A mini-review of the reported biosensor research occurring in South Africa evidences a strong emphasis on electrochemical sensor research, guided by the opportunities this transduction platform holds for low-cost and robust sensing of numerous targets. Many of the reported publications centre on fundamental research into the signal transduction method, using model biorecognition elements, in line with international trends. Other research in this field is spread across several areas including: the application of nanotechnology; the identification and validation of biomarkers; development and testing of biorecognition agents (antibodies and aptamers) and design of electro-catalysts, most notably metallophthalocyanine. Biosensor targets commonly featured were pesticides and metals. Areas  of regional import to sub-Saharan Africa, such as HIV/AIDs and tuberculosis diagnosis, are also apparent in a review of the available literature. Irrespective of the targets, the challenge to the effective deployment of such sensors remains shaped by social and economic realities such that the requirements thereof are for low-cost and universally easy to operate devices for field settings. While it is difficult to disentangle the intertwined roles of national policy, grant funding availability and, certainly, of global trends in shaping areas of emphasis in research, most notable is the strong role that nanotechnology, and to a certain extent biotechnology, plays in research regarding biosensor construction. Stronger emphasis on collaboration between scientists in theoretical modelling, nanomaterials application and or relevant stakeholders in the specific field  and researchers in biosensor design may help evolve focused research efforts towards development and deployment of low-cost biosensors. The scope for biosensor research generally in southern Africa is perhaps best understood when considering the social and economic paradigms common to most developing countries and emerging economies. per capita Gross Domestic Product (GDP) as a measure of economic productivity: most of the countries present in sub-Saharan Africa generate far less GDP than developed nations; accordingly, they tend to have significantly less money available for healthcare, both at public and private spending levels, as exemplified in the countries presented in Developing countries in Africa tend to combine a low-income population majority with less than optimal regulatory monitoring infrastructure. This is coupled to a heavy regulatory bias favouring the manufacturing/mining/agricultural industries as the primary means of employment. Using the per capita annual GDP of sub-Saharan countries  and the highest total per capita health expenditure (593 USD), still has over a quarter of its population living on less than 2 USD per capita per day, highlighting the economic inequalities present in the country and the concomitant differences in access to available healthcare. This dichotomy is one that presents African scientists across the continent, and certainly in southern Africa, with a challenge to approach research such that it caters for a wider potential, global market  against the backdrop of the overwhelming need for rapid, accurate, low-cost sensors easily operable in remote environments that are required by a large majority of the health- and environmental-care operations on the continent.The diffused nature of the healthcare institutions present in developing countries and the particular challenges those bring for sensor development is a feature that drives much of the approach to research. However, by the same token, many areas of Africa, and certainly South Africa, are blends of both developed and developing countries, where access to state-of-the-art health screening technologies match or better those in more developed economies. Tellingly, South Africa , possessThe breadth of targets identified for biosensing, combined with the diversity of technology approaches and design considerations available means that biosensor research focus in South Africa is spread across a number of areas as evidenced in this mini-review: nanotechnology and nanoscience; identification and validation of biomarkers; development of biorecognition agents (antibodies and aptamers) and design of electro-catalysts, most notably metallophthalocyanines.Nanotechnology-based approaches for sensor design are a common theme referred to in the areas under discussion, as is the strong focus on electrochemical sensor technology. In accordance with global trends of nanotechnology application in electroanalysis , and “syWhile not an exhaustive reporting of the available literature, this review serves to identify key research interests, as well as some interesting features of the research landscape currently emerging from South Africa in the development of biosensors. This review also explores in brief the positioning of the research in biosensor development within the context of national policy (most notably biotechnology and nanotechnology), the country’s infrastructure and resources.etc.Examination of the biosensor transducer types in This bias in favour of electroanalytical sensor technology is not unexpected and may be influenced by the promise thereof as a rapid and low-cost technology for qualitative testing in remote areas, given the lack of access to diagnostics in remote areas.A wide variety of materials, beyond the functional biorecognition agents that underpin biosensor technologies, have been incorporated into biosensors researched in South Africa . NanomatIn recent years, nanotechnology has been repeatedly touted as a technological route potentially capable of solving many of the current global issues. In a recent review of the future application of nanotechnology within developing countries within the sectors of water treatment, human health and agriculture , nanomaterial biosensors (largely within the agriculture sector) were specifically identified as desirable future products . This keFor the above reasons, the rational design and fabrication of nanoscale structures has been under intense investigation for application in sensors. In South Africa, these are mainly focused on the application of metal nanoparticles, largely gold, and carbon-based nanomaterials (notably carbon nanotubes). These are applied as directly to functionalise surfaces, or as composites with other materials  and freqNanomaterial use and applications are intimately associated with electroanalytical methods of sensor research in the country. The convergence of nanomaterials and electrochemical sensors in the biosensor field  is a refSpecifically, electrochemical nanocomposite electrodes offer further advantages over conventional fabrication techniques: through improving effective electrode surface area via the use of electro-conductive nanostructures; as catalysts within their own right and enhancing control of the local microenvironment . In the It is well-established that, in order to improve upon sensor response, an adequate dispersal of the catalyst(s) at/near the transducer is essential . An addiA wide variety of metallic nanoparticles ,44,47,49The catalytic properties of metallophthalocyanine (MPc) species are strongly represented as chemosensor agents in their own right by several research groups across South Africa, especially with regard to adapting their rich reduction/oxidation chemistry for electrochemical sensor purposes . ResearcMetallophthalocyanines included in composite assemblies (with enzymes) ,42 for bInterestingly no publications originating from South African groups were found utilising the combined performance enhancing characteristics of MPc-nanomaterial composites in conjunction with enzymes, antibodies, whole cells or aptamers for biosensing applications, possibly presenting a research opportunity for those groups engaged in similar research.2O2 and a wide variety of inhibitory compounds, e.g., metals, the enzyme horseradish peroxidase (HRP) has been used extensively in the design and fabrication of these electrochemical sensors  as a conductive polymer in electrochemical sensors is well-established and its continued use, both as a polymer ,25 and a sensors  [5,6,7,82 nanosphere catalysts [Additional research examined the use of polymers to aid in the dispersion of nanomaterials, e.g., ZrOatalysts , gold [4atalysts  and silvatalysts  nanopartatalysts ,38,40.Zea mays) cobs—has recently been demonstrated as a novel and inexpensive method of dispersing carbon nanotubes [Inert proteins, such as bovine serum albumin ,45 have anotubes ,11,13. PThe search for new technologies that can advance the biomedical field, particularly the early detection of disease, is a well-known driver for the development of biorecognition agents for biosensor assembly . As such3), and discrimination against the pro-hormone L-T4, was improved through the selective retention at carbon paste electrodes using anti-L-T3 antisera [While the immobilisation of suitable enzymes and catalysts are the traditional manner of producing electrochemical biosensors, the use of immunomolecules has also been improved upon. Researchers have demonstrated that the electrochemical detection of the common ELISA chromophore, 3,3′,5,5′-tetramethylbenzidine (TMB), can be used to create an electrochemical biosensor detecting the measles Antigen Protein via HRP-labelled secondary antibodies, in a similar configuration to what already occurs in conventional (colourimetric) ELISA systems . The ampantisera . Similarantisera , throughA strongly emergent thrust in South African sensor research is in the area of aptamers with researchers partnering with molecular biologists in the generation of new novel aptamers as part of an overall thrust in incorporating these into sensor assemblies . Most noAptamers are single-stranded olignoucleotides capable of selective and stringent binding to a range of compounds, generated during well-established sensor processes. In addition to the ease of synthesis, the selection of nucleic acids as biorecognition agents in biosensors is noted to have several benefits in terms of storage, e.g., An examination of A study published in 2008  demonstOf the biopolymer biosensors listed in Mycobacterium [Tuberculosis diagnosis via detection of circulating antibodies generated by the host against the mycolic acids produced by the tuberculosis acterium  was an iacterium ,38 and Hacterium  utilisinB-d-glucuronidase for Escherichia coli detection [The rapid detection of coliforms as an indicator of foecal contamination is hampered by long turnaround times, limiting rapid response to water contamination. Electrochemical biosensor configurations based on the detection of enzymes of coliform origin, etection , aimed tSensor assemblies utilising ZnO nanowires  as an inEnvironmental electrochemical biosensors for pesticides (in particular of organophosphates ) and herResearch on detection of phenolic substrates has been applied for total polyphenol content determination in wine at glassy carbon electrodes (GCE) modified with apple paste (as a source of polyphenol oxidase)  and at lGiven the current and future prospects of nanotechnology in the field of biosensors, it is important to discuss the developing interest in nanotechnology occurring in South Africa. Currently, members of the BRICS countries  publish more nanotechnology-related journal articles than any other countries within their Human Development Index Group .A leading aspect of South Africa’s growth in nanotechnology research is intimately associated with its National Nanotechnology Strategy of 2005, which has funded research, collaborative networking events , scientific meetings and, crucially, equipment. Access to state-of-the-art nanotechnology equipment through the Department of Science and Technology-led National Nanotechnology Equipment programme has been one of the key aims of the South African government in addressing equipment deficits in the country, as a means of growing nanotechnology research and development at a national level . NationaThis focus on nanotechnology funding and the provision of this budget led to the formation of Nanotechnology Innovation Centres (2007) at four sites within the country, each focusing on different aspects of the applied areas of the field . A speciThe national drive for increasing collaborative access to equipment and facilities also supports, in part, the country’s drive to innovate and develop products, as evidenced by several nationally approved strategies and development plans, most notably the country’s Department of Science and Technology Ten Year Plan for South Africa (2008–2018); (titled: Innovation towards a knowledge-based economy) and its National Research and Development Strategy. Within several of the national strategies, sensor and diagnostics are listed as either key technologies or enablers of other technology developments.In a more directed manner, national funding of the Nanotechnology Innovation Centre in Sensors, has supported a development node based in Mintek, South Africa’s national mineral research organisation, which seeks to further develop research from its higher education institutional partners into commercial products. In this regard, recent successes from Mintek include its prototypes for detection of measles  and emerThis legislative emphasis on supporting biotechnology research has, more generally, evolved research capacity and products in cognate disciplines for biosensor development such as: biomarker discovery, ; proBiosensor research in South Africa follows international trends in terms of its application of electrochemistry and nanotechnology. Many papers are aimed at new biosensor design approaches  or are aimed at conducting fundamental studies in sensor design. In terms of targets these also are not dissimilar to that studied internationally. Pesticides feature strongly (as has been noted elsewhere ), as do Indeed, the biosensor field may benefit from collaborative approaches in several areas including the development of new materials for biosensor assemblies. Substantial scope exists in this area through collaborations with scientists in nanomaterials research. Theoretical modeling of biosensor assemblies represents an additional area.Substantial scope exists for research (and associated investment) that addresses the need for point-of-care diagnostics in the healthcare setting. The Ebola outbreak of 2014 reminds us again of the need to focus both on low-cost diagnostics of relevance to the developing world, but of the need to accelerate technology transfer of such research.Despite several national strategies aimed at providing support for research commercialisation and innovation and despite the investment in fundamental research there is not yet a biosensor industry capable of commercialising biosensors for widespread use in the country. This lack of commercialisation or technology transfer is not solely a concern within the biosensor sector and may be explained by the poor links existent between research performed in higher education institutes (accounting for the greatest proportion of research papers in biosensors) and the industrial sector . HoweverBasic lateral flow diagnostics are however being produced commercially within the country and represent a key opportunity for developing low-cost rapid diagnostics that could in part support the great demand for point-of-care diagnostics in the healthcare sector. Certainly, opportunities exist to expand, for example, research in developing biorecognition agents (such as aptamers) which can be utilised in a range of biosensor assemblies and indeed in lateral flow diagnostics.


However, switching the developmental program of adult cells prior to organ regeneration is difficult in many plant species, especially in forest tree species. In these species, a decline in the capacity to regenerate shoots, roots, or embryos from somatic differentiated cells is associated with tree age and maturation. The decline in the ability to form adventitious roots from stem cuttings is one of the most dramatic effects of maturation, and has been the subject of investigations on the basic nature of the process. Cell fate switches, both in plants and animals, are characterized by remarkable changes in the pattern of gene expression, as cells switch from the characteristic expression pattern of a somatic cell to a new one directing a new developmental pathway. Therefore, determining the way by which cells reset their gene expression pattern is crucial to understand cellular plasticity. The presence of specific cellular signaling pathways or tissue-specific factors underlying the establishment, maintenance, and redirection of gene expression patterns in the tissues involved in adventitious root formation could be crucial for cell fate switch and for the control of age-dependent cellular plasticity.Cellular plasticity refers, among others, to the capability of differentiated cells to switch the differentiation process and acquire new fates. One way by which plant cell plasticity is manifested is through  Plant tissues have extensive regenerative capacity, and entire plants can be developed from single cells or small cuttings . In planDedifferentiation, i.e., the loss of a specialized form or condition previously acquired during development that can be manifested by the loss of morphological cell identity or by the re-entry into the cell-cycle of non-dividing cells, has been a central concept in plant regeneration. Although apparent dedifferentiation and re-specification of cells seems to occur , whetherde novo regeneration in non-recalcitrant model plant species demonstrate that there are many possible pathways and strategies for regeneration , WUSCHEL-RELATED HOMEOBOX (WOX), SHOOTMERISTEMLESS (STM) or LEAFY COTYLEDON (LEC), and the relevance of auxin and cytokinin signaling pathways in the regulation of key genes involved in the organization of stem cell niches have a role in de novo regeneration of Arabidopsis thaliana, Brassica napus, Medicago truncatula, or Gossypium hirsutum , was not dependent on the presence of exogenous auxin and was similar in competent and non-competent cuttings. These data suggest that the increased PAL level during the earliest stages of adventitious root formation does not seem to be a function of the rooting capacity of tissues, but rather a non-specific response to wounding. A higher transient increase of nitric oxide and a nitrate reductase gene expression were measured in juvenile cuttings of E. grandis  that has a positive effect on both adventitious and lateral root proliferation. The function of PtaERF003 is linked to the auxin signal transduction pathway, as suggested by the rapid induction and accentuated phenotypes of the transgenic plants in presence of the hormone.Among the transcripts that show significant changes in expression at the reorganization stage of adventitious root formation, several transcription factors involved in embryonic programs and cell fate determination in meristems have been identified . In poplZWILLE-LIKE, was found during the earliest stages, suggesting a role in the initiation of the root development process. Similarly, SCARECROW-LIKE gene in Monterey pine (PrSCL1) and in chestnut (CsSCL1), and a SHORT-ROOT gene in Monterey pine (PrSHR) that may play a role during the earliest stages of adventitious root induction. In Arabidopsis and other plant species, such as rice or maize, the establishment of an embryonic root meristem involves members of the GRAS family of putative transcription factors which includes SCARECROW (SCR), SCARECROW-LIKE (SCL), and SHORT-ROOT (SHR) proteins  acquisition of competence to regenerate through dedifferentiation, transdifferentiation or by use of pre-existing totipotent cells; and (2) repatterning of regenerating tissues. Searching for master regulatory genes is essential in the future of adult cell fate switch studies, but searching for master rechanneling genes, which could be among the earliest events associated with fate switching by re-directing the differentiation state, is equally important . In addied genes . The genThe author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The hypothalamic-pituitary-adrenal (HPA) axis and its end-effectors glucocorticoid hormones play central roles in the adaptive response to numerous stressors that can be either internal or external. Thus, this system has a strong impact on the brain hippocampus and its major functions, such as cognition, memory as well as behavior, and mood. The hippocampal area of the adult brain contains neural stem cells or more committed neural progenitor cells, which retain throughout the human life the ability of self-renewal and to differentiate into multiple neural cell lineages, such as neurons, astrocytes, and oligodendrocytes. Importantly, these characteristic cells contribute significantly to the above-indicated functions of the hippocampus, while various stressors and glucocorticoids influence proliferation, differentiation, and fate of these cells. This review offers an overview of the current understanding on the interactions between the HPA axis/glucocorticoid stress-responsive system and hippocampal neural progenitor cells by focusing on the actions of glucocorticoids. Also addressed is a further discussion on the implications of such interactions to the pathophysiology of mood disorders. Humans face in their daily activities unpredicted short- and long-term stressful events called stressors, which are either internal  or external   of the hypothalamus and prefrontal cortex  in a mixture with the immature newborn and mature neurons , the pituitary gland and the cortex of adrenal glands , a member of the steroid/thyroid/nuclear receptor superfamily, acts as a tissue receptor for glucocorticoids virtually in all organs and tissues including the CNS, and mediates the actions of these hormones in local tissues Kino, . GR is aMR, the closest family member to GR, also functions as a “glucocorticoid receptor” in the hippocampus Joels, . MR can Hippocampal progenitor cells maintain their lineage by self-renewal/proliferation, while they continuously differentiate and mature into neurons and other neural cell components. In addition, neural progenitor cells develop apoptosis and a majority of these cells die during their differentiation into neurons in the hippocampus. Thus, most of the regulatory activities toward hippocampal neurogenesis target these three major activities of the neural progenitor cells: proliferation, differentiation, and cell death/survival Shors, .in utero, damages the brain development of littermates during its fetal phase and changes behavior in later adult life by causing lifespan reduction of neurogenesis in the dentate gyrus and by blocking learning-induced neurogenesis in this brain area  pathway and ubiquitin-mediated degradation of cyclin D1 was suggested in glucocorticoid-induced suppression of their proliferation as potential underlying mechanisms  of this brain area, it is quite reasonable that hippocampal progenitor cells and the HPA axis/glucocorticoids stress system cooperate with each other through their mutual and multi-level interactions. Indeed, hippocampal neurogenesis is critical for the hippocampus-mediated negative control on the HPA axis (Snyder et al., The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Many respiratory viruses are mainly responsible for common cold, bronchitis, bronchiolitis, and pneumonia. Furthermore, asthma and chronic obstructive pulmonary disease (COPD) are major cause of mortality. The prevalence of asthma in developed countries is approximately 10% in adults and even higher in children Barnes, . Thus, tThis Research Topic contains 7 review articles and 3 original articles regarding pathophysiology of virus-induced asthma. As the first article, Kudo et al.  reviewedIn conclusion, we believe that these works are well summarized and informative to glimpse the field of virus-associated asthma/COPD, and may help understanding the basic and clinical aspects of the disease. We would be happy if this collection of papers will offer new stimuli and perspectives for not only researchers but also clinicians working around the exciting and emerging the e-book.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Cancer development is a multistep process in which exosomes play important roles. Exosomes are small vesicles formed in vesicular bodies in the endosomal network. The major role of exosomes seems to be the transport of bioactive molecules between cells. Depending on the cell of origin, exosomes are implicated in the regulation of several cellular events, with phenotypic consequences in recipient cells. Cancer derived exosomes (CCEs) are important players in the formation of the tumour microenvironment by (i) enabling the escape of tumour cells to immunological system and help initiating the inflammatory response; (ii) acting in the differentiation of fibroblasts and mesenchymal cells into myofibroblasts; (iii) triggering the angiogenic process; and (iv) enhancing the metastatic evolution of the tumour by promoting epithelial to mesenchymal transformation of tumour cells and by preparing the tumour niche in the new anatomical location. Since the finding that exosomes content resembles that of the cell of origin, they may be regarded as suitable biomarkers for cancer diagnosis, allowing for diagnosis and prognosis via a minimal invasive procedure. Exosome involvement in cancer may open new avenues regarding therapeutics, such as vectors for targeted drug delivery. RB1), cell cycle checkpoint related genes , genes encoding proteins involved in DNA integrity maintenance and in sustaining propagation of damaged cells , and genes involved in inhibition of apoptotic pathways  [The major modifications to normal cells that result in the formation of a tumour derive from mutational events in oncogenes or tumour suppressor genes that occur in a normal cell and will ultimately lead to uncontrolled growth . This ise.g., TP5, genes eDespite the importance of the modifications occurring at the cell level, the tumour microenvironment is also relevant for the development of cancer. In fact, in the case of epithelial tumours, it is the combination of tumorigenic cells and stromal cells that dictates the extracellular matrix composition of the carcinoma . The intIn this review we intend to highlight the relevance of exosomes in tumorogenesis, highlighting their biogenesis, composition, and main function, and then focusing on their role in cancer development and progression. Finally, we will address the potential of exosomes as biomarkers and their use for cancer therapy.Exosomes are formed in the endosomal network. The formation of the early endosomes occurs in the plasma membrane by the fusion of endocytic vesicles . The matAt the end of the maturation process, the multivesicular bodies (MVBs) composed of late endosomes together with ILVs, are situated close to the nucleus. The fusion of MVBs with the plasma membrane leads to the release of the ILVs to the extracellular environment, which are then referred to as exosomes. The releasing process of exosomes may be accomplished by the outward exosome and microvesicle budding pathway or by an inducible release, a highly regulated process that involves several components of the endocytic machinery, including the Rab GTPases, such as Rab11, Rab35, Rab27a, and Rab27b, cytoskeleton regulatory proteins, heparanase, and SNARES (soluble NSF attachment receptor) for target fusion –14. An iα), epidermal growth factor (EGF), and fibroblast growth factor (FGF), have also been found associated with exosomes [Exosomes are small vesicles ranging in size between 30 and 120 nm, composed by a lipid bilayer containing membrane proteins that surrounds a lumen comprising proteins and nucleic acids, that vary according to cell type and mechanism of biogenesis . As an eexosomes –23.Over 4600 different proteins have been associated to exosomes, including proteins from the cytosol, the plasma membrane, Golgi apparatus, and endoplasmic reticulum , 25. DueSeveral studies have shown that CCEs can alter the extracellular matrix through secretion of matrix metalloproteinases (MMPs) or activators of MMPs, such as HSPs. MMPs are zinc-dependent plasma membrane endopeptidases that can degrade extracellular matrix proteins, such as collagen, proteoglycans, fibronectin, and laminins . HakulinThe exosomal lumen is surrounded by a lipid bilayer enriched in (i) raft-associated lipids such as cholesterol; (ii) diglycerides; (iii) sphingolipids, such as sphingomyelin and ceramide; (iv) phospholipids; (v) glycerophospholipids, such as phosphatidylcholine (PC), phosphatidylserine (PS), phosphatidylethanolamine (PE), and phosphatidylinositol (PI); and (vi) polyglycerophospholipids . Interes in vitro translation of full-length proteins [One of the most distinct features of exosomes is the fact that they carry significant amounts of nucleic acids, including microRNAs (miRNAs) and mRNA, as well as mitochondrial DNA (mtDNA), piwi-RNAs (piRNAs), long noncoding RNAs (lncRNAs), ribosomal RNAs (rRNAs), small-nuclear RNAs (snRNAs), small-nucleolar RNAs (snoRNAs), and transfer RNAs (tRNAs) . Althougproteins , 34, sugproteins –39. miRNproteins . mRNAs aproteins . After iDespite the relevance of glycosylation of the membrane surface in the communication of cells with their extracellular environment, very few studies have been made to characterize the carbohydrate content of exosomes. The analysis of glycosylation patterns of exosomes derived from T cells, melanoma, and colon cancer cells revealed that the glycosylation signature seems to be conserved between exosomes and parental cell membranes . In anotThe major role of exosomes seems to be the transport of bioactive molecules between cells, with consequences in targeted cell phenotypes, such as mRNA and miRNA related to the transfer of genetic, and sometimes epigenetic, information between cells . AdditioThe importance of exosomes in tumorigenesis is emphasised by the general increased content of these vesicles in biological fluids of cancer patients relatively to healthy controls, being observed an increased content of exosomes as the tumour progresses . InteresFurthermore, CCEs have been implicated in tumour growth, survival, and spread, as well as in angiogenesis, escape from immune surveillance, stimulating tumour cell migration, conferring invasion ability to normal cells, and the preparation of distal tissues for their metastatic colonisation . The folThe mechanisms underlying interaction and fusion of exosomes with target cells remain undefined. It is believed that the uptake of exosomes by target cells may occur through three main mechanisms: (i) simple fusion of the exosome with the cellular membrane, directly releasing the content of vesicles into the cytoplasm; (ii) exosome uptake by endocytosis; (iii) uptake dependent on the presence of distinct receptor proteins that enable binding of exosomes to target cells . For theAs mentioned earlier, cancer development is a multistep process in which somatic cells experience events , accumulating genetic modifications that will ultimately result in uncontrolled growth of the cell . During An epithelial tumour mass is composed by stromal elements that frequently include an altered extracellular matrix enriched with cytokines and growth factors, fibroblasts, a scaffold composed of immune and inflammatory cells, endothelial cells, pericytes, mesenchymal cells, and in the case of more advanced tumours, blood and lymph vessels and nerves . Every sA common trait between all tumour pathologies is the phenotypic heterogeneity of the population of cancer cells within tumours. This is mainly due to genetic instability of cancer cells leading to genetic alterations, differential environmental stimuli, and stochastic processes that occur within the tumour microenvironment . It is lGenerally, early tumour microenvironment resembles the environment of wounds that never heal . In the As already referred, the communication between cells of the immunity system is mediated by direct contact or cytokine and chemokine production, which also involves exosomes . It is tβ1) and fibroblast growth factor-2 (FGF-2) [β, stressing the relevance of exosomes as effectors in the alteration of cancer stroma [Cancer associated fibroblasts (CAF) are the most prominent cell type in the tumour microenvironment of many cancers types, including colon, pancreas, and breast, and play critical roles in tumour-stromal interactions. The mechanisms inherent to CAF formation seem to involve the formation of myofibroblasts by differentiation of resident fibroblasts, epithelial and endothelial cells ), pericytes, bone-marrow-derived circulating fibrocytes and mesenchymal stem cells . This di (FGF-2) , 59 and r stroma , 61.The altered phenotype of CAFs will result in the increased production of alpha smooth muscle actin with consequences in the increased stiffness of the extracellular matrix . CAFs coβ, IL6, TNF-1a, and MMPs, that have been implicated in the induction of a stem cell phenotype in the microenvironment of tumour cells and promotion of metastasis [Another example of the role of CCEs in tumour progression is the release of exosomes containing the extracellular matrix MMPs inducer (EMMPRIN) by lung carcinoma cells . The reltastasis .β, PDGF, and IL-8 are some of the angiogenic factors that act on the regulation of quiescence, migration, and proliferation of endothelial cells, required for the stimulation of angiogenesis [The vascular formation inherent to the cancer progression may be triggered by hypoxic and nutrient depletion conditions in the tumour microenvironment, as well as by inflammatory responses, usually observed in epithelial cell carcinomas . The angogenesis .Recent studies have shown that exosomes released under hypoxic conditions contribute to the stimulation of angiogenesis . MelanomThe increased malignancy of a tumour, with several implications on cancer patient survival, consists in the formation of tumour metastasis. The metastization process is complex and involves several steps including (i) EMT; (ii) breach of the basement membrane barrier; (iii) migration of the cell through the neighbouring tissue; (iv) entry, transport, and exit from blood and lymph vessels; (v) establishment of the cell in a secondary anatomical site; and (vi) growth of the secondary tumour, by the creation of a new tumour microenvironment favourable to cancer cell growth . The rolDuring EMT, cancer cells loose the epithelial characteristics towards a more mesenchymal phenotype and acquire motility capabilities. The mechanisms inherent to this event are complex, involving cytoskeletal alterations and downregulation of expression of E-cadherin . InteresBesides involvement in EMT, exosomes seem also to be involved in the formation and preparation of the premetastatic niche at the new anatomical location. Interestingly, it was reported that exosomes of melanoma cells are preferentially taken up by sentinel lymph nodes and prepare the premetastatic niche by deposition in the extracellular matrix and vascular proliferation in the lymph nodes. Subsequently, free melanoma cells were recruited to the lymph nodes that have taken up the cancer derived exosomes . In anotAs already mentioned throughout this review, the content of an exosome depends on the cell of origin. Particularly, exosomes derived from cancer cells are enriched with proteins, mRNA, and miRNA that are more abundant in cancer cells than in normal cells . Hence, One of the major advantages of the use of exosomes as biomarkers is the possibility of a rapid pathology prognosis through a minimal invasive procedure. In fact, one of the higher challenges in the battle against tumours is an early and accurate diagnosis . CurrentFrom a therapeutically point of view, the natural role of exosomes as carriers of metabolites from donor cells to recipient cells and in inducing a biological response has been investigated by several researchers in a dual manner. Exosomes may be used both as mediators of tumour resistance and as vessels for targeted drug delivery. Several studies suggested a role of exosomes in drug resistance, by extruding hydrophilic drugs from cancer cells , 75, in α, in association with Treg cell-inhibiting treatments in patients with non-small-cell lung cancer that has been stabilised by chemotherapy [On the other side, since the first suggestions of the role of exosomes in the immune system performed by Raposo et al.  and Zitvotherapy .One other use of exosomes for cancer therapy is the possibility of using these vesicles for targeted delivery. This process may be accomplished by the targeting of cancer cells mediated by specific antibodies or ligands of highly expressed membrane receptors, further internalisation of exosomes and induced apoptosis of the cells, which may be mediated by miRNA, siRNA and anticancer drugs. Several studies have described the successful delivery and tumour inhibition using this process –88. As aCurrent knowledge of CCEs suggests that they can play an important role in the development and progression of cancer through modulation of intercellular communication within the tumour microenvironment by the transfer of protein, lipid, and RNA cargo. The complete understanding of their role in intercellular communication and tumorigenesis will be achieved by further exploration and comparison of their secretion in normal cells and during cancer development and progression, together with assessment of their specific content under these conditions. Exploration of CCEs contents may allow the development of novel diagnostic and therapeutic approaches, with minimally invasive procedures.The use of exosomes for targeted delivery may also prove to be the answer in iRNA based therapeutics, whose main drawback has been the development of an effective delivery system. Generation of synthetic exosomes—“exosome mimetics”—for drug delivery may also allow selective targeting of cancer cells (for a review see ). Althou


The striatum and the subthalamic nucleus (STN) are the main entry doors for extrinsic inputs to reach the basal ganglia (BG) circuitry. The cerebral cortex, thalamus and brainstem are the key sources of glutamatergic inputs to these nuclei. There is anatomical, functional and neurochemical evidence that glutamatergic neurotransmission is altered in the striatum and STN of animal models of Parkinson’s disease (PD) and that these changes may contribute to aberrant network neuronal activity in the BG-thalamocortical circuitry. Postmortem studies of animal models and PD patients have revealed significant pathology of glutamatergic synapses, dendritic spines and microcircuits in the striatum of parkinsonians. More recent findings have also demonstrated a significant breakdown of the glutamatergic corticosubthalamic system in parkinsonian monkeys. In this review, we will discuss evidence for synaptic glutamatergic dysfunction and pathology of cortical and thalamic inputs to the striatum and STN in models of PD. The potential functional implication of these alterations on synaptic integration, processing and transmission of extrinsic information through the BG circuits will be considered. Finally, the significance of these pathological changes in the pathophysiology of motor and non-motor symptoms in PD will be examined. The basal ganglia (BG) are a collection of interconnected subcortical nuclei, including the striatum, globus pallidus (GP), substantia nigra, and subthalamic nucleus (STN), which closely interact with the cerebral cortex and thalamus. While historically considered as key components of the motor system, the BG receive cortical projections from all functional areas of the cerebral cortex and contribute to both motor and non-motor functions  and the STN. In turn, two different populations of GPe neurons provide GABAergic innervation to all other BG nuclei. The so-called “arkypallidal” cells are the main sources of the pallidostriatal system, while the “prototypic” cells project massively to the STN, with collateral to the GPi and SNr  and associative  cortices, respectively, while the ventral striatum (nucleus accumbens and olfactory tubercle) is the main target of limbic-related inputs from the hippocampus, amygdala and medial prefrontal cortices  and parafascicular complex (Pf), which innervates preferentially the putamen or caudate nucleus, respectively , which represent 90–97% of all striatal neurons , but requires activity of the vesicular monoamine transporter 2 (vMAT2) for vesicular loading. The inhibitory GABAergic synaptic transmission from DA neurons does not depend on synthesis of GABA by either GADs  or GABA transaminase, suggesting that DA neurons inhibit MSNs by releasing GABA they acquire from the extracellular space using membrane uptake of GABA  vs. indirect (D2R-positive) striatal MSNs, recent evidence indicates that both neuronal subtypes are affected, but through different mechanisms. Some authors reported that D2R striatopallidal neurons, but not D1R striatonigral neurons, selectively lose spines in reserpine-(systemic administration) and 6-hydroxydopamine (OHDA)-treated  mice with striatal DA depletion  and massive growth of the spine apparatus Figures , in the In a recent study, Fieblinger et al.  used gluTogether, these recent findings  dysregulation likely contributes to this pathology  and related regulatory genes  participate in the loss of glutamatergic synapses and spines in the striatum  cell loss in various models of PD  of striatal neurons in 6-OHDA-treated rats and mice. However, the chronic use of L-DOPA and the subsequent development of LID in this model is linked with the development of aberrant and excessive corticostriatal axo-dendritic and axo-spinous synapses (Zhang et al., Although the striatum remains the BG structure that received most attention in studies of synaptic plasticity, recent evidence indicates that afferents to the STN are also morphologically and functionally disrupted in PD models. In MPTP-treated monkeys, a significant loss of vGluT1-containing cortical terminals has been reported, suggesting a partial degeneration of the hyperdirect corticosubthalamic pathway (Mathai et al., The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Amyotrophic lateral sclerosis (ALS)–motor neuron disease (MND) is divisible into 6 familial and 10 sporadic syndromes  . In thisThe second Mo deficiency-associated sheep disorder is inosine motor neuron syndrome (IMNS) . In thisIn SC-ALS, 30% of cases present with bulbar signs and in the remaining 70% bulbar signs usually develop during the course of the disease. Bulbar involvement in SC-ALS is often closely correlated with respiratory muscle weakness, including reduced forced vital capacity .via abnormal upper motor control over the pontomedullary respiratory center, which then affects the phrenic nerve neurons in the cervical spine; or second, via abnormal activity of the cervical phrenic nerve neurons themselves. In IMNS, the former is the case, whereas, in SC-ALS, it is more likely that that the former occurs first but the latter develops later on.Inosine ingestion during Mo deficiency is the only etiology so far demonstrated in a mammal, which can produce this type of progressive and irreversible muscle weakness. The lingual, laryngeal, and pharyngeal muscle dysfunctions that cause the bulbar effects can be generated by either abnormal upper motor control over the hypoglossal nuclei in the medulla or by abnormal functioning of the hypoglossal nuclei themselves. The diaphragm and intercostal muscle dysfunction that causes the respiratory effect can be generated in two different ways: first, The third part of SC-ALS that occurs in animals has only been observed in horses  and catsIn EMND, these motor nervous effects are caused by the degeneration of the lower motor neuron affiliated neurons in the brain stem and the spinal motor neurons in the ventral (anterior) horns. In SC-ALS similar effects occur and are caused by the same neuronal degeneration. It is noteworthy that in EMND there is no report of asymmetric limb muscle weakness, bulbar muscle weakness, or respiratory muscle weakness. The cause of EMND is thought to be dietary but no specific imbalance has been demonstrated, so far. Low levels of the antioxidant, vitamin E, are frequently identified in affected horses , 10. BasMolybdenum deficiency predisposes mammals to CNS disease because it inactivates both xanthine oxidase-dehydrogenase (the enzyme barrier that protects the CNS from dietary purine loading) and sulfite oxidase (the enzyme barrier that protects the CNS from dietary sulfite loading). Mo is an essential cofactor for these two enzymes. XMNS and IMNS are caused by purine loading, and EMND could potentially be caused by sulfite loading. Dietary sources of the purine nucleosides xanthosine, inosine, adenosine, and guanosine, and their bases xanthine, hypoxanthine, adenine, and guanine, respectively, can cause purine loading, and dietary sources of sulfur amino acids can cause sulfite loading. Purine loading leads to astrocyte degeneration , 6, and The author proposes that Mo deficiency in humans makes them susceptible to the potential neurotoxic effects of dietary xanthosine, inosine, and sulfur amino acids, and it is the combined effect of these three substances that produces SC-ALS. The following circumstantial evidence supports the notion that a large number of humans are Mo deficient, hence, at risk of this disease. The daily Mo intake per person in the US is 0.18 mg  and in tOne final aspect of Mo deficiency requires some comment. Mo deficiency can potentially produce both astrocyte degeneration and neuron degeneration. The contribution that neuron degeneration makes to the pathogenesis of SC-ALS is universally agreed upon; however, there is also an emerging awareness of the potential contribution of astrocyte degeneration to this disease . Both XMThere is an accumulation of evidence suggesting that some sporadic cases of ALS are at least partly due to genetic mutations . In the The author confirms being the sole contributor of this work and approved it for publication.The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


FragariaXananassa) crop draws increasing attention as plant model system for the Rosaceae family, due to the short generation time, the rapid in vitro regeneration, and to the availability of the genome sequence of F.Xananassa and F. vesca species. In the last years, the use of high-throughput sequence technologies provided large amounts of molecular information on the genes possibly related to several biological processes of this crop. Nevertheless, the function of most genes or gene products is still poorly understood and needs investigation. Transient transformation technology provides a powerful tool to study gene function in vivo, avoiding difficult drawbacks that typically affect the stable transformation protocols, such as transformation efficiency, transformants selection, and regeneration. In this review we provide an overview of the use of transient expression in the investigation of the function of genes important for strawberry fruit development, defense and nutritional properties. The technical aspects related to an efficient use of this technique are described, and the possible impact and application in strawberry crop improvement are discussed.Beside the well known nutritional and health benefits, strawberry ( Rosaceae family due to its small genome size, the short generation time for a perennial species  RNA interference (RNAi). This can be achieved through the introduction of a double-stranded RNA (dsRNA) homologous in sequence to the gene of interest, resulting in the degradation of the target gene transcripts and leading to a knockout or a knockdown phenotype, depending on the specific silencing efficiency . The silNicotiana benthamiana, the viral protein P19 was shown to prevent the onset of PTGS allowing high level of transient expression of genes  from the bacterial tumour-inducing (Ti) plasmid to plant cell. The target gene (either for overexpression or for RNAi silencing), inserted into the T-DNA depleted of tumor-inducing genes, can be efficiently expressed under the control of a eukaryotic promoter. Once moved into the plant cell, the T-DNA migrates to the nucleus and only a tiny part is integrated into the host chromosomes; these not-integrated T-DNA copies persist in the nuclei of transfected cells remaining transcriptionally competent for several days and leading to non-stable, transient transcripition  it does not interfere with the stability of the host genome , (ii) itt assays  each witt assays . Howevert assays . On the function . Here, tripition . This tein silico prediction. Only recently, reverse genetics approaches were used to assign gene functions .In the past, functional genomics in strawberry was mainly confined to gene expression analysis and unctions . A numbeoduction . Indeed,oduction  and seveoduction . In addioduction . For theAgrobacterium using the β-glucuronidase (GUS) reporter gene interrupted by an intron. Consistently, this system has been firstly used for the functional analysis of two strawberry β-1-4 glucanases promoters or, combined with a biolistic transformation protocol, to functionally characterize other homologous and heterologous promoters (The first report of transient transformation in strawberry fruits goes back to 2001 when romoters .Table 1). Strawberry fruit ripening is accompanied by an increase of flavonoid biosynthesis resulting in anthocyanin accumulation at ripe red stage , the key gene in the flavonoid pathway, was chosen as a reporter gene to set up an efficient protocol for RNAi-based transient gene silencing in strawberry fruit: by agroinjecting receptacles with an inoculum of Agrobacterium carrying an intron-containing self-complementary hairpin construct, a drastic reduction in mRNA levels and enzymatic activity of CHS gene was obtained, leading to the loss of pigmentation in ripe strawberry fruits , a gene encoding for the enzyme involved in the anthocyanin biosynthesis, an Agrobacterium carrying a T-DNA expressing an RNAi-FaGT1 construct was injected into midsized ripening strawberry fruits. This resulted in reduced concentrations of anthocyanin pigments and in a significant increase in epiafzelechin, the compound sinthesized by the anthocyanidin reductase (ANR) enzyme, indicating competition between FaGT1 and FaANR for the common athocyanidin substrate and showing that FaGT1 is a key gene in the anthocyanin pathway .With the aim to elucidate  pathway . More re pathway . These wFra a1 gene, was RNAi-silenced, leading to a decreased endogenous concentration of the main flavonoids responsible for the red color of fruits and to a decrease of the expression levels of genes encoding for phenylalanine ammonia lyase (PAL) and CHS. These results demonstrated that Fra a1 is directly linked to flavonoid biosynthesis and that its function may be regulatory  genes in contributing to volatile compounds production responsible for strawberry flavor was studied by modifying the anthocyanin biosynthesis pathway in strawberry fruit using a simultaneous downregulation and ovexpression of CHS and EGS genes, respectively. This led to the deviation of the flavonoid pathway to the synthesis of phenylpropene, such as eugenol, confirming the role of EGS enzyme in the production of strawberry aroma  was found to be involved in the resistance of strawberry to Botrytis cinerea (B. cinerea fungus and increase in PAL activity and content of phenolic compounds. These results indicated that this gene plays roles not only in fruit growth regulation but also in the fruit response to pathogens. More recently, Agrobacterium transient transformation was successfully applied to study the role of the gene FaMBL1, encoding for a Mannose Binding Lectin. Expression of this gene was originally found upregulated exclusively in unripe strawberries upon infection with the pathogen Colletotrichum acutatum, but not in infected ripe ones. Since, contrary to red ripe fruits, white unripe fruits are reluctant to C. acutatum infection, a role for this lectin gene in the different susceptibility of strawberries during ripening was hypothesized. To test this hypothesis, Agrobaterium-mediated transient transformation was performed to silence and overexpress FaMBL1 gene in unripe and ripe strawberries, respectively, before C. acutatum infection. FaMBL1-silenced unripe fruits showed anthracnose symptoms, whereas FaMBL1-overexpressing ripe strawberries presented a lower susceptibility to C. acutatum. These experiments allowed concluding that this gene plays a crucial role in the resistance of unripe strawberry fruits to C. acutatum (By using a VIGS RNAi approach, a β cinerea ; silenciacutatum .Agrobacterium-mediated transient transformation for studying plant pathogen interactions: Agrobacterium itself is a plant pathogen and can interfere with plant defense response to stress, resulting in misleading interpretation of data. However, by using the appropriate controls, this technique can be a powerful tool for discovering function of genes involved in strawberry–pathogen interaction, and to unravel the complex network of defense signaling pathways in this important crop.Particular attention musts be paid while using Despite the large amount of molecular information originated from the use of new high-throughput technologies, providing useful data to improve strawberry crop, the knowledge of the role of most genes involved in important physiological processes is still scarce.The advances in the last two decades of high efficiency stable and transient transformation protocols for strawberry, open new frontiers to understand the role of genes regulating important biological processes and potentially exploitable for strawberry improvement through breeding or genetic engineering.However, the high cost, also in terms of time, of developing transgenic over-expressing or silenced lines for determining gene function, limits their usefulness to the analysis of few candidate genes. The transient transformation approach represents a rapid alternative and a highly efficient method for gene function discovery, opening new scenarios at the service of genetic engineering for strawberry crop improvement. Future research focused on the development of valuable molecular tools capable to extent the effect of transient expression, and to overcome post-transcriptional gene silencing, such as, for example, the above mentioned P19 viral protein, would be very important to allow technical advances and promote wider applications of this investigation method.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Childhood obesity when associated with serum lipoprotein changes triggers atherosclerosis. Evidences suggest that the atherosclerotic process begins in childhood and that the extent of early atherosclerosis of the aorta and coronary arteries can be associated with lipoprotein levels and obesity. Furthermore, many studies in childhood demonstrate an important relationship between parameters of insulin sensitivity, body fat distribution, and the development of lipid abnormalities. This review focuses on the most recent findings on the relationship between obesity, dyslipidemia, and cardiovascular risk in children. Childhood obesity represents one of the most important public health issues, due to its associated metabolic and cardiovascular comorbidities . In spitThe prevalence of childhood obesity remains high not only in the United States . In the During the last decade, the prevalence of atherogenic dyslipidemia is increasing in obese children and adolescents , 5. DataThe atherogenic dyslipidemia is characterized by hypertriglyceridemia, very-low-density lipoprotein (VLDL) increase, small dense LDL (sdLDL) particles, and HDL cholesterol reduced levels  thus shoAmong different features that characterize MetS the lipoprotein profile plays a basic role . The ratEvidence exists suggesting that the atherosclerotic process begins in childhood , 13 and Moreover, overweight adolescents who remained overweight in adulthood had, respectively, 2.4, 3, and 8 times greater prevalence of abnormal LDL cholesterol, TG, and HDL cholesterol concentrations than those who remained lean .Although the pathogenetic mechanism of dyslipidemia is multifactorial and still debated, visceral obesity and insulin resistance represent one of the most important markers of its progression.In the present review we illustrate the most recent findings concerning the impact of insulin resistance and visceral distribution on dyslipidemia in obese children and adolescents. Furthermore, we describe the relationship between atherogenic dyslipidemia and cardiovascular disease in pediatric age and the current knowledge regarding methods to study the lipid profile.Obese children and adolescents have been observed to have a more unfavourable lipid profile than children and adolescents with normal body weight , 15. TheAlthough the pathophysiology underlying the development of dyslipidemia in obese children is multifactorial and not yet completely defined, insulin resistance has been hypothesized to play a major role in the relationship between dyslipidemia and obesity , 16. SevIt has to be acknowledged that some studies defined insulin resistance on the basis of fasting insulin values or insulin sensitivity indices. Interestingly, Burns et al.  have evaTo strengthen the relationship between lipid profile and impaired insulin sensitivity, recent evidences  have demThe precise mechanisms by which insulin resistance leads to the development of atherogenic dyslipidemia remain unknown; however several pathways have been postulated , 22. InsInterestingly, Karpe et al.  have recper se” but the distribution of fat partitioning [Obesity is the most important cause in the development of insulin resistance and several findings have shown that the critical determinant of insulin sensitivity and its related complications is not the degree of obesity “itioning . By straitioning  observeditioning , 27 haveitioning , visceraIn spite of the demonstrated relationship between visceral fat, insulin resistance, and dyslipidemia, the ectopic fat deposition in the liver is emerging as one of the most important markers of metabolic abnormalities in children .Pediatric nonalcoholic fatty liver disease (NAFLD) is becoming the most frequent chronic liver disease in obese children and adolescents. A growing body of evidence from epidemiologic studies in both adults and children has established NAFLD as an independent predictor for development of MetS, diabetes, and cardiovascular disease , 30. SimIn the study by Schwimmer et al. , the autSeveral studies , 28 haveIn accordance with the latest, Nobili et al.  have demThus, although insulin resistance plays an important role in the development of atherogenic dyslipidemia in obese children, the abdominal fat deposition is emerging as an important link between insulin resistance and lipid alterations in obese children and adolescents.Although atherosclerotic cardiovascular disease seems to be rare in the paediatric age group, the atherosclerotic process and the risk factors associated with its development begin in childhood .The coronary atherosclerosis is a complex trait that recognizes multiple risk factor variables. In the Bogalusa Heart Study the prevalence of fatty streaks was 50% during childhood and 85% during young adulthood and the prevalence of fibrous plaques increased from 8% in childhood to 70% in young adulthood .Longitudinal cohort studies from childhood conducted in Bogalusa, Louisiana (USA) , in MuscLipoproteins represent important atherosclerotic determinants and changes in the main lipoprotein fractions characterize the MetS. These changes involve a cascade of events including increased TG and sdLDL levels and decreased HDL cholesterol , despite normal LDL cholesterol levels . The corThe role of TG has been controversial for decades  and evenIncreasing TG levels cause profound changes in the physicochemical composition of HDL, VLDL, and LDL particles, and the particle core, represented by cholesterol esters, is progressively depleted and replaced by TG. The remodeling of larger to smaller lipoprotein particles is promoted by lipoprotein lipase and hepatic lipase , active A variable degree of correlation between coronary cardiovascular events and LDL size has been observed to vary with age, gender, and ethnicity . The preSdLDL show increased susceptibility to oxidation, thus promoting endothelial damage and infiltrating the arterial wall . These mMoreover, sdLDL shows a strong correlation with the common cIMT, as outlined in a prospective study conducted in healthy males submitted to B-mode ultrasound, so providing evidence of cardiovascular risk by this surrogate marker .Concerning the predictive usefulness of sdLDL on cardiovascular disease risk, results are conflicting. On the basis of multivariate analysis, and after doing adjustment for confounding variables, in particular TG and HDL cholesterol, this association was confirmed or not as an independent variable , 50, 51.per se” is questionable [If the raised sdLDL particles relevance to coronary heart disease should be ascribed to their increased number or to the particle size “tionable . Increastionable .Thus, any final relationship between sdLDL and increased cardiovascular risk is not proven and the conclusion to consider LDL size a significant and independent predictor of cardiovascular risk is still debated.In conclusion, according to ESC/EAS Guidelines, determination of sdLDL may be regarded as an emerging risk factor that may be used in the future but is not currently recommended for risk estimation .The cardiovascular risk prevention represents now a hot topic in children. Thus, several studies focus on identifying the link between unfavorable biomarkers profile and/or the presence of clinical indicators of atherosclerosis and the increased risk of cardiovascular disease , 53.Biochemical markers of atherogenic particles, including LDL cholesterol, ApoB, and non-HDL cholesterol, are available and widely analyzed in both children and adults .LDL cholesterol is considered the “gold standard” parameter for characterizing the cardiovascular disease risk when isolated hypercholesterolemia occurs, but its power decreases when TG levels are increased .Non-HDL cholesterol includes TG-rich lipoproteins, remnants of TG-rich lipoproteins, and lipoprotein(a), so the non-HDL cholesterol values represent a better predictive indicator of cardiovascular disease than LDL cholesterol one, as demonstrated by observational and intervention studies . FurtherAccording to the Expert Panel on Integrated Guidelines for Cardiovascular Health and Risk Reduction in Children and Adolescents , the evaIt has to be acknowledged that non-HDL cholesterol levels in children and adolescents vary with age, sex, and race/ethnicity . Thus, iThe pathogenic linkage between obesity and metabolic and cardiovascular diseases leads to an increased attention to the relationship between adipocytokines levels and cardiovascular pathology , 61.Among the identified adipokines related to cardiovascular disease , adiponeMoreover, Stakos et al.  have recA growing body of evidence supports the role of subclinical indicators of atherosclerosis such as increased cIMT assessed with ultrasound, increased left ventricular mass with cardiac ultrasound, endothelial dysfunction  with brachial ultrasound imaging, and the demonstration of coronary calcium on electron beam computed tomography imaging , 65, 66.The association between cIMT and atherosclerosis biomarkers has been shown by several studies in adults and in children , 65, 67.Brachial arterial flow-mediate dilatation (FMD) is further noninvasive method to evaluate functional changes in the arterial wall , 66. MosAmong the subclinical atherosclerosis markers in children, measurement of coronary artery calcifications (CAC) is emerging as important indices of future atherosclerosis . In adulWong et al.  demonstrRecently, Bacha et al.  have evaA total of 50% of patients showed CACs and adiposity as the major determinant of these vascular alterations .Further research is needed to establish how cardiovascular risk factors affect clinical sign of cardiovascular disease.Traditional analyses of lipids and FA include spectrophotometric kits to measure serum levels of TC and TG and high performance liquid chromatographic (HPLC) separation using normal-phase techniques and evaporative light-scattering detection enables the separation of major classes of lipids, such as cholesteryl esters, TG, free cholesterol, phosphatidylethanolamines, phosphatidylcholines, sphingomyelins, and lysophospholipids, as well as gas chromatography of FA composition of total serum phospholipids or cholesteryl esters, which is still a method of choice with respect to interpreting fatty acid metabolism. Other common lipid analyses methods include thin-layer chromatography (TLC), gas chromatography (GC), and nuclear magnetic resonance (NMR) spectroscopy.However, the recent expansion in research in the field of lipidomics has been driven by the development of new tools and protocols for the identification and quantification of molecular lipids in different biological samples . Mass spData analysis can be performed with different approaches, even though the most successful are those megavariate statistics incorporating existing biological knowledge into the statistical analysis, enabling the generation of integrative knowledge for systems level investigation of lipidomics .Interestingly, more recently the application of advanced integrated methodologies to analyze global lipidomics in human plasma has revealed a remarkable diversity of lipids that may represent an individual signature of the roles of lipids in health and diseases; thus it seems likely that plasma lipidome has emerged as one of the tool-sets for bringing it to practice.On the bioinformatics side, the major limitation is the virtual absence of comprehensive and integrated reference databases. Communication of results from the global analysis of cellular lipidomes is complicated by the convoluted nomenclature of lipids. Lipid Analytical Tool (LIPIDAT) and Lipid Bank provide some information on structure and nomenclature but are not comprehensive enough on functional information and the provision of links to protein and gene data. Current efforts in both the public sector  and private sector  are addressing this need. Databases and search algorithms will also aid in the annotation of known lipid metabolites and the identification of novel lipid metabolites. Future reference bases will be more integrated and take into account our knowledge of lipid biosynthetic pathways and protein-lipid interactions. The availability of such databases will be a prerequisite for future integration of lipidomics into proteomics and genomics.In conclusion, a growing number of scientific data support that atherosclerotic process begins in childhood and represents an increasing health problem in obese children and adolescents. Visceral fat distribution and insulin resistance represent important markers of its progression. Further research is needed to identify early indicators of lipid alterations and to fully define the underlying pathophysiology. The complete characterization of the mechanisms involved in the development of atherogenic dyslipidemia in children could help in the identification of preventive and therapeutic strategies and thus leads to the reduction of the associated cardiovascular complications later in life.


In this context, integration and meta-analysis approaches are gaining importance for analyses and reuse of data as, for example, those concerning the bone and cartilage biomarkers, the biomolecular factors intervening in cell differentiation and growth, the morphology and the biomechanical performance of a neo-formed tissue, and, in general, the scaffolds' ability to promote tissue regeneration. Therefore standards and ontologies are becoming crucial, to provide a unifying knowledge framework for annotating data and supporting the semantic integration and the unambiguous interpretation of novel experimental results.In the last decades, a wide number of researchers/clinicians involved in tissue engineering field published several works about the possibility to induce a tissue regeneration guided by the use of biomaterials. To this aim, different scaffolds have been proposed, and their effectiveness tested through In this paper a conceptual framework has been designed for bone/cartilage tissue engineering domain, by now completely lacking standardized methods. A set of guidelines has been provided, defining the minimum information set necessary for describing an experimental study involved in bone and cartilage regenerative medicine field. In addition, a Bone/Cartilage Tissue Engineering Ontology (BCTEO) has been developed to provide a representation of the domain's concepts, specifically oriented to cells, and chemical composition, morphology, physical characterization of biomaterials involved in bone/cartilage tissue engineering research.Considering that tissue engineering is a discipline that traverses different semantic fields and employs many data types, the proposed instruments represent a first attempt to standardize the domain knowledge and can provide a suitable means to integrate data across the field. Several tissue engineering approaches have been proposed in the last years with the aim to mimic the natural processes of tissue regeneration (such as bone and cartilage), by delivering progenitor cells capable of differentiating into osteoblasts and chondrocytes inductive molecules and bioactive three-dimensional scaffolds supporting cellular colonization and matrix deposition .in vitro and in vivo experiments have been carried out in order to test new scaffolds able to guide the process of ex vivo connective tissue formation. Experimental preclinical models showed promising results of high scientific interest. However, this approach is far from being considered optimal and mechanisms of tissue formation/remodelling are still not completely elucidated. In particular the cell differentiation process has not been clarified yet, as well as the scaffold features that can positively or negatively influence the cellular fate and, finally, the tissue growth [In the last decades, a wide number of e growth . The idee growth .The complexity of these interdisciplinary studies is even increased by the generation of various types of data at various level of detail, and by the lack of concept models and software infrastructure helping data management and exploitation. In particular, technical details about experiments reported in literature are often scarce and barely accessible to point out the functional effectiveness of tested materials. Systematic reviews and meta-analyses approaches are often based on public scientific literature, thus requiring concepts definition and standardization.http://bioportal.bioontology.org/ontologies/BCTEO.Informatics tools are necessary to organize, and favour accessibility, integration, interoperability and sharing of data, which regards multiple and diverse scientific aspects, such as the presence and/or quantification of bone and cartilage biomarkers, the biomolecular factors intervening in cell differentiation and growth, the morphology and the biomechanical performance of a neo-formed tissue, and, in general, the scaffolds' ability to promote tissue regeneration. In this work a conceptual framework is presented for bone and cartilage tissue engineering domain: authors have defined practical guidelines to drive a structured, reliable and complete method for publishing and sharing  data coming from tissue engineering experiments. Moreover, an ontology, defined as a set of representational primitives suitable to model a domain of knowledge , has beeData standardization is a rapidly growing area of research, especially in life sciences domain. It represents the basis for data exchange, queries support, publication of reusable knowledge bases and interoperability facilitated across multiple and heterogeneous systems and databases. Standards for structuring data related to the biological field have been proposed, to treat different types of data. In particular, guidelines have been suggested for many aspects of molecular biology defining the minimum set of information (metadata) needed to unambiguously describe experiments and data (for reproduction and interpretation). Major examples of this approach are presented in Table For what concerns ontologies, biomedical research area is rich of structured vocabularies and tools to manage them. Ontologies have been developed for genes, proteins, tissues, cells, chemical compounds, anatomy etc. The most used facilities for ontologies consulting and screening are OboFundry , NCBO BiNevertheless, no standardization approaches have been developed for tissue engineering. The only semantic instruments provided in this field at the moment seem to be: (1) for what concerns bone domain, the Bone Dysplasia Ontology , a comprAdult stem cells demonstrated their multipotency leading to the formation of cartilage, bone, muscle, connective tissue or fat .Autologous stem cells are routinely used in conventional tissue engineering ex vivo in combination with biomaterials to create a cell-based scaffold for replacing or improving the tissue regeneration in vivo. Briefly, cells are typically harvested from donor tissues, isolated and expanded in culture and then associated with biomaterials both of synthetic and biological origin. The cell-material construct may be either cultured in vitro or implanted in vivo, using selectively an ectopic or orthotropic animal model.In this context, biomaterials have to provide informative microenvironments: material intrinsic nature and structure will anyway transmit a signal that has to be decoded by the colonizing cells and converted into active cellular response. A simplified schema of bone/cartilage tissue engineering approach is shown in Figure The list of minimum information to explain experiments about connective tissue growth on substitute materials has been identified by means of the expertise that authors gained in tissue engineering and in data management and integration. Relying on this experience, literature was manually overviewed for identifying recurrent concepts in papers. A primary list was then enriched with additional information that is often lacking in many published contributions but that appears basic for a correct and complete data report.On the base of the identified guidelines, which schematically point out macro-areas of the domain, BCTEO has been designed. An ontology is the formal specification of a conceptualization of domain, which consists of a common vocabulary and logical structure providing knowledge framework for annotation, semantic integration, knowledge-based searching and unambiguous interpretation of data . It provBCTEO was developed with the OBO-Edit software in the OBO format , the mosTo improve the process of ontology creation, authors followed the steps provided by Noy and McGuinness  and consFor what concerns ontology validation, two aspects have been considered: structure and concepts validation. Structure validation, that regards the consistency of the relations among terms, is guaranteed by the exploitation of the OBO-Edit internal "reasoner", a tool that automatically helps to identify formal errors in the ontology. Concepts validation, which is related to the evaluation of the correctness of the terms inserted in the ontology and their definitions, has been assured by authors' expertise in tissue engineering and strengthened by supervision of skilled end-users .Due to the multidisciplinarity of tissue engineering field, many terms crucial for BCTEO were found already classified and defined in other ontologies. The identification of cross-references among ontologies was aimed to avoid redundancy and terms duplication, to simplify vocabularies exploitation and link among partially overlapping domains. Structured vocabularies containing useful concepts for BCTEO development, and intervening in it, are described in Table Terms already included in other ontologies have been treated as follows: (1) the value of their namespace has been inherited from the already existing ontology; (2) the term definition is retrieved from the existing one; (3) the term identifier of the already existing ontology is recorded in the 'xref' field, in order to indicate term reuse.The value of biological data and the wish to reuse information to infer novel knowledge brought to increase data sharing and exchange in various biological fields. Other than influencing the creation of always more efficient storage infrastructures, this led to demand for standards and vocabularies suitable to enable data compatibility and comparability. Currently, the tissue engineering research field is completely unsupplied with informatics instruments to homogenize information and promote meta-analysis. For filling this lack, (1) an information model, for domain structuring, and (2) an ontology, for domain description, have been developed for bone/cartilage tissue engineering applications.Authors' experience in tissue engineering enabled generating a critical view about publications in this research field. Commonly, provided data and metadata do not rely on standardized information sharing schemas, limiting possibilities of data exchange, replication and reuse. To answer the need for standardization, a conceptual framework has been proposed, which provides a structure for communicating observations and improving the quality of the contents exposed in a scientific paper. This would facilitate the comprehension of the exposed concepts by readers, the information extraction and the classification of knowledge. Data generated in tissue engineering field are usually complex, due to the high number of variables intervening in the study, and would be meaningless unless a defined standard states which types of data are needed to support and verify conclusions. The identified macro-areas are: (1) 'Material': the identity of the scaffold material and its properties; (2) 'Cell': the type of used cells and the donor; (3) 'In vitro experiments': cells treatment before seeding including quantities, times and growth factors addition; (4) 'In vivo experiments': host and implant model; (5) 'Results': outcome description, consisting in the evaluation and report of the matrix deposition/tissue generation, the material resorption/degradation or adverse effects, such as cytotoxycity; (6) 'Tissue/matrix characterization': morphology, mechanical, biomolecular and biochemical tests to characterize the neo-formed tissue/matrix and to obtain a qualitative and quantitative evaluation of the scaffold performances.On this basis, the set of minimum information needed to communicate experiments' setup and results has been developed, and detailed in Table In order to provide roadmaps for data structuring in connective tissue engineering field, a finer informatics tool has been developed: BCTEO. Ontologies represent a source of standardized and recognised descriptive terms: a term belonging to a controlled vocabulary is more precise in the meaning in respect to free-text, since it presents a well established, commonly accepted definition, and a set of synonyms. Furthermore, an ontology is characterized by the relationships among terms, that help for data classification and annotation, semantic based information search , and even inference of new relations among data.The core of BCTEO conceptually refers to the guidelines proposed in the previous section and it is organized into five main classes: Biomaterial, Experiments, Organism, Tissue, Cellular response. Classes interact at various levels to model the knowledge about bone and cartilage substitute materials.For correctly describing the interactions among ontology classes, new relations have been defined. Additionally to Relation Ontology (RO)  terms , little contributions were provided by existing structured vocabularies to describe and define investigations on the in vitro and in vivo outcomes. As emerges from Figure Biomaterials have been classified on the basis of their chemical composition into 'ceramics', 'polymers', 'metals', 'gels' and 'composites'. Materials are even characterized by specific features, which define their behaviour considering two perspectives: (1) the intrinsic properties describe chemical and physical aspects and can be associated directly to each material; (2) the scaffold-related properties depend on the size, the shape and the surface of the graft and are clustered into mechanical and morphological aspects.BCTEO and the proposed guidelines have been validated through the consensus of experts. A survey has been designed and distributed to 12 skilled tissue engineers from all over the world  would enable their better interpretation, automatic collection and aggregated analysis, thus easily achieving the inference of scaffolds' performance and their comparison. Unfortunately, few analysis techniques report results quantification on numerical basis, while a widely exploited approach for results presentation consists in showing figures reporting images (i.e. for what concerns histology data) or diagrams (such as histograms showing genes or proteins expression under different conditions). Therefore, results evaluation and comparison are often performed visually, obtaining not quantifiable data. For example, histological data showing the potential of scaffolds to support newly formed tissue formation is always reported as a stained image, although it should be shown as a numerical value, besides the histological section. To this aim, in the last years image processing approaches have been published, addressed to quantify the newly formed tissue and/or the matrix deposited within the implant, through segmentation algorithms for either histological -19 or x-In addition to the guidelines an ontology has been proposed, whose terms mainly map on information and concepts suggested in the roadmap sections. BCTEO represents the first instrument for semantic approach in tissue engineering field: among other major applications, this ontology can be used as the standardized semantic layer when developing structured collection of data in this domain. Relying on ontologies, database technologies can, for example, promote data comparison and enable faster and more effective queries: characterized by a hierarchical structure, a query on the term 'metal' will retrieve even children terms such as 'stainless steel' and 'titanium', that would be excluded from the results not considering the semantic approach. Another interesting aspect of BCTEO concerns its devotion to a multidisciplinary scientific field that results in the integration of the biological perspective , with the materials science details  and the measurement methods (regarding different techniques to test the neo-formed tissue): this generates a wide presence of cross-references among BCTEO terms and definitions from external ontologies.Both the developed tools have been validated through a survey proposed to a panel of expert tissue engineers. Some consideration emerges from resulting data. Interestingly, all experts involved in the validation assigned the highest scores to concepts belonging to the "cellular response" section, thus highlighting the importance the scientific community feels about reporting complete details concerning this experimental aspect. Surprisingly, in fact details about molecular, biochemical, mechanical analysis after cell-biomaterial interaction are often missing or incomplete; moreover, only in few cases results are shown as numerical values in scientific papers.Another interesting observation related to the end-users validation was the disagreement about the features that describe the "cell" section: although experts claim to appreciate a wide number of details about cell source and expansion/culture protocols, they often disagree on the type of required information. This outcome reflects the scientific literature scenario, where experimental data are typically reported in heterogeneous, not standardized mode. In the here presented tools a wide number of features have been considered, in order to integrate different needs and finally obtain a complete set of mandatory features.Regulation of bone and cartilage tissue formation and remodelling is one of the main targets of tissue engineering research field. Lots of experiments have been performed in this context, testing the effectiveness of novel substitute materials that promote tissue regeneration. Although widely investigated, it remains a not completely elucidated research field, due to its multidisciplinarity and the complexity of the factors intervening in the experiments. To help scientists in highlighting advantages and disadvantages of biomaterials a theoretical roadmap for experiments description and an ontology for semantic approaches have been designed. Both act in the perspective of information standardization and data sharing, aiming to facilitate data comprehension, helping information classification, recognition and annotation, promoting comparison between works to better evaluate materials response, and hypothesising novel graft features and compositions. The overall platform has been validated by means of the end-user perspective.Considering that tissue engineering is a discipline that concerns many scientific aspects, traverses many scales (micro-macro levels), and employs many data types, BCTEO represents a first attempt to standardize this variegated domain knowledge, and can provide a suitable mean to integrate data across the field. In particular, database technology can be usefully integrated with an ontology layer, thus improving data integration and simplifying information searching.Finally, the framework described in this paper has been applied in the context of bone and cartilage tissue engineering: it represents one of the most widely investigated fields of regenerative medicine, due to the constant increasing of articular-skeletal diseases/trauma, mostly in young and sportive people. However, tissue engineering is not restricted to bone and cartilage aspects, and they just represent a starting point to develop informatics tools for regenerative medicine. Therefore, it is worth noting that the here presented tools (guidelines and ontology) can be generalized, thus becoming a useful instrument for semantic approach in the entire tissue engineering field.AFM: Atomic Force Microscopy; BCTEO: Bone/Cartilage Tissue Engineering Ontology; BTO: Brenda Tissue Ontology; CARO: Common Anatomy Reference Ontology; CHEBI: Chemical Entities of Biological Interest; CL: Cell Type; DMA: Dynamic Mechanical Analysis; ERO: eagle-i research Resource Ontology; GO: Gene Ontology; IEV: EVENT-INOH pathway ontology; IHC: ImmunoHistoChemistry; IHF: ImmunoHistoFluorescence; MI: Protein-protein interaction; MIAME: Minimum Information About a Microarray Experiment; MIAPE: Minimum Information About a Proteomics Experiment; MicroCT: Micro-Computed Tomography; MIMIx: Minimum Information required for reporting a Molecular Interaction experiment; MIRIAM Registry: Minimum Information Required in the Annotation of Models; MMO: Measurement Method Ontology; MO: Microarray and Gene Expression Data Ontology; MSH: Medical Subject Headings (MeSH); NCBI: National Center for Biotechnology Information; NCBO: National Center for Biomedical Ontology; NCI: National Cancer Institute; NPO: Nanoparticle Ontology; OBI: Ontology for Biomedical Investigations; OBO: Open Biological and Biomedical Ontologies; OLS: Ontology Lookup Service; PATO: Phenotype and Trait Ontology; PCR: Polymerase Chain Reaction; PDQ: Physician Data Query; RO: Relation Ontology; SEM: Scanning Electron Microscopy; SNOMEDCT: Systematized Nomenclature Of Medicine Clinical Terms; TEM: Transmission Electron Microscopy.The authors declare that they have no competing interests.FV designed the ontology and wrote the paper; SS was involved in drafting the manuscript, contributed to ontology generation and developed the survey; AO contributed in BCTEO development and paper revision; LM coordinated the project and maintained the bioinformatics resources. All authors read and approved the final manuscript.