The present article reviews the literature on the functional neuroanatomy of developmental dyslexia across languages and writing systems. This includes comparisons of alphabetic languages differing in orthographic depth as well as comparisons across alphabetic, syllabic, and logographic writing systems. It provides a synthesis of the evidence for both universal and language-specific effects on dyslexic functional brain activation abnormalities during reading and reading-related tasks. Specifically, universal reading-related underactivation of dyslexic readers relative to typical readers is identified in core regions of the left hemisphere reading network including the occipito-temporal, temporo-parietal, and inferior frontal cortex. Orthography-specific dyslexic brain abnormalities are mainly related to the degree and spatial extent of under- and overactivation clusters. In addition, dyslexic structural gray matter abnormalities across languages and writing systems are analyzed. The neuroimaging findings are linked to the universal and orthography-dependent behavioral manifestations of developmental dyslexia. Finally, the present article provides insights into potential compensatory mechanisms that may support remediation across languages and writing systems. Developmental dyslexia is a disorder characterized by severe and persistent problems in the acquisition of literacy. Performance in reading skills is markedly below the age-adequate norm – in the absence of problems regarding intelligence, motivation, vision, or educational environment . It has Using neuroimaging techniques such as functional magnetic resonance imaging (fMRI), cognitive neuroscientific research has identified brain circuits crucially involved in typical and dyslexic reading. These studies have converged on a coarse functional neuroanatomical model of reading and developmental dyslexia. The model proposes abnormal brain activation in dyslexic readers in the left posterior temporo-parietal (TP) cortex , the left occipito-temporal (OT) cortex , and the left frontal cortex .As identified by meta-analyses, the most consistent finding is dyslexic underactivation relative to typical readers in the left TP and OT cortex. In addition, dyslexic underactivation was identified in the left inferior frontal gyrus (IFG) and dyslexic overactivation in the adjacent left precentral gyrus . OccasioAlthough we have convincing evidence that the functioning of the above mentioned left TP, OT, and IFG cortical regions is altered in developmental dyslexia during reading and reading-related tasks, it is still an open question how the presumed functional and gray matter (GM) structural impairments in these regions lead to the severe and persistent reading problems of dyslexic readers. In other words, the question is not so much of whether and if so, where in the brain dyslexic abnormalities exist, but rather on how these brain regions might underlie reading- and spelling-related cognitive processes in typical and dyslexic readers. The present review article aims at providing an integrative overview and synopsis of the functional and structural brain abnormalities in dyslexic readers across languages and writing systems.Specifically, the goal here is to focus on functional activation and GM structure; and on the commonalities and differences in these measures in developmental dyslexia across languages and writing systems. First, the functional neuroanatomy of developmental dyslexia across alphabetic languages differing in orthographic depth will be discussed. Second, the neurobiology of developmental dyslexia will be compared across alphabetic, syllabic, and logographic writing systems. Third, GM structural brain abnormalities in developmental dyslexia will be discussed. Finally, there will be a section on potential compensatory mechanisms that may support remediation across languages and writing systems.Research on the relationship between functional activation and GM structure and their effects on reading development is of crucial importance but still scarce. Therefore, innovative approaches using intervention studies and longitudinal research will also be discussed. With respect to functional and structural connectivity in developmental dyslexia – which is beyond the scope of the present review – the reader is referred to other recent studies and meta-analyses e.g., .Orthographic depth (OD)   is a welThe predominant view proposed a “cultural diversity and biological unity” account of developmental dyslexia, claiming a universal neurocognitive basis of the disorder across languages. This position was based on a seminal PET study comparing the brain activation of Italian, French, and English adult dyslexic readers in response to explicit and implicit reading tasks . The uniA qualitative summary and critical discussion of the Consequently, As predicted from the cross-language literature , universThe direct statistical comparison between the two sets of fMRI studies revealed higher convergence of dyslexic underactivation relative to typical readers for DO compared with SO in the bilateral inferior parietal cortex. Interestingly, this abnormality was no longer found when foci reported with stronger dyslexic task-negative activation  were not included in the meta-analysis. Furthermore, higher convergence of dyslexic underactivation relative to typical readers for DO compared with SO was found in the triangular part of the left inferior frontal gyrus (IFG), the left precuneus, and the right STG. Higher convergence of dyslexic overactivation relative to typical readers was identified in the left anterior insula.Higher convergence of dyslexic underactivation for SO compared with DO was identified in the left FFG, left TP cortex, the orbital part of the left IFG, and left frontal operculum. On the contrary, higher convergence of dyslexic overactivation relative to typical readers was found in the left precentral gyrus. In sum, the findings are in line with the view of a biological unity of developmental dyslexia – with a core deficit in the left OT cortex and additional orthography-specific variations. Different patterns of reading-related dyslexic overactivation are assumed to reflect different compensatory mechanisms across languages. The results of the meta-analysis by Importantly, common dyslexic underactivation in alphabetic orthographies was found in the left OT cortex, including the visual word form system. The universal left OT cortex dysfunction, most probably reflecting the phonological speed deficit characteristic of developmental dyslexia, is in line with evidence showing that in typical readers this area subserves both lexical whole-word recognition and sublexical serial decoding e.g.,  – at leaIn addition to the functional neuroimaging studies on reading and dyslexia in alphabetic orthographies, there have been studies on reading in syllabic , morpho-syllabic , and logographic  writing systems. In their meta-analysis of these studies on typical readers, The separate reading-related activation patterns of the different writing systems also varied to a certain extent, particularly regarding the spatial configuration of the activation clusters. Specifically, the meta-analysis by The first evidence for a specific brain dysfunction in Chinese dyslexic reading that was previously not reported for alphabetic writing systems was put forward by The left MFG was also identified in a direct cross-linguistic comparison between dyslexic and typical readers of Chinese and English using a semantic word matching task . DespiteA remarkably similar brain network was identified by Seminal neurological examinations on the neural basis of acquired reading problems were already conducted in the nineteenth century , 1892. Iin vivo. In short, VBM provides a measure of local GM volume or density of a voxel. It is an established method in cognitive neuroscience and has been used to investigate pre-reading children with a familial risk for dyslexia . In more up-to-date conceptions on the neurology of speech and language e.g., , the funAcross different alphabetic orthographies, the left STS is assumed to play an important role in the integration of auditory and visual information e.g., . TherefoCross-linguistic comparisons have proven to provide extremely valuable information on the neurobiology of reading and developmental dyslexia. The focus, up to now, was largely on the comparison of dysfunctions in the form of reading-related dyslexic underactivation relative to typical readers. In contrast, the patterns of dyslexic overactivation relative to typical readers were rarely compared across languages and writing systems. This is probably because there is larger inter-individual variability with respect to overactivation compared with underactivation in developmental dyslexia and, in turn, less consistency across studies (and activation tasks). From the results reported by In principle, the dyslexic overactivation patterns might be informative on potential compensatory mechanisms supporting language-specific or language-universal remediation strategies. First evidence  points tOne way of providing this kind of evidence is via intervention studies and longitudinal research. These longitudinal brain imaging studies would also be helpful for a better understanding of the relationship between brain function and brain structure and their respective effects on reading development across languages. Unfortunately, such cross-linguistic longitudinal studies are extremely challenging to conduct and to analyze, and therefore, do not exist yet. Certainly, more fundamental research on the interplay between the developmental changes in brain function, brain structure and literacy acquisition is required in order to put forward comprehensive brain-based models of typical and dyslexic reading development.Across alphabetic writing systems, OD has an influence on the relative importance of different underlying cognitive processes required for fluent reading, and accordingly on the degree and spatial extent of brain activation clusters of typical readers. Consequently, the neuroanatomical dysfunctions of dyslexic readers are associated with an emphasis on different elements of the core reading network, reflected in stronger or weaker under- and overactivation relative to typical readers depending on OD. For example, in the case of the logographic Chinese writing system, a crucial role is assigned to the left MFG, which possibly subserves the working memory processes required for the successful recognition of written characters.The existing evidence, up to now, suggests that the functional neuroanatomy of developmental dyslexia is similar across languages and writing systems, with some orthography-specific peculiarities. Specifically, underactivation  in core regions of the left hemisphere reading network including OT, TP, and IFG regions in response to reading or reading-related tasks seems to be a universal signature of developmental dyslexia. At least parts of the core network were also identified with structural neuroanatomical abnormalities in dyslexic readers – sometimes even before the onset of formal reading instruction . Consequently, these core regions are language-universal prime candidates to be targeted by intervention programs.FR conceived and wrote the manuscript.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.


Atherosclerosis is a chronic inflammatory disease of arteries and it affects the structure and function of all three layers of the coronary artery wall. Current theories suggest that the dysfunction of endothelial cells is one of the initial steps in the development of atherosclerosis. The view that the tunica intima normally consists of a single layer of endothelial cells attached to the subendothelial layer and internal elastic membrane has been questioned in recent years. The structure of intima changes with age and it becomes multilayered due to migration of smooth muscle cells from the media to intima. At this stage, the migration and proliferation of smooth muscle cells do not cause pathological changes in the intima. The multilayering of intima is classically considered to be an important stage in the development of atherosclerosis, but in fact atherosclerotic plaques develop only focally due to the interplay of various processes that involve the resident and invading inflammatory cells. The tunica media consists of multiple layers of smooth muscle cells that produce the extracellular matrix, and this layer normally does not contain microvessels. During the development of atherosclerosis, the microvessels from the tunica adventitia or from the lumen may penetrate thickened media to provide nutrition and oxygenation. According to some theories, the endothelial dysfunction of these nutritive vessels may significantly contribute to the atherosclerosis of coronary arteries. The adventitia contains fibroblasts, progenitor cells, immune cells, microvessels, and adrenergic nerves. The degree of inflammatory cell infiltration into the adventitia, which can lead to the formation of tertiary lymphoid organs, correlates with the severity of atherosclerotic plaques. Coronary arteries are surrounded by perivascular adipose tissue that also participates in the atherosclerotic process. Atherosclerosis is a chronic inflammatory disease of arteries. Despite extensive research on atherosclerosis in the past years, the complex pathogenesis of disease is still not completely clear. Nevertheless, new techniques and methods provide new clues about the development of atherosclerosis and challenge the traditional assumptions.The coronary arteries (CAs) supply the heart with blood. They originate at the base of the aorta from openings located just behind the aortic valve leaflets. The wall of CAs consists of three layers: the innermost layer (tunica intima), the middle layer (tunica media), and the outermost layer (tunica adventitia or externa); all encapsulated in perivascular adipose tissue (PVAT) ,2.Atherosclerotic changes in the vessel wall characterize coronary artery disease (CAD). Atherosclerosis affects the structure and function of all three layers of the CA wall ,3.The intima consists of a lining layer of longitudinally oriented endothelial cells that cover the subendothelial layer of thin connective tissue with sparsely cellular matrix and the internal elastic membrane (lamina) ,5. The eThe intima of a newborn is composed of a single-cell layer, and with age, it gets thicker and becomes multilayered , dependiThe subendothelial layer formed in the intima consists of vascular smooth muscle cells (VSMCs) and the extracellular matrix (ECM) that is rich in longitudinally oriented elastic fibers and proteoglycans . DendritIn the early stage of atherosclerosis, called PIT , lipids According to the response-(to-neovascularization)-to-retention hypothesis, the initial events of coronary atherosclerosis are enlarged intimal thickness, cell hypoxia, and intimal neovascularization by vasa vasorum, followed by lipoprotein accumulation . Vasa vaThe response-to-injury of vasa vasorum hypothesis suggests that the leading cause of atherosclerosis is specifically the injury of vasa vasorum . AccordiThe atherosclerotic lesions are mostly located in atheroprone regions. These regions are predominantly found at the branches and curvatures of large vessels. The blood flow in branches and curvatures is disturbed with nonuniform and irregular low wall shear stress, which is detected by endothelial primary cilia sensitive to the shear stress ,33. ThisIn the intima, monocytes acquire characteristics of proinflammatory macrophages, which express the scavenger receptors and internalize modified lipoproteins. The infiltration and proliferation of macrophages is a characteristic feature of the progression of PIT to fibroatheroma stage . MacrophThe next step in atherosclerosis progression is the formation of thin-cap fibroatheroma. This structure has a thin fibrous cap, infiltrated with macrophages and T cells, and a large necrotic core. These advanced atherosclerotic plaques also show intraplaque neovascularization, where the extravasation of platelets and red blood cells into plaques contributes to the formation and expansion of the necrotic core. Thin-cap fibroatheroma is prone to rupture and thrombosis. After the erosion or rupture of the plaque, injured endothelial cells excrete increased amounts of thrombotic factors  and decreased amounts of antithrombotic factors  leading to atherothrombosis ,47-49.In atherosclerotic lesions, various cytokines and modified lipoproteins influence the phenotype of macrophages, affecting the course of atherosclerotic lesions ,39. ProiThey exhibit either proinflammatory (proatherogenic) or antiinflammatory and immunoregulatory (antiatherogenic) properties. T helper (Th) cells are known to modulate the formation of atherosclerotic lesions. It was shown that Th1, Th9, Th22, and natural killer (NK) cells have a proatherogenic function, while Th2, Th17, and T regulatory (Treg) cells have an atheroprotective function in the development of atherosclerosis ,49. ProaThe media of CAs consists of multiple layers of VSMCs  and the Except in special circumstances, VSMCs are the only cells in the media and have both contractile and synthetic functions ,58. TheyVSMCs are not terminally differentiated and display phenotypic plasticity . In respDuring atherogenesis, VSMCs also undergo phenotypic changes and selectively proliferate to and accumulate in the vessel wall . They loIn response to a variety of stimuli, VSMCs can also display markers and features of osteoblasts, chondrocytes, adipocytes, and macrophage-derived foam cells. The transformation from the normal contractile type of VSMCs into osteogenic/chondrogenic phenotype leads to calcification of the vessel wall . In advaAt birth, the intima is thin and the media is avascular . With agThe media is immunoprivileged – it develops active and passive protective mechanisms against inflammation. Passive mechanisms include the presence of elastic membranes and the rarity or absence of blood and lymphatic vessels . In actiEarly in the immune response, infiltrated innate leukocytes can stimulate VSMCs and endothelial cells to produce proinflammatory cytokines, including IL-1, IL-6, and TNFα , which rThe adventitia consists of connective tissue with collagen and elastic fibers, vasa vasorum, adrenergic nerves, and lymphatic vessels. The thickness of the adventitia in CAs ranges from 300 to 500 µm . LongituFibroblasts are the main cells in adventitial connective tissue  3,76]. ,76. 3,76The CAs are surrounded by PVAT  85]. Th. Th85]. Other PVAT-derived vasoactive factors can attenuate endothelial-dependent relaxation or increase vascular contractility . PVAT prFollowing the discovery of the correlation between the degree of inflammatory infiltration in the adventitia and the severity of atherosclerotic plaques in CAs, numerous studies have investigated the components of adventitial cell aggregations, in humans and animal models ,80,95-98In the abdominal aorta of Apoe−/− mice, ATLOs were associated with atherosclerotic lesions in the intima, which indicated that a crosstalk between the intimal lesions and ATLOs occurs via the media ,103. TheMany studies investigated the role of different cell types, associated with inflammation in the adventitia, in the development of atherosclerosis. Th1 cells are proatherogenic and secrete proinflammatory cytokines such as IL-2, TNFα, and IFNγ. NK cells also display proatherogenic function. B2 cells play a proatherogenic role to stimulate Th1 cells and dendritic cells and to secrete immunoglobulin G (IgG). Proatherogenic cells are also innate response activator (IRA) B cells, which release stimulating factors for dendritic cells. Th2 cells and Th17 cells have both proatherogenic and atheroprotective function, while Treg cells and B1 cells play an atheroprotective role. Treg cells release antiinflammatory cytokines  and B cells secrete IgM .Endothelial cell damage is an important early stage in the development of atherosclerosis. Migration of VSMCs to the intima is a prerequisite for the advancement of the disease. However, previous research also suggests that a multilayered intima consisting of VSMCs is normal adaptation of CAs to rheological conditions rather than pathology on its own. The onset of atherosclerosis is endothelial dysfunction followed by other pathological processes, such as the transformation of cells in the vascular wall that leads to atherosclerosis. The thickening of the arterial wall is accompanied by the development of vasa vasorum, to meet the need for additional supply of nutrients and oxygen. Endothelial dysfunction of vasa vasorum contributes to the development of atherosclerosis. Atherosclerotic lesions are predominant in the intima, although atherosclerosis can affect all three layers of the vascular wall. The characteristic feature of atherosclerotic lesions is the accumulation of lipoproteins in the subintimal layer, migration of inflammatory cells to the intima, and loss of media immune privilege. The VSMCs in the intima can transform from the contractile phenotype to synthetic, chondrocyte, osteocyte, adipocyte, or macrophage-foam cell type, and the fibroblasts transform into myofibroblasts. PVAT also contributes to the proatherogenic state, by activating immune cells in ATLOs to secrete proinflammatory and proatherogenic cytokines.Since each cell type contributes to the development and progression of atherosclerosis, each cell type represents a potential therapeutic target. By modulating these cells to change their function from proatherogenic to antiatherogenic, we may be able to alter, slow down, or mitigate the course of atherosclerosis. In addition, vasa vasorum may be a particularly important target in the treatment of atherosclerosis, as atherosclerosis usually affects the segments of vessel wall supplied by vasa vasorum.


The increasing number of patients with chronic wounds caused by diseases, such as diabetes, malignant tumors, infections, and vasculopathy, has caused severe economic and social burdens. The main clinical treatments for chronic wounds include the systemic use of antibiotics, changing dressings frequently, operative debridement, and flap repair. These routine therapeutic strategies are characterized by a long course of treatment, substantial trauma, and high costs, and fail to produce satisfactory results. Biomaterial dressings targeting the different stages of the pathophysiology of chronic wounds have become an active research topic in recent years. In this review, after providing an overview of the epidemiology of chronic wounds, and the pathophysiological characteristics of chronic wounds, we highlight the functional biomaterials that can enhance chronic wound healing through debridement, anti-infection and antioxidant effects, immunoregulation, angiogenesis, and extracellular matrix remodeling. It is hoped that functional biomaterials will resolve the treatment dilemma for chronic wounds and improve patient quality of life. Owing to an increasing number of patients with chronic diseases such as diabetes, there are more than 6.5 million patients with chronic wounds worldwide, and the overall medical cost for treating chronic wounds exceeds 25 billion US dollars per year  and elastin, are derived from fibroblasts. The main cell types of the subcutaneous tissue are fibroblasts and adipocytes  and methicillin-resistant S. aureus (MRSA)  network structure containing water as a dispersion medium. Chitosan (CS)-based dressings have many advantages, including excellent degradability and compatibility; they are also non-toxic and exhibits excellent antibacterial activity  have been widely used in the treatment of chronic wounds owing to their broad-spectrum antimicrobial properties and safety. AgNPs inactivate pathogenic microorganisms by breaking disulfide bonds in proteins, thereby changing the tertiary structure of the proteins , and hydroxyl radicals (·OH), serve as signaling messengers to regulate gene expression. Low level of ROS are beneficial for maintaining cellular environmental homeostasis  (PPCN) hydrogel through continuous polycondensation and free radical polymerization reactions. The antioxidant diol-citrate esters endowed this temperature-sensitive PPCN hydrogel with antioxidant properties. Compared with the PBS group, the antioxidant PPCN hydrogel group more effectively promoted the regeneration of the epidermis and the appendages. Moreover, the PPCN content of the hydrogel could be adjusted to obtain better encapsulation and slow release of angiogenic chemokine stromal cell derived factor-1 (SDF-1), which achieved the desired functions of antioxidation and promotion of neovascularization  to produce PCN. PCN-miR was fabricated by electrostatic adsorption with negatively charged antagomiR-26a. The design of this drug delivery system ensured that antagomiR-26a had direct contact with the ceria nanozymes, preventing ROS from destroying its activity, as shown in In vivo experiments showed that this self-protecting hydrogel effectively promoted the repair of non-healing wounds in diabetes  can induce macrophages to differentiate into M2 phenotypes by inducing the accumulation of nano-clusters of the mannose receptor on the macrophage surface. Without containing any drugs, the material itself has an anti-inflammatory effect and promotes wound healing  (PVA)/CS-based hydrogel using a freezing-thawing method. In vivo experiments showed that this bee venom-loaded PVA/CS hydrogel increased the gene expression levels of hydroxyproline and glutathione and reduced the level of IL-6, and its anti-inflammatory effect was comparable to that of the diclofenac hydrogel , a non-sulfated glycosaminoglycan, is the main component of ECM. HA promotes angiogenesis through controllable mild inflammation caused by degradation products  (PLLA)-oriented electrospun dressing. As shown in In vitro tests confirmed that it increased gene expression levels in human umbilical vein endothelial cells, and in vivo experiments showed that it promoted wound healing and neovascularization in diabetic mice  is a small molecule that promotes angiogenesis. To solve the problem of its rapid inactivation in the body, Ren et al. used nano-mesoporous silicon as an intermediate carrier to prepare a DMOG-loaded poly /CS film had a good ability to promote wound healing in diabetic mice and caused a steady increase in Col content. These results demonstrate the therapeutic potential of silencing MMP-9 gene expression in chronic wounds (Castleberry et al., Local silencing of sequence-specific MMP genes using RNA interference is another important strategy for regulating MMP, which avoids the disadvantages of using MMP inhibitors such as low efficiency and side effects on skeletal muscle (Overall and Kleifeld, In addition to the above commonly used functional dressings, other dressings, such as those used for diabetic foot neuropathy and scars, also need attention.Diabetic neuropathy-related ulcers often occur on the plantar surface, and local ulcers often occur on parts of the foot that are under long-term pressure. The main clinical treatment is to use pressure-reducing shoes (Zimny et al., The formation of chronic wound scars often presents a trend of increasing within a year after the wounds healing. Scar formation often leads to repeated ulceration, infection, and even cancer of the wounds. Some researchers have added epidermal growth factor and silver sulfadiazine to silk protein biomaterials to make dressings. The material encapsulates the drug in three forms: a silk membrane, a layered porous silk membrane, and an electrospun nanofiber. It promotes the proliferation of dermal cells and the synthesis of Col, and reduces the formation of scars, thereby providing a feasible strategy for the treatment of chronic wounds (Gil et al., In the past two decades, functional biomaterials that can change the wound microenvironment, such as the continuous inflammation, have had a profound impact on the development of dressings for the treatment of chronic wounds. However, many basic studies have been carried out in rodent models, which are characterized by a predominance of M2 macrophages and tend to contracture healing. Thus, the results of these studies need to be further verified using large mammalian models. Current commercial dressings do not meet the needs of patients with chronic wounds, and cost-effective alternative designs containing various types of materials should be developed. Given the clinical problems of patients with chronic wounds, an important research direction is the development of diagnostic functional dressings that clarify the condition of the wound, especially the early symptoms of the subcutaneous tissues with temporary intact skin. As the skin healing process represents the repair process of most tissues, it is widely used in orthopedics and nerve repair technologies, such as 3D printing, and is also suitable for chronic wounds. Therefore, according to the actual characteristics of the patient's wound, such as the position, size, depth, and surrounding tissue of the wound, a more diverse and personalized functional dressing can be produced using computer-aided design. In short, as the application of functionalized biomaterials in chronic wounds is further developed, their therapeutic effects on chronic wounds will be greatly improved, and these materials will replace more clinically invasive operations.XZ wrote the manuscript. XZ, WS, QY, and WQ revised the manuscript. YW and RL designed this work of review and revised the manuscript.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.


With an estimated three to five million human cases annually and the potential to infect domestic and wild animal populations, influenza viruses are one of the greatest health and economic burdens to our society, and pose an ongoing threat of large-scale pandemics. Despite our knowledge of many important aspects of influenza virus biology, there is still much to learn about how influenza viruses replicate in infected cells, for instance, how they use entry receptors or exploit host cell trafficking pathways. These gaps in our knowledge are due, in part, to the difficulty of directly observing viruses in living cells. In recent years, advances in light microscopy, including super-resolution microscopy and single-molecule imaging, have enabled many viral replication steps to be visualised dynamically in living cells. In particular, the ability to track single virions and their components, in real time, now allows specific pathways to be interrogated, providing new insights to various aspects of the virus-host cell interaction. In this review, we discuss how state-of-the-art imaging technologies, notably quantitative live-cell and super-resolution microscopy, are providing new nanoscale and molecular insights into influenza virus replication and revealing new opportunities for developing antiviral strategies. In humans, influenza viruses cause acute infections in the respiratory tract. Every year, three to five million people develop severe seasonal influenza with up to 650,000 deaths, globally . AlthougInfluenza virions consist of an RNA genome encased in a lipoprotein membrane . The viral genome is made up of eight RNA segments, wrapped by the viral nucleoproteins and the RNA polymerase , termed viral ribonucleoprotein complexes (vRNP). The envelope is derived from the host cell plasma membrane and contains two major glycoproteins, hemagglutinin (HA) and neuraminidase (NA), together with the proton channel M2. The M1 protein forms a proteinaceous matrix underneath the envelope, as seen in To understand the mechanisms underlying influenza virus replication and of cell-to-cell viral transmission, with a view to developing new anti-viral strategies, one emerging requirement is to be able to directly observe virions and virus-host cell interactions. The small size of influenza virions (~120 nm diameter) has made electron microscopy (EM) the method of choice for these observations. For example, EM has revealed that influenza virus particles can be pleiomorphic, taking shapes from ~120 nm diameter spherical particles to long filamentous forms up to several µm long and ~90 nm in diameter , but In recent years, the development of advanced and quantitative fluorescence microscopy and, importantly, that of super-resolution microscopy (SRM)  has openIn this review, we present examples of the exciting new advances in the study of influenza virus permitted by novel optical imaging tools. We limit our scope to SRM, single-molecule imaging and quantitative live-cell analysis, with a particular emphasis on SRM, since observing virus assemblies at the nanoscale is central to understanding virus replication. Therefore, we first describe these technologies and then present recent studies in the context of the different aspects of influenza viral replication, from entry to viral budding. We finally provide an outlook of what these techniques may unravel in the future.We identified three key opportunities for advanced fluorescence microscopy techniques to provide novel insights into influenza virus biology, as highlighted in In SRM comprises optical imaging techniques that offer resolution beyond the diffraction limit of light (~250 nm). SRM was developed in the mid-1990s, first with stimulated emission depletion (STED) microscopy . STED isA second technique called structured illumination microscopy (SIM)  relies odSTORM (direct STORM) . I. I22]. I viruses , showingSRM microscopy techniques can also be combined with advanced quantitative analysis to quantify the structural diversity of virions within a virus population. In particular, TIRF-SIM  was used in combination with machine learning to develop a high-throughput methodology capable of classifying influenza viruses based on their shape . This auAlthough STED has not been used to study the structure of individual influenza viruses, it has allowed the demonstration that HIV envelope protein (Env) distribution on virions is regulated by the structural polyprotein Gag proteolysis/maturation, a rearrangement that is key for efficient receptor engagement and viral entry . AdditioThe entry of the influenza viruses into cells is a multi-step process. Following the engagement of HA with sialic acid-decorated cell surface receptors, influenza virions undergo endocytosis through clathrin-mediated mechanisms , or macrAdvanced microscopy, and especially SPT, has provided essential insights into the dynamics and pathways exploited by influenza virus for entry . Here, tWithin early endosomes, influenza viruses exploit retrograde trafficking along the cell’s microtubule network to reach perinuclear late endosomes where fusion and uncoating occurs. Vesicular identity  can be obtained by simple colocalization microscopy studies, using well-established markers, and this can lead to a better understanding of the dynamics of trafficking. However, the higher resolution provided by SRM and colocalization at the nanoscale can discriminate between virions exploiting specific endosomal carriers as opposed to being in close proximity. In addition, SRM can be used to observe possible nanoscale structural re-organisations of endosomes and other organelles as a consequence of viral infection. Focusing on early trafficking events through the endosomal pathway, STED was exploited to study the entry dynamics of spherical influenza particles in human dendritic cells  see Figc. HoweveRecently, the use of QDs was shown to provide a powerful way to label viral components for long term tracking within living cells, allowing the dynamics of viral infection to be studied in near real-time over extended periods. This approach was used to label the viral envelope and track viruses inside living infected cells as they traffic along the microtubule network towards the perinuclear region, revealing the complex range of motion exhibited by the virus during trafficking towards the nucleus, and highlighting the impact of the virus on molecular transport processes within infected cells ,63.SRM can, thus, be used to interrogate multiple aspects of virus entry and trafficking including the identity and organisation of viral receptors, the identity of specific organelles and dynamics of the various events. However, the modulation of these phenomena with respect to dynamics and viral structure remain poorly understood despite viral entry being a critical step to target for anti-viral therapy.Upon reaching the perinuclear region of a cell, influenza viruses need to release their genetic material into the cytoplasm. This release requires the fusion of the viral membrane with the limiting endosomal membrane and disassembly of the M1 proteinaceous layer lining the inside of the viral envelope (uncoating). The viral envelope fusion with the endosome membrane is HA-mediated and occurs through a multi-step process involving a hemifusion intermediate, where only the apposed leaflets of the membranes merge, followed by the formation of a pore and finally complete fusion. To study the dynamics of fusion under the microscope, it is necessary to have access to a readout of the state of the viral envelope in real-time. A common approach involves observing the dequenching of the fluorescence signal from a lipophilic dye incorporated into the viral envelope . For insBy contrast, recent smFRET revealed the dynamics of fusion associated conformational changes of HA in intact virions . The stuFollowing fusion, influenza virus uncoats progressively: first the matrix layer of M1 proteins dissociates followed by the release of the 8 vRNPs into the cytosol. A recent study has shown that influenza viruses can exploit the cellular aggresome to promote uncoating following M2-mediated acidification of the viral core . For thiAfter uncoating in late endosomes, vRNPs need to travel through the cytoplasm and into the nucleus where viral genome replication and expression occur. Mathematical modelling of the key steps of viral trafficking suggested that vRNP uptake into the nucleus was shown to be a critical bottleneck for efficient infection due to the potential for degradation of incoming RNA in the cytoplasm . The modThe diffusion of the vRNPs from the release site to the nucleus has been studied using SPT and a QD-labelling strategy  Figure d. By usiThus, the capacity of advanced quantitative live-cell imaging to perform fast-tracking of multiple viral components simultaneously can provide dynamic information that enhances our understanding of the underlying biophysical processes. Fast, sensitive live-cell imaging of early infection events from trafficking, nuclear import to uncoating was also used in an elegant study of HIV uncoating . SpecifiAlthough fusion and uncoating involve fundamental structural reorganisations of viruses at the nanoscale, SRM has yet to be used to observe these phenomena in the context of influenza. This may be a consequence of the difficulty of observing these events as they occur since the highest-resolution SRM techniques like SMLM mainly work in fixed rather than live samples. To produce new infectious virions, the essential viral proteins and the viral genome must be transported from their sites of replication, the cytoplasm, ER and nucleus, respectively, to the plasma membrane where virions assemble. To study the trafficking events leading to virus assembly, it is necessary to be able to follow the spatio-temporal dynamics of viral components. It is particularly important to understand how the segmented genome is capable of bundling into infectious virions, since genomic reassortment is linked to the appearance of emerging viral strains, with pandemic potentials.In this regard, a seminal technical development has been multi-colour FISH  approachOn the other hand, advanced live-cell imaging can provide direct information about the dynamics of transport of viral component to assembly sites and the mechanisms involved. Fast 3D tracking using light-sheet microscopy was exploited to follow the co-transport of multiple influenza vRNA segments (using GFP-tagged PA)  and the In some cases, following the assembly process in living cells through the visualisation of newly expressed viral proteins can constitute a challenge for fluorescence labelling since genetic tagging of viral proteins can lead to loss of infectivity and/or assembly defects. Therefore strategies for non-invasive labelling  of newly expressed viral proteins were designed. Notably, a split green fluorescent protein (GFP) approach  was usedInfluenza virus assembly, budding and release is also closely tied to HA, NA and M2 distribution on the host cell plasma membrane . PreviouInterfering with these dynamic microdomains can potentially be used as a therapeutic intervention. STORM imaging of budding filamentous influenza viruses revealed that antibodies against the extracellular part of M2 (M2e) were able to disrupt virus production . InteresAdvanced microscopy can be used to investigate the role of specific host proteins during infection. A study unveiled CD81, a tetraspanin previously shown to be a co-receptor for hepatitis C virus entry , as a kedSTORM have been used to study how Env is recruited to Gag protein clusters during HIV assembly at the plasma membrane [It is worth highlighting that SRM has proven very powerful in the study of budding mechanisms for HIV. In particular, PALM and membrane , and 3D membrane ,20. Moremembrane . These sHost cell restriction and innate immune responses are important intrinsic suppressors of viral replication. The interferon-induced antiviral protein IFITM3 has previously been identified as an influenza restriction factor that inhibits viral entry prior to uncoating  and incrUncoating is a critical step in viral replication essential for release of the viral genome into the cytoplasm of a new host cell. However, this release exposes the vRNA to cellular innate immune sensors that may initiate interferon responses and viral restriction. Influenza virus is able to suppress the RIG-I-mediated interferon response through a specific amino-acid motif in the viral polymerase proteins PB1/PA . Multi-cAdvanced microscopy, and especially quantitative microscopy, may also offer insights into the effects of antiviral drugs at the single-virus level. By studying the structures of live influenza viruses shed from cells treated with the NA inhibitor oseltamivir, quantitative microscopy and the use of non-invasive labelling showed that oseltamivir preferentially decreases the release of filamentous viruses as opposed to smaller spherical ones . This suIn the context of drug development for influenza antiviral therapy, high-throughput microscopy approaches offer great promise for quantitative and large-scale analysis of compounds in the search of specific cellular and viral phenotypes. For instance, a high-throughput quantitative imaging methodology was developed to provide assays capable of probing different stages of the early influenza infection: Initial binding, endocytosis, viral membrane fusion, uncoating, nuclear import and translation . The imaDespite the demonstrated applications of advanced microscopy presented here, the full potential of these techniques remains limited by shortcomings that are necessary to understand. In particular, it is important to note that, although resolution with SMLM can reach ~20 nm, an observed colocalization at such a spatial range does not directly indicate molecular interactions. SRM techniques can only suggest spatio-temporal interactions and require confirmation via biochemical or FRET assays.Another clear limitation of SRM is its compatibility with live-cell imaging. Although technically feasible, performing live-cell imaging at molecular resolution (< 50nm) remains challenging due to both the acquisition times required (as in the case of SMLM) and phototoxicity (as in the case of both SMLM and STED). For instance, due to the technical nature of SMLM, exposure times of the order of minutes to hours are needed for optimal image reconstruction, thus limiting the observation of fast biological processes. In fact, the live-cell fPALM studies that demonstrated nanoscale cluster dynamics  could onPhototoxicity can also be limiting when studying light-sensitive phenomena with SRM, notably for SMLM and STED ,117,118.Additionally, with SRM, greater attention should be paid to the methods and types of fluorescent labelling used. When the instrument achieves resolutions of the order of the size of the label or its linker (a single IgG antibody is of the order of 10–15 nm long), then the data should be interpreted by keeping in mind that what is visualised is the marker and not the molecule of interest. The effect of linker length can have a significant effect on the quantification of structural parameters derived from SRM ,121. AnoFinally, the availability of SRM and other advanced microscopy systems is still a limitation preventing researchers from leveraging the power of SRM and advanced imaging. This is especially true for live imaging of viral replication which may require high levels of containment for safety reasons.Our review summarizes recent applications of SRM and quantitative advanced microscopy techniques to study influenza virus replication. It highlights important techniques such as SRM for structural studies of molecular assemblies either in the virus particles  or in inAn important underlying principle to all these microscopy methods is the combination of imaging with quantitative data analysis to determine, e.g. the relative levels and spatial distributions of envelope proteins in filamentous viral particles  or the sFor the first time, these new imaging technologies allow us to follow the fate of individual virions in live cells, to understand how the choice of entry pathway or assembly may be modulated depending on the cellular  and viral context , and to quantify the effect of potential anti-viral drugs at the single-virus level. We expect the combination of these advanced imaging technologies with new labelling methods, and new computational approaches, including machine learning, will reveal new knowledge of influenza virus replication enabling the development of effective therapeutics. These technical advances will be important tools in understanding virus-host interactions that determine infection outcomes and pandemic potential.


The ability of the cells to sense mechanical cues is an integral component of ”social” cell behavior inside tissues with a complex architecture. Through ”mechanosensation” cells are in fact able to decrypt motion, geometries and physical information of surrounding cells and extracellular matrices by activating intracellular pathways converging onto gene expression circuitries controlling cell and tissue homeostasis. Additionally, only recently cell mechanosensation has been integrated systematically as a crucial element in tissue pathophysiology. In the present review, we highlight some of the current efforts to assess the relevance of mechanical sensing into pathology modeling and manufacturing criteria for a next generation of cardiovascular tissue implants. Pathologic evolution of the cardiovascular system is not only accompanied by modifications in cellular metabolism, aging, epigenetics and risk condition-associated defects but also by fundamental changes in cells/tissues architecture, motion and geometry. As an example, the chronic setting of ischemic disease in the heart results in variations in the physical–chemical characteristics of the extracellular matrix, which lead to changes in the structure of the myocytes contractile apparatus (the sarcomere) causing, alternatively, systolic or diastolic heart failure . AnotherApart from neo-vascularization observed in consequence of local activation or repair mechanisms , the cardiovascular system is not endowed with extensive regeneration ability. A typical example is the adult myocardial tissue, where the presence of cardiogenic cells able to replenish the contractile cells after injury has been dismissed after tense discussions lasting for about two decades . This haAfter the initial enunciation of the tissue engineering principles by Langer and Vacanti in 1993 , researcCardiac fibrosis, characterized by accumulation of extracellular matrix (ECM) production and degradation, results in a progressive evolution of myocardium into a non-contractile fibrotic tissue. Indeed, this pathological remodeling leads to a replacement of myocytes with a stiff fibrotic tissue, affecting the myocardial compliance and accelerating the progression to heart failure. This process is mediated by cardiac fibroblasts (CFs), which physiologically are quiescent cells responsible for ECM homeostasis . FollowiTo date, the only possible therapy for patients with end-stage heart failure consists of implantation of a left ventricular assist device (LVAD) or transplantation of an entire donor heart. However, none of these solutions allows recovering structural and/or physiological integrity of the cardiac muscle—for example by restoring coordinated contraction and coherent electrical coupling. Lower vertebrates such as zebrafish demonstrate an impressive natural capacity for cardiac regeneration throughout life. This has been also well documented in other various non-mammalian vertebrate’s species ,22. By cDirect cardiac reprogramming has emerged as a promising therapeutic approach for regenerating the damaged heart by directly converting endogenous cardiac fibroblasts into cardiomyocyte-like cells using viral vectors overexpressing cardiac master genes ,27 or miAn alternative to the reprogramming approach is the delivery of drugs promoting re-activation of the CM cell cycle . In receHow could manipulation of Hippo signaling be exploited to regenerate the heart? YAP in adult myocytes is sequestered out of the nucleus, not only by the activity of the Hippo kinase pathway, but also by association of the protein to components of the cytoskeleton involved in extracellular stiffness sensing  ,42. GiveAnother way to harness the Hippo pathway to treat cardiac diseases  depends on the function of the YAP/TAZ transcriptional complex in cardiac fibroblasts . In thisSeveral miRNAs are known for their cardiac anti-fibrotic activities and are dysregulated in myocardial injury models . Among tNot only YAP, but also other mechanical sensors seem to be implicated in regulating cardiac function and pathological progression of cardiac diseases. For example, integrins and the angiotensin II type I (AT1) receptors are involved in mechano-dependent cellular responses, such as differentiation, ECM remodeling, inflammation ,53 and tBoth viral- and non-viral-based vectors have been used to delivery genetic materials to the heart. However, gene therapy has shown various limitations and severe side effects. As an example, none of the tested gene delivery systems is 100% cardiac specific and can activate the innate immune system with significant dose-related toxicity . AdditioTissue engineered scaffolds and hydrogels might represent an alternative to drug delivery vehicles to treat cardiac disease. Engineered cardiac patches can be used as off-the-shelf products to mimic the native ECM; they further provide mechanical support and can release drugs into the region of infarction. Acellular injectable scaffolds show different advantages, such as a limited immune reaction, low cost and extended shelf life. Because of their tissue-like biological, chemical and mechanical properties, hydrogels represent potential carriers to release drugs/miRNAs into cardiac tissue. Hydrogels are 3D hydrophilic polymer networks with tunable, compliant and biomimetic behavior well suited for drug delivery . “IntellBased on this evidence, we hypothesize that the use of “intelligent hydrogels”, supporting mechanical myocardial and releasing drugs/miRNA able to inhibit cardiac fibroblast activation and/or induce reprogramming into CMs could represent the awaited solution to prevent post-infarct myocardial remodeling and promote myocardial regeneration in patients with cardiac ischemia .The aortic valve is composed of three leaflets attached to a fibrous ring integrated in the aorta; each of these leaflets is composed of a thick extracellular matrix, organized into three layers named, respectively, lamina fibrosa, lamina spongiosa and lamina ventricularis. All three layers have peculiar matrix compositions, which fulfill different mechanical functions. The lamina ventricularis is richer in elastin and is important for the recoil of the tissue during the transition from the open (systolic) to the closed (diastolic) conformation. The lamina spongiosa contains principally proteoglycans, and it is the softest and most amorphous layer with numerous cells, the valve interstitial cells (VICs) . FinallyAlthough VICs are embedded in the most protective layer in the leaflet, they are continuously exposed to mechanical stress by wear and tear. In their normal function, these cells are engaged in renewing the extracellular matrix subject to continuous mechanical stress. However, in consequence of inflammation due e.g., to lipid accumulation, they can modify their phenotype into that of pro-inflammatory cells, potentially affecting the matrix structure and biophysical characteristics. As a matter of fact, the pathological evolution of the aortic valve—calcific aortic valve disease (CAVD)—is characterized by extensive deposition of hydroxyapatite nodules in the leaflet layers , which aCAVD is regulated also by hemodynamic cues , in partEpigenetics refers to several mitotically and meiotically transmissible DNA/chromatin modifications, which do not affect the primary DNA sequence, but its function. Epigenetics controls genome functions through modifications of the chromatin structure, including interactions with miRNAs and long-coding RNAs (lnc-RNAs) . GrowingTaken together, this evidence holds the promise that a pharmacological therapy for CAVD progression could be endeavored by treating with drugs impacting on the progression of the disease by targeting the ability of VICs to decrypt mechanical cues and, at the same time, maintaining a permissive epigenetic landscape for physiological activity of the valve cells. Compared to other cardiovascular disorders, there are still extremely limited options for drug-based therapeutic approaches to limit progression of valve diseases, most of which—e.g., statins ,85,86,87The field of cardiac valve tissue engineering dates about 30 years back with the demonstration of the growth of endothelial cells onto the surface of glutaraldehyde-fixed animal valves (reviewed in ). The inTissue engineered heart valves (TEHVs) have been proposed historically as a solution for some of these requirements, such as the ability to self-renew and be manufactured with cells of the patients, thus eliminating rejection or long-term immune responses ,90. MoreProblems inherent to the loss of tissue structure and mechanical coherence are the major limitations of TEHVs in the long term. These modifications are due to the biological activity of the cells embedded in the initial constructs, and those from the host that gradually populate the constructs over time after implantation. ”Compaction” and ”retraction” are the two most recurrently observed modifications of TEHV leaflets. The first effect is determined by the uncontrollable matrix remodeling activity of the cells that in the long term determine shrinking and thickening of the leaflets. The second effect is relative to loss of mechanical coherence of the leaflets that cause valve regurgitation. From a biological point of view, transformation of the cells of the valve tissue constructs into active myofibroblasts is one of the most important causes of both effects. Myofibroblasts, in fact, have potent matrix remodeling activity and exert strong pulling forces dependent on stress fibers  potentiaIn the in situ approach scenario, cell-free prostheses, ideally manufactured with biologically compatible polymers, are provided as ”off-the-shelf” implants ready to be used without pre-cellularization  and are Natural polymers, such as collagen, fibrin, hyaluronic acid, show important properties since are constituted by macromolecules involved in tissue homeostasis and repair, minimizing, in this way, chronic inflammation and immunological reactions . HoweverAnother solution to overcome the adverse remodeling of cell-free valve implants could be to directly modulate the activity of genes involved in cell mechanosensation and thus reduce the intracellular transmission of mechanical cues, thereby reducing the transition of cells toward a matrix remodeling and compacting phenotypes. Examples may include the employment of materials with finely tuned viscoelastic properties to maintain a relatively low level of intracellular stress fiber tensioning ,79 or deThe in vitro approach to manufacture TEHV consists of the employment of decellularization techniques  to produThe third approach is represented by the in vivo method, which consists of the implantation of the scaffold directly in the human body, used as a bioreactor. However, this process has possible disadvantages: the collagen-based membrane, formed after the subcutaneous implantation, could be thrombogenic; scar-like tissue formation might cause a lack of valve ECM proteins, such as elastin, affecting tissue architecture and functionality; valve tissue formation and maturation may be long-term .In summary, by integrating various manufacturing technologies with material discoveries and long-term biological characterization, it is possible to realize a customize scaffold able to regulate the cell behavior and to provide the formation of a functional heart valve tissue durable under biomechanical loading.As we have already discussed in another contribution , mechanUnder physiological conditions, laminar shear stress (LSS), a tangential force generated by the flowing blood, acts on the endothelial cells surface by promoting vaso-protective effects, through the release of small molecules and cytokines with antithrombotic and vasodilator effects ) ,110. IndDisturbed blood flow also affects the stability of the plaque. In this respect, it has been demonstrated that forming plaques create, inside vessels, sections with perturbed flow, which contribute significantly to their rupture .Vascular cells are subjected not only to shear stress but also to hydrostatic pressure and cyclic strain due to pressure patterns, which involve the deeper layers of the vessel wall, such as the media and the adventitia. In particular, the position and the arrangement of vascular smooth muscle cells (VSMCs) inside the vessel wall affect their ability to sense and respond to this oscillatory pulsatile pressure . An inteTaken together, this evidence suggests the importance of a better understanding of the mechanical characteristics of the vessel in order to realize biomaterials suitable for vascular tissue engineering, which take in consideration shear stress, cyclic strain and matrix stiffness.Despite several technological advances in bioengineering in recent years, there is still intense research to find suitable methods and materials to manufacture a definitive tissue engineered vascular graft (TEVG) with the ability to replicate the functionality of natural vessels ,125. TheMechanical sensitivity of the cells employed to engineer TEVGs might be exploited to obtain better performances of engineered vascular grafts. For example, a line-oriented scaffold topography along the flow direction is beneficial to achieve cellular orientation along the direction of the flow and, therefore, enhance the formation of a confluent monolayer of endothelial cells at the lumen of the grafts ,129. In Concerning materials, one of the most investigated for manufacturing vascular structures is silk protein component fibroin ,133. ComAs in the case of TEHVs, TEVGs can also be manufactured with various strategies depending on the desired application. In a first approach, cells are allowed to grow inside the tubular scaffolds to achieve a full level of cellularization before transplantation . In suchDifferently from TEHVs, where in situ cellularization with recipient cells able to form the multi-layered structure of the leaflets is still relatively inconsistent, the process of TEVG cellular colonization in vivo leads to a more complete and stable vascular maturation of the constructs. This depends on reparative inflammatory pathways that are activated in the hosts following interposition of the graft in native vessels  and by tSummarizing, a functional vascular graft material should have: proper mechanical characteristics, in order to withstand pressure and flow forces; suitable suturability and ease of use; a broad availability in different sizes, a good porosity, reasonable manufacturing cost and long term patency . AdditioIn conclusion, the ideal graft will enforce a biomimetic topography and tunable stiffness providing the proper signaling cues for the cells in order to promote a correct tissue remodeling and to prevent vascular occlusion in consequence of wall strain/stress due to hemodynamic forces.The present article describes findings supporting the integration of basic knowledge on cell mechanosensation into criteria of bioartificial tissue manufacturing, with the aim at evolving more effective cardiovascular tissue generation/regeneration approaches. This emerging evidence indeed suggests that homeostatic control of cardiovascular tissues depends not only on paracrine pathways acting on cellular renewal vs. pathological programming but also on correct distribution and sensing of mechanical forces by the cells. Alongside the direct consequences of cell motion directly impacting cellular physiology, the passive biophysical characteristics of the tissues,  and the three-dimensional distribution of cell adhesion and cell-to-cell communications establish crucial mechanical-dependent ”checkpoints” ensuring proper cellular integration and self-renewal of bioartificially designed tissues. Finally, the possibility to integrate specific drugs and factors affecting the pathways involved cell mechanoperception in the 3D scaffold design provides a realistic hope to achieve proper arrangement of instructive signals regulating cellular differentiation and reprogramming in a spatially controlled manner. In our view, this approach will contribute to increasing the performance of cardiovascular advanced therapies and will open new avenues for the delivery of durable replacement tissues, overcoming the existing limitations.


Despite the fact that an educational programme for parents of youth tennis players has been launched recently, there is a need to empower parents of athletes in sustaining the combination of education and sport careers  of their talented and elite athletes across sports. The aim of this study was to explore the parents’ view of their role as dual career supporters and their need for educational support in this area.In total, 115 parents  of athletes (14–23 yrs) engaged in full time academic education (high school/university) and competitive  sports  in five European Member States  took part in national workshops. The workshops involved discussing five themes and agreeing statements relevant to assist parents supporting their children as dual career athletes: 1) the athletes’ needs; 2) the sports environment; 3) the academic environment; 4) dual career-related policies and services; 5) The educational methods for parenting dual career athletes.A final list of 80 agreed statements were identified: 25 statements mainly related to the sports entourage; 23 to information on dual career-related policies and services; 22 to the athletes’ needs; 17 to the academic entourage, and 8 to the relevant educational resources to parenting dual career athletes, respectively.This cross-national qualitative research synthesized the parents’ perspectives about their needs and the most relevant content of an educational programme for parenting dual career athletes. The findings of this research will help influence the formulation of effective education strategies on parenting dual career athletes to ensure an optimal supportive environment for the successful combination of high-level sport and education careers. In Europe, competitive sports are mainly structured at a club level, with limited relationship with the educational system resulting in possible cultural and organizational divergences and potentially placing talented and elite athletes at risk of academic or sports dropouts . In the Among the key stakeholders involved in establishing a supportive entourage for dual career , 10 and Parenting dual career athletes is a long-term process, often requiring more than a decade of involvement, spanning from the identification of a talented athlete to her/his developmental years and eventual elite sport stage as well as academic requirements at high school and university levels. Thus, dual career stages and transitions are characterized by significant variations in typology, volume, intensity, organization and planning of training and study demands, all which have an impact on the psychological, social and financial development of the athlete , 28. In The extant literature providing information collected from parents themselves on their experience as key influencers and dual career supporters of athletes involve a limited number of participants from few countries and sports , 35–38. To gain a thorough understanding of the educational needs of parents supporting the dual career of their talented and elite progeny, a consortium of ten European university and sporting institutions from six Member States  engaged in a Collaborative Partnership to structure an Education Model for Parents of Athletes in Academics (EMPATIA), under the Erasmus+ Programme of the European Commission for the 2018–2020 period (590437-EPP-1-2017-1-SI-SPO-SCP). In including European countries spanning from dual career state-centric regulations  to countries with laissez faire/no formal dual career structures  , the EMPTherefore, the purpose of the study was to investigate through the use of focus groups the current experiences of student-athletes’ parents and their educational needs to support their parental role. It was hypothesized that parents might face various difficulties  in supporting the student-athletes, in connecting with different dual career constituents , and in being informed on the dual career policies and best practices in place in the countries where the student-athletes live or in other countries where they intend to relocate for sport or academic purposes.The purpose of the focus groups that we conducted with the parents of dual-career athletes was to enable participants to collectively stimulate, build upon, and question ideas and concepts through discussion , with thWhen using qualitative research methods –54 in a person: mother, father, or guardian role; process: how parents/guardians interact with the sport and academic environments of their child; context: dual career policies in place at the meso and macro dual career dimensions; and time: academic and sport levels of the student-athletes) were used as a theoretical framework for interpretation and understanding [Finally, to understand the role of parents in supporting student-athletes in their dual career paths over time, Bronfenbrenner’s four systems of influence  To ensure an appropriate representation of parents aligning with agreed inclusion criteria define by the EMPATIA project team a purposeful sampling was deemed appropriate and a core strength to gain comprehensive meaningful and practical knowledge on parenting dual career athletes . Thus, pIn total, 115 parents  of athletes competing in 12 individual  and 9 team  sports, provided written consent to obtain information on how they perceive their parental role and challenges, and their perceived needs in terms of an educational support for parenting dual career athletes. To maintain a manageable sample size for in-depth qualitative discussion , 12 focuAt the beginning of the focus groups, the organizers provided a 5–10 min presentation on the EMPATIA project and its aims, the expected contribution of participants, and the standard operating procedures of the focus group. Then participants were organized around a table in sub-groups of 5 people with the question to be addressed clearly written on a flipchart. One parent was assigned a leading position, with a researcher as facilitator acting and an observer manually registering supplementary observational data . The open-ended questions allowed parents freedom to interact directly, questioning one another, recalling personal anecdotes, building upon one another’s views, sharing personal experiences, and agreeing or disagreeing with opinions. Each participant was encouraged to write on the flipchart the statements regarding the most important factors considered relevant for answering the question. After a 15-minute discussion, the sub-group leaders had to report the statements to all participants and a 5/7-minute general discussion was allowed. Then, the composition of the sub-groups was changed. When all the five questions had been addressed, a 20/30-minute general discussion with all the participants identified and agreed a final list of statements from their focus group deemed relevant to parenting dual career athletes.Any statement  emerging from each of the five research questions  during the National focus groups was recorded. To produce a conceptually and semantic equivalent translation of the original statement, two members of the research team from each country performed independent translations in English and agreed on a combined version, which was subsequently backward translated by an English reviewer using a blind translation procedure. According to the literature , the besTo validate the clarity of the statements, during the 2018 European as Athlete conference on dual career, 32 dual career experts from 11 countries  were requested to score the list of statements by means of a 5-point Likert scale , with a clarity threshold ≥ 4.0 pt. In light of the potential cultural differences within the European contexts, the consensus method was deemed especially important to maintain the collegiality of decisions .From the 211 descriptive statements of parenting dual career athletes established in an open-ended and non-judgmental fashion, a list of 80 statements was synthesized, validated, and analysed. to information on dual career-related policies and services, 22 to the athletes’ needs, 17 to the academic entourage, and 8 to the relevant educational resources to parenting student-athletes, respectively. Fifteen statements  loaded on two content units. For the athletes’ needs, 10 statements  pertained to aspects related to health and wellbeing, 7 statements  related to values, and 5 statements  concerned life management and logistics. Statements associated with the sports entourage referred mostly to the sport clubs and/or federations , whereas 8 statements  related to the parental role. Out of the 17 statements linked to the academics entourage, the majority of them concerned the schools and universities  and only 4 statements  referred to the parental role. For dual career-related policies and services included several statements requesting more information on existing policies/provisions  or envisaging new policies , whereas 4 statements referred to requests of provisions  and 3 to needs of dual career cooperation/partnerships . Finally, for the relevant educational resources for parenting dual career athletes, statements related to information technologies/social media were less represented  with respect to more traditional educational means .Relative to the five content units, the research team assigned 25 statements to the sports entourage, 23 The current study contributes to foster the dual career culture by presenting the views of 115 European parents of dual career athletes competing in 21 sports in five European Member States, which differ in terms of history, culture, economy, sport preferences, and dual career policies in place. In including countries with well-established structures and policies required to assist athletes and others with fragmented dual career policies , this reFurthermore, both traditional and technological resources for dual parenting education were identified.In this study, parents recognized the need of an education on parenting student-athletes to attain a clear awareness of their parental role and perspective in guiding the athletes’ choices in education and sport careers. In fact, parents recognized that they play a crucial role in providing motivation, encouragement, and support when prioritizing relevant athlete-centred informed short- and long-term plans for sound educational and sport decisions, which could have immediate and/or longer-term implications. In fostering expectations of continuing education viewed as non-negotiable, parents contribute decisively to the formation of the athletes’ attitudes towards dual career that help them not questioning their dual paths, maintaining their motivation to achieve in both domains, being resilient in case academic obligations conflict with their sport duties emerge and extract their potential both in athletics and academics , 38, 61.A holistic perspective is needed to assist the student-athletes coping with positive and more challenging aspects of their individual dual career pathways –64. To eDespite the literature claiming that parents have a consistent readiness to provide a solution-focused logistical support for the student-athletes , 22, 23,To help them supporting the needs of the athletes and their family, some parents envisaged an access to a national dual career coordinator with high levels of contextual intelligence within this domain. Indeed, a cooperation with a dual career specialist, who would serve as a point of contact, is strongly envisaged to help parents learning to recognize early symptom and effective strategies to resolve the problems the athlete is experiencing in the sport, educational and social contexts , 40, 69,The sport entourage encompasses several actors  who might perceive problematic the involvement of parents in sport-related decision-making, especially in presence of differences in cultural, educational, and social background . DespiteIn this study a need of specific education on the parent’s roles, presence, and function at sport level emerged to set realistic and achievable dual career goals, especially when quality coaching is lacking. To foster the advancement of awareness of dual career related-issues, provision of dual career tutorship, mentoring, services, and opportunities, parents envisaged their involvement in the decision bodies of sport clubs and sport federations. In fact, the sport system tends to be resilient to take responsibility in dual career, considering educational aims secondary or problematic for high-performance , 74, 75.Several of the aspects emphasized by parents have been recommended to sports club/training sport centres to increase their awareness in dual career with respect to parents, education, business, and their own coaches . FurtherSimilarly, to the sport entourage, the academic system should not care only for professional knowledge , but also attempt to be adaptable for developing multi-culturally educated and engaged citizens in an era of globalization . In genePrevious research emphasises a lack of consistency in educational support  between and within Member States , 79. In European strategies and approaches to dual career are still in a developmental phase and require a continuous dialogue and exchange of best practices between stakeholders for further implementation, which should include the contribution of parents who are well placed to present clear insights into the support needs of the athletes. In general, the parents involved in the current study were unaware of the European guidelines, policies, services and best practices in place in other Member States. Interestingly, almost half of the statements included in this content unit loaded also on other content units, with a series of issues discussed previously in the sport (n = 6) and educational (n = 4) entourages. These findings emphasize the deep link between legislative regulations and practical actions in the parents’ view of dual career.www.dualcareer.eu) represents the knowledge hub for educational bodies  and sport organisations  involved in envisaging, advancing, and putting into practice the advancements dual career culture [A compelling demand for cooperation between academic, sport, and labour market emerged from several statements, reiterating also the European recommendations in dual career , 9, 30,  culture .To guarantee a full recognition of athletes as students at national and international levels, parents highlighted the need of a clear definition of dual career status. This aspect is very critical because different requirements and eligibility criteria for dual career programmes and services determine unequal treatments of elite European student-athletes, especially those who migrate for sport and/or academic purposes in a Member State different from their native one , 9. PareEducational resources encompass traditional face-to-face and self-directed web-based delivery education, the latter used to reach out large audiences, to eliminate logistical constraints, to allow learning by repeating activities and review educational content at an individual pace . In lighWhilst a quantitative approach to nomothetic theory generation usually results in highly generalizable theories, a qualitative approach is better suited to generate mid-range theories through a hermeneutic path that allows balancing generality and specificity of the generated assumption . The preThis broad knowledge in the understanding the educational needs of parents of student-athletes uncovered the complex phenomenon of parental support for athletes but two main limitations are still present. First, despite the adopted eminence-based approach did offer possible connections between factors, it is important to bridge the fundamental gap of considering relevant factors as standing on their own by means of systematic way of compiling and synthetizing this qualitative information is needed to manage and frame it in contextual and cultural lenses. In particular, further research should consider a concept mapping methodology involving a system-based approach to integrate ideas and knowledge across stakeholder, which could be valuable to improve theory development as a sound basis for directing not only effective educational programmes but also dual career policies and interventions , 87. ThuSecond, the data from the present research are intended as a base to design an EMPATIA educational programme for parents of student-athletes. However, in considering that parenting dual career athletes is a complex multifaceted phenomenon, it is conceivable that the limited sample of dual career experts, university professors, coaches, and club directors from six European Member States would develop educational material not necessarily fitting the needs of parents of student-athletes across and beyond Europe. Thus, further research should consider the opinions of a representative cohort of parents piloting a specific programme for parenting dual career athletes and providing insights for future revisions.The current cross-national qualitative research synthesized the parents’ perspectives about the needs of an education for parenting dual career athletes, which are not available in studies carried out in a single country. The present findings highlight four key dual career partners: the student athlete, the academic staff, the sport staff, and the parents themselves, who are often inexperienced regarding operational strategies and have limited information on specific dual career policies and services currently available. Indeed, parents envisaged consistent and shared dual career values with athletes, coaches and teachers to determine coordinated efforts in ensuring quality experiences during the holistic development of the student-athletes , 36, 40.S1 File(XLSX)Click here for additional data file.


Thanks to omic disciplines and a systems biology approach, the study of essential oils and phytocomplexes has been lately rolling on a faster track. While metabolomic fingerprinting can provide an effective strategy to characterize essential oil contents, network pharmacology is revealing itself as an adequate, holistic platform to study the collective effects of herbal products and their multi-component and multi-target mediated mechanisms. Multivariate analysis can be applied to analyze the effects of essential oils, possibly overcoming the reductionist limits of bioactivity-guided fractionation and purification of single components. Thanks to the fast evolution of bioinformatics and database availability, disease-target networks relevant to a growing number of phytocomplexes are being developed. With the same potential actionability of pharmacogenomic data, phytogenomics could be performed based on relevant disease-target networks to inform and personalize phytocomplex therapeutic application. The use of plant extracts dates back to the ancient Mediterranean populations. Aromatic plants, essences, and oils have been used for ages in traditional medicine, ceremonies, beauty care, food preservation, and perfumes. They have also been the basis for herbal and botanical medicines and remedies contributing, together with other traditional medicinal preparations, to the development of pharmaceuticals . The earEOs are generally extracted by low/high-pressure distillation. Other processes include solvent extraction, absolute oil extraction, resin tapping, wax embedding, cold pressing, liquid carbon dioxide, or microwaves . Most ofEOs are known for multiple biological activities, among which antioxidants, antiseptic, antifungal, analgesic, anti-inflammatory, spasmolytic, and anesthetic properties ,10,11,12EOs biological activities are usually tested on pharmacological experimental models, and the activity is normally attributed to the most meaningful molecule(s) based on the composition. Nevertheless, when used alone, the same purified single molecules usually do not possess the same biological activity. This is usually attributed to the presence of many different molecules, many with similar structures, that can collectively affect the biological activity. As a result, pharmaco-toxicological parameters such as IC50, although useful to screen different EOs with respect to specific biological activities , can harEO research has often used bioactivity-guided fractionation to identify fractions enriched with the pharmacological activity of essential oils or other phytocomplexes, an approach that has often led to the identification of molecules or families further developed to obtain drugs ,20,21. PIn general, the reductionist, magic-bullet oriented approach of identifying single molecules to strike a pharmacological target, has revealed with time its intrinsic limits when dealing with phytocomplexes. In most cases, the biological activity of the isolated compound does not correspond to the initial biological activity of the phytocomplex, where multiple synergies and antagonisms between molecules and between molecules and molecular targets occur and contribute to the biological activity as a whole . By remoHerbal products have dominated the pharmacopeia for hundreds of years and have provided large amounts of medicines ,32,34. MDiseases with a multifactorial etiology are today increasingly treated using different drug combinations, aiming at different targets  . It is rSystems biology integrates information about individual components of a biological system. Large databases from various sources and dedicated software can be used to predict the effects of substances on human health . In netwOmics technologies allow the simultaneous detection of entire molecular families in a given biological system. At the same time, bioinformatics provide different software tools to collect, classify, network, and view a large number of analytical data. Systems biology offers the system-level framework and a holistic approach to all biologic phenomena, based on the analysis of molecular networks in their dynamic interactions within highly interconnected pathways ,50,51,52Phytocomplexes exert their biological activity by influencing the steady-state of a large number of components in a biological system and their interactions. Biomolecules create tightly integrated networks, and biological responses derive from the behavior of such networks . PhytocoThe multimolecular systems approach of network pharmacology provides a strategy based on bioinformatics tools  to map the multiple simultaneous interactions of the meaningful molecular clusters of the phytocomplex with their biological targets, highlighting pharmacologically relevant pathways. By informing the therapeutic potential of the phytocomplex, the analysis of relevant interactions with pharmacological network nodes empowers the actionability of its applications.This field is currently being extensively used in pharmacology. Identifying in silico the most promising compounds or mixtures of compounds for the desired molecular targets on virtual platforms is currently one emerging strategy in the discovery of new drugs to be used for the treatment of multifactorial diseases . CurrentImportant results have been obtained with the analysis of herbal preparations from traditional Chinese medicine (TCM), where plants are used as blended herbal medicines in formulas that comprise mixtures of mixtures, with each herbal component supposed to exert its specific role, either as an effector, an enhancer, or a mitigator ,65. TCM These TCM studies are based on knowledge from the traditional use of herbal formulae. In most cases, the evidence is a body of traditional medical observations collected during millennia of practical experience, guided by a holistic philosophical framework. The medical approach focuses on wellness based on maintenance of balance between opposite and complementary principles, linked by a flow of energy (Qi) extending as a continuum from cosmos to individuals. Notwithstanding the non-scientific framework, TCM has been successfully using herbal formulae for centuries, and these are still used in integration with clinical, scientifically sound therapeutic interventions. The recent introduction of omic analytical techniques, endowed with bioinformatics and a holistic systems biology approach, has opened to the possibility of re-interpreting complex TCM herbal formulae within a scientific framework. Indeed in the last ten years, numerous network pharmacology studies have produced a wealth of experimental data that today provides a new interpretation supporting the use of phytocomplexes from TCM formulae ,67.A network pharmacology approach involves a functional reconstruction of the phytocomplex based on its molecular components. Their association with relevant molecular targets is the first layer of the network, while the further association to disease and pharmacological effects completes the network  characterization to verify their bioavailability to reach biologically significant targets. Once drugability has been evaluated, computational methods and databases applied in herbal medicine can be used to identify potential drug targets from multiple therapeutic areas. Identifying drug-target interactions provides essential elements for a network construction where drugs and targets are represented as nodes and the interactions as edges, where all the elements are connected to one or more nodes. The prediction of target profiles and pharmacological actions can then lead to the drug-target-disease co-module associations ,82,83,84Essential oils can differently synergize, antagonize, and/or interact with the human body by numerous mechanisms. Their pharmacological efficacy is obtained thanks to integrated multimolecular systems that analyze different biological phenomena leading to a collective effect of clinical significance . Herbal Methods of multivariate statistics can evaluate chemical fingerprints to classify samples and predict their quality. Chemometric methods can be used for assessing data relating to the quality of herbal products . TechniqPistacia genus on human tumor cell lines. The biological activity of different samples from various species of Pistacia was plotted versus the molecular fingerprint of the EOs, and several clusters of molecules resulted in associating significantly better with the biological activity. In particular, PCA was performed on a Pearson correlation matrix, computed with the contribution of each compound to the IC50 of each oil sample, taking into consideration the intrinsic contribution of the compound to the cytotoxic effect of the phytocomplex. PCA allowed the identification of 46 compounds in the phytocomplexes correlated with potential biological activity, distributed within different clusters of molecules potentially cooperating to achieve the cytotoxic activity on the cell lines. The analysis does not take into account the contribution of the single molecules, but the final result of their presence in the biological environment, providing an inductive, and at the same time, holistic reading of the experimental evidence. Merging the chemical composition data and the biological results by a multivariate approach allows evaluating the bioactivity of complex mixtures. At the same time, it highlights the cooperating clusters of bioactive molecules  and principal component multivariate analysis (PCA) to study the cytotoxic activity of essential oils from various species of the ules see  68]. Th. ThPistaOnce significant clusters of bioactive molecules are identified within the framework of their original poly-molecular complex, the appropriate analytical steps need to be pursued to translate the molecular knowledge into a potential therapeutic context. The best possible characterization is needed for direct and indirect molecular mechanisms of action that together can contribute to a therapeutic effect. As previously suggested, this step can be achieved using a network pharmacology approach, with the construction of the phytocomplex-target-disease network based on the known molecular targets of the experimentally bioactive molecules in the EO. With the characterization of the disease-relevant target network, it is possible to rationalize the multiple effects of the essential oil components maintaining the integrity of the poli-molecular nature of the phytocomplex and standardize its use to treat or prevent specific medical conditions. The therapeutic actionability of this information finally depends on the experimental strength of available data on the phytocomplex, so that proper clinical trials can be carried out.Individual genetic profiling is becoming more common in the clinic and pharmacogenomic data can be used to personalize therapeutic interventions. Pharmacogenomics is used to identify key molecular assets of pharmacological interest, providing essential pharmacokinetic, pharmacodynamic, and toxicological information. Personalization of therapies can be based on the genetic characteristics of the individual, affecting how drugs are absorbed, distributed, metabolized and excreted, how the pharmacological targets respond to treatment, and how susceptible the individual is to toxic effects. Individual molecular characteristics can thus be identified for prominent risk factors and treatments tailored using targeted therapies. The identification of networks between phytocomplexes and targets highlights the molecular mechanisms that collectively are associated with the herbal effect and depend on molecules whose genetics can significantly affect the efficacy of the herbal treatment . DiffereIn conclusion, the high-throughput power of omic disciplines together with bioinformatics, multivariate analysis, and in silico methodologies, rationalized within the holistic framework of network pharmacology, provide a fast-growing and unprecedented number of tools and a new strategy to study multifactorial biological environments. This introduces a multidisciplinary scientific approach to study complex mixtures that have long been approached with an experience-based view or a reductionist single active-molecule isolation quest. Currently, it is becoming increasingly possible to bridge together direct experimental data with the real multimolecular composition of biologically active mixtures, thus contributing to an evidence-based study of phytocomplexes. Such an improved insight into phytocomplexes can promote the development of drugs based on essential oil bioactivities, as well as new strategies for activity-driven drug development based on multi-target and molecular cooperation of drug combinations. In this context, the pharmacological activity of known drugs can be improved by the addition of other molecules, thus guiding the conception of new multimolecular drugs, exploiting their synergy in multi-drug combination therapies, or multitarget drugs ,111,112.