As cancer care is transitioning to personalized therapies with necessary complementary or companion biomarkers there is significant interest in determining to what extent non-invasive liquid biopsies reflect the gold standard solid biopsy. We have established an approach for measuring patient-specific circulating and solid cell concordance by introducing tumor touch preparations to the High-Definition Single Cell Analysis workflow for high-resolution cytomorphometric characterization of metastatic colorectal cancer (mCRC). Subgroups of cells based on size, shape and protein expression were identified in both liquid and solid biopsies, which overall displayed high inter- and intra- patient pleomorphism at the single-cell level of analysis. Concordance of liquid and solid biopsies was patient-dependent and between 0.1-0.9. Morphometric variables displayed particularly high correlation, suggesting that circulating cells do not represent distinct subpopulations from the solid tumor. This was further substantiated by significant decrease in concentration of circulating cells after mCRC resection. Combined with the association of circulating cells with tumor burden and necrosis of hepatic lesions, our overall findings demonstrate that liquid biopsy cells can be informative biomarkers in the mCRC setting. Patient-specific level of concordance can readily be measured to establish the utility of circulating cells as biomarkers and define biosignatures for liquid biopsy assays. Colorectal cancer (CRC) is the third most common cancer globally . The livWith the increasing range of approved drugs and the recognition that targets may bridge across different organ sites, the need for companion and complementary diagnostic biomarkers is becoming a more frequent requirement. Development of these diagnostics within specific clinical contexts requires an understanding of underlying mechanisms to decipher the complexity of tumor evolution. While many current biomarkers are tissue-based, requiring a solid biopsy with limited sampling frequency, there is significant interest in determining whether minimally invasive liquid biopsies that utilize measurements of factors that exist in the blood, such as CTCs, can reflect the state of solid tumors to complement, or substitute, solid biopsies. To faithfully capture cell heterogeneity, personalized oncology is furthermore moving towards single cell profiling for more informed clinical decision making, as opposed to homogenized cell populations in conventional bulk biopsy analysis . To thisHigh-Definition Single Cell Analysis (HD-SCA) is a workflow for detailed characterization of circulating rare cells by high resolution imagery of individual cells. By screening all nucleated cells within a sample, it is a ‘no cell left behind’ approach enabling both increased sensitivity compared to most other CTC assays, as well as inherent morphometric profiling of all candidate tumor cells. Importantly, downstream analyses are designed for a standard slide format and include genomic profiling of individual cells by isolation and sequencing , 7, or mThe primary objective of the study was to explore the relationship of liquid and solid cell morphology and to develop an approach for assessing the concordance of liquid biopsy cells to the solid tumor. The tumor touch prep format was used as representative solid biopsy samples as it could be readily integrated in the existing HD-SCA platform originally developed for high-resolution characterization of CTCs. Unlike FFPE samples, the HD-SCA solid and liquid biopsy processing provides fresh and intact cells enabling a comparable downstream single whole cell characterization. The harmonized workflow is outlined in An assay was developed specifically for characterization of CRC using primary (SW480) and metastatic (LoVo) CRC cells and negative hepatocellular carcinoma (HepG2) cells spiked in normal donor whole blood and processed according to standard HD-SCA procedure. CDX2 expression was higher in metastatic than in primary CRC cells, and absent in hepatocellular carcinoma . FollowiIn total, pre-surgery blood and one or more solid biopsies were collected from 43 mCRC patients undergoing hepatic metastasectomy with curative intent . FifteenDespite the pleomorphism observed within the HD-CTC compartment, liquid and solid biopsy cells displayed an overall similar intra-patient morphology . Based oPrincipal component analysis (PCA) was applied on combined data from CRC liquid and solid biopsy cells, WBCs segmented from the same images for a subset of cells, as well as HD-CTCs from a prostate cancer (PrC) patient that were imaged and segmented using the same method. Since the PrC sample had been stained with a different 4-color marker (androgen receptor), measurements based on that channel were excluded from the analysis. Based on the remaining features CRC liquid and solid cells and the PrC liquid cells clustered together and separate from WBCs, even though overlap was observed, showing that CRC HD-CTCs more resembled solid (primary and metastatic) CRC cells and genomically and morphologically well characterized HD-CTCs from a PrC patient  than surThe computed features could be loosely grouped by type of measurement, i.e. object size, shape or biomarker distribution. Spearman correlation coefficients were computed based on the entire dataset, and features were ordered by hierarchical clustering . As expeThe liquid and solid biopsy concordance was assessed using hierarchical clustering of features normalized within each patient . For patTo assess the intra- and inter- patient concordance of cells and to further describe subgroups of cells present in the cohort, metaclusters were defined by mean values of each cluster from the individual patients . The 10 Although the primary objective of the study on the HD-CTC positive subset of patients was the cytomorphometric comparison of solid and liquid cells, the association of HD-CTC concentration and clinical features was also evaluated using the full set of 43 patients. HD-CTC levels was significantly higher in patients with high tumor burden, i.e. primary CRC tumor and hepatic metastasis, or >4 hepatic tumor lesions present . HD-CTC While solid tissue biopsy histology remains the gold standard for tumor diagnosis, the interest in using liquid biopsies for diagnostic and prognostic purposes is gaining more ground with the increasing demands of companion and complementary biomarkers for individualized treatments, for non-invasive longitudinal monitoring of disease evaluating response to approved agents, new drugs, or drug combinations . Bulk anOne challenge for CTC assays has been the lack of studies showing correlation to the solid biopsy, which remains the gold standard for tumor characterization. The concordance of expression profiles of CTCs and solid tumor cells has previously been quantified using real time qPCR, but the studies have been hampered by low detection rate of genes in CTCs , 20. In Despite the observed pleomorphism in the liquid biopsy cell compartment, the distribution of several features was equivalent in HD-CTCs and solid cells. In general, features based on size and shape were more similar in the two, while measurements based on biomarkers showed larger variation . The obsThe traditional seed-soil theory has long overshadowed the possibility that blood biopsies might not fully reflect the solid tumor. More recently, it has been suggested that only specific subsets of tumor cells, e.g., cancer stem cells or cells undergoing epithelial to mesenchymal transition, have the survival benefits required to exist in circulation , 24. OurThe ratio of 35% HD-CTC positive patients in this cohort was comparable to detection rates of CTCs in patients with widespread mCRC using CellSearch® –28. The In conclusion, we demonstrate that HD-SCA imaging of single cells from liquid and solid biopsies from multiple mCRC patients reaches the analytical resolution required to quantitatively describe single cell pleomorphism within tumor samples of individual patients and across cohorts. To this end, we established a new approach within the validated HD-SCA platform, introducing quantification of liquid and solid biopsy cell concordance. While the study was limited by the current lack of genomic and/or multiplex proteomic single-cell data, the morphometric characterization of >1000 single cells supports that liquid biopsy cells are related to solid tumor cells in mCRC, and that subgroups of cells with characteristic morphology are present across patients and the liquid and solid cell compartments. The association with tumor presence shows that HD-CTCs have the potential to be used as markers for disease and to monitor disease burden over time. Analysis of tumor touch preparations in parallel with the conventional liquid biopsy cell profiling within the HD-SCA platform can be readily introduced to the workflow to assess the level of concordance within a patient, and to determine the applicability of liquid biopsies for tumor characterization for each individual case. In line with the recently clinically approved AR-V7 predictive test for prostate cancer, we foresee that our approach could pave the way for developing additional CTC assays measuring a condensed set of features of liquid biopsy cells for addressing clinical decision-making.Patients >18 years of age, diagnosed with surgically resectable hepatic mCRC were enrolled on harmonized, IRB approved clinical protocols at Scripps MDAnderson Cancer Center , USC Norris Comprehensive Cancer Center/Keck School of Medicine  and Baylor College of Medicine . Systemic chemotherapy was not administered for at least 21 days prior to resection. Samples were collected at the day of surgery, except for patient CRC712 where the pre-surgery sample was collected 1 week prior. Study data were managed using REDCap. Venous peripheral blood was collected in Cell-free DNA Blood Collection Tubes®  prior to surgery (before anesthesia) and post-surgery. Five touch prep slides were collected from each piece of resected tumor tissue immediately after resection, by gently touching the tissue against standard microscope glass slides . At the 6 cells/slide  before cryobanking. Slides were stained in batches of 50, using the validated, standard HD-SCA protocol with a cocktail of pan-cytokeratin (CK), CK-19, CD45 antibodies and DAPI [Matching blood and tumor touch prep samples were processed in parallel within 24 hours after collection as previously described for peripheral blood , with nuand DAPI  complemeThe CRC assay was developed using primary (SW480) and metastatic (LoVo) CRC cell lines and a hepatocellular carcinoma cell line (HepG2) as positive and negative controls, respectively, spiked in whole blood from normal blood donors. Two rabbit monoclonal CDX2 antibody clones (SP54 and EPR2764Y) were evaluated and confirmed to have no cross-reactivity with the standard HD-SCA reagents. The preferred antibody based on signal-to-noise ratio (EPR2764Y) was titrated in 6 dilutions combined with 2 dilutions of the secondary Alexa Fluor 488 goat anti-rabbit antibody  in 3 independent experiments and used at 100X dilution with 500X dilution of secondary goat anti-rabbit Alexa488 in the final assay Non-CRC cells , endothelial cells (HUVEC) and white blood cells (WBCs) from normal blood donors showed no CDX2 expression when stained with the assay. The analytical performance in CRC was demonstrated by ability and reproducibility in identifying spiked CRC cells and by comparing intensity levels in model CRC cells and patient HD-CTCs.effsize package in R . Cohen’s effect sizes were calculated for each feature and patient using the age in R . Mean efrom 8-20 , thus itggplot2 package. Boxplots were created from raw measurements of individual features, using the geom_boxplot and geom_jitter functions. The caret package was applied for preprocessing, using the BoxCox, zv, center and scale functions on a per patient basis, and the pca function in the FactoMineR package for PCA. Hierarchical clustering and heatmaps were displayed using the heatmap.2 function. Dendrograms were analyzed by third level branching to define clusters of cells within each patient. Mean values of features were used for each cluster as input to a metacluster analysis to pinpoint cell groups of different characteristics. Main “clusters of clusters” were defined by second level branching. Spearman correlation of features were computed using the cor function and visualized using the corrplot package. For correlation analysis of liquid and solid biopsy cells within each patient, the raw measurements were scaled to a 0-1 range. Correlation of liquid and solid biopsy cells was assessed by Pearson correlation coefficients of the profiles of (average) scaled values within each patient. Intraclass Correlation Coefficents (ICC) were calculated using the ICC package. Individual p-values for intra-patient liquid versus solid biopsy cells as well as HD-CTCs from CRC versus PrC were calculated using two-tailed, heteroscedastic Student’s t-tests, and pre- and post-surgery samples were compared using 2-sided paired t-test.All data analyses were conducted in R and displayed using the


An 80-year-old woman with normal-tension glaucoma was transferred to our clinic 9 years ago. She exhibited progressive visual field defect despite intraocular pressure in both eyes remaining stable in the low teens after treatment with prostaglandin-derivative eye drops. Increased short-term fluctuation in optic nerve head (ONH) blood flow was detected using laser speckle flowgraphy. After the patient was administered kallidinogenase tablets, the fluctuation was reduced and her visual field defect was ameliorated. However, the fluctuation increased and the visual field defect deteriorated after the patient discontinued the medication. The increased short-term fluctuation in ONH blood flow seemed to be associated with the development of glaucomatous visual field defect in this case. Global surveys indicate that glaucoma is the second leading cause of visual impairment . Most asIn the current report, we present a case of advanced-stage NTG with increased short-term fluctuation of optic nerve head (ONH) blood flow detected using laser speckle flowgraphy (LSFG).An 80-year-old woman had complained of bilateral visual field defect and had been diagnosed with NTG by an ophthalmologist in 2004. She had been receiving treatment with eye drops (unoprostone isopropyl) when she was transferred to our clinic 3 years after the first diagnosis. Her personal medical history included multiple cerebral infarctions and left mild atrial hypertrophy . She underwent surgeries for cataracts in both eyes at our clinic in in the next year. She exhibited progressive visual field defect despite the IOP in both eyes remaining stable in the low teens after treatment with topical application of tafluprost.When she visited our clinic on 21 October 2013 , her best-corrected visual acuity was 18/20 in the right eye (OD) and 14/20 in the left eye (OS); her IOP was 11 mmHg OD and 13 mmHg OS; and the cup-to disc ratio was 0.8 OD and 0.6 OS . DeterioKallidinogenase tablets  were prescribed for her starting in September 2014. Approximately 3 months later (Day 3), the fluctuation of ONH blood flow was noticeably reduced in both eyes , Table 1To the best of our knowledge, this is the first report indicating increased short-term fluctuation of ONH blood flow in a case of NTG, detected by the use of LSFG. This case exhibited progressive visual field defect in spite of stable IOP in the low teens.We measured ONH blood flow using LSFG-NAVI, an updated model of LSFG. The principle and method for determining ONH blood flow with LSFG-NAVI have been described elsewhere ,18. Mean


Evasion of immune surveillance is an important feature of cancer, which expresses certain molecules that affect immune functions such as immune checkpoints . Among tAlthough the expression of CD137 and its functional role in immune cells have been intensively studied, only limited information is available regarding the expression of CD137 in tumor cells. Several reports have described the expression of CD137 in various types of malignancies including lung cancer , leukemiCD137 transcription in cancer cells. The relative contributions of the MAPK and NF-κB pathways in promoting the CD137 expression in cancer are likely cell type-dependent, as evinced by the findings that specific suppression of each pathway affected CD137 expression differently in different cell types [For cancer cells with activating K-ras mutations, the MAPK signaling and NF-κB pathway seem to function in parallel to promote CD137 expression. Although both pathways are driven by K-ras activation, each pathway appears to operate independently and is likely able to compensate each other if one of them is suppressed. The NF-κB pathway seems mainly activated by extracellular IL-1α , whose expression and secretion are enhanced by K-ras . In contll types . Both pall types . This suThe expression of CD137 in immune cells and its role in promoting immune function against cancer have been well-characterized. In fact, CD137 agonists have been tested as anticancer agents with some success . HoweverSince CD137 is an important molecule known to be expressed in immune cells and modulates immune functions, the discovery that K-ras-driven cancer cells also express CD137 should prompt further investigation in this area. The important questions that remain to be answered are: (1) what is the overall impact of CD137 expression in tumor cells on immune functions against cancer? This question could be addressed using genetic approaches and tumor formation study in immuno-competent animal models; (2) what are the specific effect of tumor CD137 on the functions of APC, T cells, or macrophage? (3) In addition to K-ras, could other oncogenes such as c-Myc and Bcr-Abl regulate the expression of CD137 expression in cancer cells? If so, what are the regulatory mechanisms? The answers to these questions would provide important new information on the role of CD137 in cancer immunity with potential clinical implications.


However, in the field situation it is increasingly apparent that pathogen co-infections within the same host are a common occurrence. For example, chronic infection with pathogens including malarial parasites, soil-transmitted helminths, Mycobacterium tuberculosis and viruses such as HIV may affect a third of the human population of some developing countries. Increasing evidence shows that co-infection with these pathogens may alter susceptibility to other important pathogens, and/or influence vaccine efficacy through their effects on host immune responsiveness. Co-infection with certain pathogens may also hinder accurate disease diagnosis. This review summarizes our current understanding of how the host's immune response to infection with different types of parasites can influence susceptibility to infection with other pathogenic microorganisms. A greater understanding of how infectious disease susceptibility and pathogenesis can be influenced by parasite co-infections will enhance disease diagnosis and the design of novel vaccines or therapeutics to more effectively control the spread of infectious diseases.Parasites have evolved a wide range of mechanisms that they use to evade or manipulate the host's immune response and establish infection. The majority of the  The important host-parasite interactions that influence the progression and control of infection to individual pathogenic microorganisms have been studied in much detail, especially in laboratory mice. However, since many pathogens are acquired through similar routes of exposure  infection of natural host species in field situations and humans with multiple pathogens is common, especially in regions with poor sanitation and/or limited access to clean drinking water. In order to sustain chronic infections, the parasitic microorgansisms have evolved a diverse range of mechanisms to enable them to evade or manipulate the host's immune response. As a consequence, by-stander effects due to infection with certain parasite species can significantly alter susceptibility to other important pathogens, and/or influence the development of pathology. Discussed throughout this review are examples of the many studies that have investigated the effects of parasitic helminth infections on host susceptibility to co-infection with a diverse range other pathogenic microorganisms.Ascaris lumbricoides; whipworms, such as Trichuris trichiura in humans and T. muris in mice; hookworms, such as Necator americanus; flukes (trematodes), such as Facsiola hepatica and Schistosoma mansoni; and cestodes (tapeworms). Many of these helminth parasites are soil-transmitted and cause gastrointestinal infections following ingestion of pasture or water contaminated with their eggs. The schistosomes, in contrast, can establish chronic infection within the host's bloodstream. Infections with helminth parasites often cause significant pathology, for example as they migrate through host tissues following infection via the skin , or feed on the gut epithelium  (+ T helper cell type 2 (Th2) polarized immune responses. These are typically characterized by increased expression of cytokines such as interleukin-4 (IL-4), IL-13, eosinophilia, production of immunoglobulin E (IgE), and stimulation of alternatively activated (M2) macrophages and type 2 innate lymphoid cells (ILC2) , such as is spp.) . These c+ Th1 cells, CD8+ T cells, and NK cells and the stimulation of pro-inflammatory classically activated (M1) macrophages , IL-12, increased levels of CD4rophages .Mycobacterium bovis. Current measures to control this disease include the regular testing of cattle and removal of infected stock from herds. A widely used diagnostic test for this disease is based on the detection of M. bovis-specific IFNγ production by peripheral blood lymphocytes. However, as helminth infections can modulate host IFNγ responses, co-infections with these parasites could have serious consequences for the reliable detection of other important pathogens (see section Mycobacterium tuberculosis).An increasing body of data indicates that alterations to these responses due to co-infection with other pathogens can dramatically influence disease susceptibility. Many of the examples presented below suggest links between parasite-induced disturbances to the Th1/Th2 balance and/or macrophage phenotype  and altered susceptibility or pathogenesis following subsequent co-infection with other pathogens. The effects of the parasite infection on the host's immune response may also have serious implications for the accurate diagnosis of other infections within the same individual. Bovine tuberculosis is an important infectious disease of cattle caused by the pathogenic bacterium This review therefore discusses how the induction of an immune response to infection with distinct types of parasites may modulate the development of effective immunity and/or disease pathogenesis to co-infections with other important pathogenic microorganisms. Many of the data obtained in the studies described below was derived from experimental mouse models, but studies from humans and natural host species are included where data were available.Plasmodium parasites, is an important infectious disease of humans and animals that inflicts significant morbidity and mortality in tropical and sub-tropical regions throughout the world. Infections are transmitted through the deposition of sporozoite parasite stages into the skin via the bite of infected mosquito vectors. These then travel via the bloodstream to the liver, where they infect hepatocytes and undergo morphological change and replication before subsequently infecting erythrocytes. The parasites then undergo extensive cycles of replication and infection in erythrocytes leading to anemia. During the erythrocytic stage of malaria infection, IFNγ production from CD4+ Th1-cells and CD4+ T-cell help for the B-cell response are each required for effective control and elimination of the parasitaemia. The actions of CD4+ T cells are also important for controlling the pre-erythrocytic stages of infection through the activation of parasite-specific CD8+ T cells [for in-depth review see . As a consequence, the co-infected mice were less able to control the malaria infection, displaying increased parasitemias during the early phase of P. berghei infection and increased fatality  suggests that this also plausible. This study showed that the baboons that were chronically infected with S. mansoni had significantly lower P. knowlesi malaria parasite burdens and were protected from anemia  model show that co-infection with Chikungunya virus can impede the sequestration of malaria parasites into the brain and provide some protection against ECM  seven weeks before infection with P. berghei malaria parasites was similarly shown to reduce the severity of ECM . When mice were co-infected with malaria parasites 4 weeks after S. mansoni infection (prior to the onset of the Th1/Th2 switch), no improvements to ECM pathogenesis were observed  and the in vitro IFNγ assay are commonly used to identify M. bovis infected cattle. However, certain parasite co-infections can reduce the sensitivity of these assays. A large-scale epidemiological study in England and Wales showed that in dairy herds with a high incidence of F. hepatica fewer animals were detected as positive reactors against BTB (F. hepatica reduces the sensitivity of the standard BTB tests (SICCT test and IFNγ test) through reduced M. bovis-specific Th1 immune responses , caused by infection with the bacterium inst BTB . Experimesponses . Howeverted cows . These dM. bovis-specific Th1 immunity in cows co-infected with F. hepatica, mycobacterial burdens were shown to be reduced  than those where this nematode species was not detected (C. oncophora enhanced their susceptibility to lungworm (Dictyocaulus viviparus) infection  is important for parasite clearance. However, during the chronic phase of schistosomiasis high levels of arginase-1 are instead expressed by alternatively activated macrophages. In the S. mansoni co-infected mice, the reduced protection against T. cruzi coincided with the reduced production of IFN-γ and NO  co-infection did not affect the induction of a protective Th1 response to T. gondii infection  and these can significantly influence the virulence and severity of the disease in different host species  is a leading cause of waterborne diarrhoeal disease. The characteristic signs of Giardiasis include diarrhea, abdominal pain, nausea, vomiting, and anorexia, with some individuals also developing extra-intestinal and post-infectious complications. Little is currently known of the effects of parasite co-infections on susceptibility to giardiasis. A study of 3 year old children in the Quininde district of Ecuador has shown that those co-infected with the gastrointestinal helminth A. lumbricoides had plasma cytokine profiles indicative of an increased Th2/Th1 cytokine bias, and significantly lower plasma levels of IL-2 and TNF-α when compared to those infected with G. lamblia alone  genes  genes . This stSalmonella (NTS) bacteraemia is a common cause of community-acquired bacteraemia in the human populations of several regions of sub-Saharan Africa , a chronic disease affecting ~2 billion people worldwide. This infection typically affects the lungs, and the majority of individuals infected with M. tuberculosis have latent infections and are asymptomatic. However, data from various clinical studies in humans have raised the hypothesis that helminth co-infections may affect TB susceptibility and the risk of developing latent M. tuberculosis infection, as co-infected patients often displayed more advanced disease  similarly presented with more severe types of leprosy  can induce an alternatively activated phenotype in human macrophages, reducing their ability to control M. tuberculosis infection in vitro  may reduce susceptibility to gastrointestinal co-infection with certain pathogenic bacteria. These studies also highlight how differences in the anatomical niches that the parasites inhabit may also have a significant influence on the disease pathogenesis of a bacterial co-infection. When pathogens infect the same niche such as the gastrointestinal tract, infection with Giardia spp. may provide protection against bacterial co-infection  had increased viral loads and reduced levels of virus-specific CD4+ T cells expressing IFNγ and TNF-α when compared to mice infected with norovirus alone , and co-infection with hookworms correlated with much lower peripheral blood CD4+ T cell levels than those infected with HIV alone .In contrast to the above reports, it is plausible that in some circumstances that co-infection with helminths may enhance protection against viruses. Respiratory syncytial virus (RSV) is a major respiratory pathogen, and nearly all infants are infected with this virus by the age of 2 years old. However, because the virus does not induce lasting immunity recurrent RSV infections can occur throughout life. A study has shown how a gastrointestinal helminth infection can promote protective antiviral effects in the lung . Mice inEchinoparyphium spp.) significantly reduced viral loads following co-infection with ranavirus . The cellular and molecular mechanisms by which helminth co-infection mediated these effects on ranavirus pathogenesis remain to be determined.All the above studies have described parasite virus co-infections in mammals or birds, but similar interactions have been reported in experimental amphibians. Prior infection of the larval stages (tadpole) of four distinct amphibian species with trematode parasites (anavirus . FurtherSc) of the host cellular prion protein (PrPC), co-purifies with prion infectivity in diseased tissues (neuroinvasion)  –104.Teladorsagia circumcincta at monthly intervals from 6 to 11 months old and effects on prion disease determined  and pathogen susceptibility, raising the hypothesis that many of the effects of co-infection are immune mediated. For example, alterations to the polarity of the T-cell response or macrophage phenotype induced by the parasite infection could affect the induction of protective immunity to co-infection with another pathogen. Further scrutiny of these studies shows that in many instances definitive demonstrations that the effects are indeed immune-mediated are lacking. Addressing these issues in natural host species is technically challenging. However, a large array of murine Laboratory mice housed to high microbiological status in specific-pathogen free conditions have proved to be highly tractable model systems in which to study the pathogenesis of many infectious diseases. How representative these mice are to natural host species in field conditions is questionable, since wild mice are typically infected with numerous micro- and macro-parasite species. The immune status of laboratory mice and wild mice differs significantly . Wild miMany of the above examples suggest links between alterations to Th2/Th1 polarity or the nature of the innate immune response and susceptibility to pathogen co-infection. However, the underlying rules that dictate whether these interactions are likely to confer increased susceptibility or protection are not always apparent and are likely influenced by multiple factors. For example, susceptibility to these co-infections may be dependent on the individual niches that each pathogen inhabits in the host e.g., the same niche, or mucosal  vs. systemic. In other situations, the induction of a strong pro-inflammatory response to a parasite infection may exacerbate susceptibility and/or pathology following co-infection with another pathogen that induces a similar pro-inflammatory response. Chronic helminth infection in mice also promoted the reactivation of a latent virus infection. But not all the effects on co-infection appear to be directly immune mediated, as disruptions to the metabolic profile within the gastrointestinal tract following helminth infection promoted susceptibility to co-infection with certain pathogenic bacteria. As well as influencing host susceptibility to pathogen co-infection, the immune response to certain parasite infections may have other important health issues by negatively affecting the induction of antigen-specific immunity to vaccine antigens, reducing vaccine efficacy.A greater understanding of how infectious disease susceptibility and pathogenesis are influenced by concurrent parasite infections will help the design of more effective treatments to control the spread of infectious diseases. For example, some helminth-derived ES products possess potent immunoregulatory properties, and these could be sufficient to suppress allograft rejection . WhetherThe author confirms being the sole contributor of this work and has 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.


CanAssist-Breast is an immunohistochemistry based test that predicts risk of distant recurrence in early-stage hormone receptor positive breast cancer patients within first five years of diagnosis. Immunohistochemistry gradings for 5 biomarkers  and 3 clinical parameters  of 298 patient cohort were used to develop a machine learning based statistical algorithm. The algorithm generates a risk score based on which patients are stratified into two groups, low- or high-risk for recurrence. The aim of the current study is to demonstrate the analytical performance with respect to repeatability and reproducibility of CanAssist-Breast.All potential sources of variation in CanAssist-Breast testing involving operator, run and observer that could affect the immunohistochemistry performance were tested using appropriate statistical analysis methods for each of the CanAssist-Breast biomarkers using a total 309 samples. The cumulative effect of these variations in the immunohistochemistry gradings on the generation of CanAssist-Breast risk score and risk category were also evaluated. Intra-class Correlation Coefficient, Bland Altman plots and pair-wise agreement were performed to establish concordance on IHC gradings, risk score and risk categorization respectively.CanAssist-Breast test exhibited high levels of concordance on immunohistochemistry gradings for all biomarkers with Intra-class Correlation Coefficient of ≥0.75 across all reproducibility and repeatability experiments. Bland-Altman plots demonstrated that agreement on risk scores between the comparators was within acceptable limits. We also observed > 90% agreement on risk categorization (low- or high-risk) across all variables tested.The extensive analytical validation data for the CanAssist-Breast test, evaluating immunohistochemistry performance, risk score generation and risk categorization showed excellent agreement across variables, demonstrating that the test is robust.The online version of this article (10.1186/s12885-019-5443-5) contains supplementary material, which is available to authorized users. Only 15% of patients with early-stage breast cancer benefit from adjuvant chemotherapy . IdentifMost tests that are currently available to predict risk of distant recurrence for breast cancer utilize quantitative gene expression-based platforms –8. TheseCAB uses IHC based evaluation of expression levels of 5 key biomarkers  and three clinicopathological prognostic parameters tumor size, tumor grade and node status  to arrive at a “CAB-Risk Score”. “CAB Risk Score” classifies patients into two categories, either low- or high-risk for distant recurrence. The detailed rationale for choosing these biomarkers is explained in the prior publication describing the development of CAB test . BrieflyThe steps involved in the CAB test are described in Fig. The Evaluation of Genomic Applications in Practice and Prevention Working Group (EGAPP) has postulated recommendations for validation of gene expression-based prognostic and predictive tests in breast cancer that includes both clinical and analytical components . SeveralSince the CAB risk score generation depends on the cumulative gradings of five different biomarkers, it is important that we evaluate the effect of variability in the gradings of individual biomarkers on risk scores and risk category assignments. To address this, precision analysis was further extended to the CAB risk score generation and risk categorization.To analyze the effect of factors influencing IHC performance and gradingsTo assess the effect of the factors on CAB risk score generation and risk categorizationThus, analytical validation study described here aims to establish repeatability and reproducibility of the CAB test and is performed in conjunction with CAP recommendations and CLSI guidelines:Tumor samples were obtained from patients diagnosed with invasive breast carcinoma. The sample selection criteria for the current analytical performance study were similar to that used for the development of CanAssist-Breast . FurtherThe FFPE blocks were physically examined for proper labelling. Integrity of block was assessed by a senior technician for any kind of processing artifacts. The blocks were sectioned into 3-μm sections for H & E staining and IHC experiments. The H & E stained slides were assessed for tumor content and tissue artifacts like necrosis and hemorrhages. The blocks with tissues of necrosis/hemorrhage greater than 10% were rejected. IHCs were performed on tissue slides within a week of sectioning.According to CAP and CLSI guidelines, optimal antibody concentration and antigen retrieval conditions were established as integral components of standardizing IHC based assays. Immunohistochemistry was performed on five serial sections of the FFPE tumor blocks for the 5 CAB biomarkers. Positive and negative controls were included in every IHC experiment. The detailed IHC staining protocol for the 5 CAB biomarkers has been performed as described previously . The detPositive control used is a previously tested breast cancer tissue sample that expressed high levels (> 60% staining) of the biomarkers. Either an isotype matched non-specific IgG, instead of the specific antibody or negative reagent control were used as negative controls. All IHC slides were graded by qualified Pathologists who have been trained to grade these slides in our central laboratory where all tests are performed.We did not assign a specific cut-off value for assessing positivity of IHC staining for any of the five biomarkers. Each biomarker was graded as absolute value of percentage of the tumor cells stained at a specific location (membrane or cytoplasmic) on a continuous scale of 0–100 and based on the intensity of the staining on a scale of 0–3 . CD44, ATo establish precision of CAB test (repeatability and reproducibility), variations due to operator, run and observer (pathologist) Fig.  were evaThe experimental design for investigating the various sources of variabilities is shown in Fig. This was assessed using Intra-class Correlation Co-efficient (ICC) using the MedCalc software. Correlation co-efficients were measured for IHC gradings for each of the 5 biomarkers. Comparisons were made on the grading by each observer with the average grading of 3 observers (inter-observer); each grading by an observer with average of triplicate gradings by the same observer (intra-observer); grading by a single observer for each IHC experiment with the average of gradings from triplicate IHC experiments done by the same operator (intra-operator/intra-run); grading by a single observer for each IHC experiment performed by an operator compared to the average gradings of the independent IHC experiments performed by 3 operators (inter-operator); each grading by the same observer from IHC experiments done by the an operator with the average gradings of IHC experiments done on three consecutive days by the same operator (inter-run). Thus, three ICC values were obtained for every category. The final ICC values were expressed as an average of these three ICC values. ICC values below 0.4 indicate poor agreement; between 0.4 and 0.59 indicate fair to moderate agreement; between 0.6 and 0.74 indicated good agreement and greater than 0.75 indicated almost excellent agreement .+ 2SD) was considered to have good agreement between two measurements that were analyzed [Bland Altman plots were used to assess the agreement on risk scores across various variables tested. For all comparisons done in this analytical study, individual reading/score is correlated to average readings/scores. Risk scores computed from the gradings of the 5 CAB biomarkers done by one observer was compared to average risk score obtained by gradings from three observers (inter-observer) or three gradings by same observer (intra-observer) or gradings obtained by a single observer on IHC performed by three different operators (inter-operator) or gradings obtained by triplicate IHC experiments done by the same operator and graded by a single observer (intra-operator/intra-run) or gradings done by the same observer on IHC performed by a single operator on three non-consecutive days (inter-run). In these plots, presence of samples between the two dotted lines , and each individual observers’ prediction was compared to the consensus prediction and pairwise agreement was assessed. Statistical analysis for inter-observer variation was performed by calculating Overall Percent Agreement  Negative Percent Agreement  and Positive Percent Agreement  as described in the CLSI guidelines [Pairwise agreement was calculated using the majority call/consensus method as described at idelines . 95% conAll the five biomarkers showed a range of expression across tumor samples in our patient cohort Fig. . The typThe quantification of IHC results is dependent on the subjective evaluation of percentage and intensity of staining even by trained observers , 17. VarIntra-observer variation was assessed on a sample size of 162 for each of the 5 biomarkers. Triplicate gradings for the same slide by a single observer with a washout period of two weeks between 2 gradings showed strong agreement across all the 5 biomarkers, with ICC of ≥0.8 (Table n = 42), and the resulting slides were graded by a single observer. Strong agreement was indicated across all the 5 biomarkers for three operators with an ICC of ≥0.85 . There was a strong agreement for each of the CAB biomarkers within runs with an ICC of ≥0.97 (Table Intra-run/intra-operator variation was assessed as a measure of repeatability by running triplicates within a single run n = 4. There wn = 309) used in this study had risk scores distributed across the whole range of CAB test from 0 to 100  and intra- (n = 33) observer variability using Bland Altman plots for samples with risk scores around the clinical decision point (CAB scores of 15.5 ± 2.5). The data showed strong agreement between inter- , high-risk (PPA) and over-all (OPA) predictions with tight 95% confidence intervals Table .Table 2CCanAssist-Breast is a laboratory developed test that uses a well-accepted technology, IHC along with a complex algorithm to predict risk of cancer recurrence. Demonstration of the analytical performance is a critical requirement for all the diagnostic tests to ensure that accurate results are reported for every patient. Clinical utility of a test not only requires that the test is clinically validated but also is analytically validated for its precision, repeatability and reproducibility. EGAPP has reported that many of the tests lack either analytical validation or corresponding available data is insufficient . ExperimOne of the challenges inherent in demonstrating the analytical validity of any IHC based test is the lack of comprehensive evaluation criteria and a gold standard of reference. To overcome this, we adopted the evaluation criteria set by CAP and CLSI recommendations on thorough analysis of steps involved in validation of new IHC based tests , 13. CLS> 0.75  helped us to bring down the cost of CAB test to a fraction of the cost of currently available multi-gene tests, thus making it more affordable even in low-resource settings. Furthermore, the CAB test has been clinically validated using a large cohort of 900 retrospective patient samples in a global, multi-center study  which esWe have thus conclusively shown that CanAssist-Breast, multi-marker IHC-based test is robust across several repeatability and reproducibility variables tested making it a reliable, cost-effective and accurate prognostic test to stratify patients with early stage HR+ breast cancer into low- or high-risk for distant recurrence.Additional file 1:Additional information on methods. (DOCX 14 kb)Additional file 2:Figure S1. Representative IHC images of the CanAssist-Breast biomarkers: IHC images of CanAssist-Breast biomarkers captured at 40X magnification a. CD44, b. ABCC4, c. ABCC11, d. N-cadherin, e. Pan-cadherin respectively. (TIF 10398 kb)Additional file 3:Figure S2. Analysis employed for assessing ‘Precision on risk scores’. Schematic representation of the analysis employed in assessing precision on risk scores for all variables tested (a-e). (TIF 347 kb)


Depression is a common comorbidity in type 2 diabetes. Studies have consistently shown that major depression is associated with decreased diabetic self-care behaviour. People with subthreshold depression experience greater functional impairment, have a poorer quality of life and use health services more than those without depressive symptoms. Although subthreshold depression impacts self-care behaviour, the relationship between subthreshold depression and diabetes self-care behaviour has not been systematically reviewed. The objective of this systematic review is to determine the association between subthreshold depression and self-care behaviour in adults with type 2 diabetes.This protocol will follow the guideline of the Preferred Reporting Items for Systematic Review and Meta-Analysis Protocols 2015 (PRISMA-P-2015). A systematic search of literature will be conducted for observational studies reporting the association between subthreshold depression and self-care behaviour in adults aged 18 years or over and diagnosed with type 2 diabetes. Electronic databases including MEDLINE, EMBASE, PsycINFO, Emcare and CINAHL will be searched using predefined search terms. Title and abstract, full-text screening and data extraction of identified articles will be done by two reviewers independently. Discrepancies will be resolved by a third author. The methodological quality of the included studies will be assessed using The Joanna Briggs Institute (JBI) risk of bias tools. The review results will be presented in the form of narrative synthesis, and if sufficient studies are available and variability among the studies is low, a random effects meta-analysis will be done to quantify the result.This review will synthesise evidence on the association between subthreshold depression and self-care behaviour in type 2 diabetic adults. The findings will be useful to researchers and policymakers to determine the most effective approach to overall diabetes management. The review will also identify research gaps in the current literature and provide direction for future research in this area of study.PROSPERO CRD42018116373The online version of this article (10.1186/s13643-019-1084-7) contains supplementary material, which is available to authorized users. Diabetes is a complex and progressive metabolic disorder that affects approximately 425 million people worldwide . DepressSubthreshold depression is a less severe form of depression, that occurs when an individual experiences depressive symptoms that do not meet the diagnostic threshold for a major depressive disorder in regard to frequency, severity and/or duration of symptoms , 6. CommAlthough subthreshold depression has less severe symptoms than a major depressive disorder , it causDiabetes is a complex disease that requires regular medical care for its effective management . Self-caFor a patient with depression, self-management can be particularly challenging . A meta-Reviews published to date have largely focused only on major depression and self-care behaviour. Despite the increasing interest in subthreshold depression and its significant impact in both clinical and community population, to the best of our knowledge, the relationship between subthreshold depression and self-care behaviour has not been systematically reviewed. Therefore, the objective of this systematic review is to assess the association between subthreshold depression and self-care behaviour in type 2 diabetic population.This protocol complies with the guideline of the Preferred Reporting Items for Systematic Review and Meta-Analysis Protocols 2015 (PRISMA-P-2015) . The detAny observational studies Studies conducted in adults aged 18 years or over and diagnosed with type 2 diabetesStudies that reported on the association between subthreshold depression and any one’s diabetes self-care activitiesStudies published in EnglishThe following inclusion criteria will be applied to all stages of the review: Title and abstract screening and full-text screening. The inclusion criteria are:Qualitative studies, review articles and non-peer-reviewed (grey) literature will be excluded. Although exclusion of grey literature may limit some of the valuable data, the validity of the non-peer-reviewed literature is difficult to be determined. The self-care behaviours that will be included in the study are healthy eating, being physically active, monitoring of blood glucose, taking medication and risk-reduction behaviours  . There iA systematic search of databases including MEDLINE, EMBASE, PsycINFO, Emcare and CINAHL will be carried out to identify all relevant articles. Search terms related to “type 2 diabetes”, “depression” and “self-care” will be used. The databases will be searched using the various combinations of medical subject headings (MeSH) and keywords. Appropriate Boolean operators , proximity operators  and truncation will be incorporated into the search strategy to cater for the different use of terms. In consultation with a medical librarian experienced in systematic review database searching, a comprehensive MEDLINE (Ovid) search strategy has been developed . Covidence is a software package developed to help in the management of systematic reviews. Two reviewers (MS and FA) will independently screen titles and abstracts according to the eligibility criteria. A third reviewer (AG) will resolve any disagreements between MS and FA. Full texts of potentially relevant articles will then be retrieved and again reviewed by two reviewers. The final decision regarding the inclusion of the studies will be made based on a thorough review of the full articles and joint discussion with the review team. The study selection process and the reason for exclusion will be documented. The outcome of the screening process will be reported in a PRISMA flowchart [Citations identified from different databases will be imported into the reference manager software programme EndNote X9 40). After removing duplicates, citations will be imported into Covidence systematic review software , study design , study setting , sample size, sample size calculation, sampling technique, data source (survey or secondary data), definition of subthreshold depression, measure used , analysis, confounder variables adjusted and key observation of the study. For each study that meets the inclusion criteria, data extraction will be done independently by two reviewers (MS and FA). Any discrepancy between two reviewers during the data extraction process will be resolved through discussion and if necessary, a third (AG) reviewer will be consulted for the final decision. Authors will be contacted through emails to obtain missing or other relevant information if needed.The Joanna Briggs Institute (JBI) Critical Appraisal Checklist tool will be used to assess the quality of studies. The JBI has separate checklists for cross-sectional (8 criteria), case-control (10 criteria) and cohort studies (11 criteria). Ratings will be made for the following criteria: eligibility criteria, study population, sample size, exposure measure, exposure time, exposure level, exposure before the outcome, confounders, outcome measures, follow-up and statistical analysis. Each component will be rated yes, no, unclear or not applicable. The summary of the quality of each study will be given. The studies will not be excluded based on their quality appraisal. Two reviewers (MS and FA) will independently assess the quality of each study. Any disagreement will be resolved by discussion and consensus within the review team.A systematic narrative synthesis of the findings from the included studies will be conducted. Tables and narrative summaries will be used to present the study characteristics and findings of all the included studies. The relationship between subthreshold depression and each self-care behaviour including the overall self-care behaviour will be discussed.χ2) and quantified with the I2 statistics [I2 is the proportion of total variation provided by between-study variation, and I2 values of 25%, 50% and 75% will represent low, moderate and high heterogeneity, respectively [If sufficient studies are available and variability among studies is low, a random effects meta-analysis will be undertaken to compute the pooled estimate. Although the studies may be comparable for pooling the effect sizes, there may be certain variations in effect sizes across the studies due to different populations and study characteristics . So, a ratistics . I2 is tectively . We willSubthreshold depression is a common comorbidity among type 2 diabetic patients. The effects of major depression on different self-care behaviours including physical activity, diet, blood glucose monitoring and foot care have been clearly documented. In this review, we intend to determine the relationship between subthreshold depression and self-care behaviour among type 2 diabetic people. Assessing the association between subthreshold depression and diabetes self-care is a critical step in identifying the most effective approach to overall diabetes management. Identifying an association between subthreshold depression and self-care activities may imply a need to integrate subthreshold depression screening in the treatment of type 2 diabetic patient. The effective management of comorbid subthreshold depression in people with type 2 diabetes could potentially enhance self-care behaviour resulting in better glycaemic control among other benefits.The inclusion of studies only in the English language, the exclusion of grey literature and not undergoing peer review of the search strategy might be a potential limitation of the review. The heterogeneity between studies regarding the terminology used to label and define subthreshold depression and measures used to screen subthreshold depression and self-care behaviour may potentially limit to deriving a conclusion on the association between subthreshold depression and self-care behaviour. The review is expected to identify research gaps in the current literature and provide direction for future research in this area of study.Additional file 1:Preferred Reporting Items for Systematic Review and Meta-Analysis Protocols 2015 (PRISMA-P-2015). (DOCX 33 kb)Additional file 2:MEDLINE search strategy. (DOCX 15 kb)


Suprathermal electrons are routinely generated in high-intensity laser produced plasmas via instabilities driven by non-linear laser-plasma interaction. Their accurate characterization is crucial for the performance of inertial confinement fusion as well as for performing experiments in laboratory astrophysics and in general high-energy-density physics. Here, we present studies of non-thermal atomic states excited by suprathermal electrons in kJ-ns-laser produced plasmas. Highly spatially and spectrally resolved X-ray emission from the laser-deflected part of the warm dense Cu foil visualized the hot electrons. A multi-scale two-dimensional hydrodynamic simulation including non-linear laser-plasma interactions and hot electron propagation has provided an input for ab initio non-thermal atomic simulations. The analysis revealed a significant delay between the maximum of laser pulse and presence of suprathermal electrons. Agreement between spectroscopic signatures and simulations demonstrates that combination of advanced high-resolution X-ray spectroscopy and non-thermal atomic physics offers a promising method to characterize suprathermal electrons inside the solid density matter. Suprathermal electrons in laser-generated plasmas are potentially useful in many plasma environments. Here the authors show the characterization of suprathermal electrons in laser-generated Cu plasma using a high-resolution X-ray spectroscopy combined with hydrodynamic and atomic modeling. Hot electrons can cause degradation in the performance of ICF capsules by fuel preheat and reduced compressibility of the capsule7. In the fast ignition scheme8, laser coupling to fast electrons9 and their subsequent transport11 determine the efficiency of the energy delivery to the ignition region.Hot  electron (HE) production driven by instabilities in laser plasma interaction12 the fuel is ignited from a central hot spot heated by a strong spherically convergent shock. The laser intensities required to launch this shock exceed the threshold of parametric instabilities (such as stimulated Raman scattering (SRS) or two–plasmon decay), which couple a significant fraction of the laser energy to hot electrons14.In the shock ignition scheme17. In spherical geometry experiments, the shock strength was shown to be significantly enhanced by the generation of hot electrons; their total energy reached up to 19.Shock ignition experiments in the planar geometry reported pressures up to 26. Moreover, optimized 28.Hydrodynamic simulations of laser-plasma interactions for pulse durations of the order of 24 whereas 30. The problem of detection of the temporal evolution of hot electrons inside near solid density matter with high-resolution X-ray spectroscopy  X-ray emission spectroscopic data can also be understood as a benchmark for the implementation of non-linear laser-plasma interaction in large-scale hydrodynamic simulations.In this paper we address critical timing issues of hot electron generation and propagation inside near solid density matter via state-of-the-art high-resolution X-ray spectroscopyThe spatially and spectrally resolved X-ray emission from Cu foil irradiated by kJ-ns-laser , which originates from higher charge states. The first emission group S1 corresponds to open M-shell configurations, higher energy emission groups The data in Fig. S1) extends up to about The simultaneously achieved high-spectral and high-spatial resolution allows also a temporal reconstruction of the suprathermal electron production from a time integrated original data set. As can be seen from the X-ray images  compared to the emission of the groups Figure entclass1pt{minimaFigure In order to discus the potential of high-resolution X-ray spectroscopy coupled to non-thermal atomic physics to validate complex large scale hydrodynamic simulations  we consider two spectral simulations with hot electron temporal distributions artificially described by a Gaussian profile with FWHM = 250 ps and peak maxima at either 0 or +400 ps with respect to the laser maximum. The spatial profiles of the S1, the relative contribution of higher charge states in panel b is larger. This analysis demonstrates the potential of these emission groups to evaluate the absolute number of hot electrons and to validate corresponding predictions from large scale hydrodynamic simulations.In order to demonstrate the sensitivity of the present non-thermal atomic physics analysis with respect to the hot electron fraction, Fig. We have demonstrated a spectroscopic approach to measure the suprathermal electron evolution inside dense laser-heated plasma. Hot electrons are characterized via the detailed non-thermal atomic physics modeling of the spatially resolved X–ray emission based on the data obtained from large scale hydrodynamic simulations including non-linear plasma interactions. Our analysis shows very good overall qualitative agreement with the experiment and indicates a time delay of about Concerning temporal evolution of suprathermal electrons  crystal cylindrically bent to a radius of 76 mm. This spectrometer provided simultaneously high-spatial resolution with The Cu K–shell emission was observed by the Vertical geometry Johann Spectrometer (VJS) mentclass2pt{minim36. Hot electron fluxes and temperatures are derived from the laser intensity in plasma calculated with the paraxial complex geometrical optics model coupled to the hydrodynamic code CHIC37 in the two-dimensional axially symmetric geometry. The simulation used the experimentally measured laser pulse energy, temporal and spatial profile. Examples of the spatial distribution of the plasma temperature and density for the times The theoretical model considers simultaneous and concurrent electron generation by the processes of resonant absorption, SRS and two-plasmon decay (TPD)26.The CHIC hydrodynamic simulation provided also the number and mean energy of hot electrons generated by plasma instabilities with temporal and spatial resolution for the use in non-thermal atomic physics40 at each time step. This approach is justified if the parameter 26. For an ion density of The coupling of hydrodynamic simulations to atomic physics has been realized by interpolating the plasma and hot electron parameters from the CHIC non-structured Lagrangian mesh onto a regular Eulerian grid to calculate local quasi–stationary non–Maxwellian population kinetics and synthetic spectra by using the code FLYCHK41. It can be seen that numerous 42. The strong emission of the group The ionization states relevant for emission of the L-K transitions are presented in Fig. entclass1pt{minima26The Thermal emission, i.e., the Figure These regimes are clearly seen in the synthetic spectra, Fig. Production of the 43.This explains the origin of efficient The streak camera was positioned perpendicular to the plasma expansion direction and only used for expansion velocity estimates derived from the plasma emission. Figure 36 requires in the ideal case methods that are independent of laser-plasma simulations. As outlined above, this can be realized with atomic physics  and high-resolution X-ray spectroscopy. Let us therefore discuss some particular critical issues in large scale hydrodynamic simulations and start with Fig. The validation of large scale hydro simulations is a very complex and challenging problem under the conditions where non-linear processes play an important role. Validation of numerous approximations described in ref. The time evolution of the total number of hot electrons as predicted by the hydrodynamic simulations show fine-scale temporal fluctuations (about 10 ps), cf. Fig. 33. However, this is only important for relativistic electrons with a stopping range of The delayed


Fibrillin microfibrils are evolutionarily ancient, structurally complex extracellular polymers found in mammalian elastic tissues where they endow elastic properties, sequester growth factors and mediate cell signalling; thus, knowledge of their structure and organization is essential for a more complete understanding of cell function and tissue morphogenesis. By combining multiple imaging techniques, we visualize three levels of hierarchical organization of fibrillin structure ranging from micro-scale fiber bundles in the ciliary zonule to nano-scale individual microfibrils. Serial block-face scanning electron microscopy imaging suggests that bundles of zonule fibers are bound together by circumferential wrapping fibers, which is mirrored on a shorter-length scale where individual zonule fibers are interwoven by smaller fibers. Electron tomography shows that microfibril directionality varies from highly aligned and parallel, connecting to the basement membrane, to a meshwork at the zonule fiber periphery, and microfibrils within the zonule are connected by short cross-bridges, potentially formed by fibrillin-binding proteins. Three-dimensional reconstructions of negative-stain electron microscopy images of purified microfibrils confirm that fibrillin microfibrils have hollow tubular structures with defined bead and interbead regions, similar to tissue microfibrils imaged in our tomograms. These microfibrils are highly symmetrical, with an outer ring and interwoven core in the bead and four linear prongs, each accommodating a fibrillin dimer, in the interbead region. Together these data show how a single molecular building block is organized into different levels of hierarchy from microfibrils to tissue structures spanning nano- to macro-length scales. Furthermore, the application of these combined imaging approaches has wide applicability to other tissue systems.  •Extracellular matrix fibrillin microfibrils assemble to form ocular ligaments.•Individual beaded fibrillin microfibrils are highly symmetric biological polymers.•Zonule fibers are composed of aligned, organized arrays of fibrillin microfibrils.•Bundles of zonule fibers are wrapped by large fibers providing structural support.•Fibrillin organization shows how a single building block constructs an elastic tissue. Fibrillins form extracellular matrix microfibrils found in many mammalian tissues such as the eye, skin, lung and vasculature where their extensible properties endow long-range elasticity Fibrillin microfibrils also play a key role in tissue homeostasis through their interaction with growth factors such as transforming growth factor-β (TGFβ) and bone morphogenetic proteins Fibrillin-1, a ~ 350-kDa glycoprotein, comprises epidermal growth factor-like (EGF) domains interspersed by TGFβ-binding like (TB) and hybrid domains. It has been suggested that arrays of domains have a linear, rod-like structure, modeled from high-resolution structures of domain pairs and triplets Fibrillin microfibrils form a number of tissue-specific higher-order structures Therefore, we aimed to investigate on the macroscale how the zonule fibers in the ciliary zonule are organized into bundles, on the microscale how the zonule fibers are constructed from fibrillin microfibrils, and on the nanoscale how the fibrillin microfibril is composed of fibrillin molecules. To address each level of hierarchy, different imaging techniques were used. Serial block-face scanning electron microscopy (SBF-SEM) imaging and thick-section electron tomography were used in an integrated manner between macro- and microscales to create three-dimensional (3D) reconstructions of the bovine ciliary zonule to interrogate zonule fiber and microfibril bundle structures, respectively. These approaches were coupled with negative-stain transmission electron microscopy (TEM) with single-particle averaging of tissue-purified microfibrils to generate a nanoscale 3D model of the fibrillin microfibril repeating unit. This approach has allowed the modeling of individual microfibrils and their organization in fibers within the zonule, and which covers length scales across 5 orders of magnitude has the potential to be applied to other hierarchical tissue structures.To determine how fibrillin microfibrils are organized into ciliary zonule fibers, the ciliary zonule was dissected from bovine ocular tissue and the ciliary apparatus . Sections were imaged using an FEI G2 Polara TEM operating at an accelerating voltage of 300 kV. Low-magnification images are shown in SI Fig. 1. Areas of interest were selected, and tilt series were collected from + 65° to − 65° at a magnification of 23,000 ×. Tilt series were aligned and tomograms were reconstructed using weighted back projection in IMOD. A representative tomogram collected on a region close to the basement membrane is shown in To investigate microfibril packing in different regions of the ciliary zonule, including the electron-dense areas at the periphery of zonule fibers observed by SBF-SEM , sub-tomTo determine the 3D structure of the fibrillin microfibril repeat, microfibrils were purified from ciliary zonules and imaged using negative-stain TEM A. MicrofThe class averages and rotational average of the 3D model reveal the distinct asymmetrical fibrillin banding pattern with clearly defined bead, arm and interbead regions C, as desTo overcome potential heterogeneity caused by the flexibility of microfibrils and with the objective of increasing the resolution of the microfibril reconstruction, separate sub-models of specific microfibril regions  were created by refining the initial cylindrical starting model of the full microfibril . Sub-models were created by aligning particles to these specific regions using binary masks. The reconstructions were then projected at 5° increments and cross-correlated against the initial data set using FindEM. All the microfibril regions showed a strong correlation at 180°, suggesting that all regions of fibrillin microfibrils have two-fold symmetry along the microfibril axis and further correlation at 90° and 45° suggesting 4- and 8-fold pseudo-symmetry in some regions of the microfibril A. This iThe model of the bead region has a complex arrangement of fibrillin molecules with two distinct features, a central core and outer ring structure. Using segmentation in UCSF Chimera, the bead can be separated into the bead core region and the ring region for clarity B. If theSeparate refinement of the interbead region revealed a structure with three distinct features D. At theThe 3D reconstructions of the arm and interbead regions show very well-defined structure with a clear symmetric arrangement. Although only 2-fold symmetry was applied during the data processing, four clearly delineated strands can be visualized A. AlthouFibrillin is an evolutionarily ancient elastic molecule that provides long-range elasticity to mammalian elastic tissues and is essential for the extracellular regulation of growth factor signalling. We wanted to understand how a single molecular building block is used to construct elastic tissues with nano- to macroscales of hierarchical structure to provide these unique functional properties. Therefore, to determine how individual fibrillin microfibrils are arranged in higher-order structures, bovine ciliary zonules were imaged using SBF-SEM and electron tomography. We show that parallel zonule fibers are encircled by circumferential wrapping fibers, and although microfibrils are highly aligned in the region close to the basement membrane, microfibril bundles are not parallel to the basement membrane. We show individual microfibrils peeling off from the bundle to run in parallel and make multiple contacts with the basement membrane. Microfibrils also make contacts with each other and binding proteins, and we show that these contacts predominantly occur at the bead region of microfibrils. Our 3D reconstructions of purified microfibrils confirm that microfibrils contain a central cavity and reveal that it is only found in the interbead region, which explains the light/dark appearance of microfibrils in tissue section images. We also show that throughout all regions of the microfibril , there is a pseudo-8-fold symmetric arrangement of fibrillin molecules.Ciliary zonule fibers were roughly cylindrical and consistent with sizes observed previously Using electron tomography, the same tissue blocks were imaged following SBF-SEM. Tomograms revealed that there was a range of microfibril diameters in the zonule with a mean diameter of 11.4 nm, similar to tissue microfibril measurements The microfibrils imaged by electron tomography in the zonule were tubular in appearance, and the bead and arm regions could be identified in some repeats B. This iFlexibility was observed along the axis of the microfibril E, resultExample cbEGF domains and a TB domain were modeled into the interbead region of the microfibril, which could be fitted in an extended but non-linear array. At this resolution, it is not possible to determine the location of specific domains, which means that the precise molecular packing within the microfibril cannot be distinguished yet. However, the models show that the fibrillin molecules take a non-linear path through the microfibril repeat. The bead region has been shown by immunolocalization to contain the N- and C-terminal regions of fibrillin. Furthermore, the initial step in microfibril assembly is thought to be head-to-tail interaction between the N- and C-terminal regions of fibrillin and the recombinant C-terminal half of the fibrillin molecule can assemble to give bead-like structures Here we used SBF-SEM imaging and thick-section electron tomography on the same tissue block to bridge the macro- and micro-scales. The combination of these techniques has broad applicability to samples containing structural information spanning multiple length scales including other ocular tissues, tendons and the vasculature. Furthermore, since microfibrils could be purified from zonules, we coupled these approaches with negative-stain TEM to span 6 orders of magnitude from nanometer to millimeter, enabling the modeling of individual microfibrils in the zonule and their organization into bundles and fibers . These dCiliary zonules were dissected from bovine eyes and prepared for SBF-SEM and electron tomography as previously described www.ccpem.ac.uk). The initial template was an average image of the unaligned stack of particles. An initial 3D model was constructed by creating a cylindrical average model from the sum average of the aligned particle set, using SPIDER, filtered to 20 Å; this was subsequently refined using a projection matching approach as we described for collagen VI microfibrils Fibrillin microfibrils were purified for negative-stain TEM as previously described Supplementary figuresImage 1Serial block-face SEM data set of the ciliary zonule. A movie showing a representative sub-set of the SBF-SEM image stack analyzed in Movie 13D rendering of ciliary zonule fibers. The movie shows 2D slices through the SBF-SEM data set shown in Movie 23D segmentation of an individual ciliary zonule fiber. A movie of a 3D rendered segmented ciliary zonule fiber. The ciliary zonule fiber is shown in blue, and the more electron-dense fiber around the perimeter is shown in orange. A 2D orthogonal slice of the SBF-SEM data is also shown.Movie 3The following are the supplementary data related to this article.