Over the recent years, much has been unraveled about the pro-inflammatory properties of various mitochondrial molecules once they are leaving the mitochondrial compartment. On entering the cytoplasm or the extracellular space, mitochondrial DAMPs  can become pro-inflammatory and initiate innate and adaptive immune responses by activating cell surface and intracellular receptors. Current evidence indicates that uncontrolled and excessive release of mitochondrial DAMPs is associated with severity, has prognosis value in human diseases, and contributes to the dysregulated process observed in numerous inflammatory and autoimmune conditions, as well as in ischemic heart disease and cancer. Herein, we review that the expanding research field of mitochondrial DAMPs in innate immune responses and the current knowledge on the association between mitochondrial DAMPs and human diseases. Innate immunity represents the first line of defense of the human body against infection. This system can detect pathogens through the recognition of conserved molecular motifs, called pathogen-associated molecular patterns (PAMPs). The bindings of these molecules to pattern recognition receptors (PRRs), such as toll-like receptors (TLRs) and NOD-like receptors (NLRs), activates the immune system and triggers a response against the pathogen. Interestingly, it has been shown in the recent years that the human body is using this same system to signal tissue damage in absence of infection in a theory developed by Matzinger in 2004 . Thus, iIt has recently been demonstrated that “danger” signals present in the cytoplasm in context of an active infection (PAMPs) or cellular damage (DAMPs) could also be detected by intracellular sensors of the innate immune system either directly or indirectly as a result of loss of homeostasis and trigger a pro-inflammatory immune response through the formation of inflammasomes , 6. InflRecently, mitochondrial DAMPs have been identified as important mediators of the innate immune response and implicated in various conditions such as trauma, sepsis, and autoimmune disorders Table . AccordiTherefore, it is not really surprising that the release of mitochondrial molecules such as mitochondrial DNA (mtDNA) into the cytosol or the extracellular space triggers such a significant pro-inflammatory response.To prevent the leak of pro-inflammatory mitochondrial molecules into the cytosol or the extracellular space in context of mitochondrial dysfunction, cells have developed various prevention systems. One of those systems is autophagy that enables cells to recycle damaged mitochondria and prevent leakage of its content into the cytosol or extracellular space.However, in context of major cellular stress and tissue injury, the system may be overwhelmed, and mitochondrial DAMPS are released into the cytoplasm or the extracellular space, triggering a pro-inflammatory response.There is a growing body of literature suggesting that mitochondrial DAMPs are implicated in various human diseases . Indeed,Herein, we review the nature and the role of the different mitochondrial DAMPs and discuss their interactions with different organs in the settings of sterile inflammation.Mitochondrial DNA contains 37 genes coding for 2 ribosomal nucleic acids, 22 transfer RNAs, and 13 essential protein subunits of the oxidative phosphorylation system , 119. SpMitochondrial DNA can be recognized by three important PRRs of the innate immune system, TLR9, cytosolic inflammasomes, and type I interferon response, and interactions of mtDNA with its receptors trigger a pro-inflammatory response Figure .TLR9 is a member of the TLR family and is expressed mostly in immune cells including dendritic cells, macrophages, and naturel killer cells. TLR9 is widely referred as a DNA-sensing PRRs since it is able to detect hypomethylated CpG motifs of DNA present in both bacterial , 121 andvia the TLR9 pathway. mtDNA injection in mice causes severe inflammatory response in the lungs (tlr9 −/− and MyD88 −/− mice (tlr9 −/− mice)  . These avia TLR9  and AM2 being the inflammasomes the most involved in mtDNA sensing.via NF-κB activation by a TLR or NOD2 agonist such as lipopolysaccharide (LPS), followed by an activation signal produced by crystals, aggregated β-amyloid, extracellular ATP, or microbial toxins that prompts NLRP3 to assemble the inflammasome complex  protein family and binds to DNA t domain , whereast domain , 132.The groups of Nakahira et al.  and ShimRecent reports have shown that mtDNA could also be recognized by another DNA sensing pathway of the innate immune system, namely, the cyclic GMP-AMP synthase (cGAS) and stimulator of interferon genes (STING) axis , 27 thatThese results suggest that the apoptotic cascade play a critical role to render mitochondrial apoptosis immunologically silent by suppressing DAMP signaling, such as mtDNA.via mitochondrial membrane permeability transition (MPT). One of the first description of the MPT dates from the late 1970s when Haworth and Hunter showed in a series of publications . Patrushev et al.  targeting either peroxisomes  or lysosvia death receptors, such as Fas or tumor necrosis factor-alpha (TNF) receptor leading to the activation of the receptor-interacting protein kinase 1 or 3 (RIP1/RIP3)  –151. In a et al.  demonstra et al.  and indua et al. . Interesa et al.  found thIn a recent study, Caielli et al. brought some insights on the mechanism regulating mtDNA extracellular release in neutrophils . First, To prevent the leakage of the highly pro-inflammatory mtDNA both into the cytosol and the extracellular milieu, various cellular mechanisms have been described for the removal of mtDNA and damaged mitochondria. Autophagy, already mentioned in this review, represents a well described cellular mechanism for the removal of damaged organelles and is defined as mitophagy for the specific removal of damaged mitochondria. Numerous studies have demonstrated that blockade of autophagy results in the accumulation of dysfunctional mitochondria in the cells with increase mitochondrial ROS production and cytosolic release of mtDNA with NLRP3 activation , 153.DNase II is an acid DNAse found in lysosome that plays an essential role in the degradation of DNA in apoptotic cells by macrophages after phagocytosis . StudiesJust like DNA, bacterial RNA constitutes other potent PAMP that is able to trigger an innate immune response. Numerous studies have demonstrated that bacterial RNA can be detected by cell type-specific endosomal and cytoplasmic receptors –159. In Adenosine triphosphate is a nucleotide that has the particularity to play two distinct roles in the intracellular and in the extracellular spaces. ATP constitutes the main source of energy for most cellular processes. It is produced in mitochondria through the activation of the glycolysis pathway and the tricarboxylic acid (TCA) cycle. When found in the extracellular space, ATP plays a key role in cell signaling, as well as in the regulation in renal blood flow, vascular endothelium, and inflammatory responses. Moreover, it has been demonstrated that ATP released by injured or dying cells served as a DAMP to alert the immune system of tissue damage binding to a P2 purinergic receptors. ATP release seems to play key roles in interglia afferent signaling (including pain), in the regulation of renal blood flow, vascular endothelium, and in inflammatory responses. When ATP is released into the extracellular space, it constitutes important signaling molecules that has been involved in numerous inflammatory disorders by binding to purinergic P2 receptors. P2 purinergic receptors have a widespread expression throughout different tissues and are subdivided into P2Y receptors (P2YR) that are G-protein-coupled receptors and P2X receptors (P2XR), which are ligand-gated ion channels Figure .2R signaling is the most studied and best characterized. A striking point that came out from all those studies is the complex and ambivalent effect that P2Y2R signaling can mediate, depending on the environment. On one hand, P2Y2R activation improves mucociliary clearance, bacterial clearance, and wound healing, but on the other hand, it contributes to a pro-inflammatory response in context of asthma et skin hypersensitivity  by human eosinophils and airway epithelial cells followed by a Th2-type innate immune response in mice exposed to allergens . Unlike P2YR, which can bind different ligands , P2XRs respond primarily to extracellular ATP. Binding to P2XR allow sodium and calcium influx and potassium efflux resulting in activations of p38 MAPK or phospholipase A2 mediated by the increased intracellular calcium level . Among avia NOX2 , a member of the high mobility group box (HMGB) family, is essential for maintenance of mtDNA. It can bind to mtDNA in a sequence-independent manner and regulate both mitochondrial transcription initiation and mtDNA copy number , 174. FuTFAM is stabilizing mtDNA through the formation of nucleoids. Several reports have demonstrated that TFAM can promote a pro-inflammatory response both intracellularly or when it is released into the extracellular space. West et al.  demonstrThe N-formyl peptide (NFP) fMet-Leu-Phy is a very potent chemoattractant for immune cells , 175 andvia the TCA cycle that plays an essential role in mitochondria function. Recent studies suggest that succinate, in addition of its crucial role ATP production, can also act as DAMPs and induce inflammation  that is highly expressed on the surface of dendritic cells  as well Cardiolipin is a lipid dimer consisting of two phosphatidyl group bridged by glycerol localized in the inner mitochondrial membrane that plays a critical role in diverse range of mitochondrial functions including mitochondrial respiration and mitochondrial biogenesis , 180. InCytochrome-c, a nuclear encoded, small soluble electron carrier located in the inner mitochondrial membrane, transfers electrons from complex III to IV , 188. CyNumerous studies  have shoThere is a growing body of literature demonstrating that circulating mtDNA levels are elevated in large variety of human diseases, including sepsis , 192, trin vivo and may exacerbate inflammation by stimulating the release of pro-inflammatory cytokines, thereby creating a vicious cycle of sterile inflammation and cell destruction with further release of immunogenic mtDNA.Increased mtDNA levels were also detected in plasma and body fluid of patients presenting with a chronic inflammatory disorder , 59, 61.in vitro results by showing that not only neutrophils isolated from patient with SLE had an increase in oxidized mtDNA aggregates in their mitochondria compared to neutrophils from healthy patients or juvenile dermatomyositis patients but also that oxidized mtDNA represented an SLE autoantigen since they detected anti-oxidized mtDNA autoantibodies in SLE sera (Although innate immunity plays a major role in the pathogenesis of systemic lupus erythematosus (SLE), recent reports have suggested that dysregulation of intra- and extracellular DNA degradation may be involved in human SLE , 197. InSLE sera . FurtherSLE sera  detectedvia P2X7R were critical steps for the success of the treatment of certain types of cancers  with mitochondrial alteration being observed in pulmonary arteries and in the right ventricle in context of PH associated with a metabolic shift from mitochondrial oxidative phosphorylation to glycolysis . InteresAtp8b1 was associated with severe pneumonia in humans and mice.Under normal conditions CL constitutes only 1–2% of alveolar surfactant but its level increases in animal model of lung injury , 202. ThExtracellular release of cytochrome-c had been confirmed in various conditions characterized by cell death. Indeed, increased serum cytochrome-c level was reported in patients with myocardial infarction  as well via MDVs or autophagy requires further clarification. Also, while our understanding of the function and regulation of each individual mitochondrial DAMPs is constantly improving, additional studies are warranted to clarify the biological relevance of the interactions between different mitochondrial DAMPs and other extracellular molecules. The interplay between the various DAMPs is also a key to mediate sterile inflammation. Although not discussed herein, DAMPs also frequently synergize with pathogen-associated molecular pattern.In this review, we discussed the function and molecular regulation of various mitochondrial DAMPs and their role in human diseases. This review highlighted the complexity of the interaction between mitochondria, inflammation, and the immune system. It has become increasingly evident that mitochondrial DAMPs play critical role in sterile inflammation and in various diseases such as those characterized by chronic inflammation. However, many questions remain unanswered. These include the mechanisms for mtDNA release to the outside of the cells and the origin of cell free circulating mtDNA, particularly in critical ill patients. Although, an increasing number of studies have demonstrated that mitochondrial DAMPs can be actively released by non-necrotic cells exposed to an external stimulation, the mechanism for extracellular release for example Finally, therapeutic immunomodulation by targeting mitochondrial DAMPs and their signaling pathways is promising in various inflammatory and auto-immune diseases. The measurement of mitochondrial DAMPs in body fluids as a biological marker of disease may also be of utility and bear prognostic values.SG wrote the manuscript, and JP edited and provided direction for the article.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.


Background:The field of parathyroidectomy (PTx) is complex and brings together many specialists. Even if the surgical approaches changed from classical to minimally invasive PTx, a good outcome is correlated with an adequate localization before and during PTx, while blood assays, such as parathormone (PTH) or 25-hydroxyvitamin D, become useful additional markers.Aim. Specific aspects related to parathyroidectomy and vitamins D (VD) were introduced.Material and Method. The article represents a PubMed-based narrative review.Results. The growing evidence regarding the high prevalence of hypovitaminosis D and early detection of primary hyperparathyroidism (HPT) requires a particular attention to the association of these two disorders, which may be incidental, but some common pathogenic links are displayed. Low VD stimulates PTH production as a secondary or even tertiary type of HPT diagnosis. VD deficiency is associated with larger parathyroid adenomas and higher levels of PTH before and after surgery for primary HPT. Asymptomatically and normocalcemic forms of primary HPT, which are not immediately referred to PTx, require a normalization of the VD levels. VD supplements are safe under some serum calcium cutoffs and offer a better outcome after PTx. However, primary HPT is cured by surgery and, if the indication is well established, this should not be delayed too long to replace VD. Up to half of PTx cases may experience increased PTH levels after surgery, but most of these are transitory if rapid VD correction is done and only a few remaining cases will eventually develop persistent / recurrent primary HPT.Conclusion. A close following of 25-hydroxivitamin D represents one of the keys for a good outcome in the field of parathyroid surgery. Abbreviations:HPT = hyperparathyroidism, MEN = Multiple Endocrine Neoplasia Syndrome, PTx = parathyroidectomy, PTH = parathormone, VD = Vitamin D. Once the removal of the parathyroid adenoma or hyperplasic glands is needed, a surgery and anesthesia team will be necessary [2]. Further on, the postoperative outcome needs endocrinology surveillance of the calcium and PTH levels variations because of the risk of hypoparathyroidism, on one hand, or, on the other hand, persistent/recurrent HPT, which may be found as specific endocrine anomalies of the parathyroid [3]. In cases with genetic backgrounds, such as Multiple Endocrine Neoplasia (MEN) Syndrome (type I or II), the thyroid or adrenal tumor diseases will eventually require specific surgery [4-6].The indications of parathyroidectomy vary, like the presence of high calcium levels, secondary osteoporosis, kidney stones to young patients’ age . Even if the surgical approaches changed from classical to minimally invasive PTx, the key for a good outcome is an adequate localization before and during PTx, while blood assays such as PTH or 25-hydroxyvitamin D become useful additional markers .The field of parathyroidectomy (PTx) is complex and it brings together different specialists starting with endocrinologists who first establish the diagnosis of primary hyperparathyroidism (HPT) and then collaborate with the imagery team to have an adequate localization of the parathormone (PTH) excess source. Based on a regular feedback mechanism, the VD deficiency stimulates the cells causing secondary HPT, which is a common fact in some geographical areas with low sun exposure, in the elderly communities or in cases with renal failure  [12-14].Recent studies pointed the fact that in patients with primary HPT, a secondary component of high PTH levels is brought by vitamin D deficiency, regardless of its cause or seasonal anomalies .If low levels of VD are not corrected before PTx, a part from higher levels of PTH, parathyroid adenomas seem larger while the phenotype in primary HPT is more severe, especially regarding bone involvement [17-19].In order to supplement vitamin D before PTx,the exact thresholds of 25-hydroxivitamin D are not very well established  [20-22].Moreover, intra-operative PTH kinetics after PTx is not changed if VD deficiency is not corrected but PTH is displayed at higher values . Thus, hypovitaminosis D therapy,such as daily oral supplements before PTx, will prevent the development of post-operatory normocalcemic HPT, which is found in up to 43% of the cases in some studies [25].However, other factors have been found in correlation with the lack of PTH normalization after surgery, a part from the VD levels, one of them being the correct identification and further removal of the adenoma; for instance, a six-years study showed that focused-approached surgery had higher PTH levels two weeks after PTx compared to the four-gland exploration [26].The other parameters like total blood calcium, parathormone, bone turnover markers such as alkaline phosphatase,which are higher on patients with primary HPT and severe VD insufficiency, have been studied, while one of the best prognosis tools remains the intra-operative morning PTH, being correlated with the post-operatory eucalcemic state more accurately than the VD status itself (based on some observations) [27].Overall, an elevated PTH value is expected after PTx (the percents depend on study from 9% to 62%), but this is not necessarily the failure of surgery,however, it may be an uncorrected VD deficiency, hungry bone syndrome in conditions with a long medical history before PTx or, exceptionally, a PTH resistance or reduced peripheral sensitivity to the hormone, or even unrecognized mild chronic kidney disease with inadequate function [28]. These causes are easy to adjust if they are correctly identified, mostly by VD supplementation  .Most of these situations with elevated PTH will eventually correct while a small number of the remaining cases will need a close check-up since a persistent or recurrent HPT should be assessed [31-33]. VD has specific receptors at the level of PTH producing parathyroid cells, displaying a direct inhibitor effect regarding hormonal secretion . The importance of VD supplementation before parathyroid surgery is still a matter of debate and many clinicians are actually afraid of offering VD replacement to a patient who probably already has a high calcium level but the VD replacement is actually safe in most situations  . Some observations showed that in primary HPT the conversion of 25-hydroxyvitamin D to a 24-hydroxylated component is increased, thus, the prevalence of VD deficiency might be even higher [37].But, on the contrary, a long standing severe VD deficiency hyper-stimulates parathyroid glands to produce PTH on an autonomic basis as seen in tertiary HPT [38].Inadequately low VD means an additional PTH increase and a more aggressive phenotype of primary HPT [39].Even if the correction of VD insufficiency in mild forms of primary HPT will not exacerbate hypercalcemia or hypercalciuria and will not eventually cure the PTH-producing tumor as PTx does, some authors consider that the adenoma excision should not be delayed in order to restore the VD pool [39-41]. However, not all the cases of primary HPT are referred from the start to surgery, especially asymptomatically and normocalcemic variants and these particular situations will benefit from the VD supplementation when 25-hydroxivitamin D is decreased .VD deficiency is largely known and an incidental overlap with primary HPT might be expected since both of the conditions have been recently widely recognized and early detected due to accessible assays  .Separately from the PTH excess, low VDmight act as a confounding factor knowing its correlation to skeleton health and the underlying pathways of many others non-bone morbidities [46-48].A connection between low VD and cardio-metabolic impairment, such as arterial hypertension, obesity, or type 2 diabetes mellitus has been described although the direct cause-effect relationship is not completely understood yet [49-52]. This aspect might interfere with cardiovascular alterations, which are already presented in primary HPT [53-55].Other observations are related to the VD implications on immune system, which interfere with anesthesia practice and a longer hospitalization, and associated costs with a higher rate of complications and even mortality have been found in patients with chronic conditions requiring surgery if their VD level was low [56].Critically ill patients commonly lack an adequate VD, and this may become a hidden enemy of a good outcome and new protocols of case finding strategies as well as nutritional improvement are necessary .The major motivation of relating VD deficiency to primary HPT and the need to analyze the outcome after PTx in this situation is related to new data regarding its high prevalence in heterogeneous populations, including patients with parathyroid disorders [The growing evidence regarding the high prevalence of hypovitaminosis D and the early detection of primary HPT requires a particular attention to the association of these two disorders, which may be incidental but some common pathogenic links are displayed. VD deficiency, otherwise a commonly recognized condition, is associated with larger parathyroid adenomas and higher levels of PTH before and after surgery for primary HPT. VD supplements are safe under some serum calcium cutoffs and offer a better outcome after PTx. However, primary HPT is cured by surgery and, if the indication is well established, this should not be delayed too long to replace VD. A close following of 25-hydroxivitamin D represents one of the keys for a good outcome in the field of parathyroid surgery. Conflict of interestThe authors have nothing to declare.AcknowledgementNone.


Bile acids are best known as detergents involved in the digestion of lipids. In addition, new data in the last decade have shown that bile acids also function as gut hormones capable of influencing metabolic processes via receptors such as FXR (farnesoid X receptor) and TGR5 (Takeda G protein-coupled receptor 5). These effects of bile acids are not restricted to the gastrointestinal tract, but can affect different tissues throughout the organism. It is still unclear whether these effects also involve signaling of bile acids to the central nervous system (CNS). Bile acid signaling to the CNS encompasses both direct and indirect pathways. Bile acids can act directly in the brain via central FXR and TGR5 signaling. In addition, there are two indirect pathways that involve intermediate agents released upon interaction with bile acids receptors in the gut. Activation of intestinal FXR and TGR5 receptors can result in the release of fibroblast growth factor 19 (FGF19) and glucagon-like peptide 1 (GLP-1), both capable of signaling to the CNS. We conclude that when plasma bile acids levels are high all three pathways may contribute in signal transmission to the CNS. However, under normal physiological circumstances, the indirect pathway involving GLP-1 may evoke the most substantial effect in the brain. Bile acids are synthesized in the liver from cholesterol and released in the intestinal lumen upon food intake. They are predominantly known for their role as nutritional detergents that dissolve lipids and lipid-soluble vitamins. However, a growing body of recent literature describes bile acids as versatile signaling molecules  and chenodeoxycholic acid (CDCA); in mice the dominant bile acids are CA and muricholic acid (MCA). Subsequently, these bile acids are conjugated with the amino acids glycine (mainly in humans) or taurine (mainly in mice). Bile acids are transported from the hepatocytes through the bile canaliculi and stored, together with cholesterol and phospholipids, in the gallbladder. Following food intake, the presence of nutrients  in the stomach triggers gallbladder emptying which results in the release of bile acids into the duodenum. When bile acids pass through the intestinal tract, they contribute to the absorption of lipids and fat-soluble vitamins. In the intestine, gut microbiota deconjugate and dehydroxylate the primary bile acids, converting them into secondary bile acids and enhancing the diversity of the bile acid pool  protects the brain from potentially harmful circulating molecules  seem to be mostly derived from the periphery by passive diffusion as brain levels correlate with serum levels and intraperitoneally injected DThe most studied bile acid receptors are FXR  15/19 Figure . FXR is de novo bile acid synthesis by inhibition of Cyp7a1. Liver and intestinal FXR KO models have shown that Cyp7a1 inhibition depends mostly on intestinal FXR activation via FGF15  did not become diabetic or obese  mice showed that these mice still produce GLP-1 and seemed not different from wild type mice  cells in the NTS and in the intermediate reticular nucleus within the hindbrain  is released from intestinal L-cells in response to nutrients and bile acids. In addition, also in the brain GLP-2 is released from the preproglucagonergic neurons in the brainstem together with GLP-1. GLP-2 acts via its own G protein-coupled receptor, GLP-2R which is mainly expressed in the gastro-intestinal tract and CNS. In the gut, GLP-2 functions in intestinal mucosal health and stimulates nutrient absorption, and in this way influences energy homeostasis (Baldassano et al., In this review we discussed three different pathways via which bile acids could signal to the CNS. In the direct pathway Figure , bile acThe indirect pathway mediated by FXR-FGF15/19 Figure  could exThe indirect pathway mediated by TGR5-GLP-1 Figure  can signAll authors contributed to the design and concept of the review. KM drafted the manuscript and provided the figures. AK, MS, and HE critically reviewed the manuscript and attributed with important intellectual content. All authors approved the final version of the manuscript and agree to be accountable for all aspects of the work.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.


Pulmonary arterial hypertension (PAH) shares many hallmarks with cancer. Cancer cells acquire their hallmarks by a pathological Darwinian evolution process built on the so-called cancer cell “identity crisis.” Here we demonstrate that PAH shares the most striking features of the cancer identity crisis: the ectopic expression of normally silent tissue-specific genes. This detrimental rise in pulmonary blood pressure is due to distal pulmonary artery occlusion driven by unregulated proliferation of pulmonary arterial smooth muscle cells (PASMC) and endothelial cells (PAEC) within the foci of complex vascular lesions. During the last decade, landmark studies demonstrated significant parallels between PASMC/EC in cancer and PAH,2 Similar to what is observed in cancer, epigenetic modifications have been shown to predispose and participate in PAH pathogenesis.5 Indeed, germ line mutations in the bone morphogenetic protein receptor II (BMPRII)4 and potassium channel subfamily K member 3 (KCNK3)6 genes have been demonstrated to participate in PAH development. Furthermore, somatic mutations, single nucleotide polymorphisms, microsatellite instability, and aneuploidy have also recently been found to increase predisposition to PAH.8 For example, two sisters with heritable PAH carry the same missense mutation in Krüppel-like factor 2 (KLF2), a gene known to participate in nitric oxide vascular homeostasis.9 In another PAH patient carrying a BMPRII mutation, Aldred et al.7 identified a deletion in chromosome 13 resulting in the deletion of mothers against decapentaplegic homolog 8 (Smad-8). In both cases, these somatic mutations could participate in cancer and PAH pathogenesis. In addition, increased DNA damage followed by increased somatic mutation acquisition has recently been demonstrated in PAH.11 Finally, there is growing evidence for genome-wide epigenetic modifications in PAH that lead to chromatin remodeling and gene expression alterations. These modifications include varied expression of epigenetic modifiers and readers including histone deacetylases (HDACs)12 and BRD4,13 DNA methylation modifications,15 and microRNAs.16These hallmarks include an increased proliferation rate, evasion of growth suppressor, resistance to apoptosis, cellular energetic deregulation, angiogenesis in foci of complex vascular lesions, sustained inflammation, genetic mutations, and epigenetic modifications.17 These modifications in gene expression lead to a loss of the cells’ identity (i.e. phenotype) and the development of a full identity crisis (aberrant cell commitment). In cancer, the most striking example of this identity crisis is the activation of normally silent tissue-specific genes, such as male germline or placenta-specific genes.17 One hypothesis is that the development of this identity crisis provides a fertile ground for initiating a pathological Darwinian evolution process,18 leading to the acquisition of cancer hallmarks  concomitant with the aberrant activation of “off-context” genes  .17 Theseallmarks . In thisIn this study, we analyzed the lungs of one male and one female with IPAH and one female with PAH associated with systemic scleroderma  and compared them to three non-PAH female lungs (mean age = 64 years). Individuals previously gave signed informed consent in accordance with Laval University and the IUCPQ Biosafety and Ethics Committee (CER20773).17 Briefly, using publicly available transcriptomic data from normal human tissue, a list of 506 genes with an expression restricted to the germline or the placenta tissue was defined. An extended list of genes was obtained by adding genes with a predominant expression in the germline, embryonic stem (ES) cells, or in the placenta on the basis of their mean expression level in one of these tissues being above a threshold defined as the mean expression in a series of 112 adult non-germline tissues + 3*standard deviations.17 The aberrant expressions of the abovementioned genes were systematically detected using the transcriptomic data of the samples from patients with PAH and a threshold value corresponding to the mean value in the three control samples + 3*standard deviations.The detection of genes ectopically expressed from the transcriptomic analysis was carried out with a similar strategy as described by Rousseau et al.20 Nextbio allows user-friendly access to microarray data using a consistent analysis pipeline between datasets as described by Kupershmidt et al.20 We found three relevant microarray datasets in this microarray repository database; namely, GSE53408 comparing the lung gene expression of 11 controls to 12 patients with severe PAH, GSE21284 comparing primary cultured PASMC from two control to two PAH patients, and GSE15197 comparing the lung gene expression of 13 control to 18 PAH patients and eight patients with pulmonary hypertension associated with pulmonary fibrosis.In order to confirm the expression of genes found to be ectopically expressed in our microarray analysis, we investigated their expression by meta-analysis using the public Illumina microarray repository database, Nextbio.Our analysis revealed an aberrant expression of 40 genes in all three human PAH lungs . Among tThese results were further confirmed by meta-analysis using Nextbio. Indeed, bio-informatics research in the Nextbio database revealed that, despite using a different microarray platform (Illumina vs. Affymetrix array), different approaches and different populations, three previous microarray studies also documented that 18 of the genes identified above have aberrant expression in PAH  and 1e. 1216 as a mechanism to provide fertile ground for a pathological Darwinian evolution process.To our knowledge, this is the first study demonstrating the aberrant activation of normally silent tissue-specific genes in the lungs of human PAH patients. Indeed, 23 of the altered genes have already been demonstrated in PAH, but this is the first time that a systematic investigation of off-context gene expression has been done in the context of PAH. This demonstration of an ectopic gene expression in PAH gives the first strong evidence of a cancer-like identity crisis in PAH. This also reinforces the potential role of epigenetic landscape modification in PAH pathogenesis21Interestingly, while all the PAH patients we tested showed ectopic gene expression, they did not re-express the same set of genes. In our view, this differential re-expression is due to stochastic gene expression modification driven by the Darwinian evolution process. Thus, in this view, it is not the re-expression of a defined set of off-context genes which is of interest but rather the process of re-expression itself which reflects the underlying pathological mechanism that gives rise to the identity crisis. Nonetheless, a potential role for some of these ectopically expressed genes cannot be excluded, particularly for the genes we found expressed in all of our patients including embryonic stem cell genes which could participate in recruitment of progenitor cells known to play a role in PAH pathogenesis.17 The generation of false-negative results cannot be controlled in our study setting; however, if this bias occurs, it will not nullify our finding of an identity crisis in PAH as false-negative results may only affect the number of genes found but not the demonstration of an off-context gene expression in PAH.Other studies are needed to investigate the specific role of these genes in a larger cohort of PAH patients. Indeed, the number of patients used in our study as well as their heterogeneity  could lead to the generation of false-positive and false-negative results. Nonetheless, the generation of false-positive results is unlikely, considering the nature of genes investigated in the present study are epigenetically tightly locked in somatic cells.22Taken together, our results demonstrate an off-context expression of normally silent genes which supports our hypothesis that an identity crisis is occurring in PAH. Much like in cancer, this off-context gene expression could pave the way for potential new predictive markers in PAH. In cancer, the ectopic expression of cancer testis antigens (CTAs), a class of immunogenic proteins with a testis- and tumor-restricted pattern of expression, are already use as predictive markers.1216 This observation of an identity crisis in PAH strengthens the parallels that can be established between the PAH condition and cancer where a similar finding has been reported.In conclusion, our study brings the first evidence of an identity crisis occurring in the lungs of PAH patients by demonstrating the aberrant and off-context expression of normally silent, tissue-specific genes. In our view, this ectopic gene expression is due to a pathological Darwinian evolution process evolving on the fertile ground of genome-wide epigenetic modifications already suggested in PAH.


The Tasmanian devil, a marsupial species endemic to the island of Tasmania, harbours two contagious cancers, Devil Facial Tumour 1 (DFT1) and Devil Facial Tumour 2 (DFT2). These cancers pass between individuals in the population via the direct transfer of tumour cells, resulting in the growth of large tumours around the face and neck of affected animals. While these cancers are rare, a contagious cancer also exists in dogs and five contagious cancers circulate in bivalves. The ability of tumour cells to emerge and transmit in mammals is surprising as these cells are an allograft and should be rejected due to incompatibility between Major Histocompatibility Complex (MHC) genes. As such, considerable research has focused on understanding how DFT1 cells evade the host immune system with particular reference to MHC molecules. This review evaluates the role that MHC class I expression and genotype plays in allowing DFT1 to circumvent histocompatibility barriers in Tasmanian devils. We also examine recent research that suggests that Tasmanian devils can mount an immune response to DFT1 and may form the basis of a protective vaccine against the tumour. Cells transferred between individuals should be rejected in a robust immune response due primarily to differences in Major Histocompatibility Complex (MHC) genes between individuals Gorer . Tumour In the context of a contagious cancer, cells passing as an allograft should be recognised by T-cells stimulated by allogeneic MHC class I and/or class II molecules present on the cancer cells (Gould and Auchincloss Canis lupus), Tasmanian devils (Sarcophilus harrisii) and four species of bivalves , cancer cells are able to pass as an allograft between individuals , mussels (M. trossulus), cockles (C. edule) and golden carpet shell clams (P. aureus), suggests that the transmission of cancer cells may be relatively common among bivalves   is a transmissible cancer that arose in an ancient species of dog or wolf more than 10,000 years ago Novinski . CTVT isThe Tasmanian devil is a carnivorous marsupial that is endemic to the island of Tasmania south of mainland Australia and is the only mammal in which two genetically distinct contagious cancers have emerged  contigs  and tammar wallaby (Macropus eugenii)  cells . This data fits with the finding that there is no IFNγ response element in the promoter region of Saha-UD, in contrast to the other MHC class I heavy chain genes , based on sequencing of part of exon 2 of the class I gene from DFT1 biopsies. More recently, it was reported that DFT1 cells have four class I alleles, SahaI*35, SahaI*46, SahaI*90 and SahaI*28 . Although it is difficult to assign these alleles to specific loci, phylogenetic comparison of the DFT1 alleles to all described devil class I sequences suggests that SahaI*35 and SahaI*46 belong to the Saha-UA gene and SahaI*90 and SahaI*28 belong to the Saha-UB and Saha-UC genes, respectively (Fig. SahaI*32 is an established allele for the Saha-UD gene (Siddle et al. Saha-UK is expressed by DFT1 cells, but as this gene is not polymorphic, it is likely not relevant in this context. In addition, there is no significant expression of the non-classical MHC class I genes, Saha-UM or CD1 in DFT1 cells (Cheng and Belov The MHC class I genotype of DFT1 has not been determined conclusively, and the MHC class I alleles reported in DFT1 cells have varied between studies Table  have repThe expression of MHC molecules is also of importance in the progression of CTVT. CTVT has a different pattern of progression to DFT1, once CTVT cells are transmitted, there is a growth period followed by a stationary phase and or regression of the tumour Cohen . The immThere have been a number of isolated cases of tumour cells successfully passing between individuals in humans. These cases have occurred during transplantation and across the maternal/foetal barrier (Isoda et al. As invertebrates, bivalves do not have MHC or antigen receptors and phagocytosis is the primary clearance mechanism used to protect against pathogens. However, terrestrial molluscs are capable of recognising differences in cell surface molecules of self and non-self tissue, rejecting allografts through a macrophage and perforin-induced death (Furuta and Yamaguchi The devil immune system does not respond effectively to DFT1 in the majority of cases, as evidenced by the lack of immune cell infiltration (Howson et al. When MHC class I molecules are upregulated on DFT1 cells, mismatches in MHC alleles between the tumour and host become relevant. As an allograft, DFTs should provide additional antigenic stimuli to host devils when compared to single organism tumours. In humans, the position and number of mismatches at specific HLA correlates with graft failure (Petersdorf


Patients with dementia present epilepsy more frequently than the general population. Seizures are more common in patients with Alzheimer’s disease (AD), dementia with Lewy bodies (LBD), frontotemporal dementia (FTD) and progressive supranuclear palsy (PSP) than in other dementias. Missense mutations in the microtubule associated protein tau (MAPT) gene have been found to cause familial FTD and PSP, while the P301S mutation in MAPT has been associated with early-onset fast progressive dementia and the presence of seizures. Brains of patients with AD, LBD, FTD and PSP show hyperphosphorylated tau aggregates, amyloid-β plaques and neuropil threads. Increasing evidence suggests the existence of overlapping mechanisms related to the generation of network hyperexcitability and cognitive decline. Neuronal overexpression of tau with various mutations found in FTD with parkinsonism-linked to chromosome 17 (FTDP-17) in mice produces epileptic activity. On the other hand, the use of certain antiepileptic drugs in animal models with AD prevents cognitive impairment. Further efforts should be made to search for plausible common targets for both conditions. Moreover, attempts should also be made to evaluate the use of drugs targeting tau and amyloid-β as suitable pharmacological interventions in epileptic disorders. The diagnosis of dementia and epilepsy in early stages of those diseases may be helpful for the initiation of treatments that could prevent the generation of epileptic activity and cognitive deterioration. Epileptic seizures are more frequent in patients with AD than in patients with other dementias ..A  and the generation of epileptic seizures ,187,188,Multiple experimental models have been generated overexpressing mutant human APP, presenilin and tau proteins, and combinations of these proteins. Convulsive or non-convulsive seizures have been found in almost every AD mouse line, particularly in those transgenic for APP and PSENs. In APP/Aβ transgenic models, high levels of amyloid-β expression were enough to produce epilepsy prior to the appearance of neurodegeneration and neuronal loss, but this effect did not take place in the absence of the wild type tau protein ,68,108. As was mentioned above, overexpression of FTDP-17 tau in VLW mice  producesData obtained from experimental models of AD, as we mentioned above, suggest a key role for the tau protein in amyloid-β toxicity. EEG analysis of several APP/Aβ transgenic mouse models revealed the presence of cortical and hippocampal synchronous intermittent epileptiform discharges and non-convulsive generalized seizures . In the The tau A152T mutation increases risk for tauopathies, including AD and FTD diseases, such as PSP and CBD ,201,202.Hyperphosphorylated tau in NFTs is also found in traumatic brain injury, in long-term and drug resistant epilepsy ,76. In eEpileptic seizures are more common in patients with dementia than in the general elderly population and their appearance has been associated with the progression of cognitive impairment. Both abnormally phosphorylated tau and amyloid-β overexpression in mouse models produce aberrant excitatory activity, although amyloid-β excitotoxicity depends on the presence of tau. On the other hand, hyperphosphorylated tau regulates excitotoxic damage and increases GABAA receptor-mediated hyperexcitability in the absence of abnormal amyloid-β peptide. The connection between abnormally increased network excitability and cognitive deterioration has driven a novel field of research focused on searching for plausible common targets for both conditions. The efficacy of various antiepileptic drugs has been demonstrated in the prevention of cognitive impairment in cases of patients with AD and, most extensively, in experimental models of epilepsy and AD. Moreover, attempts should also be done to evaluate the use of drugs targeting tau hyperphosphorylation and amyloid-β accumulation, as well as microtubule-stabilizing agents, as suitable pharmacological interventions in epileptic disorders. Subsequently, the diagnosis of dementia and epilepsy in early stages of the diseases may be helpful for the initiation of treatments that could prevent the generation of epileptic activity and the progression of cognitive decline. Long-term video-EEG recording should be considered to properly evaluate the presence of epileptiform activity in patients with dementia.


Polymers have been emerging to be the cornerstones for therapeutic applications as well as the largest and versatile class of biomaterials. Synthetic polymers can be designed and synthesized with a broad variety of structures and appropriate physical and chemical properties, which are of increasing interest in a wide range of biomedical applications as diverse as tissue engineering, drug delivery, therapeutics, diagnostics, and so on.Since the last decade, the methods of polymer synthesis, processing, and characterization are developing rapidly, which bring both challenges and opportunities to design novel polymeric biomaterials as well as to understand the biological behaviors between biological systems and polymeric materials. Therefore, we launch this special issue, including two review articles and four research articles, to summarize the application of synthetic polymers in biomedical engineering and to illustrate the new development of polymeric biomaterials.One review article “Strain and Vibration in Mesenchymal Stem Cells” focuses on the effect of various culture conditions and strain or vibration parameters to review the response of mesenchymal stem cells to vibration and cyclic tension and then discuss how polymer scaffolds influence cell response to vibration and strain. The other review article “Scaffolds for Pelvic Floor Prolapse: Logical Pathways” highlights the recently developed macroporous monofilament meshes and electrospinning emerged method, which may fill the gap in the market to treat pelvic organ prolapse. These two review articles indicate the importance of synthetic polymer scaffold in basic research and therapeutics, respectively. In Vitro Degradation.” The mechanical and physicochemical properties of the PGA plates are evaluated in detail during in vitro degradation. G. Rijal et al. fabricated 3D porous scaffolds via PLGA and another biodegradable synthetic polymer polycaprolactone (PCL) in the article “Application of Synthetic Polymeric Scaffolds in Breast Cancer 3D Tissue Cultures and Animal Tumor Models.” It has proven that cancer cells grown on 3D polymeric scaffolds exhibit distinct survival, morphology, and proliferation compared to those on 2D polymeric surfaces. Tumor models produced via these 3D scaffolds have obvious advantages in anticancer drug screening, which can facilitate the observations of cancer biomarker expression, molecular regulation of cancer progression, and drug efficacies across tumors at similar sizes and developmental stages. Polyethylene glycol (PEG) is another one of the most commonly used synthetic polymers for biomaterials. In the article “Efficient Self-Assembly of mPEG End-Capped Porous Silica as a Redox-Sensitive Nanocarrier for Controlled Doxorubicin Delivery,” porous nanosilica particles are modified with PEG shell via disulfide bridges and supramolecular interaction for drug delivery, with benefits of enhanced drug loading capacity and decreased risk of systemic toxicity.The research articles in this issue extend the application of synthetic polymers in both basic research and therapeutics. Synthetic poly(lactic-co-glycolic acid) (PLGA) is widely considered as a base material for biomedical applications due to its good biocompatibility and degradability. In the article “Influence of Processing Conditions on the Mechanical Behavior and Morphology of Injection Molded Poly(lactic-co-glycolic acid) 85:15,” an overview is provided among processing conditions, morphology, and mechanical property relationship of injection molded PLGA. Based on the study of mechanics, PLGA is further processed by injection molding as craniofacial bioresorbable medical devices in the article “Effect of Injection Molding Melt Temperatures on PLGA Craniofacial Plate Properties duringIn summary, this special issue connects the synthetic polymers to biomaterials science and engineering. We sincerely hope that the readers enjoy reading the presented original research work in this special issue and get inspired for their future studies.


The particle properties can be modified to promote different and more flexible applications, resulting in consumer benefits, particularly in medical, cosmetic, and industrial applications. As this applied science matures and flourishes, concerns have arisen regarding potential health effects of exposures to untested materials, as many newly developed products have not been adequately evaluated. Indeed, it is necessary to ensure that societal and commercial advantages are not outweighed by potential human health or environmental disadvantages. Therefore, a variety of international planning activities or research efforts have been proposed or implemented, particularly in the European Union and United States, with the expectation that significant advances will be made in understanding potential hazards related to exposures in the occupational and/or consumer environments. One of the first conclusions reached regarding hazardous effects of nanoparticles stemmed from the findings of early pulmonary toxicology studies, suggesting that lung exposures to ultrafine particles were more toxic than those to larger, fine-sized particles of similar chemistry. This review documents some of the conceptual planning efforts, implementation strategies/activities, and research accomplishments over the past 10 years or so. It also highlights (in this author’s opinion) some shortcomings in the research efforts and accomplishments over the same duration.Nanotechnology is an emerging, cross-disciplinary technology designed to create and synthesize new materials at the nanoscale  many different nanomaterial types, 2) various potential routes of exposure, 3) nanomaterial characterization issues, 4) limitations in research methodologies, such as time-course and dose-response issues, and 5) inadequate The term “nano” arises from the Greek term, referring to “dwarf”. What makes the technology intriguing is that particle properties may often become altered as they are reduced below 100 nm in size. For example, gold particles may transform to blue or red pigments. Electrical insulating functions of certain particle types may become conductors at the nanoscale. In short, particle properties can be modified to promote different and more flexible applications, resulting in consumer benefits, particularly in the areas of medical and industrial applications. From a material science perspective, this represents an exciting challenge because as the particulate material size decreases in the direction of the nanoscale (particularly below 100 nm), the physical properties of particles are known to be altered and can be harnessed to create new products and applications1.Nanotechnology continues to be an emerging multidisciplinary science platform that is often defined as creating products and applications based primarily upon the synthesis of molecules in the nanoscale , nanoscale titanium dioxide particulates, or zinc oxide particles for health-related sunscreen applications. However, as the commercialization and promotion of the technology has become more widespread to consumers, concerns have been raised about safety issues with questions relating to health effects and impacts on the environment. Questions initially were raised about the inhalation route of exposure, i.e. can inhaled ultrafine particles have adverse pulmonary impacts when compared to fine particles? But, equally important, the safety of the other two major routes of exposure, i.e. ingestion/oral and dermal, has also been raised. Therefore, it is critical to assess the health impacts of nanoparticle exposures for all routes when considering exposures to both occupational workers and consumers.During the past 10 years, several research strategies and challenges have been proposed to facilitate and direct “verification”, i.e. the safe handling of and exposures to individual forms of nanoparticles and nanotechnology in general. A major problem is that all of the different nanoparticle types cannot be effectively evaluated for safety and environmental effects in a timely manner owing to 1) the vast numbers of different nanoparticle types, 2) the numerous variations within specific nanoparticle types , 3) the overwhelming expense required to adequately test each individual nanoparticle type, 4) the inadequate time that would be required to test each of the nanoparticle types  the vast expense can easily cost approximately 2 million dollars for a single chronic inhalation study.As a result of this large but important dilemma, efforts have been expended by numerous authors, groups of research investigators, entire workshops, and millions of dollars/euros of grant funds to provide “guidance” on developing research health and safety challenges and strategies and to support or sponsor inter-laboratory research validation efforts as well as funding for investigators to study the hazards and environmental impacts (toxicology) of different nanoparticle exposures. It is hoped that an understanding of both health effects and environmental hazards of nanoparticle exposures  as well as environmental effects could lead to improved health and environmental risk evaluations.Given the potential beneficial impact of nanotechnological innovations in our society, this brief review identifies and documents many of the initial conceptual grand challenges, discussions, workshop activities, and, finally, collective research efforts that have been expended, organized, promulgated, and published in an attempt to encourage, rather than to discourage, the development of the technology; and to foster the implementation of risk assessment strategies following nanoparticle exposures relative to the normal routes of entry, i.e. inhalation, oral, and dermal. Basically, the question is how best to approach this difficult but important set of issues.Some of the original research efforts, discussions, and challenges/workshop issues/conclusions that have been proposed and implemented during the past 10 years or so are presented; for example, some of the more prominent European Commission FP7-sponsored programs on nanosafety issues followed by some pertinent conclusions of the US National Academy of Science Committee on Research Progress of Environmental Health and Safety Aspects of Engineered Nanomaterials. In addition, the implementation components of a risk framework for nanomaterials is briefly outlined. The NanoRisk framework was a collective 2-year effort fostered by ongoing discussions of a non-governmental organization (NGO)  and a company synthesizing and commercializing products derived at the nanoscale (the DuPont company). The objectives of the framework were to develop and produce a systematic and disciplined procedure to identify, manage, and reduce the possible environmental safety and health risks associated with engineered nanomaterials throughout every stage of a product’s life-cycle. The scope of the framework provides an informed counsel regarding many of the essential issues that should be considered by an organization when developing the applications of nanomaterials and regarding research findings in order to deliver solid risk evaluations and risk-management decisions. The target audiences represent companies and research institutions that are actively working with nanomaterials to develop applications or products.For completeness, additional sections of this mini-review include discussion of important and controversial issues related to 1) the evaluation of the strengths and weaknesses of the entire scientific literature on nanosafety research over the past 10 years by an expert who has read and critically assessed over 6,000 studies out of more than 10,000 publications selected on human health effects or biological studies; and 2) important discussions on “biokinetics and biodistribution” of nanomaterials following pulmonary, dermal, or oral exposures .Finally, a study design and the implementation of a subchronic (13-week) inhalation toxicity study with CNFs in rats are described. This discussion serves to demonstrate and emphasize the difficult challenges, time dependency, expenses, and toxicological complexity that are required to generate meaningful data and provide accurate perspectives on the issues related to developing adequate risk assessments for the variety of nanomaterial types that currently exist in commerce and to which workers and consumers are exposed.2. These investigators recommended five generalized grand challenges for nanotechnology research, which were conceptually designed to serve as a blueprint to stimulate nanosafety research strategies.One of the earlier efforts to stimulate risk-related research was proposed as a series of “Grand Challenges” by Maynard and colleagues in 2006The five grand challenges were described in the following manner and they addressed the following needs:1)Advancement of equipment over the next 5–15 years to estimate or determine aerosolized and water exposures to engineered nanomaterials2)Techniques and approaches to assess the hazards of engineered nanomaterials3)Developing predictive models to gauge the potential hazardous effects of nanomaterial exposures on environmental and human health systems 4)Developing methods for estimating the EHS impacts of nanomaterial exposures over a lifespan5)Facilitating methodologies to assess strategically programs that could be utilized to implement relevant risk-focused research3 assessed the progress made during the subsequent 10-year period. It was concluded that appreciable progress had been made in the advancement and funding of important programs to initiate health-related research regarding the safety of nanomaterials. Specific organizations that were identified included 1) the US-based National Academy of Sciences, which published a comprehensive research plan for nanosafety research, 2) the European Union, as shown by FP7 and Horizon 2020 programs, which sponsored numerous research-based consortia, and 3) the Organization for Economic Cooperation and Development (OECD) projects, which developed protocols to standardize and validate test methods for conducting toxicity studies on nanomaterials. It should be noted that the success of this first initiative was related primarily to the funding of safety-related projects and could be measured, in part, by the numbers of publications addressing two of the previously listed grand challenges: 1) hazard studies on nanomaterials and 2) life-cycle assessments. In contrast, however, little significant progress was made on developing instruments to monitor airborne or waterborne engineered nanomaterials or predictive methodologies for modeling applications.Following the publication of five grand challenges for developing “safe nanotechnology” in 2006, two of the authors (Maynard and Aitken)4. The participants were charged with addressing the following primary questions related to nanomaterial health effects: what can we do today and what do we need tomorrow? The three major topics to be addressed at the workshop were 1) the need for enhanced efforts in the characterization of nanoparticles, 2) development of methods for the evaluation of aerosolized and internal exposures to nanomaterials, and 3) evaluation of the hazard potential following pulmonary or dermal routes of exposures. The workshop participants concluded that physical factors can influence toxicity, including particle composition, surface area, and characteristics such as size and shape. With regard to a testing approach for human health effects, a first step might be to gauge potency hazards, and it was recommended to includein vitro screening assessment strategies to evaluate possible reactivity, biomarkers, inflammation, or cellular uptake indices.During the same time, the European Centre for Ecotoxicology and Toxicology of Chemicals (ECETOC) convened a workshop in Spain of 70 scientific and clinical experts from all relevant sectors  in November 20055. The ITS framework was considered to be a process that promotes the risk of nanomaterials to be evaluated accurately, effectively, and efficiently, thereby obviating the need to test each and every nanomaterial type, on a case-by-case basis. The major topics were determined, by consensus, to represent 10 different commercially available nanomaterials. Physicochemical characterization, exposure identification, hazard assessments, and modeling—deemed to represent key priorities for research areas—were modeled in a “stepping stone” matrix. This stepping stone matrix was represented by hexagonal diagrams which served to provide tools for individual stakeholders to formulate enhanced reliability criteria for developingin silico approaches. A manuscript written to summarize the meeting results described an appraisal of how this particular framework compares with current risk assessment approaches and how future methodologies could adapt to accommodate new approaches. According to the authors, the ITS-NANO project expressed a detailed, flexible, and stakeholder-driven research strategy tool which describes and prioritizes specific research needs for dealing with a wide variety of nanomaterial types, including newly discovered or synthesized materials.A series of workshops were convened for expert stakeholder groups  with the intention of proposing, discussing, and ultimately conceptually implementing an “intelligent testing strategy” (ITS) designed to assess the risk of nanomaterials on a case-by-case basis6. The authors evaluated the findings of numerous nanotoxicology-based research publications up to that date and reflected on the progress and advancement made regarding risk assessment conclusions for nanomaterial exposures. Several subtopics were described and evaluated, including 1) nanomaterial selection, 2) nanomaterial physicochemical characterization, 3) nanomaterial dispersion techniques, 4) dose level selection and concentrations for implementing experiments, 5) identification of target organ sites and endpoints, 6) development of animal alternative methodologies, and 7) nanomaterial risk assessment methodologies. The authors considered the value of this review article to be in its informing of a discussion of these various important issues and providing guidance on improving upon the utility of current methods, thus achieving and fulfilling enhanced relevant strategies and experimental designs which could better inform upon the wide range of relevant nanomaterial risk assessments.This review paper summarized the results from the European project entitled Particle Risk, which was one of the first multidisciplinary projects funded by the European Commission’s Framework Programme 7. The project reviewed and considered results and interpretations of a wide range ofin vitro andin vivo studies. The main objectives of the ENPRA project were 1) to assess the physicochemical characteristics of 10 commercially available nanomaterials, 2) to evaluate the hazards of these nanomaterials usingin vitro toxicology testing methods with cells sourced from pulmonary, cardiovascular, hepatic, renal, and developmental systems and to examine five potential mechanisms of toxicity including cytotoxicity, oxidative stress, inflammation and immune response, genotoxicity, and potential fibrogenicity, 3) to confirmin vitro findings with relevantin vivo testing using acute (24-hour) intratracheal exposure of mice, 4) to utilize data generated from this project in an “exposure–dose-response” relationship paradigm via “mathematical modeling”, and 5) to develop and implement a strategy for the dissemination of findings for risk assessment and risk management determinations.ENPRA (Risk Assessment of Engineered NanoParticles) was an early multidisciplinary European Commission FP7-funded project designed to assess the risks to human health following nanomaterial exposure specifically when considering pulmonary, cardiovascular, hepatic, renal, and developmental systemsin vitro andin vivo methodologies. One of the major goals was to apply the ENPRA completed objectives to achieve a framework for quantitative risk assessment, particularly in occupational workplaces. Alternatively, some of the shortcomings of the ENPRA projects were related to the use of non-physiologically relevant intratracheal particle exposures  concomitant with the lack of a full time-course experimental regimen (i.e. the protocol for the studies utilized only a 24-hour post-exposure regimen) which did not permit a full accounting of risk assessment evaluations.The project generated substantial fruitful data and, in some cases, achieved similar comparative findings utilizing bothin vitro test methods for hazard of toxicity evaluation in order to promote the development of an ITS (see above description)8. In protocols similar to other European Commission FP7 projects (using round-robin laboratory comparisons for uniformity of results), six representative oxide-type nanomaterials provided by the European Commission Joint Research Center (JRC) were tested usingin vitro-type methodologies in nine different research laboratories using 12 different cellular models incorporating six different target organ systems. According to the authors, a hazard ranking could be established for the representative nanomaterials tested . The investigators proposed that their testing approach could be implemented for the development of an ITS strategy suitable for nanomaterial risk assessments.The FP7-MARINA  project aimed to identify and evaluatein vitro approaches to evaluate the pulmonary fibrogenic potential of aerosolized nanomaterials9. In an attempt to develop substitutein vitro strategies to replace 90-day inhalation studies, given the monetary, ethical, and scientific concerns with thisin vivo test, an international expert panel was convened in Washington, DC, to discuss and design possible alternative approaches to assess the inhalation toxicity of MWCNTs using alternative,in vitro-type strategies. Inhalation exposures to MWCNTs in rats are known to produce pulmonary fibrosis as a key adverse outcome. Given that the development of lung fibrotic responses can require weeks or months to developin vivo, it was postulated that anin vitro test system might serve to replace inhalation studies in a more rapid fashion by measuring and documenting pro-fibrotic precursors/mediators such as relevant cytokines and growth factors that have been implicated in the development of adverse lung outcomes. Accordingly, the workshop discussions dealt primarily with recommendations for designing and monitoring necessary indicators for such anin vitro system, utilizing relevant co-cultured lung cells, preferably exposed at a relevantin vitro aerosolized exposure system and utilizing air–liquid interface (ALI) methodologies. The investigators acknowledged that future planning is a necessary prerequisite for ultimate substitution of inhalation studies in rats within vitro methodologies; the current effort represents only a very early step in the process and has many hurdles ahead for any serious consideration ofin vitro substitution as a viable and achievable system for replacing inhalation toxicity studies.More recent attempts have been made to obtain consensus on10. A major focus of the committee’s report was to provide consideration and guidance on the development of research strategies for developing basic toxicity studies for the wide variety of current and future nanomaterials, owing, in large part, to time and expense considerations. Obviously, there exists a critical need for implementing reliable and validated screening tools to identify and confirm toxicity pathways for health and environmental effects and to address important mechanistic issues, given that the reliable testing of individual nanoparticle types is not practical. In addition, toxicity results obtained from a variety ofin vitro studies often have limited value for determining relevant health hazards, in large part because toxicological studies with engineered nanomaterials are often conducted at extremely higher doses than might be encountered in real-world exposures, concomitant with a focus on short-term toxicological responses. Accordingly, time-course experiments would better reflect potential realistic effects following exposures to nanomaterial types.Recently, a National Academy of Sciences committee undertook an assessment of research progress on EHS aspects of engineered nanomaterialsin vivo experiments, the application of a spectrum ofin vitro investigations could provide some useful mechanistic insights into toxicity pathways, although several shortcomings to currentin vitro methodologies need to be addressed. Optimizing the relevance ofin vitro studies to real-world toxicity considerations should require the utilization of experimental designs that require dose-response behaviors over a full range of doses and should also require the duration of exposure using relevant cell types (focusing on route of exposure regimens), including time-course evaluations concomitant with comparisons and validation with correspondingin vivo systems.Alternatively, given the limitations of animal testing going forward, and with a greater resistance to conductingTo summarize the suggested framework and research strategy, implementation of the following considerations would significantly improve the methodology for the development of reliable and validated screening tools:1)Rigorous physicochemical characterization of nanoparticle types and behavior through the life-cycle2)Dose-response characterization and careful attention to reliable dosimetry at relevant human exposure levels3)in vitro studies, selection of relevant cell types and cell models that reflect the route of human exposuresParticularly for4)Time-course assessments that span acute to chronic exposure durations5)11Application of proper benchmark controls to improve the interpretation of toxicological outcomesin vitro techniques. In addition, numerous studies have reportedin vitro toxicity results using only single cell types 10. More recently, complexin vitro systems are being developed, which can, in part, better simulate particle phagocytosis, an important aspect in the simulation of lung defense responses to particles13. The development of suchin vitro techniques expedites the transition from the current animal-based inhalation testing system to one that is based primarily on human cell lines andin vitro assays. Such reproducible, accurate, and validated cell-basedin vitro screening assays for assessing pulmonary and genetic toxicity will have important benefits  and may provide experimental designs to address mechanistic questions.The lung is a complicated organ system made up of a number of different cell types ; thus, the pulmonary microenvironment and the complex interactions in the lung which occur following inhalation and subsequent deposition of particles are difficult to simulate when employingin vitro methodological approach, several steps have been designed to better represent the physiology of the distal lung microenvironment, concomitant with the implementation of an aerosol exposure system. Accordingly, this transition from a traditional “submerged” cell culture system to a more physiologically relevant ALI cell culture system, using Transwell® Permeable Support devices (microporous membranes), is useful for providing and maintaining pulmonary cell co-cultures  and exposing the co-cultures to particulate aerosols in a humidified atmosphere.To improve the efficacy of anin vitro aerosol pulmonary toxicity study. These considerations include, but are not limited to, the following actions:Many fundamental issues require consideration for optimization when transitioning from an animal inhalation toxicity set-up to anDetermining the cell types to be used in a co-culture or tri-culture system in order to better simulate the lung microenvironmentTransitioning from primary cell types collected from animals to immortalized cells derived from cell lines and tested for biological functionalityDetermining the number or ratio of lung epithelial cells to alveolar macrophages in a co-culture plateDetermining the aerosol generation method for particles/nanoparticles and appropriate, reproducible, and quantifiable dose metrics to be utilized for particle inhalation deposition assessments and for comparisons of the results of one study to anotherhttps://www.nanopartikel.info/en/projects/completed-projects/nanocare/ver-nanocare).The NanoCare project (2006 to 2009) was one of the first big projects (before the European Commission started with the increase of the funding money within the seventh framework program) to demonstrate the good cooperation between industry and academia and resulted in some very important results concerning the fiber paradigm and metal oxides (the complete list of publications can be found here:https://www.nanopartikel.info/en/projects). All information is combined within a specific website (www.nanoobjects.info) which delivers not only a literature criteria checklist but also a summary of SOPs to be used by everyone.As a result of this activity, a funding program called the same (NanoCare) was born and is still running in Germany with many projects funded  and a commercial company (the DuPont company), which commercializes products containing nanomaterialswww.nanoriskframework.com. Briefly, the six steps are outlined below:The NanoRisk framework is composed of six fundamental measures which align with stages of development and is a repetitive process. This framework can be downloaded at the following website address:Step 1. Careful physicochemical characterization of the materialStep 2. Delineate life-cycle(s)2A. Nanomaterial physicochemical properties2B. Nanoparticle toxicity findings2C. Nanoparticle exposuresStep 3. Assess risksStep 4. Evaluation of risk managementStep 5. Decide, document, and actStep 6. Review findings and readjustwww.NanoRiskFramework.com.2 (uf-TiO2) particle type were reported by Warheitet al.15. These hazard assessments were composed by focusing upon general potential routes, including acute lung instillation studies, oral toxicity tests, dermal irritation and sensitization investigations, acute ocular (eye) irritation tests, genetic toxicology assessments, and screening aquatic toxicity guideline studies. Lung bioassay tests were conducted using a well-designed protocol, including dose-response and time-course parameters. The acute dermal irritation tests were carried out using rabbits according to standardized OECD 404 guidelines. The local lymph node assay in mice was performed to investigate skin sensitization (OECD 429 guideline). The acute oral toxicity test was conducted in rats according to a standardized OECD 425 guideline. Anin vitro Ames mutagenicity assay was conducted using a standardized bacterial reverse mutation test (OECD 471) concomitant with anin vitro mammalian chromosome aberration test using Chinese hamster ovary cells (OECD 473). Acute aquatic toxicity assays were carried out to test for potential environmental effects using prototypical aquatic organisms, including rainbow trout (fish), daphnia (invertebrates), and green algae.15  were also evaluated at 3-months post-exposure (i.e. recovery) by clinical and histopathological methods, bronchoalveolar lavage (BAL) analysis, and epithelial cell turnover effects. Cell proliferation (CP) studies with BrdU were conducted to gauge different anatomical compartments of the respiratory tract, including the following anatomical regions of the respiratory tract: terminal bronchiole (TB), alveolar duct (AD), and subpleural regions. The results demonstrated that aerosol exposures of rats to 0.54 (4.9 f/cc), 2.5 (56 f/cc), and 25 (252 f/cc) mg/m3 of VGCFTM-H produced concentration-related small, detectable accumulation of extrapulmonary fibers (outside the respiratory tract) with no adverse tissue effects outside of the lungs. Histopathological observations revealed that at the two highest concentrations tested, minimal (2.5 mg/m3) and slight (25 mg/m3) neutrophilic-based inflammation was observed at the anatomical junctions of the TBs and ADs (known as the AD bifurcations). These were the same anatomical sites wherein fiber-containing lung macrophages had migrated and accumulated. The impact of this exposure at high concentrations was described histologically by accumulations of neutrophilic inflammatory cells and some thickening of interstitial compartments and hypertrophy/hyperplasia of pneumocyte type II epithelial cells and was graded as slight for the rats exposed to the 25 mg/m3 (highest) concentration. Lung lavage fluid and CP endpoint increases versus air-exposed controls were quantified at 25 mg/m3 CNFs but were not different from control values at the 0.54 or 2.5 mg/m3 exposure concentrations. It is noteworthy that greater than 90% of CNF-exposed, lavage-recovered pulmonary macrophages from rats exposed to 25 and 2.5 mg/m3 CNFs had phagocytized CNFs (>60% for 0.54 mg/m3). The percentages of phagocytic macrophages exposed to CNFs recovered in BAL fluids are enumerated in3 CNFs. Alternatively, the low-level pulmonary tissue alterations at this intermediate exposure level of 2.5 mg/m3 can be regarded as relatively normal physiological responses to subchronic inhalation exposures of particulates. The histopathological profile as well as the BAL fluid  and lung parenchymal cell turnover findings were consistently similar at the high-exposure concentration (i.e. 25 mg/m3), but at the intermediate concentration (2.5 mg/m3) there was a lack of compatibility when reconciling the histopathological observation of minor lung inflammation to the more sensitive BAL fluid (both inflammatory and cytotoxicity data) and cell turnover findings  by focusing on four core topics17. The assessment focused on three uptake pathwaysin vivo: the respiratory tract, the gastrointestinal tract, and the dermal pathways, i.e. skin. A fourth and major target was the comparability of findings following inhalation versus intratracheal instillation exposures to better gauge pulmonary exposures.Krug evaluated the scientific literature on nanosafety over a period of 10–15 years (during the period of 2000–2014) by undertaking an assessment of more than 10,000 publications concentrating on either human health effects or biological endpoints in bothThe major findings from this comprehensive evaluation of the scientific literature was that following pulmonary exposures, only a minor fraction of the inhaled or instilled dose translocated from the lung to the pulmonary or systemic circulation and ultimately reached secondary organs within the body. Moreover, the very small fraction of studies that claimed nano-specific effects related to toxicokinetic distribution could not be attributed to so-called nano effects owing to flaws in study design.When comparing particle results of lung instillation studies versus inhalation studies (more relevant route of exposure), the findings of instillation studies were necessarily conducted at high dose overload (bolus) conditions. Krug concluded that these pulmonary inflammation effects following particle deposition were not related to a specific “nano particle” effect but occurred in response to particle exposures, per se.A third aspect noted by Krug was that engineered nanomaterials  can dissolve either slowly or more rapidly in body fluids following exposures. This has often been misinterpreted as a form of nanotoxicity but can relate to either solubility or a more general form of toxicity that is not unique to nanoparticles.A fourth major finding by Krug was that many, if not most, of the published studies did not have adequate physicochemical characteristics of the test nanoparticle—and, in the author’s opinion, this critical shortcoming limits the impact or reliability of the findings—rendering the results “totally meaningless”.2 particles in rats following exposures via three main routes, namely intravenous, intratracheal (pulmonary), and oral exposure (intragastric intubation)20. The investigators used radiolabeled TiO2 nanoparticles which permitted monitoring the overall biodistribution of radiolabeled TiO2 into various tissues over 28 days post-exposure and to implement accurate mass balance estimates. These studies represent “state-of-the-art” approaches to determine the fate and clearance of nanoparticles following various routes of exposure. It was noteworthy that the biodistribution and biokinetics of nanoscale TiO2 exposures following pulmonary and gastrointestinal routes were relatively similar but distinctly different from the intravenous route of administration.In a series of three publications, Kreyling and coworkers studied the biokinetics and translocation or biodistribution of a single dose of nanoscale TiOIn conclusion, this mini-review has focused on select hazard and risk assessment strategies and research efforts that have been proposed or conducted during the past decade with the intention of better estimating potential adverse health effects following nanoparticle exposures. Although some research progress has been made on the “conceptual” and organizational front, the challenges of assessing the health effects of specific nanoparticle types remain a daunting task. Below is a listing of take-home messages that should be considered critical for future research endeavors:Robust and sufficient characterization of the material is an essential requirement before toxicity studies can beginStandardized protocols and guideline studies are necessary for the validation of methods and research resultsRound-robin inter-laboratory studies are necessary for the validation of test methods and toxicity resultsThe funding for establishing protocols and standardizing methodologies should be derived from governmental organizationsIt is necessary to test commercially relevant nanoparticle types (to which humans will be exposed): hazard studies using laboratory-made or exotic-type nanoparticles have little value for determining relevant health effectsThe results of acute-type studies conducted at high doses provide little useful hazard informationin vitro andin vivo studies concomitant with time-course assessments that span acute to chronic exposure durations are critically necessary to gauge the medium- and/or long-term effects of relevant nanoparticle exposuresDose-response characterization at relevant dose levels for bothin vivo effects toin vitro effects; this requires the selection of relevant cell types and cell models that reflect the route of human exposures and should be validated by a variety of research groupsGiven that nanoparticle-based hazard studies will inevitably be transitioned from the use of experimental animals to animal alternatives, it is necessary to develop a more effective relevant protocol to transition fromin vitro toxicity methodologies or cell culture techniques to better simulate longer-term effects, including more realistic dosimetry, repeated dosing schedules, and better particle characterization techniquesDevelopment is required forFrom a regulatory standpoint, more useful definitions for defining nanomaterials are necessary, as the current European Union definition is arbitrary and has little significance with respect to health effects; indeed, any definition needs to be supported by a reliable method of assessmentThe question of whether nanoparticles are more hazardous than fine particles of similar or identical chemistry has not been adequately addressed; this is important because there is a substantial database informing on the toxicity of numerous fine particle types, and this could obviate the need to conduct extensive studies on the nanoparticle forms of fine particles, for which there are significant toxicity data available2 exposures but need to be conducted for many other nanoscale materialsGreater research emphasis should be focused on the biokinetics/toxicokinetics of nanoparticles at relevant human exposure levels when considering the major routes of nanoparticle exposures ; the studies by Kreyling and coworkers have been highlighted for nano TiOThe application of proper benchmark control particles or nanoparticles is necessary to improve the interpretation of toxicological outcomesin vitro methodologies that can be validated for representingin vivo effects in standardized guideline toxicity testing.As discussed throughout this mini-review, a significant amount of planning and effort has been given to the challenge of establishing safety following exposures to nanomaterials and, also acknowledged, some progress has been made regarding the standardization of research protocols and generation of useful data. A number of European FP7-sponsored projects have provided some successful data-generated outcomes for hazard assessments. The NanoCare project has also been very successful in providing useful toxicological data, demonstrating that dermal exposures to nanomaterials are generally safe. Significant concerns should be raised, however, by Krug’s evaluation of more than 10,000 publications, demonstrating significant deficiencies in many toxicological publications. In taking a “snapshot” of the status of nanomaterial safety efforts, it seems clear that numerous challenges yet remain in providing accurate health and environmental decisions on a variety of nanomaterial types. Significant improvements can be made in the near term to advance better information on 1) the various different routes of exposure , 2) better, standardized nanomaterial characterization issues, including a better definition for nanomaterial, that can be scientifically justified, 3) improvements in research methodologies, protocols, and implementation of studies, such as time-course and dose-response issues (because acute high-dose exposure protocols generate meaningless data), and 4) the development of robust


Investigators from the Department of Functional Neurology, Epileptology and Epilepsy Institute (IDEE), and the Lyon’s University Hospital examined the clinical determinants of ADHD severity in children with epilepsy (CWE) along with the response to treatment with methylphenidate (MPH). Investigators from the Department of Functional Neurology, Epileptology and Epilepsy Institute (IDEE), and the Lyon’s University Hospital examined the clinical determinants of ADHD severity in children with epilepsy (CWE) along with the response to treatment with methylphenidate (MPH). A total of 167 patients, aged 6-16, with ADHD and epilepsy entered into a multi-center prospective observational study and were followed over a 12-16-week period. Baseline severity and evolution of ADHD symptoms were assessed using the ADHD Rating Scale-IV. Each subject was assessed for ADHD severity at baseline, response to MPH, and risk of seizures aggravation in those who received MPH. MPH was administered with a dosage of 0.3-1.0mg per kilogram either two to three times per day (short-acting MPH) or once per day (slow-acting MPH). At baseline, 61 out of 167 patients received MPH, whereas the remainder were followed without specific pharmacological intervention. Epilepsy was considered to be well controlled in 91 patients at study entry. Among these patients, 6 demonstrated relapse during follow-up, with 4 being in the MPH treatment group. At study conclusion, 41 subjects treated with MPH and 39 subjects who did not receive pharmacological intervention demonstrated a >25% reduction in ADHD Rating Scale-IV total score. [COMMENTARY. Up to 40% of CWE have comorbid ADHD  and bothSimilar to other studies , 4, thisThis study provides support for efficacy and safety of MPH use in CWE due to statistically significant decrease of ADHD symptoms in 75% of treated subjects. Proportion of seizure relapse was not significantly different between the subjects treated with MPH compared to those who were not. However, neither initiation of MPH or follow up assessment used randomized controlled approach, thus may have been affected by the selection bias amongst other limitations. In addition, there was also a lack of EEG data gathered at baseline or follow-up to assess the subclinical ictal abnormalities and changes during the course of treatment.To further advance this very important question of the specific association of ADHD and epilepsy as well as efficacy and safety of treatment with MPH, we need to have more rigorous evidence obtained from the randomized double blind controlled treatment studies.


Mutations that reduce Kv3 currents limit the firing rate frequency range. Mutations of Kv1 channels act mainly on inhibitory interneurons, generating excessive GABAergic signaling onto PCs, resulting in episodic ataxia. Kv4.3 mutations are responsible for a complex syndrome with several neurologic dysfunctions including ataxia. Mutations of either Cav or BK channels have similar consequences, consisting in a disruption of the firing pattern of PCs, with loss of precision, leading to ataxia. Another category of pathogenic mechanisms of ataxia regards alterations of synaptic signals arriving at the PC. At the parallel fiber (PF)-PC synapse, mutations of glutamate delta-2 (GluD2) or its ligand Crbl1 are responsible for the loss of synaptic contacts, abolishment of long-term depression (LTD) and motor deficits. At the same synapse, a correct function of metabotropic glutamate receptor 1 (mGlu1) receptors is necessary to avoid ataxia. Failure of climbing fiber (CF) maturation and establishment of PC mono-innervation occurs in a great number of mutant mice, including mGlu1 and its transduction pathway, GluD2, semaphorins and their receptors. All these models have in common the alteration of PC output signals, due to a variety of mechanisms affecting incoming synaptic signals or the way they are processed by the repertoire of ionic channels responsible for intrinsic membrane properties. Although the PC is a final common pathway of ataxia, the link between specific firing alterations and neurologic symptoms has not yet been systematically studied and the alterations of the cerebellar contribution to motor signals are still unknown.Purkinje cell (PC) dysfunction or degeneration is the most frequent finding in animal models with ataxic symptoms. Mutations affecting intrinsic membrane properties can lead to ataxia by altering the firing rate of PCs or their firing pattern. However, the relationship between specific firing alterations and motor symptoms is not yet clear, and in some cases PC dysfunction precedes the onset of ataxic signs. Moreover, a great variety of ionic and synaptic mechanisms can affect PC signaling, resulting in different features of motor dysfunction. Mutations affecting Na In this review, we focus on the molecular and cellular mechanisms responsible for the alterations of basic physiological functions of Purkinje cells (PCs), including intrinsic membrane properties and synaptic signaling, commonly observed in animal models of ataxia. The PC output is generated by processing of incoming synaptic signals, which reach the cerebellar cortex by two pathways: the mossy fiber-granule cell-parallel fiber (PF) pathway and the climbing fibers (CFs) contacting directly PCs. Several studies indicate that both systems can be shaped by sensory experience and neuromodulation Ito, , 2016. SThere is plenty of evidence that ion channel mutations affecting intrinsic membrane properties are responsible for numerous forms of ataxia Figure , some ofIn addition to intrinsic membrane properties, several mutations affecting the development and/or function of synaptic circuits, particularly of PF and/or CF to PC synapses, have been revealed in hereditary and acquired forms of ataxia Figure . Such muin vivo spontaneously fire action potentials also in resting conditions and under anesthesia  are very important for setting the threshold for action potential initiation, axonal propagation of action potentials and integration of the synaptic inputs impinging on the cell. Ten genes encoding for α subunits (the pore-forming subunit) of NaV channels have been described  sensitive , which controls NaV channels’ membrane expression and localization by direct interaction with their C-terminal domain , which in other types of neuron is essential for pacemaker activity, in PCs is not required for this function  revealed an ataxic phenotype with alterations of gait parameters in the footprinting test . Furthermore, the injection of stronger depolarizing current was necessary to evoke action potential firing in NaV1.1 homozygous and heterozygous KO mice relative to their wild-type littermates , are associated with cerebellar atrophy and ataxia  carrying a missense mutation  and/or granule cells  disrupts PC high frequency firing, which is rescued by its re-introduction by the dynamic clamp technique  was created by Herson et al.  showed an increased frequency and amplitude of inhibitory postsynaptic potentials (IPSPs), causing a reduced PC firing frequency. Mutations of Kv1 channels, in addition to effects on the axon terminals of basket cells, might also directly alter the dendritic excitability of PCs. In fact, application of a selective Kv1 blocker causes random transient increases of PC firing, driven by an uncontrolled action of dendritic Ca2+ spikes  have a double gating mechanism. In fact, the voltage-dependance curve of their gating is shifted to negative potentials by an increase in intracellular Ca2+ concentration, allowing them to open whenever a depolarization is associated with Ca2+ entry . BK−/− mice exhibited abnormal gait with shorter stride length and irregular step pattern in the footprinting test . PC-BK−/− mice displayed motor alterations similar to those observed in global BK−/− mice although with a reduced severity  are responsible for Ca2+ entry in the cell in response to membrane depolarization. There are different subclasses of CaV channels, which exert important roles in different cell types  is widely expressed in the brain , accounting for more than 90% of Ca2+ currents, with the remaining fraction due to CaV1 (L type) channels , so that only the first few events undergo summation with a marked suppression of subsequent summation , and to spinocerebellar ataxia type 6 . SCA684Q mice developed motor coordination deficits in adult age , or classic ataxia following introduction of CAG repeats (SCA6). The different functional alteration of the channel might be responsible for the specific phenotype of the disease. It is interesting to note that ataxia due to Cav2.1 mutation causing reduced CaIn the cerebellum, the delta-2 receptor (GluD2) is expressed post-synaptically on PCs at the PF-PC synapse  semi-dominant point mutation  autosomal recessive mutations . In fact, GluD2−/− mice have an impaired PF synaptogenesis, with a reduction in the number of spines between PF-PC, and an increase of free spines ; IP3 induces Ca2+ release from intracellular stores  expansion in the ataxin-1 protein  stores in PCs  before the onset of overt ataxia and PC morphological alterations  as well as staggerer mice (with the mutation in the gene encoding ROR-α), while the AMPA receptor-mediated synaptic transmission was still preserved in PCs of both phenotypes  or evoked responses to electrical or optogenetic stimulation . This fact imposes serious limitations to the understanding of the mechanisms of motor control in ataxia. Future studies concerning the alterations of cerebellar output signals during the performance of motor tasks would give better insight into the specific mechanisms of motor control failure in the different types of ataxia.MCM and FT designed the outline of the article. EH, IB, MCM and FT wrote the manuscript. EH and IB prepared the figures.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.