medicine Institute, Monash University, VIC 3800, Australia ; Systems Biology Institute (SBI) Australia, Monash University, VIC 3800, Australia.

Heart failure is one of the leading causes of death worldwide [1-4]. Current therapeutic strategies are inefficient and cannot cure this chronic and debilitating condition [5]. Ultimately, heart transplants are required for patient survival, but donor organs are scarce in availability and only prolong the life-span of patients for a limited time. Fibrosis is one of the main pathological features of heart failure [6,7], caused by inappropriate stimulation of fibroblasts and excessive extracellular matrix production. Therefore, an in-depth understanding of the cardiac fibroblast is essential to underpin effective therapeutic treatments for heart failure [5]. Fibroblasts in general have been an underappreciated cell type, regarded as relatively inert and providing only basic functionality; they are usually referred to as the  'biological glue' of all tissues in the body. However, more recent literature suggests that they actively participate in organ homeostasis and disease [7,8]. We have recently uncovered a unique molecular identity for fibroblasts isolated from the heart [9], expressing a set of cardiogenic transcription factors that have been previously associated with cardiomyocyte ontogenesis. This signature suggests that cardiac fibroblasts may be ideal for use in stem cell replacement therapies, as they may retain the memory of where they derive from embryologically. Our data also revealed that about 90% of fibroblasts from both tail and heart origins share a cell surface signature that has previously been described for mesenchymal stem cells (MSCs), raising the possibility that fibroblasts and MSCs may in fact be the same cell type. Thus, our findings carry profound implications for the field of regenerative medicine. Here, we describe detailed methodology and quality controls related to the gene expression profiling of cardiac fibroblasts, deposited at the Gene Expression Omnibus (GEO) under the accession number GSE50531. We also provide the R code to easily reproduce the data quantification and analysis processes.
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http://dx.doi.org/10.1016/j.gdata.2014.10.006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4536021PMC
December 2014

VISIONET: intuitive visualisation of overlapping transcription factor networks, with applications in cardiogenic gene discovery.

BMC Bioinformatics 2015 May 1;16:141. Epub 2015 May 1.

Systems Biology Institute (SBI) Australia, Monash University, Clayton, VIC, 3800, Australia.

Background: Existing de novo software platforms have largely overlooked a valuable resource, the expertise of the intended biologist users. Typical data representations such as long gene lists, or highly dense and overlapping transcription factor networks often hinder biologists from relating these results to their expertise.

Results: VISIONET, a streamlined visualisation tool built from experimental needs, enables biologists to transform large and dense overlapping transcription factor networks into sparse human-readable graphs via numerically filtering. The VISIONET interface allows users without a computing background to interactively explore and filter their data, and empowers them to apply their specialist knowledge on far more complex and substantial data sets than is currently possible. Applying VISIONET to the Tbx20-Gata4 transcription factor network led to the discovery and validation of Aldh1a2, an essential developmental gene associated with various important cardiac disorders, as a healthy adult cardiac fibroblast gene co-regulated by cardiogenic transcription factors Gata4 and Tbx20.

Conclusions: We demonstrate with experimental validations the utility of VISIONET for expertise-driven gene discovery that opens new experimental directions that would not otherwise have been identified.
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http://dx.doi.org/10.1186/s12859-015-0578-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4426166PMC
May 2015

Breast cancer incidence after hormonal treatments for infertility: systematic review and meta-analysis of population-based studies.

Breast Cancer Res Treat 2015 Apr 6;150(2):405-13. Epub 2015 Mar 6.

S.C. Oncologia Medica, E.O. Ospedali Galliera, Via Volta 6, 16128, Genoa, Italy,

The increasing practice of hormonal infertility treatments (HITs) raised concerns about their effects on breast cancer (BC) risk. Available evidence reported conflicting results. The aim of this study was to assess the potential association between HITs and BC risk. The literature was searched through November 2014. Eligible studies included cohort studies reporting BC incidence in women undergone HITs. Data were analyzed with standard meta-analytic techniques. Subgroup analyses were performed by type of intervention (IVF vs. NO IVF), follow-up duration (<10 vs. >10 years), and type of control (population vs. infertile). 20 eligible studies (207.914 women, 2347 BC) were retrieved: no increased risk was detected (SRR = 1.05, 95 % CI 0.96-1.14), with a significant heterogeneity (I (2) = 59 %, p = 0.001) among studies. In the seven studies with the in vitro fertilization (IVF) procedure, no increase in BC risk was observed (SRR = 0.96, 95 % CI 0.80-1.14); in the three NO IVF studies, an increased BC risk was identified (SRR = 1.26, 95 %CI 1.06-1.50). A borderline interaction between type of intervention (IVF vs. NO IVF) and BC risk was observed (p = 0.06). An increased risk with longer follow-up (≥10 vs. <10 years) was detected (SRR = 1.13, 95 % CI 1.02-1.26 vs. SRR = 0.95, 95 % CI 0.85-1.06). Overall, HITs are not associated with an increased BC risk. In particular, no increased risk was observed in women undergoing IVF. Conversely, an increased in BC risk cannot be ruled out with older treatment protocols based on clomiphene. The long-term administration of clomiphene outside the current indications should be discouraged because of a possible increase in BC risk.
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http://dx.doi.org/10.1007/s10549-015-3328-0DOI Listing
April 2015

IVF cycle cost estimation using Activity Based Costing and Monte Carlo simulation.

Health Care Manag Sci 2016 Mar 22;19(1):20-30. Epub 2014 Apr 22.

Ospedale Evangelico Internazionale, Corso Solferino, 1A, Genova, Italy.

The Authors present a new methodological approach in stochastic regime to determine the actual costs of an healthcare process. The paper specifically shows the application of the methodology for the determination of the cost of an Assisted reproductive technology (ART) treatment in Italy. The reason of this research comes from the fact that deterministic regime is inadequate to implement an accurate estimate of the cost of this particular treatment. In fact the durations of the different activities involved are unfixed and described by means of frequency distributions. Hence the need to determine in addition to the mean value of the cost, the interval within which it is intended to vary with a known confidence level. Consequently the cost obtained for each type of cycle investigated (in vitro fertilization and embryo transfer with or without intracytoplasmic sperm injection), shows tolerance intervals around the mean value sufficiently restricted as to make the data obtained statistically robust and therefore usable also as reference for any benchmark with other Countries. It should be noted that under a methodological point of view the approach was rigorous. In fact it was used both the technique of Activity Based Costing for determining the cost of individual activities of the process both the Monte Carlo simulation, with control of experimental error, for the construction of the tolerance intervals on the final result.
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http://dx.doi.org/10.1007/s10729-014-9282-2DOI Listing
March 2016

Cardiogenic genes expressed in cardiac fibroblasts contribute to heart development and repair.

Circ Res 2014 Apr 20;114(9):1422-34. Epub 2014 Mar 20.

From the Australian Regenerative Medicine Institute (M.B.F., M.W.C., E.A.P., E.S., A.R.P., A.C., N.A.R.), Department of Anatomy and Developmental Biology (A.R.P., R.B.), and Monash Biomedical Imaging (J.P.), Monash University, Melbourne, Victoria, Australia; Baker IDI Heart and Diabetes Institute, Melbourne, Victoria, Australia (N.T.L., D.M.K.); Department of Pediatrics, Indiana University School of Medicine, Indianapolis (P.S., S.J.C.); and Victor Chang Cardiac Research Institute, Darlinghurst, New South Wales, Australia (R.P.H.).

Rationale: Cardiac fibroblasts are critical to proper heart function through multiple interactions with the myocardial compartment, but appreciation of their contribution has suffered from incomplete characterization and lack of cell-specific markers.

Objective: To generate an unbiased comparative gene expression profile of the cardiac fibroblast pool, identify and characterize the role of key genes in cardiac fibroblast function, and determine their contribution to myocardial development and regeneration.

Methods And Results: High-throughput cell surface and intracellular profiling of cardiac and tail fibroblasts identified canonical mesenchymal stem cell and a surprising number of cardiogenic genes, some expressed at higher levels than in whole heart. While genetically marked fibroblasts contributed heterogeneously to interstitial but not cardiomyocyte compartments in infarcted hearts, fibroblast-restricted depletion of one highly expressed cardiogenic marker, T-box 20, caused marked myocardial dysmorphology and perturbations in scar formation on myocardial infarction.

Conclusions: The surprising transcriptional identity of cardiac fibroblasts, the adoption of cardiogenic gene programs, and direct contribution to cardiac development and repair provoke alternative interpretations for studies on more specialized cardiac progenitors, offering a novel perspective for reinterpreting cardiac regenerative therapies.
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http://dx.doi.org/10.1161/CIRCRESAHA.114.302530DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4083003PMC
April 2014

Effect of oxygen on cardiac differentiation in mouse iPS cells: role of hypoxia inducible factor-1 and Wnt/beta-catenin signaling.

PLoS One 2013 12;8(11):e80280. Epub 2013 Nov 12.

Heart Failure Research Group, Baker IDI Heart and Diabetes Institute, Melbourne, Australia.

Background: Disturbances in oxygen levels have been found to impair cardiac organogenesis. It is known that stem cells and differentiating cells may respond variably to hypoxic conditions, whereby hypoxia may enhance stem cell pluripotency, while differentiation of multiple cell types can be restricted or enhanced under hypoxia. Here we examined whether HIF-1alpha modulated Wnt signaling affected differentiation of iPS cells into beating cardiomyocytes.

Objective: We investigated whether transient and sustained hypoxia affects differentiation of cardiomyocytes derived from murine induced pluripotent stem (iPS) cells, assessed the involvement of HIF-1alpha (hypoxia-inducible factor-1alpha) and the canonical Wnt pathway in this process.

Methods: Embryoid bodies (EBs) derived from iPS cells were differentiated into cardiomyocytes and were exposed either to 24 h normoxia or transient hypoxia followed by a further 13 days of normoxic culture.

Results: At 14 days of differentiation, 59 ± 2% of normoxic EBs were beating, whilst transient hypoxia abolished beating at 14 days and EBs appeared immature. Hypoxia induced a significant increase in Brachyury and islet-1 mRNA expression, together with reduced troponin C expression. Collectively, these data suggest that transient and sustained hypoxia inhibits maturation of differentiating cardiomyocytes. Compared to normoxia, hypoxia increased HIF-1alpha, Wnt target and ligand genes in EBs, as well as accumulation of HIF-1alpha and beta-catenin in nuclear protein extracts, suggesting involvement of the Wnt/beta-catenin pathway.

Conclusion: Hypoxia impairs cardiomyocyte differentiation and activates Wnt signaling in undifferentiated iPS cells. Taken together the study suggests that oxygenation levels play a critical role in cardiomyocyte differentiation and suggest that hypoxia may play a role in early cardiogenesis.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0080280PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3827186PMC
July 2014

Functional characterization of a novel mutation in NKX2-5 associated with congenital heart disease and adult-onset cardiomyopathy.

Circ Cardiovasc Genet 2013 Jun 9;6(3):238-47. Epub 2013 May 9.

Developmental and Stem Cell Biology Division, Victor Chang Cardiac Research Institute, Darlinghurst, NSW, Australia.

Background: The transcription factor NKX2-5 is crucial for heart development, and mutations in this gene have been implicated in diverse congenital heart diseases and conduction defects in mouse models and humans. Whether NKX2-5 mutations have a role in adult-onset heart disease is unknown.

Methods And Results: Mutation screening was performed in 220 probands with adult-onset dilated cardiomyopathy. Six NKX2-5 coding sequence variants were identified, including 3 nonsynonymous variants. A novel heterozygous mutation, I184M, located within the NKX2-5 homeodomain, was identified in 1 family. A subset of family members had congenital heart disease, but there was an unexpectedly high prevalence of dilated cardiomyopathy. Functional analysis of I184M in vitro demonstrated a striking increase in protein expression when transfected into COS-7 cells or HL-1 cardiomyocytes because of reduced degradation by the Ubiquitin-proteasome system. In functional assays, DNA-binding activity of I184M was reduced, resulting in impaired activation of target genes despite increased expression levels of mutant protein.

Conclusions: Certain NKX2-5 homeodomain mutations show abnormal protein degradation via the Ubiquitin-proteasome system and partially impaired transcriptional activity. We propose that this class of mutation can impair heart development and mature heart function and contribute to NKX2-5-related cardiomyopathies with graded severity.
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http://dx.doi.org/10.1161/CIRCGENETICS.113.000057DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3816146PMC
June 2013

Cardiac systolic dysfunction in doxorubicin-challenged rats is associated with upregulation of MuRF2 and MuRF3 E3 ligases.

Exp Clin Cardiol 2012 Sep;17(3):101-9

Programa de Biologia Molecular e Estrutural, Instituto de Biofísica Carlos Chagas Filho; ; Programa de Biologia Molecular e Biotecnologia, Instituto de Bioquímica Médica, Universidade Federal do Rio de Janeiro; ; Ecodata Exames Médicos Ltda; ; Instituto Nacional para Pesquisa Translacional em Saúde e Ambiente na Região Amazônica, Conselho Nacional de Desenvolvimento Científico e Tecnológico/MCT;

Doxorubicin (DOXO) is an efficient and low-cost chemotherapeutic agent. The use of DOXO is limited by its side effects, including cardiotoxicity, that may progress to cardiac failure as a result of multifactorial events that have not yet been fully elucidated. In the present study, the effects of DOXO at two different doses were analyzed to identify early functional and molecular markers of cardiac distress. One group of rats received 7.5 mg/kg of DOXO (low-dose group) and was followed for 20 weeks. A subset of these animals was then subjected to an additional cycle of DOXO treatment, generating a cumulative dose of 20 mg/kg (high-dose group). Physiological and biochemical parameters were assessed in both treatment groups and in a control group that received saline. Systolic dysfunction was observed only in the high-dose group. Mitochondrial function analysis showed a clear reduction in oxidative cellular respiration for animals in both DOXO treatment groups, with evidence of complex I damage being observed. Transcriptional analysis by quantitative polymerase chain reaction revealed an increase in atrial natriuretic peptide transcript in the high-dose group, which is consistent with cardiac failure. Analysis of transcription levels of key components of the cardiac ubiquitin-proteasome system found that the ubiquitin E3 ligase muscle ring finger 1 (MuRF1) was upregulated in both the low- and high-dose DOXO groups. MuRF2 and MuRF3 were also upregulated in the high-dose group but not in the low-dose group. This molecular profile may be useful as an early physiological and energetic cardiac failure indicator for testing therapeutic interventions in animal models.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3628421PMC
September 2012

Probing transcription factor diffusion dynamics in the living mammalian embryo with photoactivatable fluorescence correlation spectroscopy.

Nat Commun 2013 ;4:1637

European Molecular Biology Laboratory (EMBL) Australia, Australian Regenerative Medicine Institute, Monash University, Clayton, Victoria 3800, Australia.

Transcription factors use diffusion to search the DNA, yet the mechanisms controlling transcription factor diffusion during mammalian development remain poorly understood. Here we combine photoactivation and fluorescence correlation spectroscopy to study transcription factor diffusion in developing mouse embryos. We show that the pluripotency-associated transcription factor Oct4 displays both fast and Brownian and slower subdiffusive behaviours that are controlled by DNA interactions. Following cell lineage specification, the slower DNA-interacting diffusion fraction distinguishes pluripotent from extraembryonic cell nuclei. Similar to Oct4, Sox2 shows slower diffusion in pluripotent cells while Cdx2 displays opposite dynamics, suggesting that slow diffusion may represent a general feature of transcription factors in lineages where they are essential. Slow Oct4 subdiffusive behaviours are conserved in embryonic stem cells and induced pluripotent stem cells (iPS cells), and lost during differentiation. We also show that Oct4 diffusion depends on its interaction with ERG-associated protein with SET domain. Photoactivation and fluorescence correlation spectroscopy provides a new intravital approach to study transcription factor diffusion in complex in vivo systems.
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http://dx.doi.org/10.1038/ncomms2657DOI Listing
October 2013

Timing, characteristics and determinants of infertility diagnostic work up before admission to eleven second-level assisted reproductive techniques (ART) centres in Italy.

Eur J Obstet Gynecol Reprod Biol 2013 Mar 23;167(1):53-8. Epub 2012 Nov 23.

Dipartimento di Fisiopatologia preconcezionale e prenatale, Ospedale Galliera, Genoa, Italy.

Objective: To describe the time-course of infertile couples not conceiving spontaneously or with medical or surgical therapies before assisted reproductive techniques (ART).

Study Design: Multicentre study of consecutive couples seen for the first time in eleven second-level infertility centres in Italy. A total of 464 couples entered the study and completed a structured questionnaire with the assistance of a clinician. Information was collected on general characteristics, reproductive and gynaecological history, and presumed causes of infertility. Further information was collected on: date of first trying for pregnancy and first consultation for infertility; doctor first consulted by the couple and who decided the diagnostic work-up; instrumental and laboratory tests performed during the diagnostic work-up.

Results: The first medical consultation for infertility occurred after an average of 13 months of unprotected intercourse. This interval was statistically significantly longer for women with low educational level. The median interval between the first medical consultation and the consultation in a second-level infertility centre was 9.5 months. This interval was shorter when the first clinician consulted was a specialist in infertility working in a first-level public centre. Moreover, this interval was longer among women with low educational level. At the time of the consultation in a second-level infertility centre, most of the female patients had already been examined for ovarian, hypophyseal and thyroid function, but only 12% had undergone an anti-Mullerian hormone (AMH) determination: 56% had microbiological culture performed. Nearly 40% had been studied for tubal patency and karyotype. More than 50% of the male partners had not a complete semen evaluation, but 46.0% had second-level examinations.

Conclusion: Educational level is linked to a higher possibility of recognizing fertility problems. The referral process to a second-level centre is quicker in the public sector.
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http://dx.doi.org/10.1016/j.ejogrb.2012.10.022DOI Listing
March 2013

Complex SUMO-1 regulation of cardiac transcription factor Nkx2-5.

PLoS One 2011 12;6(9):e24812. Epub 2011 Sep 12.

Developmental and Stem Cell Biology Division, Victor Chang Cardiac Research Institute, Darlinghurst, New South Wales, Australia.

Reversible post-translational protein modifications such as SUMOylation add complexity to cardiac transcriptional regulation. The homeodomain transcription factor Nkx2-5/Csx is essential for heart specification and morphogenesis. It has been previously suggested that SUMOylation of lysine 51 (K51) of Nkx2-5 is essential for its DNA binding and transcriptional activation. Here, we confirm that SUMOylation strongly enhances Nkx2-5 transcriptional activity and that residue K51 of Nkx2-5 is a SUMOylation target. However, in a range of cultured cell lines we find that a point mutation of K51 to arginine (K51R) does not affect Nkx2-5 activity or DNA binding, suggesting the existence of additional Nkx2-5 SUMOylated residues. Using biochemical assays, we demonstrate that Nkx2-5 is SUMOylated on at least one additional site, and this is the predominant site in cardiac cells. The second site is either non-canonical or a "shifting" site, as mutation of predicted consensus sites and indeed every individual lysine in the context of the K51R mutation failed to impair Nkx2-5 transcriptional synergism with SUMO, or its nuclear localization and DNA binding. We also observe SUMOylation of Nkx2-5 cofactors, which may be critical to Nkx2-5 regulation. Our data reveal highly complex regulatory mechanisms driven by SUMOylation to modulate Nkx2-5 activity.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0024812PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3171482PMC
February 2012

Detection of the first gross CDC73 germline deletion in an HPT-JT syndrome family.

Genes Chromosomes Cancer 2011 Nov 11;50(11):922-9. Epub 2011 Aug 11.

Hereditary Endocrine Cancer Group, Spanish National Cancer Research Centre (CNIO), Madrid, Spain.

Hereditary primary hyperparathyroidism (HPT) may develop as a solitary endocrinopathy (FIHP) or as part of multiple endocrine neoplasia Type 1, multiple endocrine neoplasia Type 2A, or hereditary HPT-jaw tumor syndrome. Inactivating germline mutations of the tumor suppressor gene CDC73 account for 14 and 50% of all FIHP and HPT-JT patients, respectively, and have also been found in almost 20% of apparently sporadic parathyroid carcinoma patients. Although more than 60 independent germline mutations have been described, to date no rearrangement affecting the CDC73 locus has been identified. By means of multiplex-PCR we found a large germline deletion affecting the whole gene in a two-generation HPT-JT family. Subsequently array-CGH and specific PCR analysis determined that the mutation spanned ∼ 547 kb, and included four additional genes: TROVE2, GLRX2, B3GALT2, and UCHL5. Although no clear mutation-specific phenotype was found associated to the presence of the mutation, further studies are needed to assess whether the loss of the neighboring genes could modify the phenotype of carriers. There was complete absence of nuclear staining in the two HPT-JT-related tumors available. The finding of the first rearrangement affecting the CDC73 gene warrants screening for this tumor suppressor gene inactivation mechanism not only in high-risk CDC73 point mutation-negative HPT-JT families, but also in FIHP patients.
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http://dx.doi.org/10.1002/gcc.20911DOI Listing
November 2011