The genus Tityus Koch, in the family Buthidae, responsible for severe/fatal accidents elsewhere in northern South America and the Amazonia, is represented in Ecuador by 16 species, including T. asthenes, which has caused fatalities in Colombia and Panama, and now in the Ecuadorian provinces of Morona Santiago and Sucumbíos. Underestimation of the medical significance of scorpion envenoming in Ecuador arises from the fact that Centruroides margaritatus (Gervais) (family Buthidae) and Teuthraustes atramentarius Simon (family Chactidae), whose venoms show low toxicity towards vertebrates, frequently envenom humans in the highly populated Guayas and Pichincha provinces. This work also updates the local scorpion faunal endemicity (74.5 %) and its geographical distribution, and reviews available medical/biochemical information on each species in the light of the increasing problem of scorpionism in the country. A proposal is hereby put forward to classify the Ecuadorian scorpions based on their potential medical importance.Ecuador harbors one of the most diverse Neotropical scorpion faunas, hereby updated to 47 species contained within eight genera and five families, which inhabits the “Costa” ( There aruroides .Chactas Teuthraustes so far described (n = 24) have been collected in the Andean mountains of Ecuador, Peru and Colombia, and in the Amazonian highlands of Venezuela and Brazil, with the highest species diversity corresponding to Ecuador. Even though the taxonomic validity of some of these taxa will probably be the subject of further scrutiny, the outstanding concentration of species in Ecuador is realistic , and recognized by their generally clear and yellowish coloration, produces intense pain in humans without further complications, although some venoms contain neurotoxins that can produce autonomic effects  but they are not considered medically significant due to the low amount of venom injected , “Tityus androcottoides” [T. (A.) ythieri], or “Tityus asthenes” [T. (A.) asthenes]. These species seem to be restricted to lowland rain forests of the piedmont east and west of the Andes. Most severe/lethal envenoming cases in northern South America, the Amazonian region, and Central America are due to species in this subgenus , with the exception of T. (T.) gasci. This complex contains species mostly inhabiting the inter-Andean valleys in the “Sierra” region  inhabiting the Galápagos archipelago are not considered dangerous to humans although their sting is painful [Species of the genera  painful .According to the species involved, their areas of distribution and the available knowledge of the venom action and composition of allied taxa, as presented in this annotated checklist, we propose a classification of the Ecuadorian scorpion fauna as follows:Tityus antivenoms are available: anti-Tityus (Atreus) discrepans (Venezuela), anti-Tityus (Tityus) serrulatus (Brazil), and anti-Tityus (Tityus) trivitattus (Argentina) [As severe envenoming cases have been recorded from east of the Ecuadorian Andes, a study of the immunological reactivity of venoms from medically significant species towards available scorpion antivenoms is required to establish their neutralization capacity or whether new antibodies are needed . In thisgentina) , 55. Morgentina) .


Diabetes mellitus is associated with greater risks for cardiovascular diseases (CVD). Multiple noninvasive screening tools for CVD including cardiac CT, carotid intima-media thickness test, myocardial perfusion imaging have been examined in those with diabetes, but the prognostic value of these tests vary and issues remain regarding their cost-benefit ratios, potential harms of radiation, and how they fit into screening algorithms for CVD. We discuss in this report the needs and criteria for screening tests and summarize the evidence from observational studies and clinical trials. We also explore whether there should be more sensitive screening modalities to better detect both short and long-term cardiovascular risk among asymptomatic patients with diabetes. Although the prevalence of DM remains high in developed countries, developing countries now comprise the greatest increases in DM prevalence and burden of accompanying comorbidities. Patients with type 2 DM have up to four times the risk of atherosclerotic cardiovascular disease (ASCVD) events compared with those without DM2. Not only do patients with DM have an increased risk of developing ASCVD, but it is often silent4. However, once ASCVD becomes clinically identified, both shorter- and longer-term outcomes in persons with DM are worse than those in persons without DM, and this may be the result of other features associated with DM . These observations establish the theoretical foundations for the early screening of ASCVD among those with DM. Although the concept for systematic ASCVD screening in DM is appealing, the benefits of such an approach have not been fully demonstrated. As there is ambiguity in outcomes regarding the benefits versus harm of such screening, major organizations, including the American Diabetes Association, currently do not recommend routine screening in those with DM5. Therefore, in this report, we summarize current evidence of pros and cons of screening modalities and propose a framework for screening for subclinical coronary atherosclerosis among asymptomatic patients with DM.Globally in 2014, 8.3% of the world population or nearly 400 million people had diabetes mellitus (DM)6. Refining risk estimates in patients with DM may aid in implementing prevention strategies in an efficient and cost-saving manner. Although the Framingham and European risk scores and more recently the American Heart Association/American College of Cardiology (AHA/ACC) Pooled Cohort risk scores emphasize the classic ASCVD risk factors, they are only moderately accurate for the prediction of short- and long-term risk of CVD events7. In addition, the United Kingdom Prospective Diabetes Study (UKPDS) risk engine shows no better performance than FRS and tended to overestimate the coronary heart disease (CHD) risk9. Diabetes in fact can often attenuate the protective effect of optimal levels from other risk factors , and thus the number of traditional risk factors may not be useful in identifying risk in those with DM10. On the other hand, directly examining for subclinical ASCVD, such as by coronary artery calcium (CAC), carotid intima-media thickness (CIMT), endothelial dysfunction, and myocardial ischemia, holds the potential to more accurately discriminate risk in those with DM12.Despite the higher ASCVD risk among persons with diabetes, they are a highly heterogeneous population and diabetes often is not a coronary artery disease (CAD) equivalent. We have previously noted from data in the United States National Health and Nutrition Examination Survey that 32% of men and 48% of women with DM were deemed by the Framingham Risk Score (FRS) to be at low to intermediate risk7. Additional criteria that have been proposed include: (1) ensuring the test identifies a high enough prevalence of disease so that a reasonable number of persons can be identified for intervention and (2) exhibiting high cost-effectiveness13. Currently used modalities may not satisfy all of the criteria completely and instead may vary in providing support for each criterion, thus warranting more studies to provide further validation.A good screening test should have the following features: (1) accurately discriminate low- and high-risk persons, (2) produce reliable and reproducible results, (3) provide incremental value to risk predicted by office-based risk assessment, and (4) detect individuals for whom early intervention is likely to have a beneficial impact14. CAC scanning has emerged as the most powerful tool for refining risk assessment on top of global risk assessment in asymptomatic individuals15. In those subjects with DM, a CAC score of 0 is associated with ASCVD event rates as low as or lower than those of many persons without DM and increasing CAC scores are associated with progressively higher ASCVD event rates; those with DM who have CAC scores of at least 400 have 10-fold greater event rates (CHD incidence of 4% per year) than those with CAC of 0 (38% of our DM subjects)11. Subjects who undergo scanning for CAC also appear to have improved outcomes in terms of improved risk factor control, including blood pressure, low-density lipoprotein cholesterol, and waist circumference, compared with those not scanned16. This observation may be explained by greater adherence to lifestyle modifications and medical therapy on the basis of visualizing their disease20. Also, CAC screening has been noted to be more cost-effective than myocardial perfusion imaging (MPI); it was estimated that CAC scanning can prevent one event at a cost of $71,249, about a third of the cost of MPI and half that of no screening (treating everyone)21.CAC assesses the extent of calcified atherosclerotic plaques in the coronary arteries and is exquisitely sensitive for detection of atherosclerosis23. The 2012 American Association of Clinical Endocrinology (AACE) Lipid Management Guideline also stated that CAC can be used in those with DM to refine risk stratification and the need for more aggressive preventive strategies24. Most recently, the ACC/AHA guideline on the assessment of cardiovascular risk25 identifies CAC screening  as a tool that can be used when, after quantitative risk assessment, a risk-based treatment decision is uncertain. Although current guidelines recommend that all DM patients who are 40 or over be on statin therapy, the intensity of therapy (or possible consideration of therapy in those younger than 40 years of age) may be guided by the use of such testing.Most guidelines have suggested that CAC be considered for screening and risk stratification of patients with DM. Both the 2010 AHA risk assessment guidelines (level IIa) and the 2014 position statement of the Brazilian Diabetes Society (level A) recommend CAC scanning for those who are at intermediate risk or who have diabetes27. However, the Detection of Ischemia in Asymptomatic Diabetics (DIAD) randomized clinical trial demonstrated that screening patients with DM does not improve clinical outcomes28, even when ischemia is present upon repeat testing (at 3 years)29. The negative result may be due to the low prevalence of CAD and thus a low incidence of coronary events. Although some guidelines have advocated screening for silent myocardial ischemia in high-risk asymptomatic patients with DM22, it is no longer routinely recommended in current guidelines23.Observational investigations of MPI have shown high sensitivity (86%) in those with DM and even higher sensitivity among those at higher risk (94%)30. Some of these features are associated with a higher likelihood of near-term major adverse cardiovascular events. Several studies have shown the prognostic value of CCTA findings in subjects with asymptomatic DM32. Whether clinical outcomes can be impacted by CCTA screening, however, was only recently reported by the FACTOR-64 randomized clinical trial, which enrolled 900 asymptomatic patients with diabetes; while showing a 20% reduced risk of subsequent adverse cardiovascular events in those screened with CCTA, the findings were not statistically significant and this was due in part to the fairly low event rates33. Due to a lack of sufficient supportive evidence, CCTA is not conventionally recommended for screening asymptomatic individuals with DM in current guidelines36.Coronary computed tomography angiography (CCTA) allows the evaluation of the full spectrum of CAD from totally normal arteries to non-obstructive disease to significant coronary stenosis and total occlusion. It also allows plaque characterization, including calcified and non-calcified plaque, spotty calcification, positive remodeling, and the napkin ring sign37, the meaning of measuring CIMT alone has recently been questioned, as meta-analysis and pooled cohort studies showed that the addition of common CIMT to traditional risk models was associated with only a modest improvement and is unlikely to be of clinical importance39. Similar findings in a cohort of 4,220 patients with DM demonstrated that common CIMT did not add predictive value to the FRS during a median follow-up of 8.7 years40. However, if combined with CAC, ankle brachial index, high-sensitivity C-reactive protein, and family history, the predictive ability for future CHD events may supersede the traditional FRS and UKPDS risk engine41. Importantly, the presence of carotid plaques alone and in combination with CIMT does add to risk prediction beyond FRS, as demonstrated by Nambi and colleagues42, although this question was not specifically evaluated in those with DM in that study.CIMT is an indicator of atherosclerosis in the carotid artery, measuring the combined thickness of the intima and media with B-mode ultrasound. Although CIMT is related to higher CVD event risk43. Patients with DM require individualized risk assessment before appropriate intensity of treatment can be implemented. Current screening methods have proven to be effective in predicting future coronary events, but limitations remain in that: (1) few studies concerning the cost-effectiveness of various scanning modalities have been carried out; (2) large randomized clinical trials should be designed to directly look into the impact of screening tests on CVD outcomes as well as the impact on downstream clinical decisions, risk factor changes, and the total medical costs; and (3) few screening methods have directly compared predictive efficacy. The results of such trials will allow us to better identify which screening methods should be employed in patients with DM and help inform therapeutic decision making.The appropriate use of multimodality screening in those with DM depends on: (1) clinical history of other risk factors, (2) whether the use of a second or third method can address residual risk not addressed by the first method, and (3) determining the correct order to conduct such tests to maximize clinical utility and cost-effectiveness. For instance, CAC scores can accurately reflect the possibility of abnormal stress MPI findings (and with much less radiation), suggesting a role for CAC scoring as a gatekeeper for patients who may benefit from further risk stratification with stress MPI. Expert consensus opinion recommends stress testing imaging in individuals whose CAC score exceeds 400, given that 25% of such subjects will have significant asymptomatic ischemia on MPI. In addition, CAC screening and MPI are complementary for risk assessment since CAC is usually an indicator of anatomic CAD whereas MPI is a physiological test for CAD. Although different algorithms of screening have been proposed, more complete and detailed protocols should be developed and tested for effectiveness to be used in guidelines. Persons with DM are at an increased mortality risk because of CVD, but many receive inadequate treatment for CVD risk factors


Endoplasmic reticulum aminopeptidase 1 and 2 (ERAP1 and ERAP2) are key components on the pathway that generates antigenic epitopes for presentation to cytotoxic T-lymphocytes (CTLs). Coding single nucleotide polymorphisms (SNPs) in these enzymes have been associated with pre-disposition to several major human diseases including inflammatory diseases with autoimmune etiology, viral infections, and virally induced cancer. The function of these enzymes has been demonstrated to affect CTL and natural killer cell responses toward healthy and malignant cells as well as the production of inflammatory cytokines. Recent studies have demonstrated that SNPs in ERAP1 and ERAP2 can affect their ability to generate or destroy antigenic epitopes and define the immunopeptidome. In this review, we examine the potential role of these enzymes and their polymorphic states on the generation of cytotoxic responses toward malignantly transformed cells. Given the current state-of-the-art, it is possible that polymorphic variation in these enzymes may contribute to the individual’s pre-disposition to cancer through altered generation or destruction of tumor antigens that can facilitate tumor immune evasion. The immune system plays a dual role in cancer, on one hand suppressing tumor growth by eliminating cancer cells or inhibiting their outgrowth, and on the other hand promoting tumor progression by selecting tumor cells able to survive inside an immunocompetent host –7. The dIn the elimination phase, cells of innate and adaptive immunity work together to identify and destroy nascent cancer cells, before they become clinically apparent tumors. If a cancerous cell variant is not eliminated in this phase, it may enter the equilibrium phase and remain in a state of functional dormancy under control of cells and mediators of the adaptive immunity. In this phase, the immune system controls tumor growth but does not eliminate it. Cancer cells that acquire mutations can become: (i) invisible to adaptive immunity, i.e., antigen-loss variants or tumor cells that develop defects in antigen-processing or presentation, (ii) insensitive to immune effector mechanisms, or (iii) induce an immunosuppressive state within the tumor microenvironment, enter the escape phase and grow progressively eventually becoming visible tumors. The achievement of the last phase is indicative of a failure in the adaptive immune cells to provide protection from tumor development due to selection of poorly immunogenic tumor cell variants during the equilibrium phase.Many different cells belonging to the innate and adaptive immunity play an active role in cancer control, from the earliest stages of transformation to the terminal phase of widespread metastasis. The immune cells most involved in the host protection from development of cancer are natural killer (NK) cells and cytotoxic T-lymphocytes (CTLs).NK cells provide the first immune defense against infections and tumor transformation through recognition and killing of aberrant cells. Their function is finely tuned by the interaction of activating and inhibitory receptors with their specific ligands expressed on target cells . ActivatCTLs constantly monitor peptide-MHC (pMHC) class I complexes on the cell surface and eliminate virally infected or transformed cells expressing novel peptides derived from abnormal gene products. The generation of these peptides is central in the regulation of CTL and NK cell responses against altered cells. Aberrant antigenic peptide generation can lead to either immune evasion or to immune responses against normal cells, initiating or sustaining autoimmune reactions. Antigenic peptides are generated by the concerted action of multiple components of a biochemical pathway termed MHC class I antigen-processing and presentation.Antigen generation generally starts with the ubiquitin–proteasome pathway where proteins are tagged for proteolytic destruction by the proteasome although other cytosolic peptidases often play important roles . The immThe key role of ERAP1 and ERAP2 in the generation of antigenic epitopes has been repeatedly demonstrated in several cell lines and mouse models. Reduction of ERAP1 expression by RNA interference results in drastically defective presentation for some antigenic epitopes , 20, 21 in vivo studies emerged that ERAP1 plays an important role in immune response to viruses, either enhancing or reducing CTL responses to particular viral epitopes and, thereby, helping establish immunodominance hierarchies  specifically inhibited surface expression of MHC class I, but did not affect the expression of MHC class II molecules . The exprarchies . Undauntgenicity , 26.ERAP2, the second aminopeptidase demonstrated to be involved in antigen trimming in the ER, is highly homologous to ERAP1 but has distinct specificity –29. ERAPOverall, the exact effect of the ERAP1 and ERAP2 activities on antigen presentation can be highly variable and difficult to predict. Any factor that can influence the generation of the immunopeptidome may contribute to this, including the cell line used, the MHC class I alleles, whether the cell contains immunoproteasomes or constitutive proteasomes, the activities of cytosolic aminopeptidases and the sequence of the epitope studied. Regardless, ERAP1 and ERAP2 are undoubtedly important factors that influence the generation of the immunopeptidome, with ERAP1 having a dominant role , 22, 25.J Innate Immunity, in press). This secreted form of ERAP1 was found to be enzymatically identical to the ER-retained form and only differ in glycosylation patterns consistent with maturation through the secretory pathway. Since ERAP1 does not contain an ER retention signal it has been hypothesized that it is normally retained inside the ER through interactions with specific ER-resident proteins and can be secreted when these interactions are saturated or disrupted , type I IL-6 receptor (IL-6Ra), and type II IL-II decoy receptor –33. Addiisrupted .+ and CD8+) anti-tumor responses. This rejection does not depend on a simple quantitative reduction in surface MHC class I expression, but is rather the result of changes in the MHC class I-peptide repertoire, because replacement of endogenous peptides with high-affinity mature antigenic peptides was sufficient to rescue the inhibitory activity of NK cells  in their genes have been associated with pre-disposition to a variety of human diseases primarily of autoimmune etiology Figure –49. The The association of ERAP1 with diseases in the context of specific HLA class I alleles is also observed in the cases of Behçet disease (BD) and Psoriasis. A GWAS study in a Turkish population, where BD is frequently encountered, identified rs10050860-T 575N) and rs17482078-T (725Q) as increased risk variants, with odd ratios significantly higher for HLA-B51 positive individuals . Similar5N and rsERAP2 has been associated with AS independently of ERAP1. Two functional SNPs have been described: coding rs2549782 (K392N) and rs2248374, a SNP that greatly influences ERAP2 expression . The ERAOverall, a very strong genetic link between ERAP1/2 SNPs and HLA-associated autoimmunity has been established and has contributed to our understanding of the pathogenesis of these diseases. The genetic variability of ERAP1 and ERAP2 appears to constitute part of the natural variability of immune responses and may therefore contribute to pre-disposition to any number of diseases actively fought by the adaptive immune response.The multitude of genetic and population studies linking ERAP1 and ERAP2 SNPs to pre-disposition to autoimmunity and viral infections prompted several research groups to examine the effects of the identified polymorphic variations on the enzyme’s biological function and molecular mechanism. To date, most studies have focused on the most well disease-associated SNPs, namely K528R, D575N, R725Q, and Q730E.in vitro studies have shown that the 528R variant exhibits less enzymatic activity, compared to the 528K  ensured efficient peptide trimming and a higher HLA-B27 stability, compared to the ERAP1 with the AS-protective SNPs  , 73. It in vitro cell-based analysis showed that the 392N variant is much more effective in trimming hydrophobic N-terminal residues from antigenic peptide precursors compared to 392K. In contrast, the rs2248374-G allele can determine ERAP2 expression levels by inducing mRNA instability and non-sense mediated decay  and the non-coding rs2248374. ERAP2 polymorphism N392K alters the activity and specificity of ERAP2 in a manner much more pronounced compared to the effects described for all coding SNPs of ERAP1 -induced cervical carcinoma.In the first study, the authors identified two common ERAP1 polymorphisms, R127P and Q730E, significantly associated with increased risk of cervical cancer . A haploIn a subsequent study, the same group investigated which genetic ERAP1 variation affected tumor progression and overall survival in cervical carcinoma patients, and provided the first indication of association of ERAP1 SNPs with ERAP1 protein expression . GenotypThe association between ERAP1 SNPs and cervical carcinoma risk or patient survival may be explained by different mechanisms, all related to altered ERAP1 function. Genetic variations at these individual SNPs have been found to affect the expression and stability of transcripts and proteins, to reduce trimming activity or modify substrate specificity , 64, 76.CTL and NK cell responses against malignantly transformed cells are important for eradicating tumor cells often at the early stages of carcinogenesis.Establishment of solid tumors often requires adaptive measures from the transformed cells that lead to the evasion of immune responses. Such adaptive measures include changes in antigen-processing and presentation in order to suppress the presentation of tumor-specific antigenic epitopes while also evading NK-cell recognition.Polymorphic variation in ERAP1 and ERAP2, alters their function and their ability to generate antigenic peptides and controls cytotoxic reactions against antigen-presenting cells. This is now well established in autoimmunity and viral infections.ERAP1 and ERAP2 expression levels, which undoubtedly affect effective enzyme activity, are consistently altered in tumors, presumably as an adaptive measure for immune evasion.ERAP1 function can regulate innate immune responses and the production of inflammatory cytokines, a function that can either help eradicate cancer cells or contribute to localize inflammation that can promote tumor growth.Current knowledge on the roles of ERAP1 and ERAP2 in the human immune response have gradually led to the maturation of the hypothesis that the naturally occurring polymorphic variation in ERAP1 and ERAP2 may play significant roles in the pre-disposition to certain cancers, as well as their progression and prognosis. More specifically, the rational behind this hypothesis is based on the following points:As a result, the polymorphic variability in ERAP1 and ERAP2 may affect the severity of early cytotoxic responses toward transformed cells and influence their chances to accrue genetic adaptations that will allow them to evade the immune system (Figure In addition to the possible role of the activity of ERAP1 and ERAP2 on the normal immune control of cancer, a series of recent studies have highlighted that these two enzymes may be important pharmacological targets for boosting immune responses to established cancers. In one study, the genetic down-regulation of ERAP1 in cancer cells that establish solid tumors in mice resulted potent NK cell-mediated cytotoxic responses . In anotIn summary, we review the state-of-the-art on the role of ERAP1 and ERAP2 in adaptive and innate immune responses and their role on disease pathogenesis. Combination of knowledge on the role of polymorphic variation on those enzymes and disease pre-disposition with their role in cancer development has led to the formulation of a hypothesis on their direct role on cancer pre-disposition and prognosis. If this hypothesis holds to be true, haplotype analysis of components of the antigen-processing machinery may contribute to our understanding of cancer pre-disposition and complement other genetic findings on biochemical pathways that control malignant transformation. Because of the genetic heterogeneity of tumors however, it may be difficult to discern such effects at the tumor level and population studies may have to focus on the overall influence of this genetic locus on pre-disposition to specific cancers. Ongoing and future GWAS studies on cancer patients may yet reveal the importance of immune system variation to developing and fighting cancer, contributing to personalized treatments.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.


Drosophila have now demonstrated that the vesicle proteins Synaptotagmin-1 (Syt1) and Rab3 also regulate glutamate release by shaping differentiation of the CAZ ultrastructure. We review these unexpected findings and discuss mechanistic interpretations of the reciprocal relationship between synaptic vesicles and AZ states, which has heretofore received little attention.Neurotransmitter is released from synaptic vesicles at the highly specialized presynaptic active zone (AZ). The complex molecular architecture of AZs mediates the speed, precision and plasticity of synaptic transmission. Importantly, structural and functional properties of AZs vary significantly, even for a given connection. Thus, there appear to be distinct AZ states, which fundamentally influence neuronal communication by controlling the positioning and release of synaptic vesicles. Vice versa, recent evidence has revealed that synaptic vesicle components also modulate organizational states of the AZ. The protein-rich cytomatrix at the active zone (CAZ) provides a structural platform for molecular interactions guiding vesicle exocytosis. Studies in  Chemical synapses are important regulators of neuronal information transfer. At these specialized intercellular contact sites the arrival of an action potential at the presynaptic terminal triggers the release of neurotransmitter onto a postsynaptic cell, where subsequent receptor activation gives rise to signal transduction. Remarkable electrophysiological work by Bernard Katz and colleagues on the quantal nature of neurotransmitter release in the mid-20th century set the basis for interpreting morphological features of the synaptic ultrastructure  influx through voltage-gated Ca2+ channels (VGCCs). To ensure spatial precision of exocytosis, molecular interactions spanning the synaptic cleft align the AZ membrane exactly opposite the postsynaptic receptor field. The impressive speed and precise timing of neurotransmitter release, in turn, is provided by the coordinated interplay of individual protein-protein interactions occurring at the AZ  and by clustering AZ VGCCs (via its N-term) Brp incorporates the functions of several vertebrate AZ proteins thereby ensuring efficient excitation-secretion coupling generation play an important role in shaping short-term plasticity , molecular mechanisms underlying related modifications of AZ states have been studied in considerable detail. Here, a homeostatic enhancement of Ca2+ influx through VGCCs is mediated by RIM-BP and plasma membrane insertion of epithelial sodium channels , which requires the CAZ constituent RIM1α and the vesicle protein Rab3A  NMJs have uniform dimensions and they posses comparable numbers of AZs with similar Brp content. In agreement with these observations, focal electrophysiological measurements report indistinguishable evoked excitatory postsynaptic current amplitudes at proximal and distal locations.The vesicular protein Synaptotagmin-1 (Syt1) plays a decisive role as a Car Figure . Recent It is recognized that CAZ components can affect the organization of synaptic vesicles at the AZ. For example, filamentous proteins concentrate vesicles in the vicinity of the AZ membrane by tethering them to the CAZ. Such a function is performed by the C-terminal end of Brp to promote rapid vesicle recruitment during high frequency synaptic activity (Hallermann et al., 2+ -dependent control of exocytosis. In contrast, an additional influence of Syt1 on neuronal differentiation and AZ architecture has remained largely unaddressed. With increasing knowledge of the molecular makeup of the CAZ (Figure Work on Syt1 has focused on its role in regulating the final stages of the synaptic vesicle cycle and specifically on its CaZ Figure  and infoZ Figure . To thisZ Figure .KDsyt and rab3 mutant NMJs are dissimilar, indicating different pathways, and other manipulations of vesicle release do not parallel the Brp layout seen e.g., in rab3 mutants (Graf et al., There is strong evidence that the CAZ is a plastic structure and that its dynamic rearrangement gives rise to different functional properties of AZs. The observations that synaptic vesicle proteins can influence this plasticity are puzzling (Graf et al., syt1 to identify protein domains relevant for shaping CAZ ultrastructure and neuronal morphology. Notably, Rab3 is enriched at AZs of the Drosophila NMJ. Its punctate clustering requires the presence of Brp and is quite different to the more homogeneous distribution of other synaptic vesicle proteins (Graf et al., Drosophila, Brp is co-transported along the axon with RIM-BP (Siebert et al., Alternatively, the structural abnormalities may be directly linked to the involvement of Syt1 and Rab3 in the synaptic vesicle cycle. Consistent with this notion, a recent study of mutant alleles has demonstrated that normal AZ differentiation depends on a typical vesicle tethering mechanism of Rab3 (Chen et al., rab3 and syt1 in other organisms not reported structural AZ defects similar to those observed in Drosophila? One possibility is that the stereotypic morphological layout of the fly NMJ, including its developmental synaptic differentiation, facilitates the quantification of parameters pertaining to neuronal structure and AZ ultrastructure, which are more difficult to measure in other systems. In particular, the T-bar as a prominent marker of the CAZ and the characteristic modular assembly of Brp support analyses of the AZ nanostructure (Kittel et al., Drosophila NMJ or an evolutionarily conserved feature, it will be worthwhile to investigate other synapses with prominent CAZ architectures.Why have genetic studies of Finally, these new results have important implications for our current understanding of Rab3 and Syt1 functions. Single-synapse resolution is rarely attained in electrophysiological recordings and therefore structural data, e.g., concerning the number of sampled synapses, must be taken into account when extending functional interpretations to the single synapse level. Evidently, we still lack fundamental information on the mechanisms guiding the dynamic organization of AZ states. As we continue filling the gaps old players may be seen in a new light.RJK and MH conceived and wrote 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.


We carried out a mutational analysis of 3,594 genes coding for cell surface proteins (Surfaceome) in 23 colorectal cancer cell lines, searching for new altered pathways, druggable mutations and mutated epitopes for targeted therapy in colorectal cancer. A total of 3,944 somatic non-synonymous substitutions and 595 InDels, occurring in 2,061 (57%) Surfaceome genes were catalogued. We identified 48 genes not previously described as mutated in colorectal tumors in the TCGA database, including genes that are mutated and expressed in >10% of the cell lines . Analysis of these genes uncovered important roles for FGF and SEMA4 signaling in colorectal cancer with possible therapeutic implications. We also found that cell lines express on average 11 druggable mutations, including frequent mutations (>20%) in the receptor tyrosine kinases AXL and EPHA2, which have not been previously considered as potential targets for colorectal cancer. Finally, we identified 82 cell surface mutated epitopes, however expression of only 30% of these epitopes was detected in our cell lines. Notwithstanding, 92% of these epitopes were expressed in cell lines with the mutator phenotype, opening new venues for the use of “general” immune checkpoint drugs in this subset of patients. Colorectal cancer is the most common gastrointestinal cancer in the world, with approximately one million new cases being diagnosed and more than 500,000 deaths occurring yearly. Approximately, one in five patients is diagnosed with metastatic disease, and an additional 30%–40% develop metastasis during the course of their disease. Unfortunately, only a minority of the patients with metastatic disease is amenable to curative resection and remains free of disease recurrence . Even thGenetic analysis of colorectal tumors over the past 30 years allowed the characterization of distinct molecular pathways altered during the development and progression of this disease . More reCell surface proteins are involved in a variety of cellular functions, including nutrient and ion transport, adhesion and signaling. These proteins also play important roles in pathological conditions such as diabetes, neurological disorders and cancer. They represent approximately 18% of all protein-coding genes in the human genome  and, dueWe have recently used a combined bioinformatics approach to generate a catalog of genes coding for transmembrane proteins located on the surface of human cells . BrieflyTo define the mutational profile of the Surfaceome in colorectal cancer, we target sequenced the coding regions of the 3,594 cell surface protein genes in a panel of 23 tumor cell lines  that altSomatic point mutations were detected using an in house computational pipeline based on SAMtools mpileup calling Figure . As matcA total of 184 (5%) putative non-synonymous substitutions were nonsense, and 529 (89%) of the InDels introduced a frame-shift alteration in the mutated protein Table . To furtTo further address the biological significance of the uncovered point mutations, we have incorporated gene expression data available for the cell lines (RNAseq and microarray) and restricted our downstream analysis to mutated and expressed genes. A list of genes coding for cell surface proteins that are mutated and expressed in >10% of the 23 cell lines analyzed is provided in Semaphorin 4C (SEMA4C) mutations were detected and validated by Sanger sequencing in 4 cell lines . Two of these mutations occur in the SEMA domain, a highly conserved sequence of approximately 500 amino acids critical for inducing targets of Semaphorin signaling. A third mutation occurs in the plexin-semaphorin-integrin (PSI) domain, another highly conserved domain, enriched in cysteine residues  alterations were detected and validated by Sanger sequencing in 4 cell lines . Three cell lines carry frameshift mutations and the remaining cell line carries a non-synonymous point mutation with a predicted damaging effect, indicating a loss of function of the FGFRL1 protein in colorectal cancer  are mutated in >10% of the colorectal cancer cell lines, literature searches did not reveal evidence of the functional role or therapeutic potential of these genes in colorectal cancer. Nevertheless, recurrent mutations (>3%) in FAM38A, SLC36A1 and WDR81 have been reported for other primary tumors in the TCGA database, and further functional studies will be necessary to address their involvement in colorectal tumorigenesis.In order to identify putative druggable mutations in cell surface proteins, we searched for mutated genes present in the Drug-Gene Interaction Database (DGIdb), which integrates drug-target information from 13 different sources, including the literature and previously established databases . We geneA significant fraction (34%) of these mutations occurred in membrane transporters. Membrane transporters, including solute carriers (SLCs) and ABC transporters, control the uptake and efflux of amino acids, sugars, lipids and vitamins, and their expression and activity are frequently altered in cancer as a consequence of the higher energy and nutritional requirements of the tumor cells . MembranWe then focused on expressed genes with putative druggable mutations that were not previously considered as potential therapeutic targets for colorectal cancer, but for which specific inhibitors have previously been developed. We particularly focused on surface proteins with kinase activity, as they represent a significant fraction of the genes mutated in cancer and are highly amenable to targeting by rationally designed small molecule inhibitors. Two druggable RTKs (AXL and EPHA2) were found to be frequently (>20%) altered in our cell lines.Five point mutations in the kinase domain and/or with predicted functional impact in the AXL Receptor Tyrosine Kinase (AXL) were detected and validated by Sanger Sequencing in 22% 5/23) of our cell lines   were detected and validated by Sanger sequencing in 3 cell lines . Three of these mutations are located in the tyrosine kinase domain and one in the Ephrin-ligand binding domain  sequences. Our local pipeline for immunogenic epitope prediction was based on two algorithms RANKPEP and NetMHC as described in Materials and Methods. Mutated epitopes were required to have a binding affinity to the HLA*0201 molecule that was at least 20% higher than the reference epitope as predicted by both algorithms. A total of 82 putative mutated epitopes were identified (73 epitopes from non-synonymous mutations and 9 epitopes from frameshift mutations). However, when we combined gene expression data with epitope prediction analysis, we found that only 30% (25/82) of the predicted epitopes are expressed, and that 92% (23/25) of these epitopes are expressed in a subset of the cell lines with the mutator phenotype. These results suggest that the use of potentially immunogenic mutations in cell surface proteins for personalized T-cell based immunotherapy in colorectal cancer is limited, as only 30% of the mutated epitopes are expressed and less than half (11/23) of the tumors cell lines express mutated epitopes.One of the major objectives of cancer genome sequencing projects is to identify therapeutically targetable mutations. This objective has been achieved with repeated success in cancer therapy, resulting in the introduction of new treatment protocols in the clinical practice. The use of Imatinib, for chronic myeloid leukemia and other solid tumors, of Trastuzumab and Lapatinib, for ERBB2 positive breast cancer, and of Vemurafenib, for BRAF mutant melanomas, are emblematic examples of how genomic alterations can be used to target cancer cells . Over thCell surface proteins constitute optimal targets for directed therapies and represent two-thirds of the protein-based drug targets , 35. Surin vitro propagation of the cell lines, our results are in agreement with a recent mutation saturation analysis of 4,742 sequenced tumors, across 21 cancer types [We found that a significant 57%) fraction of the Surfaceome is reshaped by somatic point mutations in colorectal cancer cell lines. Our analysis identified 48 genes coding for cell surface proteins that were not previously described as mutated in primary colorectal tumors in the TCGA database [7% fractier types . This ster types .in vitro and in vivo and is comparable to that elicited by other well-known angiogenic molecules, such as VEGF-A, HGF and bFGF [SEMA4C mutations were found in 17% of the cell lines and recurrent mutations in SEMA4G (17%) and SEMA4D (22%) were also observed. The effects of Semaphorins and their receptors in cancer are broad, context dependent and complex . SEMA4C and bFGF , 39. OurInactivating mutations in FGFRL1, the most recently discovered member of the FGFR family, were detected in 17% of our cell lines. FGFRL1 binds with high affinity to heparin and FGF ligands, but it does not possess an intracellular protein kinase domain and, therefore, cannot signal by trans-auto-phosphorylation . FGFRL1 in vitro, as well as in tumor angiogenesis and metastasis in vivo [Higher mutation frequencies in the RTKs AXL (22%) and EPHA2 (17%) were detected in our panel compared to those reported in the TCGA database for primary colorectal tumors (3.51% AXL and 2.63% EPHA2) . AXL has in vivo . Moreove in vivo , Nilotin in vivo  and Lapa in vivo . In lung in vivo .In colorectal cancer, AXL expression is associated to increased invasiveness of tumor cell lines with overexpression of the chemokine receptors CXCR4 and CXCR7, and AXL knock-down in these cell lines significantly hampered tumor cell invasion . Considein vitro [Somatic alterations in EPH receptors were also frequently observed in our cell lines, including frequent mutations in EPHA1 and EPHA2. Point mutations in EPHA2 and EPHA1 have not so far been described in the literature for colorectal cancer. Nevertheless, mutations in the kinase domain of EPHA3 was reported in 5% of colorectal cancer cell lines  and EPHAin vitro . On the in vitro , 31.Non-synonymous and frameshift mutations in tumor cells can generate unique T-cell mutated epitopes and induce tumor antigen-specific immune response . There iNotwithstanding, we observed that mutated expressed epitopes are predominantly found in colorectal cell lines presenting a mutator phenotype and that this specific subset of cell lines express a total of 23 mutated epitopes. In this context, it was recently demonstrated that patients with tumors showing naturally occurring immunogenic mutations presented higher cytotoxic T-cell infiltration and improved overall survival and, based on these observations, the use general immune modulators that block immune regulatory checkpoints such as anti-CTLA4 and anti-PD1 was proposed as a treatment strategy for patients with immunogenic mutations . AccordiTo the best of our knowledge, this is the first systematic and focused screen of point mutations in genes coding for cell surface proteins in colorectal cancer. By combining high-throughput sequencing, bioinformatics tools, data integration and literature searches, we have successfully discovered novel altered pathways and druggable mutations for targeted therapy in colorectal cancer. We have also uncovered the potential use of existing RTK inhibitors and immune checkpoint target drugs in specific subsets of colorectal cancer patients. Results presented here are encouraging, however our study also presents some limitations.in vitro propagation of the cell lines or to very rare germline polymorphisms not represented in public databases, nor in individuals sequenced by the 1000 Genomes Project. However, we believe that these possibilities do not significantly affect our results, since we have previously shown that the rate of mutation accumulation during in vitro propagation is not significant [in vitro growth. Second, the functional consequences of the uncovered genetic alterations were predicted primarily using computational tools, and confirmation with functional in vitro assays is further required. Similarly, additional experiments to evaluate the effects of pharmacologic inhibition of the altered pathways using pre-clinical models are compulsory to translate our findings to the bedside. Finally, although we suggest potential molecular therapeutic targets in colon cancer, it is important to recognize that a recent study matching targeted therapy with specific molecular abnormalities for patients with advanced colorectal cancer failed to confer significant clinical benefit [First, although we have described novel druggable mutations occurring in a representative panel of colorectal cancer cell lines, it will be important to confirm the prevalence of these alterations in clinical samples matched with normal tissue. At present, we cannot completely exclude the possibility that some of the alterations reported in this study correspond to mutations acquired during nificant  and stri benefit . We belioC and 5% CO2. Cell lines were authenticated and tested for Mycoplasma contamination as previously described [The panel of 23 colorectal cancer cell lines used in this study was obtained from different sources . CACO2, escribed .We target-sequenced the coding regions of 3,594 cell surface proteins in 12 cell lines . Surfaceome-capture and sequencing were performed using Sure Select Target Enrichment baits (Agilent Technologies) and the SOLiD 4.0 sequencing platform (Life Technologies), respectively. For the remaining cell lines  whole-exome capture was performed using the TruSeq Exome Enrichment Kit (Illumina) and paired-end sequencing was performed using Illumina HiSeq 2000. A local pipeline was then developed to extract the genomic sequences corresponding to the Surfaceome targeted region from whole-exome data.kinase.com/human/kinome) was used to identify genes coding for cell surface proteins with kinase activity.Exome-capture sequencing data on colorectal tumors were retrieved from TCGA and used to identify novel mutated genes and to determine mutation frequencies in colorectal cancer primary tumors. The DGIdb  was usedwww.novocraft.com). A local pipeline for point mutations was developed using Samtools mpileup and bcftools [For single nucleotide variations (SNVs) detection, SOLiD 4.0 and Illumina reads were aligned to the human reference genome sequence (GRCh37/hg19) using BioScope (Life Technologies) and BWA , respectbcftools . Duplicabcftools , PolyPhebcftools  and Mutabcftools  were useRNA-Seq data was generated for 12 cell lines  using the 5500XL sequencing platform to a depth of >100 million reads. Sequences were aligned to the human reference genome sequence (GRCh37/hg19) using TopHat  with sta50 was ≤ 500nM. To check for predicted cleavage, sequences were then analyzed using the NetChop algorithm [Peptide sequences corresponding to non-synonymous mutations and InDels, flanked by 10 aminoacids on either side, were used for epitope prediction by applying a similar approach to that described by Segal et al. 2008 . The samlgorithm , and onl


One of the most challenging questions in neuroscience is to dissect how learning and memory, the foundational pillars of cognition, are grounded in stable, yet plastic, gene expression states. All known epigenetic mechanisms such as DNA methylation and hydroxymethylation, histone modifications, chromatin remodelling, and noncoding RNAs regulate brain gene expression, both during neurodevelopment and in the adult brain in processes related to cognition. On the other hand, alterations in the various components of the epigenetic machinery have been linked to well-known causes of intellectual disability disorders (IDDs). Two examples are Down Syndrome (DS) and Fragile X Syndrome (FXS), where global and local epigenetic alterations lead to impairments in synaptic plasticity, memory, and learning. Since epigenetic modifications are reversible, it is theoretically possible to use epigenetic drugs as cognitive enhancers for the treatment of IDDs. Epigenetic treatments act in a context specific manner, targeting different regions based on cell and state specific chromatin accessibility, facilitating the establishment of the lost balance. Here, we discuss epigenetic studies of IDDs, focusing on DS and FXS, and the use of epidrugs in combinatorial therapies for IDDs.  Intellectual disability disorders (IDDs) are complex multifactorial illnesses involving chronic alterations in neural circuit structure and function as well as likely abnormalities in glial cells. Converging evidence indicates that epigenetic control of gene expression is pivotal to learning and memory, as underscored also by the range of intellectual disabilities and behavioural deficits increasingly traced to a staggering number of epigenetic modulators. This review focuses on the importance of epigenomics in neuroscience, especially in neurodevelopment and cognition. Since epigenetic mechanisms are reversible, they are targets of interest in conceiving new therapies for the treatment of IDDs. We will specifically address two genetic intellectual disabilities, Down Syndrome (DS), caused by trisomy 21 [Since the first definition of epigenetics  the meanThe family of enzymes called DNA methyltransferases (DNMTs) catalyse the most studied modification of DNA. DNMTs transfer a methyl group from S-adenyl methionine (SAM) to a cytosine residue to form 5-methyl-cytosine (5mC). Cytosine methylation occurs especially at CG dinucleotides (CpG sites) which are underrepresented in the genome since 5mC tends to deaminate into thymine . Those sIn mammals, three main DNMTs exist: DNMT1 is called the “maintenance DNMT” since it usually binds to hemimethylated sites avoiding passive demethylation during DNA synthesis and DNMT3a and DNMT3b are the so-called de novo DNMTs . InteresSeveral studies highlight regulation by DNA methylation at the promoters of key genes involved in cognition. Interestingly, following contextual fear conditioning, one of the most used models for studying memory in rodent models, DNMTs are upregulated in the hippocampus during memory formation and this results in an increase in DNA methylation at the promoter of the memory suppressor gene PP1 and a decrease in the methylation at the promoter of the synaptic plasticity gene RELN during memory consolidation. Accordingly, inhibition of DNMTs resulted in PP1 demethylation and problems in memory consolidation . The samMoreover, the brain shows particularly high levels of two other methylation types: non-CpG methylation  and hydroxymethylation (5hmC), suggesting a specific neural role for these modifications , 20. WhiHeyward and Sweatt  proposedBut how can this transient mark lead to memory storage, where memories can last a lifetime? There should be a self-perpetuating mechanism. Many studies on DNA methylation investigated the hippocampal role in memory formation and consolidation but not the further consolidation of this information in remote memory. According to a well established model, bursts of activity called “sharp-waves” would promote cortical plasticity, transferring memories from the hippocampus to the neocortex . HeywardHistones are the main protein component of the chromatin and come in 4 flavours: H2A, H2B, H3, and H4. These basic proteins strongly associate with the DNA forming an octamer called nucleosome, along which 147 bp of the DNA helix wrap around. Additional compaction is performed by the H1 linker histone, which binds the nucleosome at its entry and exit site. Importantly, long protruding tails depart from each histone core and their posttranslational modifications (PTMs) regulate the level of chromatin compaction . There aHistone acetylation has a positive effect on transcription by relaxing the chromatin compaction. The acetyl group neutralizes the positive charges on Lysine (K) and Arginine (R) residues, decreasing the electrostatic interactions between the nucleosome and the DNA. The writers of this epigenetic modification are called histone acetyl transferases (HATs), while the erasers are called histone deacetylases (HDACs) .Histone acetylation has emerged as a key mechanism of memory regulation. One of the first studies showed how novel tastes induce long-lasting Lysine acetylation through ERK/MAP pathway activation in the insular cortex ; the samSeveral HDAC isoforms can regulate histone acetylation levels in the adult brain. For instance, while HDAC5 is important in the nucleus accumbens, the reward centre of the brain and its disruption result in a hypersensitive response to chronic drug abuse ; HDAC2 wHistone acetylation often correlates with histone phosphorylation. For example, H3 phosphorylation at serine (S) 10 (H3S10P) together with acetylation of H3K9 is induced during spatial memory formation and facilitates the early gene activation  of the ERK/MAPK pathway .The second most studied histone modification is methylation. While histone acetylation always results in transcriptional activation, histone methylation effects depend on the protein complexes docking on the different modifications. For example, H3K4 methylation and monomethylation of H3K9 (H3K9me1) result in transcriptional activation, whereas H3K9me2 and H3K9me3 result in transcriptional silencing. Histone methylation can occur at either Lysine (K) or Arginine (R) and is performed by a group of proteins containing SET domains called histone methyl transferases (HMTs). Despite being conceived initially as a static histone modification, whose half-life coincides with the histone turn over itself, histone methylation has shown to be dynamically regulated through the action of histone demethylases (HDMs) such as LSD1 for H3K4me and H3K4me2 and JMJD1a for H3K9me and H3K9me2 . Mll, a H3K4 methyltransferase, show defects in contextual fear memory formation [H3K4me3 is usually present in the proximity of the transcription start site of active genes and it has been shown to be induced one hour after contextual fear conditioning, activating promoter regions of memory genes such as ZIF268 and BDNF, to return to baseline levels at 24 hours, underlining a role in memory formation. A similar dynamic was observed for the transcriptional repressive H3K9me2 mark. Interestingly, mice deficient inormation . In paraormation , acting ormation . Moreoveormation . Finallyormation .Although less studied, several other histone methylation marks play an important role during cognitive processes. For example, H3K36me3, marking the 3′ end of transcribed genes, is immediately induced during object recognition memory in both the hippocampus and prefrontal cortex and is reactivated after activation of recent (24 h) and remote (7 days) memory, with hypermethylation of the ZIF268 promoter [Future research should focus on integrative analysis showing how those marks crosstalk and what is the precise dynamic of their activation.Nucleosome remodelling complexes (NRCs) alter nucleosome positioning in an ATP-dependent way, promoting nucleosome sliding, eviction, or histone variants exchange. In the brain the most studied NRC is the neuron-specific Brg1/hBrm Associated Factor (nBAF) complex, a multiprotein complex belonging to the SWI/SNF family that regulates gene expression in both development and adult cognition. Of particular importance in neurodevelopment is the upregulation of the BAF45b and BAF45c subunits and the switch between the BAF53a and BAF53b, which begins at E12.5 and is exclusive to postmitotic neurons, being essential for BRG1's ATPase activity . This coNoncoding RNAs (ncRNAs) are transcripts that are not translated into a protein. They include two broad categories: small RNAs and long noncoding RNAs (lncRNAs). The first comprehends micro-RNAs (miRNAs) that generally inhibit gene expression by complementarity to their targets and PIWI interacting RNAs (piRNAs), involved in transposon repression through RNA mediated DNA methylation. The function of long noncoding RNAs is less known; while initially thought to be “transcriptional noise,” recent studies suggest that lncRNAs can regulate gene expression by acting as “guide” or scaffold RNAs, targeting epigenetic changes to specific genomic locations. Many noncoding RNAs have been identified in the brain, and approximately 40% of them are not found in other tissues . While fThe complex picture of epigenetic regulation in the brain can be puzzling, with some epigenetic changes enhancing cognition and other impairing neural activities. However the take-home message is that every kind of epigenetic change has been found associated with neural activity, indicating that a correct balance of the epigenetic machinery is needed for a proper neural function. Moreover, epigenetic changes should not be seen as distinct and isolated events. Repressive modifications tend to occur together and the same is true for permissive modifications. As an example, several methyl binding proteins recruit HDACs allowing cytosine methylation and histone deacetylation to act in concert to repress gene transcription . That meMany intellectual disability disorders arise from mutations affecting the function of the epigenetic regulators discussed in Besides the epigenetic syndromes arising by direct perturbation in the functions of key epigenetic molecules, several, if not all, other syndromes and IDDs have probably an epigenetic component or origin. Epigenetics means dynamics and reversibility, and thus a lack of epigenetic coordination may lead to defects in neurodevelopment with consequent defects in cognition. In this context it is obvious that an early therapeutic intervention is preferential, but given the reversibility of epigenetic processes, it is in theory possible to restore a proper neuronal function ameliorating the cognitive impairment in this developmental IDDs. To what extent this is feasible, together with the efficacy and duration of the effect for this approach, remains to be elucidated. The best therapeutic strategies will likely consist in combinatorial therapies using both neuromodulators and “epidrugs” as cognitive enhancers.In the end of the section we will specifically focus on two developmental genetic disorders, DS and FXS, showing how both development and cognition are interconnected and how epigenetic regulation is essential in both processes as a gateway for processing inputs received from the environment.Epigenetic effectors involved in intellectual disability developmental disorders are likely interacting with fundamental players in neuronal maturation. For instance, nBAF complexes regulate genes essential for dendritic outgrowth and spine formation , and theConverging evidence indicates that epigenetic control of gene expression is also pivotal to learning and memory through its crosstalk with neuronal activity and synaptic plasticity mechanisms, as underscored also by the range of intellectual disabilities and behavioural deficits increasingly traced to a staggering number of epigenetic modulators. Specifically, several epigenetic modifications act as key signalling relay in the integration of synaptic inputs, as vividly shown for histone acetylation in CREB-dependent changes triggered during NMDA-receptor mediated long-term potentiation (LTP) , but als (CANTAB), originally developed at the University of Cambridge in the 1980s [The amount of information obtained from cellular and molecular neuroscience of cognitive processes is overwhelming. However, the connection between this information and mechanistic conclusions at the cognitive level relies on important assumptions and generalizations. For example, while several molecular events in neurons signal plasticity mechanisms, the link between these mechanisms and the formation and loss of memories is only correlational. Even so, to verify whether the studied mechanisms are involved in cognition we still use behavioural tests in normal and diseased animal models. Thus, critical aspects for the assessment of rodent models of IDDs and the study of the underlying molecular mechanisms are face and predictive validity of the tests , 79. In he 1980s . Deepenihttp://aaidd.org/) and epigenetic disturbances are expected to have widespread downstream consequences  has been linked to a number of well-known causes of IDDs , 87. Intequences . Rett SyAnother IDD directly arising from mutations in epigenetic players is Rubinstein-Taybi syndrome (RTS). Most of the RTS patients have mutations in the gene encoding for the cyclic AMP-responsive element binding protein (CREB) binding protein (CBP) , while iSeveral HMTs have been associated with congenital IDDs. A deletion containing the GLP/EHMT1 gene (euchromatin histone methyltransferase 1) causes Kleefstra syndrome, a developmental severe IDD, with defects in learning, motivation, and environmental adaptation. GLP/G9a is essential for regulating H3K9 dimethylation levels and regulates brain function through maintenance of the transcriptional homeostasis in adult neurons . In minoSeveral mutations have been described in subunits of the nuclear remodelling complex nBAF, which are linked to IDDs and autism spectrum disorders (ASDs) . The mosSince affecting epigenetic mechanisms, these mutations would lead theoretically to the deregulation of a very broad and nonspecific set of genes; however they surprisingly give rise to well defined syndromes, suggesting that they conversely lead to specific dysregulation of key genes. However, all these IDDs share common clinical features, indicating that they share common molecular pathways, deregulated upon epigenetic imbalance, which could be targeted therapeutically.Note that even though these IDDs arise from mutations/deletions in specific components of the epigenetic machinery, the common molecular phenotype is a global epigenetic imbalance, affecting several epigenetic mechanisms. Histone modification and/or DNA modifications always occur in concert, with nuclear remodelling complexes bringing various epigenetic players at the regulatory regions of the genome. Moreover, several other disorders, even if not arising from direct impairment of the epigenetic machinery, show a strong epigenetic component, such as foetal alcohol spectrum disorders, neurodegenerative diseases , poly-Q disorders , autism spectrum disorders (ASDs), addiction, schizophrenia, stress, and Friedrich ataxia , 93, 94.Down Syndrome (DS) is the most common genetic intellectual disability arising from the total of partial trisomy of chromosome 21, leading to a developmental disorder characterized by various defects, including impairments in language, memory, learning, and a higher frequency of developing Alzheimer Disease (AD) ) has shoEpigenetic deregulation due to triplicated genes could explain the genome-wide change of gene expression, as chromosome 21 contains genes regulating all epigenetic aspects discussed in DNMT3L is encoded on chromosome 21 and stimulates the activity of DNMT3a and DNMT3b . SeveralMany HSA21 genes influence specific histone modifications. An example is the phosphotyrosine kinase DYRK1A, which is able to regulate several proteins involved in epigenetic mechanisms. It promotes both histone deacetylation by phosphorylating SIRT1  and histHSA21 encodes for five miRNAs: mir-99a, mir125b2, mir155, mir802, and let-7c. Interestingly, mir155 and mir802 downregulate the methyl binding protein MeCP2 . FurtherSumming up, even though DS is caused by a precise genetic defect (trisomy 21), epigenetic mechanisms are globally dysregulated through various mechanisms. One of the main problems for obtaining a complete picture of epigenetic contributions in DS is that different studies performed analyses on different cell types and tissues, in different developmental stages . Since epigenetics is involved in differentiation and cell fate, this has led to different results that are often difficult to compare, as the epigenetic differences related to development and cell differentiation could mask the differences due to the trisomy.Fragile X Syndrome (FXS) is the most common monogenic cause of intellectual disability, where a “CGG” triplet expansion at the 5′-UTR of the FMR1 gene is responsible for the loss of the Fragile X mental retardation protein (FMRP), a synaptically expressed RNA-binding protein regulating translation . FMRP acWhen the CGG repeat expands between 56 and 200 (permutation), the FMR1 gene is upregulated with increased histone acetylation in the promoter , while iInterestingly, DNA demethylation with demethylating agents such as 5-azadeoxycytidine (5-azadC) reactivates FMR1 transcription in full mutation patients, with restoration of euchromatic marks (H3K4 methylation and acetylation) and partial reduction of the repressive H3K9 methylation , 125. EvOne of the most debated questions is if this epigenetic deregulation has a genome-wide effect or affects only the FMR1 locus. A recent work succeeded in detecting differential DNA methylation only at the FMR1 locus, which is wholly affected. However, the study does not discriminate DNA methylation and hydroxyl-methylation  and used the HumanMethylation450 BeadChip kit, taking into account just a specific subset of the genome . HDAC4/5, NCOR1–3, and CBP [Indeed, a global deregulation is possible since a lot of FMRP target mRNAs are involved in chromatin remodelling such as and CBP  and seve and CBP , 130, wh and CBP . Interes and CBP . Once agFMRP has been shown to be involved in dendritic mRNA localization, synaptic protein synthesis, and synaptic plasticity. The mechanism relies on mGluR signalling in glutamatergic postsynaptic sites. When mGluR channels are active in a synapse, a phosphorylation cascade is triggered that affects the LTP pathway and triggers rapid local protein synthesis of preexisting dendritic mRNAs, including FMRP, around the active synapse . As a reDS and FXS show striking similarities and differences. Both intellectual disabilities are common genetic developmental disorders characterized by specific defects in structural and synaptic plasticity due to alterations in specific molecular pathways. However, those alterations are often opposite, with the common final outcome of cognitive impairment . DS patiαCaMKII [Although presenting opposite phenotypes, DS and FXS share defects in dendritic spine morphology due to alterations in local protein synthesis. Both HSA21 RCAN1 and FMRP regulate calcineurin (CaN) activation, which is important for cofilin dephosphorylation. RCAN1 normally keeps calcineurin inactive; this increases phosphorylated cofilin that facilitates actin polymerization at the spine level. The enlarged spine heads observed in DS patient are probably due to RCAN1 overexpression. As a matter of fact, RCAN1-overexpressing mice show a phenotype similar to DS, with reduced volume and neuron number in the hippocampus, defective neurogenesis, enlarged spine heads, enhanced local protein synthesis of dendra, and impaired LTP , 142. OnαCaMKII . This fup < 0.0005, hypergeometric test) and included 9 HSA21 genes. Among those genes, APP is involved in Alzheimer Disease, which as we already stated has an early onset in DS patients [ SYNJ1/synaptojanin regulates neurotransmission together with two other HSA21 genes, intersectin/DAP160 and RCAN1 [ Tiam1 and Ttc3 are involved in neurogenesis [Recent analyses from our group  show the link between DS deregulated molecular pathways and affected proteins in FXS by comparing 324 genes found to be consistently deregulated in DS in a published meta-analysis , with a patients , 147; SYnd RCAN1 , and is ogenesis ; and NRIogenesis  . EGCG has a plethora of different effects and has thus been investigated in studies from various research areas, including cancer research [The flavonoid epigallocatechin-3-gallate (EGCG) is the most abundant polyphenol extracted from green tea. Strikingly, this molecule is effective in both mouse models of AD and DS. In AD mice, EGCG decreased beta-amyloid levels and plaques via ADAM10-mediated promotion of the alpha-secretase proteolytic pathway and modulates tau-profiles with final cognitive improvements . Similarresearch . Howeverresearch  and clasresearch , 175; itresearch  and afferesearch . The proresearch . In addiresearch . Remarkaresearch , underliDevelopmental disorders are often characterized by intellectual disability due to defects in structural and synaptic plasticity, with impaired activity-dependent cognitive-related molecular processes such as local protein synthesis, long-term potentiation, and long-term depression. In this context, it is difficult to discern what can still be rescued in cognitive developmental disorders and what is irreversibly lost. Epigenetics is not only indirectly needed for cognition by regulating neurodevelopment, but, as we amply discussed in this review, directly regulates experience-based cognitive processes. Epigenetics intercalates in development, cognition, and aging/neurodegeneration, playing a key regulatory role in all these processes. For instance, DNA methylation allows cells to be “programmed” and differentiate during development, is dynamically regulated during cognitive processes, and increases gradually with aging. In contrast to genetic alterations, epigenetic modifications are reversible and this gives a great therapeutic potential to epigenetic drugs to at least partially revert the phenotype associated with IDDs. We have reviewed how epigenetic treatments restore cognitive deficits in various models of cognitive impairment, restoring a correct balance among writers and erasers of epigenetic modification. Of course an early treatment will maximize the efficacy of epidrugs, since the more differentiated a tissue, the less reversible the phenotypes.Two main concerns are associated with epigenetic treatment: genome-wide nonspecific effects and toxicity. As regards the genome-wide effects, it would be worth conceiving ways to deliver epigenetic drugs such as DNMTi and HDACi to the cell types that actually show the epigenetic imbalance. Moreover, the epigenetic editing approach might be a promising solution, allowing directing the epigenetic drug to the loci of the key genes involved in the IDD. Noticeably, the genome-wide action of epigenetic drugs may also be an advantage, since it makes it possible to restore the epigenetic balance in disorders such as DS and FXS, where a similar cognitive impairment is originated by different morphologic phenotypes affecting common altered synaptic plasticity pathways. In this case the same molecule will work in a context specific manner on different loci, since the “substrate” of their action (the syndromic chromatin state) will be different in both cases, restoring the impaired epigenetic balance.However, the epigenetic reversibility property is a double-edged sword. Since epigenetic changes are reversible, some epigenetic drug formulations are simply not long lasting. That would, for example, account for what has been found in the pilot clinical trial involving EGCG and the rescue of the cognitive impairment in DS patients, where stopping EGCG treatment leads to the reappearance of the impaired phenotype . To overTo this end a deeper understanding of all the epigenetic, transcriptional, and molecular cascades activating upon cognition in both physiological and pathological contexts is needed. Future integrative studies will combine epigenetic data, transcriptional data, and molecular data to get new insights into the pathogenesis of IDDs, focusing both on common altered pathways and to specific mechanisms. The development of new technologies and the increase in high-throughput data will allow in a near future elucidating the cognitive processes that are dysregulated in IDDs. Most has yet to come. For instance, as regards DNA methylation, one of the first epigenetic modifications that has been studied, it would be important to discriminate among 5mC and 5hmC, since both are highly present in the adult brain and have opposite outcomes on transcriptional regulation. A new technique called TET Assisted Bisulfite sequencing (TAB-seq) in conjunction with bisulfite sequencing, allows discerning between 5mC and 5hmC at single base resolution ; howeverThe main problem with previous studies is that they lack cell-specificity. For example, many studies on DS focus on different types of cells  and different animal models, taking cells at different developmental stages, with results that are difficult to compare. Moreover, the brain is probably the most complicated organ, where several different cell types such as excitatory pyramidal cells, inhibitory interneurons, and glial cells compose even a reasonably delimited part, such as the hippocampus. Since epigenetic changes are responsible for cell fate, the epigenetic variations associated with cell type determination will sum up to the epigenetic changes associated with the syndromic state or to the cognitive processes, and this will dramatically reduce the power of the studies. Differences in cell type composition in compared samples of complex tissues will result in difficulties to distinguish treatment or disease specific changes from “epigenetic noise” caused by cell type specific marks varying based on cell type fractions. Therefore, cell type specific studies using techniques such as cell sorting to focus on single cell populations will have an increased power in detecting epigenetic differences and underpinning new key mechanism of regulation.We speculate that epigenetic drugs, such as EGCG in combination with other cognitive enhancers and specific drugs interfering with the cell and disorder specific molecular targets, will allow the recovery of the epigenetic balance lost in IDDs such as DS and FXS, making the healing of the cognitive impairment possible.


A reduced bone mineral density (BMD) is observed in several rheumatic autoimmune diseases, including Systemic Sclerosis (SSc); nevertheless, data concerning the possible determinants of bone loss in this disease are not fully investigated. The aim of this study is to evaluate the relationship between BMD, body mass composition, skin sclerosis and serum Vitamin D levels in two subsets of SSc patients. 64 post-menopausal SSc patients, classified as limited cutaneous (lcSSc) or diffuse cutaneous (dcSSc) SSc, were studied. As control, 35 healthy post-menopausal women were recruited. Clinical parameters were evaluated, including the extent of skin involvement. BMD at lumbar spine, hip, femoral neck and body mass composition were determined by dual-energy X-ray absorptiometry. Serum calcium, phosphorus, alkaline phosphatase, urine pyridinium cross-links, intact parathyroid hormone and 25-hydroxyvitamin D (25OHD) were measured. BMD at spine, femoral neck and total hip was significantly lower in SSc patients compared to controls. In dcSSc subset, BMD at spine, femoral neck and total hip was significantly lower compared to lcSSc. No differences in both fat and lean mass were found in the three study groups even if patients with dcSSc showed a slightly lower total body mass compared to healthy controls. Total mineral content was significantly reduced in dSSc compared to both healthy subjects and lcSSc group. Hypovitaminosis D was observed both in healthy post-menopausal women and in SSc patients, but 25OHD levels were significantly lower in dcSSc compared to lcSSc and inversely correlated with the extent of skin thickness. These results support the hypothesis that the extent of skin involvement in SSc patients could be an important factor in determining low circulating levels of 25OHD, which in turn could play a significant role in the reduction of BMD and total mineral content. Systemic sclerosis (SSc) is a rare chronic autoimmune disease characterised by an increased synthesis and deposition of extra-cellular matrix in skin and internal organs and widespread macro and micro- vascular damage. Depending on the extent of skin fibrosis, SSc can be classified in two main subtypes, the limited cutaneous form (lcSSc) and the diffuse cutaneous form (dcSSc) .Several previous studies showed a reduced bone mineral density (BMD) in patients with SSc ,3 but daIn the latest years, there has been growing interest concerning the role of Vitamin D both in bone metabolism and in several clinical manifestations of chronic autoimmune diseases –7. Low VThe aim of this study is to evaluate BMD, body mass composition, and Vitamin D levels in two skin subsets (limited or diffuse) of SSc patients and to assess the possible correlation with the extent of skin sclerosis.64 consecutive post-menopausal SSc patients  classified according to Leroy as having limited cutaneous systemic sclerosis (lcSSc) or diffuse cutaneous systemic sclerosis (dcSSc), were enrolled in the study. As control group 35 healthy post-menopausal women matched for age  and years from menopause were recruited.2) and thus expressed as Kg/m2.For both subsets of patients and healthy controls body mass index (BMI) and menopausal status (age at onset and duration) were assessed. BMI was calculated as body weight (Kg) divided by square of height  measurements were performed in all participants using a total body scanner  to evaluate BMD, body mass composition and T-score. BMD was evaluated both at lumbar spine (L1-L4) and hip  and was expressed as g/cmWeekly physical activity was assessed by the Rapid Assessment of Physical Activities (RAPA) questionnaire .Total, tSerum levels of 25 OH Vitamin D (25OHD) were measured using the commercial kit (LIAISON 25-OH Vitamin D assay—DiaSorin), considering as normal a range between 30–100 ng/ml. In all recruited subjects, 25OHD serum concentration was evaluated in the period between June-September. None of the recruited subjects received vitamin D supplementation and other medication, including bisphosphonates, with a specific effect on bone metabolism in the previous year. Laboratory parameters of bone metabolism were measured in all recruited subjects and included serum calcium, phosphorus, alkaline phosphatase, urine pyridinium cross-links, serum intact parathyroid hormone (PTH). All blood samples were analysed at a single laboratory.For each patient, the extent of skin fibrosis was evaluated by the modified Rodnan Skin Score (RSS), and all patients were evaluated also for internal organ involvement.Particularly, pulmonary involvement was evaluated by chest radiograph, high resolution CT (HRCT) and pulmonary function test with diffusing lung capacity for carbon monoxide adjusted to haemoglobin (DLCO). Cardiac involvement was assessed by electrocardiogram; all patients were screened for pulmonary arterial hypertension (PAH) with doppler echocardiogram and when appropriate, according to DETECT algorithm , selectePatients were investigated for renal failure or previous history of scleroderma renal crisis. Musculo-skeletal involvement (arthritis and/or myositis), nailfold video-capillaroscopy, erythrocyte sedimentation rate (ESR), anti-nuclear antibodies (ANA) were also assessed. Corticosteroids treatment (doses and duration) was evaluated.All the investigations were performed at Hospital as part of routine clinical evaluation of SSc patients. Written informed consent was obtained from all the participants to the study. The study was approved by the Ospedali Riuniti University Hospital’s Ethics Committee for Medical Research  and carried out in accordance with the Helsinki Declaration.Continuous variables were reported as means and standard deviations while categorical variables were summarized as percentages.Differences between groups were assessed using the Student’s t-test for continuous variables. Comparison of percentages between groups was performed by Fisher’s exact test. Pearson correlation coefficient was used to evaluate the correlation between selected continuous variables . Multiple logistic regression was used to assess the relationship between selected demographic and clinical variables  and BMD values / Vitamin D levels.Values of p<0.05 were considered to be significant.The demographic and clinic characteristics of the two SSc subgroups (limited and diffuse cutaneous form) are shown in 2 in control group vs 0,853 g/cm2 in dcSSc at spine, p<0,001; 0,932 g/cm2 in control group vs 0,755 g/cm2 in dSSc at total hip,p<0,001) whereas there was no significant difference between healthy subjects and lSSc patients. BMD at femoral neck was lower both in dcSSc and lcSSc patients compared to healthy subjects We found that compared to healthy controls, BMD at spine and total hip was significantly lower in dcSSc patients  and the whole group of SSc patients  and between the diffuse and limited SSc groups .No differences in BMI were observed between healthy subjects , whereas total body mass was significantly lower in dSSc patients compared to healthy controls. Patients with dcSSc also showed a slightly lower total body mass compared to lcSSc which did not reach statistical significance . No diffWhen measuring the circulating levels of 25OHD in healthy subjects and the two cutaneous subsets of SSc, we found that in all groups they were below the lower limit of normal range levels (30 ng/ml). Nevertheless the circulating levels of 25OHD in healthy subjects  were significantly higher compared to the whole group of SSc patients ; furthermore, comparing the two cutaneous subset of SSc patients, we found that in dcSSc patients, 25OHD levels were significantly lower compared to the lcSSc ones  and disease duration between the limited and the diffuse cutaneous subset of SSc patients excepted for interstitial lung disease whose prevalence was significantly higher in the dSSc subset . A total of 12  lcSSc and 11 dcSSc  patients were discontinuously treated with corticosteroids for musculoskeletal symptoms  for a mean duration of 36 and 42 months years respectively. Only 5  lcSSc and 7  dcSSc received 25–50 mg/day of prednisone equivalents for treatment of lung severe complicances  for a short time . No significant differences in the cumulative dose of corticosteroids was detected between lcSSc and and dcSSc.No significant relationship was found between internal organ involvement  and 25OHD serum levels, PTH serum levels, BMI values in the whole group of SSc patients. 25OHD levels were independent from the duration of disease and the presence of anticentromere or antitopoisomerase I autoantibodies.BMD values and the presence of osteoporosis in SSc patients have been assessed in various reports . Lower BThe reduction of total bone mineral content is associated with a defect in mineralization processes, which in turn could be related to a lower levels of circulating 25OHD due to its well-known role in regulating calcium and phosphorus metabolism, bone formation and mineralization. Further, more recently, in addition to these important metabolic activities, Vitamin D has been recognized to affect the regulation of immune system. Decreased serum levels of 25OHD have been reported in various chronic rheumatic diseases –25. CaraThe causes of low serum 25OHD levels in subjects affected by SSc may be various and could include reduced sun exposure due to disability or vascular complications, or intestinal involvement and malabsorption, or renal insufficiency. Skin thickening with capillary damage could also play a role in determining hypovitaminosis D, as it can be involved in a reduced drawing of pre-vitamin D3 synthesized from 7-dehydrocholesterol by UVB radiation in the epidermis. Nevertheless, to date few studies investigate the possible relationship between the extent of skin fibrosis, serum levels of 25OHD and BMD in SSc, with discordant resultsIn this study, circulating 25OHD was found to be low in control healthy group also, but in SSc patients the observed hypovitaminosis D was more serious; further the levels of 25OHD we found in the diffuse cutaneous form were significantly lower compared to the limited cutaneous form, suggesting that the extent of skin fibrosis could be related to the import of hypovitaminosis D. To correlate serum 25OHD levels to skin involvement, we evaluated skin fibrosis trough Rodnan Skin score and we found a significant inverse relationship between the degree of skin thickness and circulating levels of 25OHD. This data partially agree with a previously published report in which there was no statistical difference in the serum levels of 25OHD between lcSSc and dcSSc, but patients with Rodnan skin score of 10 or less showed a higher mean 25OHD concentration compared with patients with higher score (above 10) .Table 1, when considering Raynaud phenomenon the first sign of disease. Concerning lung involvement, SSc patients were considered affected by interstitial lung disease also when minimal bi-basal interstitial fibrosis, even if asymptomatic, was detected by lung HRTC. This could explain the high prevalence of interstitial lung disease observed in SSc patients recruited in the study, considering that interstitial abnormalities on HRTC have been reported in up to 90% of SSc patients [No differences about internal organ involvement were observed between the limited and the diffuse cutaneous subset of SSc , even if the prevalence of PAH and lung fibrosis resulted to be higher in respect to what reported in larger cohorts of patients. As the patients included in the presented studied were all post-menopausal women, the high prevalence of PAH could be explained by the older age and a longer disease duration of recruited SSc in respect to the larger systemic sclerosis cohorts which included young patients and patients with early disease. Furthermore it is possible that the duration of disease (defined as years from the first sign/symptoms of disease other than Raynaud phenomenon) is greater than those reported in the patients . We founpatients , 29.Also no correlation was found between malabsorption and 25OHD levels/BMD values. Of note that the reported prevalence of malabsorption in patients with SSc can vary widely depending on the case-series and the diagnostic tools employed to evaluate it. The prevalence of SIBO has been reported to be 30–62% in SSc patients and was associated with biochemical signs of subclinical malabsorption [We found no correlation between BMD and 25OHD levels and autoantibody profile, musculo-skeletal involvement, corticosteroid treatment, duration of disease of the whole cohort of SSc patients. These findings are consistent with previous published reports , 26, 31.Plasma PTH levels did not show any correlation with the degree of skin fibrosis, even if a non- significant trend toward a direct relationship was observed. Although usually PTH circulating levels inversely correlate with 25OHD levels, the observed data can be explained by the normal levels of serum calcium detected in all SSc recruited patients, as calcium and not 25OHD regulate PTH secretion.At present, Vitamin D status of SSc patients has not been sufficiently investigated. Even if in some studies a high prevalence of Vitamin D deficiency has been demonstrated among SSc patients, findings concerning the potential correlation between low Vitamin D serum levels and other clinical aspects in SSc patients or the possible determinants of hypovitaminosis D have been inconclusive due to the small size of the studied populations. Further, there is large discrepancy in the literature about BMD and Vitamin D levels in SSc and it is very difficult to compare the different studies on this topic because of the different sample size, the wide age range, the different time course of the recruited patients.Our results show a significantly lower BMD and total mineral content in the dcSSc subtype; circulating levels of 25OHD were significantly lower in dcSSc patients and inversely correlated with the extent of skin fibrosis. Skin thickness could be one of the factors determining hypovitaminosis D, which in turn could contribute to reduced BMD and total mineral content in these patients.Further, it should be highlighted that it is well know that Vitamin D affects the immune system at many levels and with different mechanisms, thus deficiency might influence severity and activity disease. Recent studies on murine cells have clearly shown a direct anti-fibrotic effect of Vitamin D. Vitamin D is able to decrease the expression of TGFß1, a pro-fibrotic cytokine, and collagen I and collagen III synthesis in mesenchymal multi-potent cells, and to enhance the expression of some anti-fibrotic factors such matrix metalloproteinase 8 . It has The main limitations of the presented report is represented by the relatively small sample size, even it should be highlighted that SSc is a rare disease; further studies are required on the possible causes of Vitamin D deficiency in SSc patients. Studies on the effect of Vitamin D supplementation in SSc patients could confirm the role of Vitamin D not only on BMD and bone metabolism, but also in the in determining the expression various clinical aspect of this disease.S1 TableIndividual values of SSc groups (dcSSc and lcSSc) of data not shown in the manuscript.(XLSX)Click here for additional data file.S2 TableIndividual values of healthy controls group of data not shown in the manuscript.(XLSX)Click here for additional data file.