ACTA2, FBN1, and TGFBR2 genes. Moreover, in 8 patients with BAV and thoracic aortic dilation, not fulfilling the clinical criteria for MFS, FBN1 mutations in 2/8 patients were identified suggesting that FBN1 or other genes involved in syndromic conditions correlated to aortopathy could be involved in BAV. Beyond loci associated to syndromic disorders, studies in humans and animal models evidenced/suggested the role of further genes in non-syndromic BAV. The transcriptional regulator NOTCH1 has been associated with the development and acceleration of calcium deposition. Genome wide marker-based linkage analysis demonstrated a linkage of BAV to loci on chromosomes 18, 5, and 13q. Recently, a role for GATA4/5 in aortic valve morphogenesis and endocardial cell differentiation has been reported. BAV has also been associated with a reduced UFD1L gene expression or involvement of a locus containing AXIN1/PDIA2. Much remains to be understood about the genetics of BAV. In the last years, high-throughput sequencing technologies, allowing the analysis of large number of genes or entire exomes or genomes, progressively became available. The latter issue together with the development of “BigData” analysis methods improving their interpretation and integration with clinical data represents a promising opportunity to increase the disease knowledge and diagnosis in monogenic and multifactorial complex traits. This review summarized the main knowledge on the BAV genetic bases, the role of genetic diagnosis in BAV patient managements and the crucial challenges for the comprehension of genetics of BAV in research and diagnosis.Bicuspid aortic valve (BAV) is a common  congenital heart defect with increased prevalence of aortic dilatation and dissection. BAV has an autosomal dominant inheritance with reduced penetrance and variable expressivity. BAV has been described as an isolated trait or associated with syndromic conditions . Identification of a syndromic condition in a BAV patient is clinically relevant to personalize aortic surgery indication. A 4-fold increase in BAV prevalence in a large cohort of unrelated MFS patients with respect to general population was reported, as well as in LDS patients (8-fold). It is also known that BAV is more frequent in patients with thoracic aortic aneurysm (TAA) related to mutations in  Bicuspid aortic valve (BAV) represents a common congenital heart defect   mutations in patients with BAV and aortic dilation/aneurysm in whom MFS and other more severe type 1 fibrillinopathies were clinically excluded  and TGFBR2  have been described in MFS-like conditions and have been consistently associated with LDS  mutations have been firstly associated with aortic valve abnormalities, such as aortic valve calcium deposition, suggesting their potential role in cardiac disease in humans. These studies suggested that mutations in this gene, identified in a small number of families, may represent the genetic basis for hypoplastic left heart syndrome in some patients  allowed the identification of 14 NOTCH1 mutations , 10 out of 11 families and 1 out of 3 isolated cases showed BAV ] than among sporadic forms [3/280 (1%)] of LS-CHD. The Notch signaling pathway is highly conserved across species. NOTCH1 encodes a large protein containing an extracellular domain with 36 tandem epidermal growth factor (EGF)-like repeats and three cysteine-rich Notch/LIN-12 repeats, an intracellular domain with six ankyrin repeats, and a transactivation domain . EMT, occurring in the developing cardiac valves, represents an important process determining the transition from primordial to mature valves , which partly stall the progression of aortic valve calcification  allowed to deepen the knowledge concerning the genetic bases of the disease, thus evidencing a role for other candidate loci not previously associated with syndromic BAV. Nos3 pathway has been implicated as a regulator of BAV formation in animal models. Nos3 is expressed in endocardial cells of the heart and is shear-stress-dependent. Nos3−/−mice were reported to carry CHD in early studies  as possible regulator in BAV formation was also supported by other studies showing a significant reduction of its expression in patients with BAV .Besides Notch, which appears to be critical for normal tricuspid formation, NOTCH1, GATA5 has been linked to BAV in humans as a number of studies reported several rare sequence variants of this gene in BAV and its associated aortopathy  that completely abolished its interaction with GATA5 was found to segregate with disease in a family with BAV and a small proportion (11%) of mice carrying cardiac homeobox Nkx2-5 haploinsufficiency  and GATA6  were observed in human congenital heart defects , whose expression has been demonstrated to be down-regulated in BAV patients. Its product is a component of a multi-enzyme complex involved in the degradation of ubiquitin fusion proteins during embryogenesis, with an important role in the development of ectoderm-derived structures; it has been observed to be diminished in BAV patients  in a man with BAV, aortic valve stenosis, and coarctation and calcification of the aorta was identified  in a replication cohort consisting of 346 additional probands with a broad range of cardiovascular malformation phenotypes revealed another missense mutation (p.Pro415Leu) in an infant with BAV and moderate aortic stenosis. Gene network analysis identified haplotypes for ENG , a gene known to be important in heart valve formation, and for AXIN1  and PDIA2  to be associated with BAV in a cohort of 68 probands  has been performed on 48 BAV patients  were identified in 7 family members with aortic aneurysms and dissection, of whom 3 had BAVs, their aortic tissue displaying increased proteoglycans accumulation, fragmentation, loss of elastic fibers, and decreased numbers of smooth muscle cells, consistent with aortic wall degeneration. These findings could lead to the hypothesis of a unique pathogenetic basis for BAV-TAA patients (as BAV has been observed to occur more frequently in patients with TAA who have mutations in his gene)  not previously associated with human BAV  approaches on nine genes previously associated with BAV  investigating a wide number of genetic variants in genes supposed to be associated with the disease added a further support in identifying novel candidate loci  sequencing principle. In this approach clonal template populations are generated after sample DNA fragmentation followed by ligation with an oligonucleotide adaptor, complementary to an oligonucleotide fragment immobilized on the surface of a capture bead, and emulsion PCR (emPCR), carried out in aqueous droplets  platform  approach  is shared by a large number of instruments, ranging from lower throughput benchtop units, such as MiniSeq (<7.5 Gb) or MiSeq v3 (<15 Gb), to ultra-high-throughput instruments, such as HiSeq × (800–900 Gb per flow cell) , thus offering a better efficiency in providing information concerning complex /repetitive DNA regions. Nevertheless, although their overall error higher rate in non-homopolymeric DNA regions could be considered comparable with that of other platforms, a higher prevalence of false positives in insertion/deletion (indel) variants detections is more commonly observed  sequencing approach, in which the generation of a clonally amplified DNA fragments population is no longer required in the sequencing protocol. The most widely adopted SMRT approach is that developed by Pacific Biosciences , at whose transparent bottom a polymerase is fixed, thus allowing labeled dNTP incorporation on each single molecule template. After the fluorescence signal is recorded by the instrument imaging system, the enzyme removes the fluorophore from the nucleotide and permits the next labeled dNTP to be added. Of interest, the present approach uses a unique circular DNA template, thus allowing to consecutively sequence the same DNA molecule several times  developed a handheld sequencer based on nanopore sequencing technology, the MinION system Table . Data frde novo genomes' assembly and in exploration of structural variants by nanopore technology application  with a strong pathogenetic effect, in the large part of BAV patients the disease is likely to have a multifactorial nature. The latter condition is the result of complex interactions among genetic alterations , hemodynamic shear stress , and other environmental and stochastic factors. To further complicate the pathogenic complexity, BAV often accompanies syndromic disorders, such as MFS or LDS thus being difficult to distinguish whether the pathogenetic variants determining the syndromic picture are also responsible for BAV or they represent two different pathogenetic conditions due to genetic variants in different genes , and make decisions on the best options and timing for aortic surgery  as well as on global patients management.All the Authors substantially contributed to (1) the conception or design of the work, acquisition a revision of literature data; (2) drafting the work or revising it critically for important intellectual content; (3) final approval of the version to be published; AND All Authors agree to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


We have recently identified the expression levels of Sequestosome1/SQSTM1 (p62) and activating molecule in Beclin 1-regulated autophagy protein 1 (AMBRA1) as novel independent prognostic biomarkers for early stage melanomas. While increasing followed by subsequent decreasing levels of p62 expression reflects the paradoxical role of autophagy in melanoma, expression levels additionally define a novel prognostic biomarker for AJCC stage II tumors. Conversely, loss of AMBRA1 in the epidermis overlying primary melanomas defines a novel prognostic biomarker for AJCC stage I tumors. Collectively, the definition of AMBRA1 and p62 as prognostic biomarkers for early stage melanomas provides novel and accurate means through which to identify tumors at risk of disease progression, facilitating earlier patient therapeutic intervention and stratification tools for novel personalized therapeutic approaches to improve clinical outcome.Prognosis and survival for malignant melanoma is highly dependent on early diagnosis and treatment. While the American Joint Committee on Cancer (AJCC) criterion provides a means of staging melanomas and guiding treatment approaches, it is unable to identify the risk of disease progression of early stage tumors or provide reliable stratification for novel adjuvant therapies. The demand for credible prognostic/companion biomarkers able to identify high-risk melanoma subgroups as well as guide more effective personalized/precision-based therapy is therefore of paramount importance. Autophagy, the principle lysosomal-mediated process for the degradation/recycling of cellular debris, is a hot topic in cancer medicine, and observations of its deregulation in melanoma have brought its potential as a prognostic biomarker to the forefront of current research. Key regulatory proteins, including Atg8/microtubule-associated light chain 3 (LC3) and BECN1 (Beclin 1), have been proposed as potential prognostic biomarkers. However, given the dynamic nature of autophagy, their expression  Malignant melanoma, the most aggressive form of skin cancer arising from the malignant transformation of melanocytes, is an increasing public health concern worldwide with incidence rates doubling every 10–20 years  2009 staging criterion, the most comprehensive staging system for melanoma to date, which remains the international standard for disease staging and as a guide for treatment approaches. AJCC staging combines several prognostic factors for melanoma, including the depth of invasion (Breslow depth), rate of mitoses, presence of ulceration (loss of the epidermis overlying the tumor), evidence of metastatic spread, and changes in serum lactate dehydrogenase (LDH) to allow risk stratification of morbidity and mortality at the initial diagnosis . In geneWhile early stage melanoma is largely curative by surgical excision, metastatic disease represents the cause of death from melanoma in the vast majority of cases due to a lack of consistently beneficial treatment regimens for late stage disease. Furthermore, despite its comprehensiveness, AJCC staging as a prognostic biomarker is limited by the inability of its criteria to accurately identify high-risk melanoma subgroups that will go on to progress; a particular problem in seemingly “low risk” AJCC stage I melanomas where up to 10% of tumors subsequently metastasize. This emphasizes the urgent need for novel credible biomarkers to identify high-risk tumors as well as the stratification of such patients for more efficacious and earlier therapeutic approaches .Observations of deregulated autophagy in many cancers, including melanoma, have brought this key signaling mechanism to the forefront of much research , 8, inclin vitro, autophagy is activated in line with its pro-survival role; however, when autophagy is inhibited exogenously, this leads to increased cell death /extracellular signal-regulated kinase (ERK) cellular growth pathway . Activatll death , 18. Conll death , 18, 19.To date, several markers of autophagy, including LC3 and Beclin 1, have been identified as potential prognostic biomarkers for melanoma. Under normal homeostatic conditions, exogenous LC3 is cytoplasmic but upon autophagy induction becomes conjugated to phosphatidylethanolamine (PE) to form the membrane bound form LC3-II, thus acting as a marker of autophagy induction . Increasin vivo, an important concept in view of emerging autophagy modulator therapies  is a component of the Beclin 1/VPS34 complex and involved in the formation of PI3K rich membranes during the nucleation phase of autophagy . Again,Since the current and universally adopted AJCC staging system is unable to identify the risk of disease progression in seemingly “low risk” early stage melanomas, such tumors are only identified after the onset of metastatic disease progression, at which point treatment options are limited and frequently ineffective. Critically, identifying, refining, and validating prognostic biomarkers for early stage melanomas such as the proposed biomarkers of autophagy will thus enable the identification of high-risk tumor subgroups. Both p62 and AMBRA1 expression exemplify how autophagy can be harnessed as prognostic biomarkers for melanoma, each providing clinically relevant information over and above AJCC staging, which in particular will be useful for refining the risk of melanoma progression in patients with AJCC stage I or II melanomas. Ultimately, further validation of these biomarkers will allow application in a clinical context, facilitating both earlier therapeutic intervention and the refinement of personalized therapies for malignant melanoma to improve clinical outcome and the prevention of premature loss of life.DT: main author of the paper and corresponding author involved in all aspects of authorship. RE: substantial contributions to the acquisition, analysis, and interpretation of data for the work including production of figures for the paper. PL: senior author, contribution to the analysis of data and the writing of the manuscript; accountable for all aspects of the work. All the authors have undertaken final approval of the final manuscript version to be published.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.


The ability of astrocytes to rapidly clear synaptic glutamate and purposefully release the excitatory transmitter is critical in the functioning of synapses and neuronal circuits. Dysfunctions of these homeostatic functions have been implicated in the pathology of brain disorders such as mesial temporal lobe epilepsy. However, the reasons for these dysfunctions are not clear from experimental data and computational models have been developed to provide further understanding of the implications of glutamate clearance from the extracellular space, as a result of EAAT2 downregulation: although they only partially account for the glutamate clearance process. In this work, we develop an explicit model of the astrocytic glutamate transporters, providing a more complete description of the glutamate chemical potential across the astrocytic membrane and its contribution to glutamate transporter driving force based on thermodynamic principles and experimental data. Analysis of our model demonstrates that increased astrocytic glutamate content due to glutamine synthetase downregulation also results in increased postsynaptic quantal size due to gliotransmission. Moreover, the proposed model demonstrates that increased astrocytic glutamate could prolong the time course of glutamate in the synaptic cleft and enhances astrocyte-induced slow inward currents, causing a disruption to the clarity of synaptic signalling and the occurrence of intervals of higher frequency postsynaptic firing. Overall, our work distilled the necessity of a low astrocytic glutamate concentration for reliable synaptic transmission of information and the possible implications of enhanced glutamate levels as in epilepsy. The role of astrocytes in the excitability and hyperexcitability of neurons is a subject which has gained a lot of attention, particularly in the pathology of neurological disorders including epilepsy. Although not completely understood, the control of glutamate homeostasis is believed to play a role in paroxysmal neuronal hyperexcitability known to precede seizure activity. We have developed a computational model which explores two of the astrocytic homeostatic mechanisms, namely glutamate clearance and gliotransmission, and connect them with a common controlling factor, astrocytic cytoplasmic glutamate concentration. In our model simulations we demonstrate both a slower clearance rate of synaptic glutamate and enhanced astrocytic glutamate release where cytoplasmic glutamate is elevated, both of which contribute to high frequency neuronal firing and conditions for seizure generation. We also describe a viable role for astrocytes as a “high pass” filter, where astrocytic activation in the form of intracellular calcium oscillations is possible for only a certain range of presynaptic neuronal firing rates, the lower bound of the range being reduced where astrocytic glutamate is elevated. In physiological terms this perhaps indicates not only neuronal but also astrocytic glutamate-mediated excitation in the neural-astrocytic network. Glutamate is the most abundant excitatory neurotransmitter in the brain  and due Astrocytes perform the role of glutamate homeostatic maintenance through a combination of glutamate clearance by excitatory amino-acid transporter EAAT2 (GLT-1)  and rapi2+ elevation in response to presynaptic neuronal firing; the astrocytic Ca2+ elevation stimulating the release of astrocytic glutamate ; thnsmitter  and synansmitter . The connsmitter –31, and nsmitter –36. Thesnsmitter ,26,37. Insmitter –36 descrnsmitter , an inhinsmitter  or alter+) concentration. We incorporate our EAAT2 model within a modelling framework of gliotransmission described by syn, [Glu-]syn, [K+]syn and [H+]syn are the synaptic (extracellular) concentrations and [Na+]ast, [Glu-]ast, [K+]ast and [H+]ast are astrocytic (intracellular) concentrations of Na+, Glu-, K+, H+, respectively, R is the universal gas constant, F is Faraday’s constant and T is the temperature. All parameter values used are given in the Methods section and EAAT model derivation described in supplementary material This model was fitted to experimental data  to calcuribed by , 39Vrev-). The current density can be converted to an ionic gradient-supported rate of uptake, VEAAT, with the equation:ast describes the astrocytic membrane surface area at the synapse  of 1.5mM, 5mM and 10mM.The results of model simulation are described in two different time-scales: the first is the slower astrocytic response to the presynaptic stimulation and subsequent glutamate release and the second is the much faster post-synaptic neuronal response to astrocytic activation. For each simulation three cases are considered, reflecting three basal astrocytic glutamate concentrations  into the astrocytic cytoplasm both in the soma and perisynaptic process  glutamate and Naand 10mM . Upon cland 10mM , inset. + and synaptic K+ concentrations  and membrane potential (Vast) across the three conditions. The concentrations and Vast were determined not only by EAAT2-mediated currents, but by the ubiquitous sodium-potassium pump (NaK-ATPase), sodium-calcium exchanger (NCX) and concentration-balancing membrane leak currents. In analysing the concentration dynamics  pathway. In turn, the presence of IP3 allows a release of Ca2+ from ER stores in the astrocytic soma, therefore initiating a [Ca2+] elevation. The large flux of [Ca2+] from ER stores have been seen to affect concentrations in the far processes of the astrocyte, therefore we model each compartment (soma and process) individually. The model used for IP3 and Ca2+ dynamics at the soma utilised a proposed mixture of amplitude and frequency modulation (AFM)  as is likely in a physiological context  activation where the time course of glutamate affects the astrocytic response to lower presynaptic firing stimulation where [Glu]ast,eq is higher. Thus the system behaves as a high pass filter for astrocytic activation, possibly reflecting not only a hyperexcitable neuronal response to prolonged time course of glutamate in the synaptic cleft, but also excessive astrocytic activation, an effect which is far-reaching within the brain by means of the astrocytic network.In this paper we have proposed a model for glial-neuronal communications which accounts for some of the physiological conditions observed in MTLE: increased extracellular glutamate and non-neuronal provoked intervals of rapid postsynaptic firing. The model presented illustrates both the rate of glutamate uptake from the synaptic cleft following presynaptic release and the concentration of astrocytic-released glutamate by gliotransmission as a function of intracellular astrocytic glutamate concentration. It is likely that this fluctuation of uptake rate occurs in the functional brain as a result of transient ionic perturbations. However, following the downregulation of enzyme activity GS, as in MTLE, there would follow a higher basal concentration of astrocytic glutamate and therefore the EAAT function would be compromised. We have illustrated the effects of the altered synaptic glutamate clearance for both neuronal and astrocytic signalling and report changes to the postsynaptic firing activity as a result. The model also takes into account enhanced gliotransmission for postsynaptic neuronal firing rates and predicts SIC-mediated intervals of higher frequency (up to 65 Hz) firing where the astrocyte-release content was increased. We also reported that lasting synaptic glutamate affects mGluRs-mediated astrocytic  (μM) described below and all other parameters are enumerated in In these equations 3 in the astrocytic cytoplasm allows the opening of Ca2+ channels located on the ER, which allows an influx of Ca2+ into the cytoplasm. Similar to  increases past a threshold Cθ concentration (0.5 μM), glutamate is released by the astrocytic into the extra-synaptic compartment. Here we implement a model ast,eq.Stability diagram of astrocytic Na(TIF)Click here for additional data file.S1 TableList of values for the parameters used in the model simulations.(DOCX)Click here for additional data file.


With nearly 42 million mild traumatic brain injuries (mTBIs) occurring worldwide every year, understanding the factors that may adversely influence recovery after mTBI is important for developing guidelines in mTBI management. Extensive clinical evidence exists documenting the detrimental effects of elevated temperature levels on recovery after moderate to severe TBI. However, whether elevated temperature alters recovery after mTBI or concussion is an active area of investigation. Individuals engaged in exercise and competitive sports regularly experience body and brain temperature increases to hyperthermic levels and these temperature increases are prolonged in hot and humid ambient environments. Thus, there is a strong potential for hyperthermia to alter recovery after mTBI in a subset of individuals at risk for mTBI. Preclinical mTBI studies have found that elevating brain temperature to 39°C before mTBI significantly increases neuronal death within the cortex and hippocampus and also worsens cognitive deficits. This review summarizes the pathology and behavioral problems of mTBI that are exacerbated by hyperthermia and discusses whether hyperthermia is a variable that should be considered after concussion and mTBI. Finally, underlying pathophysiological mechanisms responsible for hyperthermia-induced altered responses to mTBI and potential gender considerations are discussed. This public health problem is underestimated because an estimated 50% of people with a concussion do not seek medical attention2. This places the annual incidence of mTBI and concussion at about 42 million people worldwide3. The current definition of concussion as recommended by the 4th International Conference on Concussion in Sport in 2012 is “concussion is a brain injury and is defined as a complex pathophysiological process affecting the brain, induced by biomechanical forces”4. This dysfunction is mediated by a cascade of pathophysiological responses to the injury that ultimately alters cerebral function without necessarily causing concurrent macrostructural damage4. For the purposes of this review, we will use concussion and mTBI synonymously, as a non-penetrating head injury that alters brain functioning with no or brief loss of consciousness5. Other than management of symptoms, the patient with mTBI is typically recommended to rest until symptoms resolve, followed by a gradual increase in return to normal activity6. Concussions and mTBIs are a significant clinical problem yet lack effective evidence-based therapeutics2.Mild traumatic brain injuries (mTBIs) and concussions occur in about 100 to 300 people per 100,000 annually worldwide7. However, several studies have demonstrated that mTBI may also result in chronic cognitive impairments lasting for weeks after injury9. Additionally, many have more chronic physical symptoms such as headaches, vestibular issues, irritability, depression, and fatigue, collectively referred to as post-concussion syndrome12. Although there is some variability in the recovery trajectory, for 50%–60% of individuals with mTBI, symptoms will typically resolve in 7 to 10 days8. However, imaging and more objective clinical assessments indicate that abnormalities in the brain may persist beyond 30 days after mTBI13.Concussions and mTBIs can result in significant cognitive, psychosocial, and physical issues, although many of these symptoms resolve rapidly. Clinical assessments of learning and memory ability have revealed that a single concussion can result in significant impairments in working memory, attention and concentration issues, and decreased processing and reaction time15. If the patient is hypothermic, outcome is improved, but if the patient is hyperthermic, outcome is greatly worsened. However, hyperthermia has not been typically considered to be an important variable in mTBI studies despite the potential co-occurrence of hyperthermia with mTBI.An aspect of mTBI that has been understudied and may affect recovery is the effect of temperature. It is well known in severe and moderate TBI that brain temperature has a profound effect on outcome18. Exercise-induced hyperthermia slowly recovers once an athlete has stopped exercising20. If core temperature has risen to 39–40°C, this temperature naturally returns to normothermic levels within 60 minutes to 3 hours after exercise has ceased21. The brain in particular is slow to cool, and published studies have found that after 1 hour of bicycle exercise, the jugular venous blood, a close approximation of brain temperature, is still significantly hyperthermic even 60 minutes after exercise has ceased  both pre- and post-injury resulted in a significant exacerbation of pathology , which is released from injured astrocytes, and ubiquitin C-terminal hydrolase L1 (UCH-L1)42. However, both are limited in utility because of the relatively short half-life of the biomarkers. Other promising biomarkers that are being evaluated for their diagnostic utility for mTBI include auto-antibody to GFAP, auto-antibody to S100β, tau, select microRNAs (miRNAs), and plasma soluble cellular prion protein47. Preclinical and clinical studies have demonstrated that several of these biomarkers are sensitive to therapeutic hypothermia in moderate to severe TBI. In particular, GFAP and UCH-L1 are downregulated after therapeutic hypothermia in preclinical models of moderate TBI49. Therapeutic hypothermia decreases levels of GFAP in adult patients with severe TBI but not in pediatric patients with TBI51. However, both GFAP and UCH-L1 are limited in utility for mTBI because of the relatively short half-life of these biomarkers. Other potential biomarkers that are temperature-sensitive in moderate to severe TBI include neuron-specific enolase, S-100, brain-specific creatine kinase, and several miRNAs54. Whether they are regulated by temperature after concussion or mTBI remains to be established. Pro-inflammatory cytokines are highly sensitive to temperature manipulations after TBI, including hyperthermia59. However, cytokines are limited in utility for mTBI since mTBI often can be complicated by systemic injuries and these biomarkers do not differentiate well between brain-derived versus systemically derived sources. Biomarkers known to be sensitive to temperature in other studies include MMP-9 and HSP70 and merit study for mTBI63.The failure of multiple phase 3 clinical trials for TBI has directed National Institutes of Health and US Department of Defense working groups to conclude that non-invasive measures of therapeutic efficacy need to be developed to demonstrate that the therapy is engaging its proposed molecular target(s) and resulting in a biological effect66. In one study, female rats after moderate fluid-percussion injury (FPI) demonstrated significantly smaller contusion volumes compared with male rats66. In that study following ovariectomy, contusion volumes in females were not significantly different than those in males. These and other data emphasize the importance of estrogen and progesterone in the pathogenesis of TBI64. Recent studies have also demonstrated that alterations in post-traumatic temperature have gender-specific effects on outcomes after moderate TBI. For example, Suzuki and colleagues67 reported that while post-traumatic hypothermia following moderate FPI provided neuroprotection, no significant effect on contusion volume was seen in female rats. Thus, there appear to be gender-specific effects of temperature manipulations in preclinical models of brain injury.The importance of gender on traumatic and ischemic outcome has been documented in various models of brain injury66 reported that an induced period of mild hyperthermia after moderate TBI increased contusion volumes, cortical neuronal cell death, and axonal damage in intact female rats. Interestingly, in that study, the effects of post-traumatic hyperthermia were more pronounced in ovariectomized animals. These results therefore emphasize that hyperthermia worsens outcomes after moderate TBI in female rats and that neural hormones may protect against secondary hyperthermic insults. Future studies are required to determine whether gender is also an important factor in the pathogenesis of mTBI or concussion and the detrimental effects of hyperthermia on functional outcomes.In reference to the present discussion, more recent studies have evaluated the effects of post-traumatic hyperthermia in models of moderate TBI. In this regard, Suzuki and colleagues68. In regard to post-traumatic hypothermia, numerous reports have shown that lowering brain temperature after a moderate to severe TBI reduces excitotoxicity, free radical generation, apoptosis, and neuroinflammation71. In the area of inflammation, post-traumatic hypothermia after moderate FPI reduces blood-brain barrier permeability to both large and small tracers and the infiltration of CD68-positive cells72. Other studies have also shown that whereas hypothermia reduced the accumulation of polymorphonuclear leukocyte infiltration after TBI, post-traumatic hyperthermia increased inflammatory cell accumulation73. These studies are also in agreement with post-traumatic temperature modifications 74.The pathophysiology of TBI is complex and involves multiple injury cascades that have been reported to be temperature-sensitive75. In that study, temperature-sensitive effects on the various subsets of pro-inflammatory (M1) and anti-inflammatory (M2) microglia and macrophages were determined by using flow cytometry and reverse transcription–polymerase chain reaction. This study provided a link between temperature-sensitive alterations in macrophage/microglia activation and polarization toward an M2 phenotype with hypothermia that could be permissive for cell survival and repair. Studies to determine whether mild hyperthermic TBI also alters microglial and macrophage polarization are under way. Taken together, these studies emphasize the importance of post-traumatic temperature on pro-inflammatory signaling after brain injury.Most recently, the effects of post-traumatic hypothermia on microglial and macrophage phenotype polarization have been investigated after moderate TBI78. Previous studies have reported the importance of post-traumatic temperature in the frequency of damaged axons by using a variety of immunocytochemical techniques such as beta-amyloid precursor protein (β-APP). For example, after moderate TBI, hypothermia reduced the frequency of axonal damage at early post-traumatic time periods in several animal models79. In reference to the effects of hyperthermia on DAI, Suzuki and colleagues66 reported that post-traumatic hyperthermia after moderate TBI significantly increased axonal damage as indicated by β-APP in both intact and ovariectomized female rats. Following mTBI or concussion, evidence for white matter perturbations has been reported in animal models as well as clinical investigations82. Future studies are required to determine the effects of hyperthermia on DAI in models of mTBI and concussion.Diffuse axonal injury (DAI) is a common consequence of mild and moderate TBI and is considered to be significantly related to the resulting functional deficit84. Whether temperature at the time of injury alters this prevalence rate has yet to be studied. Interestingly, in an animal model of mTBI, core-temperature regulation was found to be disrupted in the post-injury recovery period during exercise85. Understanding the interaction of pituitary dysfunction and temperature at the time of mTBI may facilitate clinical guidance in identifying which patients with mTBI need to be screened for potential pituitary dysfunction.An important cause of persistent behavioral problems after mTBI is pituitary dysfunction. Hypopituitarism has a prevalence rate of 25%–50% of patients with TBI overall and was found to occur in 37.5% of patients with mTBImTBI or concussion is a serious and fairly common medical problem that can affect all age groups and produce both short- and long-term consequences. Because of the high incidence of concussion in athletes and military personnel, a great appreciation of the detrimental effects of single and multiple concussions has led to more research on this particular type of brain injury. Recent studies have implicated gray and white matter pathologies that result in a spectrum of neurological symptoms. Complicated concussions that lead to long-term disturbances in memory function or other problems require patients to undergo extensive rehabilitation strategies to retrain the nervous system. In parallel, new treatment strategies that target selective neurotransmitter systems and secondary injury mechanisms are being tested in animal models and in some clinical studies.It now appears from experimental and clinical work that small alterations in brain temperature at the time of an mTBI or concussion may be among several factors that can affect the brain’s response to injury through altering secondary injury mechanisms. Therefore, mild levels of hyperthermia that can occur in people during periods of strenuous activity or exercise may be considered a risk factor for more severe or long-term functional problems. In regard to potential treatment strategies, normalizing brain temperature after a hyperthermic brain injury appears to reduce the degree of structural damage and reduce behavioral outcomes. In this regard, new technologies are being used to develop cooling helmets to reduce brain temperature in acute injury settings such as brain trauma. Also, established targeted temperature management approaches currently being used in severely injured patients should be considered for individuals with milder brain injuries. In the future, new pharmacological strategies that target specific injury cascades that have been shown to be aggravated with elevated temperature need to be identified and tested in clinically relevant concussion models. In this regard, it might be reasonable to consider first testing US Food and Drug Administration–approved drugs that have shown some promise in clinical studies and trials for severe TBI. Only through continued research will new prevention and treatment strategies be discovered and tested to minimize the potentially devastating consequences of mTBI and concussion.


Coronary artery disease (CAD) is a major cause of morbidity and mortality worldwide. Coronary artery bypass graft (CABG) surgery is the revascularisation strategy of choice in patients with diabetes mellitus and complex CAD. Owing to a number of factors, including the ageing population, the increased complexity of CAD being treated, concomitant valve and aortic surgery, and multiple comorbidities, higher-risk patients are being operated on, the result of which is an increased risk of sustaining perioperative myocardial injury (PMI) and poorer clinical outcomes. As such, new treatment strategies are required to protect the heart against PMI and improve clinical outcomes following cardiac surgery. In this regard, the heart can be endogenously protected from PMI by subjecting the myocardium to one or more brief cycles of ischaemia and reperfusion, a strategy called “ischaemic conditioning”. However, this requires an intervention applied directly to the heart, which may be challenging to apply in the clinical setting. In this regard, the strategy of remote ischaemic conditioning (RIC) may be more attractive, as it allows the endogenous cardioprotective strategy to be applied away from the heart to the arm or leg by simply inflating and deflating a cuff on the upper arm or thigh to induce one or more brief cycles of ischaemia and reperfusion (termed “limb RIC”). Although a number of small clinical studies have demonstrated less PMI with limb RIC following cardiac surgery, three recently published large multicentre randomised clinical trials found no beneficial effects on short-term or long-term clinical outcomes, questioning the role of limb RIC in the setting of cardiac surgery. In this article, we review ischaemic conditioning as a therapeutic strategy for endogenous cardioprotection in patients undergoing cardiac surgery and discuss the potential reasons for the failure of limb RIC to improve clinical outcomes in this setting. Crucially, limb RIC still has the therapeutic potential to protect the heart in other clinical settings, such as acute myocardial infarction, and it may also protect other organs against acute ischaemia/reperfusion injury . Although advances in surgical and cardioprotection techniques have resulted in improved clinical outcomes following CABG surgery, changes in patient demographics have meant that higher-risk patients are now undergoing CABG surgery, with an increased risk of perioperative myocardial injury (PMI), which is detected by the release of serum cardiac biomarkers such as CK-MB, troponin I, and troponin T, and a higher operative mortality risk of 5–6%3. These changes include (a) the ageing population , (b) the presence of co-morbidities such as diabetes and hypertension , (c) more complex CAD is being operated on, and (d) concomitant valve and aortic surgery. Therefore, new treatment strategies are required to protect the heart from PMI during cardiac surgery in order to improve clinical outcomes in these higher-risk patients5. In this regard, the endogenous cardioprotective phenomenon of ischaemic conditioning has been investigated as a treatment strategy for protecting the heart and improving clinical outcomes in patients undergoing cardiac surgery.Coronary artery disease (CAD) remains a leading cause of morbidity and mortality worldwide. For patients with complex multi-vessel CAD, coronary artery bypass graft (CABG) surgery is the revascularisation strategy of choice, as it offers survival advantage when compared to multi-vessel percutaneous coronary intervention (PCI)8. The concept of IPC was first discovered in a seminal study by Murryet al. in 19866, when they made the surprising observation that four 5-minute episodes of regional myocardial ischaemia and reperfusion could dramatically reduce myocardial infarct (MI) size following a lethal period of ischaemia. IPC has since been reported to exist in every species and organ tested9. The IPC stimulus elicits two windows of cardioprotection: the first one  begins immediately following the IPC stimulus and lasts for 2–3 hours6, and the second one (termed the “second window of protection” or SWOP and first described in 199311) appears 12–24 hours after the IPC stimulus and lasts for 48–72 hours. The mechanisms underlying classical IPC have been extensively investigated, are complex, and involve the activation of plasma membrane receptors , the recruitment of a number of signal transduction pathways , the inhibition of mitochondrial permeability transition pore (MPTP) opening25, and the prevention of necrotic and apoptotic cell death. The delayed cardioprotective effect of the SWOP has been shown to be mediated by the transcription of several new proteins such as inducible nitric oxide synthase, heat shock proteins, and cyclo-oxygenase-226.The myocardium possesses an innate ability to protect itself from the detrimental effects of acute ischaemia/reperfusion injury (IRI). This can be harnessed by subjecting the heart to one or more non-lethal cycles of brief (5–10 minutes) ischaemia and reperfusion, a phenomenon that has been termed “ischaemic preconditioning” (IPC)et al. in 200327 found that applying three 30-second cycles of ischaemia and reperfusion to the canine heart at the onset of reperfusion following a period of index ischaemia reduced MI size to a level on a par with IPC, a phenomenon that was termed “ischaemic postconditioning” (IPost) and that has provided a therapeutic strategy to protect the heart following AMI28. The signalling pathways underlying IPost are similar to classical IPC, although there are some differences31.One major disadvantage of IPC is the need to apply the stimulus prior to the index ischaemic insult, which is not possible in acute myocardial infarction (AMI). In this regard, Zhaoet al.32 made the intriguing discovery that applying the IPC stimulus (four 5-minute cycles of ischaemia and reperfusion) to the circumflex coronary artery could reduce MI size following a sustained occlusion of the left anterior descending coronary artery, demonstrating that the protection elicited by ischaemic conditioning could be transferred from one region of the heart to another, a phenomenon which has been termed “remote ischaemic conditioning” (RIC)35. Subsequent experimental studies demonstrated that the heart could be protected against AMI by applying the IPC stimulus to an organ or tissue remote from the heart, extending the concept of RIC to inter-organ ischaemic conditioning. The discovery that RIC could be induced by applying one or more cycles of brief ischaemia and reperfusion to the hind limb to reduce MI size37 facilitated the translation of RIC into the clinical setting with the use of a blood pressure cuff placed on the upper arm or thigh to induce one or more cycles of brief ischaemia and reperfusion to the limb (termed “limb RIC”)38. The mechanisms underlying limb RIC are not known, especially those conveying the cardioprotective signal from the limb to the heart. The current paradigm suggests that the limb RIC stimulus generates a blood-borne transferrable factor, which then activates protective signal transduction pathways common to IPC and IPost, but the identity of the factor or factors remains unknown39. Several potential candidates have been proposed, including nitrite40, miRNA14441, and SDF42, but conclusive evidence for their role as the mediators of RIC is lacking. Interestingly, the neural pathway to the limb has to be intact for RIC to be effective44, suggesting that the underlying factor or factors may be a neurotransmitter or neuropeptide.Crucially, both IPC and IPost require an invasive stimulus to be applied directly to the heart, thereby limiting their clinical application. In 1993, Pryzklenket al. in 199345; they demonstrated that subjecting the heart to two 3-minute cycles of global ischaemia and reperfusion by clamping and unclamping the aorta was able to preserve myocardial ATP levels45 and reduce PMI46 following cardiac surgery. Since this pioneering study, a number of clinical studies have confirmed the cardioprotective effect of direct IPC in patients undergoing cardiac surgery, and a subsequent meta-analysis found that IPC was able to reduce ventricular arrhythmias, lower inotrope use, and shorten intensive care unit stay when compared to control47. In 2007, Luoet al.48 were the first to apply IPost to the setting of cardiac surgery when they showed that applying IPost at the time of aortic unclamping, by re-clamping the aorta after 30 seconds and then unclamping it for 30 seconds, a cycle that was repeated twice, reduced PMI in children undergoing cardiac surgery for Tetralogy of Fallot. A number of clinical studies have confirmed the efficacy of this IPost protocol in children and adults undergoing cardiac surgery50. Given the invasive nature of the IPC and IPost protocols and the risk of thrombo-embolism from serial clamping and unclamping of the aorta, neither IPC nor IPost has been applied in the clinical setting.The first study to translate IPC into the clinical setting was by Yellonet al. in 200051. They found that limb RIC, comprising two cycles of 3-minute arm ischaemia and 2-minute arm reperfusion, did not reduce PMI during cardiac surgery. In 2002, Kharbandaet al.38 characterised the use of a blood pressure cuff to non-invasively deliver limb RIC (three 5-minute cycles of ischaemia and reperfusion), demonstrating MI size reduction in a porcine model of acute myocardial IRI and improved endothelial function in human volunteers. The first clinical study to report a cardioprotective effect with limb RIC (three 5-minute cycles of arm ischaemia and reperfusion) was by Cheunget al.52, who found less PMI in children undergoing corrective cardiac surgery for congenital heart disease. Our group was the first to demonstrate less PMI (43% reduction in serum troponin T release over a 72-hour postoperative period) in adult patients undergoing CABG surgery with limb RIC (three 5-minute cycles of arm ischaemia and reperfusion) when compared to control53. Since these early studies, there have been a number of small positive studies confirming the cardioprotective effect of limb RIC in the setting of cardiac surgery, although there have also been several neutral studies  reduced PMI and actually reduced all-cause mortality at 1.5 years by 73% when compared to control. However, this study was not prospectively designed or powered to test the effects of limb RIC on major clinical outcomes following cardiac surgery.The first clinical trial to investigate limb RIC as a cardioprotective intervention in the setting of cardiac surgery was a small study of only eight patients by Günaydinet al.60. They found in adult patients that limb RIC (four 5-minute cycles of ischaemia and reperfusion administered twice to the upper limb before and after cardiopulmonary bypass) failed to improve the large primary composite endpoint . The German RIPHeart clinical trial did not find any improvement in the in-patient primary composite endpoint  with limb RIC (four 5-minute cycles of arm ischaemia and reperfusion) in 1,385 adult patients undergoing cardiac surgery 61. Finally, the UK ERICCA clinical trial randomised 1,612 higher-risk adult patients undergoing CABG with or without valve surgery (Additive Euroscore ≥5) to either limb RIC (four 5-minute cycles of arm ischaemia and reperfusion) or control and failed to find any improvement in the 1-year primary composite endpoint 62.The effect of limb RIC on clinical outcomes following cardiac surgery has been recently investigated in three large prospective multicentre randomised controlled clinical trials, all of which failed to demonstrate any benefit with limb RIC on either PMI or major clinical outcomes. The first of these was a South Korean clinical study of 1,280 patients undergoing cardiac surgery , published in 2014 by Hong The potential reasons why the three large clinical trials failed to find any reduction in PMI or improvement in short-term and long-term clinical outcomes following cardiac surgery with limb RIC include the following:63. Moreover, patients undergoing concomitant valve surgery may be less amenable to RIC cardioprotection when compared to CABG surgery alone owing to the larger surgical trauma. Furthermore, RIC has been demonstrated in experimental studies to protect the heart mainly against acute IRI, whereas during cardiac surgery the causes of myocardial injury are multi-factorial and include inflammation (from cardiopulmonary bypass), direct handling of the heart, and coronary micro-embolisation. As such, limb RIC may be more likely to be effective in the setting of AMI, in which the target for cardioprotection is greater. In this regard, several clinical studies have reported a reduction in MI size with limb RIC applied prior to either thrombolysis64 or primary PCI71 in ST-elevation MI (STEMI) patients, and a large European multicentre randomised controlled clinical trial  is underway investigating whether limb RIC can reduce cardiac death and hospitalisation for heart failure at 1 year72.CABG surgery may not be the optimum clinical setting to test the cardioprotective effects of limb RIC given that the extent of acute myocardial injury sustained in this clinical setting is relatively small and the fact that cardioprotection has been optimised by improvements in surgical and anaesthetic techniques and the use of myocardial preservation strategies such as hypothermia and cardioplegia62. Whether this is the optimal limb RIC protocol for cardioprotection in the setting of cardiac surgery is not known, as the RIC protocol has not been fully characterised in either the experimental animal or the clinical setting of acute myocardial IRI. Further work is therefore needed to investigate the most effective limb RIC (this has recently been done in mice73), a task which would be made easier if a biomarker could be discovered, which can be used to assess the cardioprotective efficacy of limb RIC. However, this will be difficult given that the mechanisms underlying limb RIC remain unclear. It has also been suggested that the failure to fully blind the limb RIC protocol may have contributed to the positive results of the smaller clinical studies74. Achieving full blinding of the limb RIC protocol in the setting of cardiac surgery can be challenging but is possible using a cuff attached to a dummy arm beneath the surgical drape76.Both the ERICCA and RIPHeart studies used a limb RIC protocol comprising four cycles of arm ischaemia and reperfusion77. Furthermore, the experimental studies have for the most part included healthy, juvenile, small and large animals, making them far removed from the clinical setting of the typical middle-aged patient with IHD and multiple co-morbidities and co-medications (see below)78.The majority of experimental studies demonstrating cardioprotection with limb RIC have used an experimental animal model of MI based on external occlusion of a coronary artery and have not tested limb RIC using a more relevant experimental animal model of cardiopulmonary bypass80. Although some experimental studies have been able to recapitulate one individual co-morbidity  when assessing cardioprotection, most patients have multiple co-morbidities and, furthermore, they are often on multiple treatments for their co-morbidities —reproducing this in animal models will be extremely challenging58.A number of co-morbidities  have been shown in experimental animal studies to attenuate the cardioprotection induced by IPC and IPost, and emerging data suggest that limb RIC is also susceptible to this phenomenon78. Of these, it has been suggested that the use of the intravenous anaesthetic propofol may have contributed to the failure of limb RIC to reduce PMI and improve clinical outcomes in the ERICCA and RIPHeart studies, given that over 90% of patients received propofol62; however, the data supporting this proposition are not conclusive. The first clinical study to draw attention to the potential confounding role of propofol on limb RIC was by Kottenberget al.81, who showed that limb RIC was cardioprotective in the presence of isoflurane anaesthesia (n=19 patients), but not propofol anaesthesia (n=14 patients) in the setting of CABG surgery. Interestingly, propofol anaesthesia in the absence of limb RIC had no cardioprotective effect, suggesting that propofol was somehow antagonising the cardioprotective effect of limb RIC in the setting of cardiac surgery. More recently, Bautinet al.82 showed in 48 patients (12 per group) undergoing aortic valve replacement surgery that the cardioprotective effect of limb RIC observed with sevoflurane anaesthesia was absent in the presence of propofol. In contrast, there have been several clinical studies reporting cardioprotection with limb RIC in cardiac surgery patients in the presence of propofol anaesthesia84. Furthermore, there are experimental data suggesting that propofol itself can reduce MI size85 and is cardioprotective in a porcine model of cardiopulmonary bypass86 through anti-oxidant and mito-protective mechanisms. Therefore, a suitably powered prospective randomised controlled clinical trial is required to test whether propofol anaesthesia antagonises the cardioprotective effect of limb RIC in the setting of cardiac surgery when compared to volatile anaesthesia.Patients undergoing CABG surgery receive a number of different medications, many of which have the potential to interfere with the cardioprotection elicited by limb RIC—these include anti-diabetic medications, statins, angiotensin-converting enzyme inhibitors or angiotensin receptor blockers, calcium antagonists, beta-blockers, nitrates, morphine, inhaled anaesthetics, and propofolIschaemic conditioning has been investigated as an endogenous cardioprotective strategy for protecting the myocardium against PMI and improving clinical outcomes following cardiac surgery. Of these, IPC and IPost have been reported to reduce PMI, but, as they require direct application of the cardioprotective stimulus to the heart and because of the potential thrombo-embolic risk from repetitive clamping of the aorta, their clinical application has been limited. In this regard, limb RIC, which allows the cardioprotective stimulus to be applied to the arm or leg by simply inflating a blood pressure cuff placed on the upper arm or thigh, has facilitated RIC’s use in the clinical setting of cardiac surgery, where it has been shown to reduce PMI. However, three large multicentre clinical studies have failed to find improved short-term and long-term clinical outcomes with limb RIC following cardiac surgery, questioning the role of limb RIC in the setting of cardiac surgery. Further studies are required to investigate why limb RIC failed to improve clinical outcomes in this clinical setting. However, limb RIC still has therapeutic potential to protect the heart in AMI patients and may also protect non-cardiac organs  from acute IRI.


Axons can be several orders of magnitude longer than neural somas, presenting logistical difficulties in cargo trafficking and structural maintenance. Keeping the axon compartment well supplied with energy also presents a considerable challenge; even seemingly subtle modifications of metabolism can result in functional deficits and degeneration. Axons require a great deal of energy, up to 70% of all energy used by a neuron, just to maintain the resting membrane potential. Axonal energy, in the form of ATP, is generated primarily through oxidative phosphorylation in the mitochondria. In addition, glial cells contribute metabolic intermediates to axons at moments of high activity or according to need. Recent evidence suggests energy disruption is an early contributor to pathology in a wide variety of neurodegenerative disorders characterized by axonopathy. However, the degree to which the energy disruption is intrinsic to the axon vs. associated glia is not clear. This paper will review the role of energy availability and utilization in axon degeneration in glaucoma, a chronic axonopathy of the retinal projection. Glaucoma is the leading cause of irreversible blindness worldwide , the summation of all action potentials after stimulation, in freshly isolated D2 optic nerves. CAP amplitude decreased significantly by 6 months of age  how energy is produced and utilized in axons, (2) how energy is associated with axon degeneration, (3) the nature of axon degeneration in glaucoma, and (4) how axon degeneration might be prevented. As discussed below, the entry points for the final destruction of the axon vary between neurodegenerative disorders but have much in common beyond initiation. The focus here is energy depletion, which arises from a number of circumstances, including ischemia, mitochondrial dysfunction, age-related changes in the NAD+-sirtuin-PGC-1α axis, loss of axon support factors, and activation of axon degeneration factors. Below we review the current understanding of energy production in white matter and possible intervention points in glaucoma and, by extension, other chronic axonopathies.Energy is produced both within axons and around them using substrates delivered by the circulation and glial cells. Axons obtain most of their energy from ATP through oxidative phosphorylation in mitochondria. Glial cells that contact axons (oligodendrocytes and astrocytes) are capable of providing energy substrates to axons as well. This energy is necessary for axon function.The central nervous system prefers glucose as its substrate; glucose enters the brain (and retina and optic nerve) through glucose transporters. Neurons express GLUT3, a high-affinity glucose transporter  lay waste to the cytoskeleton, disrupting axon transport, physiology, and other vital axon maintenance processes. Extracellular Ca++ influx is both necessary and sufficient for axon degeneration  mouse. Wlds mice carry a naturally occurring mutation that greatly slows axon degeneration, allowing axons that are separated from the cell body to maintain structural and conduction integrity for 2 weeks after transection in mice  with nicotinamide mononucleotide adenylyltransferase-1 (NMNAT1) , a MAP3K upstream of JNK, is necessary and sufficient for RGC death after optic nerve crush  after transection but retrogradely (from the synapse back toward the injury) after crush injury in peripheral nerve  signaling concomitant with growth factor provision has proven to be supportive of RGC survival  and pre-pathological D2 optic nerve. That relationship is abolished in the transport dysfunctional D2 optic nerve, for which we observed axon volumes that were not matched by appropriate mitochondrial volumes  nullizygous for NMNAT2 showed truncated RGC axons; the axons did not reach the optic chiasm, and there was no optic tract. RGC bodies appeared normal, with proximal axons that formed the optic nerve , have had variable impact on degeneration in the CNS, their activation effective at delaying neurodegeneration in some situations, but not all  and those with glaucoma  polymerase-1] can inhibit mitophagy by disrupting the NAD+-sirtuin-PGC-1α axis in cells developed from xeroderma pigmentosum group A patients  led to significant protection of FluoroGold-positive retrogradely labeled RGCs, as well as the b wave of multi-focal ERG  or acarbose .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.


The development of childhood solid tumours is tied to early developmental processes. These tumours may be complex and heterogeneous, and elucidating the aberrant mechanisms that alter the early embryonic environment and lead to disease is essential to our understanding of how these tumours function. MicroRNAs (miRNAs) are vital regulators of gene expression at all stages of development, and their crosstalk via developmental signalling pathways is essential for orchestrating regulatory control in processes such as proliferation, differentiation and apoptosis of cells. Oncogenesis, from aberrant miRNA expression, can occur through amplification and overexpression of oncogenic miRNAs (oncomiRs), genetic loss of tumour suppressor miRNAs, and global miRNA reduction from genetic and epigenetic alterations in the components regulating miRNA biogenesis. While few driver mutations have been identified in many of these types of tumours, abnormal miRNA expression has been found in a number of childhood solid tumours compared to normal tissue. An exploration of the network of key developmental pathways and interacting miRNAs may provide insight into the development of childhood solid malignancies and how key regulators are affected. Here we present a comprehensive introduction to the roles and implications of miRNAs in normal early development and childhood solid tumours, highlighting several tumours in depth, including embryonal brain tumours, neuroblastoma, osteosarcoma, Wilms tumour, and hepatoblastoma. In light of recent literature describing newer classifications and subtyping of tumours based on miRNA profiling, we discuss commonly identified miRNAs, clusters or families associated with several solid tumours and future directions for improving therapeutic approaches. Caenorhabditis elgans) in 1993 .Fig.Fig47].FThere are a large number of miRNAs that have been confirmed to regulate components in essential developmental pathways. Crosstalk between these signalling pathways and miRNAs emphasises that miRNAs contribute largely during embryogenesis and early development. An example is the Wnt signalling pathway, which is a major contributor in controlling biological processes and is extensively researched. There is supportive evidence of miRNAs regulating WNT signalling components and being associated with various cancers in the literature. The signalling pathways critical in early development are interwoven with the function of numerous miRNAs and the affect of aberrant miRNA expression could have widespread consequences in the development of a specific organ or body system. Additionally, mice lacking the essential miRNA-processing enzyme, Dicer, are embryonically lethal before embryonic day (E) 7.5, suggesting the roles of the components of the machinery which produce functional miRNAs, and the miRNAs themselves, are essential in embryogenesis .RAS oncogenes. Mutations in RAS oncogenes are present in around 25–30% of all human tumours. In vitro experiments on a pulmonary adenoma cell lineage indicate that let-7 is able to inhibit cell proliferation through RAS, which suggests it may play a role as a tumour suppressor [There are a number of mechanisms that may affect the miRNA profile in a specific tissue or group of cells. These events can alter expression in both oncomiRs and tumour suppressor miRNAs, or on a more global scale, mutations in components of the miRNA biogenesis pathway may affect larger groups of miRNAs, and their ability to mediate repression of target mRNA Fig. . The letppressor . From thAlthough the links between miRNAs and the initiation and progression of cancer are supported, the area which remains difficult to interpret and requires further exploration is the exact mechanism by which miRNAs function to cause downstream effects, or conversely, how miRNA genes are influenced by other events which may alter the normal miRNA profile. Often miRNA expression is repressed in cancer compared to normal tissues; contrarily, there are also some miRNAs overexpressed in cancer .In addition to the deletion and mutation of miRNA genes, aberrant methylation may cause epigenetic inactivation with subsequent transcriptional suppression. Aberrant hypermethylation in mir-9-1, mir-124a, mir-148, mir-152 and mir-663 was observed in up to 86% of cases in a cohort of 71 primary breast cancer tissues . AdditiomiR-17/92 cluster induced by the MYC oncogene, the upregulation of which modulates the anti-apoptotic and proliferative actions of E2F1, mediating the MYC proliferative effect [Cancer-related transcriptional control of miRNAs has also been demonstrated by the observation of transcriptional activation of the e effect . AlteredIn childhood cancers, the pathogenesis of tumours is often tightly linked with the processes of development such as organogenesis, tissue growth and maturation –58. ThisThe most common types of childhood cancers are leukaemia, brain and central nervous systems tumours and lymphomas. In addition to these cancers, are the embryonal solid tumours of the kidney (Wilms tumour), neural crest (neuroblastoma), muscle (rhabdomyosarcoma), eye (retinoblastoma), and the liver (hepatoblastoma). Embryonal solid tumours of childhood are more commonly sporadic than heritable. In some cases they are associated with various syndromes such as Beckwith-Wiedemann syndrome (BWS) and in patients with familial adenomatous polyposis (FAP) . The parIt is essential to continue research to build a better understanding of the role miRNAs in the development of childhood cancers. It is clear that many of these cancers are tightly associated with irregular events occurring during development, and what we can learn from the inclusion and exploration of these vital regulatory elements may be the key to a deeper understanding of the complex nature of how childhood cancers arise. Another complex issue involving childhood cancers, where miRNAs may provide valuable insight, is the development of treatment resistance. It is important to identify aberrant miRNA expression associated with pathogenesis and also drug resistance, especially chemoresistance, as this may be critical in providing lasting and effective treatments for children .The following sections review several types of childhood solid tumours, describing common features and key developmental pathways that have been identified to contribute to embryogenesis and disease. The following childhood cancers were selected based on their association with a variety of affected organs or body systems, , and because recent literature has uncovered unique links to miRNAs and the development of these solid tumours. In addition, the childhood cancers discussed are those where the etiology remains unclear and the involvement of miRNAs could help to explain some of the unique qualities of the disease and may allow for a better understanding of new opportunities for targeted therapies. Young children are most at risk for developing debilitating late effects from the regimented chemotherapy courses. Through knowledge gained from these studies, we may be able to make better predictions about prognosis.A number of studies in recent times have indicated the frequent involvement of miRNAs in the progression and development of childhood brain tumours, and that this tumour has unique characteristics among rare tumour types. Brain tumours are among the most common cancers in children. The different tumour types and classifcations are generally based on cell structure, composition, growth rate of the tumour and a range of further characteristics. Central nervous system primitive neuro-ectodermal brain tumours (CNS-PNETs) are a heterogeneous group of CNS neoplasms. These tumours are composed of poorly differentiated neuroepithelial cells and, some variants are associated with aggressive clinical behaviour and poor outcome.LIN28 expression. In addition, recent identification of Tweety family member 1 (TTYH1):C19MC gene fusions in embryonal tumours with multi-layered rosettes (ETMRs) have been identified and observed to be associated with very high expression of specific miRNAs. ETMRs are a rare and deadly form of paediatric brain tumours, and are characterized by high levels of amplification of C19MC. ETMRs, cell lines and xenografts all demonstrated specific DNA methylation signatures distinct from other tumours and normal tissues, very high overexpression of a previously uncharacterized isoform of DNMT3B, which originates from an alternative promoter, and which is interestingly only active in the first weeks of neural tube development. This research uncovers a potential oncogenic re-activation of an early developmental signalling program in ETMR via an epigenetic alteration mediated by the brain specific DNMT3B isoform, which has a known embryonic origin [Chromosome 19q13.41 contains the largest human miRNA gene cluster ; the miRNAs comprising this cluster have been identified as oncogenic . C19MC mc origin .Pediatric pilocystic astrocytoma (PA) is a Word Health Organisation grade I glioma. It is a brain tumour, which often arises in the cerebellum or close to the brainstem in the hypothalamic region or the optic chiasm. However it is possible for it to occur in any location where astrocytes are present . While often these tumours are slow growing and benign, the neoplasms can be cystic and grow very large, causing multiple problems due to the increased intracranial pressure. miRNA profiling has been performed in a group of 43 PA tumours and 5 non-neoplastic brain controls. Differentially expressed miRNAs were identified between PA and normal brain tissue; 13 miRNAs were underexpressed in PA and 20 were over expressed compared to normal brain tissue  are the third most common group of brain tumours in children. These tumours comprise four entities; the most common are grade II and grade III, most of which are located in the posterior fossa, with infiltration into vital brain structures. Due to the difficult location of these tumours surgical resection is highly limited. Additionally there are currently no effective prognostic features aside from how accessible they are to surgical intervention. Childhood ependymomas usually don’t have genomic imbalances, further making it difficult to establish molecular prognostic features. Recently, however, three miRNAs were demonstrated to differentiate between grade II and III ependymomas. These include miR-17–5p, miR-19a–3p and miR-106b–5p, and the expression of these miRNAs was also significantly correlated with EZH2 expression (a suggested marker for PFA ependymomas). Survival analysis indicated overall and event free survivals were reduced with higher expression levels of miR-17–5p .CDK6 is of interest as it has previously been identified as an adverse prognostic marker in MB because of its role in upregulating cell cycle progression and blocking differentiation [miR-17-92 cluster is also implicated in MB. Mouse MB models have demonstrated overexpression of several members of the miR-17-92 cluster, three of which, , were also overexpressed in human MBs with a constitutively activated Sonic Hedgehog signaling pathway, and not in other forms of the disease . These mice formed MBs in orthotopic transplants with complete penetrance, but in similarly engineered cells from lnk4−/−; p53−/− mice there was no MB formation. These findings point toward a possible functional connection between the miR-17-92 cluster, the Sonic Hedgehog signaling pathway, and the development of MBs in both mice and humans [Medulloblastomas (MBs) are a common brain tumour in children, thought to originate in cerebellar granule neuron progenitors, which have failed to undergo normal cell cycle exit and differentiation. In MB miR-218 expression is downregulated, and low expression of this miRNA has been identified in other cancer types. In addition, reduced expression has been observed in glioma cells  (Table 1ntiation . The miRd humans .Neuroblastoma (NB) is an extracranial solid childhood tumour, and is the most common cancer in infants. The cancer is a neuroendocrine tumour, stemming from neural crest elements of the sympathetic nervous system; it is an ectodermally derived tumour, which reflects its developmentally derived origin . While imiR-17-5p-92 cluster of miRNAs, , is found to be expressed at higher levels in NB cells lines exhibiting overexpression of MYCN [. Tumours with amplification of the MYCN transcription factor and/or loss of the distal chromosome 1p and gain of 17q are a major genetic subtype of metastatic NB that has a particularly poor prognosis (known as MNA). It has also been demonstrated that MYCN proteins c-Myc/N-Myc can bind directly to the promoter of the miR-17-5p-92 cluster, initiating transcription and resulting in the up-regulation of this group of miRNAs from a single transcription unit [MYCN amplified NBs. However, previous research has shown that therapy resistant NB can be abolished in vivo using antagomir-17-5p [Recent work in this area suggests the dysregulation of miRNAs may play an important role in the pathogenesis of NB. For instance, the  of MYCN . Tumoursion unit , 78. A pir-17-5p .DICER and DROSHA were identified in high-risk tumours. The low expression subsequently accounted for the overall global reduction of miRNA expression observed in the advanced disease state and correlated with a poor outcome for these patients [MYCN.miR-34a is located on chromosome 1 and it is not surprising that lower levels are expressed in tumours with/or due to a 1p deletion [MYCN. miR-34a is also involved in the regulation of other genes associated with cell proliferation and apoptosis such as E2F3, BCL2, CCND1, and CDK6 [WEE1. Expression of miR-497 in MNA tumours is significantly lower than other tumour types, and high WEE1 levels (low miR-497) are significantly associated with poor overall survival and event free survival in NB  is an aggressive malignant neoplasm of the bone that originates in primitive transformed mesenchymal cells . This caThe molecular etiology of OS remains elusive; miRNAs have been investigated in association with many diseases due to their properties that may allow for widespread effects on development and disease progression. In prior investigations four miRNAs were found to be differentially expressed in OS cell lines compared to normal human osteoblast cell lines . The miRIn a recent investigation, a miRNA signature reflecting the pathogenesis of OS was described using surgically procured samples from human patients . These sWilms tumour (WT), or nephroblastoma is a solid tumour occurring in the kidneys of children. Embryonal renal neoplasms are thought to develop in nephrogenic rests (NRs) with morphological and molecular analogies to kidney development. Most commonly WTs are unilateral with a single tumour affecting the kidney. However multiple tumours can arise in one of the kidneys, or the cancer may occur bilaterally in both kidneys , 105.MYCN, SMARCA4 and ARID1A, missense mutations were discovered in the miRNA processing enzymes DROSHA and DICER1. Further examination of tumour miRNA expression using in vitro processing assays and genomic editing of cell lines demonstrated that these mutations in DROSHA and DICER1 greatly influence miRNA processing and likely cause down regulation in mature miRNAs, which generate an altered miRNA expression profile in these WT patients. Further, there were distinct mechanisms also identified; DICER1 RNase IIIB mutations preferentially affected the processing of miRNAs derived from the 5’ arm of pre-miRNA hairpins. Conversely DROSHA RNase IIIB mutations were found to cause global inhibition of miRNA biogenesis through a dominant-negative mechanism. Mutations in these enzymes also impaired the expression of tumour suppressor miRNAs such as the let-7 family; this is an important regulator of genes such as MYCN, LIN28 as well as other previously identified WT oncogenes [In WT relatively few driver genes have been identified . Recentlncogenes .miR-17-5p-92) was also identified as being up-regulated in the subtype of NB known as MNA [LIN28 [LIN28, and it is a potent regulator of stem cell self-renewal and differentiation. A murine model has shown that directed overexpression of both LIN28A and LIN28B in the renal lineage results in the formation of WTs [LIN28 expression in mutant mice revealed that terminal differentiation only occurred in nephron progenitor cells after LIN28 had been completely withdrawn. It is plausible that the overexpression of LIN28 may cause an imbalance in proliferation and differentiation in these specific cells, rather than a complete blockage of differentiation, resulting in the formation of these types of tumours [A number of up regulated miRNAs were also identified in WT; up-regulation of miR-17-5p, miR-18a, miR-19b, miR-92 and miR-20a was reported in WT compared to other kidney tumours or normal kidney tissues . This isn as MNA   is a relatively rare disease, although it is the most common childhood liver cancer. It is often diagnosed as an asymptomatic abdominal mass . The mospleiomorphic adenoma gene 1 (PLAG1) has been characterised in HB, and has been demonstrated through RNA interference analysis combined with miRNA arrays to strongly influence miR-492. Additionally, it was revealed that miR-492 could originate from the coding sequence of the HB marker gene keratin 19 (KRT19). Significantly elevated levels of co-expressed KRT19 and miR-492 were identified in metastatic HB tumour samples [Since HB is a rare cancer, research is challenging and often relies on formalin fixed paraffin embedded (FFPE) samples, which are known to provide obstacles in the extraction of total RNA due to degradation. However, recent research has demonstrated miRNA expression is relatively stable and well preserved in these valuable archival samples which is an important factor for future research in this field , 119. Pr samples . This is samples .MYCN-amplified and others) [Other potential prognostic miRNAs have been identified in a recent study, which showed that histological subtypes of a group of 20 tumour samples did not correlate with survival. However this investigation did identify that the levels of several miRNAs were independently prognostic for HB with significantly increased overall survival. These miRNAs included the high relative expression of miR-21 and low relative expression of miR-222 and miR-224  Tables . In a re others) . It is o others) . Serum l others) . Express others) . These sThe role of miRNAs in the regulation of critical signalling pathways such as Wnt/β-catenin, Notch, and TGFβ/BMP, has emerged as an important area in the exploration of childhood cancer development. Childhood solid tumours often display heterogeneous embryonic features, pointing to an early origin of tumourigenesis. While research into miRNA expression at very early stages of embryonic development (preimplantation) is largely unexplored at this stage, there is evidence that miRNAs regulate key components of several developmental pathways following these stages, and that expression of miRNAs directs development and differentiation of specific cells and tissues, which becomes more complex as the embryo continues to develop Fig. . If disrmiR-17-92 cluster is implicated in several childhood solid tumours and is highlighted in this review in NB, WT, HB and MB; it is a well-researched topic and is known to be vital in early development [In the childhood tumour types discussed above, there are miRNAs (or clusters) that are clearly implicated in more than one type of tumour Tables , 2 and 3elopment . Memberselopment . AnotherC. elegans [It is critical to uncover the mechanisms by which aberrant developmental signalling occurs and how these processes may be manipulated to regress into a more regulated path without causing additional disruption in embryonic development. Since these events may occur very early, it has thus far been challenging to study miRNA expression in embryo preimplantation. However, miRNA expression in the stages following implantation, at the beginning of embryonic development, is vital and has been widely researched. The discovery of miRNAs themselves were uncovered in the investigation of developmental timing in  elegans .While it is hopeful that elucidation of how miRNAs contribute to the formation of solid tumours can help revolutionise the approach to treating patients, it adds yet another layer of complexity to an already entangled web of interactions occurring both at the genetic and epigenetic level. Currently there are over 2,500 mature human miRNA products identified in humans . Given t


Although the importance of dendritic cells has been demonstrated in many animal models, their contribution to human immunity remains relatively unexplored Given their central role in infection, autoimmunity, and malignancy, dendritic cell deficiency or dysfunction would be expected to have clinical consequences.in vivo. The contribution of dendritic cell and monocyte dysfunction to the pathogenesis of primary immunodeficiency disease phenotypes is becoming increasingly apparent. However, dendritic cell analysis is not yet a routine part of primary immunodeficiency disease workup.Human dendritic cell deficiency disorders, related to GATA binding protein 2 (GATA2) and interferon regulatory factor 8 (IRF8) mutations, have highlighted the importance of dendritic cells and monocytes in primary immunodeficiency diseases and begun to shed light on their nonredundant roles in host defense and immune regulation Widespread uptake of dendritic cell/monocyte screening in clinical practice will facilitate the discovery of novel dendritic cell and monocyte disorders as well as advancing our understanding of human dendritic cell biology in health and disease. The analysis of dendritic cells in primary immunodeficiency disease (PID) has provided new diagnostic tools and revealed new clinical syndromes in addition to providing a unique opportunity to probe the function of human dendritic cells In this review, we explore what is known about dendritic cells and monocytes in PID, highlight the recently described dendritic cell deficiency syndromes related to GATA binding protein 2 (GATA2) and interferon regulatory factor 8 (IRF8) mutations, suggest a practical solution to dendritic cell analysis in clinical practice, and speculate how best to further our understanding of dendritic cells in PID and immunity in general. Dendritic cells are bone marrow derived, specialized antigen-presenting cells (APCs), positioned within the immune system to bridge innate and adaptive immunity . They arBased on in-vitro observations , it was A recent consensus study  has defiDendritic cells and monocytes develop in the bone marrow under the control of distinct transcription factors. They develop discrete functions owing to their expression of particular pattern recognition receptors (PRRs) and elaboration of specialized secretory products Fig. .cDC1 are dependent on BATF3 and IRF8 for development , have sucDC2 require IRF4 and KLF4 expression for differentiation ,18. They+ responses and may induce Treg differentiation after viral infection and in the thymus . In adults, primary immunodeficiency may be a more prominent feature with susceptibility to mycobacterial, viral (human papillomavirus) and fungal infection, autoimmunity, and respiratory compromise due to recurrent bacterial infections and/or pulmonary alveolar proteinosis. Development of MDS/AML (in approximately 50% of patients) is associated with the acquisition of additional cytogenetic (monosomy 7/8) or molecular genetic abnormalities .IRF7 mutation, recently described in one compound heterozygous patient, results in severe influenza infection, attributed to impaired IFN type I and III production by pDCs ▪▪.The potential of dendritic cell and monocyte analysis to assist in the clinical diagnosis and understanding of PID is only just being realized . SimplisDiagnosis of known dendritic cell deficiency syndromes: Myeloproliferation of biallelic IRF8 deficiency may mask the absolute monocytopenia on automated blood counters but absence of dendritic cells and monocytes in this context is, to date, pathognomonic. Depth of mononuclear cell loss, together with increase in serum Flt3L, in GATA2 mutation may inform ‘stage’ of disease and associate with development of symptoms or progression to bone marrow failure.Discovering novel dendritic cell deficiency syndromes: Analysis of the recently described ‘human gene connectome’ (HCG)  has idenUnderstanding dendritic cells in known primary immunodeficiency diseases: Dendritic cell deficiency or dysfunction may contribute to the diagnosis and phenotype of many known PIDs, in which functions of the mutated gene have not yet been explored beyond lymphocytes, for example TLR3 , TRAF3 , and ADADendritic cells are too rare to enumerate morphologically by cytological examination of whole blood and automated blood counters are unable to distinguish dendritic cells from lymphocytes by light scatter properties Fig. a. ClassiFlow cytometry lends itself well to clinical dendritic cell/monocyte analysis as it allows accurate identification and enumeration of rare populations of cells from clinically appropriate volumes of blood. A minimum of six channels is required to identify all monocyte and dendritic cell subsets, including human leukocyte antigen-DR (HLA-DR) expressed on all APCs, lymphocyte lineage markers to exclude HLA-DR+ T-, B-, NK-cells, and CD34+ progenitor cells, CD14 and CD16 for monocyte analysis, CD123 or CD303/CD304 for pDC, CD141 or Clec9A for cDC1, and CD1c/BDCA1 for cDC2  features of global monocyte and dendritic cell loss can be inferred from IRF8 and GATA2 mutations and these would include susceptibility to tuberculosis and atypical mycobacteria, viral infection (particularly HPV and herpes simplex virus), pulmonary pathology , and autoimmunity. Specific features of these mutations may also be sought, such as myeloproliferation in biallelic IRF8 mutation and MDS or extrahematopoietic features in GATA2 mutation.Second, it is difficult, at present, to define the pretest risk without a better picture of the prevalence, which will only be achieved through much greater uptake of dendritic cell and monocyte enumeration by diagnostic laboratories.We suggest, therefore, that a survey level analysis (six-color flow cytometry) should be performed in all new patients with suspected immunodeficiency alongside, for example, T and B cell subset analysis. An abnormal screen or a high index of suspicion should prompt full immunophenotyping of myeloid mononuclear cells.This will facilitate the discovery of novel dendritic cell and monocyte disorders as well as build a picture of the dendritic cell/monocyte landscape in PID specifically and in immunity generally.Mouse models have suggested that dendritic cells are critical to initiate and maintain immunity and tolerance, yet little attention has been paid to their contribution in PID. Recent descriptions of dendritic cell deficiencies and dysfunctions in the context of immunodeficiency have highlighted their importance in this field. Clinical level screening for dendritic cell and monocyte defects will allow the identification of novel disorders and aid the diagnosis and pathophysiological understanding of immunodeficiency.in vivo, looking to harness their immune potential for therapeutics across wider fields of medicine.From a research perspective, through the coordinated investigation of dendritic cells in patients with immunodeficiency, the PID community is in a unique position to advance our understanding of the function of human dendritic cells and monocytes https://bridgestudy.medschl.cam.ac.uk/pid.shtml) and 100,000 Genome Project (https://www.genomicsengland.co.uk), with the support of cohesive research networks such as the Genomics England Clinical Interpretation Partnership (GeCIP), will likely yield fruitful results.Aligning these efforts with those of next generation sequencing consortia such as the NIHR BRIDGE Project (PID)  for their vital contributions to this work.This work was supported by Wellcome Trust (V.B.), BrightRed , and BloodWise (M.C.).There are no conflicts of interest.▪ of special interest▪▪ of outstanding interestPapers of particular interest, published within the annual period of review, have been highlighted as: