Correlative light-electron microscopy (CLEM) is a powerful technique allowing localisation of specific macromolecules within fluorescence microscopy (FM) images to be mapped onto corresponding high-resolution electron microscopy (EM) images. Existing methods are applicable to limited sample types and are technically challenging. Here we describe novel methods to perform CLEM and immuno-electron microscopy (iEM) on cryostat sections utilising the popular FM embedding solution, optimal cutting temperature (OCT) compound. Utilising these approaches, we have (i) identified the same phagosomes by FM and EM in the retinal pigment epithelium (RPE) of retinal tissue (ii) shown the correct localisation of rhodopsin on photoreceptor outer segment disc like-structures in iPSC derived optic cups and (iii) identified a novel interaction between peroxisomes and melanosomes as well as phagosomes in the RPE. These data show that cryostat sections allow easy characterisation of target macromolecule localisation within tissue samples, thus providing a substantial improvement over many conventional methods that are limited to cultured cells. As OCT embedding is routinely used for FM this provides an easily accessible and robust method for further analysis of existing samples by high resolution EM. The localisation of macromolecules and the spatial mapping of their interactions is key to understanding how cellular signalling is regulated and how it can become dysregulated in disease. The most accessible methodology for subcellular localisation for most researchers employs immunofluorescence microscopy (iFM). Although the effective resolution of fluorescence microscopy has been enhanced by the development of super-resolution methods it is still far below that achievable by the more technically demanding immuno-electron microscopy (iEM). With both methodologies, macromolecules are typically labelled with antibodies conjugated to a fluorophore (for iFM) or gold particle (for iEM). There are multiple advantages and disadvantages of both methodologies. EM allows the location of macromolecules to be visualised in the context of all subcellular organelles, which can be identified on the basis of their ultrastructure . In contrast FM relies on the presence of additional dyes/fluorescent probes to identify subcellular organelles. FM potentially allows a far greater cell/tissue volume to be analysed, since EM relies typically on ≤100nm sections, which must be searched at high magnification to identify 5-15nm diameter gold particles. Sensitivity of labelling tends to be lower for EM and the various embedding and sectioning methods to preserve antigenicity of the specimen involve compromise in cell/tissue preservation and can require highly specialised equipment. An increasing range of dyes and fluorescent probes have been developed for FM that allow the highly sensitive detection of proteins, lipids and protein:protein interactions, but many of these are unsuitable for EM.CLEM makes it possible to take advantage of both methodologies and allows detection of very small quantities of the target macromolecule in relatively large specimen volumes by FM that can then be located by EM, either with or without additional iEM staining –5. ExistTo test the CLEM methodology, we first determined whether the same organelle could be identified by FM and EM in sections of retinal tissue. As part of a daily process that is essential for maintaining retinal health, the distal tips of photoreceptor outer segments (OS) are shed and then phagocytosed by the closely apposed retinal pigment epithelial (RPE) cell layer . The OS in vitro model to study photoreceptor physiology and disease [in vivo equivalent as they have a shorter less developed OS. This could be due to the lack of RPE and other intraocular tissue [To further illustrate the CLEM methodology and include dual FM and iEM labelling, we examined the localisation of rhodopsin in induced pluripotent stem cell (iPSC) derived optic cups. These 3D tissue-like cultures are self-organised structures that develop a laminated organisation with all the major retinal cell types, resembling the human retina, and providing an effective  disease . Even thr tissue , but cour tissue . In healr tissue . The useFinally, we used a non-correlative approach to examine the localisation of peroxisomes in the RPE of retinal tissue. As highly abundant organelles in the RPE, peroxisomes have been implicated in general lipid homeostasis and oxidative degradation of long-chain fatty acids from phagocytosed photoreceptor OS ,16. How The mouse eyes used were existing OCT-embedded samples from five-month-old wild type C57Bl6 untreated mice that had been sacrificed by cervical dislocation at a Schedule 1-approved designated establishment in accordance with Animals (Scientific Procedures) Act 1986 (United Kingdom) and Home Office (United Kingdom) guidance rules, adhering to the Association for Research in Vision and Ophthalmology Statement for the Use of Animals in Ophthalmic and Vision Research.Control BJ inducible pluripotent stem cells (iPSC) were differentiated into photoreceptor optic cups as described . Four opWe used whole eyes from mice sacrificed 1.5 hours (2 eyes) or 5.5 hours (one eye) after light onset or optic cups, all of which had previously been fixed immediately after extraction in 4% paraformaldehyde (PFA) in PBS for 1 hour at room temperature before infusing with 30% sucrose (cyroprotectant) in PBS overnight at 4°C. Samples had then been embedded in OCT compound and frozen using a bath of acetone cooled to -78°C using dry ice. 5–20 um sections of the frozen samples were cut at -20°C with a microtome blade and placed onto slides using a cyrostat. If sections were not used straightaway they were stored at -80°C.Cyrostat sections were permeabilised using either 0.004% digitonin or 0.02% saponin  in 1% BSA in PBS for 30 mins at room temperature followed by incubating in blocking solution consisting of 1% BSA and 0.1% acetylated BSA in PBS for 30 mins at room temperature. Antibody labelling was performed by incubated the sections with antibodies against rhodopsin , laminin-1  and PMP70  in blocking solution overnight at 4°C. Secondary antibodies bound to an Alexa Fluor dye (ThermoFisher Scientific used at 1 in 500), FluoroNanogold (goat anti-rabbit from Nanoprobes 7204 used at 1 in 100) or Nanogold (goat anti-mouse from Nanoprobes 2002/AB_2637031 used at 1 in 200) in addition to fluorescent phalloidin (abcam ab176759 used at 1 in 500) in blocking solution were added to the sections for 2 hrs at room temperature. In between all antibody labelling steps, the sections were washed three times in blocking solution for 30 mins at room temperature on a rocking platform. Sections immunolabelled with a fluorescent secondary antibody were mounted in PBS (instead of mounting media as it is easier to remove the coverslip after FM imaging) and visualised for FM using a Zeiss 700 confocal and high and low magnification images were taken at regions of interest.Cyrostat sections were re-fixed in 2% PFA, 2% glutaraldehyde in 0.1M cacodylate buffer for 1 hr at room temperature. If Nanogold or FluoroNanogold secondary antibodies were used the sections were incubated with a gold enhance solution which was prepared in accordance with manufacturers specifications (Nanoprobes) on ice at 4°C. The sections were incubated in 1% osmium tetroxide, 1.5% potassium ferrocyanide in distilled water for 1 hr in the dark at 4°C. To provide extra contrast to the sections they were incubated in aqueous uranyl acetate for 1hr at room temperature. The sections were dehydrated in increasing concentration of ethanol  and in a mixture of propylene oxide:epon (1:1) for 30 mins at room temperature. All the propylene oxide:epon was carefully removed by applying four changes of epon ever 20 mins at room temperature before embedding in epon overnight at 60°C. 100 nm sections were cut and examined on a JEOL 1010 EM. An outline of the method is shown in Microsoft Image Composite Editor (ICE) was used to generate montages from low magnification FM and EM images and Photoshop was used to overlay the EM on top of FM images. ImageJ was used to quantify the number of melanosomes or phagosomes in contact with peroxisomes when apposing membranes were within 30 nm of each other.The mouse eyecup is a multi-layered tissue up to 0.5mm thick and so its preservation and infiltration with embedding reagents used for conventional EM can be challenging. In this study, we found that 10–50 μm cryostat sections of OCT embedded mouse retinal tissue could be embedded for conventional EM with a high degree of preservation . OrganelFollowing the method outlined in As phagosomes and blood vessels are relatively large compared to a single macromolecule, IF staining of these structures can be mapped onto EM images without the need for immuno-gold labelling. When examining macromolecules that are dispersed throughout a cell or localised to small structures, dual FM and EM immunolabelling may be required. The recent development of secondary antibodies conjugated to both a fluorophore and a nanogold particle allows this, as the same primary antibody molecule can be imaged by both FM and EM. Cryostat sections of optic cups derived from iPSC  were staAs a preliminary investigation of peroxisomes in the RPE, eyes that had been extracted from a mouse at 9.30 am and from a second mouse at 1.30 pm were examined. As peroxisomes are so numerous in the RPE, to examine them in detail CLEM analysis was not required, instead non-correlative IF and iEM was used. Peroxisomes were labelled using an antibody against the 70-kDa peroxisomal membrane protein (PMP70), and IF indicated staining clustered around phagosomes labelled with an anti-rhodopsin antibody . PerformWe present a new method of sample preparation that allows CLEM and iEM analysis of samples that were originally prepared for FM. Using this approach the specimen preservation is almost equivalent to tissue prepared using conventional methods for EM analysis. Therefore, this approach could be extremely valuable to many laboratories, as it can save time and money by minimising sample preparation as well as allowing contemporary EM analysis of old or unique cryostat samples. Additionally, most of the current CLEM methodologies are better suited for examining macromolecules in cultured cells, whereas cryostat sections are ideal for performing CLEM on tissue samples. We found that to obtain reproducibly good EM preservation and antibody labelling of cryostat samples it was important to use good quality cryostat sections, high affinity antibodies, adequate sample washing in between incubations and careful removal of all propylene oxide before embedding  many labelled phagosomes are not present in a 100nm EM section of the same region. Once matched together, the IF gave precise positioning of phagosomes as confirmed by high magnification EM images of the morphologically dense membranous phagosome structures. As rhodopsin staining and markers involved in phagosome maturation in the RPE can be readily identified by IF and further assessed in detail at the high resolution achievable by EM, this approach could be highly beneficial for future studies. These studies include examining phagosome maturation in greater detail in human tissue samples and retinal degenerative disease models to determine how this process can become defective and lead to retinal disease ,30.in vivo. This helps to validate the optic cups as a good model for analysing the molecular regulation of photoreceptor OS biogenesis and renewal, defects in which underlie multiple retinal degenerations [Our knowledge of the molecular regulation of photoreceptor OS biogenesis and renewal is incomplete, partly because of the lack of adequate photoreceptor culture systems. Therefore, the development of optic cups from stem cells potentially represents a major advance, provided that membrane traffic systems leading to OS biogenesis are functional. The FM rhodopsin staining when overlaid onto the differential interference contrast (DIC) and low magnification EM images indicate the most intense immunoreactivity at the outer region of the optic cups. By immuno-labelling with a secondary antibody bound to both a fluorophore and a gold particle to allow iEM, in addition to CLEM, we were able to show that rhodopsin localises to disc like structures in the rudimentary OS of the optic cup photoreceptors, similar to its localisation to OS discs erations –33.Using a non-correlative imaging approach, we have provided potential new insight into peroxisome-organelle interactions in the RPE. FM labelling of peroxisomes indicates that they surround basally situated phagosomes in the RPE, and when examining this positioning more closely by iEM, many of the phagosomes were in contact with at least one peroxisome. Phagosome maturation proceeds in sequential stages as they move through the RPE towards the basal surface where lysosomal fusion takes place and OS discs are lost. As peroxisomes are almost entirely absent from the apical half of the RPE the peroxisome:phagosome interactions must occur at a late stage in the maturation process. Even as a preliminary investigation, more phagosomes interacting with peroxisomes were observed at 1.30pm  compareIn the examples presented here we have used mouse retina and cultured human optic cups, but this method is potentially extendable to multiple tissue and cell samples. Combining FM and EM provides a powerful tool to accurately study macromolecule localisation, and with the advent of significantly improved confocal resolution, the benefits of using CLEM are rapidly advancing. Additionally, recent advances in cryo-EM allow samples to be high-pressure freezing before imaging by FM . This alS1 Fig(A-C) Are low magnification IF images mapped onto retinal cryostat section embedded within a resin EM block. (D-F) higher magnification EM overlay onto IF data with the box in (F) highlighting the region examined in (TIF)Click here for additional data file.S2 Fig(A) Fluorescence microscopy (FM) image of laminin-1 and actin  staining. (B) Light microscopy (LM) image of embedded section in epon resin and C) with FM overlaid on top. (D) high magnification FM image of an area of interest with (E) electron microscopy (EM) image of the same area and (F) an overlay of the FM and EM. (G–F) Higher magnification of the boxed regions highlighted in (D–F) showing blood vessels (asterisks) as visible by the laminin-1 staining in (G & I) and the EM ultrastructure in H). Scale = (A–C) 500um and (D–F) 50 um (G—I) 10 um.(TIF)Click here for additional data file.S3 FigPeroxisomes are almost completely absent between the cell medial and apical surface. The regions of RPE include the basal infoldings (BI) and the apical processes (Ap) and just beyond the RPE basal surface is Bruch’s membrane (Br). Scale = 1um.(TIF)Click here for additional data file.S4 FigOther organelles include melanosomes (Me) and mitochondria (M). Scale = 250nm.(TIF)Click here for additional data file.S5 FigScale = 250 nm.(TIF)Click here for additional data file.


The hallmark manifestations of Cushing’s syndrome (CS) are well known, but hypercoagulability is perhaps least recognized. Patients with CS are at increased risk of both spontaneous and postoperative thromboembolism, with the significant majority of events occurring in the lower extremity and pulmonary venous circulations. We present a case of portal vein thrombosis (PVT) occurring in the setting of newly diagnosed CS due to a left adrenal adenoma. Factor VIII activity was approximately 2.5-fold elevated, a known mechanism by which hypercortisolemia predisposes to venous thrombosis. Acute abdominal pain and fever responded well to unfractionated heparin and parenteral antibiotics, and CS was eventually cured by left adrenalectomy. No thromboembolic events have occurred since surgery. PVT is uncommon and usually occurs as a complication of primary or secondary hepatobiliary malignancies and cirrhosis. To the best of our knowledge, this is just the second reported case of PVT due to CS and the first published in the English language literature. Endogenous CS may be caused by corticotrope adenomas in the pituitary (Cushing’s disease), ectopic corticotropin (ACTH) production by peripheral tumors of neuroendocrine origin , or adrenal adenomas and carcinomas that autonomously overproduce cortisol. The adverse effects of excess cortisol are wide ranging and include centripetal obesity, sarcopenia, insulin resistance, hypertension, immunosuppression, and accelerated bone loss. Endogenous CS patients have a 2-fold higher rate of mortality than unaffected individuals that persists even after successful treatment.2 A subsequent multicenter cohort study documented a 4% preoperative VTE rate for CS patients, and 3.4% of Cushing’s disease patients experienced VTE after transsphenoidal surgery (TSS) despite postoperative thromboprophylaxis with low-molecular-weight heparin. No VTE occurred among control patients undergoing TSS for nonfunctional pituitary adenomas.3An important but less commonly recognized complication of hypercortisolism is hypercoagulability and venous thromboembolism (VTE). CS patients have an increased risk of both spontaneous and postoperative VTE. In a systematic review of 15 patient series, the incidence of spontaneous or postoperative VTE for CS patients was approximately 10-fold higher than the estimated incidence in an age- and gender-matched control population.4; to the best of our knowledge, this is only the second reported case of CS complicated by spontaneous PVT and the first English language case report.We present an unusual case of portal vein thrombosis (PVT) complicating CS caused by an adrenal adenoma. A PubMed search with the terms “portal vein thrombosis” and “Cushing’s syndrome” yielded only one brief French language case report3, 4.00-10.80), mild elevation of alkaline phosphatase level , and hypokalemia . Neither cholescintigraphy nor right upper quadrant abdominal ultrasound showed evidence of cholecystitis, but the left portal vein was found to be occluded by ultrasonography. No sonographic features of cirrhosis  were observed. Left PVT was subsequently confirmed by computed tomography ; deficiencies of protein C, protein S, and antithrombin III; prothrombin G2021A mutation; and activated protein C resistance was unremarkable. Factor VIII activity was significantly elevated at 365% (50% to 149%).PVT due to hypercoagulability caused by a cortisol hypersecreting left adrenal adenoma was diagnosed. Fever was felt to be a sign of pylephlebitis, so the patient was managed acutely with unfractionated heparin and the combination vancomycin, levofloxacin, and metronidazole. Fever and abdominal pain resolved quickly with anticoagulation and antimicrobial therapy. Warfarin was titrated to therapeutic international normalized ratio, and the patient was discharged home to complete a 3-month course of oral anticoagulation. Laparoscopic left adrenalectomy was subsequently performed , with gl5 Several humoral markers of endothelial dysfunction, including endothelin-1,6 homocysteine,7 osteoprotegerin,8 and vascular endothelial growth factor9 are elevated in CS, and endothelial dependent vasodilatation is also measurably impaired.10 Multiple coagulation factors are increased in CS, particularly factor VIII and von Willebrand factor (vWF).11 Glucocorticoid excess also increases plasminogen activator inhibitor type 1 (PAI-1) levels and activity.12 The combination of increased coagulation factors and decreased fibrinolytic capacity predisposes to venous thrombosis. Neither vWF nor PAI-1 was measured in this case, but factor VIII level was significantly elevated (365%). Levels above 160% are associated with a 4-fold increased risk of VTE.11 Some CS patients develop a secondary polycythemia that may cause venous stasis from hyperviscosity, and decreased activity, especially immobilization after surgery, also predisposes to venous stasis and thrombosis.11Increased risk of VTE in CS appears to be due cortisol-induced changes to all 3 components of Virchow’s triad—vascular endothelial injury, hypercoagulable state, and venous stasis.13 Ten of the 27 patients with DVT also experienced fatal or nonfatal pulmonary embolism (PE), and another patient experienced a fatal PE without reported DVT. A recently published review of 13 patient series and over 1300 cases of CS found a similar rate of VTE (8.9%), with almost all patients experiencing lower extremity DVT and/or PE.14 Slightly more cases of VTE occurred after surgery rather than spontaneously.Our patient’s case is unusual because most cases of CS-associated VTE occur in the lower extremities and PVT is rare. In a large series of 307 patients with CS, 29 (9.3%) experienced VTE, and the vast majority of patients  were diagnosed with deep vein thrombosis (DVT).15 Secondary hepatobiliary malignancy, cirrhosis, primary hepatobiliary malignancy, abdominal infections or inflammatory conditions, and myeloproliferative disorders were the major etiologies of PVT in descending order of frequency and collectively accounted for 86% of PVT cases. No cases of PVT associated with CS were reported in the Swedish autopsy series, and PubMed searches linking the terms “Cushing’s syndrome,” “hypercortisolemia,” “hypercortisolism,” “cortisol,” and “portal vein thrombosis” yielded only the single French language case report from Tunisia previously noted. There appear to be at least 2 key reasons why CS is less likely to cause thrombosis in the portal venous circulation than the lower extremity venous circulation: (1) hypercortisolemia does not cause the thrombophilia states—factor V Leiden mutation, protein C deficiency, protein S deficiency, antithrombin deficiency, and antiphospholipid antibodies—that are most commonly associated with PVT16 and (2) blood flow rate through the portal venous circulation measured by duplex ultrasonography (DUS)17 is 2- to 20-fold higher than DUS measured lower extremity venous blood flow depending on the vein evaluated.18An autopsy series from Malmö, Sweden, with nearly 24 000 patients found a 1% cumulative incidence of PVT.13 compared the rate of VTE in 75 patients who did not receive postoperative anticoagulation (group 1) to 232 patients who received unfractionated heparin for at least 2 weeks followed by warfarin for at least 4 months (group 2). VTE rate was reduced from 20% in group 1 to 6% in group 2, and survival analysis demonstrated a significantly lower rate of morbidity and mortality from thromboembolic events in group 2. Recently, Barbot and colleagues19 compared 2 groups of Cushing’s disease patients undergoing TSS; group A received fractionated heparin from postoperative day 1 to discharge or a total of 14 days plus glucocorticoid replacement, and group B was managed with 30 days of fractionated heparin, compression stockings, early ambulation, and omission of glucocorticoids. There were no VTE events in group B compared to 3 in group A, though the difference was not quite statistically significant. The hypercoagulable state of CS may persist well beyond the time that cortisol levels return to normal. Markers of hemostasis and fibrinolysis failed to change significantly after 80 days in a group of patients with Cushing’s disease managed medically to biochemical remission of hypercortisolemia as indicated by normalization of 24-hour urine-free cortisol.20 Though PVT was our patient’s initial thromboembolic event, she was managed with a 6-month course of warfarin due to occurrence of VTE in the setting of CS.Perioperative thromboprophylaxis appears to reduce the risk of postoperative VTE for CS patients, though relatively little is published on the topic and there are no clear recommendations regarding the type or duration of anticoagulation. Boscaro and colleaguesElevated serum cortisol induces changes in hemostatic and fibrinolytic pathways that create a prothombotic state, and this manifests clinically as increased risks of spontaneous and postoperative VTE. Lower extremity DVT and PE account for the significant majority of thrombotic events in CS patients. We present a case of CS due to an adrenal adenoma as the etiology of PVT, a rare clinical event that mostly occurs as a complication of cirrhosis or hepatobiliary malignancy. This appears to be only the second reported case of PVT complicating CS and the first in the English language peer-reviewed literature. The patient responded well to initial treatment with unfractionated heparin and antimicrobial therapy followed by laparoscopic adrenalectomy and a 6-month course of warfarin. Prolonged anticoagulation for patients with CS and VTE appears advisable given potential persistence of a hypercoagulable state beyond initial resolution of hypercortisolemia. Though lower extremity DVT and PE are the most likely complications of hypercoagulability in CS, this case illustrates that CS also has the potential to cause PVT.


A bioanalytical method using inductively-coupled plasma-mass spectrometry to measure endogenous levels of strontium in human serum was developed and validated.This article details the experimental procedures used for the method development and validation thus demonstrating the application of the inductively-coupled plasma-mass spectrometry method for quantification of strontium in human serum samples. The assay was validated for specificity, linearity, accuracy, precision, recovery and stability. Significant endogenous levels of strontium are present in human serum samples ranging from 19 to 96 ng/ml with a mean of 34.6 ± 15.2 ng/ml (SD).88Sr) and is suitable for routine clinical testing of strontium in human serum samples.Calibration procedures and sample pretreatment were simplified for high throughput analysis. The validation demonstrates that the method was sensitive, selective for quantification of strontium ( Lay abstract: The study of strontium in human serum is useful to understand its biological function. Strontium is found naturally in the environment including drinking water and food. This alkali earth metal plays a biological role in bone and has been shown to have therapeutic properties for osteoporosis and to suppress chemically induced sensory irritation and inflammation. A technique called inductively coupled plasma mass spectrometry was used for its superior ability to measure low levels of strontium in biological matrices. Strontium was measured in normal human samples to demonstrate the method utility and correlate with age, gender and race. Accuracy of back calculated concentrations for calibration standards was within 10% of nominal value .A calibration standard curve is the relationship between the instrument response  and the known concentration of the analyte (in ng/ml). A calibration curve is used to demonstrate linearity, the ability of a method to elicit test results proportional to the concentration of the analytes within a given range. Since human serum contains endogenous strontium, calibration standards were prepared in a diluent/internal standard solution containing 1% HNOPrecision is the repeatability or closeness of agreements between a series of measurements for a statistically significant number of samples (multiple sampling of the same homogenous sample). The accuracy of an analytical method describes the closeness of agreement between the test results obtained by a method to the conventional true value or an accepted reference value.To determine accuracy and method precision, five sets of QC samples over three concentration levels  covering the specified range were prepared and analyzed. Accuracy and method precision were calculated for five injections of LQC (unspiked), MQC (80 ng/ml spike) and HQC (150 ng/ml spike) samples. Accuracy and method precision results obtained are presented in 3 to achieve a result (concentration) within the calibration range. To accommodate analyzing samples originally 2× above the ULOQ (250 ng/ml), recovery for diluted serum samples was validated at approximately 100 ng/ml. A 500 ng/ml spiked serum sample was prepared, fivefold diluted with a 1% HNO3 and analyzed according to the method (n = 3). The mean accuracy of diluted samples (n = 3) was within 2% of undiluted values and relative standard deviations (%RSDs) were within 0.1% (n = 3). These dilution linearity (fivefold) results obtained also indicated that there were no significant matrix effects.During the analysis of samples, if any sample result was higher than the Upper Limit of Quantitation (ULOQ) range, the sample had to be diluted with 1% HNORepeatability was determined by preparing multiple aliquots from a single homogenous lot of serum. Intraday precision is a measure of within run repeatability , determined by injection of six replicates at each QC level . Precision results obtained are presented in Interday precision is a measure of the repeatability of the concentration measurement from day-to-day and from assay-to-assay. Interday precision was determined by injection of three replicates at each QC level on three separate days . The day 2 analysis was performed 43 h after day 1 (t = 0) and the day 3 analysis was performed 67 h after day 1. Aliquots of LQC (unspiked), MQC (80 ng/ml spike) and HQC (150 ng/ml spike) samples were stored at -20°C, 3 aliquots of each sample were prepared  prior to analysis and analyzed according to the method (n = 3) on three separate days against a freshly prepared standard curve.Stability of strontium in human serum was evaluated at ambient temperature storage, frozen storage and through three freeze–thaw cycles. Stability of the prepared standards and samples  was determined at ambient temperature.Postpreparative stability was performed to define the period of time in which the prepared samples  are stable and representative of freshly prepared samples. Stability was evaluated by comparing the results from stored samples (for ≥24 h and ∼3 days at ambient temperature with caps closed) versus freshly prepared samples. Two replicates of standards  and nine replicates of samples (at each QC level) were prepared . Duplicate injections of each standard were made at 43 h and at approximately 3 days Table  against Stability of the analyte in the matrix was evaluated at ambient temperature (bench top stability) and storage stability at ≤-20°C was performed. Short-term stability of the samples was evaluated for 43 h at ambient temperature. Long-term sample stability was assessed by storing samples at ≤-20°C for approximately 1 week and 2 months. Aliquots of LQC (unspiked), MQC (80 ng/ml spike) and HQC (150 ng/ml spike) samples without IS addition were stored at ambient temperature and at ≤-20°C. Prior to analyses, samples were diluted with IS and analyzed according to the method (n = 3) against a freshly prepared standard curve. Prepreparative stability results obtained are presented in Stability of the analyte in the matrix was evaluated through three freeze–thaw cycles by freezing samples at ≤-80°C. Aliquots of LQC (unspiked), MQC (80 ng/ml spike) and HQC (150 ng/ml spike) samples without IS addition were stored at ≤-80°C. After three freeze–thaw cycles, samples were diluted with IS and analyzed according to the method (n = 3) against a freshly prepared standard curve. The mean accuracy (% of t = 0) after three freeze thaw cycles were within 5% of expected values .To verify the validity of the calibration curve and to monitor system performance throughout the analytical run, one set of QCs were injected once in every 20 study samples. For QC accuracy calculations of each assay, the measured low QC value (average of duplicate injections of low QCs before the injection of study samples) was used as the expected low QC concentration for the assay. For all analyses, correlation coefficients (R) of calibration curves were equal to or higher than 0.9999 and accuracy of bracketing QCs ranged from 90 to 105% of each nominal concentration.Analytical results of endogenous strontium levels in 24 serum samples (six samples from Bioreclamation) prepared from individual human subjects are presented in 88Sr) in human serum. The available stability data indicate that postprocessed serum samples  are stable at ambient temperature for 3 days. Preprocessed serum samples (undiluted) are stable for at least three freeze–thaw cycles, stable for 2 days (at ambient temperature) and 2 months at ≤-20°C. The validation demonstrated that this method can be used in support of routine clinical testing of strontium (88Sr) in human serum samples. Consideration of spectral and nonspectral interferences is essential to determine elements in biological matrices. A number of techniques can be employed to eliminate interferences such as calibration procedures, internal standardization, sample dilution, mathematical correction equation, chemical separation and matrix modification [88) are calcium dimers , calcium argides (48Ca40Ar), iron oxides (56Fe16O2), zinc oxides (70Zn18O) germanium oxides (72Ge16O) and doubly charged species of 176Yb, 176Lu, 176Hf at significantly higher levels [88 is free from interferences at this low levels in serum and is recommended for routine clinical analyses. When analyzing Sr88 in high calcium, iron and zinc containing samples, use of interference correction equations combined with optimized instrumental parameters such as sampling depth, ion lens settings, nebulizer and collision gas flow is recommended. However, if the intensity of analyte signal is extremely low and intensity of interfering ions is significantly high, use of mathematical correction equation is not suitable [Based on the data presented, the bioanalytical ICP-MS method developed is accurate, sensitive, precise and selective for quantification of strontium  reported in this study. In comparison of both experimental approaches, following differences appear to be particularly interesting. Zhang et al. used 7:93 (V/V) ratio of hydrogen/helium mixed gas as a collision gas at 3.75 ml/min. This current work demonstrates that helium gas mode alone at lower flow rate (1 ml/min) is sufficient for 88Sr analysis in serum. Analysis time is shorter in single gas mode analysis when compared with multiple mode analysis as longer stabilization delays are not necessary. A calibration curve ranging from 30 to 15,000 ng/ml (LLOQ set to 30 ng/ml) used in Zhang et al. method is applicable to quantitate strontium over a wide range of concentrations in clinical study samples. But to measure trace levels of strontium reliably, a high sensitive assay is required. Lower limit of quantitation established at 10 ng/ml in the present method is accurate enough to measure trace levels of strontium in serum and is reproducible with acceptable signal to noise ratio. On the other hand, current method was validated to evaluate dilution linearity, to dilute and analyze test samples with strontium levels exceeding the ULOQ. We conclude that our approach offers advantages from analytical and economical point of view for high throughput analysis. Signal to noise ratio at LLOQ can be improved significantly by reducing background contribution from the system using high purity reagents, decontaminated containers and clean operating conditions of laboratory. Limits of detection can be improved by further optimization of method, but achieving quantitation limits less than 10 ng/ml may not be important for this study as it is outside the physiologically relevant range. With minor optimization of the experimental conditions, demonstrated ICP-MS method can be tailored to detect and quantify other physiological elements present in serum at lower concentrations such as rubidium, boron, copper, zinc and aluminum. To apply the current method for routine multielemental analyses, fine tuning of instrument to obtain optimum signal intensity for intended mass range is needed. Spectral interferences associated with either the plasma, or matrix components of sample/solvent have to be compensated. Using element specific internal standards  across entire mass range of analytes is essential to minimize matrix effects. However, method detection limits will be compromised as obviously single-element monitoring yields better detection limits than multielemental analyses.Statistically similar type of experimental results were obtained by Zhang et al.  in a recet al. [A mean value of 34.6 ± 15.2 ng/ml (n = 24) found in this study is in close agreement, but slightly lower than the previously published mean value of 49.2 ± 172 ng/ml (n = 110) by Forrer et al. . The obset al. . StatistProposed methodology can be enhanced for simultaneous multielemental analyses in serum by further optimization of experimental conditions and data collection parameters. High concentrations of inorganic constitutes in clinical samples trigger matrix and spectral interference problems, hence achieving best detection limits for physiological elements  which, at ultratrace levels, can be challenging. This demands a highly sensitive, accurate assay capable of resolving matrix and spectral interferences for high throughput multielemental analyses to support bioanalytical studies. To the best of our knowledge no study has been done to determine racial, gender and geographical variation of strontium levels in human serum. The current study is limited by sample size and endogenous strontium levels determined are insufficient for statistical interpretation. Current method can be employed for further extensive studies to gain insight into strontium distribution and to establish reference ranges respective to age, race and gender.The study of strontium in human serum is useful to understand its biological function. A systematic validation of a sensitive assay capable of measuring strontium in serum at low ng/ml concentrations has not been reported. This report details bioanalytical ICP-MS method validation, developed to measure endogenous and therapeutic levels of strontium in human serum.Simplified sample preparation and calibration procedure is well suited for high throughput clinical applications related to study of strontium in human serum. Endogenous levels of strontium present in human serum samples ranged from 19 to 96 ng/ml.The method is sensitive, accurate, precise and reproducible. The study results indicate that method was successfully validated and applied for analysis of strontium in human serum samples. Results from the current study and previous studies indicate that endogenous levels varied significantly between individuals. Observed variability could possibly be explained by difference in age, sex and other geographical differences.


Single-cell heterogeneity confounds efforts to understand how a population of cells organizes into cellular networks that underlie tissue-level function. This complexity is prominent in the mammalian suprachiasmatic nucleus (SCN). Here, individual neurons exhibit a remarkable amount of asynchronous behavior and transcriptional heterogeneity. However, SCN neurons are able to generate precisely coordinated synaptic and molecular outputs that synchronize the body to a common circadian cycle by organizing into cellular networks. To understand this emergent cellular network property, it is important to reconcile single-neuron heterogeneity with network organization. In light of recent studies suggesting that transcriptionally heterogeneous cells organize into distinct cellular phenotypes, we characterized the transcriptional, spatial, and functional organization of 352 SCN neurons from mice experiencing phase-shifts in their circadian cycle. Using the community structure detection method and multivariate analytical techniques, we identified previously undescribed neuronal phenotypes that are likely to participate in regulatory networks with known SCN cell types. Based on the newly discovered neuronal phenotypes, we developed a data-driven neuronal network structure in which multiple cell types interact through known synaptic and paracrine signaling mechanisms. These results provide a basis from which to interpret the functional variability of SCN neurons and describe methodologies toward understanding how a population of heterogeneous single cells organizes into cellular networks that underlie tissue-level function. The principal biological clock in mammals resides in the suprachiasmatic nucleus (SCN) of the hypothalamus and synchronizes circadian oscillations and behavioral rhythms throughout the body. Synchronization, which enables coordinated anticipation of the 24 h daily light/dark cycle, results from coherent, rhythmic output signals generated and adjusted by the SCN in response to photic inputs. This oscillatory behavior of the SCN arises from neurons, which exhibit autonomous circadian rhythms, interacting with one another via synaptic and paracrine signaling mechanisms to form cellular interaction networks that synchronize the oscillatory behavior of individual SCN neurons  staining of neurons expressing vasoactive intestinal polypeptide (VIP), or arginine vasopressin (AVP) have shown that these neurons mainly localize within the ventrolateral (core) or dorsomedial (shell) regions of the SCN, respectively  exhibit circadian behavior. In the absence of synaptic signaling, these behaviors can become unstable, resulting in some neurons losing their intrinsic rhythmicity while others can spontaneously gain rhythmicity  were housed within 12 h light, 12 h dark cycles and given free access to food and water. Warm white fluorescent bulbs (150 lux) were used for the light cycle. After 10 days of light/dark cycle entrainment, the lights were switched off. On the second day of the constant dark period, animals were given a 1 h light exposure (150 lux of white light) at Zeitgeber time (ZT) 14, 2 h into their subjective dark period, and sacrificed 1 h later at ZT 15 (light-pulsed). SCNs were also collected from non-light-pulsed animals at ZT 15 (dark adapted). Animals were euthanized by carbon dioxide asphyxiation in dim red light, and brains were extracted in light. Hypothalamic tissue blocks were dissected and embedded in Optimal Cutting Temperature (OCT) embedding medium and frozen on dry ice. OCT-embedded tissue samples were stored at −80 C prior to sectioning and laser capture microdissection. All protocols were approved by the TJU Institutional Animal Care and Use Committee.In order to minimize RNA degradation and maintain quality of the tissue samples for laser capture microdissection we developed a rapid immunofluorescent staining protocol. OCT-embedded hypothalamus blocks were sectioned in a cryostat into 10 um thick sections and thaw-mounted onto glass slides. Sections were fixed in 75% ethanol for 30 s, then blocked and permeabilized with PBS containing 2% BSA (Sigma-Aldrich) for 30 s. Afterwards, sections were incubated with the primary antibody anti-NeuN 1:25 (Millipore®). PBS-washed slides were incubated in the dark for 3 min at room temperature with the secondary antibody Alexa-488 anti-mouse 1:50 diluted in PBS containing 2% BSA. The slides, again washed with PBS, subsequently underwent a standard dehydration process , rinsed briefly in Xylenes (Sigma-Aldrich) for 1 min, and then transferred into another bath of fresh xylenes for 5 min to further remove any trace of ethanol. Finally, the slides were air-dried for 5 min prior to laser capture microdissection.We collected 352 single SCN neurons from the light-perturbed or dark-adapted mice using laser capture microdissection . All cDNA samples underwent 20 cycles.©) according to the manufacturer's instructions. The same primer set used in the pre-amplification process was the basis for the probe-based quantitative PCR. The 96.96 dynamic gene expression arrays were used. qPCR was based on the Universal Probe Library . A detailed list of probe and primer sets is included in the supplemental .The expression level of 96 genes was measured using a high-throughput quantitative PCR platform  selected by using previously developed gene-stability evaluations across measured samples  resulting in a −ΔΔCt value that allowed the relative gene expression values to be compared across both treatment groups. Modified z-values were then calculated by means of dividing the −ΔΔCt the standard deviation within a gene assay across all single-cell samples, values that were used for data visualization using heat maps. Reference the Supplemental Information for additional information and quality control assessment of qRT-PCR data.Expression levels were normalized by using the −ΔΔCa(i) represent the average dissimilarity of member i relative to all other members within its designated cluster, the lower the value of a(i), the better the cluster assignment. Average dissimilarity values of member i relative to all other clusters are then calculated, from which the lowest average dissimilarity value is chosen as b(i). Using the values a(i) and b(i), we then calculate the silhouette score s(i) as follows:This method quantifies the consistency across members within a designated cluster. A silhouette score is calculated for each member within a cluster and then an average score is used to represent how similar members are within a cluster. Let s(i) ≤ 1From the above equation, silhouette scores fall within a range of −1 ≤ s(i) values within a group. Similar to an individual score, an average score approaching 1 indicates a well-defined cluster, –1 indicates an ill-defined cluster, and 0 represents a neutral clustering of members. Silhouette scores were determined using the silhouette function provided in the R package “cluster”  or kept in darkness for 2 days and then exposed to a light-pulse (LP) at a clock time corresponding to 2 h after lights-out of the previous 12 h light-dark cycle . Brains were collected after 1 h of light exposure in the LP group and at the corresponding clock time in the DD group , which measures gene expression over five orders of magnitude with minimal technical , which codes for the neuropeptide PACAP  occurred in approximately 25% of neurons sampled from DD mice  each gene had to each of the first three principal components to determine which genes had the largest influence on the distribution of transcriptional states of LP and DD neurons along these principal components. Genes having the largest influences  along the first three principal components included light-induced genes such as Vip, Avp, and Adcyap1  indicated that a large amount of variation within the LP neuronal dataset remained  or negative expression (−ΔΔCt ≤ median gene expression), yielded eight clusters  and rec1 Figure .Vip+ LP neurons, Vip shared correlations with only three other genes , were aVip, Per1, and Per2 expression, suggesting that these neurons responded directly to photic inputs from the RHT . Analysis of the spatial organization of these neurons revealed that they were distributed throughout the SCN  mirrored both expression behavior and ventral localization of Group 1 neurons. However, differences in Slc12a7 and Grin2m expression distinguished these neurons from those of Groups 1 and 2. Likewise, another subset of Group 5 neurons expressed increased levels of Adcyap1r1 and decreased levels of Vip and Avp, similar to Group 4 neurons. However, decreased expression of Pcks1n differentiated these sets of neurons. It is possible that Group 5 neurons may represent functional variances of neurons within Groups 1–4 and add functional robustness , which would appear to support current neurochemical criteria used to describe SCN neurons. However, the current criteria does not fully account for the underlying transcriptional states of these neurons, as evidenced by the presence of Group 5 neurons that express multiple neuropeptides and exhibit nuanced gene expression behavior. Moreover, a correlational analysis of the genes within each transcription module within Groups 1–4 revealed that of the five genes having the highest number of correlations (Pearson correlation coefficient ≥0.5), none were neuropeptide genes  paracrine source , ii) paracrine target , and iii) autocrine signaling . The regime where low peptide and receptor expression occurred were not considered to play a dominant signaling role within this defined signaling scheme and were therefore ignored  and PROK2 receptor, Prokr2, pairing  , assuming that Group 4 neurons co-express Grp, upregulated expression of gastrin-releasing peptide receptor (Grpr) in Groups 2 and 3 suggests that additional interactions occur between these groups and Group 4, further supporting its integrating role in the SCN.Multiple interactions involving Group 4 also hinted at the interconnected nature of the neuronal network within the SCN. A plausible interaction between Group 4 and 3, mediated by the PACAP–ADCYAP1R1 pairing Figure , was supBy viewing plausible neuronal interactions in this manner, we can develop a more comprehensive neuron-interaction network that builds upon the established input-oscillator-output system. In addition to recapitulating known interactions between VIP+ and AVP+ neurons  and group 2 (Avp+) LP neurons, suggests that the photic input forces a constrained transcriptional response. A diverse array of inputs, including non-hypothalamic neuronal innervations and paracrine signaling neuropeptides  that begin to reconcile the heterogeneous behavior observed in SCN neurons in dark-adapted and light-pulsed conditions. The absence of any clear anatomical organization in the DD neurons, relative to the structured anatomical organization between group 1 (Vip+ neurons and Group 2–Avp+ neurons), but also revealed additional phenotypes that have not been described previously. While there is an extensive amount of literature detailing the various excitatory and inhibitory signaling mechanisms affecting the SCN, we have focused our single-cell transcriptomic analysis on the neuropeptides and paracrine signaling mechanisms deemed to regulate the circadian clock. Within the context of this paracrine signaling network  the spatial organization of a subset of Avp+ Group 2 neurons, ii) the co-expression of Vip and Avp, and iii) the endogenous expression of Adcyap1 in neurons located throughout the SCN. One possible explanation for the larger than expected spatial distribution of Avp+ Group 2 neurons may be due to differences in the rhythmicity between mRNA and peptide expression . These neuronal circuits undergo distributed rearrangements throughout life yet are able to maintain stable behavior within an environment of continual perturbations (Maffei and Fontanini, Our results show distinct functional phenotypes that exist outside the traditional neurochemical definitions of the SCN. Even though these neurochemical criteria are useful and conveniently describe key aspects of SCN function, they fall short of fully capturing the complexity and diversity of the neuronal components driving SCN function. Although our findings are limited to specific times chosen for this study, our approach provides a unique perspective of SCN functional networks that provide plausible explanations as to how these neuronal phenotypes and neuron-neuron interactions organize under dark-adapted and phase-shifting behavior.t and −ΔΔCt values of samples passing quality control are included as supplemental text files (Table Both raw Ces Table  and S2.JP performed analysis, figure generation, and was a main contributor to the writing of the manuscript. HZ conducted experiments and guided sample collection, performed analysis, and contributed to writing and figure design. SO contributed to analysis and writing. BO was involved in initial analysis and editing. DW, JS, and RV designed the study and were involved in analysis, figure design, and editing. JP and HZ contributed equally to the study. All authors discussed the results and commented on the manuscript.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.


Increased circulating levels of prolactin have been associated with increased risk of both in situ and invasive breast cancer. We investigated whether or not physical activity had a dose–response effect in lowering plasma levels of prolactin in postmenopausal women.Four hundred previously inactive but healthy postmenopausal women aged 50–74 years of age were randomized to 150 or 300 min per week of aerobic physical activity in a year-long intervention. Prolactin was measured from fasting samples with a custom-plex multiplex assay.A high compared to moderate volume of physical activity did not reduce plasma prolactin levels in intention-to-treat (Treatment Effect Ratio (TER) 1.00, 95% Confidence Interval (CI) 0.95 – 1.06) or per-protocol analyses .It is unlikely that changes in prolactin levels mediate the reduced risk of breast cancer development in post-menopausal women associated with increased levels of physical activity.NCT01435005.clinicaltrials.gov identifier:  The primary hypothesized mechanisms underlying the associations between physical activity and reduced breast cancer include reductions in adiposity, sex hormone levels, insulin resistance and chronic inflammation . These pProlactin is a luteotropic peptide hormone involved in regular lactation which is produced by the anterior pituitary gland. Increased circulating levels of prolactin have been associated with increased risk of both in situ and invasive breast cancer . Two preThe design of the BETA study has been previously described in detail . Briefly2 max was estimated from a modified Balke treadmill test [Predicted VOill test  using thill test . Body coa priori variables including baseline body mass index (BMI (weight (kg)/height (m2)), estimated physical fitness (VO2max), and total percent body fat.We considered the comparison of means of 12-month outcomes  for the prolactin outcomes. Standard deviation was estimated from previous reports . A samplThe study protocol was approved by the Alberta Cancer Research Ethics Committee and the Conjoint Health Research Ethics Board of the University of Calgary and the Health Research Ethics Board of the University of Alberta. All participants provided written informed consent.p = 0.89). Overall, we did not observe statistically significant effects for increasing quartiles of physical activity and change in level of prolactin in the n = 384 women included in the analyses .The distribution of the study participants’ baseline characteristics was similar in the two trial arms with no meaningful differences between arms . Baselin2max) and total body fat, no treatment effect ratios in any of the subgroups were significantly different from 1.0  or per-protocol analyses (TER 1.02, 95% CI 0.93 – 1.13; Table Overall, a higher volume of exercise compared to a standard volume did not reduce prolactin levels in BETA. Moreover, the effects did not vary according to exercise adherence or baseline fitness levels, BMI, and total body fat.2 max by >5% had a statistically significant reduction in prolactin levels.This exercise intervention trial is the first study to examine the effects of a high versus moderate volume of MVPA aerobic exercise on prolactin levels in postmenopausal women. Previous studies comparing 12 months of moderate-intensity aerobic exercise to no exercise have reported null effects on prolactin levels , 4. SimiOur analyses were motivated by the strong animal and in vitro data supporting an important role of prolactin in breast carcinogenesis  and epidIn conclusion, the results from the BETA study suggest that 300 min per week of moderate-intensity aerobic exercise does not reduce prolactin levels more than 150 min per week in a year-long intervention in postmenopausal women. It is unlikely that changes in prolactin levels mediate the reduced risk of breast cancer development in post-menopausal women associated with increased physical activity at any level.


This work provides a comprehensive CpG methylation landscape of the different layers of the human eye that unveils the gene networks associated with their biological functions and how these are disrupted in common visual disorders. Herein, we firstly determined the role of CpG methylation in the regulation of ocular tissue-specification and described hypermethylation of retinal transcription factors  in a tissue-dependent manner. Second, we have characterized the DNA methylome of visual disorders linked to internal and external environmental factors. Main conclusions allow certifying that crucial pathways related to Wnt-MAPK signaling pathways or neuroinflammation are epigenetically controlled in the fibrotic disorders involved in retinal detachment, but results also reinforced the contribution of neurovascularization  in diabetic retinopathy. Finally, we had studied the methylome in the most frequent intraocular tumors in adults and children . We observed that hypermethylation of tumor suppressor genes is a frequent event in ocular tumors, but also unmethylation is associated with tumorogenesis. Interestingly, unmethylation of the proto-oncogen RAB31 was a predictor of metastasis risk in uveal melanoma. Loss of methylation of the oncogenic mir-17-92 cluster was detected in primary tissues but also in blood from patients. The Origin of Species” that the human eye was an organ of extreme perfection that pushed to the limit its theory about natural selection. In his own words: “Reason tells me, that if numerous gradations from a simple and imperfect eye to one complex and perfect can be shown to exist, each grade being useful to its possessor, as is certainly the case; if further, the eye ever varies and the variations be inherited, as is likewise certainly the case; and if such variations should be useful to any animal under changing conditions of life, then the difficulty of believing that a perfect and complex eye could be formed by natural selection, though insuperable by our imagination, should not be considered as subversive of the theory”. Its evolution represents a graduation from the simplest form (an optic nerve merely coated with pigment) to the most complex . The layers of the eye, especially the retina, are highly complex and have frequently served as a model of central nervous system development1. Development and maintenance of the complexity of the human eye proceed by the fine-tuned regulation of gene expression in space and time. Apart from the regulation by the dynamic DNA-binding transcription factor, epigenetic factors may also be central to controlling changes in gene expression in the development of the eye4. Indeed, the role of epigenetics during eye development has been described in several experimental systems: reduction in the expression levels of histone deacetylase HDAC4, in the histone methyltransferase G9a, and in the miRNA processing enzyme Dicer during normal retinal development lead to apoptosis of retinal cells7. A dynamic expression pattern of DNA methyltransferase, Dnmt1 and Dnmt3, from immature to adult retina has also been reported8.Charles Darwin wrote in “10. However, epigenetic studies are still in their infancy, and most of the research effort so far has focused on the role of epigenetics in regulating genes involved in retinal cell fate during embryogenesis, and the epigenetic alterations associated with tumoral disorders4. To date, macular degeneration is the only non-tumoral visual disorder in which CpG methylation has been explored11.Aging-related eye diseases have become a priority problem in public health services due to their increasing prevalence in the general population and the seriousness of their impact on the quality of life of patients. For this reason, in recent years increased attention has been paid to the molecular biology of ocular diseases, especially the genetic basis of these disordersversus pathological), we generated the largest CpG methylation map of embryonic and mature eye layers and their associated ocular diseases created so far. These data could be integrated to provide a comprehensive understanding of the epigenetic, genetic and environmental factors underlying visual disorders.In this study we assessed the epigenetic characterization of visual disorders from a broader perspective, investigating the involvement of genome-wide CpG methylation in ocular diseases associated with environmental factors, inflammation and aging , and in ocular tumors . Characterizing the normal epigenetic patterns that govern the maintenance of tissue-specificity of the adult mature eye is as important as profiling alterations in pathologies. Considering both approaches  iris, (ii) choroid/RPE/ciliary body, (iii) sclera and (iv) retina. The substantial involvement of CpG methylation in tissue commitment was reflected by the clear segregation of the ocular layers in an unsupervised cluster exclusively using the methylation signals of the CpGs contained in the arrays     , the SIX homeobox family  and retinal homeobox transcription factors (RAX)  Fig. . By contAX) Fig. . To compAX) Fig. . It is iAX) Fig. . In contAX) Fig. . To unraAX) Fig.  we quantAX) Fig. .Figure 2https://sph.uth.edu/RetNet/home.html). Indeed, gene set enrichment analysis (GSEA) revealed a statistically significant correlation (p < 0.001) between the hypomethylated ret-CpGs and the genes associated with retinal diseases  neuroretina from healthy donors (n = 8), (2) retina from background non-proliferative diabetic retinopathy (NPDR) with cotton-wool spots, microaneurysms but no retinal detachment (n = 8) and (3) fibrovascular membranes (FVMs) obtained from vitreoretinal surgery in patients with proliferative diabetic retinopathy (PDR) (n = 9) Fig. . Unsuper18, whereas FVM membranes are characterized by neovascularization through the presence of vascular endothelial cells19. The selective alterations involved in early vascular changes in diabetic retinopathy patients (but not in PVR) could facilitate the design of novel therapeutic approaches for preventing FVM formation in proliferative diabetic retinopathy patients. For this reason, we decided to study the CpG methylation differences between FVM and PVR membranes. Only 293 CpGs were found to be differentially methylated between FVM and PVR membranes  mainly composed of fibrotic tissue leading to retinal detachment. Another cause of retinal detachment is the formation of fibrotic membranes in proliferative vitreoretinopathy (PVR). This process can be a consequence of traumatisms or complications after retinal surgery  Fig. . Inflammin vitro PVR model to study the correlation between promoter hypermethylation and gene silencing. We obtained two vitreous samples at the outset of the vitrectomy surgery of patients with active PVR  by bisulfite sequencing . We selected three genes on the basis of their roles in tumorigenesis: integrin alpha 7 subunit (ITGA7), N-myc downstream-regulated gene 2 (NDRG2) gene and paired-like homeodomain 2 (PITX2) for bisulfite sequencing validation  and their normal retina controls n = 12) Fig. . Further2 Fig. . 22 (GSE 36064) as controls. As expected, genome-wide profiles of retinoblastoma samples from blood and primary tumors could be completely segregated by principal components analysis  in retina samples that differ from the methylation level in the other ocular tissues  . In addition, functional assays should be performed to demonstrate the causal role of epigenetics in instructing gene expression in retinal tissues.The retina is an immensely complex structure in which the orchestrated control of gene expression by epigenetic regulation is of great importance. It has been previously described that global reductions of epigenetic enzymes impair normal development of retinal cells27 and that global DNA methylation levels are associated with retinopathy in diabetic patients with progression in accordance with the severity of the disease28. In this study, we found that CpG methylation regulates pathways that are used as pharmacological targets in diabetic retinopathy progression, such as angiogenesis  . It has been previously described that low levels of serum folic acid and vitamin B12 (intermediates for DNA methylation that are found in many common nutrients) increase the risk of diabetic retinopathy16) Fig. . Althoug30. Epigenetic knowledge is also making its contribution and a clinical trial with the histone-deacetylase inhibitor vorinostat (NCT01587352) is in progress. In addition, development of appropriate clinical trials requires the use of biomarkers that predict drug response. On the basis of their gene expression profile, uveal melanomas have been classified into those at low risk of metastasis (class 1) and those at high risk (class 2)21. We found that the promoter methylation of one of the genes (RAB31) included in the panel of genes associated with metastasis was also correlated with patient survival . All donors gave informed consent before being included in the study. Surgical specimens were handled in accordance with the tenets of the Declaration of Helsinki.Human whole eyes were obtained from donors of the Banc de Sang i Teixits (BST) of the Catalan Ministry of Health . The cause of death in all adult subjects was multiorgan dysfunction and none of them had exhibited previous ocular diseases (see Supplementary Table Proliferative vitreoretinopathy (PVR) membranes were obtained from patients undergoing PVR surgery at the Retina Unit of the Bellvitge University Hospital . PVR membranes were surgically dissected from the retinal surface with Eckardt horizontal forceps in one eye of each of eight patients with a PVR diagnosis . Eight human postmortem eyes were obtained from diabetic donors free of fundoscopic abnormalities or with mild non-proliferative diabetic retinopathy in the ophthalmological examinations performed during the preceding two years. Eight eye-cups obtained from non-diabetic donors matched by age were used as a control group. The clinical characteristics of diabetic and non-diabetic donors included in the study are shown in Supplementary Table The fibrovascular membranes (FVM) obtained from diabetic patients FVMs were surgically dissected from the retinal surface in 12 eyes from 12 patients with type 2 diabetes and proliferative diabetic retinopathy at the Kyushu University Hospital (Japan), Fukuoka University Chikushi Hospital (Japan) and Bellvitge University Hospital   were obtained from patients at Bellvitge University Hospital  and the Shiley Eye Institute and Institute for Genomic Medicine . Fresh samples were immediately frozen after surgery and a fraction was conserved in the Anatomical Pathology Unit for histopathological characterization. Clinical features of the tumors are summarized in Supplementary Table Frozen retinoblastoma samples (n = 40) were obtained from patients at Sant Joan de Deu Hospital (HSJD) , Instituto de Salud Carlos III , Vall d’Hebron Hospital (HUVH) , the Tumour Bank of the Spanish National Cancer Research Centre (n = 2) and the Pediatric Ophthalmology Service of the Pediatric Hospital EHS of Canastelin  (n = 10). A collection of 27 blood samples from retinoblastoma obtained at the Pediatric Ophthalmology Service at the Pediatric Hospital EHS of Canastelin were also included in the study. Clinical features of the samples included in the study are summarized in Supplementary Table 4Cl; 1 mM KHCO3; 0.1 mM Na2EDTA) for abolishing blood cells. The resulting cells were cultured in DMEM Ham’s F12 medium (Invitrogen-Gibco) supplemented with 20% FBS 500 U/mL of penicillin, and 500 μg/mL of streptomycin. Cells were cultured at 37 °C in a humidified 5% CO2 atmosphere. After 15 days, cultures were washed with PBS buffer to remove unattached cells and then established fibroblasts were fed with fresh culture medium. Fibroblast markers were studied by RT-PCR for vimentin, fibroblast-specific protein 1 (FSP1), alpha-smooth muscle antigen (α-SMA) and ribosomal protein, large, P0 (RPLPO) genes, and by immunofluorescence, for vimentin and α-SMA. Primer sequences are listed in Supplementary Table All human vitreous specimens were obtained from patients of the Retina Unit at the Bellvitge University Hospital . Briefly, vitreous samples were obtained at the outset of vitrectomy surgery of two patients. 0.2 mL of vitreous fluid was drawn from the central portion of the vitreous cavity after dry vitrectomy and collected using a TB syringe connected to the aspiration line. After centrifugation of the vitreous sample, the cellular pellet was treated with ammonium-chloride-potassium (ACK) lysis buffer  . Cell lines were maintained in appropriate media and treated with 1 μM 5-aza-2′-deoxycytidine (Sigma) for 72 h to achieve demethylation.35 package available for Bioconductor36 in the R statistical environment37. This consisted of color bias adjustment, background level adjustment and quantile normalization across arrays35. The methylation level (β) for each of the 485,577 CpG sites was calculated as the ratio of methylated signal divided by the sum of methylated and unmethylated signals plus 100. To avoid batch effects, the function preprocessFunnorm in minfi package was used38. It is particularly useful for studies comparing conditions with known large-scale differences, such as cancer/normal studies, or between-tissue studies. It has been shown that for such studies, functional normalization outperforms other existing approaches39. After the normalization step, probes related to X and Y chromosomes, and those containing SNPs with a frequency of >1% (1000 Genome Project) in the probe sequence or interrogated CpG site were removed. Probes located in frequent copy number variant regions were also excluded. The methylation score of each CpG is represented as a β-value. DNA methylation microarray data are freely available for download from NCBI Gene Expression Omnibus under accession number GSE57362 (private link for the reviewers until the acceptance of the manuscript is https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?token = qdcvqcsyjbwpbid&acc = GSE57362).DNAs were extracted using conventional phenol:chloroform:isoamylalcohol (Sigma), quantified by Quant-iT PicoGreen dsDNA Reagent (Invitrogen) and DNA integrity was analyzed in a 1.3% agarose gel. Bisulfite modification of 600 ng genomic DNA was carried out with the EZ DNA Methylation Kit (Zymo) following the manufacturer’s protocol. Next, 4 μL of bisulfite-converted DNA were used to hybridize on an Infinium HumanMethylation450 BeadChip, following the Illumina Infinium HD Methylation protocol. The chip was analyzed using Illumina HiScan SQ fluorescent scanner and the intensities of the images were extracted using GenomeStudio (2010.3) Methylation module (1.8.5) software. A three-step-based normalization procedure was performed using the lumi40. The CpGs selected were those with adjusted values of p < 0.05 and an absolute methylation differential value of >0.33.Samples were clustered in an unsupervised manner using the 5,000 most variable β-values for CpG methylation according to their standard deviation in the CpG sites located in promoter regions by hierarchical clustering. An agglomeration method for Manhattan distances was used. Principal component analysis (PCA) was performed to analyze the effect of sample traits (age and tissue type) effect on the CpG methylation values among all sample set). Principal components analyses were run using the princomp package in the open-source R statistical software. This has been done for various sample traits such as age and tissue type. Also, projections of ages on the first two principal components (PC1 and PC2) were used for visualization of possible effects of age in distribution of the samples. For the differential methylation analysis between conditions (see main text), Wilcoxon signed-rank tests were conducted in the R statistical environment for all CpGs. The resulting p-values were corrected for multiple testing41 in the R statistical environment to search for overrepresented gene ontology biological processes using Fisher’s exact test to obtaining probabilities for each pathway, thereby to control the error rate. We corrected for multiple testing with the Benjamini and Hochberg algorithm.We employed the Bioconductor package GOStats42. When a specific comparison demanded two expression arrays from different platforms because the data were not present in the GSE to enable comparison of the two tissues, we used the Bioconductor Package virtualArray43, which enables the combination of raw expression data of different microarray platforms regardless of the platform and chip generation method used. The package generates a combined virtual array as an”expression set” object from different datasets by matching raw data entries based on probe, transcript, gene and protein identifiers. Redundancies, gaps, and batch effects are removed before proceeding with data analysis.The following expression data from the GEO Omnibus database were downloaded in order to extract the normalized expression values from the differentially methylated samples in each condition: GSE29801 , GSE51880 and GSE20986  and GSE5222 (for retinoblastoma). All data were downloaded and analyzed in the R environment using the Bioconductor package GEOquery44 using NOVOALIGN software (http://www.novocraft.com/main/downloadpage.php). Raw counts for each miRNA were extracted and analyzed under using the Rsamtools and DESeq Bioconductor packages in R.MirSeq data were downloaded from GEO Omnibus database under GSE18381 and GSE7072 for uveal melanoma and retinoblastoma samples, respectively. Fastq raw data were aligned to mature miRs from mirbaseWe determined the CpG island methylation status of the selected genes by PCR analysis of bisulfite-modified genomic DNA, which induces the chemical conversion of unmethylated, but not methylated, cytosine to uracil. For bisulfite sequencing, we used the same cohort of samples than those used in the genome-wide analysis by methylation arrays. The candidate CpG contained in the methylation array was included into the PCR amplicon. After PCR and cloning, ten clones of each sequence and sample were automatically sequenced to determine their degree of methylation. Primer sequences are listed in Supplementary Table in vitro treated with SssI methyltransferase was used as a positive control for methylated alleles. DNA from normal lymphocytes was used as a positive control for unmethylated alleles. Primer sequences are listed in Supplementary Table As a second strategy for assessing the CpG methylation status of specific genes in a panel of primary tumors from melanoma samples (n = 67) we used methylation-specific PCR using primers specific to either the methylated or modified unmethylated DNA. DNA from normal lymphocytes 2, 2 mM of dNTP, 1 µM of each primer and 3U of EcoStart DNA polymerase (Ecogen). 100 ng of cDNA were used for each PCR amplification. We also carried out PCR with GAPDH (22 cycles) to ensure cDNA quality and loading accuracy. Three biological replicates were included for each gene. Primer sequences are listed in Supplementary Table Total RNA was prepared from all samples using TRIZOL®  and further purified using RNeasy columns  according to the manufacturer’s instructions. For qPCR assays, we reverse-transcribed total RNA (2 µg) treated with DNase I (Ambion) using oligo (dT) 20 primer with ThermoScript TM RT-PCR (Invitrogen). We carried out PCR reactions in a final volume of 16 µL containing 10x PCR buffer (Ecogen), 50 mM of MgClFor immunolocalization experiments, cells were grown on coverslips and, after treatment, fixed with freshly prepared 4% paraformaldehyde for 20 min at RT or with 100% cold methanol for 10 min at 20°CC. Cells were mildly permeabilized with PBS containing 0.1% Triton and 2% bovine serum albumin at room temperature for 1 h. Thereafter, incubation with primary antibodies diluted in PBS with 0.1% bovine serum albumin was performed overnight, washed intensively and incubated with adequate secondary antibodies labeled with Alexa 488 and Alexa 568 dyes . After staining, coverslips were mounted in Mowiol  with DAPI (Sigma). Images were acquired using a confocal spectral Leica SP5 microscope . Z-projection and image processing were performed using ImageJ software . Primary antibodies and dilutions were: vimentin (Abcam) at 1:200 dilution and alpha smooth muscle actin (Abcam) at 1:200 dilution.Paraffin-embedded eye sections (7-μm thick) from diabetic and non-diabetic donors were processed with an apoptosis detection kit . TUNEL staining in the diabetic retina was compared with that in the non-diabetic retina. For this purpose, each retina was visually scanned with a high power lens (60x), which covers an area of 212 × 212 μm.https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?token = qdcvqcsyjbwpbid&acc = GSE57362).The DNA methylation microarray data reported in this article can be found at Gene Expression Ommibus (GEO) under the accession number GSE57362. (private link for the reviewers until the acceptance of the manuscript is Supplementary InformationSupplementary Dataset 1


While traditionally it was thought that Rac1 activity drives protrusive lamellipodia at the leading edge of a polarised cell while RhoA drives rear retraction, more recent work in 3D microenvironments has revealed a much more complicated picture of GTPase dynamics. In particular, RhoA activity can dominate the leading edge polymerisation of actin to form filopodial actin-spike protrusions that drive more invasive cell migration. We recently described a potential mechanism to abrogate this pro-invasive localised leading edge Rac1 to RhoA switch via manipulation of a negative feedback loop that was revealed by adopting a logical modelling approach. Both challenging dogma and taking a formal, mathematical approach to understanding signalling involved in motility may be vital to harnessing harmful cell migration and preventing metastasis in future research.Metastasis, initially driven by cells migrating and invading through the local environment, leads to most cancer-associated deaths. Cells can use a variety of modes to move  Indivr chains  that ther chains  which ger chains ,11. Whilr chains .Rho GTPases, such as RhoA and Rac1, have been recognised as master regulators of cell migration since their discovery 25 years ago ,14. Thesin vitro [a priori: the simulations suggested that either Rac1 activity simply gives way to RhoA activity following increased EGF–EGFR binding if RacGAP1 dominates the activity of Rac1 above all activators or, more interestingly, a steady state with RhoA-dominated Rac–RhoA pseudo-oscillations is reached if the Sos1-Eps8-Abi1 Rac1-specific activating complex [in vitro evidence suggested that this prediction was correct: using MEK1/2 inhibitors led to slower, more lamellipodial 2D migration, higher leading edge Rac1 and lower RhoA activity in cells migrating on 3D cell-derived matrix, more lamellipodial actin revealed by super-resolution imaging, and a significantly decreased capacity for cells to invade 3D fibronectin-rich ECM. Abrogating the formation of the Sos1-Eps8-Abi1 complex via Eps8 siRNA, however, rendered cells insensitive to MEK inhibition and returned cells to the RhoA-driven, more invasive filopodial phenotype as predicted by the model [The regulation of Rho GTPases by GEFs and GAPs is, while well known, incredibly complicated ,30. Firsin vitro  and high complex  preferen complex , Raf [38 complex , ERK1/2  complex  and p90R complex  are acti complex , which ihe model . Thus, tLife science research is undergoing a technological and information explosion at present: the tools available in regard to microscopy, mass spectrometry and analysis are of a far higher standard to what was even imaginable during the nascent cell migration and Rho GTPase research conducted 25 years ago. With the wealth of knowledge regarding cell motility growing ever larger, mathematical modelling approaches first help to formally understand and organise known priors, and then in turn make and test predictions regarding gaps in the knowledge. Despite the necessary abstractions, assumptions and simplifications, which naturally come with the Boolean approach, tangible predictions which lead to new biological insights when tested can be made; insights which arguably would never have come to light with a more traditional ‘wet lab’ approach. Using more elegant and complicated mathematical methodologies such as those based on ordinary/partial differential equations or stochastic rule-based models — despite the caveats which come with the difficulty of parameter estimation and the potential of over-determined systems — also have a role to play in move cell migration research forward given their increased quantitative potential. For example, recent modelling approaches have ranged from ordinary differential equations regarding small scale networks of Rho GTPase signalling  to more in vitro and in silico, fits within the ultimate aim of preventing fatal metastasis. A potential issue when moved to the scope of the real host human is that of migratory mechanism switching [Cancer cell migration research, both witching : a thera