After injury to the spinal cord, edema contributes to the underlying detrimental pathophysiological outcomes that lead to worsening of function. Several related membrane proteins called aquaporins (AQPs) regulate water movement in fluid transporting tissues including the spinal cord. Within the cord, AQP1, 4 and 9 contribute to spinal cord injury (SCI)-induced edema. AQP1, 4 and 9 are expressed in a variety of cells including astrocytes, neurons, ependymal cells, and endothelial cells. This review discusses some of the recent findings of the involvement of AQP in SCI and highlights the need for further study of these proteins to develop effective therapies to counteract the negative effects of SCI-induced edema. Water can move rapidly into and out of most cells of the human body. This passive, bidirectional transport of water may occur directly across biological membranes. However, the phosopholipid bilayer can restrict the rate of water flow depending on the properties of the cell . As suchThe initial discovery that AQPs were functional water channels came from transiently transfecting AQP1 into Xenopus oocytes which, when exposed to distilled water, swelled significantly when compared with control, non-transfected oocytes. This was the result of increased AQP1 levels present within the cell membrane, which allow an influx of water to occur when the extracellular solution was hypotonic to the intracellular ,10. The 9 molecules of water per second across in some water-selective AQPs, e.g., AQP1, 2, 4 and 5 [AQPs appear to exist functionally in a common homotetrameric structure  with rec 4 and 5 . The tertiary structure of AQPs is considered to be of an hourglass shape with the α-helices tilting to form a barrel  21]. Fu. Fu21]. There are 13 functional isoforms of human AQPs currently identified with each containing specific and localized expression patterns and functions. These isoforms have subtle differences in the primary, secondary, and tertiary structures of the proteins. The most important differences are those observed within the elements that line the pore of the channel especially the residues that offer capacitance or resistance to water and sometimes other small molecules. Since different tissues in the body require transport at different rates, the isoform localization of AQPs reflects this specificity of action. For example, different cell types in the kidney collectively express nearly all known human AQPs (AQPs 1–4 and 6–11) while the spleen only appears to express AQP9 . AQPs seWater permeation is a dynamic and fluctuating process in most tissues of the human body and, therefore, functional regulation of AQPs are critical to their activity. This is to ensure that water flow occurs at the appropriate locations and rates for each cell or tissue. Regulation of protein expression is generally the most common means of protein regulation since transcriptional rates of most genes physiologically fluctuate especially under stress . This haThe most common and frequently observed method for regulating AQP activity is through conformational ‘gating’. This type of regulation was first reported in plant AQPs such as those in spinach where an intracellular loop physically barricades the intracellular pore following dephosphorylation at a local serine residue ,38. Whil2), results in a G-coupled intracellular response leading to protein kinase A (PKA) activation, AQP2 phosphorylation, and the ability to translocate to the basolateral membrane .,67.65,67−/−Aqp4 knockout mice [The identification of AQP4 at these locations probably relates to the identification of crystal-like intramembranous protein ‘assemblies’ that were previously observed in the brain and termed orthogonal arrays of particles (OAPs) ,69. AQP4out mice . These Aout mice . Higher out mice . Collect−/−Aqp4 mice models, which exhibited vastly reduced levels of post-ischemic edema following an induced ischemic stroke [−/−Aqp4) [−/−Aqp4 mice, this volume reduction is enhanced [+/K+/2Cl− cotransporter 1 (NKCC1) into astrocytes. Many of the physiological functions of AQP4 have been revealed by using genetically modified mice models exposed to varied pathological conditions . One of c stroke . Furtherc stroke . More spAqp4−/−) . AdditioAqp4−/−) . It is dAqp4−/−) . Howeverenhanced . TherefoAQP4 is also present with a non-polarized distribution within fibrous astrocytes of the spinal cord white matter and optic nerve and also within Muller cells within the retina ,77,95. IMost of the studies investigating roles of astrocytic AQP4 tends to focus on either the brain or the spinal cord. At present, the translation of knowledge of the AQP4 location to physiological function is not conclusive. However, due to the highly similar cellular and sub-cellular distribution seen at different CNS locations, it is expected that the same or similar functional mechanisms of AQP4 occur throughout the whole CNS. −/−Aqp4 mice, results in sporadic hydrocephalus [AQP4 is also observed within the basolateral membrane of the ependymal cells surrounding brain-CSF barriers . This sucephalus . Howevercephalus ,96.In the normal spinal cord, AQP9 is expressed in astrocytes, catecholaminergic neurons, and endothelial cells of sub-pial blood vessels ,62,82,83A spinal cord injury (SCI) can be broadly categorized into being of traumatic or non-traumatic origin. The former is the result of external physical insult  and the latter is the result of a much more diverse range of internal insults  . The incEdema is a clinically important feature in the progression of SCI that causes secondary damage through physical contortion and/or obstruction of nerve fibers and vasculature that were, otherwise, uninjured by the primary damage . Shortly+ levels for clearance [+ K+-ATPase pumps, which drive excess Na+ down its electrochemical gradient and cause ionic imbalance [− ions is then driven into the cell to electrically neutralize it, which is followed by the influx of water into astrocyte endfeet. This results in cell swelling [Cytotoxic edema is more prominent in astrocytes than in neurons due to their sensitivity to Klearance . It happlearance ,109. Colmbalance ,110. To swelling . AQP4 is+) are altered in their normal concentration gradients. This secondarily affects the driving force for secondary molecules . This dysregulation then modulates the transendothelial concentration of Na+ where the blood concentration of Na+ is significantly higher than that of the parenchymal concentration, which creates a new Na+ gradient and causes a water flux into the parenchyma across the intact BBB. This is the first stage of CNS tissue swelling [Cytotoxic edema creates a premorbid driving force leading to ionic oedema, which is a process whereby highly concentrated extracellular ions , and SCI ,123,124.−/−Aqp4 mice exhibited 35% less cerebral edema when compared to wild-types (WT) with significantly reduced astrocyte endfoot swelling [−/−Aqp4 mice, there is also reported functional and pathological improvement after compression SCI in which mice demonstrated significantly higher motor scores and decreased edema than their WT counterparts for up to 14 days post-injury [AQP4 is the most well-characterized AQP after SCI. AQP4 protein levels appear to change biphasically after SCI, starting with an immediate decrease between onset and three days post-injury, which is followed by a significant increase seen as late as nine months post-injury . In ischswelling . Furtherswelling . In Aqp4t-injury . Collectively, studies focused on the roles of AQP4 in cytotoxic edema suggest that AQP4 is a mediator in the formation of cytotoxic edema in the CNS. However, more recently, a study showed that inhibition of AQP4 by TNG-020, which is a direct AQP4 inhibitor, following SCI reduces spinal cord water content and AQP4 expression cumulatively with the dose of the inhibitor . This su−/−Aqp4 mice, it was observed that vasogenic edema, as determined by water content, was significantly higher up to 28 days after compression SCI when compared to control levels, which was not observed in the wild-type (WT). Furthermore, these −/−Aqp4 mice also appeared to have a poorer functional recovery following SCI than their WT counterparts [−/−Aqp4 mice were subject to transectional SCI, the water content within the injured half of the spinal cord was significantly higher than the water content within the uninjured half of the spinal cord in +/+Aqp4 mice three days post-surgery. This suggests that knockdown of AQP4 enhances edema even in acute stages of SCI pathogenesis [In terparts . Anotherogenesis .It is important to consolidate and resolve the differences observed for the roles of AQP4 between cytotoxic and vasogenic edema since both of these elements have been demonstrated to occur following human traumatic SCI ,126. Des−/−Aqp4 mice examined shortly after injury appear to have reduced edema and better behavioral functionality than those examined later. This suggests that the role of AQP4 following SCI may be biphasic, having an edema-forming role immediately following injury, followed by a resolution role in later stages. Perhaps this functionally biphasic phenomenon of AQP4 is a result of it having two different roles. The pathological role of causing edema by encouraging cytotoxic and/or vasogenic oedema is followed by the physiological role of maintaining ionic homeostasis by regulating water flow. The conflicting evidence also suggests that, in addition to the role of AQP4 in different types of edema, there must also be consideration about the type of the experimental model used since different models reflect different types of human SCI including both the severity and the pathophysiological processes that occur. For example, transactional laceration SCI likely results in significantly larger amounts of vasogenic oedema due to the direct compromise of BSCB while contusion SCIs likely spare the BSCB directly, which implies that they occur through different processes ,129. FurThere is a need for clarification between the differences in the role of AQP4 in different types/stages of edema formation and resolution in different models of SCI and over the extended time-course after injury . Such inCurrently, there is only one study in rats that shows upregulation of AQP9 after SCI with myelotomy reducing SCI-induced AQP9 and correlating with reduced edema and enhanced functional recovery . HoweverThe approach to treating SCI is complex one due to the highly variable nature of the condition. Despite our increasing pathophysiological knowledge of SCI including the primary, secondary, and tertiary damage mechanisms and their consequences, current therapeutic approaches remain largely palliative. This includes pain relief, rehabilitating symptomatic consequences such as muscle spasticity or bowel problems, and/or implementing methods of patient self-care for symptoms ,132. WhiThe realization that edema can be a secondary damage-causing consequence of the initial injury is a relatively new one and, therefore, therapeutic interventions are few . CurrentMany attempts have been made at altering the genetic expression of AQP proteins to observe changes in structure and function of the CNS both physiologically and pathologically ,135. ForCurrently, the only drug used clinically in treatment following SCI is methylprednisolone (MD). When it was first investigated, it was considered to be an inhibitor of lipid peroxidation and was, therefore, considered to be beneficial following SCI by limiting secondary damage through this mechanism . HoweverAnother drug currently under investigation for the treatment of SCI edema is melatonin. Two earlier experimental studies suggested that melatonin was beneficial for treating SCI due to its apparent ability to reduce free radical damage and neutropil toxicity  and redu180 has been reported numerously to result in the inhibition of the AQP4 channel [180 phosphorylation [111 via CaMKII and PKG results in the increased activation of AQP4 in cultured astrocytes [a-transfected oocytes have reduced water permeability in response to Vasopressin [Some of the clues for pharmacologically treating cytotoxic edema acutely may still reside in biochemical studies of AQP4 and its regulation. In the CNS, the activity of astrocytic AQP4 can be regulated by both short-term and long-term mechanisms. Such short-term mechanisms include the “gating” of activity, which is controlled by phosphorylation of serine residues. PKC-mediated phosphorylation of Ser channel ,149. Howrylation . Furthertrocytes . Howeveropressin  while propressin , which s2+-Calmodulin (CaM)-PKA signaling cascade leading to Ser276 phosphorylation of AQP4, which triggers vesicular translocation between intracellular stores and the cell surface membrane [Another short-term regulatory mechanism of interest in CNS astrocytes is that of AQP4 ‘trafficking’, which is similar in principle to the trafficking of kidney AQP2 in response to Vasopressin. Earlier studies in gastric cells demonstrated that AQP4 could be shown to be internalized and subsequently recycled to the surface membrane in response to Histamine . More remembrane . TherefoThis review highlights some of the current findings about AQPs and their regulation after SCI. It is evident that AQP1, 4 and 9 play a major role in water regulation in the spinal cord but differential expression of AQPs in different cell types suggests multiple functions that require greater understanding prior to the development of therapies aimed at reducing SCI-induced edema. For example, it is evident that AQP4 is probably the most important molecule in regulating water in the spinal cord. However, knockout mouse studies have suggested that complete deletion of AQP4 while reducing acute edema and improving functional outcomes may be detrimental in the chronic stages where AQP4 is required for the clearance of water. Hypoxia and hypertonicity clearly regulate different AQPs and, therefore, may be important mediators of AQP function. Hence, greater understanding of the biology of AQPs after SCI will lead to more effective treatments for SCI-induced edema and relief from the secondary detrimental effect.


Advancements in fabrication technologies and materials engineering, have recently enabled the development of culturing platforms able to display patterns of biochemical and biophysical signals whose features change in time and space in response to external stimuli and according to selected programmes. These dynamic devices proved to be particularly helpful in shedding light on how cells adapt to a dynamic microenvironment or integrate spatio-temporal variations of signals. In this work, we present the most relevant findings in the context of dynamic platforms for controlling cell functions and fate in vitro. We place emphasis on the technological aspects concerning the fabrication of platforms displaying micro- and nano-scale dynamic signals and on the physical-chemical stimuli necessary to actuate the spatio-temporal changes of the signal patterns. In particular, we illustrate strategies to encode material surfaces with dynamic ligands and patterns thereof, topographic relieves and mechanical properties. Additionally, we present the most effective, yet cytocompatible methods to actuate the spatio-temporal changes of the signals. We focus on cell reaction and response to dynamic changes of signal presentation. Finally, potential applications of this new generation of culturing systems for in vitro and in vivo applications, including regenerative medicine and cell conditioning are presented.Biophysical and biochemical signals of material surfaces potently regulate cell functions and fate. In particular, micro- and nano-scale patterns of adhesion signals can finely elicit and affect a plethora of signaling pathways ultimately affecting gene expression, in a process known as mechanotransduction. Our fundamental understanding of cell-material signals interaction and reaction is based on static culturing platforms, i.e., substrates exhibiting signals whose configuration is time-invariant. However, cells  In fact, several routes have been developed to formulate well-defined media that enabled studying cell response to specific soluble signals systematically . Although most cells live in a three-dimension (3D) context in vitro with the introduction of dynamically changing signals would better mimic a natural context thus enabling the possibility to guide and stimulate cells with improved effectiveness.Increasing the complexity of material substrate to control cell functions and fate In this review we first illustrate the basic mechanism of cell ECM or material interactions focusing on cell adhesion processes to provide basic guidelines to engineer bioactive platforms to control cell behavior. We also discuss notable examples of cell interaction with “static” platforms to provide insights into cell's reactions and responses to specific signal arrangements, being more details on this aspect reported elsewhere , cadherins, integrins, selectins, and proteoglycans, as well as non-integrin collagen, laminin, and elastin receptors Hinek, . In partMoreover, molecules related to FAs are important signaling proteins such as kinases  and GTPase  that alter their activities when subjected to the mechanical forces exerted by the actomyosin machinery. For example, contractile forces can induce the phosphorylation of additional sites of the Src family kinases substrate p130CAS, which can eventually trigger signal transduction pathways , while a density of 1.0 × 10−14 mol/cm2 (interligand spacing of ~140 nm), was necessary to promote the formation of focal contacts and stress fibers  is among the most studied and used ligand to control cell adhesion . Literature is sometimes ambiguous on the term static and dynamic . We here refer to dynamic presentation when signals and more specifically adhesive signals significantly change configuration and or location in a way that cells recognize such a change and modify their behavior accordingly. The extent of change in signal display depends on the specific context that has to be analyzed as changes may occur on different length or time scales .Synthetic materials with properties that are static in time are often inadequate for mimicking natural cell environments, where temporal changes in chemical and physical properties are important in a wide variety of contexts including development, differentiation, morphogenesis, diseases progression, wound healing, and homeostasis , topographic and mechanical signals on cell adhesion, spreading, migration, and differentiation.Self-assembled monolayers (SAMs) are one among the first and perhaps most used and studied platforms to enable signal display dynamically. SAMs can be defined as orderly assemblies of molecules formed by the adsorption of an active surfactant on a solid surface Ulman, . The molAnother method to fabricate cell culturing substrates is polymer casting. Surfaces of cast polymer are compatible with functionalization techniques such as chemical grafting or topographic embossing 11(OCH2OCH2)3OH and HS(CH2)17CH3] on gold confined cells in specifically designed rectangles. The application of a cathodic voltage pulse (−1.2 V) caused the alkanethiols to desorb. In the absence of a protein repellent layer, soluble FN in the medium readily adsorbed on the bare gold enabling the cells to move from the initial rectangle onto the newly available adhesive regions  a photomask. This is a substantial advantage as opposed to electrical controlled surfaces for which case embossing adhesive patterns of signals could require the design of complex networks of electrodes or additional manipulations of the substrate with, for instance μCP.2 adhesive islets formed FAs on the patterned islets only, whereas they were able to stretch and spread across the non-adhesive areas. These data confirm that photomasking enables achieving sufficient spatial resolution to study cell adhesion at a FA level dynamically -2-butyl ester attached on the Asp residue of the ligand. A tetra(ethylene glycol) linker was anchored to a silica surface from the one side, whereas the caged ligand was on the tail side. 3T3 fibroblasts were seeded on the substrate before and after irradiation with UV at 351 nm. The authors found more than 10-fold increase in adhered cells on substrates exhibiting the ligand in the active form. Furthermore, the authors used photomasks to activate the substrate on selected regions and, as expected, cells adhered on the exposed ligands predominantly. However, a degree of non-specific adhesion was observed on non-irradiated areas especially at longer culturing times.In order to control cell-material adhesion at the molecular level directly and irrespective of influences arising from material properties Petersen et al. exploited a photosensitive ligand caging strategy . Conversely, a general decrease in adhesion was observed when the azobenzene was in the cis state, which caused spacer shortening and decreased accessibility to the ligand  or hidden in the SAM (in the cis configuration). The authors demonstrated that the SAM reversibly enabled/impaired adhesion of NIH3T3 in a time-frame of few hours. Also, in this case, changes in the orientation and conformation occurring at the molecular level are sufficient to alter cell adhesion. Adhering cells could be detached by supplementing the culture medium with soluble GRGDS at pH 8.0. In this condition, the soluble ligand competes with the bound one. After detachment azomolecules could be switched to the cis isomer thus hiding the ligand and reducing cell attachment consequently. After irradiating with visible light, azomolecules reverted to the trans form, which once again enabled cell adhesion. In this case reversibility is subjected to additional steps therefore this method cannot be considered as fully reversible. RGD ligand burying into a polyelectrolyte multilayer (PEM) was exploited to reversibly modulate NIH3T3 cells adhesion with UV light irradiation  caused an extensive cell detachment.Changes in azomolecule conformation were also exploited to modulate adhesion on SAMs through a host-guest approach  (PNIPAM) showing a lower critical solution temperature (LCST) around 32°C, a temperature that does not alter cell viability significantly. Okano's group pioneered the use of PNIPAM to revert the adhesion of confluent cell layers enabling their detachment from culturing plates  receptor colocalization. Conversely, hMSCs on enzyme activated substrates expressed β5 rich FAs and increased BMP signaling. Altogether, these data suggest that low (but not null) adhesion prevents metabolome activation for osteogenesis, whereas high adhesion increases BMP signaling through increased levels of intracellular tension. This work demonstrated how delicate the adhesion/tension balance is and subtle perturbations can result in differentiation of stemness maintenance. Only advanced dynamic platforms as the one here described can provide a reliable testing platform to unravel the complex mechanisms underpinning fate decisions.More recently, Roberts et al. exploited enzyme switchable substrates to investigate and manage the delicate adhesion/cytoskeleton balance regulating quiescence or differentiation of human multipotent mesenchymal stromal cells (hMSCs)  (pDR1m) as a suitable substrate to emboss topographic patterns reversibly for cell culture experiments  of 40°C  and effective, it is not truly reversible as secondary shapes cannot be reacquired. Furthermore, cells on secondary topographies need to be cultivated below the Ttrans (28°C) which might be an issue for long-term cultures. Beside adhesion and orientation, patterned SMPs were shown to dynamically regulate the structural and contractile properties of cardiomyocytes  enabled to investigate cell recognition and response to time changing topographies by exploiting consolidated and inexpensive technologies such as casting and hot embossing -based gel whose stiffness can be increased through light-mediated radical polymerization diacrylate whose crosslinking kinetics and hence stiffening dynamics was tuned to match the temporal variations of stiffness observed in developing embryos . Cells are exposed to continuous changes of the Montell, . Therefoin situ softening (from 14 to 3.5 kPa), whereas subsequent stiffening (from 3.5 to 28 kPa) increased the cell area and nuclear localization of YAP/TAZ. Authors of the same groups further elaborated the design of the photoresponsive system and successfully produced a supramolecular hydrogel constituted by hyaluronic acid functionalized with the photoresponsive guest-host pair azobenzene and β-cyclodextrin (Rosales et al., trans configuration form links with cyclodextrins that can be disrupted upon UV irradiation that promotes the cis conversion under cytocompatible conditions. Such a design possesses the advantage of decoupling changes in network mechanical properties from changes in chemical composition. This endows the system with well-defined physical/chemical characteristics suitable for studies in mechanobiology.More recently, hydrogels with reversible mechanical properties were fabricated through modular modifications of HA (Rosales et al., Adhesion mediated signaling regulates various aspects of cell behavior including proliferation, migration, and differentiation. Cell adhesions have a direct impact on cytoskeleton arrangement, cell contractility, and nucleus shape, which altogether affect gene expression and eventually cell functions (Dalby et al., Literature data has provided compelling evidence that cell functions and fate are not dictated by a single signal type, but it is rather a complex interplay of multiple signals acting on different length- and time-scales that determines the final cell state (Dalby et al., Furthermore, dynamic platforms are usually engineered and fabricated to display/conceal binding sites. However, dynamic changes of the ECM are not limited to adhesion cues and involve multiple signal changes according to different chronoprogrammes. In fact, the literature on dynamic platform mostly concerns platforms regulating adhesion/detachment and migration. Yet, recent examples demonstrated enormous potentialities of dynamic signal display, particularly in what concerns fate decisions and in the acquisition of specific tissue functions (Young and Engler, CC, LR, and MV gathered information and data from the literature and drafted the manuscript. MV and PN took the lead in organizing the final version of the manuscript. MV wrote the final version of the manuscript and edited figure panels. All authors provided critical feedback to shape the final version of 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.


This review gives an overview on these additional diagnostic techniques and the recent developments in the field of molecular genetic alterations in Spitzoid neoplasms. We also discuss how the recent findings might facilitate the diagnosis and stratification of atypical Spitzoid neoplasms and how these findings will impact the diagnostic work up as well as patient management. We suggest a stepwise implementation of ancillary diagnostic techniques thereby integrating immunohistochemistry and molecular pathology findings in the diagnosis of challenging ambiguous Spitzoid neoplasms. Finally, we will give an outlook on pending future research objectives in the field of Spitzoid melanocytic lesions.Spitzoid neoplasms are a distinct group of melanocytic proliferations characterized by epithelioid and/ or spindle shaped melanocytes. Intermediate forms that share features of both benign Spitz nevi (SN) and Spitz melanoma, i.e., malignant Spitz tumor (MST) represent a diagnostically and clinically challenging group of melanocytic lesions. A multitude of descriptive diagnostic terms exist for these ambiguous lesions with atypical Spitz tumor (AST) or Spitz tumor of uncertain malignant potential (STUMP) just naming two of them. This diagnostic gray zone creates confusion and high insecurity in clinicians and in patients. Biological behavior and clinical course of this intermediate group still remains largely unknown, often leading to difficulties with uncertainties in clinical management and prognosis. Consequently, a better stratification of Spitzoid neoplasms in benign and malignant forms is required thereby keeping the diagnostic group of AST/STUMP as small as possible. Ancillary diagnostic techniques such as immunohistochemistry, comparative genomic hybridization, fluorescence  Melanocytic neoplasms comprise a heterogeneous group of tumors that are characterized by their distinct histopathological appearance, their clinical features and their genetic aberrations. Roughly, melanocytic neoplasms can be distinguished into three major groups of (i) conventional melanocytic neoplasms including benign acquired and congenital nevocellular nevi and cutaneous malignant melanoma, (ii) dendritic, i.e., blue melanocytic neoplasms and (iii) Spitzoid melanocytic neoplasms. Each group is characterized by a distinct histomorphological appearance what is also reflected by the spectrum of underlying genetic aberrations Figure . In the BRAF V600E mutation, melanomas with NRAS activating mutation, those with NF1 mutation and the “triple wild-type” lacking mutations in the afore mentioned genes [Figure GNAQ and GNA11 [Figure According to the Cancer Genome Atlas (TCGA) cutaneous malignant melanoma comprises four molecular subtypes, i.e., melanomas with Spitzoid neoplasms are composed of large epithelioid and/ or spindle-shaped melanocytes with large nuclei that contain vesicular chromatin and often prominent nucleoli and are usually arranged in a distinctive architectural configuration Figure . SpitzoiThe unclear etiology and pathogenesis of Spitzoid neoplasms as well as the diagnostic difficulties has led to numerous additional studies aiming to better understand pathogenesis and to identify biomarkers for use in daily diagnostics.In this review, we will briefly describe the histopathological features of Spitzoid neoplasms and give a detailed overview of the current knowledge of the molecular genetic landscape of Spitzoid neoplasms. We also discuss the usefulness and the limitations of ancillary methods in ambiguous Spitzoid neoplasms to achieve a more accurate and distinct diagnosis. A reasonable integration of histopathology and stepwise performance of ancillary immunohistochemical and/ or moleculargenetic techniques should aim to minimize the gray zone in between SN and MST thereby improving diagnostic accuracy and patient management. Finally, we will give an outlook on potential future research questions and unresolved research objectives in the field of Spitzoid melanocytic lesions.The diagnosis of a Spitzoid neoplasm is primarily based on histopathological grounds. However, the diagnosis should always be made in the context of the clinical features, especially including the patient's age with lesions in adults harboring a higher suspicion for malignancy [Table A SN is usually >5 mm in diameter, shows a dome or wedge shaped symmetrical outline with sharp lateral circumscription, maturation, and zonation to the depth of the lesion and epidermal hyperplasia [Figure AST present with intermediate histological features of SN and MST. AST show increase in at least one worrisome histological feature i.e., ulceration, size >5 mm, infiltrative growth into subcutaneous tissue with “pushing” margins, increased cytonuclear atypia, increase in cell density with confluent growth, >2 dermal mitoses, absence of junctional clefts, few or no Kamino bodies and more extensive pagetoid extension Table . TherefoFinally, MST is diagnosed on the basis of the following histological criteria: ulceration, asymmetrical architecture, infiltrative growth, severe and/ or confluent cytonuclear atypia, dermal mitoses, pushing borders, epidermal effacement and pagetoid extension (Table TERTp) mutations, BRCA1 associated protein 1 (BAP1) mutations, HRAS mutations and receptor tyrosine kinase alterations  will be discussed. Furthermore, results from molecular pathology analysis using comparative genomic hybridization (CGH) and fluorescence in situ hybridization (FISH) will be reviewed and their diagnostic applicability will be discussed. We also discuss recent findings from mRNA and micro RNA (miRNA) expression studies. Finally, we will briefly focus on recent results from mass spectrometry imaging (MSI), which potentially represents a promising adjunct technique. The additional diagnostic techniques and molecular genetic targets which have been reported in the literature for Spitzoid neoplasms are summarized in Table The aim of many additional studies has been to identify criteria that enable assignment of Spitzoid lesions to a correct and distinct diagnosis of SN or MST and thus to keep the ambiguous diagnostic category of AST as small as possible. The major interest of these studies was to identify parameters that predict malignant behavior in Spitzoid neoplasms with increased risk of recurrence, risk for metastasis, and risk for death. In the following, the findings from IHC studies in Spitzoid neoplasms including cell cycle and apoptosis regulator related markers as well as application of melanocytic markers will be outlined. Recent findings from next generation sequencing (NGS) which have identified telomerase reverse transcriptase promotor  which is localized on Chr. 9p21 have been associated with an aggressive clinical behavior in AST  is a melanogenesis related protein and useful in the assessment of a lesions maturation toward the depth. HMB45 IHC shows cytoplasmatic staining and diminishes in the depth of a SN, whereas in MST positivity has been described to be trans-lesional and more uniform without limitation to the upper parts of the lesion . For ASTHRAS gene is located on the short arm (p) of chromosome 11 and copy number gains of 11p as well as activating HRAS mutations are found in approximately 20% of Spitzoid neoplasms  or an arginine (HRASQ61R). Mutations in exon 2 are infrequent. The amino acid exchange leads to constitutive activation of the MAP/ERK and the PI3K/AKT/mTOR pathway. It has been described that tumors with 11p gains often show an intradermal localization, a desmoplastic morphology with stromal sclerosis and infiltrative margins; proliferative activity is low and most often the prognosis is favorable  1 gene have been described in up to 16% of Spitzoid neoplasms spanning a spectrum from benign to malignant  than in SN [2/8 cases,  occur in a mutually exclusive pattern and are not found in BAP1 (see below) or HRAS mutated Spitzoid lesions  and lesions often show a histologically distinct epithelioid phenotype  in desmoplastic SN , chromosome 6 with the MYB region (6q23), centromere 6 (Cep6) and/ or chromosome 11 with the CCND1 region (11q13), and/or chromosome 9 with the CDKN2A region [9p21; (c-MYC region) has led to improvement in the diagnosis of equivocal Spitzoid neoplasms  with real-time PCR and compared them to benign common nevi . The conTPM3-NTRK1 (2 samples), rearrangements in TPM3, ALK and IL6R (1 sample), BAIAP2L1-BRAF (1 sample), EML4-BRAF (1 sample), and two samples containing a second fusion gene, ARID1B-SNX9 or PTPRZ1-NFAM1. Two tumor samples with metastatic disease harbored a TERTp mutation which was also confirmed with mRNA in situ hybridization (ISH).Characterization of the transcriptome of Spitzoid melanocytic neoplasms by mRNA sequencing has gained increasing attention in the last years , 99, 100Jansen et al. investigated RNA expression differences in long intergeneic non-coding RNA 518 (LINC) and preferentially expressed antigen in melanoma (PRAME) from SN and compared them to conventional malignant melanoma . From thCAPN2, CDC25B, ITGA3, LIG4, HSPA6, DUSP10, and JAK3 and downregulation of IGFR1, IDH1, HES1, BAIAP3, SUV39H2, SHC2, FGF2, and MSH6 transcripts in SN when compared to NCN. We are currently expanding the mRNA expression analysis to AST and MST samples to investigate whether there is a differential mRNA expression profile in the whole spectrum of Spitzoid melanocytic lesions (SN vs. AST vs. MST). We are also evaluating whether the identified transcripts can be identified on translational level with immunohistochemistry.Our group recently investigated the mRNA expression profile of SN in comparison to common nevocellular nevi . A signiAlthough no definitive conclusions can be drawn from the to date performed mRNA expression studies because of the limited number of studies and the relatively small numbers of investigated samples, the results from mRNA analysis are likely to reflect the actual functional state of the neoplastic cells and future studies should expand upon transcriptome sequencing in Spitzoid melanocytic neoplasms.MSI is a powerful new methodology to analyze proteomic signatures in fresh frozen and also in formalin-fixed paraffin embedded (FFPE) tissue samples with spatial fidelity. Few studies have implemented MSI in order to assess its value in the differential diagnosis of melanocytic lesions. Hardesty and colleagues found peptides that discriminate prognostic subgroups in melanoma patients . Lazova After all, the histopathological evaluation remains the initial step and gold standard in diagnosing a Spitzoid neoplasm Figure .2 and absence of ulceration, Table In cases of evident benign histomorphological characteristics with absence of any suspicious criteria , especially homozygous loss of Chr. 9p11, loss of Chr. 6q23 as well as PTEN and TERTp mutations argue for MST.If results from step one with IHC investigation are intermediate showing a MIB-1 IHC index of 2–10%, and/ or variable HMB45- and p16 IHC expression step two should be followed with implementation of molecular pathology , or y Figure , 108. Uny Figure , 107–109Over time there have been numerous approaches to classify Spitzoid neoplasms which are summarized in Figure The findings from the to date identified molecular alterations have not led to emergence of one stand-alone or a clearly defined distinct set of biomarkers that can reliably distinguish whether an ambiguous Spitzoid lesion will behave benign or malignant in the future. The urgent need remains to extend research projects and further immerge into the search of predictive biomarkers.First, the analysis of the transcriptome and further proteomic studies, which are likely to represent the actual functional state of a cell, represent a promising strategy for identification of potential robust biomarkers. In first pilot studies the miRNA profile –74 and tSecondly, Spitzoid melanocytic neoplasms are well-known to harbor prominent collagenous or fibrotic stroma with fine vascularization and presence of infiltrating lymphocytes , 111–113Finally, the lack of objective and reproducible histologic criteria to determine the malignant potential of Spitzoid tumors remains a major diagnostic challenge. Very often the morphology does not reliably reflect the biological potential of a Spitzoid lesion. Apparently, the established histopathologic criteria which are used to differentiate nevocellular nevi from conventional malignant melanoma do not account for the group of Spitzoid melanocytic neoplasms , 118. ThIn summary, the diagnosis of a Spitzoid lesion requires accurate correlation of the clinical, histological and molecular features. Yet, there are no isolated clinical, histological or molecular criteria nor a clearly defined diagnostic pattern of criteria that can distinguish AST from SN or MST with adequate certainty , 119–121In conclusion, the results from the current ancillary techniques and molecular genetic findings should be interpreted together with the clinical and histopathological data to formulate a firm integrative diagnosis and to keep the uncertain diagnostic category of AST as small as possible.VW, JV, and LH designed and developed the outline and concept of the manuscript. VW, JV, LH, and AzH supervised the writing of the manuscript and edited the figures and tables. LH, JV, MG, JB, AzH, and VW wrote, discussed and commented the manuscript. All authors approved the final 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.


Influenza is a major health burden worldwide and is caused by influenza viruses that are enveloped and negative stranded RNA viruses. Little progress has been achieved in targeted intervention, either at a population level or at an individual level (to treat the cause), due to the toxicity of drugs and ineffective vaccines against influenza viruses. MicroRNAs (miRNAs) are small non-coding RNAs that play critical roles in gene expression, cell differentiation, and tissue development and have been shown to silence viral replication in a sequence-specific manner. Investigation of these small endogenous nucleotides may lead to new therapeutics against influenza virus infection. Here, we describe our current understanding of the role of miRNAs in host defense response against influenza virus, as well as their potential and limitation as new therapeutic approaches. Orthomyxoviridae family of single-stranded, negative sense RNA viruses with segmented RNA genomes  and neuraminidase (NA)], nucleoprotein (NP), two matrix proteins (M1 and M2), three polymerase complex proteins PB1, PB2, and PA, four non-structural proteins  , 47. HA in silico analysis and a luciferase reporter assay have reported that the same conservative region of PB1 mRNAs of H1N1, H5N1, or H3N2 subtypes is targeted by miR-3145 , and laboratory of genetics and physiology 2 (LGP2) –90. RIG-New evidence has emerged on the potential role of miRNAs in IAV-induced host defense responses and in the modulation of the production of cytokines. MiR-26a significantly inhibits IAV replication by promoting the type-I IFN production and subsequent expression of ISGs in H1N1-infected A549 cells . IAV infInterestingly, certain miRNAs have been shown to have opposing effects on regulating the antiviral response when host cells are infected at different doses of specific viruses. MiR-485 is induced by IAV infection and inhibits RIG-I pathways at low levels of IAV infection, which suppresses host antiviral responses and enhances virus replication . By contAlthough miRNAs have been increasingly recognized in recent years as potential therapeutic targets for treating influenza infection, the successful miRNA manipulation can be difficult because of many factors such as short half-life of miRNAs, low cellular uptake and expression, pre-matured elimination by host immune cells, interruption of endogenous RNA processes and off-target effects. In particular, targeting a single miRNA may have limited success for treatment because an understanding of the molecular mechanisms underpinning many complex diseases  remains rudimentary. To elucidate the function of a miRNA family simultaneously, traditional antisense methods for a single miRNA are inadequate and laborious for targeting multiple miRNAs; to create genetically modified lab animals is even more arduous. As such, microRNA sponges have been designed to inhibit the function of a miRNA family by creating a single RNA sequence that consists of several tandem miRNA-binding sites for all the members . In factHow to effectively deliver miRNA sponges or mimics to manipulate host miRNAs is challenging for clinical application. For this reason, it is essential to carefully design and select candidate miRNA sequence. Non-specific responses induced by those molecules should also be examined with great caution. Any unexpected effect associated with miRNA’s target should be quantified for the safety and success of miRNA manipulation approaches. Indeed, various types of strategies have been developed to optimize the delivery methods, including chemical modification of miRNA sponge molecules or to encapsulate them with macromolecules . Furthermore, the promoters used by miRNA sponge should be strongest and most suitable for the cells of interest to achieve highest efficacy. If the targeted miRNAs are expressed in many cell types in multiple tissues, the tissue- or cell-specific promoters could be employed to achieve precise expression, which could minimize potential side effects.in vivo  vectors are also valuable to silence candidate miRNAs l models , 122. Hol models . On the l models , 123. Thl models , 123. Fuin vitro assays using luciferase reporter system and miRNA mimics and inhibitors are usually employed to verify an interesting target transcript, caution should be taken to interpret the important biological function in dynamic living systems by exclusively relying on these methods. Furthermore, as miRNAs potentially act as master regulators of disease and inflammation, ill-designed manipulation of a miRNA may generate unexpected side-effects. This is particularly important when considering the observations that different species infected with different IAV strains/subtypes generate different miRNA signatures. In addition, delivery methods that limit miRNA mimics and inhibitors precisely to the infected and inflamed tissues are also needed to be further developed as it is essential for not causing any disruption to normal function of surrounding tissues.Current understanding of the roles of miRNAs in the pathogenesis of influenza is limited by a lack of sufficient characterization of the miRNA-associated molecular pathways in the context of influenza infection, IAV replication, and host immunity. Indeed, great challenge still exists in the field of the identification of miRNA targets within a living organism and in host defense processes that have many layers of molecular and cellular elements with specific spatiotemporal patterns. One of miRNA biological features is that they target multiple mRNAs; therefore, pathophysiological outcomes observed by modifying their function may correlate with subtle changes in the levels of diverse target mRNAs. Although in silico pathway analysis to target the 3′-UTRs of leukemia inhibitory factor (LIF) and NIMA-related kinase 7 (NEK7) whose activation leads to lower H7N9 replication  into viral genomic segments such as NP, NS, or PB1. An attempt to generate novel attenuated IAV vaccine has yielded promising results by inserting a let-7b MRE into H1N1 PB1 gene, which significantly reduced viral replication in bronchial epithelial cells . AlthougManipulation of miRNAs needs to be approached with caution for the reason that intervention of miRNA function may predispose to impaired immunity, cancer, or other unforeseen biological abnormalities. However, miRNA has become more and more attractive as diagnostic biomarkers and potential clinical intervention targets as effective prevention and treatments for IAV infection is poorly available. Furthermore, direct targeting of key miRNAs that underpin IAV infection may lead to new and more specific therapeutic interventions as these small RNAs are implied in regulating specific gene clusters triggered by infection . It is particularly important to explore those miRNAs that can both degrade IAV RNAs and alleviate virus-induced inflammation, as they may concurrently control both virus replication and over-reactive immune responses. Understanding the role of miRNA in fundamental processes associated with IAV infection is necessary to fully characterize their potential in disease diagnosis and prognosis and ultimately for the treatment of disease.TN and MY wrote and edited the paper. XL, ZS, AH, and PF edited the paper.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.


Vasoplegia is the syndrome of pathological low systemic vascular resistance, the dominant clinical feature of which is reduced blood pressure in the presence of a normal or raised cardiac output. The vasoplegic syndrome is encountered in many clinical scenarios, including septic shock, post-cardiac bypass and after surgery, burns and trauma, but despite this, uniform clinical definitions are lacking, which renders translational research in this area challenging. We discuss the role of vasoplegia in these contexts and the criteria that are used to describe it are discussed. Intrinsic processes which may drive vasoplegia, such as nitric oxide, prostanoids, endothelin-1, hydrogen sulphide and reactive oxygen species production, are reviewed and potential for therapeutic intervention explored. Extrinsic drivers, including those mediated by glucocorticoid, catecholamine and vasopressin responsiveness of the blood vessels, are also discussed. The optimum balance between maintaining adequate systemic vascular resistance against the potentially deleterious effects of treatment with catecholamines is as yet unclear, but development of novel vasoactive agents may facilitate greater understanding of the role of the differing pathways in the development of vasoplegia. In turn, this may provide insights into the best way to care for patients with this common, multifactorial condition. Vasoplegia is an abnormally low systemic vascular resistance (SVR) that is manifest as profound hypotension or the requirement for therapies to avoid this, in the presence of a normal or increased cardiac output Fig. . PhysiolPatients in hospitals most commonly experience hypotension due to vasodilatation because of the administration of general or neuraxial anaesthesia, and even when transient this has been associated with adverse outcomes –3; howevIt is uncertain if it is justifiable to consider vasoplegia to be a pathophysiologically distinct entity representing uncontrolled failure of vascular homeostasis or to represent the end of a spectrum of vasodilatation.Vasoplegic shock (VS), synonymous with distributive shock, is a more significant circulatory perturbation that is best described as vasoplegia with evidence of tissue hypoperfusion which may be accompanied with hyperlactataemia . The preTo date, our treatment options are limited and do not target some of the main pathophysiological pathways. First-line vasopressor therapy is typically with catecholamines and resistance is referred to as catecholamine-resistant hypotension (CRH). Although vasopressor infusion is required in order to maintain an adequate MAP, significant variation remains in clinical practice, particularly with regard to personalised targets depending on premorbid characteristics, and current research efforts are addressing this issue . MoreoveThe tools available to clinicians to monitor the severity and impact of vasoplegia are limited , 12 and The commonest cause of vasoplegia in critical care is sepsis. The incidence is dependent upon the definition used and the patient population under consideration . ReceiptVasoplegia and VS occurring in patients following cardiac surgery are the second commonest cause. Diagnosis is more complex as there is an ever-present risk of impaired cardiac output contributing to hypotension, the cause of which must be ascertained early—differentiating between reduced preload from bleeding, impaired myocardial contractility or the occurrence of cardiac tamponade. The second main complicating factor is the frequent use of vasodilatory inotropes that directly influence vascular tone. Therefore, although no consensus definition exists, there are several working definitions that combine i) hypotension in the absence of a low cardiac output state and ii) absence of infection; additional criteria may also include the absence of vasodilatory inotropes such as dobutamine or milrinone, or presence of evidence of tissue hypoperfusion. Clinical factors that predispose to the development of vasoplegia following cardiac surgery have been described –19 and vHypotension due to vasodilatation in patients following major non-cardiac surgery is usually manifest as requirement for vasopressors to maintain an adequate MAP following appropriate resuscitation to restore euvolaemia, and its incidence is seldom reported. Reported risk factors include prolonged surgery and significant requirement for blood transfusion , 25. WheThese are conditions united by significant tissue injury, with consequent hypermetabolism, systemic inflammation and predisposition to developing organ dysfunction. Vasoplegia could be considered to be one such organ dysfunction, and is a recognised complication of polytrauma, burns –28 and, 2+) concentration. VSMC contraction is driven by a rise in cytosolic Ca2+ concentration through release of stored Ca2+ from the sarcoplasmic reticulum as well as extracellular Ca2+ influx through voltage-sensitive channels. Relaxation of the VSMC is driven by a fall in cytosolic Ca2+, due to uptake of Ca2+ by the sarcoplasmic reticulum and expulsion of potassium (K+) or Ca2+ (via K+ channels and Ca2+-ATPase pumps) into the extracellular space, resulting in cellular hyperpolarisation and vasodilation. Vascular tone is therefore dependent on the rate of Ca2+ influx versus removal, which in turn is regulated by intrinsic and extrinsic mechanisms [i.endothelial secretions ii.vasoactive metabolites iii.2)autacoids  in the tunica media. The contractile state of the VSMC is referred to as the vascular ‘tone’ and is regulated through intracellular calcium , first identified as the endothelial-derived relaxing factor (EDRF) , is a cr2) is produced by the endothelium constitutively and causes platelet aggregation [2 by prostacyclin synthase (PGIS) [2 likely offset by other prostaglandin-mediated actions [Prostacyclin  –47, whic actions .2) opposes the actions of PGI2 and promotes vasoconstriction and platelet aggregation [2 has been implicated as a potential causative factor in the increased risk of cardiac ischaemia in patients taking COX2 inhibitors [2 regulates vascular tone through binding to thromboxane-prostanoid (TP) receptors in vascular smooth muscle and, in keeping with other agents, promotes calcium influx and vascoconstriction [2 as a regulator of vasoplegia [2 and PGI2 may be important with high relative levels of TXA2 associated with worse outcome in a preliminary study of patients with sepsis [A short lived prostainoid, thromboxane A2  is the predominant isoform of the endothelin family and is a small peptide which acts as a vasoconstrictor . ET1 acttraction . Subtypetraction . In condtraction . Selectitraction .−), which acts as a powerful oxidising agent that provokes cellular dysfunction and vasoplegia [− production, which may be attenuated by antioxidants [Uncoupling of endothelial NOS enzymes may cause an increase in reactive oxygen species and mitochondrial dysfunction . The supsoplegia . Under poxidants , and reaoxidants .2S) is synthesised from the amino acid L-cysteine through vitamin B6-dependent cystathionine-β-synthase or cystathionine-γ-lyase [2S readily diffuses into the vascular smooth muscle and at low concentrations may have cytoprotective effects, although in sepsis concentrations are significantly elevated [2S contributes to the development of vasodilatory shock through a range of oxygen-dependent actions, including inhibition of cytochrome c oxidase with impairment of mitochondrial function, activation of potassium ATP channels and inhibition of endothelial angiotensin converting enzyme activity [2S interacts with NO, which may attenuate NO actions [2S has also been suggested as a potential therapeutic agent leading to the development of a cytoprotective hibernation-like state. Animals treated with H2S are protected from both lethal hypoxia [2S treatment in modulating the deleterious effects of ischaemia-reperfusion injury in experimental models, including porcine myocardial injury [Hydrogen sulphide (H-γ-lyase . H2S reaelevated . At highactivity , 65–67.  actions , 69. H2S hypoxia  and haem hypoxia . This fil injury .As indicated, efflux of potassium through ATP-sensitive potassium channels is an important mechanism for the regulation of VSMC membrane potential. Over-activation of potassium channels results in hyperpolarization of the cell, with resulting inactivation of voltage-gated calcium channels. The subsequent vasodilatation is an important driver of vascular dysfunction. In addition to endothelial-derived mediators, a number of circulating factors can drive potassium channel-mediated vascular dysfunction, including hypoxia , reducedThe development of vasoplegia may also be driven by changes in the efficacy of circulating catecholamines in generating VSMC contraction. Animal models suggest that in later stages of sepsis, alpha-1 adrenoceptor expression falls, resulting in peripheral resistance to norepinephrine , 81. In Glucocorticoids drive diverse tissue responses in inflammation, including circulating immune cell function and cytokine release . These pVasopressin acts via specific V1 receptors on the VSMC surface to promote increased intracellular calcium via G protein-coupled receptors and phospholipase C, which in turn drives contraction. In septic shock, plasma concentrations of vasopressin increase in the early stages of shock; however, after 24 h levels fall to sub-normal levels, which may be a mechanism for loss of vascular tone . This maAlthough vasoplegia is a well-recognised phenomenon, it still suffers from the lack of a unifying clinical definition. This prevents clinical trialists and translational scientists from sharing the common language necessary to facilitate research and increase understanding of this phenomenon. Certainly, we believe that a uniform approach to describing vasoplegia would reap benefits and stimulate further investigation of the underlying pathophysiological mechanisms. Vasoplegia is a complex phenomenon centred around vascular reactivity with multiple contributory potential mechanisms (outlined in Fig.


This review provides an overview of the known effects of diet, obesity, and the intake of different nutrients on systemic lupus erythematosus (SLE). It summarizes and discusses the studies in rodents that identified how different diets can regulate gene expression in the disease, together with a description of the effects of diet on lupus patients’ inflammatory state and disease severity. The identification of selected dietary candidates that can modulate SLE onset and progression is analyzed in relation to possible targeted approaches that could ultimately ameliorate the management and prognosis of this disease. Systemic lupus erythematosus (SLE) is a systemic autoimmune disease characterized by immune responses to multiple self-antigens, mostly of intracellular origin and including cell nucleus and DNA molecules. The dysregulation of innate and adaptive immune responses in SLE is secondary to several abnormalities that include an impaired clearance of apoptotic cells and an increased availability of self-antigens that induce production of autoantibodies that can form immune complexes with their cognate antigen, fix complement, and deposit in tissue where they can cause damage and impaired organ function .Genetics alone is not sufficient to explain the development of SLE, and environmental triggers influence the development and expression of the disease. Among the environmental factors that modulate the onset and progression of SLE, diet plays a critical role. Nutrients not only impact overall health but also alter the gut microbiota composition and the immune response. In other words, the host’s metabolic status, which highly depends on diet, can favor or not the development of immune abnormalities manifesting with varied symptoms and/or organ involvement that depend on the genetic backgrounds and additional factors . This exBoth lupus-prone mice and patients with SLE benefit from a dietary consumption of n-3 polyunsaturated fatty acids (n-3 PUFA). Mammals, including humans, are not able to synthesize PUFA and thus require those nutrients from exogenous sources such as fish oil .The beneficial effects of dietary n-3 PUFA have been associated with anti-inflammatory activities induced by the suppression of proinflammatory cytokine production and the promotion of antioxidant enzyme activities, in addition to a reduced immune effector activity and a decreased antigen presentation .1 (BW) mice, while it increased the expression of antioxidant enzymes [+ T cells, the helper cells in the production of autoantibodies from B cells, provided a comparison, by PCR array, of the relative expression of 84 genes in kidneys and spleens from the three different groups of mice. At week 34, 33 genes were downregulated and 3 upregulated in kidneys of DFO-fed mice as compared to HOS-fed mice, and 37 were downregulated and 2 upregulated as compared to CRN-fed mice. In the spleens, 26 genes were downregulated and 1 was upregulated in DFO-fed mice as compared to HOS-fed mice, and 25 genes were downregulated and 7 upregulated as compared to CRN-fed mice [Clinical trials have provided evidence that the consumption of fish oil benefits lupus patients ,7. Those enzymes ,9. Reduc enzymes , togethe enzymes . Further enzymes  (yet oth enzymes ). Gene e+) activation, the phenolic fraction of extra virgin olive oil increased the expression of IκBα due to the prevention of its degradation, leading to downregulation of NFκB (IκBα inhibits NFκB), together with the inhibition of ERK1/2 phosphorylation [The phenolic fraction of extra virgin olive oil has anti-inflammatory and immunomodulatory properties that associated with a reduced production of pro-inflammatory TNF-α, IL-6, IL-1β, and IFN-γ in 38 lupus patients’ peripheral blood mononuclear cells (PBMCs) . In addirylation .+ transport and NaCl homeostasis [Other studies found that high salt intake in the diet of lupus-prone MRL/lpr mice led to an increased frequency of proinflammatory Th17 and Th1 cells that associated with an increased disease severity and shortened lifespan . Intereseostasis .+ T cells had DNA hypomethylation and increased DNA hydroxymethylation that correlated with higher levels of the hydroxyltransferases ten-eleven translocation (TET)3 and TET2, the latter playing an important role in Tfh cell differentiation (and NaCl-induced Tfh cell promotion via regulation of Spn gene expression). In addition to SPN, NaCl increased the expression of SH2D1A, MAP3K1, and STAT5B in SLE patients as compared with controls [Spn and Stat5b, indicating that increased expression of SPN and STAT5B might associate with enrichment of TET2 after NaCl addition. Gene silencing confirmed that regulation of Tfh cell differentiation by Spn was a target of NaCl [+ T cells showed that NaCl induced DNA hypomethylation of genes involved in cell differentiation and development (DNA hypomethylation facilitates gene transcription). High salt-treated cells displayed decreased the expression of BLIMP  and Th1 (IFN-γ) and Th2 (IL-4 and IL-10) cytokines. The genes that were increased were instead involved in T cell differentiation and activation , including the master regulator of the germinal center reaction Bcl-6.The deleterious effects of high salt in MRL/lpr mice on the acceleration and worsening of lupus disease manifestations associated with high salt-induced promotion of T follicular helper (Tfh) cell differentiation . Tfh celcontrols . By enha of NaCl . Maps ofTogether, these studies show that high-salt diet could provide an epigenetic contribution to the pathogenesis of SLE.Dietary supplementation with selected vitamins can have beneficial effects in SLE . Low levAdditionally, the administration of the active metabolite of vitamin A, the all-trans-retinoic acid (tRA), also ameliorates SLE in both patients and mice ,20,21.A limited consumption of phenylalanine and tyrosine in lupus-prone mice or a moderate protein intake is useful in improving renal function in SLE patients . AccumulEpigenetics is the branch of biology that studies the stable, reversible, and heritable changes in gene expression resulting from DNA or chromatin modification or post-transcriptional mechanisms that do not associated with changes in DNA coding sequences . These cDiet is a key environmental component that influences host’s gene expression. Altered DNA methylation pattern and subsequent changes in gene expression have been observed in embryos from pregnant mice fed diets that were rich in methyl donors . DietaryIn addition to methylation, other epigenetic components under investigation in SLE that are influenced by diet are the miRNAs (discussed elsewhere) .Lactobacillus and Bifidobacterium) in lupus patients [Diets can also influence the host’s gut microbiome, imparting changes in the flora composition that have been associated to the pathogenesis of autoimmune diseases . In SLE,patients . On the patients .+ Tregs [Diets directly influence body weight. Food restriction and/or calorie restriction have long been known to be beneficial in reducing disease manifestations in lupus-prone mice ,39, inclAlthough the therapeutic management of SLE that frequently includes the use of corticosteroids affects the patient’s body mass significantly, an excess weight in lupus patients is problematic not only because of its association with impaired functional capacity and fatigue but also because of its implication in worsening the patient’s clinical picture and increased generalized inflammation.Measurements of the body mass index of lupus patients indicated that about 35% of SLE patients are overweight and about 27% are obese . AnotherObese lupus patients have increased gene and protein expression of several proinflammatory molecules such as the cytokines IL-23  and TNF-It has become clear over the years that lupus disease activity and severity can be modulated by dietary intervention, as summarized in However, since there are more than 100 metabolites that are altered in lupus patients , dietaryStudies in rodents where dietary regimens can be tightly controlled have been informative yet have at times resulted in pitfalls when attempting to extrapolate those results to SLE patients. Notwithstanding these considerations, some dietary approaches that target a specific metabolic pathway with a critical role in lupus pathogenesis, such as transmethylation, have shown promise and are moving from preclinical work toward possible clinical use . Further


This review shows recent trends in the use of nanoparticles as labels for electrochemical immunosensing applications. Some general considerations on the principles of both the direct detection based on redox properties and indirect detection through electrocatalytic properties, before focusing on the applications for mainly proteins detection, are given. Emerging use as blocking tags in nanochannels-based immunosensing systems is also covered in this review. Finally, aspects related to the analytical performance of the developed devices together with prospects for future improvements and applications are discussed. Among electrochemical biosensors, immunosensors, based on the principle of high biospecific affinity interactions between antigens and the corresponding antibodies, have attracted considerable attention in many fields, such as clinical diagnosis or environmental monitoring. Electrochemical immunosensors are easy to use, but usually the affinity interaction generates a small current response that is not adequate to satisfy the desired sensitivity, being essential the development of efficient signal amplification strategies. Although enzyme-based labels are the most common, there are critical problems in the construction of enzymatic-based immunosensors such as the need for harsh conditions to keep the enzymatic activity. Additionally, natural enzymes have some intrinsic drawbacks, such as low thermal and environmental stability due to denaturation, sensitivity of catalytic activity to environmental conditions, non-conductivity and difficult and expensive synthesis and purification. The shortcomings associated with the use of enzymes as labels have promoted their substitution by nanomaterials that do not present these deficiencies and have many advantages over them.Nanoparticles (NPs) have been extensively studied compared to bulk materials due to their unique features, including their catalytic, electric, optical and magnetic properties, being electrochemical analysis one of the fields with higher potential application . Thanks From the variety of immunosensors, those based on electrochemical detection are of special relevance, thanks to the inherent advantages of the electrochemistry in terms of sensitivity, simplicity, low cost of analysis and miniaturization possibilities, among others ,10.NPs have been extensively applied in electrochemical immunosensors as modifiers of electrode surfaces for improving both the electronic transference and the ability for antibody immobilization. Metallic NPs are the most widely used here, with carbon nanomaterials being also recently proposed for such purposes ,12,13. NDespite the increasing interest and number of published works on NPs as labels in electrochemical biosensing, there is a lack of recent reviews covering this field, mainly focusing on immunosensing. In this context, the aim of this review is to present a general overview about the most representative and recent applications of NPs as labels in electrochemical immunosensors, based on the aforementioned properties. Such immunosensors have been mostly applied for the detection of biomolecules with clinical interest, such as cancer biomarkers or tumour cells. The obtained results show that the developed technologies can be valid alternatives to the traditional methods and are currently the objective of extensive research.Some NPs possess redox properties that make them easy to be directly detected with electrochemical techniques, without the need for additional steps/reactions after the immunoreaction in which they act as labels. The most representative examples are detailed below.Gold nanoparticles (AuNPs) stand out from the variety of NPs used as labels in immunosensing due to their simple synthesis, narrow size distribution, optical and electrochemical properties and easy bioconjugation alternatives. The advantageous properties of AuNP-based immuno and DNA electrochemical assays have been extensively exploited in the last few years ,16. The Salmonella typhimurium through a magneto-immunoassay using both magnetic particles and AuNPs linked to specific antibodies  resulting of the NP dissolving/destruction, the need of hazardous reagents in this process has limited its practical application in immunosensors as a reliable alternative to traditional immunoassays based on enzymatic labels. In this sense, Costa-García’s group was the pioneer in the development and application of an alternative methodology based on the direct detection of AuNPs without the need for previously dissolving them in highly acidic media . The strThe excellent electroactivity of silver metal together with the well-defined sharp voltammetric peaks associated to the process of oxidation of Ag (0) to Ag (I) make silver nanoparticles (AgNPs) to be of great potential for analytical applications, as reviewed by Compton’s group . Here, tBased on that principle, Ting et al. proposed the use of Ag tags in the development of an immunosensor for prostate specific antigen (PSA) detection at fg/mL levels . In the Escherichia coli and Salmonella typhimurium bacteria, taking advantage of the affinity of Ag for cell surface macromolecules [The high susceptibility of Ag to oxidation makes easy its combination with Au to obtain bimetallic AuAgNPs  with sucolecules   are semiconductor NPs with spherical shape and a diameter between 1–12 nm. Nowadays, they are one of the most studied nanomaterials, mainly because of their unique optical and semiconductive properties. They were discovered in the early 1980s by Alexey Ekimov during his research on semiconductor nanocrystals ,32. SomeIn addition to their well-established optical properties/applications, QDs have also inherent electroactivity coming from their metallic components that make them easy to detect with electrochemical techniques. The typical strategy consists in the QDs dissolution in highly acidic/oxidative media followed by the ASV detection of the metal ions released. Moreover, the use of QDs made of different metals allows to do the simultaneous detection of different targets through the specific potential of re-oxidation of each metal. In this sense, Wang and co-workers were pioneers in the use of QDs of different metals  as labels for the simultaneous detection of different analytes through ASV analysis .ASV detection after QDs labels acidic dissolving has also extensively used in immunosensing in the last decade. As example, PSA, a biomarker for prostate cancer, has been detected using CdS QDs as labels in a sandwich-type immunoassay at clinically relevant levels of pg/mL .2) by Lu et al. [Other QDs, CdSe QDs, have been combined with zirconia NPs  in the surface of the NP, without the need of decompose the QDs . This me2 NPs) and mercury selenide nanoparticles (HgSe NPs). Copper-based metal nanoparticles (CuNPs), that have been proposed only for DNA hybridization biosensing, are also listed in this section.Some other nanoparticles with outstanding electrochemical properties, but not yet been used as labels in biosensors, deserve to be briefly included in this review due to their great potential for such application. That is the case of cerium oxide nanoparticles  is a very interesting material characterized by its high electron mobility and large electron concentration, extensively investigated in the area of optoelectronics . The eleMore recently, it has been demonstrated that selenium and mercury have a toxicological antagonism in animals, occurring normally bioaccumulation of both elements . Such coCopper-based metal nanoparticles are attracting attention in bioanalysis due to their biocompatibility, low toxicity and outstanding optical properties. Some recent approaches have taken advantage of the in situ generation of CuNPs after DNA amplification, followed by NP dissolving and Cu ions detection by stripping voltammetry. This strategy has been combined with the use of aptamers for the detection of PSA biomarker at fg/mL levels . The strOn the other hand, DNA-templated copper NPs are also considered as functional probes in bioanalysis . They arCatalysis is one of the main fields of nanoscience and nanotechnology, with the development of highly catalytic and electrochemically stable catalysts being a crucial goal in electrochemical applications. The special surface characteristics of the NPs make them ideal candidates for their use in electrocatalytic processes, mainly due to the higher proportion of atoms at the NP surface than in bulk state, that results in a high surface to volume ratio.Silver electrodeposition, hydrogen evolution reaction (HER), water oxidation reaction (WOR) and oxygen reduction reaction (ORR) are simple reactions on which the electrocatalytic activity of some NPs has been approached for electroanalytical applications.Moreover, various nanomaterials have been found to mimic the activities of natural enzymes such as peroxidase, superoxide dismutase, catalase and oxidase, being thus termed as nanozymes ,72. PartThe most representative/relevant works on the immunosensing application of the above mentioned electrocatalytic properties of NPs used as labels are described in this section.Gold nanoparticles have grabbed considerable attention in last years because of their unique catalytic properties. Although bulk gold is an inert material towards redox processes, AuNPs have shown a different behaviour due to the quantum effects related with shape and size of the NPs. AuNPs have been found to exert an excellent electrocatalytic activity towards a variety of chemical reactions, and their ease of conjugation with different biomolecules make them ideal candidates for using as labels in the electrochemical detection of different proteins . AuNPs have shown catalytic properties towards the electroreduction  of Ag (I) to Ag (0), since the presence of NPs shifts the Ag reduction potential to less negative values. A selective electrodeposition of silver only onto the AuNPs surface was achieved by applying an adequate electrodeposition potential, being then the silver re-oxidized scanning to positive potentials, generating a voltammetric peak of currents proportional to the amount of AuNPs . This seHowever, the practical implementation of this strategy is limited because of its incompatibility with screen-printed electrodes (SPEs), probably the most popular electrodes for point-of-care (POC) electrochemical immunosensing. The presence of silver in the Ag/AgCl printed pseudoreference electrode interferes in the detection of the selectively electrodeposited silver, making its discrimination difficult. Alternative methodologies compatible with SPEs, mainly the one based on the electrocatalytic hydrogen evolution, were later proposed.+) reduction from a slightly acidic medium to form hydrogen (H2), has also been proposed as an outstanding methodology for the sensitive AuNPs detection on SPEs [+ ions reduction potential to less negative values in the presence of AuNPs.The electrocatalytic effect of AuNPs towards the hydrogen evolution reaction (HER), that is the hydrogen ions  [3 cells/mL [De la Escosura-Muñiz et al. studied and optimized an AuNPs quantification methodology based on chronoamperometric measurements. By applying a constant voltage  the Hspension    also takcells/mL   and water samples, without the need of broth enrichment, achieving detection limits in the order of 300–500 CFU/mL [Leishmania DNA using primers labelled with AuNPs [The use of magnetic particle platforms in combination with AuNPs tags and the electrocatalyic HER-based reaction was extensively exploited by the same group for the immunodetection of different analytes. As examples of protein biomarkers, they proposed the detection of HIgG at ng/mL levels  Figure C, anti-h0 CFU/mL . The metth AuNPs .2O2 reduction [2O2 reduction [The peroxidase-like activity of various nanomaterials, including AuNPs , has atteduction   [2O2 reduction relied on the synergistic effect towards that reaction of Hemin and AgNPs from AgNPs@CS-Hemin/rGO nanocomposite (microporous carbon spheres loading silver nanoparticles-spaced Hemin/reduced graphene oxide porous nanocomposite) used as label (see 3O4 nanospheres and Ag@Au nanorods (Ag@Au-Fe3O4) was also proposed as electrocatalysts of H2O2 reduction for the detection of HIgG at fg/mL levels [Silver nanomaterials, particularly AgNPs, have also attracted increasing attention from the electrocatalytic perspective, due to their quantum characteristics including large specific surface area and small granule diameter, showing also catalytic activity towards the reduction of H (fg/mL) . The chrabel see B. A hybrL levels .2O2 reduction [2O2 reduction have been reported for the quantitative determination of different biomarkers. Li et al. achieved the detection of hepatitis B surface antigen (HBsAg) at fg/mL levels by using a molybdenum disulphide@cuprous oxide–platinum (MoS2@Cu2O-PtNPs) nanocomposite as signal amplification label [2-GS) as label [Noble metal nanoparticles, especially platinum (PtNPs) and palladium (PdNPs), have attracted extensive attention thanks to their superior biocompatibility, favouring the immobilization of antibodies, and their remarkable electrocatalytic activity towards Heduction ,94. Varion label   as label through the enhanced catalysis of H2O2 reduction (3O4/graphene) as electrocatalytic labels [A chronoamperometric immunosensor for PSA determination was developed by Feng et al.  using Pteduction D. The fgc labels .2O2 reduction. The most representative examples found in the bibliography are detailed in this section.Pd-based nanocomposites have also been proposed as excellent labels for immunosensing, thanks to their electrocatalytic properties towards the H2 mesoporous nanocomposite functionalized by 3-aminopropyltriethoxysilane supported Pd octahedral NPs (Pd/APTES-M-CeO2-GS), that exhibits an important electrocatalytic activity towards H2O2 reduction, was reported by Wei et al. [2-ZIF-67) nanocomposite, taking advantage of the synergistic catalytic effect of PdNPs and the electroactive NH2-ZIF-67 to decompose H2O2 [As example, an ultrasensitive immunosensor for the quantitative detection of AFP cancer biomarker (fg/mL levels) using a nanocomposite made of graphene oxide and CeOi et al. . Anotherose H2O2   were also found to have outstanding intrinsic peroxidase-like activity [Palladium-doped graphitic carbon nitride nanosheets  [SCC-Ag cancer biomarker has been detected at clinically relevant levels of fg/mL through immunoassays, using different multimetallic NPs with catalytic activity towards the Hhell NPs  and amin2-Au@Pt)  have bee2O), with an excellent catalytic activity due to the synergy between Au and Ag and Cu were successfully prepared and used as label for amplified PSA detection at fg/mL levels [2O2 reduction  nanocomposite also exhibited high electrocatalytic activity that was applied for the immunoelectrochemical determination of L levels .2+) was used as label for the early detection of CEA at levels low enough for diagnostic applications (fg/mL) [A nanocomposite made of cubic Au@Pt NPs dendritic nanomaterial functionalized with nitrogen-doped graphene loaded with copper ions (Au@Pt DNs/NG/ Cu (fg/mL)   have been proposed by Gao et al. for the detection of hepatitis B e antigen (HBeAg) at levels of clinical interest (fg/mL) [2+-CS/PPy NTs) not only showed electrocatalytic activity towards H2O2 reduction, but also was used as electroactive compound to directly amplify the current signal of Fe2+, without need of any additional substance [The combination of Au with other noble metal such as Pd has also been used in nanocomposites for its peroxidase-like activity in immunosensing. As example, gold@palladium nanoparticles loaded with molybdenum disulfide functionalized multiwalled carbon nanotubes (Au@Pd/MoS (fg/mL) . A partiubstance . Benefit2-GS) nanocomposite was used as label [In a lower extent, bimetallic Pd–Pt NPs have also been used as electrocatalytic labels in immunosensors, based on the above detailed principles. As representative example, Wang’s group reported two novel signal amplification strategies based on Pd–Pt NPs for the quantitative monitoring of PSA. In their first work a novel mesoporous core@shell palladium@platinum NP loaded by amino group functionalized graphene  have been considered as an excellent catalyst towards Hof fg/mL . 2 NPs) are interesting for biosensing thanks to their biocompatibility, catalytic activity and outstanding stability. A benefit towards other NPs is their ease of being electrocatalytically detected at neutral pH through the water oxidation reaction (WOR), which favours their direct detection in the same medium where immunoreactions usually occurs, avoiding the use of additional reagents. Even more interesting is the fact that the oxide form of the IrNPs exhibits catalytic ability towards the water oxidation reaction, as has been demonstrated under photochemical conditions . Iridium2 NPs towards the WOR in neutral media was studied by cyclic voltammetry and chronoamperometry [2 NPs on the SPE transducer ), was carried out by the same group,  reaching3O4 NPs) are quite popular in biological analysis due to their peroxidase-mimic activity, having advantages such as easier production, lower cost of synthesis, higher stability in harsh conditions and flexible storage conditions [Ferromagnetic nanoparticles, mainly made of magnetite  was linked with the corresponding secondary antibody to form his-Fe3O4@Ab2. This conjugate acted as peroxidase-like reagent, catalysing the H2O2 reduction and therefore the simultaneous oxidation of 4-chloro-1-naphthol (4-CN) present in the media to benzo-4-chlorohexadienone, that precipitates on the sensor surface causing the blocking of the electron transfer. Hereby the target antigen was quantitatively detected via the decrease in the photocurrent signal, produced by the induced catalytic precipitation  have been found to display electrocatalytic activity towards the hydrogen evolution reaction (HER) and towards the oxygen reduction reaction (ORR) [Molybdenum disulphide nanoparticles (MoSon (ORR) .2 NP conjugated with antibodies as signal-enhancing labels based on the chronoamperometric HER monitoring for rabbit IgG quantification at pg/mL levels  and the oxygen reduction reaction (ORR), which occur at neutral pH without the need of additional reagents after the immunoassay. This is of special relevance for the building of highly integrated biosensing systems.Nanoparticles are emerging materials with outstanding potential for their use as labels in electrical immunosensing. Gold, silver, palladium and platinum are the main components of such particles, thanks to their direct electroactivity (redox properties) and/or their electrocatalytic activity towards secondary reactions. While the direct detection is faster and simpler, the electrocatalytic strategies are generally more sensitive, so the choice of nanoparticle and detection route is strongly dependent on the requirements of the concrete application. Attending to the electrocatalytic routes, those based on the hydrogen evolution reaction (HER) and the HIt is also worth highlighting the recent use of nanoparticles in combination with nanochannels for immunosensing approaches, with this being especially advantageous for real sample analysis, thanks to the filtering abilities of the nanoporous-based platforms.Protein biomarkers of a variety of diseases, including tumour cells, are the target analytes on which such electrochemical immunosensors have been mostly applied. In most cases, pg/mL levels of interest for diagnostic applications are detected, being thus the sensitivity a challenge that has been overcome. However, some important issues need to be solved for the definitive implantation of such sensing systems for routine analysis. Efficiency and long-term stability of the nanoparticle-antibody conjugates is a crucial issue that is not usually addressed in most of the reviewed works. Moreover, looking at the application as an alternative to well-established ELISA assays, multidetection abilities, either through electrode arrays or by simultaneously using nanoparticles with different properties should be strongly required. Efforts in this sense should be the next at the current state of the art.Nevertheless, nanoparticle-based electrochemical immunosensors have the greatest potential in the field of the point-of-care (POC) analysis, in our opinion. The combination of the miniaturized electrochemical transducers, the cheap and portable electrochemical instruments and the well-studied properties of the nanoparticles make altogether ideal for POC applications. In this case, the most challenging and less reported issue is related to the sampling/introduction of the analytes in the immunosensor as well as the washing, etc. steps required, which difficult to have highly integrated real POC systems. Here the combination with microfluidics seems to be crucial. Considerable effort in this sense is expected in the next few years.


SNCA, one of the hallmark genes involved in PD, have become more prominent from the current perspective, than mechanisms that directly involve DNA methylation. In the absence of current epigenetic clinical targets to counteract PD progression, we also hypothesize how several mechanisms may affect local and global epigenetics in PD neurons, including inflammation, oxidative stress, autophagy and DNA repair mechanisms which may lead to future therapeutic targets.Parkinson disease (PD) is a common neurodegenerative disorder that progresses with age, with an increasing number of symptoms. Some of the efforts to understand PD progression have been focusing on the regulation of epigenetic mechanisms, that generally include small molecular modifications to the DNA and histones that are essential for regulating gene activity. Here, we have pointed out difficulties to untangle genetic and epigenetic mechanisms, and reviewed several studies that have aimed for untangling. Some of those have enabled more solid claims on independent roles for epigenetic mechanisms. Hereby, evidence that specific DNA hydroxymethylation, global hyperacetylation, and histone deacetylase (HDAC) dependent regulation of  Parkinson disease (PD) is a common neurodegenerative disorder, with a variety of motor and non-motor symptoms . The proThe low prevalence of familial PD suggests that stochastic events, for instance environmental cues, pathogens and lifetime somatic mutations of both nuclear and mitochondrial DNA may all contribute to PD. These ideas have been used as well to explain the relatively low concordance of PD that mono-zygotic twins show while they sprout from a single cell . Next toSince PD shares some characteristics with aging, like DNA and mitochondrial damage, together with an increase in (neuro)inflammation  as well epi translates ‘near’) use of the term seems to increase the usefulness of the term also for pragmatic reasons. As is exemplified by one of the most debatable members of the epigenetic family, micro-RNAs. When compared to the plethora of chemical DNA and histone modifications, micro-RNAs have a very clarifying and distinctive name that does not necessarily benefit being over-arched by a term as epigenetics. In line with this, we consider the epigenome as the pattern of chemical modifications to histone proteins and DNA.The term epigenetic has been established to appoint the intriguing phenomenon that equal genomes give rise to different gene activity states . The terFinally, changes in epigenetic marks may not always change expression states directly, or lead to measurable expression fluctuations , still, such changes we will call epigenetic changes. Therefore, we find that the broadly used definition of epigenetics by Adrian Bird complies these criteria best: “the structural adaptation of chromosomal regions so as to register, signal or perpetuate altered activity states” . To us, 2+ regulation, mitochondrial oxidation pathways and proteosomal/autophagy overload or capacity -positive cellular inclusions called Lewy bodies (LBs) are the most prominent post-mortem pathological hallmark though perhaps not essential for clinical PD  or neuronal degeneration . MoreoveSNCA gene, show largely divergent phenotypes, with the symptomatic onset varying from 18 to 77 years of age in one report, or even lacking full penetrance in another. Even when the SNCA gene is triplicated, it may take decades before the first symptoms are observed and decades more for the disease to become lethal  may find their origin during early fetal development, when on average already several 100 mutations give rise to unique genomes for each individual neuron –D Peran. At the Although the authors show in these elegant studies that the DNA repair related neurodegenerative diseases Cockayne syndrome and Xeroderma Pigmentosum have increased DNA mutation rates , 2018, iIf somatic mutations would play a role in the low PD concordance observed in monozygotic twins, somatic mutations in zygotic cells seem to be better candidates than progenitors or matured neurons. Especially when these mutations occur randomly in all genes, which they seem to do, though with a preference for exons . TherefoAnother interesting aspect herein is that PD progression regularly has a preference to develop earlier and faster on one side of the left-right axis but is most severe when progressing at both sides . To whicBased on the current knowledge, roughly 6–10 mutations will accumulate upon five zygotic cell divisions . From thSNCA point mutations and duplication is not 100%. Noteworthy, several familial mutations in genes involved in mitochondrial function or autophagy do have a penetrance of 100%  and changes in copy number variants (CNVs) have been found to exist in a mosaic pattern in the brain . AlthougFinally, another category of somatic mutations are found in the mitochondrial DNA (mtDNA). Copy numbers of mtDNA increase in SNc neurons of healthy individuals unlike other neuronal types and exceed numbers found in DA neurons of PD patients . Althougcis and trans regulatory DNA elements can affect epigenetics elsewhere, mutations could cause the loss of epigenes (genes that encode epigenetic modifiers) and even undetectable timely changes to the DNA may cause substantial epigenetic defects, like epigenetic modification following DNA repair  epigenetic and transcriptional changes or that they go hand in hand during aging even running of epigenetic clocks remain elusive prevents to determine if they are of any biological relevance.Other ‘independent’ epigenetic phenomena that gathered much attention are epigenetic clocks, that have the potential to predict chronological age very accurately in healthy aging by measure of cumulative methylation states of small, defined subsets of CpGs . StrikinOpposing to epigenetic clocks is epigenetic drift, which has been defined as “the collection of methylated CpGs that are associated with age within an individual but are not common across individuals” . Fraga aSNCA) and its regulatory elements for the following reasons: firstly, as previously mentioned, an increase of SNCA alleles resulting from multiplication correlates with an increased onset of PD and severity of the disease. Secondly, detailed in vitro and GWAS studies have proposed cis-regulatory elements that are associated to PD and potentially regulated by methylation. Thirdly, α-Syn transcript levels correlate to protein levels, together suggesting that transcriptional deregulation is key in disease development of at least a subset of PD patients  regulation of epigenetic marks seems intuitively related to neuronal homeostasis. In PD, global reduction of DNA methylation (∼30%) has been found to correlate to elevated levels of α-Syn and associated to sequestering of DNMT1 outside the nucleus, perhaps functioning as feed forward mechanism . In compFrom a different perspective, global changes in DNA methylation induced by conditional genetic models or in disease have revealed hints on the potential role of global DNA methylation defects for DA neuronal maintenance. For instance, Rett syndrome is caused by an X-linked mosaic pattern of MeCP2 expression, which binds to methylated CpGs in gene bodies of active genes, leading to miscommunication between methylated DNA and other repressive epigenetic mechanisms in the maturing brain of young children . As RettPart of the DNA methylome is expectedly rigid, but a relatively high turnover may occur especially on a selection of genes affected by direct environmental cues and neuronal activity instead. More specifically via hydroxymethylation through the activity of the ten eleven translocation (TET) enzymes that oxidize 5-methylcytosines (5mCs) and promote locus-specific reversal of DNA methylation . In a liCentral in genome wide gene regulation are histone acetylation dynamics. Hereby, negatively charged acetyl groups neutralize positively charged histone lysine residues thereby weakening the interaction with the negatively charged DNA and generally resulting in gene activity . Severalin vitro cellular stress by the administration of pesticides as well as protection against Lipopolysaccharides (LPS), α-Syn and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) using HDAC inhibitors  in intron 4 of the 2/NKX6.1 . The lac.1  agonists to repress SNCA expression (SNCA enhancing elements can be regulated in a level dependent manner by both β2AR agonists (down) and antagonists (up) , an HDAC inhibitor that has highest affinity for class I HDACs where HDAC1 and HDAC2 belong to. On top of that, VPA has been associated with PD in the past, and withdrawal of the drugs has proven to alleviate PD symptoms raising the intriguing question if some of the PD symptoms are reversible by targeting epigenetics   and confgenetics . In summA glance of epigenetic dysregulation of ClassI HDACs in neurodegeneration has been provided by Gräff and colleagues in a study where they primarily underscore the possibility that some of the loss of functions associated to neurodegeneration may actually be reversible via targeting acetylation . HoweverAltogether, in recent years there has been an increasing number of studies supporting the importance of global H3K27 acetylation state in PD, in particular via the regulation of PD associated α-Syn and via a reduction in HDAC activity, or a lack of their recruitment .SNCA will become more important in stressed situations or changed activity of epigenetic modifiers, when it seems likely that more is demanded of gene regulatory feedback mechanisms , HDAC1 and Suv39H1 (H3K9me3), or the silencing of the HATs: P300/PCAF  and SET domain containing 2 (SetD2), two H3K36 histone methyltransferases, which are linked to longevity, though not general to chromatin silencing  Figure . AltogetGenetics and Epigenetics are evidently entangled when it comes to cellular stress responses. In PD, DA metabolism, dysfunction of autophagy, mitochondrial respiration and inflammation have all been suggested to potentially elevate levels of oxidants . One of Additional mechanisms that may well be conserved to neuronal aging is the localization of fumarate to DNA damage sites to facilitate non-homologous end joining (NHEJ) DNA repair and downregulates the H3K36me2 demethylase, KDM2B, leading to increasing H3K36me2 . PerhapsSNCA, possibly caused by a reduction in HDAC activity, or a lack of its recruitment. In diseases associated to PD, findings support a comparable role for Sirt1. Pathways that induce hyperacetylation or epigenetic infidelity include neuroinflammation and endogenous cellular stress, like deregulation of autophagy, oxidation and DNA repair mechanisms (listed in In recent years there has been an increasing number of studies supporting the importance of global H3K27 acetylation in PD, in particular via deregulation of All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.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.