DNA methylation alterations are related to multiple molecular mechanisms. The DNA context of CpG sites plays a crucial role in the maintenance and stability of methylation patterns. The quantitative relationship between DNA composition and DNA methylation has been studied in normal as well as pathological conditions, showing that DNA methylation status is highly dependent on the local sequence context. In this work, we describe this relationship by analyzing the DNA sequence context associated to methylation profiles in both physiological and pathological conditions. In particular, we used DNA motifs to describe methylation stability patterns in normal tissues and aberrant methylation events in cancer lesions. In this manuscript, we show how different groups of DNA sequences can be related to specific epigenetic events, across normal and cancer tissues, and provide a thorough structural and functional characterization of these sequences. However, the mechanisms influencing the sensitivity of certain sites to epigenetic modulation are still not completely understood as many internal and external factors are thought to be involved in this process. This aspect has been extensively studied in bacteria, where particular sequences are able to favor the binding of enzymes that modify the methylation status2.Sizable efforts have characterized epigenetic signatures and their localization across the human genome4. Indeed, high density CpG clusters  are usually located in the proximity of Transcription Start Sites (TSS) and promoters of housekeeping genes in an unmethylated state, hence allowing gene transcription6. Furthermore, a model of collaborative methylation among CpG sites has been recently suggested, explaining how CpGs in CGIs could maintain a stable methylation through the generations due to their clustered localization7. In 2009, McCabe and colleagues identified distinct sequence patterns marking methylation deregulation events in cancer8.It is well known that a dependency exists between DNA-sequence composition and occurrence of methylation in mammalian genomes. A well-known example of DNA-sequence-dependent mechanism is the dependency of DNA methylation on CpGs density10. Ghorbani and colleagues11 gave an insight to the identification of the motifs underlying common methylation patterns across different cancer types. Their results paved the way to the identification of alternative cancer biomarkers, currently needed in order to expand and diversify the therapeutic possibilities for cancer patients. In this context, epigenetic marks are considered as pivotal elements in biomarker discovery. For example, epigenetic signatures are used as diagnostic, prognostic and therapy-assessment markers in colorectal cancer12. However, the genomic context of aberrant methylated CpGs in cancer is still largely unexplored, and it remains unclear whether these contexts can be exploited to track tumor progression.To date, only a few correlations between phenotypic alterations, DNA methylation alterations and DNA sequence context have been describedIn this study, we analyzed DNA sequence contexts in the form of DNA motifs characterizing: (i) interindividual CpG methylation variability in normal tissues and (ii) aberrant CpG methylation between normal and cancer tissues. By using such approach we revealed the existence of i) tissue specific motifs characterizing low and high population variability of CpG methylation in normal tissues and (ii) putative cancer-specific biomarkers, constituted by sequence motifs related to aberrant methylation across 33 cancer types. Furthermore, we investigated the regulatory consequences of aberrant methylation in selected genomic contexts, in order to give an insight on the mechanisms underlying the gene expression deregulation in the onset and progression of cancer. Our results suggest the existence of alternative biomarkers in human cancer and provide novel hints in understanding of the role of the DNA sequence context in genetic and epigenetic deregulation.13 repository and the Epic Italy cohort14. Supplementary Table sCpGs), showing variance values below the 1st percentile, and unstable CpGs (uCpGs), showing variance above the 99th percentile.To investigate the relationship between DNA context and CpG methylation stability, we retrieved pre-processed DNA methylation data by Illumina Infinium 450 k assays from The Cancer Genome Atlas (TCGA)15. Supplementary Table 16. For each considered cancer type, we further divided these sets in hyper-methylated iDMC (hyper-iDMC) and hypo-methylated iDMC (hypo-iDMC) based on the observed trend of differential methylation15.For the tumor samples, we collected CpGs from the dataset of informative differentially methylated CpGs (iDMCs) computed inDREME) tool from the Multiple EM for Motif Elicitation (MEME) suite17. In particular, for each CpG set, we provided DREME with two fasta files containing the hg19 20 bp flanking sequence of the CpGs in the set of interest and of the CpGs in the background set, respectively.Given a set of CpGs of interest  and the whole set of CpGs probed in the Illumina Infinium 450K platform as a background set, we searched for enriched motifs by using the Discriminative Regular Expression Motif Elicitation  methylated if more than 70% of the CpGs carrying the motif were hyper- (hypo-) methylated, in all other cases the motif methylation status was considered as undefined. Moreover, for each motif in each tissue, we performed a hypergeometric test to evaluate the enrichment of CpGs carrying the motif for hyper- or hypo-methylation with respect to the corresponding background set. We considered as significantly hyper- or hypo-methylated the motifs with hypergeometric p-value lower than 0.05.hg19 genes information, using the IlluminaHumanMethylation450kanno.ilmn12.hg19 R package. In particular, we counted the number of CpGs in the set related to gene regions  and CGI regions .For each discovered motif, we selected the set of CpGs containing the motif in their surrounding sequence. We then annotated each set with Tomtom tool18 from the MEME suite. For each motif, we used Tomtom to search against the JASPAR 2018 (JASPAR2018_CORE_vertebrates_non_redundant) sequence database provided by the MEME suite. We considered for subsequent analyses only Jaspar TFBS having at least 5 bp overlap with the motif sequence and an E-value lower than 10.In order to assess whether the discovered motifs encompass a Transcription Factor Binding Site (TFBS), we employed the Tomtom tool, showing binding affinity. We then considered only the transcription factors (TFs) annotated as MethylPlus or MethylMinus in19. For each TF, we retrieved the list of potential target genes using the TRRUST database20. For each motif/TF pair, we retrieved the expression profiles of the TF target genes in the associated tumor and normal tissues using the FirebrowseR package16. We then performed a test for differential expression using a Wilcoxon test and defined each gene upregulated or downregulated in cancer, if the difference in median expression values between tumor and control was positive or negative and the Wilcoxon p-value was lower than 0.05. After that, we annotated the motif/TF pairs as associated with up-regulation if more than 50% of differentially expressed target genes were up-regulated, down-regulation if more than 50% of differentially expressed target genes were down-regulated, mixed-regulated otherwise.For each motif we considered the set of TFs, derived from the sCpGs) and unstable CpGs (uCpGs).To investigate the relationship between DNA context and CpG methylation stability, we built a set of normal methylomes using DNA methylation data of normal tissues associated to 11 cancer types from TCGA and normal blood samples from the EPIC Italy cohort. Considering the variance distribution, we selected two sets of CpGs for each tissue: stable CpGs  in the 20 bp flanking regions surrounding each CpGs and we found 108 motifs characterizing sCpGs and uCpGs in different tissues corresponding to overall 77 unique motifs  and hypo-methylated iDMC (hypo-iDMC) based on their methylation trend in tumor with respect to normal. By following a similar approach as in the previous analysis we considered, for each cancer type, the entire set of iDMCs and looked for recurrent DNA motifs (not necessarily including the CpG sites) in the 20 bps flanking regions surrounding each iDMC and we discovered statistically overrepresented DNA motifs in almost all cancer types , given the background composition of input iDMCs, for the particular aberration (Supplementary Table In order to ascertain how similar signatures were distributed among different cancer types and different aberration types (hypo- or hyper-methylation), we computed a distance function based on the sequence similarity among all pairs of derived motifs and performed a hierarchical clustering  whose binding domains have a potential affinity for the discovered motifs. Our analysis highlighted that most enriched transcription factors are both related to basal transcriptional regulation as well as to epigenetic mechanisms Fig. . For insTo better characterize the regulatory role of the motifs in the malignant phenotype, we identified transcriptional regulators acting both in a specific cancer and across multiple types of cancers. As shown in Fig. Such regulators were enriched in several cancer types, as well as lung, bladder cancer and kidney renal clear cell carcinoma. On the other hand, a distinct regulatory signature sustained by the IRF family regulators shared by stomach, cervical squamous cell carcinoma and in lymphoid neoplasm diffuse large B-cell lymphoma emerged. Furthermore, our findings show a specific enrichment of the ETV family regulators in colon adenocarcinoma and uterine corpus endometrial carcinoma. While such patterns of regulation are peculiar for the mentioned cancers, we observed a remarkably complex regulation in lung and prostate adenocarcinoma as well as in esophageal cancer.19. In order to strengthen our findings from a mechanistic point of view, we tested the hypothesis that there is a concordance between the methylation status of the considered motifs, the methylation-dependent binding affinity of the transcriptional regulators, and the expression trend of their target genes. To achieve this goal, we first classified each association TF/motif as concordant if the TF was reported as MethylPlus in19 and associated to a hyper-methylated motif or if it was classified as MethylMinus and associated to a hypo-methylated motif in the corresponding cancer tissue, as discordant otherwise  is specifically occurring upstream of genes whose expression is regulated by cyclic adenosine monophosphate molecules, and its aberrant methylation is related to the loss of binding properties by ATF family regulators and subsequent transcriptional inactivation.Interestingly, structures formed by more than 4 nucleotides, such as the CACGTGAC motif significantly overrepresented in breast and kidney. Anantharaman and colleagues22 investigated the cis-regulatory role of this motif in the basal regulation of transcription highlighting the capability of such motif to bind HLH proteins in erythroid cell line differentiation. In this context, the CACGTGAC motif is known as E-box. Furthermore, early in vitro studies demonstrated that this motif constitutes the binding site for the transcription factors USF1 and USF224. These transcriptional regulators can act both as homo or heterodimer recruiting the TFIID complex to the promoter. This motif has been already described as having a high affinity binding for multiple other transcription factors such as NF-E2, GATA-1, EKLF, and Tal1, in vivo25.Moreover, we found the CCAATCA motif, enriched in the colon tissue, is localized in the region surrounding one of the most diffused cis-regulators in the basal transcription machinery, the CCAAT box. Meergans and colleagues26 clarified the role of this well conserved sequence in the expression regulation of genes coding for the histone proteins H1, the main factor guiding the nucleosome positioning. Interestingly, the so-called CCAAT box is highly conserved in all the promoters of the H1 loci and it is followed by a CA dinucleotide.The 29. CpGs proximal to a TSS show less variation (more stability) than distal CpGs6. In this context, our results support the hypothesis that stable CpG motifs are related to high CpG content and located in promoter regions, while unstable CpG motifs are related to low CpG content and located in gene body regions.A correlation between the local methylation profiles and CpGs density is known30.Similarly, in Acute Lymphoid Leukemia cells it has been shown that CpGs located outside the CGIs show a higher variation (instability) than those falling within the CGIs25. To date, the exact mechanism that modulates the sensitivity of CpGs to methylation is still unknown. A seminal study by Feltus et al. reported that aberrant methylation relies on local sequence composition31.Methylation changes occur not only during physiological processes but also in several pathological conditions including cancerHaving observed the existence of motif structures associated with CpG methylation stability in normal tissues, we ascertained whether similar structures can be found when considering aberrant methylation associated with cancer tissues.TGACTCA motif, which was statistically overrepresented in mesothelioma and in its variant TGACTMA in kidney, has been already identified as the binding motif of the transcriptional complex AP132. Seldeen et al.32, inspected the effect of constitutive modifications of the TGACTCA motif, given the important role of the Fos-Jun transcription factors in the translation of extracellular signals in gene expression regulation (by growth factors and cytokines).For instance, the 33 as CpG island methylator phenotype (CIMP). Although CIMP has been reported in several cancers, including gastric37, lung38, liver39, ovarian40, endometrial41, breast42, gliomas43 and leukemias44, most of the literature relates its effects to colorectal cancers (CRC)45. Currently, the CIMP causative factors are still unknown46. However, the investigation of CIMP in different cancers led to the hypothesis of an epigenetic-driven onset of the malignant transformation, in turn leading to the inactivation of essential genes as well as tumor suppressors. The results obtained in the present work are also in line with this hypothesis. When looking at the genomic localization, we found that enriched motifs in STAD and PRAD are mainly localized within the 200 bp sequence surrounding the transcription start site, suggesting their possible direct involvement on the corresponding gene expression activity. Once obtained a list of motifs associated with each cancer type, we investigated their potential transcription factor binding affinity.In order to disentangle the motif relatedness among different tissues from the similarity of the corresponding CpG input sets, we built a dendrogram based on the Jaccard distance among sets of iDMC for each tumor TCA-3′ sequence of the AP-1-like binding site48. This regulatory complex affects the expression of a wide range of genes. Dimerization of Fos-Jun proteins with members of the Maf and NF-E2 (CNC) families further expands the range of Fos-Jun targeting. In addition, different Jun and Fos family members can have opposite effects on AP-1-mediated transactivation51. Notably, by looking at the methylation status of the considered motifs, we noticed that this cluster of transcriptional regulators is typically related to hypomethylated motifs.For instance, the motifs encompassing the HINFP binding sites, are among the most enriched overall the considered cancers. HINFP is a crucial regulator of the expression of genes encoding the H4 histones. Therefore, its function is essential in the S phase of the cell cycle, affecting the packaging of newly synthetized DNA into condensed chromatin53.Similarly, markers of E2F-dependent transcription are mostly represented overall cancers types, as well as E2F4 and TFDP1, which regulate genes involved in key processes of malignant transformation, such as cell cycle regulation and DNA replication54. For this reason, we suppose that hypermethylation at E2F binding motifs, which we found overrepresented in colon and stomach adenocarcinoma as well as in sarcoma, could lead to an unmodulated E2F-dependent transcription of targets involved in the G1/S checkpoint and, thus, uncontrolled cell proliferation. Our results show that the TFs binding affinity for motifs marking aberrant DNA methylation regions involves both Fos-Jun regulators (as found in most of the cancers), and other specific factors, shared among few cancers, suggesting a possible crucial role of these motifs in the development of cancer phenotypes. On the opposite side, we identified motifs bound by a single transcription factor. In this case, the motif CGC, found overrepresented in cholangiocarcinoma, cervical squamous cell carcinoma and endocervical adenocarcinoma, is predicted to be bound specifically by Ahr::Arnt. Similarly, the motifs CGR (breast invasive carcinoma), CACAAR (glioblastoma multiforme), ATGWTG (lung squamous cell carcinoma), CGWCCGAA (prostate adenocarcinoma) show exclusive affinity for YY2, RUNX1, ATF4 and ZBTB7B, respectively.Deregulation of E2F-dependent G1/S transcription (i.e by certain oncogenes), leads the cells to enter the S-phase, triggering cell proliferationWe finally checked the concordance among the methylation status of the observed motifs, the TFs binding affinity for methylated or unmethylated sites and TFs target genes expression deregulation. The observed results suggest that the identified DNA methylation motifs not only represent potential biomarkers, but they contribute to disentangle the complex regulatory circuits in cancer genomes.The relationship between sequence context and DNA methylation is an important feature to gain further insights on the dynamics of methylation establishment, maintenance and alteration in pathological conditions. Here, we provided the first comprehensive catalogue of short DNA sequences that can be associated to methylation stability between individuals in normal tissues and to aberrant methylation conditions in different cancer types. We showed how different DNA contexts could predispose a CpG to be more or less prone in gaining or losing methyl groups. We characterized the genomic localization of these sequences and their relationship with transcriptional regulators in the genome. When considering motifs associated to aberrant CpGs characterizing cancer tissues, we showed that some motifs are shared by different cancer types while others are cancer specific. This latter aspect could be exploited to define more precise and effective molecular targets in gene-based therapies such as those employing epigenetic CRISPR-Cas9 strategies.Supplementary Information.


PTPN11 mutations underlying Noonan syndrome and Noonan syndrome with multiple lentigines . Although differential diagnosis between these two syndromes could be difficult, particularly in the first age of life, we underline the relevance in discriminating these two disorders in terms of affected signaling pathway to allow an effective targeted pharmacological treatment.The concomitant occurrence of hypertrophic cardiomyopathy and congenital heart defect in patients with RASopathies has previously been reported as associated to a worse clinical outcome, particularly closed to cardiac surgery. Different mechanisms of disease have been demonstrated to be associated with the two classes of  Dear Editor,PTPN11  showed an early-onset presentation of HCM and a worse clinical outcome. Indeed, out of this subgroup of eleven patients, three of them  died within 6 months from birth.We read with interest a recent research by Chen and colleagues  reportinPTPN11 mutations at codons 510 (including c.1528C > G) and 506 is drastically different from what observed for NS-causing PTPN11 mutations. Specifically, the former cause defective protein phosphatase activity and differentially affect intracellular signaling [PTPN11 mutations cluster within or close to the active site of the phosphatase, which explains their dramatic impact on phosphatase activity when compared with wild-type SHP2 and NS-causing PTPN11 mutations [PTPN11 is strictly associated to NSML. Review of published cases carrying the c.1528C > G missense change documents that most of the patients have a clinical diagnosis of NSML syndrome , which can effectively be controlled by the use of inhibitors of signal transducers functioning in this cascade  [While NS and NSML are genetic conditions with overlapping features, and clinicians experienced with these syndromes are aware of the difficulty in discriminating between these two disorders particularly in the first years of age , 22, we ibitors) , 24. On ibitors) , 25, 26.ibitors)  reportedPTPN11 mutations and NS, based on the diverse impact of these mutations on SHP2 function and intracellular signaling dysregulation, and their consequent significance in terms of patient management and future personalized therapeutic options.In summary, we should avoid the misleading causative association between NSML-causing


Despite the absence of the genome in platelets, transcription profiling provides important insights into platelet function and can help clarify abnormalities in platelet disorders. The Bloodomics Consortium performed whole-genome expression analysis comparing in vitro–differentiated megakaryocytes (MKs) with in vitro–differentiated erythroblasts and different blood cell types. This allowed the identification of genes with upregulated expression in MKs compared with all other cell lineages, among the receptors BAMBI, LRRC32, ESAM, and DCBLD2. In a later correlative analysis of genome-wide platelet RNA expression with interindividual human platelet reactivity, LLRFIP and COMMD7 were additionally identified. A functional genomics approach using morpholino-based silencing in zebrafish identified various roles for all of these selected genes in thrombus formation. In this review, we summarize the role of the six identified genes in zebrafish and discuss how they correlate with subsequently performed mouse experiments. Blood platelets are important mediators of thrombus formation. However, addressing the platelet genome is challenging, as platelets are anucleated cells derived from megakaryocytes (MKs) in the bone marrow. Platelets are not able to perform de novo transcription due to the lack of genomic DNA. However, mRNA from MKs is transferred in an organized way into platelets and platelets are able to exchange mRNA with other cells via mRNA-rich microvesicles.Bloodomics Consortium deployed two different strategies to identify novel regulators of platelet function. First, unknown genes with a specific expression on platelets and endothelial cells were revealed to study their associated phenotype. Comparative transcriptome analysis of the major nucleated blood cell elements and in vitro–differentiated MKs and erythroblasts was performed. This allowed identification of 279 genes with upregulated expression exclusively in MKs.BAMBI) was selected. BAMBI is a known nonsignaling, transmembrane receptor that associates with transforming growth factor (TGF)-β family receptors and functions as a negative modulator of multiple, often opposing, actions of TGF-β.ESAM), a cell adhesion molecule on endothelial cells colocalizing with cell adhesion proteins such as cadherins, catenins, ZO-1, occluding, and claudin-5, and associated with tight junctions in the blood capillaries, was identified for further analysis.Dcbld2impaired normal vascular development and weakened VEGF-induced VEGFR2 signaling.First, vessel wall Bone morphogenetic protein and Activin Membrane Bound Inhibitor , with SNPs associated with myocardial infarction, and Copper metabolism Murr1 domain containing 7 , with SNPs associated with platelet response.The second strategy used by the Bloodomics Consortium to identify novel platelet regulators in hemostasis was to examine a correlation between the expression of genes and platelet function. Goodall et al reported that transcript levels of 63 genes in human platelets correlated with variation in platelet response toward ADP and/or collagen-related peptide.The human platelet transcriptome was later mapped using next-generation RNA sequencing in an elegant study by Rowley et al and the presence of the transcripts of these newly identified genes, postulated to be involved in hemostasis, was demonstrated.Vwfgene created by antisense morpholino oligonucleotides furthermore resulted in a bleeding phenotype in half of the embryos, which confirms that VWF is also important in the hemostatic processes of zebrafish,An important prerequisite for using the zebrafish model in hemostasis and thrombosis research is that its hemostatic system demonstrates significant similarities to the human one.In the zebrafish laser-induced thrombosis model, thrombus formation in the caudal artery is caused by laser injury of the endothelium. Thrombocytes play a major role in occlusive thrombus formation where both the time to attachment (TTA) and the thrombus surface area (TSA) are the main quantification parameters.Bambi,Lrrc32,Esam, andDcbld2in MK and HUVECs and were the candidate genes encoding receptors with a putative role in hemostasis and/or thrombosis.Commd7andLrrfip1transcripts on the other hand were identified based on their correlation with the platelet response toward ADP and/or CRP activation and the presence of SNPs within the gene that showed a possible association with either myocardial infarction or platelet response. Consequently, the contribution of these six genes to thrombosis and hemostasis was further screened in zebrafish using the laser-induced thrombosis model.The transcriptomics study showed lineage-specific expression ofLrrc32-downregulated zebrafish after laser-induced thrombosis. Some caution, however, was warranted as knockdown by one morpholino did affect the TTA, whereas another one affected the TSA, while none did both. The evidence for the involvement of ESAM was provided as knockdown of ESAM resulted in a significantly increased thrombus size (TSA).Morpholino oligonucleotides were used to create a successful knockdown in zebrafish and laser-induced thrombosis indicated that BAMBI promoted thrombus formation. In the absence of BAMBI, thrombus formation was reduced, as the time at which the first thrombocytes attached (TTA) to the injured vessel wall was significantly delayed and the thrombus size (TSA) was significantly decreased.Overall, functional genomics demonstrated BAMBI, LRRC32, COMMD7, and LRRFIP to be positive regulators and ESAM and DCBLD2 as negative regulators in thrombus formation in zebrafish.The hemostatic system in zebrafish, despite having encouraging similarities with mammals, remains genetically and systemically quite distant from the human situation and hence functional confirmation in higher species is warranted.3-induced injury of the mesenteric and carotid arteries or cremaster muscle arterioles. With laser-induced injury, the endothelial wall is damaged by heat, resulting in a controlled location and time of injury. The injury results in a fast platelet accumulation and expression of tissue factor at the injured vessel wall and is seen as inflammatory due to the absence of endothelial denudation.3injury model, and therefore the latter can be applied in much more research facilities. Upon topical application of FeCl3, the vessel denudates as a result of free radical formation causing lipid peroxidation and destruction of endothelial cells.3was shown to decrease platelet adhesion to several adhesion proteins.Thrombus formation in mice is mostly studied using either laser- or FeClWith the discovery of the six platelet genes in 2009 and 2010, all have in the meantime been studied using in vivo murine models as described earlier and/or in ex vivo human platelet studies.−/−Bambimice that had an increased tail bleeding time, an impaired thrombus formation after FeCl3injury of the mesenteric arterioles, and a decreased thrombus stability after laser-induced thrombosis.−/−Bambimice, the latter observation pointed into the direction of endothelial rather than platelet BAMBI to be responsible for the observed effects. Consequently, radiation chimeric mice were generated to investigate the contribution of endothelial versus platelet BAMBI. Radiation chimeric mice are prepared by irradiating recipient mice to kill the bone marrow and are next injected with hematopoietic progenitor cells from donor mice with a different genotype (here WT cells in KO mice or vice versa) that then repopulate the recipient bone marrow. Although thrombus size and time to reach maximal thrombus size did not differ, mice lacking BAMBI on the endothelial cells only demonstrated a decrease in thrombus stability compared with mice lacking BAMBI on their platelets.The function of BAMBI as positive regulator of thrombus formation in zebrafish was confirmed by Salles-Crawley et al usinglrrc32knockout mice, as the fulllrrc32knockout in mice was lethal.Cregene was expressed after either thePf4or theTie2promoter.3-induced thrombosis models in the carotid and mesenteric artery or in the tail bleeding time in platelet- or endothelial-specificlrrc32-deficient mice compared with littermate controls.lrrc32knockout and control mice. Therefore, the role of LRRC32 as a positive regulator of thrombosis, as suggested in zebrafish, could not be confirmed in mice, at least for as far as LRRC32 on platelets and endothelial cells is concerned.In the knockdown zebrafish, LRRC32 was found to be a positive thrombosis regulator.garp/lrrc32deletion in platelets improves adoptive T-cell therapy as tumor treatment in mice.Although the function of platelet LRRC32 thus remained elusive, Rachidi and colleagues, however, recently reported that conditionalEsamresulted in more stable and larger thrombi upon laser injury of the cremaster muscle arterioles compared with controls.Esam−/−mice.In the same year as the zebrafish study, corroborating results in mice were published by Stalker and colleagues who demonstrated that platelet ESAM is involved in the interplatelet interaction due to its localization toward junctions between activated platelets.−/−apoEEsam−/−mice compared with+/+apoEEsam−/−mice.In humans, increased levels of soluble ESAM are associated with major cardiovascular risk factors and inflammatory markers.−/−Dcbld2mice. The−/−Dcbld2mice showed no apparent spontaneous bleeding or thrombotic events over their lifespan, and no significant difference in number of circulating platelets was measured between the−/−Dcbld2mice and+/+Dcbld2control mice. Platelets of−/−Dcbld2mice did not demonstrate abnormalities, as the expression levels of physiologically important platelet receptors such as GPIb, integrin αIIb, and GPVI were not altered on platelets at rest or after activation by thrombin. Different in vivo bleeding and thrombosis models were used to perform experiments in−/−Dcbld2mice; bleeding time was determined by the tail clipping assay, and time to artery occlusion upon FeCl3injury was determined in the large carotid artery and in the smaller mesenteric artery using a milder lower FeCl3concentration. Where only a modest reduction in thrombus formation was seen in the smaller mesenteric arteries using a milder injury inflammation, tumorigenesis, and angiogenesis. Indeed, currently platelets LRRC32 and DCBLD2, both of which were demonstrated not to be involved in hemostasis in mice, have been implicated in the control of T-cell immunity and angiogenesis, respectively.SRCandACTN1genes, were found to cause bleeding using high-throughput sequencing of a large patient cohort and the human phenotype ontology.SLFN14,RUNX1, andFLI1genes were found to be involved in bleeding.RASGRP2, involved in inherited bleeding disorders.Different platforms are introduced in the past few years to identify the underlying cause of inherited platelet disorders using next-generation sequencing. In the BRIDGE-Bleeding and Platelet Disorders (BPD) study, novel variants in different genes, such asThis demonstrates that genomics underwent an evolution in which initially platelet-specific genes were identified and investigated, followed by the identification of genes whose transcripts correlated with a phenotype, to currently end up starting with a diagnosed platelet defect. This approach pretty much resembles the one that in the early days led to the discovery of GPIb and αIIbβ3. Nevertheless, starting from the diagnosis of hereditary bleeding or platelet disorders provides much more power to provide insight into platelet function and to unravel new platelet receptors with a putative role in thrombosis and hemostasis.


In the current article, we further discuss the possibility of a closed, feed-forward loop between SOX2 and CDK1 through a long non-coding RNA, CCAT1, which would explain the sustained activation of this loop. Despite the extensive investigation of the cancer stemness as a cause of drug resistance, its role in immune evasion still requires further understanding, and hence, in this article, we further discuss the possibility of this CDK1-SOX2 axis contributing to immune resistance through modulating cell-to-cell interaction directly or indirectly in the tumor microenvironment.The concept of cancer stemness has undergone a paradigm shift during the last decade where there is wider acceptance of the idea that stemness in cancer is a more dynamic and plastic phenomenon than previously thought. However, we have yet to understand the mechanisms on how this stochastic plasticity arises and is maintained. Recently, we have shown that CDK1 plays a critical role in stochastic stemness and tumor initiation potential through regulating SOX2 phosphorylation in multiple cancer types. The phosphorylation of SOX2 affects its nuclear localization, thereby determining the transcriptional fate of its downstream targets. We have also validated the significance of these findings using clinical samples by demonstrating that CDK1 Cancer stemness is a concept that has evolved with our understanding of the nature of cancer cells. In certain cancer types such as colon cancer and squamous cell carcinoma, lineage-tracing studies using mouse models have pointed to a hierarchical model of cancer development where epithelial stem cells have been identified as the cause of tumor initiation. However, cancer cells exhibit an exorbitant plasticity in their phenotypes after the transition into a cancerous state, and even adult stem cells can exist in a state of constant flux or non-hierarchical plasticity in the tissue microenvironment, all of which implicate a shift from a hierarchical rigid model of cancer stem cells to a non-hierarchical or phenotypically plastic model .Despite numerous studies investigating the phenotypic markers and functional consequences of the plastic state of cancer cells, there is a dearth of information available on the molecular mechanisms regulating this plasticity. Our recent study sheds light on this field by elucidating a novel mechanism of CDK1 contributing to cancer stemness. We have demonstrated an upregulation of CDK1 in a subpopulation of melanoma and colon cancer cells with high MHC I expression that displayed high tumor initiating potential . The expWe have also delineated an indispensable role for the stem cell transcription factor SOX2 in CDK1-mediated stemness and tumorigenesis by demonstrating the interaction of CDK1 with SOX2 in cancer cells. SOX2 is known to be instrumental in tumorigenesis in multiple cancer types , 4; howeFurthermore, we have validated the clinical significance of our findings by unveiling the involvement of CDK1 in MYC target genes, mTOR signaling and DNA repair pathways in patient tumors from melanoma, colon and pancreatic cancer. Among these signatures, the upregulation of MYC target genes is of particular importance because MYC can bind to up to 85% of the same promoters as SOX2, amplifying its downstream targets including various CDKs . TherefoAnother emerging field of interest is the role of cancer stemness in immune evasion. Immunotherapies have shown to be effective in many cancer types including melanoma; however, only a minority of patients exhibit a durable and sustained response to immunotherapies . AccumulAltogether, our study provides a mechanistic perspective on cancer progression through CDK1-SOX2-mediated cancer stemness and immune evasion of tumor cells. Hence, targeting the CDK1-SOX2 axis would hold immense promise in future cancer therapeutics, and warrants further investigation.


Most tumor cells reprogram their glucose metabolism as a result of mutations in oncogenes and tumor suppressors, leading to the constitutive activation of signaling pathways involved in cell growth. This metabolic reprogramming, known as aerobic glycolysis or the Warburg effect, allows tumor cells to sustain their fast proliferation and evade apoptosis. Interfering with oncogenic signaling pathways that regulate the Warburg effect in cancer cells has therefore become an attractive anticancer strategy. However, evidence for the occurrence of the Warburg effect in physiological processes has also been documented. As such, close consideration of which signaling pathways are beneficial targets and the effect of their inhibition on physiological processes are essential. The MAPK/ERK and MAPK/JNK pathways, crucial for normal cellular responses to extracellular stimuli, have recently emerged as key regulators of the Warburg effect during tumorigenesis and normal cellular functions. In this review, we summarize our current understanding of the roles of the ERK and JNK pathways in controlling the Warburg effect in cancer and discuss their implication in controlling this metabolic reprogramming in physiological processes and opportunities for targeting their downstream effectors for therapeutic purposes. Cellular metabolism is the process by which a living cell converts nutrients into energy or new macromolecules through a series of biochemical reactions, known as catabolic pathways and anabolic pathways, respectively . ThroughOver the past decade, aerobic glycolysis has taken center stage in cancer research, because it is characteristic of essentially all types of cancer as well as its implications for cancer diagnosis and monitoring –6, 11–14Of the three types of MAPKs, ERKs and JNKs have been recently shown to regulate the redirecting of energy harvest to glycolysis in both malignant and highly proliferative cells by affecting the activity of key metabolic regulators. Here we provide a comprehensive overview of the functional implications and our current knowledge of the role of ERK and JNK signaling pathways in regulating glucose metabolism of highly proliferating cells in cancer and some physiological contexts, such as inflammation and immunity as well as tissue development.+ from NADH in an enzymatic reaction catalyzed by lactate dehydrogenase A (LDHA). As NAD+ is consumed by glyceraldehyde-3-phosphate dehydrogenase activity in a reaction that generates 1,3-biphosphoglycerate from GAP in glycolysis , in vitro , 75. DifSLC2A1) gene and specific glycolytic genes as well as elevated lactate production accompanied by a decline in mitochondrial activity. Additionally, flux into the PPP, which allows the synthesis of nucleotides and NADPH generation, also enhances .Fig.Figcis-tHowever, it is worth noting that c-Myc not only promotes glycolysis but also favors mitochondrial respiration by enhancing the expression of genes involved in mitochondrial structure and biogenesis , 124–126There is also striking evidence that ERK1/2 activation is critical for the switch from OXPHOS to glycolysis observed in activated T cells that, as discussed above, is essential for T cell effector differentiation and function. Indeed, pharmacologic inhibition of ERK1/2 activity blocked the increase in glucose uptake and glycolysis as well as mRNA expression and activity of the glycolytic enzyme HK induced by the ligation of the TCR and the co-stimulatory receptor CD28 . Furtherjnk1 (mapk8), jnk2 (mapk9), and jnk3 (mapk10) ..146].Moreover, it was shown that activation of the JNK pathway by the histone methyltransferase inhibitor chaetocin, which induces apoptosis in cancer cells by inducing ROS production, resulted in a reduction of glucose uptake and lactate production in glioma cells . AlthougJnk1 alleles or silencing of Jnk1 by small interfering RNA abrogates glycolysis induced by growth/survival factors, such as serum or IL-3 [However, in the literature there are also examples where activation of the JNK signaling drives aerobic glycolysis, instead of inhibiting it. Deng et al.  reported or IL-3 . Further or IL-3 . Thus, b or IL-3 . Of note or IL-3 . Activat or IL-3 .In conclusion, the information outlined here indicate that JNK signaling (more likely JNK1) acts as negative regulator of aerobic glycolysis in different types of glycolytic tumors, suggesting an intricate link between JNK and cellular metabolism. These studies also provide evidences for JNK regulating an inextricable crosstalk between apoptosis and cancer metabolism and opens up an interesting opportunity to explore the importance of understanding both the functional roles of each JNK protein in the context of tumor metabolism, in order to validate the therapeutic potential of JNK inhibition in cancer. As pointed out earlier, restraining of the JNK signaling is a common trait of glycolytic tumors , 147. LiA common characteristic of cancer cells that distinguish them from their normal counterparts is an increase in glycolysis with concomitant lactate production (the Warburg effect) , 11, 12.


The aim of this study was to assess the correlation of hemoglobin A1c (HbA1c) levels with choroidal thickness in patients with type 2 diabetes mellitus (DM) using spectral domain optical coherence tomography (SD-OCT).In this prospective case series, 180 eyes from 90 patients with type 2 DM were classified into three study groups based on HbA1c values: group 1 included patients with good glycemic control (HbA1c Mean central, nasal, and temporal choroidal thicknesses in diabetic patients (247.80, 238.63, and 239.30  HbA1c values have a significant correlation with choroidal thickness in diabetic patients, and better glycemic control with HbA1c  The clinical features and pathogenesis of DR are primarily related to retinal vascular changes; however, choroidal vascular damage may have an important role. Delayed filling of the choroidal vessels using indocyanine green angiography has been reported in eyes with DR.,4,5 Moreover, reduction in the choroidal blood flow and choroidal volume has been demonstrated in eyes with both non-proliferative and proliferative DR using Doppler flowmetry. Choroidal vascular changes, including choroidal aneurysms, choroidal vessel narrowing and tortuosity, choroidal neovascularization, and choroidal non-perfusion may occur in eyes with DR.,8Diabetic retinopathy (DR) is one of the major causes of visual impairment worldwide.in vivo imaging of the posterior segment of the eye. Nowadays, improvement of the enhanced depth imaging (EDI) software permits highly reliable measurement of the choroidal thickness.,11Optical coherence tomography (OCT) is a non-invasive method that allows high resolution  A previous study reported that at least 1% reduction in HbA1c can lead to significant reduction of the serious complications of DM, including death, myocardial infarction, and microcellular damage. Hemoglobin A1c (HbA1c) is one of the standard tools for the assessment of glycemic control and its optimum value is 5.6–7% in patients with diabetes.The aim of this study was to evaluate the correlation of HbA1c values with choroidal thickness in diabetic patients using EDI-OCT. In eyes with "no diabetic retinopathy", as the name implies, there were no diabetic changes in the fundus examination, and eyes with "mild NPDR" were characterized only by the presence of a few microaneurysms. All fundus examinations were performed by a single vitreoretinal specialist.Patients with type 2 diabetes mellitus (DM) with no evidence of DR or with mild non-proliferative diabetic retinopathy (NPDR) were enrolled in this cross-sectional study. The disease severity was determined based on the clinical findings and using the International Clinical Disease Severity Scale for DR.The exclusion criteria were the presence of DR more than mild NPDR, diabetic macular edema (DME), any other ocular disorders, history of ocular surgery, including laser photocoagulation or intraocular anti-vascular endothelial growth factor (anti-VEGF) injection, myopia more than The current study was approved by the Ethics Committee of our institute and was performed in agreement with the ethical principles of the Declaration of Helsinki. Informed consent was obtained from every enrolled subject.EDI-OCT using Heidelberg spectral domain OCT  was performed without pupillary dilation on the same day as blood sampling. Complete ophthalmic examinations were performed one or two days before EDI-OCT imaging. All OCT scans were performed by the same experienced operator between 11 and 11:45 am to avoid potential choroidal thickness changes with circadian rhythm.Central sub-foveal choroidal thickness was measured using the caliper that is present  from the hyper-reflective line corresponding to the Bruch's membrane under the retinal pigment epithelium (RPE) to the interface of the choroid and sclera, in the central horizontal B-scan passing directly through the foveal center. Then, the nasal and temporal choroidal thickness were measured 500 All subjects were enrolled into three study groups based on HbA1c values:Group 1 included patients with good glycemic control . Mean A total of 180 eyes from 90 diabetic patients and 50 eyes from 25 non-diabetic patients were enrolled in this study. Group 1 included 48 eyes from 24 diabetic patients with good glycemic control  were significantly lower than those in the non-diabetic healthy subjects (50 eyes) [Table 2]; however, the mean central macular thickness in the diabetic eyes (251.51 Choroidal thickness in diabetic patients in group 1 had no significant difference with non-diabetic patients (The mean central choroidal thickness in diabetic patients had no significant correlation with FBS (r = Since patients with DME were excluded from this study, the mean central macular thickness was not different among all study groups.In this study, we found that the choroidal thickness in non-diabetic patients was significantly greater than in diabetic patients. Moreover, this study showed that the choroidal thickness in non-diabetic patients was similar to that in diabetic patients with good glycemic control (HbA1c  They examined 203 eyes of 203 diabetic patients and 48 eyes of 48 non-diabetic controls and showed that the sub-foveal choroidal thickness was less in the eyes with NPDR or PDR than that in the healthy non-diabetic eyes; however, there was no significant difference between the diabetic eyes with no DR and the normal control eyes. Sudhalkar et al assessed choroidal thickness changes in different types of DR. They showed that diabetic patients with or without DR had a significantly thinner choroid compared to non-diabetic control group. Moreover, they found that patients with PDR had a thinner sub-foveal choroid than patients with NPDR. The authors concluded that choroidal thinning continued with increasing stages of DR.Lee and co-workers evaluated the choroidal thickness in diabetic patients using SD-OCT. Sudhalkar et al, as well as some other previous studies reporting choroidal thinning in diabetic patients compared to non-diabetic patients.,17 However, some other studies reported no significant difference in choroidal thickness between the diabetic and non-diabetic eyes.In the current study, we showed that choroidal thickness in diabetic eyes is less than that in non-diabetic control eyes. This finding is in agreement with Lee et al, which may lead to choroidal hypoxia and ischemia and finally choroidal thinning. Yazici and co-workers compared the choroidal thickness in diabetic patients with and without polyneuropathy and healthy control subjects. They reported that the central choroid is thicker in diabetic patients compared to normal subjects and also showed that there was additional choroidal thickening in diabetic patients with polyneuropathy. They concluded that choroidal thickening may be related to autonomic dysregulation secondary to diabetic neuropathy. Kocasarac et al compared the choroidal thickness between diabetic patients with nephropathy and diabetic patients without nephropathy and found that the choroid is thinner in patients with diabetic nephropathy. Additionally, Faries et al reported that in diabetic patients with microalbuminuria, central choroidal thickness is less than diabetic patients without microalbuminuria. Microalbuminuria is an indicator of generalized vascular dysfunction; therefore, choroidal thinning in these patients may be a sign of choroidal vascular damage and dysfunction.A previous study showed that abnormalities in the choriocapillaris and reduction of choroidal blood flow occurred in the early stages of DR, They reported that the sub-foveal choroid in patients with no DR was significantly thicker than that in patients with DR and without macular edema. Moreover, they reported that no significant differences in HbA1c levels were detected among the three study groups and concluded that there was no correlation between sub-foveal choroidal thickness and HbA1c values. Unlike Sahinoghlu-Keskek et al's study, we classified diabetic patients with the same DR severity based on HbA1c values and compared the choroidal thickness among them and found that the choroidal thickness in the healthy control group was nearly equal to that in patients with good glycemic control (HbA1c  Therefore, in patients with uncontrolled DM and high levels of HbA1c, the choroid is expected to be thinner. Unsal et al reported a weak to moderate negative correlation between the sub-foveal choroidal thickness and HbA1c values, which is compatible with our results.Sahinoglu-Keskek et al classified 122 eyes of 122 diabetic patients into three study groups including diabetic patients without DR, diabetic patients with DR and no macular edema, and diabetic patients with DR and macular edema. this may be due to the lower choroidal blood flow and choroidal hypoxia in the higher concentrations of blood sugar that ultimately may lead to choroidal thinning. Diabetic choroidopathy may play a major role in the pathogenesis of DR because the outer retinal layers are dependent on the choroid for nutrition and oxygenation.Poorly controlled HbA1c is associated with more severe stages of DR;,26In the current study, we found that there was no significant correlation between the duration of diabetes and choroidal thickness. This is compatible with the studies of Sahinoghlu-Keskek et al and Shen et al.We are aware of some of the limitations of our study. First, we had no axial length data; however, the patients with high refractive errors were excluded. The possibility of measurement errors due to manual measurement and measurement by a single observer constitute other limitations.In conclusion, we showed that the choroidal thickness in normal control subjects was almost equal to diabetic patients with good glycemic control (HbA1c Nil.There are no conflicts of interest.


Longevity is determined by a number of factors, including genetic, environmental and lifestyle factors. A major factor affecting longevity is the development of degenerative disorders such as cardiovascular disease, diabetes, kidney disease and liver disease, particularly where these occur as co-morbidities. In this article, we review the potential role of supplementation with coenzyme Q10 (CoQ10) for the prevention or management of these disorders. Thus, randomised controlled clinical trials have shown supplementation with CoQ10 or CoQ10 plus selenium reduces mortality by approximately 50% in patients with cardiovascular disease, or in the normal elderly population, respectively. Similarly, CoQ10 supplementation improves glycaemic control and vascular dysfunction in type II diabetes, improves renal function in patients with chronic kidney disease, and reduces liver inflammation in patients with non-alcoholic fatty liver disease. The beneficial role of supplemental CoQ10 in the above disorders is considered to result from a combination of its roles in cellular energy generation, as an antioxidant and as an anti-inflammatory agent.  Longevity depends on a number of factors, one of the most important determinants being the development of chronic degenerative diseases, particularly where these occur as co-morbid conditions. For example, a 70-year old individual with no chronic degenerative diseases will be expected to live for more than 20 years, whereas those with several such disorders would be predicted to live some 8 years fewer . In the The role of CoQ10 supplementation in the treatment or prevention of cardiovascular disease has been detailed in previous reviews ,10. AlthThe Q-SYMBIO study was carried out in some 400 patients with chronic heart failure , and the effect of CoQ10 supplementation (3 × 100 mg/day for two years) on symptoms and biomarker status  were assessed . AssessmSupplementation with CoQ10 reduced the risk of MACE by 42%, with a reduction of approximately 40% in both cardiac related deaths and all-cause mortality. There was no significant difference in adverse events between the CoQ10 treated and placebo groups over the duration of the study. CoQ10 has therefore been reported as the first novel drug  to improve heart failure mortality in over a decade.The KISEL-10 study was a double blind, randomised, placebo controlled clinical trial carried out on normal elderly individuals (70–88yrs) from the Kinda region of Stockholm ; 440 parThe KISEL-10 study demonstrated long-term supplementation with CoQ10 and selenium significantly improved quality of life and heart function, and reduced hospitalisation frequency and the risk of cardiovascular related mortality in the elderly. At a mechanistic level, the benefits of supplementation with CoQ10 and selenium are derived both from their role in cellular energy production, and as tissue protecting antioxidants. Supplementation with CoQ10 and selenium may be of particular benefit for individuals prescribed statins, since the latter are known to interfere in the production of both CoQ10 and selenoproteins such as thioredoxin reductase and glutathione peroxidase . ThioredThe KISEL-10 study supplemented two nutrients, CoQ10 and selenium, with a key role in heart function, which were also likely to be deficient in the elderly population investigated. In the case of CoQ10, most of the body’s daily requirement is obtained by endogenous synthesis. As people age, the capacity for endogenous synthesis declines, such that blood CoQ10 levels in a 65 year old are approximately half that in a 25 year old. With regard to selenium, dietary intake in many European countries, including Sweden and the UK, is known to be sub-optimal, particularly in the elderly. The above provides a rationale for the success of the KISEL-10 protocol in reducing the risk of death from heart disease.Amongst the normal elderly Swedish population, those with the lowest levels of blood selenium were found to be at increased risk of cardiovascular mortality . The ageThe potential benefit of CoQ10 supplementation in type II diabetes has been recently reviewed , from whSome 15 randomised controlled clinical trials supplementing CoQ10  in type II diabetic patients are currently listed by Medline. Although Eriksson et al.  found noSimilarly, glycaemic control and blood antioxidant levels were significantly improved in type II diabetics following supplementation (100 mg/day for three months) with the reduced (ubiquinol) form of CoQ10 . In patiSupplemental CoQ10 may benefit type II diabetes via several mechanisms, for example by promoting enhanced levels of cellular energy required for glucose metabolism, or via direct modulation of the expression of genes relevant to glucose metabolism, or via its antioxidant action . Thus, sThe use of statins (particularly simvastatin) has been associated with an increased risk of between 10% and 40% of developing type II diabetes ; this isThe role of supplemental CoQ10 in chronic kidney disease (CKD) has been reviewed , from whAlthough haemodialysis is essential for removing uremic toxins, it is a consequence of the procedure that individuals are subject to additional oxidative stress , in addition to the oxidative stress associated with CKD. A number of clinical studies have reported that supplementation with CoQ10 significantly improves outcome in haemodialysis patients by reducing markers of oxidative stress and inflammation. In a randomised controlled trial, Zahed et al.  reportedPatients with CKD are at high risk of developing cardiovascular disease, with a 10–20 fold increased risk of cardiovascular mortality compared to non-CKD individuals. Overall, approximately 50% of deaths in CKD patients result from cardiovascular disease, rather than as a direct consequence of kidney failure. Conversely, cardiovascular disease can cause CKD leading to a vicious circle in which each disorder exacerbates the other. Thus, treatment of CKD can reduce the incidence of cardiovascular disease, and treatment of cardiovascular disease can reduce further deterioration in renal function. The ratio of plasma CoQ10 versus low density lipoprotein (LDL) cholesterol + VLDL (very low density lipoprotein) cholesterol, considered to be more important in athersclerosis prevention than the ratio of HDL (high density lipoprotein ):LDL cholesterol , was sigEpicardial fat thickness, a new risk factor for cardiovascular disease, was found to be significantly greater in CKD patients undergoing haemodialysis compared to controls and correlated with reduced plasma CoQ10 levels . SimilarAlthough endogenous CoQ10 synthesis occurs throughout the body, because of its physical size and high metabolic capacity, the liver is the major site of CoQ10 synthesis. In patients with liver disease where metabolic capacity has been compromised, a reduction in CoQ10 production is likely to have a deleterious effect on heart function. Thus, non-alcoholic fatty liver disease (NAFLD) is a risk factor for cardiovascular disease, which has been reported to be one of the major causes of death in NAFLD patients . NAFLD iIn addition to reducing the risk of cardiovascular problems in patients with liver disease, CoQ10 supplementation may also benefit the disease process within the liver by reducing inflammation and oxidative stress. For example, one of the principal mechanisms by which alcohol causes liver damage is via the generation of free radicals, and the antioxidant action of CoQ10 can help to protect liver cells from such oxidative damage . Free raA number of studies in animal models have demonstrated the ability of CoQ10 to reduce or prevent the development of liver cirrhosis following a variety of toxic insults; these include exposure to medicinal drugs , toxic cThere have been relatively few clinical studies relating to CoQ10 and liver disease. Evidence for depleted CoQ10 blood levels in NAFLD patients was reported by Yessilova et al. . Two ranWith regard to clinical studies involving oral supplementation with CoQ10, one very important issue that has received relatively little attention is the question of supplement quality and bioavailability. Because nutritional supplements are not regulated in the UK in the same way as prescription medicines, there is no legal quality requirement relating to defined levels of active substances and product stability; supplements may therefore, for example, contain lower levels of CoQ10 per capsule than stated on the product packaging. The best way to avoid this problem is therefore to use a CoQ10 supplement that has been manufactured to pharmaceutical standards.There are currently more than 300 randomised controlled clinical trials relating to CoQ10 listed on Medline, including 70 on heart disease, 30 on diabetes, 10 on renal disease and six on liver disease. Whilst the majority of such studies have reported significant benefit of CoQ10 supplementation (particularly in heart disease), some studies have reported no significant benefit. Such disparity may result from a number of factors, including insufficient CoQ10 dosage or treatment duration, and inter-individual variation in the ability to absorb CoQ10, but particularly from inadequate bioavailability, especially where CoQ10 blood levels have not been determined pre- and post-supplementation. Because of the molecular characteristics of CoQ10, bioavailability is intrinsically low. As a lipid soluble substance, bioavailability is optimized via the use of a carrier oil such as soy oil; however, the key factor in determining bioavailability is the efficient dispersion of CoQ10 crystals formed during the yeast fermentation manufacturing process . SimilarAnother factor affecting efficacy is the relative bioavailability of the ubiquinone and ubiquinol forms of CoQ10. Some supplement manufacturers have claimed that the ubiquinol form is more bioavailable, based on the concept that absorption of CoQ10 into enterocyte cells was thought to require reduction of ubiquinone to ubiquinol, and presentation of supplemental CoQ10 in ubiquinol form facilitated this absorption, particularly in patients with malabsorption disorders. However, work by Judy  demonstrIn summary, we have reviewed evidence from randomised controlled clinical trials as to how oral supplementation with CoQ10, either alone or in tandem with selenium, can significantly reduce mortality risk from cardiovascular disease in normal elderly subjects, or in those with heart failure. In addition, supplementary CoQ10 may reduce mortality risk in patients with type II diabetes, chronic kidney disease or liver disease, both by beneficial effects on the primary disease process in these tissues, or on cardiovascular dysfunction secondary to these disorders.


Benign prostatic hyperplasia (BPH) is a disease of aging that impacts 50% of men in their 50s and 90% of men in their 80s. While rodent models have been invaluable in the study of lower urinary tract dysfunction (LUTD) associated with human disease, many studies recapitulate aspects of aging, steroid hormone fluctuations, and/or inflammation without using aged mice that would better correspond to the age range of patients. In this study, we examine the impact of age in the hormone-induced mouse model of LUTD, so we can better understand the contribution of age to disease initiation and progression. We’ve discovered that aged mice exhibit a level of LUTD that is further exacerbated by hormone implantation when compared to both treated and untreated younger mice. Examination of circulating levels of androgens and estrogens indicate an alteration in steroid hormone metabolism with age, suggesting an altered nuclear receptor activation within disease. Epigenetic modifications have been associated with normal aging, including an increase in DNA methylation to alter gene expression. Examination of the proximal promoter of a steroid enzyme gene, CYP7B1, responsible for the degradation of 3β-diol (an ERβ ligand) within the prostate shows an age-mediated increase in methylation. With this, there is a corresponding decrease in CYP7B1 gene expression in the aged mice. Taken together, this suggests the altered steroid hormone environment seen in LUTD is further exacerbated due to normal aging-related epigenetic modifications that need to be considered for better treatment efficacy.


In February 2018, the 6th World Symposium on Pulmonary Hypertension (WSPH) brought together experts from various disciplines to review the most relevant clinical and scientific advances in the field of PH over the last 5 years. Based on careful review and discussions by members of the different task forces, major revisions were made on the hemodynamic definition for various forms of PH and new genes were added to the list of genetic markers associated with pulmonary arterial hypertension (PAH) and pulmonary veno-occlusive disease. In addition, the use of risk stratification tools was encouraged as a strategy to reduce one-year mortality risk in PAH patients through early implementation of PAH therapies. While members of the medical community are still debating some of the proposed changes, the new WSPH guidelines advocate early diagnosis and initiation of combination therapy to reduce mortality and improve quality of life in patients with PH. Forty-five years later, the 6th WSPH took place in Nice and brought together 124 experts in 13 different task forces to review clinical and scientific advances pertinent to improving the diagnosis and management of PH3. Considering the recommendations of the various task forces, the 6th WSPH has revised the hemodynamic definition of PH for the first time since 1973. Newly discovered genes associated with hereditary and idiopathic PAH  were discussed and recommendations were made to update the clinical classification and implement risk stratification in the treatment algorithm of PH. These changes are now a topic of active debate within the scientific community. This report will summarize the highlights of the 6th WSPH, and the implications of the proposed changes to future basic and clinical research in PH will also be discussed.Following an epidemic of pulmonary arterial hypertension (PAH) cases in the 1960s in Germany, Austria and Switzerland due to the anorectic stimulant Aminorex, the World Health Organization (WHO) organized the first World Symposium on Pulmonary Hypertension (WSPH) in October 1973 in Geneva, Switzerland4,BMPR2 mutations remain the most common genetic cause of PAH, accounting for about 80% of HPAH and about 20% of IPAH. BesidesBMPR2, other transforming growth factor beta (TGF-β) superfamily genes, includingALK1/ACVRL1 (a heterodimeric partner of BMPR2),BMP9 (a BMPR2 ligand),ENG , andSMAD1,4, and9 , have been linked to both HPAH and IPAH5. In the last 5 years, next-generation sequencing technologies have been applied to genetic discovery in patient populations with IPAH, HPAH, and drug-induced PAH. For example, whole exome sequencing (WES) inBMPR2-negative HPAH patients led to the discovery of two novel genes:CAV1 6 andKCNK3 7. More recently, a WES screen of pediatric patients with HPAH revealed that, in addition toBMPR2, the most common mutation in this patient population was inTBX4, a gene linked to small patella syndrome8. Besides WES, whole genome sequencing (WGS) has now been used to characterize novel high-risk gene variants in large patient populations. A WGS study of 1048 patients with PAH by Gräf and colleagues9 screened and found novel mutations inGDF2 (which codes for BMP9) and identifiedATP13A3,AQP1, andSOX1710 as novel gene candidates, although the specific pathogenic mechanism of these in PAH remains to be determined.Since its discovery in 2000EIF2AK4 (a kinase involved in amino acid metabolism) were associated with both PVOD and PCH12. In contrast toBMPR2 mutations,EIF2AK4 mutations are autosomal recessive and completely penetrant. Detection ofEIF2AK4 in a patient with PAH can establish the diagnosis of PVOD/PCH in the appropriate clinical context without necessitating lung biopsy12.Pulmonary veno-occlusive disease (PVOD) and pulmonary capillary hemangiomatosis (PCH) are severe forms of WHO group 1 PH with a rapidly progressive clinical course and poor response to therapy. In 2014, the first of two scientific groups reported that mutations in13.The 6th WSPH Task Force on Genetics and Genomics has updated the list of gene candidates for PAH to include these new genes and strongly recommends research in understanding how they are linked to PAH pathogenesis and how this information could be used in biomarker discovery and new therapeutics13. At this point, a pedigree can be generated to identify relatives at risk, although gene testing or screening should be initiated with affected patients. There are different methods of evaluating the genetics of affected patients. In addition to commercially available diagnostic PAH/PVOD gene panels, WES or WGS may be appropriate for a patient with a negative gene panel and also have important relevance for research. However, it must be stressed that these diagnostic tests should be ordered by a specialist who is trained in genetics and who would assume the responsibility of counseling patients and their families regarding the implications of the test results.As WES and WGS become less expensive and more available, the importance of explaining these results and their significance for patients and potential offspring is critical. Given the complexities of the genetic processes underlying HPAH, this only adds nuance to the difficult task of genetic counseling to patients with a family or personal history of HPAH looking to conceive. The psychosocial concerns of genetic screening for a disease for which there is no prevention and no specific cure may weigh heavily on those affected and may cause guilt to those not phenotypically affected or who could pass disease-causing mutations onto children. After discussing the potential benefits of genetic screening , the 6th WSPH task force recommended that genetic screening be carried out under the guidance of a genetic counselor or clinical geneticist14. The authors based this recommendation on the fact that the original definition of mPAP of at least 25 mm Hg was chosen somewhat arbitrarily and does not represent the upper limit of normal mPAP in the general population. Review of all available studies on pulmonary hemodynamics in healthy individuals indicates that a normal mPAP is approximately 14 ± 3.3 mm Hg and that the upper limit (>97.5th percentile) of normal is 20 mm Hg15. Major concerns regarding inclusion of individuals with mPAP between 21 and 24 mm Hg are the risk of PH diagnosis in otherwise healthy individuals and the lack of data that treatment of this population would be safe or beneficial (or both). Despite these concerns, studies published in the past 5 years suggest that individuals with mPAP of 21 to 24 mm Hg are at increased risk of poor outcomes18 and tend to progress to “overt PH (mPAP of at least 25 mm Hg)” more often than patients with mPAP of not more than 20 mm Hg over a 2- to 3-year follow-up20. Additionally, treatment of some individuals with mPAP of 15 to 25 mm Hg in two separate chronic thromboembolic PH cohorts yielded improved clinical results22 and at least one trial is under way in patients with the same mPAP criteria in PAH associated with systemic sclerosis . Based on these data, the 6th WSPH task force recommended that a new mPAP of more than 20 mm Hg cutoff for diagnosing PH is both clinically warranted and in the best interest of the patient  of at least 25 mm Hg. During the 6th WSPH, the task force members suggested that this definition be changed to mPAP of more than 20 mm Hgpatient .14. The subcategorization and method of detecting CpcPH remain controversial. Evidence is suggestive of CpcPH as a distinct entity from PAH or IpcPH, which carries a different prognosis both before and after heart transplantation (6th WSPH on left heart disease–PH section). While PVR of at least 3 WU has strong evidence to support its diagnostic utility, other hemodynamic markers such as the transpulmonary gradient and pulmonary arterial compliance have demonstrated value in two studies for clarifying the diagnosis, and thus prognosis, in at-risk individuals24. Efforts have been made to standardize methodology to distinguish between CpcPH and IpcPH via exercise testing (CpcPH should have a greater rise in PVR than patients with IpcPH) and fluid challenge (IpcPH should have a disproportionate rise in PAWP), although these are technically difficult in the case of the former and there is a lack of consensus cutoff data for what would characterize an abnormal physiologic response in the latter.Pulmonary vascular resistance (PVR) has also taken on a new role in PH classification. Established in accordance with the 5th WSPH, PVR was considered important for diagnosing PAH but was not included in the general definition of all forms of PH, and the diagnostic criteria for combined pre- and post-capillary PH remained unclear. Now, in keeping with the 6th WSPH update, all PH (mPAP of more than 20 mm Hg) will be further subclassified as pre-capillary PH (as seen in PAH), isolated post-capillary PH (IpcPH), or combined pre- and post-capillary PH (CpcPH) on the basis of pulmonary arterial wedge pressure (PAWP) and PVR. Although the threshold and application of significant PAWP have not changed , PVR now defines the presence or absence of pre-capillary PH 26 and that, in certain situations, re-classification into a different category at follow-up on the basis of re-scoring can predict outcomes over the next year. Within the mélange of these risk stratification systems, the following parameters appear to have the greatest predictive capability: functional class, six-minute walk distance (6MWD), N-terminal pro-brain natriuretic peptide/brain natriuretic peptide (NT-proBNP/BNP) levels, cardiac index, right atrial pressure, and mixed venous oxygen saturation (SvO2)28. The guidelines acknowledge that the limitations of these tools include the inability to account for non-modifiable/ill-modifiable risk factors such as age, sex, comorbidities, and additional data available through advanced investigative techniques such as cardiac magnetic resonance and cardiopulmonary exercise testing29. In addition, most of these tools are time-consuming to use and require data points that are not routinely obtained. At present, it is also unclear which parameters define the set of patients with prolonged clinical stability. Use of machine learning and artificial intelligence in future work may assist in developing new, low-bias prediction tools that eliminate some of the above pitfalls with current prediction scores and tools31.The clinical status and trajectory of patients with PAH can be defined through the use of multiple validated risk stratification tools. The many available comprehensive risk stratification tools use a combination of parameters that assess clinical, functional, cardiac, and hemodynamic status to assign patients to low-, intermediate-, or high-risk categories. Without delving into the intricacies of the superiority of one validated assessment tool over the other, what is clear is that the baseline use of these tools can predict survival and event-free survival for up to 5 years29.Even as these risk calculators continue to evolve, they remain an important tool in assessing patients in the clinical and research setting while helping to guide management. The task force members recommend that future iterations or refinements also take into consideration accuracy and cost-effectiveness of obtaining included parameters29.The Dana Point 4th WSPH recommendations from 2008 led to a significant shift in PAH randomized control trial (RCT) design. Multiple recent trials using more clinically meaningful end-points  have led to improved confidence in, and adoption of, choices for therapeutic strategy and drug combinations. For all patients with PAH, activity within symptom limits is advised, and recent data affirm the benefits of supervised training across the spectrum of functional impairmentTreatment of WHO group 1 PAH by targeting the nitric oxide, endothelin, and prostaglandin pathways has been standard since the 2003 Venice WSPH guidelines; no drugs targeting other pathways have been approved in the interim. The 6th WSPH task force has proposed a revised treatment algorithm  that can- All patients with a diagnosis of WHO group 1 PAH should be referred to a PH center to guide treatment and ongoing management.- Monotherapy from any class based on suitability has been relegated a “residual role” in patients with the following:1) Vasoreactive PH patients who maintain reactivity, functional class I/II with sustained hemodynamic improvement after at least 1 year on calcium channel blockers only2) Patients with a low-risk profile who have historically been stable on monotherapy3) IPAH patients more than 75 years old with multiple risk factors for left heart disease4) PAH patients with suspicion or high probability of PVOD or PCH5) Patients with PAH associated with HIV, portopulmonary hypertension, or uncorrected congenital heart disease, as they were not included in RCTs of upfront combination therapy6) Patients with very mild disease defined on the basis of WHO functional class I, PVR of 3–4 WU, mPAP of less than 30 mm Hg, and normal right ventricle at echocardiography7) Combination therapy unavailable or contraindicated.- Intravenous epoprostenol received the strongest recommendation for therapy in high-risk patients because of proven mortality benefit in patients with PAH even as monotherapy.- The following dual-combination therapies are recommended on the basis of evidence:1) Macitentan and sildenafil2) Riociguat and bosentan3) Selexipag and endothelin receptor antagonist (ERA) or phosphodiesterase inhibitor (PDE5i) or both- Transition from one therapy to another thought to be clinically as efficacious should be carried out under expert guidance.- Given the lack of data and risk of decompensation, “stepping down” of therapy because of significant clinical improvement is generally advised against and should be carried out under expert guidance.- If lack of clinical efficacy is noted with selexipag or non-parenteral prostacyclins, transition to parenteral prostanoids is recommended.As data have accumulated from RCTs over the past decade, an increasing emphasis is being placed on upfront combination therapy. Longer-term data are needed to assess the cumulative impact of this strategy on long-term survival. With the drop in the mPAP criterion from 25 to 20 mm Hg, it is imperative that well-designed clinical trials be carried out to determine the impact on long-term outcomes with early intervention.14. Importantly, new clinical trials have led to an updated treatment algorithm for patients with group 1 PH, in which upfront combination therapy in treatment-naïve patients is now a recommended option33. Given the improved sophistication of our clinical data and genomic understanding, the care of patients with HPAH has entered an era of simultaneously discovering more about the complex genetic underpinnings that lead to mixed penetrance and optimizing treatment for those affected. In conclusion, the 6th WSPH has ushered in new clinical management paradigms while integrating ever-advancing scientific knowledge into the care of the patient with PH.Convincing epidemiological data have provided a rationale that justifies a revision in the hemodynamic definition of PH as mPAP of more than 20 mm Hg6MWD, six-minute walk distance; CpcPH, combined pre- and post-capillary pulmonary hypertension; HPAH, hereditary pulmonary arterial hypertension; IPAH, idiopathic pulmonary arterial hypertension; IpcPH, isolated post-capillary pulmonary hypertension; mPAP, mean pulmonary artery pressure; PAH, pulmonary arterial hypertension; PAWP, pulmonary arterial wedge pressure; PCH, pulmonary capillary hemangiomatosis; PH, pulmonary hypertension; PVOD, pulmonary veno-occlusive disease; PVR, pulmonary vascular resistance; RCT, randomized control trial; WES, whole exome sequencing; WGS, whole genome sequencing; WHO, World Health Organization; WSPH, World Symposium on Pulmonary Hypertension; WU, Wood units