Many rodent studies investigated ELS interactions in SERT knockout rodents combined with ELS. However, underlying neuromolecular mechanisms of the adaptive responses to adversity displayed by SERT rodents remain to be elucidated. Here, we provide a comprehensive review including studies describing mechanisms underlying SERT variation × ELS interactions in rodents. Alterations at the level of translation and transcription but also epigenetic alterations considerably contribute to underlying mechanisms of SERT variation × ELS interactions. In particular, SERT+/− rodents exposed to adverse early rearing environment may be of high translational and predictive value to the more stress sensitive human short-allele carrier, considering the similarity in neurochemical alterations. Therefore, SERT+/− rodents are highly relevant in research that aims to unravel the complex psychopathology of mental disorders. So far, most studies fail to show solid evidence for increased vulnerability to develop affective-like behavior after ELS in SERT+/− rodents. Several reasons may underlie these failures, e.g., (1) stressors used might not be optimal or severe enough to induce maladaptations, (2) effects in females are not sufficiently studied, and (3) few studies include both behavioral manifestations and molecular correlates of ELS-induced effects in SERT+/− rodents. Of course, one should not exclude the  possibility of SERT+/− rodents not being sensitive to ELS. In conclusion, future studies addressing ELS-induced effects in the SERT+/− rodents should extensively study both long-term behavioral and (epi)genetic aspects in both sexes. Finally, further research is warranted using more severe stressors in animal models. From there on, we should be able to draw solid conclusions whether the SERT+/− exposed to ELS is a suitable translational animal model for studying 5-HTTLPR polymorphism and stress interactions.The interaction between the serotonin transporter (SERT) linked polymorphic region (5-HTTLPR) and adverse early life stressing (ELS) events is associated with enhanced stress susceptibility and risk to develop mental disorders like major depression, anxiety, and aggressiveness. In particular, human short allele carriers are at increased risk. This 5-HTTLPR polymorphism is absent in the rodent SERT gene, but heterozygous SERT knockout rodents (SERT Major depressive disorder, also known as major depression, carries the heaviest burden amongst all mental and behavioral disorders and is globally the largest contributor to years lived with disability  variation on individual stress susceptibility  rodents. For instance, using ENU-target driven mutagenesis, a premature stop codon has been induced in the SERT gene of rats resulting in a non-functional protein product  show reduced SERT expression and function, as seen by 40–50% less SERT protein levels . In addition, intracellular 5-HT levels in SERT+/− mice and rats appear unchanged compared to SERT+/+ rodents  axis. Across species, the HPA axis provides an appropriate response system to salient environmental challenges and opportunities . Stressful stimuli activate hypophysiotrophic neurons in the paraventricular nucleus (PVN) of the hypothalamus that secrete releasing hormones, such as corticotropin-releasing factor (CRF) and arginine vasopressin, into the portal circulation of the median eminence. The releasing hormones act on the anterior pituitary to promote the secretion of adrenocorticotropic hormone (ACTH) into the systemic blood circulation  levels did not differ  mRNA levels were reduced in SERT+/− rodents are limited. Gene expression levels of all the assessed key HPA-axis players were not affected in SERT+/− rodents after ELS. Only minor adaptations in plasma ACTH levels , together with some alterations in basal gene expression levels of CRF and GR in the PVN were found when comparing SERT+/+ with SERT+/− animals , ELS had a main effect on GR expression leading to an overall decrease in mRNA levels. Again, the authors took the SERT−/− rats along in their analysis, making it difficult to separately interpret the data. However, no significant differences were found after post hoc tests between genotypes, therefore SERT+/+ and SERT+/− rats probably do not have different GR expression in the ventral hippocampus after maternal separation. Furthermore, no effects of ELS, SERT genotype or their interaction were found for GR mRNA levels in the Edinger-Westphal nucleus in the midbrain , which is important for normal development, survival, and plasticity of central nerve system neurons, is known to be altered in response to stress and is involved in the pathophysiology of psychiatric disorders , the number of stressors per day  as well as how many days the stressor is given differ among studies. Furthermore, different species (mouse or rat) or even different strains might result in discrepancies between studies. Another important notion is the fact that nearly all studies use only male rodents to minimize variation due to hormonal fluctuation. Human females are 1.7 times more likely to develop depression  stressors used might not optimal to induce maladaptations ; (2) effects in females are largely absent due to a lack of studies, (3) few studies include both behavioral outcomes and molecular correlates of ELS-induced effects in SERT knockout rodents. The question arises whether the heterozygous SERT+/− rodent creates a serotonergic subfunctional model. Although it is generally assumed that the approximately 50% remaining SERT molecules lead to differences in various processes in an adult animal, the supporting data is suggesting the opposite. Serotonergic functioning  genetic aspects in both sexes, underlying mechanisms can be directly correlated. Moreover, more severe and realistic stressors should be applied to increase the chance that a not-optimally functioning serotonergic system (the SERT+/−) might be influenced leading to lasting changes in stress sensitivity. The rodent SERT+/− × ELS model has potentially high potential, but future research is indispensable before a solid conclusion can be drawn whether this is a suitable translational animal model for ELS-induced psychopathology in psychiatric disorders.In conclusion, the exact molecular mechanisms underlying  may be:  stressorDrafting and/or revising the paper, including final approval to publish: DH, BB, EvdZ, SdB, and JO.This work was financially supported by the Marie Sklodowska-Curie IF (grant SEP-210188707) and the Swedish Society of Medicine (grant SLS-411161).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The reviewer CP and handling Editor declared their shared affiliation, and the handling Editor states that the process nevertheless met the standards of a fair and objective review.


Here, in a longitudinal study of healthy women who gave birth to healthy infants following uncomplicated pregnancies within the Canadian Healthy Infant Longitudinal Development (CHILD) cohort, we test the hypothesis that a progressively altered bias in resting innate immune status develops. Women were examined during pregnancy and again, one and/or three years postpartum. Most pro-inflammatory cytokine expression, including CCL2, CXCL10, IL-18 and TNFα, was reduced in vivo during pregnancy . Anti-inflammatory biomarkers  were elevated by ~50–100% (p<0.0001). Systemic IL-10 levels were unaltered during vs. post-pregnancy. Kinetic studies demonstrate that while decreased pro-inflammatory biomarker expression  was constant, anti-inflammatory expression increased progressively with increasing gestational age (p<0.0001). We conclude that healthy resting maternal immune status is characterized by an increasingly pronounced bias towards a systemic anti-inflammatory innate phenotype during the last two trimesters of pregnancy. This is resolved by one year postpartum in the absence of repeat pregnancy. The findings provide enhanced understanding of immunological changes that occur in vivo during healthy human pregnancy.Changes in maternal innate immunity during healthy human pregnancy are not well understood. Whether basal immune status  For healthy pregnancy to proceed to term, changes need to occur to prevent immune mediated rejection of the semi-allogenic fetus. At the same time, the immune system must maintain, or enhance, protection of mother and fetus from external pathogens. There is extensive literature concerning immunity at the maternal-fetal interface and its role in progression of fetal development –5. Similin vivo innate immune status during normal pregnancy–the healthy phenotype–will strengthen efforts to understand linkages between in vivo maternal status and the subsequent development of healthy vs. chronic inflammatory phenotypes such as asthma or autoimmunity in children, or their mothers, later in life [Surprisingly, healthy pregnancy that leads to healthy infants has not been a major research focus and is not well understood. Publications  and rece in life –13.The changes, if any, that occur in innate immune status during a healthy pregnancy are controversial. Existing evidence supports several mutually exclusive conclusions. Some data are consistent with the concept that basal maternal systemic immunity exhibits a mild bias towards inflammatory phenotypes . Others support the notion that immunosuppressed phenotypes  are normally dominant during pregnancy . A thirdin vivo during healthy pregnancy will provide better understanding of mechanisms that underlie difficult pregnancies [in vivo and clinical outcomes later in life for both mother and fetus [Interest in healthy pregnancy is driven by at least three other rationales. Exclusion of pregnant women from clinical research, while well intentioned, can be counterproductive . Their ignancies . Finallynd fetus , 17, we The Canadian Healthy Infant Longitudinal Development (CHILD) birth cohort was initiated to study the development of allergy and asthma, with a strong focus on clinical, immunologic and environmental assessments of infants and parents. The 3,624 participating infants and families are predominantly from urban centres; over 80% of the Canadian population is urban. The cohort is multicultural and ethnically varied. Its value is enhanced by extensive phenotyping of both children and parents, characterization of their environments and an extensive repository of biological samples .in vivo is transiently shifted during pregnancy. In this longitudinal study of 251 randomly selected healthy women who gave birth to healthy infants, pairwise comparisons were used to assess innate immune biomarker levels in vivo during the second/third trimester then again at one and three years postpartum. An extensive panel of pro-inflammatory cytokines that are constitutively present in most healthy individuals  was examined. While studies of inflammatory processes often include few or no anti-inflammatory regulators, endogenous levels of a broad panel of anti-inflammatory cytokines  were incorporated to provide a better immune signature of women’s health during successful pregnancy. The data reveal that extensive changes occur in vivo. Healthy pregnancy is linked to development of a constitutive systemic anti-inflammatory maternal bias that becomes increasingly intense with increasing gestational age, then self-resolves by one year postpartum.Here we test the hypothesis that resting systemic pro-/anti-inflammatory bias http://www.canadianchildstudy.ca/knowledge.html) [The Canadian Healthy Infant Longitudinal Development (CHILD) Study is a prospective national population-based longitudinal birth cohort of 3,624 neonates (ge.html) . FollowiTo increase power, rather than incorporating unrelated non-pregnant female controls, a longitudinal study design was used where a woman’s status during pregnancy was compared with her own at subsequent times. Samples were obtained once at initial recruitment  and again one year postpartum. Major clinical characteristics of the women studied are provided in in vivo pro- and anti-inflammatory cytokine expression (CCL2 and sTNF-RI). These biomarkers were chosen because published, and our own preliminary data, demonstrated that readily quantified levels are evident in >95% of healthy individuals . For more extensive analyses, 8 additional in vivo biomarkers were determined using approximately every second individual (n = 120) from the original study group. Investigators were blind to any immunological data at the time of randomization and sample selection.251 of the mothers for whom paired plasma samples were available from prenatal (PN) and one year post-partum (1YPP) time points (n = 331) were randomly selected for analysis of Among women who provided plasma samples at both one and three years post-partum, (and who did not experience pregnancy in the interim) 32 were randomly selected to further compare potential changes in selected plasma biomarker levels post-pregnancy see .Samples were processed as described . BrieflyMeso Scale Discovery  singleplex assays were used to quantify analyte concentrations following the manufacturer`s instructions. Catalog numbers for the reagent kits used are provided at For all assays, concentrations were determined based on standard curves created using serial dilutions of fresh aliquots of constant recombinant lab standards prepared in culture medium and stored at -80°C in individual 400 uL aliquots . Longitudinal sample sets from each individual were always paired on the same assay plates. Median intra-assay variation was typically below 5%.Group results are presented as medians, with each point representing the average (mean) value obtained from duplicate or triplicate analyses of an individual woman’s plasma at that timepoint. Supporting information This longitudinal study aimed to determine whether the constitutive immune status of healthy pregnant women exhibits a pro- or anti-inflammatory bias and whether it changes during pregnancy. As described in detail at Material and Methods, plasma samples were obtained at a single time point during pregnancy, again one year postpartum, and for a randomly obtained subset of individuals who did not experience further pregnancies in the interim, at three years postpartum. Two representative biomarkers of pro- and anti-inflammatory responses were assessed initially . Pro-infin vivo during pregnancy. A brief overview of the major cellular sources and biological functions of the cytokines, chemokines and receptors studied is provided in Given the large number of pro- and anti-inflammatory cytokines and inhibitors involved in human immune regulation, this analysis was extended in approximately every second woman (n = 120) to a panel of five additional pro-inflammatory biomarkers  as well as three more anti-inflammatory biomarkers . The cytokines selected were chosen on the basis of being (i) routinely present in the plasma of healthy humans and (ii) having been extensively implicated in immune regulation of inflammation in murine and human studies. These large panels of biomarkers were used in preference to relying upon a single biomarker  in order to strengthen the capacity to draw conclusions about pro- vs. anti- inflammatory bias Anti-inflammatory biomarkers  demonstrated that unlike the reductions seen in constitutive pro-inflammatory cytokine expression, anti-inflammatory biomarker levels were elevated during pregnancy  with plasma obtained from the same women at one year postpartum. One pro- and one anti-inflammatory biomarker that had exhibited differential expression during pregnancy (CCL2 and IL-1Ra) and one pro- and one anti-inflammatory biomarker that had been unchanged during pregnancy (CXCL8 and IL-10) were examined. The comparisons above test the hypothesis that resting constitutive innate immune status is distinct during healthy pregnancy. To better understand the kinetics of this shift in resting immune status, the population was next stratified by trimester of recruitment. With only a small number of women recruited during the first trimester, analysis centred on the second and third trimesters. intensity of anti-inflammatory phenotypic changes are altered with the progression of pregnancy, we assessed relationships between plasma biomarker concentrations and gestational age. sTNF-RI, IL-1Ra  are excluded from this study. Among the women studied, clinical assessments at each blood draw excluded women with active transient URT or GI infections. The data reported above, characterizing the constitutive A potential confounder to interpretation of the data obtained was day-to-day stability of biomarker expression within a given individual. We found this to have minimal impact because, as shown in in vitro and induce apoptosis of human villous trophoblast cells. Murine studies demonstrate that administration of pro-inflammatory cytokines  into pregnant mice causes increased abortion. Conversely, administration of IL-10 prevents fetal wastage in abortion-prone mice [The immunological and clinical implications that result from the increasingly pronounced anti-inflammatory phenotype that developed over the course of healthy pregnancy remains speculative. Extensive literature demonstrates associations between pro-inflammatory or type 1 cytokines and recurrent spontaneous miscarriage or preterm delivery. Exogenous TNFα and IFN inhibit the outgrowth of human trophoblasts one mice , 82. Thuone mice .in vivo expression of acute phase proteins during healthy pregnancy. Multiple studies have found increased plasma/serum CRP levels vs. non-pregnant controls [These data may stand in contrast to controls –86. The controls , 87, 88.controls , 89–92. in vivo or acute in vitro activation. Studies examining in vitro responses to various pattern recognition receptor (PRR) ligands are currently underway as a complementary approach to understanding changes in maternal health that can occur during pregnancy.In this study, we do not address putative differences in maternal immune responses upon infection in vivo during pregnancy. It underlines the need for further characterization of what constitutes a successful environment for healthy human pregnancy.With increasing attention given to defining what constitutes a healthy pregnancy , this stS1 File(XLSX)Click here for additional data file.


Central nervous system (CNS) involvement is rare but it is an increasingly recognized complication of the multiple myeloma. The craniospinal radiotherapy is a standard treatment option, however, it may be challenging to deliver due to hematologic toxicity in the patients with multiple prior systemic therapies. We report a case of CNS myelomatosis in a patient with prior stem cell transplant multiple systemic therapies treated with bone marrow-sparing proton therapy craniospinal irradiation, with the dramatic clinical response and minimal hematologic toxicity. Central nervous system (CNS) involvement in the multiple myeloma (MM) is rare and typically present in the setting of advanced disease and multiple prior therapies. The incidence may be increasing, possibly related to better detection and improved survival of myeloma patients in the era of novel systemic agents. The treatment options include intrathecal therapy, systemic therapy, and radiation therapy. Unfortunately, the prognosis remains extremely poor despite improvements in the systemic therapies. Herein, we report a case of CNS myelomatosis (CNS MM) with dramatic clinical and radiographic response to bone marrow-sparing proton therapy craniospinal irradiation (CSI).A 44-year-old male was diagnosed with International Staging System stage III, lambda light chain multiple myeloma, after presenting with progressive fatigue, hypercalcemia, and renal dysfunction. He received two months of Lenalidomide and Dexamethasone but was temporarily lost to follow-up. Approximately one year after diagnosis, he resumed therapy and completed four cycles of bortezomib, Lenalidomide, and Dexamethasone with the complete response based on normalization of his kappa/lambda ratio and negative bone marrow biopsy. He then underwent an uncomplicated autologous stem cell transplant with melphalan conditioning followed by maintenance Lenalidomide.Approximately three years and three months after initial diagnosis, he presented with left eye visual changes. The magnetic resonance imaging (MRI) of the orbits revealed contrast-enhancement and widening of the left orbital nerve concerning for malignant infiltration. In addition, two subcentimeter, enhancing parenchymal lesions were noted in the cerebellum. A lumbar puncture confirmed monoclonal plasma cells in the cerebrospinal fluid (CSF). The repeat bone marrow biopsy was negative for myeloma involvement and restaging computerized tomography (CT) scan was unremarkable.He received three cycles of high dose methotrexate (8 g/m2) followed by leucovorin rescue with persistent plasma cells in the CSF. He subsequently developed a rapid decline in neurologic status, including right-sided hemiparesis, left lower extremity radicular pain, and confusion. The repeat neuro-imaging demonstrated the progression of multiple intracranial lesions and increased leptomeningeal enhancement. Of note, there was a large mass in the left frontal region and another in the pineal region with mass effect on the cerebral aqueduct causing hydrocephalus. Imaging of the complete spine revealed diffuse leptomeningeal disease along the nerve roots of the cauda equina with multiple enhancing nodules Figure . There wGiven his history of stem cell transplant, the proton beam therapy with bone marrow-sparing was recommended to minimize the risk of hematologic toxicity Figure . To achiThe radiation therapy was fairly well tolerated with complete resolution of the neurologic deficits. His peripheral blood counts were monitored bi-weekly during the treatment and for several weeks after the treatment Figure . He requAfter completion of craniospinal irradiation, he resumed systemic therapy with pomalidomide and dexamethasone with close neuro-oncologic surveillance. At his last follow-up visit, approximately nine months after completion of proton therapy, follow-up brain MRI with contrast showed durable near complete radiographic response Figure , and he The CNS involvement is a rare but increasingly recognized complication of the multiple myeloma. Awareness of this entity is important given improved survival in the myeloma patients in recent decades, particularly in younger age groups. High-risk cytogenetics, plasmablastic morphology, and the extramedullary myeloma involvement have been associated with increased risk of CNS MM . The CNSPrognosis after the development of CNS myeloma is dismal, with survival typically in the order of one to six months -5. ThereAlthough novel systemic agents have led to improved outcomes for multiple myeloma patients, the majority of these agents has limited penetration of the blood-brain barrier and therefore, are relatively ineffective in the treatment of CNS myeloma . In a reHematologic toxicity is a potential concern when considering CSI in multiple myeloma due to prior exposure to several myelosuppressive chemotherapy agents and, in many cases, the use of autologous stem cell transplant. For example, grade three- four hematologic toxicity was seen in 33% of the patients with primary CNS tumors treated with photon CSI at the Royal Marsden Hospital . Prior rThe proton beam CSI may reduce hematologic toxicity compared to photon CSI due to superior sparing of bone marrow in the vertebral column. In a study of adults with medulloblastoma, the proton beam CSI was associated with reduced rates of grade one or higher anemia compared to photon CSI  . The relAlthough there are no standard indications for proton beam CSI, these studies suggest that this modality might be considered in special circumstances to reduce hematologic toxicity, such as in the patients with prior chemotherapy or stem cell transplant. Furthermore, the proton CSI is widely utilized in pediatric medulloblastoma both for its decreased long-term extra-CNS toxicity as well as its bone marrow sparing.In conclusion, the myeloma involvement of the CNS is rare and associated with poor prognosis. The radiotherapy in the form of CSI is an important therapeutic tool in the treatment of CNS MM and should be considered early in the disease course. Bone marrow-sparing CSI using proton therapy is a useful technique to limit hematologic toxicity, control and reverse neurologic sequelae from disease progression, and prolong survival, as illustrated in this case report.


In addition, the spatial organization of endocytic zones adjacent to active zones provides a structural basis for efficient coupling between SV exocytosis and endocytosis. Through linking different endocytosis pathways with SV fusion, these mechanisms ensure necessary plasticity and robustness of nerve terminals to meet diverse physiological needs.The intact synaptic structure is critical for information processing in neural circuits. During synaptic transmission, rapid vesicle exocytosis increases the size of never terminals and endocytosis counteracts the increase. Accumulating evidence suggests that SV exocytosis and endocytosis are tightly connected in time and space during SV recycling, and this process is essential for synaptic function and structural stability. Research in the past has illustrated the molecular details of synaptic vesicle (SV) exocytosis and endocytosis; however, the mechanisms that timely connect these two fundamental events are poorly understood at central synapses. Here we discuss recent progress in SV recycling and summarize several emerging mechanisms by which synapses can “sense” the occurrence of exocytosis and timely initiate compensatory endocytosis. They include Ca Synaptic transmission is fundamental to brain function. Nerve terminals release neurotransmitter at a different speed, depending on the types of synapses and stimulation. The rate of synaptic vesicle (SV) exocytosis directly controls neurotransmission strength. It can vary a few orders of magnitude at a single terminal   of AZs and thus impact its ultrastructure and function Figure . RemarkaDecades of research have generated a wealth of knowledge on the molecular mechanisms of SV exocytosis  studies support the ultrastructure changes of motor nerve terminals in frog neuromuscular junctions (NMJs)  , ~16% of the original AZ area . The quick-freezing EM study in NMJs , clathrin-mediated endocytosis (CME), bulk endocytosis, and ultrafast endocytosis . An SV fuses with the PM and pinches off at the same location without collapsing    , but they also have some common features .Despite the significant progress on SV exocytosis as well as endocytosis, very little is known about their temporal and spatial coupling. What triggers SV endocytosis at the right time and place remains a longstanding question. Recent work suggests that several factors are involved in SV exocytosis-endocytosis coupling, in which a synapse “senses” the SV exocytosis event and initiates endocytosis. Here, we discuss the potential means of coupling at nerve terminals.2+ classically regulates both exocytosis and endocytosis  below that Ca2+ level  allows differential endocytic regulation under weak and strong stimulations  rephosphorylates dephosphins to terminate endocytosis , amphiphysin and endophilin-A  is another Cas Figure . Intrace2+ elevation alone appears insufficient in triggering endocytosis, as demonstrated by the experiment in munc13-1 and−2 double KO synapses  do not abolish endocytosis in many studies. High sucrose stimulation also triggers efficient endocytosis   is a core protein of SNARE complex and has the highest copy number (~70 copies/SV) on SVs  and resides exclusively on SVs. Both properties make it a suited endocytosis sensor. Synaptophysin-1 KO blocks VAMP-2 endocytosis  of 0.2 μm2/s at the presynaptic PM ]. In addition to annulus EZs, other forms of EZ organization is also possible in different types of never terminals, including random distribution in the terminals or in patches at peri-AZs  is highly enriched in EZs. An annulus of F-actin is shown to surround the AZ in motor terminals of lamprey . Furthermore, insertion of SV proteins alters the rigidity of local PM, a factor playing important role in endocytosis (Hassinger et al., The local membrane stress likely facilitates the coupling between exocytosis and endocytosis Figure . The mecThe characterization of local membrane mechanics is not well-established during SV exocytosis-endocytosis, and it is likely affected by several factors including membrane tension, membrane stiffness, and local force (Hassinger et al., SV exocytosis-endocytosis coupling has a profound role in synaptic transmission, a process that is essential for neural circuit function and brain performance. Several factors emerge as sensors for endocytosis events, and we are only at an initial stage toward the mechanistic understanding of SV exocytosis-endocytosis coupling at synapses; even how SVs are born under physiological conditions remains a conundrum. The major challenge arises mainly from the complex biophysical features of exocytosis-endocytosis coupling at presynaptic terminals, such as the transient reaction, small structure, and poor accessibility. Despite these hurdles, the field is steadily moving forward with the application of new cutting-edge approaches spanning super-resolution fluorescence microscopy, single SV fusion detection, acute optogenetic manipulations (Ji et al., The author confirms being the sole contributor of this work and approved it for publication.The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Imaging biomarkers (IBs) are integral to the routine management of patients with cancer. IBs used daily in oncology include clinical TNM stage, objective response and left ventricular ejection fraction. Other CT, MRI, PET and ultrasonography biomarkers are used extensively in cancer research and drug development. New IBs need to be established either as useful tools for testing research hypotheses in clinical trials and research studies, or as clinical decision-making tools for use in healthcare, by crossing ‘translational gaps’ through validation and qualification. Important differences exist between IBs and biospecimen-derived biomarkers and, therefore, the development of IBs requires a tailored ‘roadmap’. Recognizing this need, Cancer Research UK (CRUK) and the European Organisation for Research and Treatment of Cancer (EORTC) assembled experts to review, debate and summarize the challenges of IB validation and qualification. This consensus group has produced 14 key recommendations for accelerating the clinical translation of IBs, which highlight the role of parallel  tracks of technical (assay) validation, biological/clinical validation and assessment of cost-effectiveness; the need for IB standardization and accreditation systems; the need to continually revisit IB precision; an alternative framework for biological/clinical validation of IBs; and the essential requirements for multicentre studies to qualify IBs for clinical use. A biomarker is a “defined characteristic that is measured as an indicator of normal biological processes, pathogenic processes or responses to an exposure or intervention, including therapeutic interventions” ,2. The cThe use of both imaging biomarkers (IBs) and biospecimen-derived biomarkers is widespread in oncology. In healthcare settings, biomarker uses include screening for disease; diagnosing and staging cancer; targeting surgical and radiotherapy treatments; guiding patient stratification; and predicting and monitoring therapeutic efficacy, and/or toxicityDespite some biomarkers being used extensively and others showing great potentialAll biomarkers, including IBs, must cross two ‘translational gaps’ before they can be used to guide clinical decisionsSeveral publications have described strategies for developing and evaluating cancer biomarkers, focusing mainly on biospecimen-derived biomarkers — that is, those derived from patient tissue or biofluidsAn IB is a measurement derived from one or more medical images. Many IBs are used routinely in healthcare . IBs pro99mTc]-etarfolatide folate receptor positivityApplications for IBs include the American College of Radiology Breast Imaging-Reporting and Data System (ACR BI-RADS) mammographic breast morphology18F-FDG-PET maximum standardized uptake value   18F-FDG-ke value  have beeke value  and surgMany more IBs used to measure tumour anatomy, morphology, pathophysiology, metabolism or molecular profiles are being developed in order to study the hallmarks of cancer18F-FDG-PET has improved disease staging in patients with non-small-cell lung cancer, lymphoma or melanoma by facilitating the identification of nodal and distant metastases18F-FDG-PET data to derive putative cut-off points subsequently used to identify patients with poor prognosisIBs have four key attributes. First, they are a subset of all biomarkers. Second, they can be quantitative or qualitative. Quantitative IBs  that address different research questions. This process follows two parallel and complementary tracks: in one track, technical (assay) performance is examined by addressing whether the biomarker is measurable precisely and accurately, and whether it is widely available in all geographical territories. In the other track, biological and clinical performances are examined by addressing whether the biomarker can be used to measure a relevant aspect of biology or predict clinical outcome. In reality, no biomarker is perfectly validated. Instead, strategies must be defined to identify, mitigate and quantify the uncertainty, risk and cost associated with any given biomarker in making research or clinical decisionsin vitro diagnosticsIn biospecimen-derived biomarker roadmaps, technical (assay) validation commonly occurs early in order to produce a ‘locked-down biomarker’ — that is, the data acquisition and analysis pathways used to measure the biomarker are fixed. In many cases, this stage is followed rapidly by biological and clinical validation because the locked-down biomarker enables widespread evaluation across multiple sitesMost biospecimen-derived biomarkers are molecular features found in the genome, transcriptome, proteome or metabolome that can be chosen rationally to address unmet needs in cancer medicine. This selection approach has also inspired the rational design of several novel targeted nuclear medicine or optical imaging tracersComplete technical validation is achieved when an IB measurement can be performed in any geographical location, whenever needed, and give comparable data. Technical validation does not address whether the IB measures underlying biology, or relates to clinical outcome and/or utility, but it can have a major influence on subsequent attempts at qualification because IBs with limited availability or poor reproducibility cannot sensibly undergo further clinical evaluation in large multicentre trials.in vitro and/or in vivo) using the same equipment, software and operators over a short timeframe, whereas reproducibility refers to measurements performed using different equipment, different software or operators, or at different sites and timesRepeatability and reproducibility are related measures of assay precision. Repeatability refers to measurements performed multiple times in the same subject  and transverse (R2) relaxation rates2*), diffusion anisotropy, or Ktrans , appropriate calibration phantoms are not available or poorly represent living tissue.Technical bias describes the systematic difference between measurements of a parameter and its real value15O-H2O) are highly informative, but require the presence of on-site cyclotron facilities, making worldwide translation impossible with currently available technologies, for economic reasons. Finally, specialist techniques in all modalities require advanced radiographer and technician support, which might not be available locally.IB assessments must be feasible, safe, and well-tolerated in the target population, and must have regulatory and ethical approval for use in humans. Differing regulatory approvals 18F-FDG SUVmax baseline valuesThe REMARK guidelinesKtransIBs that have demonstrated clinical validity can have important roles in drug development. For example, RECIST 1.1-defined objective response is a widely used phase II clinical trial end point that has proved useful for preliminary screening of new therapeutic agentsKtrans and tumour blood flow rate  was investigated using a terminal radiotracer-based technique in rat tumour modelsIf the Bradford Hill criteria are adopted, early preclinical imaging–pathology correlation studies18F-FDG-PET SUVmax and Ktrans  should be greater than the cost of performing the study. In healthcare, the economic test is harsher, because even well-validated IBs will not cross translational gap 2 unless they offer an advantage in terms of cost per quality adjusted life year (QALY) gainedStudies in early stages of IB discovery and validation can access conventional biomedical research funding streams, whereas evidence-gathering studies to support IB qualification can be difficult to fund. For example, large-scale multicentre reproducibility studies can be unattractive to research funders if seen as ‘incremental’ and, unless such studies create exploitable intellectual property, they are not attractive to commercial sponsors. One approach to solve this problem is to assemble consortia of commercial and not-for-profit stakeholders, such as the QuIC-ConCePT consortium13C-MRI/MRS), or by a start-up business focused on a specific technique or IB. This strategy requires careful assessment of the likely risk–benefit of the development process, and strong intellectual property positions to ensure likely financial return on the imaging device, agent or biomarkerInitial IB translation requires large-scale funding by government, charity or industry, or commercialization of the IB by an imaging company , but not for others. Nevertheless, when a biomarker is qualified in multiple settings , then the process of qualification for a new application can be expedited because most of the necessary validation requirements are likely to have been fulfilled‘Regulatory qualification’ is a more-specific term that describes a framework for the evaluation and acceptance of a biomarker for specific use in regulatory decision-makingWith this Consensus Statement we aim to accelerate IB translation. For this purpose, we have produced 14 key recommendations , accompaProposals for funding to support IB-related studies should state clearly how these will advance IB validation or qualification. Resulting journal publications should state explicitly how these aims have been achieved (recommendation 1). Study design, protocols, quality assurance processes and standard operating procedures should be reported exhaustively by making full use of supplementary materials for research publications. The software used for image processing should also be reported and be made available once intellectual property rights have been addressed (recommendation 2). These two recommendations will result in the greatest possible confidence in the reported IB-related data, will facilitate the conduct of statistically valid meta-analysis studies of imaging data, and will help investigators, reviewers, target audiences, funders and governments evaluate the risks associated with each IB for any given research or healthcare application.18F-FDG-PET in clinical trials both in Europe and North America, focusing on performing regular equipment calibration and quality assurance. Moreover, data acquisition, analysis and reporting standards have been adoptedA compelling rationale supports the accreditation of clinical imaging laboratories as being competent for measuring a given IB (recommendation 3), in line with the standards set by the biospecimen-derived biomarker communitySite accreditation is an important step towards improving technical performance in multicentre studies, but must reflect widely sought academic consensus, become adopted by international societies, and receive the backing of funders, industry and regulators for such accreditation to have value. Accreditation must simultaneously promote standardization and harmonization of IBs for multicentre use, while accommodating studies led by investigators who have scientific freedom to further develop and optimize IBs. To achieve this optimization, best-practice guidelines for each widely used IB (or related family of IBs) must be updated and reviewed regularly (recommendation 4), because biomarker drift is inevitable owing to technological advances in scanner performance . Suitable statistical methods, such as multivariate linear regression analysis and other more complex statistical approaches, must be used to adjust for changes in IBs during ongoing studies .IB precision must be demonstrated early in IB development through single-centre repeatability studies, or fewsite reproducibility studies (recommendation 5). This assessment is particularly important when testing the ability of an IB to measure the effect of therapeutic intervention. The choice of performance metric is important; for example, the coefficient of variation assumes that the standard deviation is approximately proportional to the mean. If studies of repeatability are performed under multiple conditions (such as imaging patients across different groups), then a plot of the standard deviation versus mean should be examined to determine whether this proportionality is valid. In most imaging studies of repeatability, only two replicates are acquired for each patient and thus, a Bland Altman plot will enable the evaluation of whether or not the mean of the measurements influence on variance, providing overall limits of agreementOnce the IB is shown to have sufficient technical and biological validity to cross translational gap 2, multicentre reproducibility must be evaluated (recommendation 6). Some studies have measured IB multicentre reproducibility . Analysis led by one central site can reduce data variation for studies with a moderate number of participating sitesin situ tumours, or those implanted into their orthotopic site often better recapitulate the local microenvironment of human tumoursClinical validation occurs relatively late in the development process for most IBs compared with biospecimen-derived biomarkersrho or Kendall’s tau rank correlation coefficients enable the comparison of IB values to reference standard pathology readouts within a cohort; however, if these two measurements  relate linearly to one another, regression models provide estimates of biases and the scale of the relationship.Improved methods for imaging–biology correlation are needed for IB biological validation (recommendation 9), because currently available methods often fail to account for extensive spatial heterogeneity in the IBIB studies often generate rich datasets that can be collected and banked for future re-use. These data include raw images, processed images and parameter maps, ancillary files , and essential patient metadata covering demographics, treatment history and clinical outcome . Useful data-archiving systems require financial support; collaboration between multiple academic, industry and funding partners; and ongoing curation and active management — as exemplified by the NCI Informatics Technology for Cancer Research platformCreation of animal and human cancer image repositories, following the lead of organizations including the NCI and the ESRpost hoc) should be accompanied by a description of all other exploratory analyses performed. This strategy will more-accurately reflect the potential for false-positive and false-negative findings. Adherence to these principles of reporting will help to eliminate the distortion of research findings resulting from selective reporting and failure to publish negative studies. These biases can also be reduced by registering studies and detailing their hypotheses on publically available websites  before the study is initiated or before outcome data are unblindedComplete and transparent study reporting is essential to avoid selective reporting bias and publication bias (recommendation 11). Results of all prespecified analyses  should be reported, regardless of whether they are consistent with expectations, or whether they achieve statistical significanceRobust study design and predefined statistical analysis plans are vital to ensuring that the highest quality evidence is available to qualify IBs. Late-stage multicentre clinical trials should be powered adequately to demonstrate clinically useful effects. At present, few IBs are rigorously validated or qualified as prognostic for quality of life, progression-free survival or overall survivalQualification requires rigorous and detailed statistical reporting standards. Screening and diagnostic accuracy IB studies should report sensitivity and specificity, results of receiver operating characteristic (ROC) curve analysis, and negative and/or positive predictive valuesNew models for funding and regulation should be developed for investigational devices, tracers and contrast agents, and software that have value as IBs in the research setting, but lack commercial viability as a diagnostic product in healthcare systems (recommendation 13). Imaging studies are perceived to be expensive and thus, integration of investigational IBs should be linked to existing radiological tests  whenever possible. Large-scale studies should include health-economic considerations, including measuring the cost-effectiveness of IBs versus competitor tests (other IBs or biospecimen-derived biomarkers)Clinical imaging has transformed contemporary medicineSee online article: S1 (table) | S2 (table) | S3 (box) | S4 (box) | S5 (box) | S6 (box) | S7 (box) | S8 (figure) | S9 (box)All Links are Active in the Online PdfSupplementary information S1Supplementary information S2Supplementary information S3Supplementary information S4Supplementary information S5Supplementary information S6Supplementary information S7Supplementary information S8Supplementary information S9


Sir,−1) with insufficient , vs comparing sufficient  with super-sufficient . It is worth noting that a significant association between 25(OH)D and cancer outcome was found in our review, despite this cut-off heterogeneity.We thank Dr Braillon for his interest in our recent systematic review and meta-analysis, which examines published evidence of the relationship between cancer survival outcome, vitamin D concentration and genetic variation in vitamin D-related pathways. Actually, one of the key messages of our paper was to highlight the radically different vitamin D category definitions used in the extensive discussion and supplementary materials. Our paper emphasises the need to harmonise category definition in future studies. While using clinical cut-offs of vitamin D deficiency/insufficiency , as many cancer risk factors are also associated with vitamin D deficiency. In these circumstances, demonstrated association with genetic factors involved in vitamin D metabolism offers an important new perspective that supports a causal link for vitamin D deficiency and cancer outcomes, in absence of RCTs. This is why we conclude that our findings provide powerful background rationale to instigate clinical trials to prospectively investigate the effect of vitamin D on cancer outcomes.Finally, in our view personalised medicine converges on the joint consideration of phenotypic and genotypic data, since these are biologically inseparable. Outcomes are affected by genotypes, phenotypes and also by their complex interaction. In our review, we reported on the impact of vitamin D phenotype (25(OH)D concentration) and vitamin D-related genotype on cancer outcome. Previously, we reported an interaction between genetic variation at the vitamin D receptor gene and vitamin D levels which influenced colorectal cancer survival (


Chromatin in the interphase nucleus is organised as a hierarchical series of structural domains, including self-interacting domains called topologically associating domains (TADs). This arrangement is thought to bring enhancers into closer physical proximity with their target genes, which often are located hundreds of kilobases away in linear genomic distance. TADs are demarcated by boundary regions bound by architectural proteins, such as CTCF and cohesin, although much remains to be discovered about the structure and function of these domains. Recent studies of TAD boundaries disrupted in engineered mouse models show that boundary mutations can recapitulate human developmental disorders as a result of aberrant promoter-enhancer interactions in the affected TADs. Similar boundary disruptions in certain cancers can result in oncogene overexpression, and CTCF binding sites at boundaries appear to be hyper-mutated across cancers. Further insights into chromatin organisation, in parallel with accumulating whole genome sequence data for disease cohorts, are likely to yield additional valuable insights into the roles of noncoding sequence variation in human disease. These data have provided a “locus level” view of the regulatory protein binding events and modifications to chromatin occurring  at individual genes. Meanwhile, the development of chromatin conformation capture (3C) methods, most prominently high-throughput chromosome conformation capture (Hi-C), has provided a view of the higher-order folding and nuclear organisation of human chromosomes2. These data reveal a landscape of physically interacting regions  along chromosomes and, at a larger scale, the existence of preferentially self-interacting chromatin domains, extending across hundreds of kilobases up to multi-megabase regions. However, there is no shortage of gaps in our present knowledge3. The mechanistic relationships between the hierarchical structural layers of the epigenome, from locus-level features to higher-order structures and three-dimensional nuclear architecture, are still the subject of active research. We also have an incomplete picture of how genome functions  are driven or constrained by these structures. Beyond this, there has been little evidence for any phenotypic consequences of the disruption of the interactions and domains emerging from Hi-C experiments. However, a number of recent studies have provided new insights into the consequences of pathogenic mutations acting to alter domain structures and compromise genome function.The past decade has seen a series of revolutions in the fields of human chromatin structure and regulatory genomics, driven largely by novel, high-throughput sequencing-based assays and large consortium projects. Projects such as Encyclopedia of DNA Elements (ENCODE)4 and its derivatives—4C, 5C, and Hi-C—are used to studyin vivo contact frequencies between pairs of genomic sequences, revealing the physical arrangement of DNA in the nucleus. Hi-C was developed to assess patterns of interaction between all regions of a given size across the entire genome simultaneously5. The 3C methods have continued to evolve2, but Hi-C was the original high-throughput, genome-wide variant of 3C and still accounts for the majority of 3C datasets available. The earliest low-resolution Hi-C maps showed interactions between 1 Mb regions, confirming the existence of nuclear compartments corresponding to multi-megabase regions within the accessible and transcriptionally active “A” compartment and regions within the relatively closed and inactive “B” compartment5. The highest impact of such datasets was undoubtedly the discovery of topologically associating domains (TADs), thought to represent regulatory domains, which contain many preferentially interacting subregions but very few interactions across their boundaries. TAD boundaries are often defined by relatively simple algorithms, assessing the directional (upstream versus downstream) preferences of interactions occurring along a chromosome and defining boundaries where these preferences change6. TADs were reported in multiple species as structural entities up to around 1 Mb in size in mammals  and enriched for interacting promoter-enhancer contacts8. These early studies noted the association of these domains with varying regional patterns of repressive and activating histone modifications across the genome, the enrichment of CTCF binding at boundaries, and correlations with other aspects of higher-order chromatin organisation, such as patterns of association with the nuclear lamina and replication timing domains6. About 50% of TADs appear to be cell type invariant between embryonic stem cells (ESCs) and the cortex6, and domains are often shared across species boundaries9. Later work showed differing features enriched at boundaries in different cell types, and certain enrichments  were seen across all cell types10. Although CTCF has been intensively studied as an insulator protein, it is not enriched or even detectable at all boundaries, and so the architecture(s) of TAD boundary regions and the mechanism(s) underlying their function remain elusive.The original 3C method11. Since around 60% of CTCF sites are constitutively bound12, a substantial fraction of loops may be present across tissues. The same study11 identified many TAD-like “contact domains” ranging from 40 Kb to 3 Mb in size (median length of 185 Kb), suggesting that a small fraction of boundaries were missed by previous studies with lower-resolution data. Closer investigation of Hi-C interaction matrices revealed that TADs appear to interact in clusters to form a hierarchy of nested domains, or “metaTADs”, at higher levels13. This hierarchy can be modelled as a simple tree-like structure, and rearrangements of the tree link changes in nuclear organisation to transcriptional changes at many promoters during neuronal differentiation—from ESCs via neural progenitor cells to neurons. This tree-like model of organisation provides an intuitive bridge between smaller-scale regulatory domains such as TADs and the A and B nuclear compartments13.The highest-resolution Hi-C datasets have revealed finer-scale, recurrent pairwise interactions between regions, representing “chromatin loops”, and the base of each loop is formed by two presumably interacting anchor points. Many of these loop anchor points appear to be bound by CTCF and cohesin subunits, and a majority were found to contain convergently orientated CTCF binding motifs3, and some have questioned whether TAD organisation is a cause or consequence of genome function14. As for other noncoding genomic sequences, the emphasis is shifting from the immediate biochemical roles of these new structural features to their phenotypic relevance and how their disruption impacts the fitness of real organisms15. Here, we review the latest studies of dysregulation of gene expression due to changes in chromatin conformation  can give rise to the same developmental defect19, suggesting a conserved functional role. A computational study assessed the broader importance of copy number variants (CNVs) affecting boundary elements on human developmental disorders21. The phenotypes of 922 deletion cases recorded in the DECIPHER database22 were related to the known, monogenic diseases associated with genes neighbouring the deletions. This information was used to nominate deletions likely to affect TAD boundaries and result in enhancer adoption. The results suggest that up to about 12% of deletions in the DECIPHER database resulted in enhancer adoption, based on the tissue-specific activity of a given enhancer and the tissue that was affected in the disease phenotype of the patient who carried the deletion.The extent of such disruptions in human developmental disease cases is largely unknown, but recent studies have shown that pathogenic human structural variants (SVs) overlap the boundary regions of TADs, including duplications at the SOX9 locus24, medulloblastoma25, and leukaemia27 samples, consistent with the hypothesis that TAD-disrupting SVs may act as oncogenic “driver” mutations under selection in tumour cells28. Proposed models for such phenomena include (a) deletions of boundaries to allow unusual promoter-enhancer contacts and (b) inversions that span boundaries altering the contents of neighbouring TADs; both mechanisms are thought to give rise to aberrant expression of resident genes. It also seems that boundary integrity can be compromised by hyper-methylation. Flavahanet al.29 showed that gain-of-function mutations in theIDH gene cause hyper-methylation of CpG sites as well as the GC-rich CTCF binding motif; this reduces CTCF binding at a subset of CTCF sites in mutant glioma cells. Genes most upregulated inIDH mutants as a result of this hyper-methylation included several known oncogenes, such asPDGFRA. Furthermore, CRISPR disruption of a neighbouring CTCF binding site at a TAD boundary in wild-type tumours leads to the upregulation ofPDGFRA, providing mutant cells with a growth advantage and enhancing proliferation. Intriguingly, thePDGFRA domain boundary affected by aberrant methylation in tumours can be re-established by treating mutant glioma cells with a de-methylation agent, leading to the downregulation ofPDGFRA.There have been several reports of disruptions of particular TAD boundaries in modest numbers of neuroblastomaet al.27 investigated the impact of domain boundary deletions on oncogene activation in T-cell acute lymphoblastic leukaemia (T-ALL). Using ChIA-PET  against cohesin sites, they identified around 9,000 CTCF-CTCF interactions that were shared across cell lines, demarcating active loop anchor points for “constitutive neighbourhoods”. Most genes implicated in T-ALL pathogenesis were located inside those loops. Next, they investigated the overlaps between cancer deletions (<500 Kb) in a set of T-ALL whole genome sequences and these neighbourhood boundaries. Six boundaries delineating neighbourhoods containing T-ALL pathogenesis genes were found to be recurrently deleted across tumours at unexpectedly frequent levels. Hniszet al.27 then showed that the CRISPR-engineered deletion of boundaries near two known oncogenes (TAL1 andLMO2) leads to the activation of these genes in human embryonic kidney cells, which otherwise do not express these genes. They also demonstrated changes in conformation at these boundary regions using 5C, with the intensity of contacts increasing across the boundaries following the deletion.Hnisz30. They found that recurrent SCNAs deleting a boundary of the TAD containing theIRS4 gene were associated with a marked increase inIRS4 expression in 32 samples from three different tumour types. These deletions were carefully distinguished from samples carrying focal amplifications leading toIRS4 overexpression, and the tumorigenic effects of higherIRS4 expression were validatedin vivo using mouse models. Similar to19, the authors also describe duplications mediating the formation of a new domain and driving overexpression ofIGF2 in 20 colorectal cancer (CRC) tumours. Single copy tandem duplications encompassing anIGF2 TAD boundary and an enhancer in a neighbouring TAD were found to alter conformation and activate the enhancer in CRC cell lines, leading to overexpression ofIGF2, a gene previously implicated in CRC progression. Interestingly, all duplications involving this locus were tandem duplications—rather than dispersed or inverted duplications—suggesting that the resulting head-to-tail orientation of the enhancer andIGF2 may be crucial for the gene’s upregulation31. Overall, these data suggest that enhancer adoption by oncogenes following domain boundary lesions is not a rare process, occurring at rates comparable to those of recurrent in-frame gene fusions.A larger recent study used matched expression and somatic variation data for 7,416 cancer genomes across 26 tumour types from The Cancer Genome Atlas to systematically identify somatic copy number alterations (SCNAs) likely to mediate gene dysregulation32, consistent with oncogene activation following the rearrangement of nuclear architecture. Several recent studies have also observed unexpected excesses of somatic mutations at CTCF binding sites across tumour types, including CRC33, leukaemia27, and a variety of other cancer types34. Characteristic mutational profiles were observed at positions within the CTCF binding motif, and a striking spike in mutation was seen at a central, well-conserved nucleotide, together with elevated mutation rates at sites immediately flanking the motif34. Surprisingly, these unusual patterns can be explained by selectively neutral biases in mutation rates34 and are broadly consistent with an underlying mutational mechanism involving the interference of DNA binding proteins with the replication machinery, causing elevated mutational burden at active regulatory sites35. The highest mutational loads were seen at constitutively active CTCF binding sites with roles in chromatin loops and TAD domain boundaries and were predicted to compromise CTCF binding and nuclear architecture34. These phenomena therefore appear to provide a deterministic mutation-driven process, expected to lead to altered chromatin architecture and gene dysregulation in many cancers, without invoking more complex hypotheses involving similar selective pressures across many sites and many different tumour types.CTCF hemizygous knockout mice are prone to developing cancer in a wide range of tissues34 and are underpowered to discover individual CTCF sites subject to recurrent mutation. Similarly, most WGS data for tumours lack matched RNA-seq data to study the effects of site disruption on the patterns of expression of neighbouring genes.The future will undoubtedly provide many more insights into the disruptions of chromatin and nuclear organisation underlying disease. Continuing advances in high-throughput sequencing and computational analyses are improving the resolution of Hi-C maps and allow greater precision in defining domain boundaries and other structures. Global analyses of structural disruptions in disease have generally been limited to publicly available Hi-C data, which usually are not well matched to the tissue of interest. Similar caveats apply to the available ChIP-seq data for chromatin features such as CTCF binding. With more cell- and tissue-specific Hi-C and ChIP-seq data available, many more disease-relevant disrupted interactions are likely to be found. This will also help to better distinguish “passenger” disruptions of TAD boundaries from causative “driver” mutations responsible for oncogene dysregulation. Current cancer whole genome sequencing (WGS) datasets are also inadequate for these purposes. Previous studies have examined mutational spectra at thousands of CTCF binding sites across the genome36. More complex SVs, such as inversions and translocations, are even less accurately predicted, and compound SVs (involving different overlapping or nested SVs) are beyond our reach entirely. This point is highly pertinent to cancer genomics since catastrophic rearrangements of entire chromosomes are an increasingly common observation in many cancers37, and SVs appear to be the main factors driving tumorigenesis in some cases38. Given the challenges of reconstructing SVs from standard (short read) sequencing data, it is likely that accurate resolution of all SVs present in a genome will be dependent upon novel sequencing technologies generating much longer sequence reads .In addition to improvements in the data available, there are substantial challenges ahead in WGS data analysis. Currently available algorithms for the detection of CNVs  are generally used in combinations to generate consensus predictions since no single algorithm is considered sufficiently accurate when used alone40. We also know very little about the precise physical location and composition of the protein complexes bound to domain boundaries, how the insulating effect of boundaries is achieved, and what constitutes the minimal requirements for boundary function. Higher-resolution maps derived from Hi-C or related methods will be necessary to explore the detailed features of boundary regions. The fact that some boundaries appear to lack CTCF binding sites, that many TAD boundaries change during cellular differentiation13, and that boundary compositions vary between cell types10 suggests that boundaries may vary in structure and function. The better-studied boundary components, CTCF and cohesin, appear to have distinct functions in domain formation, in that CTCF works to separate neighbouring TADs and cohesin promotes intra-TAD interactions41, but even these proteins remain the subject of ongoing research. A comprehensive picture of boundary architecture and function holds the promise of better understanding, and perhaps correcting, regulatory domain disruptions in disease.As discussed above, our knowledge of the hierarchical strata and functional inter-relationships of higher-order chromatin structure is still far from complete. Although TADs have been widely studied, we still lack detailed models of how they affect transcription. Sharing a location in the same TAD is evidently not sufficient to allow enhancer-promoter interactions to take place, and we do not understand how enhancers find their target genes within TADs. Some data suggest that a particular spacing may be needed to create a loop that successfully brings such elements together, and it is known that increasing the distance between an enhancer and a target promoter can lead to downregulation3C, chromatin conformation capture; ChIP-seq, chromatin immunoprecipitation followed by sequencing; ChIA-PET, chromatin interaction analysis by paired-end tag sequencing; CRISPR, the CRISPR-Cas9 molecular editing system allows alterations to DNA at specific locations in the genome; Hi-C, high-throughput chromosome conformation capture; RNA-seq, isolation of RNA followed by sequencing; TAD, topologically associating domain.


Bilateral diffuse uveal melanocytic proliferation (BDUMP) is a rare, paraneoplastic syndrome characterized by bilateral painless visual loss and proliferation of choroidal melanocytes in association with an underlying systemic malignancy. We report a case of bilateral diffuse uveal melanocytic proliferation associated with an underlying gynecological malignancy that also features the infrequent finding of an iris mass lesion, using multimodal imaging including ultra-widefield imaging, spectral domain and swept-source optical coherence tomography.A 59-year-old white female with a prior history of gynecological malignancy in remission presented with progressive bilateral visual loss over several weeks. The patient was noted to have a focal iris mass lesion in her right eye. Ultra-widefield color fundus photography showed a characteristic bilateral ‘giraffe pattern’ of pigmentary changes extending into the periphery as well as multiple discrete deeply pigmented lesions. Ultra-widefield autofluorescence was useful for visualizing the full extent of involvement. Indocyanine green angiography helped to demarcate the discrete pigmented choroidal lesions. Swept-source OCT clearly delineated the alternating zones of retinal pigment epithelium (RPE) thickening and RPE loss, as well as the prominent choroidal infiltration and thickening.BDUMP is an important diagnosis to consider in the presence of multiple discrete melanocytic choroidal lesions, diffuse choroidal thickening, characteristic RPE changes, iris mass lesions and exudative retinal detachment. Ultra-widefield imaging may demonstrate more extensive lesions than that detected on clinical examination or standard field imaging. Imaging with SS-OCT shows choroidal and RPE characteristics that correlate well with known histopathology of this entity. Bilateral diffuse uveal melanocytic proliferation (BDUMP) is a rare, paraneoplastic syndrome characterized by bilateral painless, profound visual loss in association with an underlying systemic malignancy . In 1966Since then, about 50 cases have been described in the literature  and the A 59-year-old white female presented with complaints of decreasing vision and photophobia over the prior month. Her medical history was notable for hypertension and chemotherapy for ovarian carcinoma 5 years prior to presentation. On examination, her best-corrected visual acuity was 20/40 OD and 20/80 OS. The anterior segment examination was remarkable for a pigmented, vascularized iris mass lesion at the superior pupillary border in her right eye Fig. . Her intSD-OCT  showed areas of subretinal fluid and discrete hyper-reflective choroidal lesions that appear to be compressing the adjacent choriocapillaris. The choroidal lesions were highly reflective on near-infrared reflectance (nIR) Fig. a–c. EnhaThe patient was referred back to her oncologist for further evaluation of a presumed recurrence of her ovarian carcinoma.We present a case of BDUMP using multimodal imaging including the use of ultra-widefield imaging, ICG angiography and SS-OCT that helped to show the full extent of the posterior segment disease. Ultra-widefield color fundus photography allowed greater visualization of pigmentary changes and multiple pigmented lesions in the periphery, while ultra-widefield AF accentuated the characteristic ‘giraffe-pattern’ fundus changes which were more difficult to appreciate on clinical examination. EDI-OCT and SS-OCT readily showed choroidal thickening and alternating zones of RPE thickening and RPE loss. These findings correlate well with the known histopathology of BDUMP. The choroid is diffusely thickened with uveal melanocytic cells including nevoid, spindle cells, and epithelioid cells. There are also aggregates of focal infiltration of pigmented melanocytes in the choroid with overlying areas of RPE destruction alternating with areas of RPE hypertrophy .Of the approximately 50 cases of BDUMP reported in literature –33, onlyVarious treatments for BDUMP, including ocular radiation , 19, 32,In summary, we show the benefit of multimodal imaging in BDUMP to demonstrate the full extent of the disease. We highlight the occurrence of iris lesions in this condition and the difficulties with treatment options.


Antegrade crossing is the most common approach to chronic total occlusions (CTOs). However, it is sometimes difficult to penetrate the proximal hard cap with guidewires, especially in the case of CTOs of anomalous coronary arteries because of a lack of support. Herein, we describe a novel, modified reverse controlled antegrade and retrograde subintimal tracking (CART) technique in which the dissection reentry was intentionally created in the proximal segment of the vessel, not within the occluded segment, using retrograde guidewire and the aid of an antegrade balloon. This technique facilitated retrograde crossing of CTOs by avoiding the proximal hard cap and may provide a viable option for patients in which conventional reverse CART is not possible. A chronic total coronary occlusion (CTO) is defined as the complete obstruction of a coronary artery that is more than 3 months old . The recA 65-year-old man with hypertension and diabetes mellitus presented to our hospital with Canadian Cardiovascular Society Class III angina despite being on optimal medical therapy, including beta-blockers and long-acting nitrates. An electrocardiogram revealed normal sinus rhythm at a rate of 66 beats/min without abnormal QS wave patterns. An echocardiogram revealed normal overall left ventricular function. The diagnostic angiography showed a CTO in the mid-segment of the anomalous right coronary artery (RCA) with a high anterior take-off from the ascending aorta and a proximal cap ambiguity of RCA-CTO because of side branches, including the sinoatrial node artery and right ventricular artery, arising near the occlusion site. The Japan-CTO score, a 5-point scoring system to assess the difficulty of CTO crossing [After discussion among the clinical heart team, PCI of a RCA-CTO was scheduled. Vascular access was obtained using bilateral femoral 7-Fr and 8-Fr long sheaths. Particularly, 7-Fr Hyperion AL 1  and 7-Fr CLS 3.5  with side holes were utilized to engage RCA and the left main coronary artery, respectively. Intravascular ultrasound (IVUS) examination from the right ventricular artery revealed that the heavily calcified plaque blocked entry of the CTO, suggesting difficulty in any guidewires penetrating into the CTO lesion .https://doi.org/10.1155/2017/3850646). Reaching the distal end of CTO, the SUOH 03 guidewire was exchanged for a Gaia Third guidewire (Asahi Intecc). It was advanced retrogradely into the subintimal space past CTO ostium  catheter was advanced into the distal LAD over a workhorse wire. Tip contrast injection revealed epicardial collateral channels. A SION guidewire (Asahi Intecc) was used as a first-line guidewire to cross the collateral channels, but it was unable to be crossed. Instead, for smoother and softer channel navigation, a SUOH 03 guidewire (Asahi Intecc) was used, which successfully traversed the collateral channels, while forming a knuckle, and thereafter a Corsair Pro catheter could follow (Movies 1 and 2 in Supplementary Material available online at O ostium . Then, aO ostium  and reacO ostium . AfterwaO ostium . The patIn this case report, we described a novel modified reverse CART technique for impenetrable CTOs in combination of epicardial collateral channels crossing using a SUOH 03 guidewire. The present case revealed that the novel modified reverse CART was feasible in CTO lesions with severely calcified proximal cap, even if an antegrade guidewire could not advance into the CTO segment, by retrogradely advancing a controllable guidewire into subintimal space past the CTO ostium, with a retrograde guidewire bypassing the calcified proximal cap of CTO.In this novel technique, dissection was intentionally created in the proximal vessel, not within the occluded segment, using an antegrade balloon inflation, and reentry was achieved in the proximal vessel, using a retrograde guidewire. Strictly speaking, we did not check if the antegrade balloon actually caused intimal disruption with intravascular ultrasound. However, a balloon that was large enough to cause intimal disruption was chosen, although the balloon-to-artery ratio should always be less than 1.19 for preventing coronary perforation .In the contemporary reverse CART technique, the connection is established within the occluded segment to minimize the length of subintimal stenting and prevent side branch loss . HoweverA new specific guidewire, SUOH 03, is of great use in crossing epicardial collateral channels, which is one of the most important steps in reverse CART. Septal collateral channels are safer than the epicardial ones and should be preferred in most cases . HoweverThere are a few limitations to this modified reverse CART technique. It cannot be applied to occluded vessels that have significant proximal side branches because subintimal reentry and subsequent stent deployment can cause side branch loss. Establishing retrograde access through collateral channels is mandatory for application of this technique but is not always successful. In addition, diffuse and very heavy calcification within CTO, which disturbs direct retrograde crossing or knuckle wiring, may prevent use of this technique.In summary, a modified, reverse CART technique combined with a retrograde approach for bypassing the calcified proximal cap was effectively and safely utilized in our patient. Successful passage of tortuous collateral channels, which is an important step of this technique, was accomplished by the use of a new specific guidewire, SUOH 03.Movies 1 and 2. A SUOH 03 guidewire successfully crossing the collaterals while forming a knuckle, followed by the advancement of a Corsair Pro catheter.Movie 3. A Gaia Third guidewire successfully penetrating the proximal true lumen and reaching the aorta in a retrograde manner.


Dentistry is a continuously changing field that has witnessed much advancement in the past century. Prosthodontics is that branch of dentistry that deals with replacing missing teeth using either fixed or removable appliances in an attempt to simulate natural tooth function. Although such “replacement therapies” appear to be easy and economic they fall short of ever coming close to their natural counterparts. Complications that arise often lead to failures and frequent repairs of such devices which seldom allow true physiological function of dental and oral-maxillofacial tissues. Such factors can critically affect the quality of life of an individual. The market for dental implants is continuously growing with huge economic revenues. Unfortunately, such treatments are again associated with frequent problems such as peri-implantitis resulting in an eventual loss or replacement of implants. This is particularly influential for patients having co-morbid diseases such as diabetes or osteoporosis and in association with smoking and other conditions that undoubtedly affect the final treatment outcome. The advent of tissue engineering and regenerative medicine therapies along with the enormous strides taken in their associated interdisciplinary fields such as stem cell therapy, biomaterial development, and others may open arenas to enhancing tissue regeneration via designing and construction of patient-specific biological and/or biomimetic substitutes. This review will overview current strategies in regenerative dentistry while overviewing key roles of dental mesenchymal stem cells particularly those of the dental pulp, until paving the way to precision/translational regenerative medicine therapies for future clinical use. Prosthodontics is “the dental specialty pertaining to the diagnosis, treatment planning, rehabilitation and maintenance of the oral function, comfort, appearance, and health of patients with clinical conditions associated with missing or deficient teeth and/or maxillofacial tissues using biocompatible substitutes”  and bone marrow-derived mesenchymal stem cells (BMMSC) with both cell types exhibiting a fibroblast-like phenotype in culture and staining positively for mesenchymal cell markers yet negatively for hematopoietic cell markers. Both BMMSC and DPSC have clonogenic and multi-differentiation potentials being able to differentiate into osteogenic, adipogenic, and chondrogenic lineages  express nestin , in addition to other neural crest stem cell (SC) markers  indicating their embryonic origin. Undifferentiated dental MSCs express markers of both neural stem/progenitor cells and mature neural cells, including SOX-2, tenascin C, ENO-2, MAP2ab, c-FOS, Nestin, Neurofilament (NEF-H and NEF-L), Glial Fibrillary Acidic Protein (GFAP), bIII-tubulin, and Microtubule-Associated Protein 2 (MAP-2)  can differentiate into functionally active neurons with the ability to express voltage-gated Na+ channels Multiple mechanisms of action involved in the neuroregenerative potential of these cells have been observed. The first is that these cells could inhibit apoptosis of neurons, astrocytes, and oligodendrocytes, which directly improved the preservation of neuronal filaments and myelin sheaths. Second, they inhibited the expression of multiple axon growth inhibitors such as chondroitin sulfate proteoglycan and myelin-associated glycoprotein, via paracrine mechanisms which directly promoted the regeneration of transected axons. They could then replace the lost cells by differentiating into mature oligodendrocytes  with passages Mesenchymal stem cells from dental origins other than pulp tissue have been shown to be affected by donor age in regards to proliferation, migration, and differentiation potential as with periodontal stem cells , basic fibroblast growth factor (bFGF), and vascular endothelial growth factor (VEGF), either in normal conditions or in response to noxious stimuli, such as injury or hypoxia. Other angiogenesis-promoting factors that have been detected in DPSCs are angiogenin (ANG), angiopoietin-1 (ANGPT1), colony-stimulating factor (CSF), dipeptidyl peptidase IV (DPPIV), endothelin-1 (EDN1), interleukin- 8 (IL-8), insulin-like growth factor binding protein-3 (IGFBP3), monocyte chemoattractant protein-1 (MCP-1), and urokinase-type plasminogen activator (uPA)  have bone marrow-derived MSC-like characteristics. An While DPSCs lose their plasticity through passaging, several investigations have provided enough evidence that SHED can retain their characteristics. Results from DNA microarray analysis revealed that there were 4386 genes that were expressed differently between DPSCs and SHED by 2.0-fold or more. SHED expressed higher levels of genes related to pluripotency , cell proliferation, and extracellular matrix, including several cytokines such as fibroblast growth factor and tumor growth factor b   DPSCs displayed comparable capacity for proliferation and differentiation as fresh-DPSCs. Cryo-DPSC transplantation was as effective as the transplantation of fresh-DPSCs in improving nerve conduction speed and blood flow in diabetic rats. DPSCs showed anti-inflammatory properties as they could ameliorate diabetic polyneuropathy upon transplantation into diabetic rats. These effects were apparent via their ability to modulate the proportions of M1/M2 macrophages in diabetic peripheral nerves  when treated with a combination of conditioned media from bladder SMCs and TGF-β1  conditions is required to address this shortage  obtained from elderly donors, and those with diabetes may have limited therapeutic potentials. This has opened the avenue for the obvious clinical advantages of universal donor cells from young healthy individuals which could be used for stem cell allotransplantation without the need for immunosuppression. Additionally, serious consequences of replicative senescence in hMSCs are that this phenomenon may impair the secretion of soluble factors in response to the inflammatory microenvironment thus resulting in loss of the immunoregulatory functions of these cells , and jaw bone MSCs, have shown strong immunomodulatory capacities  are an attractive option for exogenous stem cell therapy and have several advantages for translational cell-based therapy. Teeth are a clinically accessible source especially since many older adults possess their own teeth which could then be used as a source of viable human DPSCs for autologous transplantation. The feasibility that viable DPSCs can still be obtained up to 120 h of tooth storage in phosphate buffered saline (PBS) at 4°C post extraction from a whole tooth and can be obtained from teeth undergoing root canal treatment without the need for extraction; all support the possibility for banking these tissues for regenerative medicine applications  from teeth while avoiding any alteration of their biological properties and preserving the quality of the derived cell-based products. Cell performance is affected by cell isolation and expansion conditions and thus optimization and standardization procedures for MSC-based product manufacturing are required , further studies are required to determine more specific properties that could be used for the regeneration of human tissues, with standardized cell-based medicinal products (Ducret et al., In conclusion, as the fields of regenerative medicine and dentistry quickly move into an era of personalized and precision therapeutics, the use of dental pulp stem cells for clinical translation will soon become a feasible reality Figure . Dental All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.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.


Ixodes tick species are confirmed or strongly suspected to be vectors of zoonotic pathogens.With the resurgence of tick-borne diseases such as Lyme disease and the emergence of new tick-borne pathogens such as Powassan virus, understanding what distinguishes vectors from non-vectors, and predicting undiscovered tick vectors is a crucial step towards mitigating disease risk in humans. We aimed to identify intrinsic traits that predict which Ixodes from which many species are known to transmit zoonotic diseases to humans. We apply generalized boosted regression to interrogate over 90 features for over 240 species of Ixodes ticks to learn what intrinsic features distinguish zoonotic vectors from non-vector species. In addition to better understanding the biological underpinnings of tick vectorial capacity, the model generates a per species probability of being a zoonotic vector on the basis of intrinsic biological similarity with known Ixodes vector species.We focused on the well-studied tick genus Ixodes species with high probabilities (80%) of transmitting infections from animal hosts to humans on the basis of their traits. Distinguishing characteristics of zoonotic tick vectors of Ixodes tick species include several anatomical structures that influence host seeking behavior and blood-feeding efficiency from a greater diversity of host species compared to non-vectors.Our model predicted vector status with over 91% accuracy, and identified 14 Ixodes tick species, our model identifies particular Ixodes species with the highest probability of carrying zoonotic diseases, offering specific targets for increased zoonotic investigation and surveillance.Overall, these results suggest that zoonotic tick vectors are most likely to be those species where adult females hold a fecundity advantage by producing more eggs per clutch, which develop into larvae that feed on a greater diversity of host species compared to non-vector species. These larvae develop into nymphs whose anatomy are well suited for more efficient and longer feeding times on soft-bodied hosts compared to non-vectors, leading to larger adult females with greater fecundity. In addition to identifying novel, testable hypotheses about intrinsic features driving vectorial capacity across The online version of this article (10.1186/s12898-018-0163-2) contains supplementary material, which is available to authorized users. Ixodes, contains 244 species, of which 29 are known zoonotic vectors  by building thousands of linked classification trees that successively improve upon the predictions of the previous tree. An advantage of this approach is that it does not rely on distributional assumptions about the data, and is ideal for high-dimensional ecological data containing hidden, nonlinear interactions, and non-random patterns of missing data [Ixodes tick species as potentially novel (undocumented) vectors of zoonotic diseases, we describe a suite of features suggesting that zoonotic vectors in this group are species whose anatomy and ecology confer a fecundity advantage that results from more efficient feeding of certain life stages from a broad range of host animals.Our goal was to determine which gression , 5. Thising data . In addiIxodes based on their zoonotic vector status as established by the primary literature and GIDEON database [Ixodes tick species as vector for one or more zoonotic diseases , we searched peer-reviewed primary literature using the Latin binomial of each species , study bias can influence model results. To diagnose this possibility, we plotted the probability of a Ixodes tick species being a novel vector as a function of citation count, a proxy for study effort  that are reasonably well studied with both low and high probabilities of being zoonotic vectors  are biased by study effort , data on intrinsic features  are less subject to such biases. However, if study bias across Ixodes species, 29 species are currently recognized as vectors of human diseases in the GIDEON database. Our model was able to distinguish vector from non-vector species with over 91% accuracy on withheld test data. The best predictors of zoonotic vector status included host breadth (number of orders and families that a tick species feeds on); tarsus I length of larvae; capitulum lengths of larvae, nymphs, and female adults; scutum length of female adults; egg clutch size; and female body length , whereas those of non-vectors are generally longer. Larvae and adult female ticks have shorter capitulum lengths than non-vectors, but nymphs exhibit the opposite pattern, with nymphs of zoonotic vector species having longer capitula than non-vectors. Zoonotic vector species were also larger as female adults  and unengorged (> 2.5 mm). Adult females of zoonotic vector species also have larger clutches (> 1000 eggs) compared to adult females of non-vector species.Compared to non-vectors, Ixodes vector species that share similar trait profiles with the 29 species already recognized as zoonotic vectors. Among these, 10 species are reported in primary literature as possibly parasitizing humans . This pattern was absent at the other life stages . In addition to validating comparative patterns across species, future empirical work could explore the relationships between tarsus I and Haller’s organ sizes and their effects on host selectivity in controlled experiments.The most important predictor of zoonotic vector status in st range , 12. Sevst range . Moreovest range . If the Borrelia burgdorferi (causative agent of Lyme disease) and Francisella tularensis (causative agent of tularemia), among others [Ixodes species that transmit pathogens that cause anaplasmosis, babesiosis, and Lyme disease: of the three developmental stages, the nymphal stage is disproportionately responsible for human transmission [Ixodes tick species for zoonotic pathogens. In contrast, a shorter capitulum in larval and adult tick vectors may signal more generalized feeding that is less selective for particular host species, with host breadth ranging widely across several taxonomic groups.Another important trait distinguishing vectors from non-vectors was the capitulum length in larvae, nymphs, and adult females. Capitulum length is determined by hypostome length and salivarium size in ticks. The hypostome is a the ratchet-like anchor within the capitulum that is inserted into the host body , 15, andg others . We founsmission . With sosmission . Such stIxodes tick vectors may have a fecundity advantage over non-vector ticks [Ixodes tick vectors are most likely to be species where adult females produce a larger number of eggs, which develop into larvae that feed on a greater diversity of host species. These larvae may develop into nymphs whose capitula allow for more efficient and longer feeding times on soft-bodied hosts compared to non-vector species, leading to larger adult females with greater fecundity.Our analysis also suggest that or ticks , with laor ticks . CombineIxodes tick species that showed ~ 80% probabilities of being undiscovered vectors of zoonotic disease on the basis of their trait similarity with known Ixodes vector species (Table Ixodes acuminatus, is capable of transmitting Borrelia burgdorferi sensu lato, though it is not considered an important vector for human disease in nature, perhaps due to infrequent contact with humans [Ixodes rubicundus, causes paralysis in sheep [Ixodes vectors are understudied and some of them are already suspected to have contact with, and potential health consequences for, humans (Table Ixodes ticks vector zoonotic pathogens, our study underscores the crucial importance of basic research on ticks and other arthropod vectors, since understanding the biological underpinnings of transmission will rely fundamentally on understanding intrinsic characteristics distinguishing vector from non-vector species of Ixodes tick species.Our model identified 14 h humans . The salin sheep , but to Ixodes ticks, and suggest hypotheses about anatomical and biological mechanisms underlying vectorial capacity across Ixodes species that now require empirical validation in future work. In general, these distinguishing traits are related to the diversity of hosts species infested by ticks, and suggests that vectorial capacity could be maximized by a suite of features enabling some tick species to feed more efficiently on soft bodied hosts at particular life stages compared to non-vector species. These analyses also reveal several Ixodes species that are currently not recognized as vectors of zoonotic disease, but whose biological profile suggests they should be targets of future surveillance.This study revealed a number of intrinsic biological traits that are highly predictive of zoonotic vector status in the Additional file 1: Tables S1–S3. Tables reporting the zoonotic vector status of all 244 Ixodes ticks; A list of all variables include in the boosted regression analysis; and model parameters and performance metrics for boosted regression analyses on vector status and citation count.Additional file 2: Figures S1, S2. Figure S1 shows the relationship between citation count and the probability assigned by a boosted regression model of being a zoonotic vector. Figure S2 shows the relationship between diversity of infested hosts and tarsus I length for Ixodes tick species at larval, nymphal, and male and female adult stages.