We report the identification of a neurotropic astrovirus associated with encephalitis in a sheep. This virus is genetically almost identical to an astrovirus recently described in neurologically diseased cattle. The similarity indicates that astroviruses of the same genotype may cause encephalitis in different species. They are best known to be associated with gastroenteritis, especially in humans. Recently, reports of these viruses in association with encephalitis have increased dramatically, with reports of cases in humans  in sheep, however, the etiologic agent remains unknown . In 1 anWe used primers based on the BoAstV-CH15 genome sequence (GenBank accession no. KT956903) and the Sanger method to sequence the complete genome of the ovine strain  Figure 1A phylogenetic comparison confirmed the close relationship of OvAstV-CH16 with BoAstV-CH15  Figure 2We then analyzed brain samples of sheep with nonsuppurative encephalitis of unknown etiology (n = 47), which had been identified within the framework of active disease surveillance in Switzerland (To confirm the presence of OvAstV-CH16 in situ, we used polyclonal antisera targeted at the putative capsid protein of BoAstV-CH15 and tested formalin-fixed, paraffin-embedded brain tissues of sheep 41669 by immunohistochemistry . We obseThe importance of the link between astroviruses and encephalitis is increasingly being brought to light, but the factors determining their tropism and neuroinvasion are still unknown. Deeper epidemiologic, genetic, and molecular investigations will help to clarify these aspects of astrovirus pathology. Astroviruses were traditionally considered to be host specific, but in recent years, several reports challenged this assumption; for instance, when human astroviruses were found in fecal samples of primates (Additional materials and details for study of astrovirus associated with encephalitis in a sheep.


Dear Editor,1 recently published in journal. The investigators found a 23%prevalence of patients with acute coronary syndrome (ACS) meeting the diagnosticcriteria for major depressive disorder (MDD). Women were more susceptible to develop MDDin the sample group of ACS patients, with a three-and-a-half-time greater likelihoodthan men.1I have read with great interest the article entitled "Major Depression and Acute CoronarySyndrome-Related Factors" by Figueiredo et al.,2 many of them receiving antiplatelet therapy and oftentreated with a serotonin selective reuptake inhibitor (SSRI). Fluoxetine and fluvoxamineare potent inhibitors of CYP2C19.3Clopidogrel is a pro-drug that undergoes a two-stage activation process, which ismediated by several cytochrome P450 (CYP) hepatic enzymes. CYP2C19 is involved in bothactivation steps, raising concern that the drug that inhibit CYP2C19 may decreaseclopidogrel's effectiveness.4 Bykov etal.5 reported that treatmentwith a CYP2C19-inhibiting SSRI such as fluoxetine and fluvoxamine when initiatingclopidogrel might be associated with slight decrease in its effectiveness.Almost half of patients with ACS are affected by depression,In this context, considering not only the concomitant disorder with ACS but also thecomplicating factor due to CYP2C19-inhibiting SSRI treatment, MDD should be evaluatedmeticulously. The authors are grateful for the comments made in a letter to the editor regardingthe article Major Depression and Acute Coronary Syndrome-Related Factors, publishedin Arq Bras Cardiol. 2017; 108 (3): 217-227.We agree with the relevant comments of the author of the letter, warning about theuse of antidepressants in patients with acute coronary syndrome and theirinteraction with the antiplatelet Clopidogrel.We complement the comments with the following considerations:1) The effectiveness of Clopidogrel (a platelet P2Y12 receptor blocker), in itsaction as a platelet antiadhesive, among other known and unknown actions, isdependent on its conversion to an active metabolite by the cytochrome-dependent P450(CYP- 450), mainly CYP2C19. Its effect as a platelet antiadhesive is lower inpatients who are homozygous for non-functional alleles of CP2C19 genes, so-called"poor metabolizers". It is not recommended the use of Clopidogrel in these patientsand, when possible, to identify these cases using genetic tests. Therefore, anyother drug that interferes with the cytochrome dependent system altering theconversion of Clopidogrel to its active metabolite, mainly in the poor metabolizers,may reduce its effect.Selective serotonin reuptake inhibitor antidepressants  may interfere with the metabolism of clopidogrel, increasing theadverse effect of clopidogrel (by increasing the inhibition of the P2Y12 receptor)and, therefore, increasing the Risk of bleeding, decreasing the concentration of theactive metabolite of Clopidogrel, mainly in the genetically "poor metabolizers",reducing its effectiveness.QT interval on the ECG,ventricular arrhythmias, syndrome of inappropriate antidiuretic hormone secretion,increase of the risk of severe hyponatremias in patients already in use ofdiuretics, among innumerable adverse effects, which always more serious in patientswith acute coronary syndrome.2) Selective serotonin reuptake inhibitor antidepressants have multiple adversecardiovascular effects such as: chest pain, angina pectoris, palpitation, arterialhypertension, prolonged 3) Our article sought to initially verify the presence of the pattern (set ofvariables found in association) that is called "Major Depression" among patientswith another pattern called Acute Coronary Syndrome and hospitalized in the hospitalcoronary units environment. In addition to this identification of variable patterns(diagnostics), we intend to study how and which, among the variables collected inthese individuals, including sociodemographic variables, were associated. We do notintend to define causal associations, even because the design of the study would notallow such inferences. However, we intend to understand the interactions betweenvariables present in a certain evolutionary state of patients' lives. This allows usa better clinical understanding of the person and, therefore, greater possibility ofhelping it during its evolution in this acute period and its subsequent evolution.We do not understand causality deterministically, that is, if the variable x ispresent then y will occur. We understand a person as a complex system that interactswith the environment in which he lives. Therefore, it is more important tounderstand the organization or interaction between component variables, which we canbe identified in that complex system formed by people living in an environment, than to isolate possible "causal" variables,whether genetic or pharmacological and impute a direct causality that does not existor that we only identify in simple or more stable systems. We believe that complexthinking is the basis of clinical judgment and that the important thing is tounderstand the biological system as a whole, due to its constant evolution ordynamics. For this, only observing the system in its continuous evolution.We have chosen the linear log model to study these interactions as shown in Figures 1and 2 of the article. The previous history of depression was one of the variables ofthat model. Certainly, these patients were already exposed to antidepressant druguse during their lifetime, but we cannot conclude, and it was not our goal, thatthese drugs "cause" coronary syndrome. However, from the clinical point of view, theassociations found indicate that individuals and clinicians should be warned thatantidepressants may be involved in the interactions of variables that make up thepattern Acute Coronary Syndrome and its complications (eg. arrhythmias ), and thatthis type of drug also interacts with other drugs such as antiplatelet drugs,diuretics and others, composing other patterns such as hyponatremia syndrome ofinappropriate secretion of antidiuretic hormone, and even opposing patterns such asbleeding or loss of the efficacy of Clopidogrel.Clinical thinking needs to detach itself from the bonds in which deterministic andcausal thinking placed it and return to what has always been clinical judgment, nowwith a strong theoretical basis, complex thinking and complex adaptive dynamicsystems. To do this we have to observe the evolution of the person throughout hislife and his relationship or interaction with the environment in which he lives,including his social and cultural relations and understand the interactions betweeninfinite variables always with a degree of uncertainty and considering theappearance of emerging or unpredictable phenomena.José Henrique Cunha FigueiredoNelson Albuquerque de Souza e SilvaBasilio de Bragança PereiraGláucia Maria Moraes de Oliveira


In recent years the number of disorders known to affect amino acid synthesis has grown rapidly. Nor is it just the number of disorders that has increased: the associated clinical phenotypes have also expanded spectacularly, primarily due to the advances of next generation sequencing diagnostics. In contrast to the “classical” inborn errors of metabolism in catabolic pathways, in which elevated levels of metabolites are easily detected in body fluids, synthesis defects present with low values of metabolites or, confusingly, even completely normal levels of amino acids. This makes the biochemical diagnosis of this relatively new group of metabolic diseases challenging. Defects in the synthesis pathways of serine metabolism, glutamine, proline and, recently, asparagine have all been reported. Although these amino acid synthesis defects are in unrelated metabolic pathways, they do share many clinical features. In children the central nervous system is primarily affected, giving rise to  microcephaly, early onset seizures and varying degrees of mental disability. The brain abnormalities are accompanied by skin disorders such as cutis laxa in defects of proline synthesis, collodion-like skin and ichthyosis in serine deficiency, and necrolytic erythema in glutamine deficiency. Hypomyelination with accompanying loss of brain volume and gyration defects can be observed on brain MRI in all synthesis disorders. In adults with defects in serine or proline synthesis, spastic paraplegia and several forms of polyneuropathy with or without intellectual disability appear to be the major symptoms in these late-presenting forms of amino acid disorders. This review provides a comprehensive overview of the disorders in amino acid synthesis. In recent years exciting developments have taken place in the discovery of disorders of amino acid synthesis. Several new disorders have been reported as well as new phenotypes for already known amino acid synthesis deficiencies, which is mainly due to next generation sequencing of cohorts of patients with similar clinical phenotypes. A comprehensive overview of these recent developments in amino acid synthesis deficiencies will be presented here.We all are aware of the fact that amino acids are the building blocks for peptide and protein synthesis and that they perform important functions in intermediate metabolism. However, many amino acids have specific cellular functions of their own, in neurotransmission for instance, or energy metabolism and detoxification. Historically, the biochemical analysis of elevated levels of amino acids or their degradation products in body fluids has been the cornerstone of diagnosing inborn errors of metabolism. In Awareness of amino acid synthesis disorders is important because of the potential therapeutic consequences and the sometimes very narrow window of opportunity to alter the course of the disease and prevent neurological damage.Defects in the genes encoding the three enzymes of the L-serine synthetic pathway have been reported and, not surprisingly, they all give rise to similar clinical phenotypes. L-serine is synthesized from the glycolytic intermediate 3-phosphoglycerate via three enzymatic conversions. The enzymes involved are 3-phosphoglycerate dehydrogenase , 3-phosphohydroxypyruvate aminotransferase  and phosphoserine phosphatase .When serine deficiency disorders were first reported in paediatric patients it appeared that there were some differences in phenotype between the three defects. However, with recent insights obtained through whole-exome sequencing, it is now obvious that it is not possible to discriminate the different gene defects on clinical grounds. Molecular defects in the genes encoding the three enzymes can present with identical phenotypes ranging from a severe lethal antenatal phenotype to a milder adult onset polyneuropathy phenotype. However, recognition of serine deficiency is important because good treatment results have been reported with L-serine therapy.The severe and lethal serine deficiency phenotype was already known as Neu-Laxova syndrome (NLS). In 2014, two different groups published mutations in serine synthesis genes in patients with this Neu-Laxova syndrome . No treatment with L-serine has been reported in this severe phenotype, but given the extensive and very early onset developmental defects, successful treatment seems doubtful.The 3-PGDH knock out mouse published some years ago very much resembles the NLS phenotype and, so far, rescue of the severe developmental defects in mice with L-serine therapy has not been reported , normalization or improvement of EEG abnormalities may take much longer, in some cases as long as 6–12 months  and, for selected children with an insufficient response of their seizures to L-serine monotherapy, glycine (200–300 mg/kg/day) can be added to the treatment  leading to a defect in the ASCT 1 transporter, give rise to symptoms similar to those seen in serine synthesis disorders. Srour et al  appears to be ultra-rare among the amino acid synthesis disorders. Since this disorder was first reported by Häberle et al Glutamine is the most abundant amino acid in human plasma and CSF. It accounts for up to 20% of the total amino acid content in the human body. Similar to serine, glutamine is an important metabolite involved in maintaining nitrogen balance and cellular energy metabolism. However, glutamine synthetase also plays a key role in regulating the concentrations of ammonia and glutamate in brain tissues by converting these potential neurotoxic metabolites to glutamine.The first two patients reported with glutamine deficiency were severely affected newborns who presented with respiratory insufficiency, hypotonia, absence of spontaneous movement and primitive reflexes  was found to be disturbed in this disorder Hu et al . The depThe first family with a defect in proline synthesis was reported more than 15 years ago by Baumgartner et al . Only reALDH18A1 that lead to autosomal dominant forms of pyroline-5-carboxylate synthase deficiency (OMIM 616603) shifts our classical paradigm of autosomal recessive enzyme defects towards that of (de novo) autosomal dominant paediatric and adult-onset inborn errors of metabolism . However, the discovery of children and adults with specific heterozygous mutations in As in serine deficiency, disorders of proline synthesis can result in a broad spectrum of symptoms ranging from severe neonatal forms to adult onset spastic paraplegia.A deficiency in P5CS will result in a combined deficiency of L-proline, L-ornithine, L-citrulline and L-arginine, a disorder first reported by Baumgartner et al . In thisThe main features in P5CS deficiency are microcephaly, cutis laxa, progeroid features, mental disability, hypotonia, seizures, joint laxicity, (intra uterine) growth retardation, and cataract and corneal abnormalities. In some patients, additional movement disorders such as tremor and dystonia were also present . Patients with P5CS present more often with cataracts and corneal abnormalities, symptoms that are rare in PYCR1 deficiency and abnormalities of plasma amino acids can be observed in P5CS deficiency. The two disorders can only be discriminated by their ultrastructural abnormalities in skin biopsies.P5CS can also cause a similar cutis laxa phenotype. They reported eight paediatric patients with cutis laxa and de novo mutations in ALDH18A1. All mutations affected the same Arg138 residue of P5CS, affecting P5CS activity and leading to mislocalisation of the mitochondrial protein.Interestingly, Fischer-Zirnsak et al  demonstrAs discussed above, de novo dominant mutations are a challenge for our NGS data filtering strategies and our interpretation of NGS results. Since many of the enzymes we study in metabolic disease are also multimeric protein complexes, these observations are very relevant for other enzymes in which dominant mutations affecting dimerization can give rise to a clinical phenotype.ALDH18A1 mutations in adults with spastic paraparesis. Autosomal recessive mutations were associated with a complex spastic paraplegia combined with cognitive impairment. Surprisingly, heterozygous mutations in ALDH18A1 were also found in families with autosomal dominant spastic paraplegia as well as in sporadic spastic paraplegia patients (suggestive of de novo mutations).Coutelier et al  reportedPsychomotor disability and cognitive defects were only seen in patients with the recessive form and biallelic mutations, whereas lower limb spasticity was the predominant clinical sign in patients with dominant mutations. Less frequent findings were cataracts, as observed in paediatric cases. The skin abnormalities including cutis laxa seen in children were not reported in any of the adult patients.ALDH18A1 are the cause of spastic paraparesis type 9, a dominant spastic paraplegia associated with cataract and cyclic vomiting. Heterozygous mutations can cause this disorder because they act as dominant-negative mutations at specific sites, affecting dimerization of the enzyme complex and hence enzyme activity. This again supports the paradigm shift in our field towards (de novo) dominant inborn errors of metabolism associated with enzyme deficiencies.Panza et al  showed tAmino acid analysis in the first family reported with P5CS deficiency showed a combined deficiency of proline, arginine, citrulline and ornithine—an unusual combination of amino acids decreases. Low values of arginine, citrulline and ornithine are all observed in urea cycle defects and its related disorders, but the combination with low proline is only observed in this defect. Plasma ammonia was only mildly increased and decreased after meals in contrast to what happens in urea cycle disorders.Surprisingly, in the majority of paediatric patients reported, no abnormalities were detected in any of the plasma amino acids, and it is poorly understood why the results of biochemical testing are inconsistent in P5CS deficiency. Timing of plasma sampling might be one explanation, another might be whether the mutations are in- or outside the active sites of the enzyme complex  patients had normal plasma amino acids upon testing, and a clinical diagnosis is usually confirmed by mutation analysis. As stated before, it is important to realize that observations of (de novo) heterozygous mutations with dominant negative effects warrant careful interpretation of sequence results, particularly when NGS strategies are used, and that the presence of a de novo mutation favours a trio design of testing patient-parent trios. Confirmation of sequence results that are difficult to interpret is hampered by the absence of an accessible P5CS enzyme assay.In the first report, patients were treated with oral L-ornithine. One patient was treated from the age of five years and the sib from 12 years. Disappointingly, this treatment with L-ornithine had no clinical effect. The patient reported by Martinelli et al , receivePYCR1 were found in 35 affected patients (from 22 families) with a phenotype classified as either de Barsy syndrome, wrinkly-skin syndrome or gerodermia osteodysplastica (OMIM 179035). In none of these patients were abnormal plasma concentrations of proline detected. In another large series of patients, Dimopoulou et al  therapy.Molecular testing is the primary method for diagnosing PYCR1 deficiency. Biochemical abnormalities were not found in any of the patients reported.PYCR2 were detected because the affected patients shared the same homozygous region, and a different homozygous missense mutation was found in each family. The disorder was classified as hypomyelinating leukodystrophy-10.PYCR2 deficiency (OMIM 616420) was recently reported as a cause of secondary microcephaly and hypomyelination  were reported by Ruzzo et al  caused by defects in asparagine synthetase  progressive neurodegenerative disorder similar to PYCR2 deficiency  phenotype on one end and adult-onset neurological disease on the other.In contrast to the spectacular gain of insight into the clinical spectrum we have witnessed in amino acids synthesis disorders, reports on how to treat these disorders are still rare, but promising in the serine synthesis disorders. Treatment should therefore gain more attention given the potential benefits of supplementing missing substrates. Whole exome sequencing has proven to be invaluable for elucidation of some of these disorders. Biochemical investigation, traditionally our first approach in patients with a suspected inborn error of metabolism, is insufficient, at least for diagnosing patients with proline and asparagine synthesis defects.The observation that dominant mutations can also cause amino acid synthesis disorders will shift our classical paradigm of recessive inborn errors of metabolism. This shift requires careful interpretation of molecular diagnostics and the further development of functional assays for these amino acid disorders primarily approached by NGS techniques.


Primary myelofibrosis (PMF) is a myeloproliferative neoplasm that arises from clonal proliferation of hematopoietic stem cells and leads to progressive bone marrow (BM) fibrosis. While cellular mutations involved in the development of PMF have been heavily investigated, noteworthy is the important role the extracellular matrix (ECM) plays in the progression of BM fibrosis. This review surveys ECM proteins contributors of PMF, and highlights how better understanding of the control of the ECM within the BM niche may lead to combined therapeutic options in PMF. Primary myelofibrosis (PMF), polycythemia vera and essential thrombocythemia (ET) are the main Philadelphia chromosome negative MPNs.3PMF is described as either pre-fibrotic PMF (prePMF) or overt PMF according to the 2016 WHO diagnostic criteria. Compared to the 2008 WHO diagnostic criteria, the 2016 WHO criteria make a distinction between prePMF and overt PMF . This di5 Hallmarks of PMF include expansion of the megakaryocytic lineage, bone marrow (BM) fibrosis and extramedullary hematopoesis (EMH), which occurs predominantly in the liver and spleen, but can manifest anywhere.7 Patients with PMF can also have portal hypertension primarily through increased splenic blood flow, hepatic EMH and sinusoidal fibrosis.8 Overall survival for PMF is significantly lower than for polycythemia vera and ET, and one study showed PMF patients were also more likely to have leukemic transformation.9Although a substantial number of patients with polycythemia vera and ET are asymptomatic at presentation, patients with PMF commonly complain of fatigue and symptoms due to splenomegaly.JAK2, MPL, CALR and several other genes. Janus kinase 2 (JAK2) is a cytoplasmic tyrosine kinase engaged with numerous intracellular signaling pathways involving receptors for erythropoetin, thrombopoetin, interleukin-3, granulocyte colony-stimulating factor and granulocyte–macrophage colony-stimulating factor.10 A single acquired somatic point mutation at V617F in JAK2 causes MPN in patients.12 JAK2V617F is found in 95% polycythemia vera patients and detected in ~60% of ET and PMF patients.9 The JAK2V617F mutation affects the pseudokinase domain of JAK2 and makes JAK2 constitutively active.13Several somatic mutations have been identified in PMF, including in CALR) gene, which accounts for 25% of PMF patients.15CALR functions as an ER chaperone and its mutation activates both the thrombopoietin receptor, c-mpl and JAK2.16 Patients with PMF and CALR mutations are younger and have lower risk of death than their JAK2 and MPL-mutated counterparts, despite their higher platelet count.17 Another identified mutation that leads to 5% of PMF cases is due to a somatic gain-of-function at amino acid residues W515 (W515K/L) and S505 mutation in the transmembrane domain of c-mpl, a receptor that activates downstream JAK/STAT signaling.18Another common mutation in PMF is in the Calreticulin  had the longest survival, while CALR-ASXKL+ had the shortest survival .21 Interestingly, ASXL1, EZH2 and IDH1/2 have been shown to play a role in chromatin structure, suggesting that epigenetic dysregulation may play a role in PMF progression and leukemic transformation.22The prognosis of patients with PMF is generally poor, but depending on the mutations involved it appears that survival and adverse outcomes can vary. As mentioned before, JAK2, CALR and c-mpl are driver mutations that account for 90% of PMF cases, while 10% can be viewed as ‘triple negative'. One study found differences in median survival in patients with PMF that either had JAK2, CALR, c-mpl mutations or were triple negative. Patients with CALR-mutated PMF have a more favorable prognosis, while triple negative PMF patients have the worst prognosis .23 The BM space is evenly occupied by sinusoids. The endosteal surface of the bones and cells constitute the stem cell niche in which the hematopoietic stem cells (HSCs) reside and differentiate to different lineages.27 The BM niche is separated into two compartments. The first compartment is the osteoblastic niche found near the endosteum and the second compartment is the vascular niche near the sinusoids.28 These two niches consist of different cell types such as adipocytes, osteoblasts and smooth muscle cells, Schwann cells, reticular cells, endothelial cells and hematopoietic cells.25 However, there is no distinct separation between the two niches as HSCs can move freely and can receive inputs from the two compartments simultaneously.29 The niches also contain stromal cells and unique extracellular matrix (ECM) components that support stem cells by HSCs interaction with other cells through cell surface receptors, gap junctions and soluble factors.30 That is, the molecular crosstalk between HSCs and the cellular constituents of these niches determine the balance between HSC self-renewal and differentiation.31The BM is a spongy tissue within the central cavity of several bones of the body.in vitro (via production of granulocyte colony-stimulating factor) as well as in vivo, suggesting that osteoblasts play an important role in HSC maintenance.34 However, activated osteoblasts have also been shown to produce osteopontin and angiopoietin-1, which limit HSC expansion and contribute to their quiescence state.28 N-cadherin at the niche helps HSCs to adhere to the osteoblastic niche through tight cell–cell interactions, and overexpression of N-cadherin promotes quiescence.30 Osteoclasts also appear to be important in the osteoblastic niche by releasing calcium during bone resorption. HSCs were found to express calcium-sensing receptors. Mice deficient in calcium-sensing receptors were found to have hypoplastic BM with decreased localization of HSCs in the osteoblastic niche but had normal number of HSCs in the fetal liver.35 Interactions between the niche and hematopoietic cells are reciprocal. For instance, megakaryocytes (MKs) overexpressing BMP 2, 4 and 6 at the endosteum have been shown to stimulate osteoblasts.36 Hence, HSCs and its cell lineages are involved in bone formation and activities within the niche.37 Non-bone components are also important in the maintenance of HSCs within the osteoblastic niche. CXCL12 is a chemokine that plays an important role in the maintenance and quiescence of HSCs through its receptor CXCR4 in both the osteoblastic and vascular niches.38 Interestingly, the sympathetic nervous system appears to modulate CXCL12 expression in a circadian pattern through the release of norepinephrine which downregulates CXCL12. Circulating HSCs were found to peak at five hours after the initiation of light with ablation of sympathetic nerve fibers decreasing the numbers of circulating HSCs in mice.40 Sympathetic nerve fibers also appear to have an important role in BM regeneration as evidenced by impaired HSC recovery after treatment with cisplatin (a neurotoxic chemotherapeutic drug) in mice.41The osteoblastic niche is composed of several types of cells that aid in the maintenance of HSC. Expansion of HSCs by osteoblast factors has been shown 42 The vascular niche is located around small sinusoidal blood vessels associated with various stromal elements and together with fibronectin, type IV collagen and laminin at the ECM helps to regulate HSC differentiation and ultimately mobilization of differentiated blood cells to the peripheral circulation.30 Endothelial cells at the vascular niche secrete E-selectin and promote HSC proliferation.37 The walls of venous sinusoids consists of a layer of endothelial cells which acts as a conduit for mature blood cells and platelets to enter the blood stream from the BM compartment.43 Simultaneously, blood vessels provide a conduit for distal systemic signals, such as inflammatory and other circulatory cells into the niche.44 Besides the effect of cytokine and chemokine, a hypoxic environment in the osteoblastic niche helps to maintain HSCs in a quiescent state, while a more oxygen rich environment in the vascular niche allows HSCs to proliferate and differentiate.42The vasculature niche is located in the perivascular space and helps to activate HSCs to differentiate into other blood lineage.45 HSCs harboring mutated genes such as JAK2V16F can alter the niche to favor their clonal expansion at the expense of normal HSCs.14 One example of this is that JAK2V617F mutant HSCs secrete interleukin-1β, which activates the apoptotic pathway in mesenchymal and Schwann cells. This will, in turn, affect the survival rate of normal HSCs via disruption of the interactions between the mesenchymal cells, and injury to sympathetic nerves innervating the BM. Mice with JAK2V617F MPN had decreased numbers of Schwann cells and MPN progression was slowed by the administration of β3 adrenergic agonists that compensated for the neuropathy seen in the BM of these mice.46 In addition, mutated MKs release excessive amount of fibrotic factors, which activate mesenchymal cells, leading to MF.14 In one study of PMF patients, several genes that may be related to the maintenance of the BM niche homeostasis were found to be dysregulated. Genes such as hypoxia inducible factor 1A, CXCR4 and PAX5 were found to be downregulated in BM mononuclear cells from patients with PMF, while cyclooxygenase 2 was found to be significantly upregulated.47 Furthermore, malignant stem cells from a mouse model of MPN appear to modify the osteoblastic niche to benefit their survival at the expense of the survival of non-malignant HSC by causing mesenchymal stromal cells to overproduce osteoblastic lineage cells that promote inflammation and MF.48 Furthermore, malignant stem cells have also been shown to produce high levels of lipocalin2, which was shown to increase proliferation of PMF HSC while decreasing the numbers of normal HSC.49 Stromal cells in BM undergo changes associated with myeloproliferation which include excessive ECM deposition leading to fibrosis, neoangiogenesis and osteosclerosis.52 The JAK2V617F mutation has not only been shown in HSC and myeloid cells, but also in endothelial cells in patients with PMF and other MPNs.54 One study suggests that JAK2V617F mutated endothelial cells in the vascular niche could promote malignant stem cell proliferation.55 These changes may eventually disrupt normal HSC niches and result in the establishment of EMH.57 Although advances have been made in the understanding of how the BM niches are altered in MPNs and PMF in particular, our current understanding is incomplete.Abnormal interactions between the HSCs and its microenvironment in the BM can affect PMF disease progression.58 It is comprised of various matrix proteins, such as collagens, laminin, fibronectin, vitronectin and fibrinogen as well as soluble proteins, including cytokines and chemokines.59 These components define the biochemical and biomechanical properties of the ECM and are able to influence the attachment of cells to the ECM and directly affect their biological functions, such as cell division, differentiation, tissue polarity and cell migration.30 At the same time, cells can sense ECM compositions and transmit appropriate signals at the adhesion sites.30 The adhesion interaction requires integrins and signaling pathways, including Ras/MAPK, PI3K/Akt, RhoA/ROCK, Wnt/β-catenin and TGF-β that link the actomyosin cytoskeleton with the ECM.30 At the BM niche, the ECM provides the microenvironment for HSCs to maintain a quiescence stage or to undergo differentiate to form various progenitors.61The ECM is a three-dimensional, non-cellular structure providing physical support for tissue integrity and elasticity.62 In a study looking at ex vivo platelet production on a three-dimensional silk BM niche, the authors found that low and medium stiffness silk film supported a higher percentage of long and branched proplatelets compared to high stiffness silk film, although adhesion was not different, suggesting that increased ECM stiffness may reduce platelet production.23 Interestingly, MKs on silk films entrapped with type I collagen had a significantly reduced proplatelet formation as compared to MKs on silk films entrapped with fibrinogen.23 Another study also showed that type I collagen is a negative regulator of proplatelet formation through activation of the integrinα2β1/Rho/ROCK axis.63 It has been noted by clinicians that fibrotic tissues are usually stiff and enriched with ECM components as compared to healthy tissues.65 Furthermore, the level of tissue stiffness can be used as a predictive indicator for disease stage.66 Hence, there is a correlation between stiffness of the matrix and fibrosis, which may contribute to the clinical manifestations seen in PMF .MKs differentiation and proplatelet formation depend on the stiffness of the matrix. One study found that MKs cultured on a methylcellulose hydrogel media that mimics BM stiffness exhibited higher ploidy levels than MKs cultured on liquid media. Furthermore, MKs cultured on the stiffer media produced twice as many proplatelets when placed in liquid media than those that were cultured in the liquid media.69 MKs are known to secrete ECM components such as fibronectin, laminin and type IV collagen, which contribute to MK development and ECM homeostasis, therefore, dysfunctional MKs can potentially play a role in pathogenesis of PMF by secreting excessive amount of ECM components.71 Below we summarize the components of the ECM and their role in PMF  was identified in thrombin-stimulated platelets, and is also expressed by a variety of cells, such as endothelial cells, fibroblasts and smooth muscle cells.89 TSP-1 was significantly overexpressed in all stages of PMF independently of the degree of MF, when compared to controls. Individual follow-up biopsies showed involvement of TSP-1 during progressive MF. TSP-2 is only strongly expressed in 40% of cases with advanced MF. Interestingly, MKs and interstitial proplatelet formations were shown to be the relevant source for TSP-1 in PMF. In addition, TSP-1 inhibits the activity of matrix metalloproteinases (MMPs), which are primarily involved in proteolysis of collagens and other matrix components, which allows the accumulation of ECM.86TSP-1 is also an activator of latent TGF-β1, which is an inducer of fibroblast activation and matrix synthesis in the fibrotic response and in PMF.90 In adults, osteonectin is expressed during processes requiring ECM turnover such as wound healing and tumor progression.90 Osteonectin prevents cell spreading in vitro suggesting they play an important role in cellular proliferation and migration.91 High levels of tissue osteonectin has been associated with reduced collagen type IV deposition.92Osteonectin  is an adhesive calcium-binding ECM glycoprotein that binds various ECM components such as TSP-1 and fibrillar collagens.50 In contrast, in cases with significant stromal changes (such as PMF), osteonectin reactivity extends to stromal cells. Hence, osteonectin is part of BM response to myeloproliferation. That is, the expression level of osteonectin in BM stromal cells correlates with the degree of stromal changes and correspond to the severity of PMF.50 This is further shown in osteonectin knockout mice in which there is impairment in BM fibrosis.50Interestingly in healthy individuals and in myeloid neoplasms without associated stromal changes, osteonectin expression was confined to MKs.93 For instance, MMP2 and MMP9 are effective in degrading collagen and gelatine structures in the ECM.93 In addition, by producing MMP9, mature MKs are able to free themselves from the BM matrix at the osteoblastic niche and travel to the vascular niche. In patients with PMF, MMPs are downregulated while tissue inhibitors of MMPs (TIMP) are increased.95 This leads to decreased degradation and increased accumulation of ECM components.MMPs are a family of zinc-dependent endopeptidases and function in remodeling the ECM by its ability to degrade and cleave ECM components with wide substrate specificities.96 The crosslinking results in a dense ECM with altered elasticity.97 LOX is highly expressed in proliferating, low ploidy MKs, but its expression decreased dramatically in mature, higher ploidy mouse MKs.98 Following this observation, the Ravid laboratory was also the first to identify LOX as regulator of BM fibrosis in a mouse models of PMF.98 This link was demonstrated in a GATA-1low mouse model where LOX was found to be abundantly expressed within abnormally high levels of low ploidy MKs coupled with an extensively fibrotic ECM.99 Importantly, administration of β-aminopropionitrile (a LOX inhibitor) to the GATA-1low mice inhibited the progression of MF.98 Similarly, LOX was reported to be upregulated in human PMF cells and plasma.100LOX is a copper-dependent amine oxidase that catalyzes oxidative deamination of lysine and hydroxylysine residues on collagen and elastin precursors, leading to crosslinking within these proteins.102 PDGF, TGF-β1 and the cytokine interleukin-1β are able to increase LOX and collagen expression, all of which have been found to be elevated in PMF.102 LOX has also been shown to oxidize and activate PDGF receptor and LOX activity appears to be important for PDGF-mediated MK expansion.103 Hence, there appears to be positive feedback in which LOX is highly secreted from abnormal MKs, and soluble factors secreted by these MKs can further increase LOX level, which will enhance the fibrotic phenotype  and TGF-ß1, which can lead to fibrosis.henotype .107 Inflammatory cytokines have been found to be elevated in PMF regardless of the mutational status, including interleukin-1β and tumor necrosis factor alpha. Both of these pro-inflammatory cytokines have been shown to enhance mutant HSC proliferation and survival in vitro.108 Other cytokines released by malignant HSCs are profibrotic, such as transforming growth factor beta1 (TGF-β1), basic fibroblast growth factor, and PDGF, and angiogenic factors such as vascular endothelial growth factor (VEGF).23The ECM is maintained by several cytokines and growth factors. Dysfunctional HSCs and MKs have abnormal production and release of several cytokines and chemokines, which are associated with fibrosis and enhanced survival and proliferation of mutant HSCs, thus contributing to ECM disruption in PMF.58 For instance, TGF-β1 enhances the production of types I, III and type IV collagen and fibronectin, as well as increases the synthesis of TIMP.109 There is also a strong correlation between MK release of TGF-β1 and its activity, resulting in a dose-dependent increase of ECM component synthesis.110 In the ECM, reactive oxygen species activate a number of proteases such as MMPs and TSP1, which can digest and convert the latent TGF-β to its active form.112TGF-β1 plays a key role in regulation of genes involved in the synthesis of the ECM components and of ECM-degrading enzymes.113 Not surprising, high TGF-β concentration is found in PMF BM and is correlated to BM fibrosis in vivo.89 In PMF, TGF-β1 affects ECM biosynthesis by decreasing the amount of MMP and increasing the synthesis of TIMPs, particularly of TIMP-1.114 TGF-β leads to an increase in production of types I, III, IV and V collagens, and to overexpression of fibronectin in advanced stage of the disease, which further accelerate ECM accumulation.109 MKs and platelets are the main source of TGF-β in PMF with intraplatelet levels being 2–3 times higher in PMF patients compared to healthy controls.117TGF-β1 is involved in the pathophysiology of PMF and is a strong inducer of fibrosis.23 PDGF participates in BM fibrogenesis in PMF through its role in the proliferation and activation of medullary fibroblasts.59 VEGF functions by binding to its receptor VEGFR1 to promote MK maturation and migration from osteoblastic niche to the vascular niche where proplatelets formation and platelet release occur.118MKs are an important source of PDGF and VEGF, which contribute to MK role in the development of BM fibrosis and production of collagen.119Current management of PMF is primarily palliative and aimed at relieving symptoms of . Only allogenic hematopoietic cell transplant is curative although few patients are eligible for this treatment.120 Interestingly, response to HU may be related to the presence of JAK2V617F mutation in PMF with those harboring the mutation more likely to respond to HU.121 Anemia can be treated in PMF with red blood cell transfusions , androgens and danazol. Thalidomide or lenalidomide with prednisone can be used for persistent anemia.123 Importantly, none of the aforementioned palliative therapies have been convincingly shown to decrease BM fibrosis.122Hydroxyurea (HU) is a medication commonly used to treat symptomatic PMF and has been shown to significantly improve bone pain, constitutional symptoms and splenomegaly.125 Although the COMFORT I and II trials failed to show a survival advantage in ruxolitinib, long-term follow-up studies from COMFORT trials suggest a modest decrease in mortality in patients with intermediate-2 and high-risk disease.128 Although ruxolitinib is the only non-allo HCT treatment currently available that may improve survival, the benefits appear to be modest and leave a lot to be desired in the treatment of PMF.JAK1 and 2 inhibitors have been introduced as treatment of PMF, the only Food and Drug Administration (FDA) approved drug being ruxolitinib. In the phase 3 COMFORT I and II trials, ruxolitinib was shown to be more effective than best available therapy for reduction of splenomegaly and constitutional symptoms although it was associated with higher rates of anemia and cytopenias. Ruxolitinib also appears to reduce BM fibrosis in some patients.129 More long-term studies with larger cohort are needed but imetelstat remains a promising prospective drug.Given the poor prognosis of PMF and limitations of current treatment options, novel drugs are needed to improve the quality of life and survival of patients. One such novel drug is the telomerase inhibitor imetelstat. A recent pilot study looked at 33 patients with intermediate-2 or high-risk MF and showed that four patients had complete response and three had partial response to imetelstat. All four patients with complete response had reversal of BM fibrosis with three having molecular remission. The study also showed symptomatic spleen reduction in 35% of patients and 31% of transfusion-dependent patients before the study became transfusion-independent for at least 3 months. Myelosupression was a common adverse requiring protocol-mandated dose reduction. Interestingly, response rates were seen in 27% of patients with JAK2 mutation and 0% seen in those without.130 Mature MKs have also been shown to have reduced expression of AURKA. Interestingly, AURKA activity was found to be elevated in cells with JAK2, CALR or MPL mutations. A recent study showed that MLN8237 (AURKA inhibitor) induced differentiation of JAK2 and MPL-mutated cells as well as decreased BM fibrosis and spleen size in mouse models of MPN and MF, with no obvious myelosupression seen.133 Other novel drugs currently in early clinical development for the treatment of PMF include the hedgehog pathway inhibitor PF-0444913, JAK2 specific inhibitors (such as NS-018), and histone deacetylase inhibitors .136 Histone deacetylase inhibitors are promising given the known mutations in PMF that affect the epigenome. Combination therapy with ruxolitinib and other agents are also in development including the histone deacetylase panabinostat, which showed promise in a preclinical trial and there is a current phase I/II trial looking at this combination's benefit in patients with PMF .137MKs in PMF have an impaired ability to polypoidize, yielding another potential target for therapy. Aurora kinase A (AURKA) appears to be important in maturation and polypoidization of MKs and studies have shown that inhibition of AURKA induces polypoidization in mouse models of acute megakaryocytic leukemia.138 There is an ongoing phase 2 clinical trial looking at PRM-151 (recombinant serum amyloid P) for the treatment of MF. Preliminary data showed no significant myelosupression and improvement of BM fibrosis in six patients.139New therapeutics targeting the ECM may be beneficial in ameliorating the debilitating symptoms due to BM fibrosis. One such novel target might be LOX, considering its above-mentioned influences on BM fibrosis and on promotion of MK proliferation through PDGF-R activation. Another potential novel ECM-based therapy could be targeted against monocyte-derived fibrocytes that may play a role in the pathogenesis of BM fibrosis in PMF. One study showed that neoplastic monocyte-derived fibrocytes were overrepresented in the BM of PMF patients and inhibiting monocytes from differentiating to fibrocytes with recombinant serum amyloid P reduced BM fibrosis in two mouse models of MF.low mouse model, inhibition of tyrosine kinase of TGF-β1 receptor decreased MF, EMH and neoangiogenesis.140 Another potential unexplored treatment in PMF is nintedanib, a multikinase inhibitor that inhibits VEGFR and PDGF receptor. Although bevacizumab (anti-VEGF antibody) was ineffective in treating PMF, inhibition of multiple cytokine receptors involved in the pathogenesis may be efficacious by interrupting cytokine-mediated disruptions of the BM niches and ECM.142 TNF inhibitors have shown promise in treating the constitutional symptoms of PMF, although larger trials are necessary to assess safety and efficacy.143Inhibition of cytokines involved in PMF is another potential future ECM-based treatment of PMF. In the GATA-1Considerable advances have been achieved in the understanding of the ECM in BM and its effects on hematopoietic cell biology in health and disease. However, treatment of MF, which is found not only in PMF but also secondary to many hematological diseases, is still a challenge and options are scarce. One reason for this could be the focus of current therapies on controlling MPN cell proliferation, with the expectation that this in turn would result in amelioration of MF. At present, this approach is at best of minor efficacy in early MF and not indicated in cases of severe MF. Specific targeting of ECM dysregulation to prevent and diminish MF may prove the frontline of research and therapy development in PMF with the greatest promise of relieving symptoms and extending life expectancy of patients.


Oral polio vaccine (OPV) containing attenuated serotype 2 polioviruses was globally withdrawn in 2016, and bivalent OPV (bOPV) containing attenuated serotype 1 and 3 polioviruses needs to be withdrawn after the certification of eradication of all wild polioviruses to eliminate future risks from vaccine-derived polioviruses (VDPVs). To minimize risks from VDPVs, the planning and implementation of bOPV withdrawal should build on the experience with withdrawing OPV containing serotype 2 polioviruses while taking into account similarities and differences between the three poliovirus serotypes.We explored the risks from (i) a failure to synchronize OPV cessation and (ii) unauthorized post-cessation OPV use for serotypes 1 and 3 in the context of globally-coordinated future bOPV cessation and compared the results to similar analyses for serotype 2 OPV cessation.While the risks associated with a failure to synchronize cessation and unauthorized post-cessation OPV use appear to be substantially lower for serotype 3 polioviruses than for serotype 2 polioviruses, the risks for serotype 1 appear similar to those for serotype 2. Increasing population immunity to serotype 1 and 3 poliovirus transmission using pre-cessation bOPV supplemental immunization activities and inactivated poliovirus vaccine in routine immunization reduces the risks of circulating VDPVs associated with non-synchronized cessation or unauthorized OPV use.The Global Polio Eradication Initiative should synchronize global bOPV cessation during a similar window of time as occurred for the global cessation of OPV containing serotype 2 polioviruses and should rigorously verify the absence of bOPV in immunization systems after its cessation. Cessation of the use of oral poliovirus vaccine (OPV) represents an essential part of the global polio eradication endgame strategy , 2 becauIf the world can successfully complete serotype 1 WPV eradication, certify the world free of WPV serotypes 1 and 3, and manage the risks associated with the cessation of use of OPV containing serotype 2 polioviruses, then it will need to proceed to bOPV cessation to stop the use of all OPV containing serotype 1 and serotype 3. As of the end of 2017, no reported cases associated with serotype 3 WPV have occurred since 2012  and indiAlthough eliminating future polio cases caused by VDPVs requires the cessation of OPV use , OPV cesUsing inactivated polio vaccine (IPV) provides another option for addressing the risks associated with cessation of OPV, but it comes with limitations. While IPV provides very effective protection from paralytic polio, it remains much less effective than OPV in preventing poliovirus transmission. Individuals vaccinated with IPV without prior infection by a live poliovirus (LPV) can be infected and participate in fecal-oral poliovirus transmission to a similar degree as completely unvaccinated individuals –19. In aThe degree to which serotypes 1 and 3 OPV viruses behave like serotype 2 OPV viruses, especially regarding their ability to spread and evolve into VDPVs, is a key consideration for the planning and implementation of bOPV cessation. Similar behavior between serotype 2 and serotypes 1 and 3 would suggest a need for similar synchronization and monitoring while a serotype with substantially lower ability to spread and cause VDPVs could be withdrawn with much looser synchronization and monitoring without substantially increasing the risks associated with the withdrawal . We prev0) and low neurovirulence . Through reversion, these viruses gradually evolve to fully-reverted VDPVs with assumed R0 and paralysis-to-infection ratio values equal to those for the homotypic WPVs. Consistent with the available evidence, the model assumed serotype-specific differences for the absolute R0 values of VDPVs, for the ratios of the R0 values of the attenuated parent strains in OPV to the R0 values of the homotypic WPVs (and VDPVs), and for the reversion times. For a given population, the model calculated each of these R0 values relative to the assumed R0 value for serotype 1 WPV in that population , defined as the average number of secondary infections generated by a single infection. Rn accounts for the R0 of the poliovirus and the level of population immunity to transmission. An Rn of greater than 1 means that the corresponding poliovirus can sustain circulation. Rn values in a given population vary for different polioviruses, i.e., serotypes and reversion stages, because the corresponding R0 values differ, and they change over time due to seasonality in poliovirus transmissibility and changing population immunity to transmission. Unless an outbreak occurs in a population, Rn values continually increase following bOPV cessation due to decreasing population immunity to transmission in the absence of exposure to LPVs. The global model base case, which we adopted for analyses I and II, assumed IPV introduction in all countries by January 1, 2015, tOPV cessation on April 1, 2016, and bOPV cessation on April 1, 2019 [We conducted two analyses (I and II) related to the implication of a failure to globally synchronize OPV cessation and two analyses (III and IV) related to unauthorized post-cessation use of OPV. Analyses I and II considered the vulnerability of the various subpopulations to OPV-related viruses as a function of time after the tOPV-bOPV switch (for serotype 2) or after bOPV cessation (for serotypes 1 and 3). These analyses focus on vulnerability and do not consider the likelihood of exposure to OPV-related viruses that would occur in the event of a non-synchronous cessation, which could include exposure to OPV-related viruses in different stages of reversion. In the context of high population immunity to transmission and continued OPV use, the majority of OPV-related viruses reside in very low stages of reversion because insufficient susceptible individuals exist for the sustained transmission of OPV-related viruses needed for them to evolve to higher reversion stages. With lower population immunity to transmission, OPV-related viruses in higher reversion stages may exist in relatively greater numbers. Thus, if countries do not synchronize OPV cessation, then countries that stop OPV use earlier could import OPV-related viruses in any reversion stage, depending both on chance and the population immunity to transmission in the countries that still use OPV. To convey the range of vulnerabilities to different OPV-related viruses, we show the vulnerability to reversion stages 0 (OPV parent strain), 10 , and/or 19 (fully-reverted VDPV). Analyses I and II used the integrated global model, which divides the world into 710 subpopulations, and focused on the 520 subpopulations that used OPV in 2013 . We char 1, 2019 . The mod 1, 2019 . We expl 1, 2019 , 28.For analysis I, we compared the vulnerability after OPV cessation among the three serotypes, assuming tOPV intensification prior to the tOPV-bOPV switch and continued high maintenance of population immunity with bOPV SIAs until bOPV cessation , a hypothetical scenario of no IPV use since 2015 (labeled “No IPV from 2015”), and a scenario reflecting a decision to switch from the one IPV dose introduced in 2015 to two IPV doses at the time of bOPV cessation (labeled “Two IPV from bOPV cessation”), which assumes sufficient IPV supplies available to do so by 2021. We assumed an average per-dose take rate of IPV of 63%, i.e., corresponding to a cumulative take rate of 95% after three doses , and modn values for reversion stages 0, 10, and 19, respectively, as a function of time after the tOPV-bOPV switch (for serotype 2) or bOPV cessation (for serotypes 1 and 3). The different curves in Figs. n values among the 520 subpopulations in the global model that still used OPV in 2013 [n values at that point in time (or the average of the two subpopulations closest to the percentile). Taken together, these percentiles illustrate the full range of when subpopulations become vulnerable to ongoing transmission of the specified polioviruses, with the highest curve showing the most vulnerable subpopulation and the lowest curve showing the least vulnerable subpopulation. We emphasize that these curves only illustrate the vulnerability as a function of time since OPV cessation. The risk in the event of a non-synchronous cessation depends on both the vulnerability and the exposure to OPV-related viruses from populations that continue to use OPV to populations that already stopped using OPV.Figures  in 2013 . Thus, an values > 1 and thus become vulnerable to ongoing transmission of an OPV parent strain after OPV cessation the soonest for the serotype 2 OPV parent strain. This relates directly to the assumed high transmissibility of the serotype 2 OPV parent strain compared to the serotypes 1 and 3 OPV parent strains  curve in Fig. n curves include a decrease due to an outbreak. All decreases in Rn values for serotype 3 relate to seasonality (and consequent changes in which populations make up the different percentiles at different points in time) rather than outbreaks.Figure Figure Figure This study suggests generally similar risks associated with a non-synchronous global bOPV cessation or unauthorized bOPV use after bOPV cessation to those associated with the cessation of tOPV that occurred as part of the tOPV-bOPV switch. This finding is driven by the risks associated with ceasing use of OPV containing serotype 1 polioviruses because the risks associated with ceasing use of OPV containing serotype 3 polioviruses are lower than for OPV containing serotype 1 or 2 polioviruses. As with the tOPV-bOPV switch, post-cessation low levels of unauthorized use of bOPV could potentially continue for one or more years, depending on the area and degree of bOPV use, without causing new cVDPV outbreaks. However, this time becomes shorter in chronically under-vaccinated subpopulations within already high-risk settings . Given tThe observation from this model that the risks from cVDPVs are similar for the cessation of OPV containing serotype 1 as for the cessation of OPV containing serotype 2 is consistent with patterns in VDPVs seen before 2005 when the GPEI began to use monovalent OPV and bOPV in many SIAs instead of tOPV. For example, serotype 1 and 2 cVDPVs resulted in similar numbers of identified outbreaks and cases of polio through 2005, with 4 outbreaks and 72 cases of polio caused by serotype 1 cVDPVs and 4 outbreaks and 40 cases of polio caused by serotype 2 cVDPVs , 31, 32.Prior modeling studies have shown that preventing the creation of indigenous cVDPVs after OPV cessation requires higher population immunity to transmission for serotype 2 than for serotype 1, but higher OPV coverage for serotype 1 than for serotype 2 . This apAs long as use of all OPV containing attenuated serotypes 1 and 3 polioviruses ceases at the same time, i.e., as part of globally-coordinated bOPV cessation, the lower risks from ceasing use of OPV containing serotype 3 polioviruses will be overshadowed by the risks associated with ceasing use of OPV containing serotype 1 polioviruses. However, the characteristics of attenuated serotype 3 polioviruses become relevant if the global cessation of OPV containing attenuated serotype 3 polioviruses were separated from cessation of OPV containing serotype 1 polioviruses, perhaps in the form of a global switch from bOPV to monovalent OPV containing only attenuated serotype 1 polioviruses (mOPV1) , 36. BasSimilar to the tOPV-bOPV switch, successful bOPV cessation will require global synchronization and efforts to verify the absence of bOPV from all supply chains. Gaps of several months or more in bOPV cessation dates come with an increased risk that OPV-related viruses will spread from countries or regions that still use bOPV to countries or regions that stopped using bOPV and then transmit and evolve to cVDPVs, leading to outbreaks. Early cessation of bOPV use appears particularly problematic in high-risk countries with environments conducive to the spread of polioviruses, such as countries with inadequate sanitation and tropical climates. The larger the populations that continue to use bOPV, and the longer they continue that use after the time that high-risk countries cease using bOPV, the greater the likelihood of exportations of OPV-related viruses to countries where OPV-related viruses can easily transmit and evolve to cVDPVs. Although few documented instances of the long-range spread of cVDPVs or of cVDPVs resulting from the long-range spread of OPV-related viruses have occurred to date , the rarIn addition to the epidemiological risks involved with a failure to globally synchronize bOPV cessation, a lack of synchronization would significantly complicate bOPV supply management , 23, 28 With respect to unauthorized OPV use, environmental detections of viruses closely related to the attenuated serotype 2 polioviruses after the tOPV-bOPV switch and follow up investigations have suggested continued inadvertent use of tOPV in several countries , 21, 43.Although our models relied on an extensive calibration process to generate generic model inputs consistent with the evidence, the limitations of these models carry over to the analyses in this study , 25, 26.This analysis demonstrates that the risks associated with bOPV cessation are similar to those associated with the tOPV-bOPV switch. Therefore, the global cessation of bOPV use should incorporate multiple risk reduction measures, including relatively tight synchronization of bOPV cessation within and across countries, minimizing unauthorized OPV use after cessation, and maintaining high population immunity to poliovirus transmission until bOPV cessation . These m


The Late Pleistocene Shanidar 1 older adult male Neandertal is known for the crushing fracture of his left orbit with a probable reduction in vision, the loss of his right forearm and hand, and evidence of an abnormal gait, as well as probable diffuse idiopathic skeletal hyperostosis. He also exhibits advanced external auditory exostoses in his left auditory meatus and larger ones with complete bridging across the porus in the right meatus (both Grade 3). These growths indicate at least unilateral conductive hearing (CHL) loss, a serious sensory deprivation for a Pleistocene hunter-gatherer. This condition joins the meatal atresia of the Middle Pleistocene Atapuerca-SH Cr.4 in providing evidence of survival with conductive hearing loss (and hence serious sensory deprivation) among these Pleistocene humans. The presence of CHL in these fossils thereby reinforces the paleobiological and archeological evidence for supporting social matrices among these Pleistocene foraging peoples. Homo.Paleopathological assessments of Pleistocene human remains have increasingly identified a suite of substantial and/or systemic developmental and degenerative abnormalities among the remains, in addition to an abundance of minor traumatic and oral lesions –5. In adIn these assessments and inferences, however, there has been little consideration of impairments of the basic senses, ones which would have limited the abilities of the individuals to effectively perceive and respond appropriately to their natural and social environments. The only possible examples are post-traumatic unilateral ocular alterations in the Shanidar 1 Neandertal  and reduExternal auditory exostoses (EAE) are bony growths into the auditory canal from the tympanic and/or squamous walls of the external auditory meatus and the margins of the auditory porus ,14. TheyEAE have been extensively documented in clinical settings and Holocene skeletal samples . EAE presence has been noted in a couple of Middle Pleistocene humans ,35, seveThe ≈50 ka BP Shanidar 1 Neandertal cranium ,44 S1 T was analThe degree of development of the EAE is scored using an ordinal scale of Grade 0 (absence of EAE) to Grade 3 (large EAE that largely block the meatus)  ,25,26 s.Both of the external auditory meatus (EAM) of Shanidar 1 are well preserved; inferiorly the tympanic and adjacent portions of the temporal bone present little of note , S1 TextThe left porus  has a noThe right external auditory meatus and porus of Shanidar 1  has a moThis combination of right meatal growths completely blocks the middle of the porus, leaving only small openings anterosuperiorly and posteroinferiorly. They are then joined by a small protuberance within the meatus projecting inferiorly from the superior-most anterior tympanic bone. These large EAE, and especially the bridging of them across the middle of the porus, provide the Shanidar 1 right auditory canal with a serious Grade 3 score.Data on the shapes of the more medial canal are not fully available; the visible EAE may extend medially along the auditory canals, as is indicated by the small ones within each meatus. The medial canals adjacent to the area of the tympanic membrane are nonetheless conservatively assumed to be normal.The external auditory meatus of this moderately older Neandertal therefore present slightly different patterns of abnormalities. The left one approximates the marked patterns occasionally encountered in recent humans. The right one extends to the upper end of the four-part ordinal scale.The degree of development of the Shanidar 1 EAE is associated with conductive hearing loss (CHL) in extant humans ,24,46–49The left meatus of Shanidar 1 is likely to have accumulated sufficient material to produce CHL, given its large posterior exostoses across at least half of the lateral opening. The right one, however, would have led to the presence of cerumen and other debris behind the bony bridging that connects the anterior and posterior exostoses. It would have been essentially impossible for Shanidar 1 to maintain a sufficiently clear canal for adequate sound transmission. He would therefore have been effectively deaf in his right ear, and he likely had at least partial CHL in the left ear.Consequently, Shanidar 1 appears to have had an advanced degree of unilateral conductive hearing loss and reduced hearing acuity on both sides. In addition to a general reduction in hearing acuity, unilateral CHL limits one’s ability to discern the signal from background noise and to locate sounds in space. Among modern urban children it is associated with varying degrees of reduced academic progress , and heaIn addition to a reduced effectiveness in communication and coordinated social activities , it Shanidar 1 also experienced a suite of other degenerative difficulties ,44,65 S. He is bAs noted above, external auditory exostoses have been observed in other Pleistocene humans, but most of them are modest in size. Four other Neandertals, La Chapelle-aux-Saints 1, Krapina 39.1, Spy 1 and Tabun 1, exhibit large EAE, ones which would be scored as Grade 2 . Their bIn addition, the Middle Pleistocene (≈430 ka) Atapuerca–SH Cr.4  developeHomo. The presence of social support among non-modern Pleistocene humans, as noted above, has been inferred for a number of Pleistocene individuals with substantial abnormalities and varying degrees of loss of function . Comparon  in indicating some level of social support. Moreover, although one could discuss the degrees to which support would have been necessary for the survival of those individuals with degenerating mastication or cranial developmental defects, an individual with advanced CHL would have been highly vulnerable alone in a Pleistocene foraging context (see above). For Shanidar 1, the CHL was associated with loss of function in other aspects of his biology, all of which would have compounded his need for support, even if some of the individual deficiencies by themselves would not have required such assistance.In this context, the conductive hearing loss (CHL) of Shanidar 1 and Atapuerca-SH Cr.4 join several other Pleistocene archaic The inferred presence of social support among at least the Neandertals should not be surprising. There is abundant evidence of intentional burial of the dead , even ifThe Shanidar 1 Neandertal, in addition to his other traumatic and degenerative lesions, developed advanced external auditory exostoses, largely blocking the meatus on the left side and bridging across the auditory porus on the right side. Although a normal bony growth in the context of irritation of the external auditory canal, the extent of exostosis development in Shanidar 1 indicates a marked degree of conductive hearing loss. It joins the Middle Pleistocene Atapuerca-SH Cr.4, with its auditory atresia and associated deafness, in indicating survival of these sensorily impaired archaic humans despite the rigors and dangers of a Middle-to-Late Pleistocene foraging existence. A substantial degree of social support, especially given Shanidar 1’s plethora of other impairments, is indicated.S1 Text(PDF)Click here for additional data file.S2 Text(PDF)Click here for additional data file.S3 Text(PDF)Click here for additional data file.


The reactive oxygen species, generally labeled toxic due to high reactivity without target specificity, are gradually uncovered as signaling molecules involved in a myriad of biological processes. But one important feature of ROS roles in macromolecule movement has not caught attention until recent studies with technique advance and design elegance have shed lights on ROS signaling for intercellular and interorganelle communication. This review begins with the discussions of genetic and chemical studies on the regulation of symplastic dye movement through intercellular tunnels in plants (plasmodesmata), and focuses on the ROS regulatory mechanisms concerning macromolecule movement including small RNA-mediated gene silencing movement and protein shuttling between cells. Given the premise that intercellular tunnels (bridges) in mammalian cells are the key physical structures to sustain intercellular communication, movement of macromolecules and signals is efficiently facilitated by ROS-induced membrane protrusions formation, which is analogously applied to the interorganelle communication in plant cells. Although ROS regulatory differences between plant and mammalian cells exist, the basis for ROS-triggered conduit formation underlies a unifying conservative theme in multicellular organisms. These mechanisms may represent the evolutionary advances that have enabled multicellularity to gain the ability to generate and utilize ROS to govern material exchanges between individual cells in oxygenated environment. This review is focused on the genetic control of intercellular movement of various molecules including symplastic dye, small RNA, and proteins, and outlines the underlying mechanisms that are shared across plant and animal kingdoms.In addition to the involvement in biotic defense, these ROS-generating machineries together with antioxidants act as essential modulators of redox signaling pathways capable of effecting changes in a plethora of biological processes including, not limited to, abiotic stress, cell growth and programmed cell death, cell proliferation, and cell differentiation. For more excellent reviews, reader is referred to those by Holmström and Finkel , Río andArabidopsis, as well as in monocots and woody plants, provides indicative insights into the recognition of relative disconnection from surrounding tissues in some types of cell clusters/tissues, or namely the symplastic domain  gene encode two different RNA helicases that are involved in the very basic aspect of RNA metabolism ranging from RNA transcription, splicing to degradation, and that are localized to mitochondria and chloroplast respectively, whereas DSE1 (decreased size exclusion limit 1) gene encodes a WD-repeat protein that is distributed in both cytoplasm and nucleus. Although these genes controlling the cell-to-cell movement of symplastic tracer appear to have no apparent intrinsic coherence in light of genetic and/or biochemical pathways or even the spacial occurrence relating to PD, TEM dissection has shown these mutants have altered architecture of PD; with increased frequency of twinned and branched PD in increased tracer movement mutant or decreased frequency of twinned and branched PD in decreased tracer movement mutant.Genetic screens from the embryo-defective collection, which was predicated on the premise that defective cell-to-cell transport would result in the embryo abnormality or growth arrest, recovered mutants showing increased plasmodesmatal conductance of symplastic dye, e.g., the 10-kD F-dextran  transporter, or SID-1 which enables transport of the length of 50 bp up to 500 bp dsRNAs  mutant that shows only deficiency in the silencing movement while maintaining the production of silencing signal  enzyme. An Arg to Lys substitution in a highly conserved motif in the RCI3 peroxidase resulted in the reduced hydrogen peroxide level in the mutant line, in agreement with the function of POX which can regulate H2O2 level through peroxidative and oxidative cycle  and a reduced chloroplast redox in ise2 are all positively correlated to plasmodesmal transport whereas oxidized state of chloroplast in paraquat-treated WT plants resulted in decreased plasmodesmal transport irrespective of the redox states of either mitochondria or cytoplasm , is agnostic to CCT8 chaperonin , usually considered as counterpart to PD, have recently been discovered in a variety of cell types  through its interaction with cysteine 62 of actin   is the key to protrusion formation as specific inhibitors of p38 MAPK and ERK can nearly abolish the H2O2-induced TNTs formation , respectively, can effectively quench the TNTs induction , another type of reactive species , can drastically promote the formation of cytonemes on the surface of human neutrophil  leads to attenuated actin polymerization  gene displays elevated internal H2O2 level, and at the same time has higher peroxule frequency compared to wild type plant -activated NADPH dual oxidase (BLI-3)-maturation factor(DOXA-1) complex (Moribe et al., PLS1 (Clergeot et al., nox1/nox2 (Egan et al., P. anserina (Lambou et al., Botrytis cinerea (Siegmund et al., Epichloë festucaeis to establish mutualistic association with perennial ryegrass Lolium perenne (Tanaka et al., NOX is ROS-generating machinery, and its activity is required, at least in fungi, for multicellular formation, but the question remains how the activity is regulated in light of multicellularization. Until recently, an excellent study in For now, we have learned intercellular transport is the key underpinning to multicellularization and other forms of cellular interaction, e.g., the pathogenic and mutualistic interface, all of whom needs ROS to be involved. Strikingly, geological and archeological records together with phylogenetic analysis have shown that rise and fall of atmospheric oxygen is highly correlated with multicellular emergence and disappearance (Hedges et al., in situ ROS detection, characterization, and resolution of signaling networks, more hidden features are expected to be unearthed in this perplexing but also intriguing area.By summarizing the data from across the artificial dye movement, protein movement, gene silencing movement, and intercellular bridge formation we can readily add the role of ROS in facilitating intercellular transport into their signaling repertoire. The ROS effects on membrane protrusions are widely conserved in many different types of animal cells, although diversified signaling networks are present accordingly in different occasions. Key pathways and players involved in membrane protrusions are well-conserved in plants and animals. Superimposed on the plasma membrane, the cell wall in plants poses another challenge to intercellular communication. Remarkably, ROS are still needed for cell wall remodeling. Distinct from NOX that resides on cell membrane and is required for membrane protrusions in animals, the apoplatic player POX that is unique to land plants and located on cell walls (Passardi et al., The author confirms being the sole contributor of this work and approved it for publication.The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Recently there has been heightened interest in the potential significance of interleukin (IL)-17 and IL-23 in the development/progression of human malignancies. Here, we analyzed the systemic levels of these cytokines in 75 patients with different types of gastric neoplasms  and 42 healthy volunteers. We found that patients with all types of gastric neoplasms have significantly lower IL-23 levels. However, in comparison to the levels in healthy individuals, IL-17 concentrations were lower only in patients with types of gastric neoplasms other than carcinoma. Interestingly, IL-17 levels significantly differed between patients with early and advanced gastric carcinoma. No significant associations were detected between the systemic levels of examined interleukins and TNM staging. However, peripheral levels of IL-23 were correlated with the absolute numbers of circulating populations of bone marrow-derived mesenchymal and very small embryonic/epiblast-like stem cells in patients with gastric carcinoma. ROC curve analyses demonstrated that systemic levels of IL-17 seem to meet basic criteria for consideration as a helpful diagnostic marker in the detection of gastric carcinoma. In conclusion, our study provides translational evidence confirming the clinical significance of IL-17 and IL-23 in the pathogenesis of different types of gastric neoplasms in humans. Malignancies originating from the stomach represent an important diagnostic and therapeutic challenge in modern clinical gastroenterology1145678Cytokines are believed to play a significant role in the pathogenesis of multiple types of neoplasms, including those malignancies that originate from the gastrointestinal tract. In addition, they appear to hold promise as targets for anti-cancer therapy910111214151819Taking all of these molecular observations into consideration, in the current study, we aimed to verify an original hypothesis that an abnormal balance in systemic levels of IL-17 and IL-23 exists in patients with different types of gastric neoplasms. This is associated with both the clinical presentation of gastric tumors and with the recently reported phenomenon of intensified peripheral trafficking of selected populations of bone marrow-derived stem cells (BMSCs) in patients with gastric carcinomaTo achieve this goal, we aimed to: i) examine and compare IL-17 and IL-23 levels among groups of patients and healthy volunteers; ii) verify the potential associations between levels of examined interleukins and both clinical staging of gastric carcinoma  and absolute numbers of different populations of circulating BMSCs; and iii) estimate the preliminary clinical value that can be derived from measurements of systemic levels of IL-17 and IL-23 in patients with lesions detected within the gastric tissue, as novel diagnostic serum markers of gastric carcinoma in humans.Comprehensive evaluation revealed no statistically significant differences in anthropometric and laboratory parameters between the analyzed groups of recruited individuals . While punpublished observation).The mean peripheral levels of examined interleukins in patients with gastric carcinoma, other gastric neoplasms, and healthy individuals are presented in Next, we attempted to determine whether levels of the interleukins we examined here were associated with concentrations of other interleukins  and the values of their respective ratios that we recently reportedTo determine whether the observed alterations in systemic levels of the interleukins we examined were associated with the clinical presentation of gastric carcinoma among our patients, we performed a statistical comparison of levels of these cytokines in individuals with cancer subdivided into two groups, those with early and advanced gastric carcinoma defined according to the Japanese criteria. These analyses revealed that, in comparison to healthy individuals, patients with early gastric carcinoma had significantly higher mean values of IL-17 and lower mean values of IL-23, whereas individuals with advanced gastric carcinoma had mean IL-17 concentrations comparable to those observed in healthy individuals and IL-23 levels lower than those observed in both healthy controls and patients with early gastric carcinoma . We alsoFinally, we aimed to verify, at least on a preliminary basis, the potential diagnostic value of the examined interleukins for the detection of gastric carcinoma in humans. To determine whether systemic levels of IL-17 and IL-23 could serve as novel markers of gastric carcinoma, we constructed receiver operating characteristic curves, and determined the approximate area under the curve values to assess the suitability of these cytokines as potential novel diagnostic markers . Our anaIt has been noted for several years that interaction between various cytokines may play a significant role in the development and/or progression of malignancies. Recently, much attention has focused on the action of the IL-17/IL-23 axis, which is believed to be a major player in inflammation-related malignancies, such as gastric neoplasms910111213141516222425262728et al.31323334et al.et al.Helicobacter pylori strains present in different parts of the world36In this study, we found that in comparison to healthy individuals, patients with gastric neoplasms had altered IL-17 and IL-23 levels. Namely, in the general group of patients with gastric carcinoma, mean concentrations of IL-17 were statistically comparable to those observed in healthy controls, findings similar to those of the study by Malek-Hosseini Unfortunately, in our clinical study we were unable to define the exact molecular mechanisms of action of the examined interleukins in patients with gastric carcinoma. While there are experimental studies demonstrating that IL-17-related stimulation/activity may not be essential for the successful development and/or progression of gastric cancers, it has been shown that these molecules may promote the invasiveness of gastric cancer cells through activation of the nuclear factor-κB (NF-κB) pathway and subsequent upregulation of the expression of metalloproteinases3938Moreover, it is interesting that, in comparison to the healthy individuals, the general group of patients with gastric neoplasms other than gastric carcinoma showed significantly altered profiles of the examined cytokines. Unfortunately, the direct biochemical mechanisms associated with the development and/or progression of these rare types of gastric neoplasms remain unknown. However, our study highlights that the action of the IL-17/IL-23 axis may be a common pathway of interactions involved in the development and/or progression of all types of gastric malignancies in humans. In our recent aforementioned study, based on analysis of patients with different types of pancreatic neoplasms, we observed similar results regarding the potential significance of IL-23, in particular, in the pathogenesis of these tumors in humansFinally, we also evaluated the potential diagnostic value of the examined interleukins for eventual use as novel biochemical indicators detecting and/or differentiating gastric carcinoma from other types of gastric lesions in humans. Our analyses revealed that only measurements of IL-17 may meet initial criteria for further consideration as a potential promising marker of gastric carcinoma in humans. However, from the analyses we conducted, it seems that this cytokine will definitively not be a suitable marker for independent decision-making, as its diagnostic value is less promising than those of various cytokines previously reported2242In summary, our study supported the translational significance of IL-17 and IL-23 in the pathogenesis and clinical presentation of different types of gastric neoplasms in humans. Moreover, it highlights several associations between IL-23 and intensified BMSC trafficking in patients with gastric carcinoma. Finally, it demonstrates that systemic levels of the interleukins examined here do not possess sufficient diagnostic value to be used as independent markers for the detection of gastric carcinoma.This study was performed in accordance with appropriate regulations and guidelines highlighted in the “World Medical Association Declaration of Helsinki – Ethical Principles for Medical Research Involving Human Subjects”. The study protocol was approved by the Institutional Bioethical Committee of the Pomeranian Medical University in Szczecin, and all patients provided written informed consent prior to inclusion in the study.A total of 117 generally healthy individuals were included in the study protocol. These patients were divided into following groups: a “cancer” group of newly diagnosed gastric carcinoma patients (n = 50), an “other” malignancies group to which patients with gastrointestinal stromal tumors - GISTs (n = 5), gastric neuroendocrine neoplasms – NENs (n = 12) and primary gastric lymphomas (n = 8) were included, as well as a “control” group of healthy volunteers (n = 42).adenocarcinoma (n = 34), carcinoma mucocellulare (n = 5), signet ring cell carcinoma (n = 2), carcinoma male differentiatum (n = 2), and mixed type (n = 7).Analogically as in our previous studies21Among patients with GISTs, in 3 cases stage I low grade tumors were diagnosed, and in 2 patients the tumors were detected in stage II and III with high grade of malignant potential. In all cases GISTs were primarily localized in the fundus of the stomach. In case of patients with NEN lesions, all of the diagnosed tumors were non-functional, and were located in the fundus of the stomach. In 10 patients low grade malignancy tumors were observed (NEN G1), and in 2 cases NEN G2 neoplasms were diagnosed. None of the NEN patients presented any signs of metastasis neither to lymph nodes nor to distant solid organs. Patients suffering from primary gastric lymphomas presented following histological types: diffuse large B-cell lymphoma (n = 4), Burkitt lymphoma (n = 2), small lymphocyte lymphoma (n = 1) and mucus-associated lymphoid tissue lymphoma (n = 1). The general characteristics of the individuals enrolled in the study, together with a statistical comparison of these features between the examined groups, are presented in Peripheral blood samples (8–10 mL) were collected from all included individuals. Samples were processed immediately according to standard laboratory protocols, and plasma was separated, frozen, and stored at −80 °C until further assessment.R&D Systems, Minneapolis, MN, USA) according to the manufacturer instructions and recommendations.The systemic concentrations of interleukins (IL-17 and IL-23) were measured using commercially available, high-sensitivity ELISA kits .Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


Acute ischemic stroke (AIS) accounts for more than 80% of the approximately 610,000 new stroke cases worldwide every year. Both ischemia and reperfusion can cause death, damage, and functional changes of affected nerve cells, and these alterations can result in high rates of disability and mortality. Therefore, therapies aimed at increasing neuroprotection and neurorepair would make significant contributions to AIS management. However, with regard to AIS therapies, there is currently a large gap between experimental achievements and practical clinical solutions (EC-GAP-AIS). Here, by integrating curated disease-gene associations and interactome network known to be related to AIS, we investigated the molecular network mechanisms of multi-module structures underlying AIS, which might be relevant to the time frame subtypes of AIS. In addition, the EC-GAP-AIS phenomenon was confirmed and elucidated by the shortest path lengths and the inconsistencies in the molecular functionalities and overlapping pathways between AIS-related genes and drug targets. Furthermore, we identified 23 potential targets  and 46 candidate drugs  that may have value for the treatment of AIS. Acute ischemic stroke (AIS) is a disease that is characterized by neuronal dysfunction and apoptosis induced by the interruption of blood supply resulting from the occlusion or rupture of blood vesselsN-methyl-d-aspartate (NMDA) receptors play specific roles in pathological roles that are shared across various neurological and psychiatric disordersRecently, a new trend in drug development has been to translate the research mode from a single molecule to multiple molecules combined with biological pathways and networks that provides a new method of drug development for complex diseaseset al. proposed novel shortest path-based measurement to evaluate overlap between disease modules21Network medicine has become increasingly important for identifying novel disease mechanisms and predicting drugs1617et al.Here, according to the underlying molecular subtypes of complex diseases and the fragmentation phenomena of disease modules that were extracted from incomplete interactome datahttps://www.nlm.nih.gov/mesh/MBrowser.html) using the key words “stroke” and “infarction”. Using this approach, we ultimately identified (confirmed by the neurobiologists in our author list) 12 AIS-related MeSH headings  terminology database on the MeSH Browser website  in batch mode from the Coremine database−4) is related to glutamate release and calcium transport−3) and “Toll-Like Receptor TLR1:TLR2 Cascade”  are involved in oxidative stress and cellular necrosisTo identify the general functional characteristics of AIS-related genes, we performed GO and pathway enrichment analyses of these genes and detected the significant biological processes in which these genes participate . In an iTo identify the significant disease modules that might correspond to the temporal subtypes of AIS, we partitioned the human PPI network using a widely used community detection algorithm. We confirmed 314 modules of the whole PPI network , and no neuroprotection-associated pathways were included in the overlapping pathways  of AIS, we would be able to detect novel drug targets to promote neuroprotection in AIS. Here, we used DrugBank to filter the novel drug targets (those that have not been previously recognized as AIS drug targets) that might act via direct intervention (shortest path length< = 1) with AIS disease genes through the early-stage pathways. First, we identified 5 candidate modules from the significant identified modules that had high ratios of both AIS genes and drug targets in DrugBank. These were M58, M64, M94, M193 and M145. According to the identified functional characteristics , we found that 3 of these modules  were not associated with any neuroprotective pathways. These pathways were therefore excluded from further analysis. For example, M58 is closely related to lipid metabolism, and M94 is highly involved in immune responses. It has previously been established that the immune system contributes to the brain damage that is produced by ischemia and that the immune response is involved in all stages of the ischemic cascadeBased on our identification of potential targets, we extracted 21 approved drugs, all of which were either antagonists of NMDA receptors or inhibitors of CAMK2. The drugs are used to treat neurological diseases , psychosis , and chronic myelogenous leukemia and to induce and maintain general anesthesia during surgery  and B.2+ signaling by stimulating GPCRsThe M145 module included 149 nodes and was associated with 47 existing drug targets and 9 AIS genes. Of these, 8  targets were involved in the highest M145-enriched pathway, “G Alpha (i) Signaling Events,” which is associated with the G-protein coupled receptor (GPCR) . The cla2+ overload10Ischemic stroke is a heterogeneous disorder with a variety of clinical symptoms and causes and a diversity of disease subtypes, including CADASIL, lacunar stroke and middle cerebral artery infarction. Early stage intervention has an important impact on prognosis in ischemic stroke patients. However, few such interventions are currently available for use in a clinical setting. Investigations of the molecular network mechanisms (in term of disease modules) that contribute to ischemic stroke are key to identifying the manifestation subtypes and temporal stages of this disorder, and knowledge gained in both of these areas will increase our ability to generate tailored early-stage therapies. We identified over 1000 high-quality AIS disease-gene associations in the PubMed literature database and 29 significant disease modules in the human PPI network. We confirmed the importance of several disease modules, such as M64 and M145, which correspond to the temporal subtypes of AIS and would be highly valuable for early-stage therapies (mainly involving neuroprotection). Furthermore, we identified molecular mechanisms that underlie the gap between experimental achievements and clinical solutions for AIS treatment by measuring the difference between the pathways involving AIS genes and existing drug targets. Based on this investigation, a total of 23 potential targets and dozens of FDA approved drugs (for other disease conditions) were identified. Although the ultimate validation of these novel results will require systematic experimental and clinical studies, we searched the recent literature and found isolated studies that partially validated our results, supporting the reliability of these data. For example, both NMDA receptors and GPCR- related pathways have been identified in M64 disease modules, and related studies have shown that NMDA receptor blockers exert a neuroprotective effect while GPCR agonists are associated with cell apoptosis4546NMDA receptors are involved in a variety of neurological and psychiatric diseases4852The clinical failure of NMDA receptor antagonists is partly because of the time frame in which they are applied. Whereas in experimental models, the onset of ischemia and reperfusion can be precisely defined, this is not possible in a clinical setting, and treatment might therefore be delayed before the presence of the disease is realized2+signaling. This pathway is also related to stem cell functions. The role played by G alpha (i) signaling in pluripotent stem cells is largely unknown, but it involves the maintenance of pluripotency and the directed differentiation of human embryonic stem cellsIn addition, the significant pathways associated with M145 and G alpha (i) signaling events exert a neuroprotective effect by inhibiting the cAMP-dependent pathway, which inhibits apoptosisMoreover, ADORA3 agonists can regulate the extracellular matrix (ECM) to protect against neuronal deathhttps://www.nlm.nih.gov/mesh/MBrowser.html). Following this search, the neurobiologists in our author list ultimately confirmed 12 AIS-related MeSH headings. Using these 12 MeSH headings as disease keywords, we downloaded 1425 significant disease-gene associations (p < 0.05) in batch mode from the Coremine database.To identify AIS-related disease terms, we searched the Medical Subject Headings  terminology database using the key words “stroke” and “infarction” at the MeSH Browser website  and the American Heart Association (AHA) have classified ischemic stroke as a atherosclerotic cardiovascular disease events61Community structures are widely distributed in complex networks. Each community is comprised of nodes that densely connect its members and are sparsely connected with the nodes in other modulesGO enrichment analysis defines gene groups based on the categories in the Gene Ontology database68Pathway analysis has become an important method for gaining insight into the biological functions underlying genes and proteinsc nodes in module M1, d nodes in M2, and e edges between M1 and M2, the weight of the edge M1-M2 would be:Next, we extracted the outgoing edges between the nodes in the different modules with an OR > 2. A link was documented when two modules were found to have PPI interactions between each other. For example, if there are The heavier the weight, the closer the interaction between the two modules. Using the topological connectivity, we were able identify biological connections using the functional analysisShortest paths are significant topological and statistical quantities that are used to analyze social and biological networks. The most outstanding example of the use of these quantities is the well-known small world property of many complex networksHow to cite this article: Wang, Y. et al. Network-Based Approach to Identify Potential Targets and Drugs that Promote Neuroprotection and Neurorepair in Acute Ischemic Stroke. Sci. Rep.7, 40137; doi: 10.1038/srep40137 (2017).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.