Publications by authors named "Francesca Pistollato"

52 Publications

Current EU regulatory requirements for the assessment of chemicals and cosmetic products: challenges and opportunities for introducing new approach methodologies.

Arch Toxicol 2021 Apr 13. Epub 2021 Apr 13.

Directorate F-Health, Consumers and Reference Materials, Unit F3 Chemicals Safety and Alternative Methods, European Commission, Joint Research Centre (JRC), Via E. Fermi, 2749. TP126, 21027, Ispra, VA, Italy.

The EU Directive 2010/63/EU   on the protection of animals used for scientific purposes and other EU regulations, such as REACH and the Cosmetic Products Regulation advocate for a change in the way toxicity testing is conducted. Whilst the Cosmetic Products Regulation bans animal testing altogether, REACH aims for a progressive shift from in vivo testing towards quantitative in vitro and computational approaches. Several endpoints can already be addressed using non-animal approaches including skin corrosion and irritation, serious eye damage and irritation, skin sensitisation, and mutagenicity and genotoxicity. However, for systemic effects such as acute toxicity, repeated dose toxicity and reproductive and developmental toxicity, evaluation of chemicals under REACH still heavily relies on animal tests. Here we summarise current EU regulatory requirements for the human health assessment of chemicals under REACH and the Cosmetic Products Regulation, considering the more critical endpoints and identifying the main challenges in introducing alternative methods into regulatory testing practice. This supports a recent initiative taken by the International Cooperation on Alternative Test Methods (ICATM) to summarise current regulatory requirements specific for the assessment of chemicals and cosmetic products for several human health-related endpoints, with the aim of comparing different jurisdictions and coordinating the promotion and ultimately the implementation of non-animal approaches worldwide. Recent initiatives undertaken at European level to promote the 3Rs and the use of alternative methods in current regulatory practice are also discussed.
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http://dx.doi.org/10.1007/s00204-021-03034-yDOI Listing
April 2021

A Tau-Driven Adverse Outcome Pathway Blueprint Toward Memory Loss in Sporadic (Late-Onset) Alzheimer's Disease with Plausible Molecular Initiating Event Plug-Ins for Environmental Neurotoxicants.

J Alzheimers Dis 2021 Apr 3. Epub 2021 Apr 3.

ToxGenSolutions, Maastricht, The Netherlands.

The worldwide prevalence of sporadic (late-onset) Alzheimer's disease (sAD) is dramatically increasing. Aging and genetics are important risk factors, but systemic and environmental factors contribute to this risk in a still poorly understood way. Within the frame of BioMed21, the Adverse Outcome Pathway (AOP) concept for toxicology was recommended as a tool for enhancing human disease research and accelerating translation of data into human applications. Its potential to capture biological knowledge and to increase mechanistic understanding about human diseases has been substantiated since. In pursuit of the tau-cascade hypothesis, a tau-driven AOP blueprint toward the adverse outcome of memory loss is proposed. Sequences of key events and plausible key event relationships, triggered by the bidirectional relationship between brain cholesterol and glucose dysmetabolism, and contributing to memory loss are captured. To portray how environmental factors may contribute to sAD progression, information on chemicals and drugs, that experimentally or epidemiologically associate with the risk of AD and mechanistically link to sAD progression, are mapped on this AOP. The evidence suggests that chemicals may accelerate disease progression by plugging into sAD relevant processes. The proposed AOP is a simplified framework of key events and plausible key event relationships representing one specific aspect of sAD pathology, and an attempt to portray chemical interference. Other sAD-related AOPs (e.g., Aβ-driven AOP) and a better understanding of the impact of aging and genetic polymorphism are needed to further expand our mechanistic understanding of early AD pathology and the potential impact of environmental and systemic risk factors.
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http://dx.doi.org/10.3233/JAD-201418DOI Listing
April 2021

Exposure to human relevant mixtures of halogenated persistent organic pollutants (POPs) alters neurodevelopmental processes in human neural stem cells undergoing differentiation.

Reprod Toxicol 2021 03 14;100:17-34. Epub 2020 Dec 14.

European Commission, Joint Research Centre (JRC), Ispra, Italy. Electronic address:

Halogenated persistent organic pollutants (POPs) like perfluorinated alkylated substances (PFASs), brominated flame retardants (BFRs), organochlorine pesticides and polychlorinated biphenyls (PCBs) are known to cause cancer, immunotoxicity, neurotoxicity and interfere with reproduction and development. Concerns have been raised about the impact of POPs upon brain development and possibly neurodevelopmental disorders. The developing brain is a particularly vulnerable organ due to dynamic and complex neurodevelopmental processes occurring early in life. However, very few studies have reported on the effects of POP mixtures at human relevant exposures, and their impact on key neurodevelopmental processes using human in vitro test systems. Aiming to reduce this knowledge gap, we exposed mixed neuronal/glial cultures differentiated from neural stem cells (NSCs) derived from human induced pluripotent stem cells (hiPSCs) to reconstructed mixtures of 29 different POPs using concentrations comparable to Scandinavian human blood levels. Effects of the POP mixtures on neuronal proliferation, differentiation and synaptogenesis were evaluated using in vitro assays anchored to common key events identified in the existing developmental neurotoxicity (DNT) adverse outcome pathways (AOPs). The present study showed that mixtures of POPs (in particular brominated and chlorinated compounds) at human relevant concentrations increased proliferation of NSCs and decreased synapse number. Based on a mathematical modelling, synaptogenesis and neurite outgrowth seem to be the most sensitive DNT in vitro endpoints. Our results indicate that prenatal exposure to POPs may affect human brain development, potentially contributing to recently observed learning and memory deficits in children.
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http://dx.doi.org/10.1016/j.reprotox.2020.12.013DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7992035PMC
March 2021

Effects of caloric restriction on immunosurveillance, microbiota and cancer cell phenotype: Possible implications for cancer treatment.

Semin Cancer Biol 2020 Nov 30. Epub 2020 Nov 30.

Dipartimento di Scienze Cliniche Specialistiche ed Odontostomatologiche, Sez. Biochimica, Università Politecnica delle Marche, Ancona, Italy; International Research Center for Food Nutrition and Safety, Jiangsu University, Zhenjiang, 212013, China. Electronic address:

Fasting, caloric restriction and foods or compounds mimicking the biological effects of caloric restriction, known as caloric restriction mimetics, have been associated with a lower risk of age-related diseases, including cardiovascular diseases, cancer and cognitive decline, and a longer lifespan. Reduced calorie intake has been shown to stimulate cancer immunosurveillance, reducing the migration of immunosuppressive regulatory T cells towards the tumor bulk. Autophagy stimulation via reduction of lysine acetylation, increased sensitivity to chemo- and immunotherapy, along with a reduction of insulin-like growth factor 1 and reactive oxygen species have been described as some of the major effects triggered by caloric restriction. Fasting and caloric restriction have also been shown to beneficially influence gut microbiota composition, modify host metabolism, reduce total cholesterol and triglyceride levels, lower diastolic blood pressure and elevate morning cortisol level, with beneficial modulatory effects on cardiopulmonary fitness, body fat and weight, fatigue and weakness, and general quality of life. Moreover, caloric restriction may reduce the carcinogenic and metastatic potential of cancer stem cells, which are generally considered responsible of tumor formation and relapse. Here, we reviewed in vitro and in vivo studies describing the effects of fasting, caloric restriction and some caloric restriction mimetics on immunosurveillance, gut microbiota, metabolism, and cancer stem cell growth, highlighting the molecular and cellular mechanisms underlying these effects. Additionally, studies on caloric restriction interventions in cancer patients or cancer risk subjects are discussed. Considering the promising effects associated with caloric restriction and caloric restriction mimetics, we think that controlled-randomized large clinical trials are warranted to evaluate the inclusion of these non-pharmacological approaches in clinical practice.
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http://dx.doi.org/10.1016/j.semcancer.2020.11.017DOI Listing
November 2020

Integrating biokinetics and in vitro studies to evaluate developmental neurotoxicity induced by chlorpyrifos in human iPSC-derived neural stem cells undergoing differentiation towards neuronal and glial cells.

Reprod Toxicol 2020 12 1;98:174-188. Epub 2020 Oct 1.

Istituto Superiore di Sanità, Environment and Health Department, Mechanisms, Biomarkers and Models Unit, Rome, Italy.

For some complex toxicological endpoints, chemical safety assessment has conventionally relied on animal testing. Apart from the ethical issues, also scientific considerations have been raised concerning the traditional approach, highlighting the importance for considering real life exposure scenario. Implementation of flexible testing strategies, integrating multiple sources of information, including in vitro reliable test methods and in vitro biokinetics, would enhance the relevance of the obtained results. Such an approach could be pivotal in the evaluation of developmental neurotoxicity (DNT), especially when applied to human cell-based models, mimicking key neurodevelopmental processes, relevant to human brain development. Here, we integrated the kinetic behaviour with the toxicodynamic alterations of chlorpyrifos (CPF), such as in vitro endpoints specific for DNT evaluation, after repeated exposure during differentiation of human neural stem cells into a mixed culture of neurons and astrocytes. The upregulation of some cytochrome P450 and glutathione S-transferase genes during neuronal differentiation and the formation of the two major CPF metabolites (due to bioactivation and detoxification) supported the metabolic competence of the used in vitro model. The alterations in the number of synapses, neurite outgrowth, brain derived neurotrophic factor, the proportion of neurons and astrocytes, as well as spontaneous electrical activity correlated well with the CPF ability to enter the cells and be bioactivated to CPF-oxon. Overall, our results confirm that combining in vitro biokinetics and assays to evaluate effects on neurodevelopmental endpoints in human cells should be regarded as a key strategy for a quantitative characterization of DNT effects.
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http://dx.doi.org/10.1016/j.reprotox.2020.09.010DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7772889PMC
December 2020

Alzheimer's Disease, and Breast and Prostate Cancer Research: Translational Failures and the Importance to Monitor Outputs and Impact of Funded Research.

Animals (Basel) 2020 Jul 14;10(7). Epub 2020 Jul 14.

European Commission, Joint Research Centre (JRC), 21027 Ispra, Italy.

Dementia and cancer are becoming increasingly prevalent in Western countries. In the last two decades, research focused on Alzheimer's disease (AD) and cancer, in particular, breast cancer (BC) and prostate cancer (PC), has been substantially funded both in Europe and worldwide. While scientific research outcomes have contributed to increase our understanding of the disease etiopathology, still the prevalence of these chronic degenerative conditions remains very high across the globe. By definition, no model is perfect. In particular, animal models of AD, BC, and PC have been and still are traditionally used in basic/fundamental, translational, and preclinical research to study human disease mechanisms, identify new therapeutic targets, and develop new drugs. However, animals do not adequately model some essential features of human disease; therefore, they are often unable to pave the way to the development of drugs effective in human patients. The rise of new technological tools and models in life science, and the increasing need for multidisciplinary approaches have encouraged many interdisciplinary research initiatives. With considerable funds being invested in biomedical research, it is becoming pivotal to define and apply indicators to monitor the contribution to innovation and impact of funded research. Here, we discuss some of the issues underlying translational failure in AD, BC, and PC research, and describe how indicators could be applied to retrospectively measure outputs and impact of funded biomedical research.
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http://dx.doi.org/10.3390/ani10071194DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7401638PMC
July 2020

Assessment of developmental neurotoxicity induced by chemical mixtures using an adverse outcome pathway concept.

Environ Health 2020 02 24;19(1):23. Epub 2020 Feb 24.

European Commission, Joint Research Centre (JRC), Ispra, Italy.

Background: In light of the vulnerability of the developing brain, mixture risk assessment (MRA) for the evaluation of developmental neurotoxicity (DNT) should be implemented, since infants and children are co-exposed to more than one chemical at a time. One possible approach to tackle MRA could be to cluster DNT chemicals in a mixture on the basis of their mode of action (MoA) into 'similar' and 'dissimilar', but still contributing to the same adverse outcome, and anchor DNT assays to common key events (CKEs) identified in DNT-specific adverse outcome pathways (AOPs). Moreover, the use of human in vitro models, such as induced pluripotent stem cell (hiPSC)-derived neuronal and glial cultures would enable mechanistic understanding of chemically-induced adverse effects, avoiding species extrapolation.

Methods: HiPSC-derived neural progenitors differentiated into mixed cultures of neurons and astrocytes were used to assess the effects of acute (3 days) and repeated dose (14 days) treatments with single chemicals and in mixtures belonging to different classes (i.e., lead(II) chloride and methylmercury chloride (heavy metals), chlorpyrifos (pesticide), bisphenol A (organic compound and endocrine disrupter), valproic acid (drug), and PCB138 (persistent organic pollutant and endocrine disrupter), which are associated with cognitive deficits, including learning and memory impairment in children. Selected chemicals were grouped based on their mode of action (MoA) into 'similar' and 'dissimilar' MoA compounds and their effects on synaptogenesis, neurite outgrowth, and brain derived neurotrophic factor (BDNF) protein levels, identified as CKEs in currently available AOPs relevant to DNT, were evaluated by immunocytochemistry and high content imaging analysis.

Results: Chemicals working through similar MoA (i.e., alterations of BDNF levels), at non-cytotoxic (IC/100), very low toxic (IC), or moderately toxic (IC) concentrations, induce DNT effects in mixtures, as shown by increased number of neurons, impairment of neurite outgrowth and synaptogenesis (the most sensitive endpoint as confirmed by mathematical modelling) and increase of BDNF levels, to a certain extent reproducing autism-like cellular changes observed in the brain of autistic children.

Conclusions: Our findings suggest that the use of human iPSC-derived mixed neuronal/glial cultures applied to a battery of assays anchored to key events of an AOP network represents a valuable approach to identify mixtures of chemicals with potential to cause learning and memory impairment in children.
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http://dx.doi.org/10.1186/s12940-020-00578-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7038628PMC
February 2020

Rheumatoid arthritis research in the 21st century: Limitations of traditional models, new technologies, and opportunities for a human biology-based approach.

ALTEX 2020 17;37(2):223-242. Epub 2019 Dec 17.

Nutrition and Food Science Group, Department of Analytical and Food Chemistry, CITACA, CACTI, University of Vigo, Vigo, Spain.

Rheumatoid arthritis (RA) is a chronic systemic autoimmune inflammatory disease characterized by progressive bone and cartilage destruction, functional impairment, and long-term disability. Although RA has been described in the medical lit­erature for over two hundred years, its etiology and pathophysiology are insufficiently understood. The current treatment of RA is mainly empirical or based on drugs that interfere with generic steps of the immune response, with limited efficacy and/or significant side effects. Much of RA research has been traditionally based on animals and simplistic in vitro models, which have been shown to poorly recapitulate human RA etiopathogenesis and drug responses. A revolution in science and technology has produced a new generation of more relevant and predictive tools. These tools, which include patient-derived cells, innovative 3D cell culture systems, computational analyses and models, together with omics and large-scale epidemiological studies represent novel and exciting approaches to enhance and forward RA research in a human biology-based perspective. After considering some pitfalls and flaws of traditional models, in this review we discuss novel tools applicable to design human-oriented RA research, while fostering the need for a more holistic and pre­ventative approach to the disease. Our goal is to stimulate discussion, both at scientific and public level, on the need to explore new avenues in RA research and to support a paradigm-shift from animal-based towards human biology-based systems to better understand human pathophysiology and to develop more effective targeted therapies for personalized treatment and prevention.
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http://dx.doi.org/10.14573/altex.1910011DOI Listing
January 2021

Oral microbiota and Alzheimer's disease: Do all roads lead to Rome?

Pharmacol Res 2020 01 30;151:104582. Epub 2019 Nov 30.

Applied Biotechnology Research Center, Baqiyatallah University of Medical Sciences, Tehran, Iran. Electronic address:

Alzheimer's disease (AD) is a progressive neurodegenerative pathology affecting milions of people worldwide associated with deposition of senile plaques. While the genetic and environmental risk factors associated with the onset and consolidation of late onset AD are heterogeneous and sporadic, growing evidence also suggests a potential link between some infectious diseases caused by oral microbiota and AD. Oral microbiota dysbiosis is purported to contribute either directly to amyloid protein production, or indirectly to neuroinflammation, occurring as a consequence of bacterial invasion. Over the last decade, the development of Human Oral Microbiome database (HOMD) has deepened our understanding of oral microbes and their different roles during the human lifetime. Oral pathogens mostly cause caries, periodontal disease, and edentulism in aged population, and, in particular, alterations of the oral microbiota causing chronic periodontal disease have been associated with the risk of AD. Here we describe how different alterations of the oral microbiota may be linked to AD, highlighting the importance of a good oral hygiene for the prevention of oral microbiota dysbiosis.
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http://dx.doi.org/10.1016/j.phrs.2019.104582DOI Listing
January 2020

Pharmacological, non-pharmacological and stem cell therapies for the management of autism spectrum disorders: A focus on human studies.

Pharmacol Res 2020 02 30;152:104579. Epub 2019 Nov 30.

Nutrition and Food Science Group, Department of Analytical and Food Chemistry, CITACA, CACTI, University of Vigo - Vigo Campus, Vigo, Spain; International Research Center for Food Nutrition and Safety, Jiangsu University, Zhenjiang 212013, China; Department of Odontostomatologic and Specialized Clinical Sciences, Faculty of Medicine, Polytechnic University of Marche, Ancona, Italy. Electronic address:

In the last decade, the prevalence of autism spectrum disorders (ASD) has dramatically escalated worldwide. Currently available drugs mainly target some co-occurring symptoms of ASD, but are not effective on the core symptoms, namely impairments in communication and social interaction, and the presence of restricted and repetitive behaviors. On the other hand, transplantation of hematopoietic and mesenchymal stem cells in ASD children has been shown promising to stimulate the recruitment, proliferation, and differentiation of tissue-residing native stem cells, reducing inflammation, and improving some ASD symptoms. Moreover, several comorbidities have also been associated with ASD, such as immune dysregulation, gastrointestinal issues and gut microbiota dysbiosis. Non-pharmacological approaches, such as dietary supplementations with certain vitamins, omega-3 polyunsaturated fatty acids, probiotics, some phytochemicals (e.g., luteolin and sulforaphane), or overall diet interventions (e.g., gluten free and casein free diets) have been considered for the reduction of such comorbidities and the management of ASD. Here, interventional studies describing pharmacological and non-pharmacological treatments in ASD children and adolescents, along with stem cell-based therapies, are reviewed.
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http://dx.doi.org/10.1016/j.phrs.2019.104579DOI Listing
February 2020

Beyond the 3Rs: Expanding the use of human-relevant replacement methods in biomedical research.

ALTEX 2019 ;36(3):343-352

Johns Hopkins University, Bloomberg School of Public Health, Center for Alternatives to Animal Testing (CAAT), Baltimore, MD, USA.

This year marks the 60th anniversary of Russell and Burch's pioneering book, The Principles of Humane Experimental Technique. Their 3Rs framework has helped to inspire humane and scientific progress in experimental technique. However, it is time to update its strategic application. The 21st century has already seen the development of promising, high-tech non-animal models, such as organs-on-a-chip and computational approaches that, in our view, will replace animals as the default option in biomedical experimentation. How fast this transition will take place will depend on the pace at which these new models are optimized to reflect the biology of humans, rather than that of non-human animals. While the new methods are likely to reshape all areas in which animals are currently used in science, we particularly encourage their application in biomedical research, which accounts for the bulk of animals used. We call for the pursuit of a three-prong strategy that focuses on (1) advancing non-animal methods as replacements of animal experiments, (2) applying them to biomedical research, and (3) improving their relevance to human biology. As academics and scientists, we feel that educational efforts targeted at young scientists in training will be an effective and sustainable way to advance this vision. Our strategy may not promise an imminent end to the use of animals in science, but it will bring us closer to an era in which the 3Rs are increasingly perceived as a solution to a receding problem. Russell and Burch themselves surely would have welcomed these positive changes.
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http://dx.doi.org/10.14573/altex.1907031DOI Listing
January 2020

Effects of phytochemicals on thyroid function and their possible role in thyroid disease.

Ann N Y Acad Sci 2019 05 1;1443(1):3-19. Epub 2018 Nov 1.

Dipartimento di Scienze Cliniche Specialistiche ed Odontostomatologiche, Sez, Biochimica, Università Politecnica delle Marche, Ancona, Italy.

About 1 of 10 women, particularly those older than 60 years of age, shows some degree of thyroid hormone deficiency. Thyroid diseases are generally characterized by perturbations of thyroid signaling homeostasis. The most common examples of thyroid diseases include hypothyroidism, hyperthyroidism, and several types of thyroid cancers. Phytochemicals have been shown to have either beneficial or detrimental effects on thyroid function. Some flavonoids have been reported to affect the expression and the activity of several thyroid-related enzymes and proteins, and for this reason some concerns have been raised about the possible thyroid-disruptive properties of foods enriched in these substances. On the other hand, the beneficial effects of some plant-derived compounds, such as myricetin, quercetin, apigenin, rutin, genistein, and curcumin, and their possible role as adjuvants for the treatment of thyroid cancers have been described. Here, the role of phytochemicals in thyroid signaling modulation and their possible beneficial or detrimental effects on thyroid disease risk are discussed.
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http://dx.doi.org/10.1111/nyas.13980DOI Listing
May 2019

Investigating cell type specific mechanisms contributing to acute oral toxicity.

ALTEX 2019 12;36(1):39-64. Epub 2018 Jul 12.

EU Commission Joint Research Centre (JRC), Ispra, Italy.

The replacement of animals in acute systemic toxicity testing remains a considerable challenge. Only animal data are currently accepted by regulators, including data generated by reduction and refinement methods. The development of Integrated Approaches to Testing and Assessment (IATA) is hampered by an insufficient understanding of the numerous toxicity pathways that lead to acute systemic toxicity. Therefore, central to our work has been the collection and evaluation of the mechanistic information on eight organs identified as relevant for acute systemic toxicity (nervous system, cardiovascular system, liver, kidney, lung, blood, gastrointestinal system and immune system). While the nervous and cardiovascular systems are the most frequent targets, no clear relationship emerged between specific mechanisms of target organ toxicity and the level (category) of toxicity. From a list of 114 chemicals with acute oral in vivo and in vitro data, 98 were identified with target organ specific effects, of which 93% were predicted as acutely toxic by the 3T3 neutral red uptake cytotoxicity assay and 6% as non-toxic. This analysis will help to prioritise the development of adverse outcome pathways for acute oral toxicity, which will support the assessment of chemicals using mechanistically informed IATA.
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http://dx.doi.org/10.14573/altex.1805181DOI Listing
April 2019

Nutritional patterns associated with the maintenance of neurocognitive functions and the risk of dementia and Alzheimer's disease: A focus on human studies.

Pharmacol Res 2018 05 16;131:32-43. Epub 2018 Mar 16.

Dipartimento di Scienze Cliniche Specialistiche ed Odontostomatologiche, Sez. Biochimica, Università Politecnica delle Marche, Ancona, Italy. Electronic address:

Ample epidemiological evidence suggests a strong correlation among diet, lifestyle factors and the onset and consolidation of dementia and Alzheimer's disease (AD). It has been demonstrated that AD, diabetes, obesity, insulin resistance, and cardiovascular disease are strongly interconnected pathologies. Preventive strategies and nutritional interventions seem to be promising approaches to delay neurocognitive decline and reduce the risk of AD and other non-psychiatric co-morbidities. In this regard, healthy dietary patterns, characterized by high intake of plant-based foods, probiotics, antioxidants, soy beans, nuts, and omega-3 polyunsaturated fatty acids, and a low intake of saturated fats, animal-derived proteins, and refined sugars, have been shown to decrease the risk of neurocognitive impairments and eventually the onset of AD. Here we review the role of some nutrients and, in particular, of healthy dietary patterns, such as the Mediterranean diet and other emerging healthy diets, DASH (Dietary Approach to Stop Hypertension) and MIND (Mediterranean-DASH dietIntervention for Neurodegenerative Delay), for the maintenance of cognitive performance, focusing specifically on human studies. The beneficial effects associated with overall diet composition, rather than single nutrient supplementations, for the prevention or the delay of AD and dementia are discussed.
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http://dx.doi.org/10.1016/j.phrs.2018.03.012DOI Listing
May 2018

Strategies to improve the regulatory assessment of developmental neurotoxicity (DNT) using in vitro methods.

Toxicol Appl Pharmacol 2018 09 22;354:7-18. Epub 2018 Feb 22.

European Commission, Joint Research Centre (JRC), Ispra, Italy.

Currently, the identification of chemicals that have the potential to induce developmental neurotoxicity (DNT) is based on animal testing. Since at the regulatory level, systematic testing of DNT is not a standard requirement within the EU or USA chemical legislation safety assessment, DNT testing is only performed in higher tiered testing triggered based on chemical structure activity relationships or evidence of neurotoxicity in systemic acute or repeated dose toxicity studies. However, these triggers are rarely used and, in addition, do not always serve as reliable indicators of DNT, as they are generally based on observations in adult rodents. Therefore, there is a pressing need for developing alternative methodologies that can reliably support identification of DNT triggers, and more rapidly and cost-effectively support the identification and characterization of chemicals with DNT potential. We propose to incorporate mechanistic knowledge and data derived from in vitro studies to support various regulatory applications including: (a) the identification of potential DNT triggers, (b) initial chemical screening and prioritization, (c) hazard identification and characterization, (d) chemical biological grouping, and (e) assessment of exposure to chemical mixtures. Ideally, currently available cellular neuronal/glial models derived from human induced pluripotent stem cells (hiPSCs) should be used as they allow evaluation of chemical impacts on key neurodevelopmental processes, by reproducing different windows of exposure during human brain development. A battery of DNT in vitro test methods derived from hiPSCs could generate valuable mechanistic data, speeding up the evaluation of thousands of compounds present in industrial, agricultural and consumer products that lack safety data on DNT potential.
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http://dx.doi.org/10.1016/j.taap.2018.02.008DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6095942PMC
September 2018

Nrf2 as regulator of innate immunity: A molecular Swiss army knife!

Biotechnol Adv 2018 Mar - Apr;36(2):358-370. Epub 2017 Dec 22.

Applied Biotechnology Research Center, Baqiyatallah University of Medical Sciences, Tehran, Iran. Electronic address:

Organisms are constantly exposed to a broad range of pathological and stress-inducing agents, allergens and environmental chemicals that can induce infections, toxicity or other undesirable reactions. Our immune system has evolved over time in order to efficiently respond to these exogenous insults and maintain homeostasis. In particular, the innate immune system acts as primary barrier to prevent the entrance of invasive agents or allergens. This system is comprised of a diversity of cell types that are rapidly activated by recognition of common structures present in many potential pathogens known as pathogen-associated molecular patterns (PAMPs). The nuclear factor erythroid 2 related factor 2 (Nrf2) is a relevant basic leucine zipper (bZIP) transcription factor that is essential in the regulation of cell cycle homeostasis, cytoprotection, and innate immunity when cells are under stressful conditions. Although the role of Nrf2 in activating the expression of protective genes - such as antioxidant or anti-inflammatory - is known, its role in innate immunity and immune-related gene expression remains not yet clear. The present review summarizes current knowledge on Nrf2 signaling pathway structure and activity under both physiological state and upon oxidative stress. In addition, the relation between Nrf2 signaling pathway and the innate immune system is discussed, highlighting the potential therapeutic effects of diverse natural and synthetic compounds as Nrf2 regulators.
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http://dx.doi.org/10.1016/j.biotechadv.2017.12.012DOI Listing
February 2019

The use of natural compounds for the targeting and chemoprevention of ovarian cancer.

Cancer Lett 2017 12 7;411:191-200. Epub 2017 Oct 7.

Centre for Nutrition and Health, Universidad Europea Del Atlántico (UEA), Santander, Spain; Dipartimento di Scienze Cliniche Specialistiche Ed Odontostomatologiche, Sez. Biochimica, Università Politecnica Delle Marche, Ancona, Italy. Electronic address:

Among gynaecological cancers, ovarian cancer represents the leading cause of death in women. Current treatment for ovarian cancer entails surgery followed by combined chemotherapy with platinum and taxane, which are associated, particularly cisplatin, with severe side effects. While this treatment approach appears to be initially effective in a high number of patients, nearly 70% of them suffer a relapse within a few months after initial treatment. Therefore, more effective and better-tolerated treatment options are clearly needed. In recent years, several natural compounds (such as curcumin, epigallocatechin 3-gallate (EGCG), resveratrol, sulforaphane and Withaferin-A), characterized by long-term safety and negligible and/or inexistent side effects, have been proposed as possible adjuvants of traditional chemotherapy. Indeed, several in vitro and in vivo studies have shown that phytocompounds can effectively inhibit tumor cell proliferation, stimulate autophagy, induce apoptosis, and specifically target ovarian cancer stem cells (CSCs), which are generally considered to be responsible for tumor recurrence in several types of cancer. Here we review current literature on the role of natural products in ovarian cancer chemoprevention, highlighting their effects particularly on the regulation of inflammation, autophagy, proliferation and apoptosis, chemotherapy resistance, and ovarian CSC growth.
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http://dx.doi.org/10.1016/j.canlet.2017.09.050DOI Listing
December 2017

Fetal Bovine Serum (FBS): Past - Present - Future.

ALTEX 2018 9;35(1):99-118. Epub 2017 Aug 9.

Division of Physiology, Medical University Innsbruck, Innsbruck, Austria.

The supplementation of culture medium with fetal bovine serum (FBS, also referred to as "fetal calf serum") is still common practice in cell culture applications. Due to a number of disadvantages in terms of quality and reproducibility of in vitro data, animal welfare concerns, and in light of recent cases of fraudulent marketing, the search for alternatives and the development of serum-free medium formulations has gained global attention. Here, we report on the 3rd Workshop on FBS, Serum Alternatives and Serum-free Media, where regulatory aspects, the serum dilemma, alternatives to FBS, case-studies of serum-free in vitro applications, and the establishment of serum-free databases were discussed. The whole process of obtaining blood from a living calf fetus to using the FBS produced from it for scientific purposes is de facto not yet legally regulated despite the existing EU-Directive 2010/63/EU on the use of animals for scientific purposes. Together with the above-mentioned challenges, several strategies have been developed to reduce or replace FBS in cell culture media in terms of the 3Rs (Refinement, Reduction, Replacement). Most recently, releasates of activated human donor thrombocytes (human platelet lysates) have been shown to be one of the most promising serum alternatives when chemically-defined media are not yet an option. Additionally, new developments in cell-based assay techniques, advanced organ-on-chip and microphysiological systems are covered in this report. Chemically-defined serum-free media are shown to be the ultimate goal for the majority of culture systems, and examples are discussed.
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http://dx.doi.org/10.14573/altex.1705101DOI Listing
August 2018

Protocol for the Differentiation of Human Induced Pluripotent Stem Cells into Mixed Cultures of Neurons and Glia for Neurotoxicity Testing.

J Vis Exp 2017 06 9(124). Epub 2017 Jun 9.

Directorate F - Health, Consumers and Reference Materials, Joint Research Centre;

Human pluripotent stem cells can differentiate into various cell types that can be applied to human-based in vitro toxicity assays. One major advantage is that the reprogramming of somatic cells to produce human induced pluripotent stem cells (hiPSCs) avoids the ethical and legislative issues related to the use of human embryonic stem cells (hESCs). HiPSCs can be expanded and efficiently differentiated into different types of neuronal and glial cells, serving as test systems for toxicity testing and, in particular, for the assessment of different pathways involved in neurotoxicity. This work describes a protocol for the differentiation of hiPSCs into mixed cultures of neuronal and glial cells. The signaling pathways that are regulated and/or activated by neuronal differentiation are defined. This information is critical to the application of the cell model to the new toxicity testing paradigm, in which chemicals are assessed based on their ability to perturb biological pathways. As a proof of concept, rotenone, an inhibitor of mitochondrial respiratory complex I, was used to assess the activation of the Nrf2 signaling pathway, a key regulator of the antioxidant-response-element-(ARE)-driven cellular defense mechanism against oxidative stress.
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http://dx.doi.org/10.3791/55702DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5608344PMC
June 2017

Nrf2 pathway activation upon rotenone treatment in human iPSC-derived neural stem cells undergoing differentiation towards neurons and astrocytes.

Neurochem Int 2017 Sep 13;108:457-471. Epub 2017 Jun 13.

Directorate F - Health, Consumers and Reference Materials, Joint Research Centre, Ispra, Italy. Electronic address:

Activation of Nrf2/ARE signaling pathway occurs ubiquitously in most cell types upon induction of oxidative stress. Rotenone, an inhibitor of mitochondrial complex I, can be used to trigger oxidative stress, stimulate the activation of Nrf2 pathway in neuronal and astrocytic cells and assess neurotoxicity. We have previously demonstrated that an acute treatment with rotenone can induce Nrf2 activation, which leads to astrocyte activation and dopaminergic (DA) neuronal cell death in a mixed neuronal/astrocytic cell model derived from human induced pluripotent stem cells (hiPSCs). In this study, we characterized the effects of a repeated dose treatment with rotenone (14 days) on hiPSC-derived neural stem cells (NSCs) undergoing differentiation, assessing the expression and the activation of the Nrf2 pathway. Our results show that Nrf2 signaling increases during NSC differentiation. Moreover, we observed that rotenone treatment induced a progressive activation of Nrf2 signaling together with a induction of astrocyte reactivity, a reduction of neurite length leading to neuronal cell death, in particular of DA neurons. Altogether these data indicate that hiPSC-NSC models are relevant test systems for the evaluation of Nrf2 pathway activation upon induced oxidative stress, allowing further understanding of the molecular mechanisms underlying exposure to (developmental) neurotoxicants.
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http://dx.doi.org/10.1016/j.neuint.2017.06.006DOI Listing
September 2017

Assessment of acute and chronic toxicity of doxorubicin in human induced pluripotent stem cell-derived cardiomyocytes.

Toxicol In Vitro 2017 Aug 26;42:182-190. Epub 2017 Apr 26.

Directorate F - Health, Consumers and Reference Materials, Joint Research Centre, European Commission, Ispra, Italy.

The present study assesses acute and chronic toxicity of doxorubicin in human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs), with the aim to obtain in vitro biomarkers that can be used as readouts to predict in vivo cardiotoxicity. Possible acute toxicity was investigated by assessing effects on the beating rate and the field potential duration (FPD) of doxorubicin-exposed cardiomyocytes by measuring electrical activity using multi-electrode array (MEA) analyses. No effects on the beating rate and FPD were found at concentrations up to 6μM, whereas at 12μM no electrical activity was recorded, indicating that the cardiomyocytes stopped beating. Acute and chronic effects of doxorubicin on mitochondria, which have been reported to be affected in doxorubicin-induced cardiotoxicity, were assessed using high content imaging techniques. To this end hiPSC-CMs were exposed to 150 or 300nM doxorubicin using both single dosing (3h and 2days) and repetitive dosing (3 times, of 2days each), including washout studies to assess delayed effects (assessment at day 14) and effects on cell number, mitochondrial density, mitochondrial membrane potential, mitochondrial superoxide levels and mitochondrial calcium levels were assessed. No effects of doxorubicin were found on mitochondrial density and mitochondrial superoxide levels, whereas doxorubicin reduced cell survival and slightly altered mitochondrial membrane potential and mitochondrial calcium levels, which was most profound in the washout studies. Altogether, the results of the present study show that concentrations of doxorubicin in the micromolar range were required to affect electrical activity of hiPSC-CMs, whereas nanomolar concentrations already affected cell viability and caused mitochondrial disturbances. Integration of these data with other in vitro data may enable the selection of a series of in vitro biomarkers that can be used as readouts to screen chemicals for possible cardiotoxicity.
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http://dx.doi.org/10.1016/j.tiv.2017.04.023DOI Listing
August 2017

Alzheimer disease research in the 21st century: past and current failures, new perspectives and funding priorities.

Oncotarget 2016 Jun;7(26):38999-39016

Physicians Committee for Responsible Medicine, Washington, DC, USA.

Much of Alzheimer disease (AD) research has been traditionally based on the use of animals, which have been extensively applied in an effort to both improve our understanding of the pathophysiological mechanisms of the disease and to test novel therapeutic approaches. However, decades of such research have not effectively translated into substantial therapeutic success for human patients. Here we critically discuss these issues in order to determine how existing human-based methods can be applied to study AD pathology and develop novel therapeutics. These methods, which include patient-derived cells, computational analysis and models, together with large-scale epidemiological studies represent novel and exciting tools to enhance and forward AD research. In particular, these methods are helping advance AD research by contributing multifactorial and multidimensional perspectives, especially considering the crucial role played by lifestyle risk factors in the determination of AD risk. In addition to research techniques, we also consider related pitfalls and flaws in the current research funding system. Conversely, we identify encouraging new trends in research and government policy. In light of these new research directions, we provide recommendations regarding prioritization of research funding. The goal of this document is to stimulate scientific and public discussion on the need to explore new avenues in AD research, considering outcome and ethics as core principles to reliably judge traditional research efforts and eventually undertake new research strategies.
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http://dx.doi.org/10.18632/oncotarget.9175DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5129909PMC
June 2016

Role of gut microbiota and nutrients in amyloid formation and pathogenesis of Alzheimer disease.

Nutr Rev 2016 10;74(10):624-34

F. Pistollato, S.S. Cano, I. Elio, M.M. Vergara, F. Giampieri, and M. Battino are with the Centre for Nutrition and Health, Universidad Europea del Atlántico, Santander, Spain. S.S. Cano and I. Elio are with the Universidad Internacional Iberoamericana (UNINI), Campeche, Mexico and the Fundacion Universitaria Iberoamericana (FUNIBER), Barcelona, Spain. M.M. Vergara is with the Universidad Internacional Iberoamericana (UNINI), Arecibo, Puerto Rico, USA. F. Giampieri and M. Battino are with the Dipartimento di Scienze Cliniche Specialistiche ed Odontostomatologiche, Sez. Biochimica, Università Politecnica delle Marche, Ancona, Italy.

It has been hypothesized that alterations in the composition of the gut microbiota might be associated with the onset of certain human pathologies, such as Alzheimer disease, a neurodegenerative syndrome associated with cerebral accumulation of amyloid-β fibrils. It has been shown that bacteria populating the gut microbiota can release significant amounts of amyloids and lipopolysaccharides, which might play a role in the modulation of signaling pathways and the production of proinflammatory cytokines related to the pathogenesis of Alzheimer disease. Additionally, nutrients have been shown to affect the composition of the gut microbiota as well as the formation and aggregation of cerebral amyloid-β. This suggests that modulating the gut microbiome and amyloidogenesis through specific nutritional interventions might prove to be an effective strategy to prevent or reduce the risk of Alzheimer disease. This review examines the possible role of the gut in the dissemination of amyloids, the role of the gut microbiota in the regulation of the gut-brain axis, the potential amyloidogenic properties of gut bacteria, and the possible impact of nutrients on modulation of microbiota composition and amyloid formation in relation to the pathogenesis of Alzheimer disease.
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http://dx.doi.org/10.1093/nutrit/nuw023DOI Listing
October 2016

Evaluation of the rotenone-induced activation of the Nrf2 pathway in a neuronal model derived from human induced pluripotent stem cells.

Neurochem Int 2017 Jun 9;106:62-73. Epub 2016 Sep 9.

Directorate F - Health, Consumers and Reference Materials, Joint Research Centre, Ispra, Italy. Electronic address:

Human induced pluripotent stem cells (hiPSCs) are considered as a powerful tool for drug and chemical screening and development of new in vitro testing strategies in the field of toxicology, including neurotoxicity evaluation. These cells are able to expand and efficiently differentiate into different types of neuronal and glial cells as well as peripheral neurons. These human cells-based neuronal models serve as test systems for mechanistic studies on different pathways involved in neurotoxicity. One of the well-known mechanisms that are activated by chemically-induced oxidative stress is the Nrf2 signaling pathway. Therefore, in the current study, we evaluated whether Nrf2 signaling machinery is expressed in human induced pluripotent stem cells (hiPSCs)-derived mixed neuronal/glial culture and if so whether it becomes activated by rotenone-induced oxidative stress mediated by complex I inhibition of mitochondrial respiration. Rotenone was found to induce the activation of Nrf2 signaling particularly at the highest tested concentration (100 nM), as shown by Nrf2 nuclear translocation and the up-regulation of the Nrf2-downstream antioxidant enzymes, NQO1 and SRXN1. Interestingly, exposure to rotenone also increased the number of astroglial cells in which Nrf2 activation may play an important role in neuroprotection. Moreover, rotenone caused cell death of dopaminergic neurons since a decreased percentage of tyrosine hydroxylase (TH) cells was observed. The obtained results suggest that hiPSC-derived mixed neuronal/glial culture could be a valuable in vitro human model for the establishment of neuronal specific assays in order to link Nrf2 pathway activation (biomarker of oxidative stress) with additional neuronal specific readouts that could be applied to in vitro neurotoxicity evaluation.
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http://dx.doi.org/10.1016/j.neuint.2016.09.004DOI Listing
June 2017

Good Cell Culture Practice for stem cells and stem-cell-derived models.

ALTEX 2017 23;34(1):95-132. Epub 2016 Aug 23.

Center for Alternative to Animal Testing, Johns Hopkins University, Baltimore, MD, USA.

The first guidance on Good Cell Culture Practice (GCCP) dates back to 2005. This document expands this to include aspects of quality assurance for in vitro cell culture focusing on the increasingly diverse cell types and culture formats used in research, product development, testing and manufacture of biotechnology products and cell-based medicines. It provides a set of basic principles of best practice that can be used in training new personnel, reviewing and improving local procedures, and helping to assure standard practices and conditions for the comparison of data between laboratories and experimentation performed at different times. This includes recommendations for the documentation and reporting of culture conditions. It is intended as guidance to facilitate the generation of reliable data from cell culture systems, and is not intended to conflict with local or higher level legislation or regulatory requirements. It may not be possible to meet all recommendations in this guidance for practical, legal or other reasons. However, when it is necessary to divert from the principles of GCCP, the risk of decreasing the quality of work and the safety of laboratory staff should be addressed and any conclusions or alternative approaches justified. This workshop report is considered a first step toward a revised GCCP 2.0.
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http://dx.doi.org/10.14573/altex.1607121DOI Listing
February 2017

Associations between Sleep, Cortisol Regulation, and Diet: Possible Implications for the Risk of Alzheimer Disease.

Adv Nutr 2016 07 15;7(4):679-89. Epub 2016 Jul 15.

Center for Nutrition and Health, European University of the Atlantic (UEA), Santander, Spain; Department of Specialized Clinical Sciences and Dentistry, Marche Polytechnic University, Ancona, Italy

Accumulation of proteinaceous amyloid β plaques and tau oligomers may occur several years before the onset of Alzheimer disease (AD). Under normal circumstances, misfolded proteins get cleared by proteasome degradation, autophagy, and the recently discovered brain glymphatic system, an astroglial-mediated interstitial fluid bulk flow. It has been shown that the activity of the glymphatic system is higher during sleep and disengaged or low during wakefulness. As a consequence, poor sleep quality, which is associated with dementia, might negatively affect glymphatic system activity, thus contributing to amyloid accumulation. The diet is another important factor to consider in the regulation of this complex network. Diets characterized by high intakes of refined sugars, salt, animal-derived proteins and fats and by low intakes of fruit and vegetables are associated with a higher risk of AD and can perturb the circadian modulation of cortisol secretion, which is associated with poor sleep quality. For this reason, diets and nutritional interventions aimed at restoring cortisol concentrations may ease sleep disorders and may facilitate brain clearance, consequentially reducing the risk of cognitive impairment and dementia. Here, we describe the associations that exist between sleep, cortisol regulation, and diet and their possible implications for the risk of cognitive impairment and AD.
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http://dx.doi.org/10.3945/an.115.011775DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4942871PMC
July 2016

Inhibition of PI3K Signalling Selectively Affects Medulloblastoma Cancer Stem Cells.

Biomed Res Int 2015 18;2015:973912. Epub 2015 Oct 18.

Istituto di Ricerca Pediatrica Città della Speranza (IRP), Corso Stati Uniti 4, 35127 Padova, Italy ; Department of Woman and Child Health, University of Padova, Via Giustiniani 3, 35128 Padova, Italy.

Medulloblastoma is the most common malignant brain tumor of childhood. Although survival has slowly increased in the past years, the prognosis of these patients remains unfavourable. In this context, it has been recently shown that the intracellular signaling pathways activated during embryonic cerebellar development are deregulated in MDB. One of the most important is PI3K/AKT/mTOR, implicated in cell proliferation, survival, growth, and protein synthesis. Moreover, a fraction of MDB cells has been shown to posses stemlike features, to express typical neuronal precursor markers (Nestin and CD133), and to be maintained by the hypoxic cerebellar microenvironment. This subpopulation of MDB cells is considered to be responsible for treatment resistance and recurrence. In this study, we evaluated the effects of PI3K/AKT pathway inhibition on primary cultures of MDB and particularly on the cancer stem cell (CSC) population (CD133(+)). PI3K inhibition was able to counteract MDB cell growth and to promote differentiation of stemlike MDB cells. Moreover, PI3K/AKT pathway suppression induced dramatic cell death through activation of the mitochondrial proapoptotic cascade. Finally, analysis on the stem cells fraction revealed that the MDB CSC population is more sensitive to PI3K targeting compared to the whole cancerous population and its nonstem cell counterpart.
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http://dx.doi.org/10.1155/2015/973912DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4628705PMC
August 2016

The Use of Neuroimaging to Assess Associations Among Diet, Nutrients, Metabolic Syndrome, and Alzheimer's Disease.

J Alzheimers Dis 2015 ;48(2):303-18

Centre for Nutrition and Health, Universidad Europea del Atlántico (UEA), Santander, Spain.

In the last decade, specific dietary patterns, mainly characterized by high consumption of vegetables and fruits, have been proven beneficial for the prevention of both metabolic syndrome (MetS)-related dysfunctions and neurodegenerative disorders, such as Alzheimer's disease (AD). Nowadays, neuroimaging readouts can be used to diagnose AD, investigate MetS effects on brain functionality and anatomy, and assess the effects of dietary supplementations and nutritional patterns in relation to neurodegeneration and AD-related features. Here we review scientific literature describing the use of the most recent neuroimaging techniques to detect AD- and MetS-related brain features, and also to investigate associations between consolidated dietary patterns or nutritional interventions and AD, specifically focusing on observational and intervention studies in humans.
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http://dx.doi.org/10.3233/JAD-150301DOI Listing
June 2016