Here we demonstrate that serglycin plays a critical role in the protein cargo loading of tumor-derived exosomes. Serglycin was detected in exosomes derived from cell culture supernatants of human myeloma cell lines and serum of myeloma patients. Mass spectrometry analysis of exosomal proteins identified significantly fewer protein components within exosomes derived from serglycin-knockdown myeloma cells than within exosomes from control cells. On gene ontology analysis, exosomes derived from serglycin-knockdown cells, but not from control cells, lacked many proteins that are required for mediating different cellular processes. In functional assays, exosomes from serglycin-knockdown cells failed to induce an invasive phenotype in myeloma cells and failed to promote migration of macrophages. These findings reveal that serglycin plays an important role in maintaining the protein cargo in tumor-derived exosomes and suggest that targeting serglycin may temper the influence of these exosomes on cancer progression.Tumor cells produce and utilize exosomes to promote tumor growth and metastasis. Tumor-cell-derived exosomes deliver cargos that partially mimic the contents of the parent cell to nearby or distant normal or abnormal cells, thereby reprogramming the recipient cells to support tumor progression. Mechanisms by which tumor-derived exosomes subserve the tumor are under intense investigation. Here we demonstrate a critical role of the chondroitin sulfate proteoglycan serglycin in regulating the protein cargo and functions of myeloma cell-derived exosomes. Previous studies have shown that serglycin, the only known intracellular proteoglycan, functions mainly in the storage of basically charged components within the intracellular granules/vesicles  One factor driving multiple myeloma is the interactions between myeloma cells and the bone marrow microenvironment. Recent progress in the management of multiple myeloma, a malignancy of plasma cells, has led to the realization that future therapies should aim to disrupt these interactions to improve patient outcomes , 2. ExosGlycosaminoglycans (GAGs) attached to proteoglycans (PGs) play an important role in the biology of exosomes. For example, heparan sulfate GAGs bind exosomes and function as cell surface receptors to mediate exosome internalization . Furtheret al observed that secretory granules of bone marrow cells from serglycin knockout animals lacked exosomes filled with electron-dense materials, compared to exosomes from their normal littermates [We previously reported that the CS GAGs attached to the core protein of serglycin constitute the major component of the myeloma glycocalyx . Though termates . This obIn the study reported here, we discovered that serglycin is present in exosomes derived from the cell culture supernatants of human myeloma cell lines and from the serum of myeloma patients. More importantly, consistent with the findings from serglycin-knockout animals, we discovered that exosomes from myeloma cells with serglycin knockdown had significantly fewer proteins than exosomes from serglycin-expressing control cells. Additionally, compared to serglycin containing exosomes, serglycin-null exosomes were less effective in altering tumor and host cell behavior. Our findings provide the first evidence of a critical role of serglycin in regulating the cargo and functions of tumor-derived exosomes and have a broad significance since a role for serglycin in different cancer progression  is recently becoming apparent.http://research.themmrf.org), with the objective of assessing the effects of serglycin gene expression in 664 myeloma patients who have data available [To determine the extent of serglycin expression in myeloma patients, we analyzed the GEP data bases from the CoMMpass  database interim analysis IA9  by ultrTo determine if serglycin regulates the protein composition of exosomes, exosomes were isolated from equivalent volumes of conditioned medium from control (referred to as SRGN-exosomes) and SRGN-KD (referred to as SRGN-null exosomes) myeloma cells, layered on the top of a 40% iodixanol cushion, and centrifuged. Material excluded by the iodixanol layer was subjected to mass spectrometry for proteomic analysis. Interestingly, the protein contents in SRGN exosomes and SRGN-null exosomes were very different. Significantly fewer proteins were present in SRGN-null exosomes than in SRGN-exosomes Figure . The VenWe next conducted a gene ontology (GO) enrichment analysis of the 223 proteins that were present in SRGN exosomes but not in SRGN-null exosomes using DAVID version 6.8 . InteresBecause we previously showed a critical role of serglycin in myeloma cell adhesion to bone marrow stromal cells and collagen I , we nextFollowing the observation that serglycin can regulate the protein cargo of myeloma-derived exosomes, we studied the effect of SRGN exosomes and SRGN-null exosomes on tumor and host cell behavior. We previously demonstrated that myeloma-derived exosomes can impact the behavior of both tumor and host cells , 12. HerHaving confirmed the differential impact of SRGN and SRGN-null exosomes on tumor cells, we next assessed if these exosomes differentially impacted host cell behavior. Since myeloma-derived exosomes support the recruitment of macrophages, we investigated the effect of exosomes on the migratory properties of macrophages using a transwell migration assay. We found that, compared to no treatment, SRGN exosomes significantly increased the migration of macrophages through an 8-micron-pore membrane Figure . HoweverIntercellular communication is a hallmark of multiple myeloma progression. Despite an emerging appreciation of the role of tumor-derived exosomes in mediating intercellular communication and tumor progression, no therapeutic strategies have been developed that target the impact of tumor exosomes. In the present study, we showed an important role of the CSPG serglycin in regulating the protein cargo and functions of myeloma-derived exosomes. We established for the first time that serglycin is present in exosomes derived from myeloma cell lines and the serum of myeloma patients. We also showed that knockdown of serglycin in myeloma cells decreased the number of exosomal proteins and reduced the impact of these exosomes on tumor and host cells. Our findings provide a novel mechanism by which an intracellular molecule regulates intercellular communication in myeloma.We found that serglycin is required for maintaining the protein cargo of myeloma-derived exosomes, but not required for their biogenesis. Consistent with our findings, studies using serglycin knockout animals and their control littermates have shown that these animals have the same number of secretory granules in their mast cells, but their morphology are different , 34. Whivia serglycin’s negatively charged GAG chains [O-sulfated disaccharides [CHST11 , a sulfotransferase gene responsible for the 4-O-sulfation of CS chains, is highly upregulated in myeloma patients compared to healthy donors [CHST11 could shut down sulfation of CS chains and thereby decrease the protein cargo and functions of exosomes. Therefore, further studies are required to understand whether modulating the anionic charge density on the CS chains of serglycin by silencing CHST11 expression myeloma cells may be an alternative approach to target myeloma exosomes. We would also like to stress that, though anionic GAGs of serglycin may be responsible for the storage of proteins within exosomes, we are still not sure whether this accounts for the absence of the 223 proteins in SRGN-null exosomes. We therefore speculate that serglycin may also regulate the exosomal protein cargo by additional mechanisms, such as by regulating the protein sorting pathway or by binding and retaining exosomal proteins through their core protein. Studies have shown that the core protein of serglycin from myeloma cells can bind to proteases like MMP-9  and MMP-13 [Previous studies have shown that serglycin binds and stores granular components within the secretory granules G chains , 27. We charides , 21. Inty donors . Since td MMP-13 -38.GO analysis showed that the biological pathways associated with the proteins absent from SRGN-KD exosomes were mainly in categories such as translation, cell adhesion, RNA processing, and intracellular protein transport. These pathways were previously reported to be enriched in exosomes . Our finRPMI 8226, OCIMy5, and CAG human myeloma cells were cultured as described previously . SRGN-KD7) where incubated with protease-free bacterial chondroitinase ABC (2U/ml)  for 2h at 37°C before use in western blotting for serglycin. For mass spectrometry analyses, equal numbers of exosomes obtained by ultracentrifugation were layered on the top of a 40% iodixanol cushion (Sigma) and centrifuged for 16 h at 28,000 rpm. The remaining exosome fraction excluded by the cushion was pelleted by ultracentrifugation at 100,000 × g for 70 min and used for analysis, as described [Exosomes from cell culture supernatants (by ultracentrifugation) and serum of myeloma patients were isolated and characterized using electron microscopy and nanoparticle tracking analysis using a ZetaView analyzer, as described by us previously . Exosomeescribed .P < 0.05) were identified.Protein identifications by LC-MS/MS analysis were accepted if they could be established at > 90.0% probability and contained at least 2 identified peptides. Peptides with zero counts were considered absent in exosomes. GO enrichment analysis was performed using DAVID  version 6.8  to under6 particles/ml) were added to CAG cells, and proliferation was measured using the MTT reagent.The role of exosomes in myeloma cell spreading and adhesion to BMSCs and collagen I were studied as described previously , 18. Mig


Bos taurus indicus cattle are more resistant to ticks and tick-borne pathogens compared to purebred European Bos taurus taurus cattle. Genetic studies identifying Quantitative Trait Loci markers using microsatellites and SNPs have been inconsistent with very low percentages relating phenotypic variation with tick infestation. Several skin gene expression and immunological studies have been undertaken using different breeds, different samples , infestation protocols and geographic environments. Susceptible breeds were commonly found to be associated with the increased expression of toll like receptors, MHC Class II, calcium binding proteins, and complement factors with an increased presence of neutrophils in the skin following tick feeding. Resistant breeds had higher levels of T cells present in the skin prior to tick infestation and thus seem to respond to ticks more efficiently. The skin of resistant breeds also contained higher numbers of eosinophils, mast cells and basophils with up-regulated proteases, cathepsins, keratins, collagens and extracellular matrix proteins in response to feeding ticks. Here we review immunological and molecular determinants that explore the cattle tick Rhipicephalus microplus-host resistance phenomenon as well as contemplating new insights and future directions to study tick resistance and susceptibility, in order to facilitate interventions for tick control.Ticks are able to transmit tick-borne infectious agents to vertebrate hosts which cause major constraints to public and livestock health. The costs associated with mortality, relapse, treatments, and decreased production yields are economically significant. Ticks adapted to a hematophagous existence after the vertebrate hemostatic system evolved into a multi-layered defense system against foreign invasion (pathogens and ectoparasites), blood loss, and immune responses. Subsequently, ticks evolved by developing an ability to suppress the vertebrate host immune system with a devastating impact particularly for exotic and crossbred cattle. Host genetics defines the immune responsiveness against ticks and tick-borne pathogens. To gain an insight into the naturally acquired resistant and susceptible cattle breed against ticks, studies have been conducted comparing the incidence of tick infestation on bovine hosts from divergent genetic backgrounds. It is well-documented that purebred and crossbred  Vector-borne pathogens cause diseases with a great impact on public and veterinary health and have accounted for 22% of emerging infections between 1940 and 2004  and due to the global migration of B. t. taurus European breeds for dairy production during the eighteenth–nineteenth centuries, this tick spread across tropical and sub-tropical regions of the world , as well as R. australis and R. annulatus  breeds which were introduced into regions in which R. microplus is endemic failed to resist tick infestation to the same extent as tropical B. t. indicus breeds, which have developed an effective anti-tick immune response  whereas Asian bovines (zebuine breeds or resistant hosts) co-evolved with ticks , antibodies have been shown to mediate inflammatory reactions by triggering effector-cell recruitment and cellular immune response as a consequence of both Fc receptor activation of leukocytes and complement activation that are harmful to the tick, also an immune mechanism in human auto-immune disease syndromes , and during blood meals ticks ingest a substantial amount of IgG  as compared to resistant cattle (B. t. indicus Brahman) with a positive correlation between the level of tick infestation and the level of IgG in susceptible hosts  and B. t. indicus (3/8)], in which there is wide variation in host resistance to ticks  was recognized by resistant bovines and not susceptible, suggesting that RMS-3 could be a protective antigen  were higher in resistant cattle  secreting cells (consistent with a Th1 response) in tick-susceptible Holstein–Friesians compared with tick-resistant Brahman cattle. In contrast, expression of the Th2-associated cytokine interleukin-4 (IL4) was lower in Holstein–Friesian (susceptible) cattle when compared with Brahman (resistant) cattle  was identified from GWAS in dairy and beef cattle  were significantly associated with tick burden and explained about 1.5% of the variation in the trait. Finally, the potential functional role of allelic variation in a gene identified by the same GWAS studies  was further examined using knock out mice  and skin tick bite sites. These studies have included host qPCR, EST libraries, microarray analyses and cDNA library/next generation sequencing. The findings are summarized in Table R. microplus, while susceptible cattle demonstrated cellular and gene expression profiles consistent with innate and inflammatory responses to tick infestation , calcium channel protein transient receptor potential vanilloid 6 (TRPV6) and cysteine proteinase inhibitor (CST6) were highly expressed in susceptible cattle compared to resistant cattle   the acute phase response appeared impaired but this study confirmed the up-regulation of calcium ion control genes which correlates with the calcium binding proteins reported by Nascimento et al.  up-regulated the expression of fewer genes encoding enzymes producing volatile compounds that render them less “attractive” to ticks compared with susceptible cattle (B. t. taurus Holstein breed) , an inducer of pro-inflammatory activators  that correlates with the activity of the complement C1Q identified by Franzin et al. (BCL10 (B-cell CLL/lymphoma 10) was up-regulated in susceptible cattle  and BCL10 (B-cell CLL/lymphoma 10) and several complement factors. IL10 is considered an anti-inflammatory cytokine perhaps induced early in susceptible cattle but IL10 appears to be associated with long term resistance in B. t. indicus Gyr cattle in Brazil  have shown that ticks respond differentially according to whether they are sensing or feeding on a tick-susceptible or tick-resistant breed of cattle. A microarray study based on a Acquired immunity to tick infestation is established after a period of susceptibility to a primary infestation Wagland, . As confB. t. taurus Bonsmara cattle) is associated with an immediate type hypersensitivity reaction. On the other hand, the resistance phenotype was linked to a delayed hypersensitivity reaction in B. t. indicus Nguni breed  and B.t.indicus Brahman (resistant) cattle. Table Granulocytes (or polymorphonuclear leukocytes) are white blood cells characterized by the presence of granules in their cytoplasm and which perform different immune functions. They include neutrophils (most abundant), eosinophils, mast cells, and basophils. The inflammatory profile of the host skin contributes to resistance or susceptibility to tick infestation. Marufu et al.  showed tB. t. taurus) and Nelore cattle   in Brazil and Australian Shorthorn breed   are a multifunctional cell population involved in maintaining local homeostasis of connective tissue, control of blood coagulation and defensive functions of innate and adaptive immunity. In addition mast cell dysfunction is associated with several chronic allergic/inflammatory disorders  and B. t. indicus (Brahman) breeds  in the skin of tick resistant cattle could further be examined to attempt to correlate immunity with gene expression studies described above. The existence of tick histamine-binding salivary lipocalins have been associated with inhibiting histamines from their receptors . High throughput genomics is increasingly affordable and thus prior to the identification of tick resistance markers, it would be practical to first determine the genomic differences between and within breeds under study.Understanding the mechanisms behind genetic resistance to ticks and tick-borne diseases in livestock could improve breeding programs to develop cattle that are more resistant and productive  stimulates bovine neutrophils and potentiates platelet activating factors thus modulating the innate immune response  until tick resistance is achieved after several infestations. The studies reported most likely are hampered by the costs associated with long cattle experiments. This could be why many studies held cattle in tick infested pastures during artificial tick infestations as this is the most economical option for long term studies.Tick vaccines can potentially protect the host from tick infestations and tick borne diseases. This review concentrated less on the development of tick vaccines as it was considered that to develop a successful tick vaccine it would be wise to understand the most common immune effectors to emulate this outcome when using a new anti-tick vaccine candidate(s). Perhaps CRISPR/Cas9 gene editing technologies  may help to identify new vaccine candidates that resist ticks and tick-borne pathogens. By understanding the tick:host interface and the most common denominators of immunity to ticks, this acquired immune response could be manipulated to improve the efficacy of novel anti-tick vaccines. Conversely, the knowledge obtained may assist in the selection of tick resistant cattle or the manipulation of susceptible cattle to develop a protective tick immune response. Furthermore, the in depth analysis of host microbiota and volatile organic compounds could lead to probiotic or diet changes or inhibitory chemicals which could render susceptible cattle less attractive to ticks. Future research may lead to a combination of several of these technologies as novel tick and tick-borne disease control options by first identifying viable biological targets and dissecting pathways leading to vaccination or pharmaceutical therapies or cattle management opportunities for tick control.AA, GR, and AT conceived the research review. AT prepared the comparative Tables/Figures and with NJ revised all sections written by other co-authors. GRG, AZ, TM and NJ wrote or contributed to specific sections within the review.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Two classification systems are now at the forefront of clinical psychiatricresearch: (1) Diagnostic and Statistical Manual, Fifth Edition and (2) theNational Institutes of Mental Health Research Domain Criteria. Herein, wepropose that these two classification systems are complementary rather thanmutually exclusive, and when combined provide important information forunderstanding aspects of the pathophysiology related to Generalized AnxietyDisorder (GAD). The neurobiological literature for GAD and one relevant researchdomain criteria component, sustained threat, are reviewed from multiple units ofanalysis . It ishypothesized that generating a comprehensive, biologically based understandingof the relationship between GAD, sustained threat, and the measureable units ofanalysis will provide information critical to design the most effectivetreatments. RDoC provides a freshperspective on new ways to approach anxiety research , but because of its clinical utility, the DSM-5 maintains itsposition as the diagnostic system that guides clinical practice .Yet, the biological and nosological characterization of DSM-5 anxiety disorders,particularly generalized anxiety disorder (GAD), and other comorbid disorders  are unclear and demonstrate high correlations andunreliability.57 It ishypothesized that an RDoC-based conceptualization and investigation may be able toprovide answers to these shortcomings of the DSM-5 by developing a transdiagnostic,neurobiological model of anxiety that provides a more valid distinction from otherpsychiatric RDoC constructs . Because of the primary clinical andresearch perspectives of the DSM-5 and RDoC classification systems, respectively, weprovide a selective review of the neurobiological literature associated with GAD(DSM-5) and one relevant RDoC construct, chronic stress .Notably, other RDoC constructs are also likely to be relevant to the neurobiology ofGAD ;8 however, sustained threat is used here to provide an illustration of how aconnection between DSM-5 and RDoC classification systems can be made. The reader isreferred to recent reviews of GAD neurobiology912 and RDoC8 for additional background information beyond the scope of this review.Two—not necessarily opposing—classification systems are now at the forefront ofclinical research: (1) Diagnostic and Statistical Manual, Fifth Edition ,which an individual finds difficult to control. The worry is impairing acrossvaried contexts . Symptoms which are required fordiagnosis include feeling restless, being easily fatigued, difficultyconcentrating or mind going blank, irritability, muscle tension, and sleepdisturbance (note: only one symptom needs to be present in children). GAD hashigh rates of comorbidity, particularly between GAD and MDD that ranges from 40%to 98% in treatment studies.1418 In fact, the GAD/MDDcomorbidity may occur more often than MDD or GAD alone.19 In addition to high comorbidity, DSM-5 field trials have highlighted thepoor diagnostic reliability of GAD (kappa = 0.34).5 Other findings indicate differences between illness predictors andsymptoms of MDD and GAD.2023 GAD is often a precursorof MDD and if GAD is treated effectively, it lowers the risk for development ofMDD and some individuals with MDD never develop GAD.24 Because of these mixed findings, cross-cutting and distinct symptoms ofGAD need to be investigated in accordance with transdiagnostic and RDoCperspectives; however, diagnostic categories  should also beexamined due to their clinical relevance and adherence to the currentnosological system (DSM-5).GAD is one of the most common psychiatric disorders, occurring in up to 21% ofadults in their lifetime.25 Forexample, the amygdala has demonstrated increased activity in both anxiety anddepression groups when compared to healthy controls,25 and twin studies indicatehigh genetic correlations between MDD and GAD(r = .74–1.0).27 However, MDD and GAD canbe differentiated with respect to biology,2830 particularly withresting-state functional magnetic resonance imaging (R-fMRI).31Given the clinical ambiguities, it is not surprising that there is a limitedbiological information regarding how similar or different GAD is from otherdisorders . At the biological level, studies that have pursued abiological conceptualization from one unit of analysis  haveoften highlighted similar findings in depression and anxiety.As the current literature lacks clarity regarding the clinical and biologicaldifferences between GAD and other disorders, such as MDD, both DSM-5 and RDoC (cross-cutting constructs) approaches offercritical information for understanding the differences and overlap between GADand other disorders. Thus, the present literature review examines aspects of theneurobiological literature from both a categorical (GAD) and dimensional(chronic stress) perspective in order to build an initial understanding of thediagnostic differences versus dimensional intersections  related to GAD.33 Over time,if an individual is subjected to short-term acute stressors that are repeatedlytriggered, the resulting chronic maladaptive stress responses  may develop into interfering psychiatric difficulties ordisorders.3436 Thisresulting chronic stress impacts biological systems across multiple “units ofanalysis”—neuroendocrine, autonomic, and behavioral—when triggered by perceivedor actual threat.37 Chronic stress can significantly impact the homeostatic biological systemby increasing the allostatic load .39 Increased cumulative effects of stress on the human body are linked to many adverse health consequences, includingpsychiatric illnesses such as GAD.Chronic stress may be one cross-cutting construct or dimension  related to GAD, as well asother diagnoses (such as highly related MDD). In general, a stress response maybe adaptive in the short-term when faced with acute challenges.40), hereditary factors, and stress history.4144 For example, childhoodtrauma and life experiences increases the risk of onset and recurrence ofdepression and anxiety disorders.45 Childhood trauma, childhood life experiences, as well as genetic andenvironmental factors 47 may reduce an individual’s“tolerance” or resilience to increased allostatic load caused by repeatedstressors; thus, increase the risk for the development of psychiatric disorders,including anxiety.Individuals may be able to remain resilient  to different amounts of cumulative stress based on their early-lifeexperiences  was associated with the highest risk for experiencingsuicidal thoughts and behaviors across adolescence and into adulthood.49 Not only was GAD diagnosis associated with the highest risk trajectoryfor suicidal thoughts and behaviors, broader anxiety symptoms were as well.Combined with the shocking 24% increase of suicide mortality in the past 15years in the U.S.,50 these trajectory findings stress the impact of chronic stress andpsychiatric diagnosis on future psychopathology.Early diagnosis (and treatment) of a psychiatric disorder also may contribute toan individual’s resilience and ability to cope with repeated stress. Forexample, carrying a diagnosis of depression or anxiety in childhood can increaserisk of an individual being diagnosed with GAD in adulthood.51 breaks down cross-cutting constructs into five domains, includingnegative valence system domain, which includes constructs related to anxiety. In the present review, we exploreRDoC sustained threat as a highly relevant cross-cutting construct related toGAD and chronic stress. Although potential threat has been considered—and ishighlighted in the RDoC matrix—as being related to anxiety, it is sustainedthreat (or the experience of chronic stress), whether potential or actual, thatappears to have the most significant health consequences related to GAD . RDoC defines sustainedthreat as “an aversive emotional state caused by prolonged  exposure to internal and/or external condition(s), state(s), or stimulithat are adaptive to escape or avoid…” This description includes that the threatmay be “actual or anticipated.” Alongside this description are the multilevel(molecules to behavior) neurobiological components hypothesized to be involvedin the construct of sustained threat  related to sustained threat, or othermeasurable RDoC mechanisms, may generalize and target the chronic anxietydemonstrated in individuals with GAD. For example, implementing a pharmacogenomicsor metabolomics protocol to inform medication selection may improve pharmacotherapytreatment outcomes. Not only can neurobiological markers provide insight fordeveloping personalized medicine protocols and offer biological objective markers oftreatment efficacy, the implementation of successful treatment strategies may bedependent on timing. There are critical neurobiological developmental windows inchildhood that may be most ideal to target to produce the best outcomes whentreating neuropsychiatric disorders, including GAD .52Here, we review the neurobiological literature associated with GAD and with the RDoCdomain, sustained threat. As noted prior, this literature review is not intended toinclude an exhaustive review of all RDoC domains relevant to GAD  but rather provide a demonstration of how both DSM-5 categoricalperspectives and RDoC domains can be linked. Our proposed model see  includesOur conceptual model  organize54 to the development of ananxiety disorder. Conversely, development of an anxiety disorder due tonon-genetic factors is approximately 50% to 70%. Environmental factors  likely contribute to the development of anxiety disordersthrough epigenetic mechanisms that could impact the development of anxiety evenbeginning in utero. For example, mothers diagnosed with an anxiety disorder whodid not receive medication for anxiety have demonstrated altered DNA methylationof the glucocorticoid receptor gene (NR3C1) promoter region in cord blood andthe genome56 and may increase risk of their child developing an anxietydisorder.It is estimated that genes contribute 30% to 50%12 One genome-wide association study of GAD symptoms found genome-widesignificant association between a single-nucleotide polymorphism (rs78602344)intronic to thrombospondin 2 (THBS2) and GAD symptoms in apopulation-based sample of Hispanic/Latino adults .57 Although not specific to GAD, a study of anxious versus non-anxiousadults  found that DNAmethylation indicated that global methylation levels were higher in anxiousadults relative to non-anxious adults58 . Hospital Anxiety and Depression Scale-Anxiety scores in the anxiousgroup were also significantly correlated with the expression of the DNAmethyltransferases DNMT1/3A, such that greater anxiety severity was associatedwith more DNA expression. Notably, the generalizability of the findings of thisDNA methylation study is limited due to the small sample size.Understanding the genetic and epigenetic mechanisms of GAD is important forbuilding more comprehensive neurobiological conceptualizations to develop themost effective preventative and treatment strategies. Despite the potentialimpact of understanding these mechanisms, few human studies of anxietydisorders, including GAD, exist relative to animal studies.5961 particularly reducedresting-state functional connectivity (RSFC) between the amygdala and prefrontalcortex (PFC) in both adults and adolescents with GAD.6264 Yet, differences betweenGAD and healthy control groups, both in structural and functional neuroimaging,not only exists in frontolimbic areas but also in a downstream projection fromthe amygdala: the anterior cingulate cortex.61 Findings suggest that across adolescence and adulthood, decreasedconnectivity between the amygdala and PFC is associated with the diagnosis ofGAD.6264 The PFC iscritical for the effective regulation of emotion, particularly ventromedialregions that appear to control negative emotion,65 such as anxiety. Fittingly, the ventrolateral PFC activity has been shownto increase (perhaps increasing regulation over limbic structures) following allpharmacological and psychological intervention in individuals with GAD.66The disrupted coordination of brain activity has been proposed as one of the coreneurobiological features of GAD,59 Specifically, a recent neuroimaging study investigated the relationshipbetween both central nervous system  connectivity and autonomic in individuals with GAD pre- andpost-perseverative cognition task.59 At baseline, individuals with GAD demonstrated less connectivity betweenthe PFC and amygdala; however, after the perseverative cognition task, thisPFC-amygdala connectivity increased in the GAD group whereas healthy controlsdecreased in this area (at a trend level). After the perseverative cognitiontask, HRV reduction (increase in autonomic activation) was predicted bydecreased RSFC between the left amygdala-subgenual cingulate cortex and betweenthe right amygdala-caudate nucleus. These findings provide initial evidenceregarding specific neural modulation of differential psychophysiologicalresponses between GAD and healthy control groups.Frontolimbic structures not only impact anxiety-related responses in GAD but alsohave been demonstrated to differentially regulate autonomic mechanisms inindividuals with GAD.68 andindividuals with other anxiety disorders.69 Yet, this hyperarousal psychophysiological pattern in GAD is notuniversally observed.70 It is possible that psychophysiological differences between GAD andcontrols are context specific. For example, increased sympathetic nervous systemactivity and reduced HRV was observed in high trait worry individuals.72Additionally, worrying prior to fearful imagery exposure appears to diminish anyassociated cardiovascular reactivity, rather than relaxation or neutral stimuliprior to fearful exposure that exposes heightened cardiovascular reactivityduring a fearful exposure in GAD participants.74 Thus, it appears thatrelative to controls, GAD participants demonstrate heightened physiologicalarousal at baseline , and these individuals are likelyto demonstrate the greatest cardiovascular reactivity to an anxiety-provokingstimulus when asked to relax or view neutral stimuli. This may suggest thatindividuals with GAD demonstrate chronic physiological arousal at baseline anddemonstrate an exaggerated physiological reactivity to fearful stimuli.Similarly, when compared to other anxiety disorders , GAD is characterized by elevated baseline startle,suggesting the presence of anxious thoughts in the absence of threat.69 Overall, it appears that relative to healthy controls and individualswith other anxiety disorders, individuals with GAD may exhibit a uniquepsychophysiological signature that is characterized by hypervigilantphysiological response at baseline, as well as greater reactivity to threat.In general, the psychophysiological literature demonstrates that individuals withGAD are in a more physiologically dysregulated state  at baseline relative to controlsFindings across genetics, neuroimaging, and psychophysiology indicateneurobiological signatures related to the diagnosis of GAD. When examined inmore detail, findings appear more closely related to elevated baselineneurobiological states that are likely correlated with increased thoughts ofworry/fear without the presence of an immediate threat. The durability ofphysiological and cognitive changes—whether related to actual or perceivedthreat—potentially contributes to the neurobiological differences highlightedabove. This sustained threat  is oneparticularly relevant component in GAD. Please see 75 Serotoninergic functioning and variability appear related to individualdifferences in responding to threat, particularly the variation of the serotonintransporter gene (5-HTTLPR), which is related to neuralpatterns associated with anxiety, including GAD. When shown fearful and angryfaces, 5-HTTLPR short-allele carriers show bilateral amygdalahyperactivation compared to 5-HTTLPR long-allelehomozygotes.7678Additionally, decreased connectivity between the amygdala and pregenualcingulate, as well as decreased gray matter volume in both brain structures, hasbeen observed in 5-HTTLPR short-allele carriers.79Prior findings suggest that serotonin (5-HT) regulation is related to emotional stability.5-HTTLPR short-allele carries ,77 some studies do not findthe 5-HTTLPR × stress interaction to be significant.815-HTTLPR short-allele carriers appear to be sensitive toenvironmental cues, which may contribute to anxious thinking whenstressors/threats are present.75 This highlights a propensity of these individuals to be sensitive toacute threat cues in the environment, which may contribute to a hypervigilanceor sustained threat over time. In support of this notion,5-HTTLPR short-allele carriers have been shown to have apositive correlation between life stress and resting activity of the amygdalaand hippocampus,82 such that more lifetime stress is associated with more resting activitybetween the amygdala and hippocampus suggesting a sustained threat response.Though studies have demonstrated the relationship between amygdala hyperactivityin As highlighted in 83 Similarly, in humans, the BNST has been associated with sustained threat/anxiety.84 An fMRI study examined neural response to negative and neutral picturesat predictable and unpredictable intervals. The amygdala demonstrated reactivityto negative pictures that was not dependent on predictability, whereas the BNSTshowed a linear increase of activation across conditions as a function of anxiety;84 thus, identifying the BNST as related to sustained threat/anxiety versusacute stress .Changes in the neuroendocrine system related to sustained threat  have been demonstrated in both animaland human studies. For example, in one preclinical study, rats receivedintraventricular infusions of CRF and were placed in a sustained threat(sustained exposure to bright light) or acute threat condition. Resultsdemonstrated that longer duration (sustained threat) threat responses wererelated to the BNST and could be differentiated from acute threat response  by their sensitivity to CRF-RI receptor antagonists.85 which measures the fronto-centrally maximal negative deflection in theevent-related potential, as individuals reporting high levels of worry havedemonstrated enhanced error-related negativity during a stressor task (Stroop task).86 Further, peripheral nervous system literature highlights a heightenedsensitivity to unpredictable threat in individuals with lower respiratory sinusarrhythmia (a measure of high frequency heart rate variability),87 which is consistent with the literature reviewed above that demonstratethat individuals with impairing levels of anxiety, as indicated by a diagnosisof GAD, demonstrate more sympathetic activity  at baseline and greater cardiovascular reactivity tounpredictable threat. Contrary to these cardiac findings, however, skinconductance responses to unpredictability appear to induce a physiologicalinhibition indicated by weaker skin conductance responses to cue (stimuli presented) andtemporal  unpredictability.88 In general, it appears that the unpredictability may maintain a sustainedthreat response that is correlated with a heightened baseline physiologicalstate as well as elicit greater reactivity when a threat is presented.Psychophysiological responses related to sustained threat have been hypothesizedto be measured by error-related negativity,5-HTTLPR short allelecarriers to be at-risk for development of GAD (likely over the course of repeatedstress) and when anxiety/sustained threat responses reach a level of impairment,global methylation levels are increased . Thesegenetic profiles or changes appear to influence RSFC, particularly involvingamygdala. The chronicity of sustained threat—to the point of the development ofGAD—appears to also affect baseline psychophysiological functioning  and place anindividual in a hypervigilant physiological state .Across GAD (DSM-5) and sustained threat (RDoC-based) studies, findings provideinitial evidence of the biological system overlap (see 57); however, two studies did investigate the link between genetics/epigeneticsand neuroimaging82 as well as neuroimaging and psychophysiology.59 The investigation of both GAD and sustained threat primarily from onebiological system highlights a significant gap in the literature. In fact, novelcomputational methods have recently been designed to examine the link betweengenetics, neuroimaging, and clinical data89 and could be expanded to include psychophysiology . In line with RDoC goals,study of the integration of the biological systems, and their integration withclinical data, will provide an important understanding of the biological processesthat relate not only to psychiatric disorders but also to the cross-cuttingconstructs, such as sustained threat, that are hypothesized to provide criticalevidence for the development of new, targeted treatments.3Despite the compatibility of results across biological systems, many studies that wereviewed investigated one biological system , such as making studydata sets publically available, can greatly aid investigators in their pursuit ofstudy replication. Future directions in personalized medicine will not only need totake into account treatment targets and optimal timing  butalso carefully consider the limitations of this research that argue for replicationand improved systems that encourage data sharing.Going forward, it will be important to continue to seek a comprehensive, biologicallybased understanding of GAD, particularly in the context relevant RDoC anxietyconstructs . Further, treatments will need tobe developed from a comprehensive neurobiological framework, and timing of treatment implementation will have to beempirically tested to understand the most impactful timing to produce the optimaltreatment trajectory. This is particularly important for anxiety disorders, as theyoften emerge in school-age children when other key processes in neurodevelopment areoccurring .


Adult neurogenesis mainly occurs at the subventricular zone (SVZ) on the walls of the lateral ventricle and the subgranular zone (SGZ) of the dentate gyrus (DG). However, the majority of newborn neurons undergo programmed cell death (PCD) during the period of proliferation, migration, and integration. Stroke activates neural stem cells (NSCs) in both SVZ and SGZ. This process is regulated by a wide variety of signaling pathways. However, the newborn neurons derived from adult neurogenesis are insufficient for tissue repair and function recovery. Thus, enhancing the endogenous neurogenesis driven by ischemia and promoting the survival of newborn neurons can be promising therapeutic interventions for stroke. Here, we present an overview of the process of adult neurogenesis and the potential of stroke-induced neurogenesis on brain repair. Stroke is one of the leading causes of morbidity and mortality worldwide. In addition, about two-thirds patients had neurologic impairment and disability, based on a population study of follow-up of stroke survivors at five years . Therefo3 into adult rats and cats to tag the newborn cells and found that the labeled glia cells and neurons are present in various regions of the normal adult mammalian brain. In the 1990s, bromodeoxyuridine (BrdU) was applied to label newborn cells in neurogenesis research. With the application of this new technique, two areas of the adult neurogenesis were found: the DG and the SVZ. In the DG, new neurons continue to be generated from NSCs in the SGZ. NSCs also reside and proliferate in the SVZ and differentiate into neuroblasts. These neuroblasts migrate through the rostral migratory stream (RMS) to the olfactory bulb (OB) and integrate into OB circuits. Recently, some noncanonical sites of adult neurogenesis, such as neocortex, striatum, corpus callosum, amygdala, and hypothalamus, have been found in different species [Adult neurogenesis (AN) is a process that is continuously producing new neurons which integrate into existing circuits in adult age and have different mechanisms compared with fetal and early postnatal development . AN was  species .The process of maturation of new neurons encompasses the proliferation of resident NSCs and their subsequent differentiation, migration, survival, and functional integration into the preexisting circuitry . AN is mβ-III-tubulin (TuJ1), and polysialylated neural cell adhesion molecule (PSA-NCAM) are the unique markers of type A cells [In mammalian animals, new OB neurons are derived from SVZ, on the walls of the lateral ventricles. The SVZ have five main cell types: B1 astrocytes (type B1 cells), B2 astrocytes (type B2 cells), transit-amplifying cells (type C cells), neuroblasts (type A cells), and ependymal cells (type E cells) . MicroglNSCs in the SVZ correspond to type B1 cells. Asymmetric division of type B1 cells produce self-renewed type B1 cells and type C cells , 12, whi+ PGCs are derived from ventral NSCs, whereas superficial GCs and tyrosine hydroxylase (TH)+ PGCs are derived from dorsal NSCs. NSCs from medial wall produce calretinin + GCs and CalR+ PGCs [There are two principal types of adult-born OB neurons: periglomerular cells (PGCs) in the glomerular layer (GL) and granule cells (GCs) in the granule cell layer (GCL). Deep GCs and calbindin lR+ PGCs   that, in turn, generate intermediate neural progenitors (type 2 cells). These cells are immature neuroblasts that can be further differentiated into neuroblasts. Neuroblasts can be further divided into more differentiated cells (type 3 cells) , 23. TypType 1 cells are located in the SGZ and have a triangular-shaped soma. A strong apical process extended into the molecular layer of DG is the typical characteristic of type 1 cells. Type 1 cells have some astrocyte features that may contact blood vessels through the end-feet . RecentlPCD is the death of a cell in any form, mediated by an intracellular program that mainly occurs during embryo/adult development and in some pathologic conditions . The maj−/−bax mice has no difference compared with that in wild-type (WT) mice, whereas at 12 months, the number of DG neurons is doubled in −/−bax mice [Neuroblasts from SVZ migrate through RMS to the OB and differentiate into GCs and PGCs. GCs are mature at 15–30 days and PGCs at 4 weeks after birth. There are around 30,000 newborn interneurons integrated into OB neural circuits daily in adult mice , 29, 30.−/− mice . In adul−/− mice . These d−/−bak−/−bax mice show resistance to a series of apoptotic stimuli and are accumulated in the SVZ and SGZ. Bax single-deficient NSCs are resistant to apoptosis induced by staurosporine [Mcl1 conditional knockout mice, NSCs in the SVZ are more vulnerable to apoptotic cell death. However, overexpression of Mcl-1 reduces the apoptotic rate by about 50% in NSCs from the SVZ [bim or puma deficiency significantly enhanced the survival of adult-born cells but have no change on NSC differentiation [Puma deficiency also increases the survival of SVZ NSCs. Puma is required for p53-induced apoptosis in NSCs of DG [Trp53 enhances slow and fast proliferation in SVZ populations and associates with their differentiation toward neuronal and glial cell lines [Trp53 and p53 deficiency induces apoptotic brain lesion. These p53-deficient mice have thinner isocotex and enlarged ventricle compared with wild-type mice [Growth factors secreted in the SVZ niche are essential for the survival of NSCs. Thus, NSCs that lack neurotrophic signals are more sensitive to apoptosis stimuli. NSCs from adult osporine . While i the SVZ . Regardintiation . Puma deCs of DG , 40. Besll lines . Howeverype mice . Therefoype mice –45.bax-deficient mice, a large number of abnormal neuroblasts accumulate in the RMS [Errors during migration also induce apoptosis in the adult-born neuroblasts. In  the RMS . A simil the RMS . However−/−bax mice, apoptosis is inhibited in immature and mature neurons of DG, and the size of DG neurons enlarges continuously with age [bax-deficient mice, the pattern separation function of the hippocampus is enhanced. However, knockout bax seems to have no effects on other major hippocampal functions [−/−bax mice, the normal olfactory learning behavior is improved, and the perturbations of newborn cell migration result in imbalance of neural circuits that destroy the olfactory learning ability. Besides, the bax-deficient mice show no significant changes on olfactory sensation [The most extensive apoptosis of newborn neurons occurs during the integration into neural circuits. 30–70% of immature neurons are eliminated by apoptosis during the formation of synaptic contacts , 31, 47.with age . Synaptiwith age . All thewith age . Howeverunctions . It seemunctions . A similensation , 56. BasAlthough the majority of cell types that undergo PCD are immature neurons and neuroblasts, mature neurons also have lower levels of PCD in the OB and DG. The purpose of PCD in mature neurons is to renew the preexisting neural circuits , 57. At Adult NSCs in neurogenic regions can be activated by different stimuli such as learning  and runnIn models of transient global cerebral ischemia, cerebral blood flow is reduced throughout the whole brain . The hipIn focal brain ischemia, middle cerebral artery occlusion (MCAO) is the most frequently used focal brain ischemia model, which produces consistent infarcts in the ipsilateral hemisphere of the cerebral cortex, hippocampus, and striatum . NeurogeAbove studies have shown convincing evidence of neuroblasts migrating from the SVZ or SGZ to the ischemic areas. However, several studies have proposed the possibility that there exist other stem cell niches in the adult brain , 86. In AN has arisen great interest as it can be applied for new therapies to replace damaged neurons and treat severe neurological deficits after stroke and other neurological diseases. However, the endogenous neurogenesis failed in producing adequate amounts of newborn neurons that can survive and integrate to restore the function recovery. Stimulating or enhancing the endogenous neurogenesis driven by ischemia can be a promising therapeutic intervention for stroke. The vast majority of the newborn neurons die between 2 and 5 weeks, which may be caused by the unfavorable environment that is exposed to the detrimental injury niche, lacking appropriate trophic support and failed connections with other neurons after stroke . Reducinα, ciliary neurotrophic factor (CNTF), and platelet-derived growth factor (PDGF) have all been proposed to play essential roles in the adult neurogenesis response to ischemia stroke [A wide variety of signaling pathways are related to NSC activity during proliferation, migration, differentiation, and their maintenance in the adult neurogenic regions. As survival is the first factor of newborn neurons, neuroprotective agents or manipulations attempt to benefit from neuronal survival preserving the property to support neurogenesis in long term . Adminisa stroke –111. Reca stroke –116. Thea stroke . Though Majority of stroke patients suffer from serious morbidity and never regain full functional independence. The limited result of stroke treatment has driven the search for stem cell therapies directed at restoring neurological function. However, both technical and ethical issues limit the development of exogenous stem cell therapy . The fin


Zootermopsis nevadensis. Termites are the most ancient social insects, and developmentally distinct from highly-studied, hymenopteran social insects. We used replicated bisulfite-sequencing to investigate patterns of DNA methylation in both sexes and among castes of Z. nevadensis. We discovered that Z. nevadensis displayed some of the highest levels of DNA methylation found in insects. We also found strong differences in methylation between castes. Methylated genes tended to be uniformly and highly expressed demonstrating the antiquity of associations between intragenic methylation and gene expression. Differentially methylated genes were more likely to be alternatively spliced than not differentially methylated genes, and possessed considerable enrichment for development-associated functions. We further observed strong overrepresentation of multiple transcription factor binding sites and miRNA profiles associated with differential methylation, providing new insights into the possible function of DNA methylation. Overall, our results show that DNA methylation is widespread and associated with caste differences in termites. More generally, this study provides insights into the function of DNA methylation and the success of insect societies.Epigenetic inheritance plays an important role in mediating alternative phenotype in highly social species. In order to gain a greater understanding of epigenetic effects in societies, we investigated DNA methylation in the termite  Phenotypic plasticity is a highly important mechanism whereby a single genotype can produce multiple phenotypes based upon environmental variation. Social insects represent excellent models for studying phenotypic plasticity. In insect societies, individuals can develop distinct social phenotypes (castes), usually through the integration of information from the environment1Phenotypic plasticity, such as that displayed in insect societies, requires epigenetic information. Epigenetic information is not coded in the standard complement of DNA bases but nevertheless results in heritable changes in gene expression4de novo DNA methylation (DNMT3) was shown to have a direct impact on the production of castes in the honey bee811DNA methylation has been implicated as an important component of the determination of social insect casteHowever, the function of DNA methylation in social insects remains controversial6915Termites (epifamily Termitoidae) represent an entirely novel origin of sociality in insects, and are distinct from the Hymenoptera in many ways1720Termite developmental plasticity is largely informed by hormonal (endogenous) and environmental (exogenous) cues222321Zootermopsis nevadensis. This species is common in the cooler coastal and higher elevations of the western USAZ. nevadensis frequently engage in anal feeding as part of their social interactions. Workers eventually develop into either alates, neotenic reproductives or the altruistic soldiersZ. nevadensis therefore show different levels of inbreeding depending on the number of replacement reproductives. Z. nevadensis thus represents an interesting termite system in which to study DNA methylation in the context of sociality.In order to gain a greater understanding of the function, evolution, and importance of epigenetic information in social insects, we investigated the distribution and levels of DNA methylation and gene expression in both sexes of the final larval instars (workers) and winged reproductives  of the termite Overall, we found that DNA methylation was considerably higher and targeted to more genes in termites than in any other social insects studied to date. Moreover, patterns and levels of DNA methylation were more similar to non-insect invertebrates than to other previously-analyzed holometabolous insects. We also found that termite morphs displayed many differences in DNA methylation, the great majority of which occurred between castes. Differential methylation was also related to alternative splicing, and showed enrichment for multiple regulatory sites. Thus, our results provide insight into the evolution and regulatory function of DNA methylation in insects and expand our understanding of DNA methylation’s link to phenotypic plasticity in insect societies.Z. nevadensis termite colony. Methylome sequencing was replicated twice, and transcriptome sequencing was replicated three times, for each sample type . This provided four replicates for comparisons of DNA methylation between caste and four replicates for comparisons of DNA methylation between sexes. Methylomes and transcriptomes were derived from whole body minus guts. Methylome data were obtained from pooled individuals, whereas transcriptome data were obtained from individual termites.We sequenced the methylomes and transcriptomes of male workers, female workers, male alates, and female alates  from a single We found exceptionally high levels of DNA methylation in termites  and S1. Z. nevadensis, with considerable methylation in introns . We found that the most highly methylated genes  showed functional enrichment of terms associated with more fundamental regulatory processes such as chromatin modification, protein binding, helicase activity, metabolic processes, and regulation of gene expression, while the most lowly methylated genes (bottom 3 DNA methylation deciles) were functionally enriched for more dynamic terms such as “signaling receptor activity”, transmembrane transport of various molecules, cell periphery, and circadian behavior  and non-genic repeats separately. We found that, a low proportion of non-genic repeats showed at least some DNA methylation, which was higher within non-genic-repeat elements than in surrounding regions , and at higher levels .Unlike many insects examined to date, the C. floridanus and A. mellifera were also methylated in Z. nevadensis . Moreover, despite the lineage specific methylation of these genes, their mean methylation level was considerable . However, genes of intermediate methylation level were the most highly, and ubiquitously expressed  and within-morph replicate variation , as well as intergenic distance, average exon count and general measures of gene conservation. We also leveraged the stranded nature of our RNA-seq protocol to produce a measure of anti-sense expression, which was incorporated into our combined model.We found the strongest predictors of levels of DNA methylation in the combined model framework were expression CV and between-sample expression difference, which were both strongly negatively associated with genic DNA methylation level . Interes10We next examined DNA methylation’s relationship with gene expression among recently-expanded gene familiesOverall, we found that a very high proportion of genes possessed regions differentially methylated between phenotypes (castes or sexes). In total, 2,749 genes  possessed significant differentially methylated regions (DMRs), defined as 200 bp windows showing significant differential methylation, between phenotypes. Interestingly, relatively few genes were differentially methylated between sexes (368 genes). Instead, the vast majority of differentially methylated genes (DMR-containing genes: DMGs) arose between the reproductive and worker castes . We first performed statistical tests examining the relative enrichment of existing D. melanogaster TF motif profiles  to identify any enriched motifs in the vicinity of DMCs, independent of any a priori motif sets. We identified several such motifs in the Z. nevadensis genome. The majority of identified motifs were long (>15 bp) and exhibited considerable similarity to known D. melanogaster miRNAs . Interestingly, previous studies, even in mammals, have observed a negative relationship between intragenic DNA methylation and gene expression noise1636Strikingly, we found the relationship between DNA methylation and gene expression level or breadth to be much stronger among gene families having undergone recent duplication. This suggests that DNA methylation may play a novel role in regulating the expression of recently-duplicated genesZ. nevadensis colony. Thus further analyses of DNA methylation from more colonies would help determine if methylation differences are affected by genetic and environmental variation arising from colony differences.We compared replicated DNA methylation between males and females and between reproductive and worker termite castes. We found substantial differences in methylation between castes, and to a lesser degree sexes, demonstrating that developmental phenotypes of termites do display methylation differences. We assayed DNA methylation at the whole organism level. Therefore, the methylation differences we uncovered may reflect allometric variation between castes or sexes arising from whole body analyses. Thus further analyses of tissue-specific methylation would be of interest and help in ascertaining the exact tissues causing the differences between termite phenotypes. In addition, analyses of time matched samples would help isolate the effects of age on DNA methylation, which may also be a contributing factor to the methylation differences we observed. We also note that our analyses were centered on a single Our finding of differential methylation between social insect phenotypes stands in contrast to recent studies that did not detect methylation differences between castesdifferential splicing between castes and sexes, respectively. Notably, differences in DNA methylation in bees and ants have been linked to differences in alternative splicing, and it is hypothesized that this relationship may underlie DNA methylation’s impact on caste determination in social insects89Z. nevadensis only but not Hymenoptera.We found that the majority of differences in DNA methylation existed between castes, with far fewer existing between sexes. Both caste- and sex-specific differentially methylated genes (DMGs) were less variably expressed, but showed considerably higher levels of alternative splicing than non-DMGs. Importantly, we also found that both caste- and sex-DMGs exhibited higher proportions of Gene body DNA methylation differences have been experimentally implicated in the regulation of differential splicing in mammals4040424145Z. nevadensis, suggesting that methylation is linked to alterations in TF binding in termite genes. We further found that many of these binding sites exhibited enrichment in regions differentially methylated only between caste or sex. Notably, many of these enriched TFBSs were associated with developmental processes. Therefore, in termites, differential methylation is targeted to many regions potentially bound by important regulators of development. Thus DNA methylation may represent an important evolutionarily-conserved molecular mechanism that impacts developmentally-important TF binding.Interestingly, we found that multiple transcription factor binding site (TFBS) motifs were associated with differential methylation in Z. nevadensis genome. Although the canonical role of miRNAs is the regulation of translation, emerging evidence suggests that miRNAs can also impact the epigenome as well as the process of transcription474849Drosophila in a smRNA-guided manner4346Surprisingly, we also found multiple mature miRNA or miRNA-like sequences associated with differentially-methylated cytosines of the D. melanogaster. For example, two of the top TFs (Eip74EF and forkhead), as well as one miRNA (miR-125), in this category are directly associated with the regulation of ecdysone in D. melanogater21Z. nevadensis  levels of DNA methylation were much higher in termites than in the majority of other insects, (ii) DNA methylation was associated with gene expression, (iii) many significant differences in methylation existed between castes but few between sexes, (iv) caste and sex specific differentially-methylated genes showed higher levels of alternative splicing differences, and (v) regions surrounding differentially methylated cytosines were enriched for multiple regulatory motifs. Thus our study greatly increases the current knowledge of DNA methylation’s relationship to alternative phenotype across insects, and further provides a possible mechanism whereby DNA methylation may effect differential gene regulation in insects. Finally, our results highlight the general utility of termites as a developmental and evolutionary contrast to hymenopteran social insects, and illustrate termites as an excellent model for future molecular studies of epigenetic underwriting of insect caste and development.Zootermopsis nevadensis nuttingi was collected in its entirety within a wood log from Pebble Beach near Monterey, California, United States in October 2013. The colony was kept in the original log in a large bin in the laboratory. The log was sprayed with water once per week and kept at 22˚ Celsius with a 12 h light/dark cycle. Species identity was confirmed by analyzing the cuticular hydrocarbon profiles from workers using gas chromatography-mass spectrometry56A colony of Individual male and female alates and workers  were collected for nucleic acid extraction five months following the original collection date. Individuals for RNA extraction were dissected in RNAlater, while individuals for DNA extraction were dissected in distilled water. The guts of all samples were carefully dissected out under a microscope and not included in nucleic acid analyses. Wings were removed from alates as well. Sex was determined based on external abdominal morphology.® Reagent (Life Technologies) according to manufacturer’s instructions. The extracted RNA was suspended in 40 μL Nuclease Free Water (Ambion) and was treated with TURBO DNA-free™ kit (Ambion) to remove the genomic DNA contamination. RNA concentration was measured by Qubit ®2.0 fluorometer  and RNA integrity was checked by Agilent RNA 600 nano kit .Total RNA was extracted from single individual termites in 1 ml of Trizol®2.0 fluorometer (Life Technologies).One termite was used per sequencing library for each RNA-seq sample and five termites were used per sequencing library  for each BS-seq sample. Genomic DNA was isolated using MasterPure™ Complete DNA and RNA Purification Kit (Epicentre). The extracted DNA was suspended in 35 μL TE buffer. DNA concentration was determined by Qubit http://dna.uga.edu), and sequencing was performed using the Illumina NextSeq500 platform. These methods allowed for the detection of methylation at cytosine nucleotides (5mC) but did not allow for the detection of 5-hydroxymethylcytosine (5hmC)Bisulfite converted DNA and mRNA library preparation was performed in Georgia Genomics Facility  and then mapping these read to their respective genomes  using Bismark. CpG DNA methylation was then quantified and associated with features using the same methods as for Z. nevadensis. For C. floridanus, we only utilized a subset of samples  in order to ensure similar BS-seq coverage between species. Species-level comparisons of DNA methylation were performed with datasets produced by pooling all considered samples within species. Orthologous relationships between genes were then established using OrthodbMethylation data from MethylsigZ. nevadensis official gene annotation (v2.2), and each DMR was assigned the genic feature it overlapped most. Only DMRs exhibiting >15% absolute methylation difference and a FDR of <0.05 were utilized to establish differentially methylated genes.We also performed Methylsig analysis for all relevant caste and sex pairs  to produce a more conservative list of caste- and sex-associated DMR-containing genes that significantly differed between both pairs of a given comparison. Differentially methylated regions were then intersected with genic elements from the We compared the number of DMRs or DMCs obtained by methylsig to the number of produced when shuffling replicates between comparisons . In order to quantify large-scale patterns of gene gain and loss we also utilized orthodbZ. nevadensis copy number to average cross-insect copy number  for each gene family with representation in Z. nevadensis. This allowed for the estimation of large-scale evolutionary patterns for each ortholog group, in lieu of alternative evolutionary metrics that are complicated by the absence of a closely related species with genome data.We utilized Orthodb ortholog relationships for all ortholog groups with 1-to-1 representation in We developed DMR test sets by extracting 150 bp of genomic sequence surrounding confidently differentially methylated cytosines . Our control sets were constructed in the same manner, but were constructed around non-significantly differentially methylated cytosines (methylation level >30%) falling within 1.5 kb up- or down-stream of (but not overlapping) tested DMCs in order to account for variation in CpG content among the focal regions. This produced approximately 2-4x the number of control sequences for each test set.We performed transcription factor binding site (TFBS) motif enrichment tests with the program CloverD. melanogaster miRNA motifs over-represented in differentially methylated regions of the Z. nevadensis genome .Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


Asthma is a chronic airway inflammation with a potential systemic impact. Atherosclerosis is a chronic inflammatory artery disease. The aim of our study was to prove if there is a correlation between the occurrence of asthma and increased atherosclerotic vessel disorders. Vessel status was compared between mild-to-moderate, severe allergic asthma and matched controls. Measurements of artery stiffness were calculated by central pulse wave velocity, ultrasonographic strain imaging and ankle-brachial index. Atherosclerotic plaque burden was assessed by colour-coded duplex sonography. Additionally, analysis of cardiovascular and asthma blood markers was conducted. Arterial stiffness expressed as an increased central pulse wave velocity and decreased circumferential and radial strains as well as the prevalence of media sclerosis were significantly higher among asthma patients compared to controls. Atherosclerotic plaque burden was relevantly increased in asthma groups vs. controls . Except for the elevated IgE and fibrinogen concentrations as well as leukocyte number there were no relevant differences in the blood parameters between the groups. Allergic asthma is associated with distinct atherosclerotic artery changes compared to the respectively matched control collective. The severity of asthma correlates with more pronounced pathological vessel alternations. Asthma is a chronic inflammatory disease of the airways. A growing body of evidence shows that asthma may affect also extrapulmonal tissue. Patients with asthma demonstrate an increased risk for cardiovascular complications . MoreoveThe research was conducted according to the principles of the Declaration of Helsinki and was approved by the local ethics committee at the Medical Faculty of the University of Bonn. Written informed consent has been obtained from all subjects. The study has been conducted between October 2015 and July 2016. A total of 82 consecutive outpatients with allergic asthma have been enrolled in the study during control visits in our pneumological department. Allergic asthma was defined as an increase in forced expiratory volume in 1 second (FEV1) of more than 12% following administration of short acting ß2-agonist or a decrease in FEV1 of more than 200ml in bronchial challenge test with methacholine and detection of at least three allegro-allergens in specific IgE blood measurements or in skin prick test. All patients presented stable disease without clinical exacerbation at the time point of the study performance. Patients with immunodeficiency or a history of autoimmune or other than asthma chronic inflammatory diseases were excluded. Asthma patients have been divided into two groups: mild-to-moderate (n = 24) and severe asthma (n = 58). Mild-to-moderate asthma patients were under inhaled therapy with no present or past treatment with omalizumab or oral cortisone. In contrast, severe asthma was characterized by a maximal inhaled therapy combined with a present or past administration of omalizumab and/or cortisone oral. 21 non-asthmatic patients served as controls. There were no significant differences in sex, age, body mass index and cardiovascular risk factors such as arterial hypertension, hypercholesterolemia, diabetes mellitus, nicotine abuse or history of artery disease between these three groups. Lung function showed significantly decreased absolute and predicted FEV1 values as well as significantly increased absolute and predicted total airway resistance values in asthma patients compared to controls. The exact baseline characteristic of the patients and controls is presented in the Classical parameters of vessel stiffness have been calculated by means of central pulse wave velocity (cPWV) and ankle-brachial index (ABI). Assessment of central pulse wave velocity (cPWV) was performed using AngE Pro8® . Ankle-brachial index (ABI), defined as the ratio of the systolic blood pressure (SBP) measured at the ankle to that measured at the brachial artery , was calUltrasonographic strain imaging included global average circumferential strains (% of vessel circumference changes), global average radial strains , radial displacement , global average circumferential strain rates (vessel circumference changes per second) and global average radial strain rates . Common carotid artery was shown in the two-dimensional short axis (Philips iE33). Four electrocardiography (ECG)-triggered cardiac cycles were stored for the offline analysis. Following creation of a region of interest, the frame-to-frame displacements of the speckles representing tissue movements were analyzed by using of a computer workstation equipped with a speckle-tracking software package . This method has been previously described , 13.The following cardiovascular risk and asthma markers in blood have been examined using standard laboratory tests: total cholesterol, HDL- and LDL-cholesterol, lipoprotein(a), C-reactive protein (CRP), interleukin-6, soluble interleukin-2 receptor (sIL-2R), immunoglobulin E (IgE), fibrinogen, d-dimer, leukocytes, lymphocytes, eosinophils, hemoglobin, thrombocytes.Mean values of the determined parameters ± standard error of the mean (SEM) were compared by means of ANOVA for three groups and by means of the Student's t-test for two selected groups. Potential differences between categorical variables were calculated by using of the chi-square test. P<0.05 was considered as statistically significant.As a key finding, the angiological results have shown an increased arterial stiffness and more manifest atherosclerosis in patients with asthma, especially in those with its severe form, compared to the baseline matched controls. In detail, central pulse wave velocity differed significantly across the groups studied  with the highest values in severe asthma collective  in comparison to control  showed significant elevated levels of fibrinogen  and leukocytes  with a reduced lymphocyte fraction  and a slightly increased number of eosinophils  compared to control  and thrombocytes in asthma patients  compared to controls  vs. patients on omalizumab without oral cortisone therapy  has been conducted. Baseline characteristics were similar in both groups. The only difference was that patients from group 1a had significantly lower FEV1 values in comparison with the group 1b : 67.0±5.2% vs. 85.8±3.4%, resp., p = 0.003). Angiological examinations have shown worse vessel status with a significantly higher percentage of atherosclerotic plaques in the group 1a vs. group 1b . Blood analysis demonstrated except for increased IgE levels and most likely cortisone-associated changes in the white blood cell count in group 1a  no relevant differences between both groups.Our data show pathological artery changes in asthma patients compared to the control collective. Specifically, asthma was not only associated with preatherosclerotic vessel alternations, such as higher arterial stiffness, but much more with increased prevalence of manifest atherosclerosis compared to non-asthma individuals. Indeed, asthma has already been described to be a risk factor for stroke and heart diseases ,15,1. GeChlamydia pneumoniae [As traditional cardiovascular risk factors, such as: arterial hypertension, diabetes mellitus, hypercholesterolemia, obesity and nicotine abuse were similar between asthma and control individuals, we aimed to determine other potential risk markers in the blood. As a result, only IgE and fibrinogen levels as well as leukocyte number and subtypes differed significantly between the groups. Fibrinogen is independently linked to enhanced rates of cardiovascular complications . It has eumoniae  are commIn our study, all vessel changes were more pronounced in severe asthma compared to mild-to-moderate asthma. Interestingly, patients on the add-on combined therapy of oral corticosteroid and omalizumab (group 1a) showed even greater atherosclerotic artery alternations than the patients with the same therapy except for oral corticosteroids (group 1b). Since the lung function was more impaired in group 1a vs. group 1b, it is difficult to judge if the severity of asthma, differences in the treatment or both influenced this finding. In this context, the role of antiasthmatic medication in the process of vasculopathy is not clear. Some works reported on the beneficial effects of inhaled glucocorticosteroids on the vessel changes . One couIn our present study, we did not observe any relevant cortisone-related side effects, such as: increase in blood pressure, induction of hypercholesterolemia or diabetes mellitus. One may assume that asthma-specific activity and adverse effects of a long-term high dose cortisone therapy may have led to the worst vessel status in patients with severe asthma under oral cortisone therapy.In conclusions, our data implicate an association between asthma, in particular severe asthma, and pathological artery changes, including a significantly increased atherosclerotic plaque load.


Supplemental Digital Content is available in the text Intracranial hypertension (IH) is a neurological disorder characterized by increased intracranial pressure. It is a poorly understood syndrome that most commonly manifests nonspecific symptoms such as stroke-like headache, vision changes, nausea, vomiting, and papilledema. IH has been reported in young cancer patients but never in association with gastric signet-ring cell carcinoma.Here, we discuss the case of an 18-year-old girl with gastric signet-ring cell carcinoma in which IH was the primary symptom accompanied by the even rarer symptom of cutaneous metastases. We also present a review of the relevant literature. The patient experienced frequent headaches, vomiting, and blurred vision but showed no abnormal findings on cranial imaging studies. Further examination showed multiple skin nodules on the abdomen. Then pathological and immunohistochemical examination of gastroscopic specimens and the biopsied subcutaneous nodules were done.Pathological and immunohistochemical examination of gastroscopic specimens and the biopsied subcutaneous nodules confirmed gastric signet-ring cell carcinoma with skin metastases.To our knowledge, this is the first reported case of gastric signet-ring cell carcinoma primarily presenting IH and accompanied by subcutaneous metastases. This case emphasizes the importance of excluding malignancy from the differential diagnosis of IH. IH has been reported in cancer patients,5 but never in association with gastric signet-ring cell carcinoma. Further, gastrointestinal tumors presenting neurological problems are especially rare, and diagnosis in such cases is often delayed because of the nonspecific symptoms.Intracranial hypertension (IH) is a neurological disorder characterized by increased intracranial pressure. It is a poorly understood syndrome that most commonly presents as stroke-like headache, vision changes, nausea, vomiting, and papilledema.Here, we report the case of an 18-year-old girl whose main symptoms were IH and subcutaneous nodules in the abdomen. Pathological and immunohistochemical reports of gastroscopic specimens and biopsy examination of the subcutaneous nodules confirmed gastric signet-ring cell carcinoma with skin metastases. To our knowledge, this is the first case of gastric signet-ring cell carcinoma primarily presenting IH and accompanied by subcutaneous metastases.2An 18-year-old girl was admitted to the emergency room with frequent headaches, vomiting, and blurred vision that had persisted 2 months. She had also had low-grade fever (37.0–38.0 °C) for the previous 15 days. On neurological examination, she was fully alert and suspected neck rigidity, and the remainder of neurological examination was normal.2O). Cranial magnetic resonance imaging (MRI) conducted at our hospital showed no obvious abnormalities either, although a suspicious enhanced signal was noted at the frontal and parieto-occipital regions of the scalp on both sides  scans as well as gastroscopic examination, none of which showed any obvious abnormalities. Cerebrospinal fluid (CSF) was first examined at the emergency department, and the results were normal except that the pressure was high . The results of serum tumor marker examination showed that both carcinoembryonic antigen (12.2 ng/mL) and cancer antigen 125 (43.7 U/mL) levels were mildly to moderately elevated. Other routine laboratory tests were normal (Supplementary Table 1). Abdominal CT showed multiple lymph nodes behind the peritoneum. A biopsy specimen was obtained from a subcutaneous nodule in the abdomen.2O); further, the glucose content of the CSF was mildly elevated (4.50 mmol/L). After the second lumbar puncture, we increased the dosage of dehydrating agents, from 250 mL of glycerol fructose sodium chloride ivgtt Q12H and 125 mL mannitol ivgtt Q8H to 250 mL of glycerol fructose sodium chloride ivgtt Q12H and 125 mL mannitol ivgtt Q6H. Other treatments included agents for protecting the gastric mucosa and nutrition supplements. However the pathological results of the biopsy specimen showed invasive growth of malignant signet-ring cells  is a member of the EGFR family, breast cancer patients who test positive for this receptor are generally expected to have a poor prognosis.,18 Overexpression of HER2 in patients with gastric cancer ranges from 6% to 23%,,20 and its prognostic significance in gastric cancer is controversial. The immunohistochemical results obtained in the present study confirmed that the nature of gastric cancer in this case was signet-ring cell carcinoma.Signet-ring cells are characterized by a slightly curved nucleus found near the cell membrane and a mucin-filled vacuole that occupies most of the cell. The mucin appears clear in stains, but it is diastase resistant, periodic acid-Schiff and mucicarmine positive.,23 but primary symptoms involving the central nervous system are rare. Erdoğan et al reported a case of IH due to duodenal signet-ring cell tumor, but it was not accompanied by cutaneous metastases. Lee et al reported a patient with gastric signet-ring cell tumor showing leptomeningeal involvement. Table Gastric signet-ring cell carcinoma has been known to present many different clinical symptoms, including cutaneous metastases,,40 and hypersecretion is known to occur in cases of choroid plexus papilloma, a rare pediatric tumor. Further, the arachnoid granulations are believed to be responsible for CSF clearance, and subarachnoid hemorrhage or meningitis can result in communicating hydrocephalus, which may cause IH, since CSF absorption depends on the pressure gradient between the venous sinus and the subarachnoid space, and an increase in venous pressure warrants a concomitant increase in CSF pressure to maintain absorption rates.,43,44 Third, increased cerebral arterial pressure and cerebral venous pressure lead to IH. Normally, intracranial pressure is maintained by cerebral arterial pressure, which itself is subject to cerebral autoregulation, because of which intracranial pressure remains constant over a wide range of systemic arterial blood pressures. If a tumor invades the arteries or capillaries, it may affect cerebral autoregulation and consequently lead to IH. Additionally, the increase in venous sinus pressure resulting from venous sinus stenosis is an obvious mechanism underlying IH.,44 Lastly, cerebral edema, obesity, and pseudotumor cerebri could also underlie IH. Pseudotumor cerebri is defined as idiopathic or secondary IH with iatrogenic causes  or caused by associated disorders .The pathophysiology of IH remains poorly explained, although various mechanisms have been proposed. The first mechanism is related to the volume compensation that occurs in the cranium when an intracranial mass occurs. The cranium is a rigid structure and mainly contains the brain (80%), blood (10%), and CSF (10%). When an intracranial mass appears, a compensatory change in volume must occur through a reciprocal decrease in venous blood or CSF to keep the total intracranial volume constant. However when the mass is very large and cannot be compensated for in this manner, IH occurs. This may be the most common cause of IH. Second, hypersecretion and improper drainage of CSF may cause IH. The choroid plexus is responsible for CSF secretion,Clinically, CT and MRI are useful methods for assessing the intracranial masses, hydrocephalus, and ventricular enlargement causes of IH. However, as mentioned above, cranial MRI showed no obvious abnormalities in the present case, and laboratory examinations also did not yield any clues regarding other possible causes of IH. they tend to diffusely infiltrate the stroma and have a propensity toward vascular invasion, lymph node involvement, and distant widespread metastasis. However, the cranial images of our patient showed no such indications. Other causes of IH are meningeal carcinomatosis and paraneoplastic syndrome,,46 but the latter was not identified on CSF testing in the present case. Additionally, cranial MRI showed a suspicious enhanced signal in the frontal and parieto-occipital scalp regions on both sides, but we could not confirm whether it was related to meningeal carcinomatosis.On the monism of medicine, we consider that the IH is related to the gastric signet-ring cell carcinoma preferentially. As signet-ring cells are a group of malignant cells that have naturally lost cell–cell adhesion properties and consequently present as individual cells or loose clusters,,48 and this may have been the cause of IH in the present case. Magnetic resonance venography or cerebral digital subtraction angiography could have confirmed this possibility, but we did not perform these tests.In many situations, meningeal carcinomatosis does not appear in imaging studies, and it may have been the cause of IH in the present case. In addition, malignant carcinoma tends to induce the hypercoagulable state, leading to cerebral venous sinus stenosis,In summary, we presented the case of gastric signet-ring cell tumor presenting IH and cutaneous metastases in an 18-year-old girl. This case emphasizes the importance of excluding malignancy from the differential diagnosis of IH.


Emerging evidence indicates that Circular RNAs (circRNAs) exert post-transcriptional regulation of gene expression. A subclass of circRNA was found enriched with miRNA target sites. This evidence suggests that this kind of circRNA functions as natural miRNA sponge. Noticing the potential impacts of circular RNA research, we were motivated to identify novel circRNAs as well as putative circRNA-miRNA interactions through retroactive sourced transcriptome sequencing samples.Through the analysis in 465 RNA-seq runs and 22 reports published in recent years, putatively circRNA sponged miRNA that had been experimentally verified targeting circRNA host gene were found. From this observation, supporting evidence of the competitive endogenous relationship of circRNAs and miRNAs targeting circRNA host genes can be observed. Given the self-regulation and self-induction nature of these circRNAs, this kind of hypothetical phenomenon was hereby called Ouroboros Resembling Competitive Endogenous Loop (ORCEL) in circular RNAs.The fact that miRNA sponge circRNA originated from region miRNA target sites enriched regions, while genes encoded from these regions are conserved to be miRNA targets rationalize the existence of ORCEL.The online version of this article (10.1186/s12864-018-4456-9) contains supplementary material, which is available to authorized users. More than 50 years have passed since H. Harris deduced that most nuclear RNAs are likely to be non-protein-coding in 1959 , it is pCircular RNAs (circRNAs) represent a type of regulatory noncoding RNA whose head 3′ and tail 5′ ends covalently bond together to result in a circular form. The circular form was verified with electron microscope in 1979 . In 2012In the following years, extended identification of circRNAs in mouse , fly 1515 and otAware of the significance of circRNAs research, in 2015, we constructed a database called CircNet  to not oTo acquire an enriched collection of human circular RNAs, previously reported and newly identified human circRNAs were collected in this study. Reported human back-spliced junction sites were also collected from 22 recent studies –39. In aCircular RNA back-spliced junction sites were identified within these samples. To acquire the expression patterns of circRNAs within these samples, a pipeline was developed to annotate the sequence of the circRNAs. With the putative sequences of circRNAs, miRNA target prediction was conducted on these sequences. Transcripts abundance within the samples was conducted through the transcript deconvolution algorithm .P-value of the circRNA and miRNA pairs are lower than 0.005. From this observation, existence of competitive endogenous loop of circRNAs and their host gene can be observed. Given the self-regulation and self-induction nature of these circRNAs, it was hereby named Ouroboros Resembling Competitive Endogenous Loop (ORCEL) in circular RNAs.Through analyzing the collect circRNA, combined with the annotation and miRNA sponge prediction, we found that in 728 human circRNA host genes, the generated circRNAs were found to have high affinity to sponge miRNAs which were reported to target these genes. The calculated Through the expression profiling of circRNA, combined with the previously reported back spliced junction sites, we found 2747 circRNAs originated from 2693 back spliced junction sites that meet the defined threshold. From the analysis, we found that in 728 human circRNA host genes, including 909 circRNA transcripts, the generated circRNAs were found to have high affinity to sponge miRNAs. Meanwhile these miRNAs were reported to target the circRNA originated genes.From our analysis, we found 2693 back splice junction sites that meet the defined threshold on human genome. The combined amount of peer review reports and RNA-Seq samples in which these junction sites were found or reported ranges from 10 to 126. The miRNA sponge CDR1as  in late With the observation of most circRNAs originated from circularization of coding gene exons, it was deducted that circRNA biogenesis competes with pre-mRNA splicing . As an iAn enrichment analysis of the 728 ORCEL genes was conducted through DAVID . In the From the network illustrated in Fig. In this study, putative roles of circRNAs serving as endogenous miRNA sponges were investigated through analysis of transcriptome sequencing data sets and published results. With the head-tail junction structure, circular RNAs are more stable than other kind of long noncoding RNAs. Hence circRNAs are hypothetically easier to acuminate in cells. The longer half-lives of circRNAs allow the presence of miRNA target sites increase within the cells. Through statistical analysis of the abundance of the miRNA target seeds on circRNA sequences, putative circRNA serving as miRNA sponges were identified. Recent years experiment results suggest certain threshold of miRNA target sites needs to be reached for the ceRNAs to have physiological effects , 47, henThrough the bioinformatics analysis it was found that for certain subset of circRNAs, putatively sponged miRNA had been experimentally verified targeting circRNA host gene. From this observation, the existence of competitive endogenous loop of circRNAs and their host gene can be observed. Given the self-regulation and self-induction nature of these circRNAs, this kind of phenomenon was hereby called Ouroboros Resembling Competitive Endogenous Loop (ORCEL) in circular RNAs.P-values < 0.005 were collected. Expression profiling of the circRNAs within each of the samples collected from SRA was conducted in two different approaches: normalized counts of reads spanning the back spliced junction sites SRPBM [The data analysis process of this research is summarized in Fig. es SRPBM  and normReported human back-spliced junction sites were collected from 22 recent studies –39. In a For the back spliced junction sites locate on exact “head” and “tail” locus of exons from same transcript from RefSeq , 52, allThe method was described in our previous study . The met(2) For those back spliced junction sites flanked multiple isoforms from RefSeq , 52, exi(3) For those circRNAs associated with these back-spliced junction sites having small misalignments to exon locations, flanked exons and a small portion of intron sequence locating in the head or tail locations were considered as parts of the isoforms.(4) For the junction sites that were found to be located in intergenic positions while others, despite overlapping with certain genes, localized to their antisense strands, the entire flanked sequence was considered as the sequence of the circRNA.The resulted sequence annotation was took for expression analysis and miRNA target search. The annotation along with the gene transcripts was recorded in gtf format for the expression profiling.Developed from results of our previous study , 54, to The miRNA target sequences deemed typical: 6mer, 7mer-A1, 7mer-m8 and 8mer sequences  were extN = 6 for 6mer, N = 7 for 7mers and 8 for 8mer. With this formula, four frequency numbers can be acquired from each pair of circRNA and miRNA. To distinguish circRNA from linear isoforms, frequency values were also calculated for gene transcripts mRNA. We calculated all the frequency value of the circRNAs as well as the linear isoforms pairing to miRNAs, and then converted the Z-score of the normal distribution into one tail P-value through survival function.Where the ‘N’ is the length of the seed. P-value < 0.005 was considered high regulatory potential between the circRNA and miRNA. The miRNAs and experimentally verified gene targets were collected from miRTarBase [The circRNA-miRNA pair with RTarBase .As previously described, the abundance of the circRNA within the collected samples were estimated through the transcript deconvolution algorithm of the Cufflinks pipeline . To furtTo normalize the amount of the normalized sequence reads spanning the junction sites, a concept of spliced reads per billion mapping (SRPBM) was applied . Amount RNA-seq aligner STAR  was applFrom the result of recent year comparison study , we deduThe amount of previous peer review reports in which the back spliced junction sites were reported.The amount of samples among the 465 collected samples in which the back spliced junction sites were found meeting the criteria defined in find_circ , 49, 50.Only the circRNAs with the combined amount of these two values over 10 were considered in the analysis of this report.Additional file 1:A detail list of these circRNAs, as well as their corresponding SRPBM and FPKM values within the samples. (XLSX 528 kb)Additional file 2:A complete list of ORCEL (Ouroboros Resembling Competitive Endogenous Loop) reported in this article. (XLSX 59 kb)Additional file 3:A complete table of the enrichment of ORCEL genes. (XLSX 90 kb)