Extracellular vesicles (EVs) have numerous potential applications in the field of healthcare and diagnostics, and research into their biological functions is rapidly increasing. Mainly because of their small size and heterogeneity, there are significant challenges associated with their analysis and despite overt evidence of the potential of EVs in clinical diagnostic practice, guidelines for analytical procedures have not yet been properly established. Here, we present an overview of the main methods for studying the properties of EVs based on the principles of fluorescence. Setting aside the isolation, purification and physicochemical characterization strategies which answer questions about the size, surface charge and stability of EVs (reviewed elsewhere), we focus on available optical tools that enable the direct analysis of phenotype and mechanisms of interaction with tissues. In brief, the topics on which we elaborate range from the most popular approaches such as nanoparticle tracking analysis and flow cytometry, to less commonly used techniques such as fluorescence depolarization and microarrays as well as emerging areas such as fast fluorescence lifetime imaging microscopy (FLIM). We highlight that understanding the strengths and limitations of each method is essential for choosing the most appropriate combination of analytical tools. Finally, future directions of this rapidly developing area of medical diagnostics are discussed. The first observation of mammalian vesicle-like structures was in 1967 by P. Wolf in platelet-free plasma after ultracentrifugation .Mammalian EVs are secreted membrane-delimited structures that contain material from the cytosol, subcellular organelles and/or nuclear components, encapsulated in a lipid bilayer. Although it is unknown whether all cell types can secrete EVs , diverseFrom the biochemical perspective, EVs and their cargoes comprise several components, ranging from membrane lipids and cytosolic proteins to nucleic acids. More specifically, several techniques including Western blotting and immune electron microscopy have revealed the presence of proteins on the surface as well as the interior of EVs, some of which are ubiquitous  while others vary significantly depending on the cell type from which they originate, mainly lineage delineating proteins and disease-specific markers. A few examples of tissue-specific proteins are, among others: major histocompatibility complex MHC class II and CD86 for EVs derived from antigen-presenting cells in vitro , human eBecause of their cargoes, EVs can be considered as vehicles for disease biomarkers which have advantages over routine blood biomarkers in common use . Additioi. small size and biochemical complexity;ii. pre-analytical methodologies for isolation and purification, as the accuracy of the measurements often depends on the purity of the sample, especially for techniques with high sensitivity;Some of the most common difficulties in analysing EVs are:Typical isolation methods include gel permeation chromatography, ultracentrifugation, immunoaffinity capture or polymer-facilitated precipitation, among others . A varieiii. heterogeneity of EV populations;iv. sub-populations of different phenotypes;v. unknown concentrations;vi. correlating specific EVs with specific origins;More challenges in studying EV include:A number of the above hurdles can be addressed by optical methods, on which this review focuses. Optical methods use light in order to read the information encoded in the sample. The fundamental advantage of optics-based techniques over calorimetric or chemical readout methods is that, because light appears in a spectrum of wavelengths, multiplexed analysis is permitted, which is essential for clinical samples where assessment of multiple molecules may be required. Moreover, with the selection of the appropriate fluorophore and target molecule, the different populations and origins of EVs can be studied, while it is also possible to precisely detect and localize biomolecules using super-resolution microscopy, excluding particles which cause impurities. Additionally, we acknowledge that non-optical technologies (of which there are many) can answer critical questions and common approaches incorporate both; non-conventional tools and future approaches will also be discussed briefly later (see Emerging fluorescence methods).The aim of this review is to summarize the optical tools researchers have used in analysing the phenotype and functions of EVs, emphasizing the seemingly endless potential of fluorescence-based approaches. In general, optical methods provide unique opportunities for real-time, multiplexed and rapid analysis in complex environments. As the methods for EV isolation and size measurement appear well accepted among the research community (and have been well reviewed elsewhere), we herein focus on the biological properties of EVs and the specific analytical techniques which can be applied on the nanoscale. In parallel, with the rapid development of labelling technologies and fluorescent dye chemistry, fluorescence is nowadays a versatile tool that offers multiple options for a variety of analytical and imaging modalities. As there is not a standardized workflow for phenotypic studies of EVs, we present an overview of the classic as well as the latest approaches used in the field . In brieFrom the techniques listed, fluorescence measurements can be generally divided into two subgroups: A) steady-state and B) time-resolved analysis. A) Steady-state fluorescence spectra result from the excitation of the sample with a continuous light beam and the recording of the emission intensity. In practice, steady-state measurements average the recorded parameters from the initiation until the completion of the phenomenon. B) In time-resolved techniques, data are collected in a time-dependent manner, resulting in a fluorescence decay curve representative of not only the steady-state aspects but also transient information on the phenomenon under study  . Most meFluorescence provides information about a range of processes, such as the interaction of fluorophores with the solvent or rotational freedom and molecular distances . Here wein the form of a dynamic suspension , flow cytometry, fluorescence anisotropy, live microscopy);captured on a surface ;Analysis of intact EVs, in which the sample can be:Lysed EVs for molecular analysis (PCR microfluidic systems).A simple categorization of the physical state of the sample and the associated optical analytical approaches is as follows:Most techniques analyse either sizing  or molecular functionality  and there is a need for advanced techniques in the areas of fluorescence NTA, microscopy and microfluidics that offer both size and functional information simultaneously . The potIn order to utilize imaging and spectroscopic techniques based on fluorescence, either direct or indirect labels need to be introduced to the EV target sample. Examples include the following options for labelling strategies: (i) staining via the sequestering of free dyes directly into the lipid bilayer via hydrophobic interactions or by genetically encoding fluorescent proteins, (ii) selection of fluorophore for the formation of a covalent chemical bond with a probe which has affinity for a vesicular compartment, and (iii) the selection of a suitable affinity probe such as immunoglobulins, nanobodies or aptamers. The target components can be nucleic acids , membraA broad categorization of the main groups of labelling options mentioned earlier is given here in more detail:(i) staining via hydrophobic interactions and genetically encoded fluorescent proteinsFree fluorescent dyes are commonly used to stain the total EV population non-specifically, either by lipophilic interactions with the membrane or by diffusion and retention inside the vesicles. Some dyes associating with the membrane of human or bacterial EVs used for flow cytometry measurements and fluorescence confocal microscopy for cellular uptake tracking and trafficking include, among others, BODIPY-PC (conjugated with phosphatidylcholine), DiD, Dil, DilC, Octadecyl rhodamine B chloride (R18)  dyes froFor membrane and lipid rafts studies, there is a large variety of fluorophores which intercalate into the lipid bilayer. These are currently used widely on cells and have potential application to vesicles. The most common category is the BODIPY range of dyes which can bind to different parts of the acyl chain of membrane lipids, thus providing images from different depths of the lipid bilayer ,31. Envi(ii) fluorophores used for labelling an affinity probeRed/near-infrared (NIR) fluorescent dyes offer a number of advantages for EV analysis. In particular, their longer-wavelength excitation usually minimizes both the background endogenous fluorescence from the sample under study and the detection of Rayleigh scattered excitation [citation . Some adcitation ,37. Red/citation . In addicitation .Dyes intended for super-resolution microscopy based on stochastic activation of fluorophores such as Stochastic Optical Reconstruction Microscopy (STORM) are special organic molecules or proteins which have blinking properties (reversible photoswitching) such as the AlexaFluor and the ATTO range of molecules [olecules . Techniqolecules . More deLuminescent nanoparticles such as inorganic QDs absorb light across a specific wavelength region and emit narrower spectral bands of light depending on the composition, size and surface properties of the nanocrystal. Their spectral versatility and capacity for surface modification have made them popular for biomedical applications [ications . Apart fications . Pairingications . QDs areications .(iii) options for affinity-binding reagentsNanobodies are fragments engineered from antibodies found naturally in camelids  and cartilaginous fish (sharks). They are single-domain components of the variable parts of an antibody, with a weight of 15–25 kDa, significantly smaller than Fab fragments (around 50 kDa) and whole immunoglobulins (around 150 kDa) [Although antibodies and Fab domains are being used for specific EV labelling, extrapolating their potential from their well-tried use on cells, the size of EVs suggests that smaller labelling probes can be more effective, as they can potentially increase the label-to-EV ratio. 150 kDa) . Besides150 kDa) . Fusion 150 kDa) , as well150 kDa) . Conjuga150 kDa)  or even 150 kDa) , as well150 kDa) –54, thusNucleic acid aptamers are synthetic single-stranded DNA or RNA sequences that are selected based on their high specificity for a target species and can be custom-engineered with different labels to amplify the optical signal associated with EV interaction. DNA dendrimers are a type of 3-D macromolecular structures with numerous binding sites, incorporating DNA sequences and have been used as part of an EV capture and detection platform, where the capture of EVs on CD63 magnetic beads triggers the release of a DNA probe which hybridizes on other hairpin structures and initiates a cascade of signal which is finally amplified by the dendrimers [ndrimers . CD63 apndrimers  and as pndrimers . A methondrimers .Nanoparticle tracking analysis (NTA) is a relatively recently developed (2006) technique, now distributed by Malvern Panalytical Ltd (UK), which can be generally applied to many types of nanoparticles ranging from 30 nm to 1 μm, thus encompassing many EVs ,60. In mThis technique is one of the most widely accepted methods for EV size and concentration measurements, and can also be used for EV phenotyping using fluorescent markers. Because of the prolonged exposure of the sample in the laser beam, the most popular fluorescent labelling is via QDs, as they are generally photostable compared to other commercial fluorophores and this strategy is overall considered more sensitive than flow cytometry for particles in the sub-micron size range . A numbeRegarding the relative advantages of NTA over other techniques discussed later, NTA measurements are relatively fast (require a few minutes), with non-sophisticated sample preparation procedure and provide real-time imaging of unlabelled or labelled particles. It is also particularly sensitive for samples containing small populations of larger particles because the intensity of the scattered light is proportional to the sixth power of the diameter. At the same time, this means that size determination could be less accurate, especially in populations of smaller particles, where dust particles or protein aggregates can interfere in the measurement. This poses an additional point of limitation, related to the sample purity. The presence of large particles reduces the number of the small particles the software can detect . DiffereA versatile technique to characterize labelled or unlabelled cells and particles derived from biologic samples is flow cytometry. The working principle of this technique is the sensing of cells or particles in suspension, streamed through a laser excitation path . Once thFlow cytometry was originally designed for larger particles , substantially larger than the smaller end of the EV size spectrum, but has been used to count, sort and investigate the phenotype of EVs, although their small size raises a matter of controversy for this technique ,72. As iAnother commonly used approach for flow cytometry of EVs is immunocapture on magnetic or non-magnetic beads to form a larger complex which can be detected within the conventional size detection limits of cytometers ,81. SomeFlow cytometry is a popular method for clinical analyses as it offers the advantages of a rapid and versatile tool for which fluorescent probes are well established and optimized. However, there are concerns regarding the sensitivity of the system as applied to EVs. Alternative techniques such as NTA which are designed for sub-micron particles and offer more sensitive phenotype analysis  demonstrWith EV diameters ranging from a few nanometres up to a few hundreds of nanometres, the diffraction-limited resolution capabilities of a microscope  in the study of such small particles are unworkable. However, although the small size of many EVs does not allow their visualization in an unlabelled state by brightfield illumination, the attachment of a fluorophore which emits light enables the imaging of EVs and their interactions with live cells. Imaging by microscopy is particularly useful for the study of the localization of the particles in cells or tissues, or presence of a fluorescently labelled target. The majority of the cases discussed in this section refer to confocal microscopy, in which only a small defined area of the sample is exposed at each time excluding all the out-of-focus light, in contrast to wide-field imaging where the total area of the specimen is illuminated and the precision of the fluorophore localization is lower .et al. by using zebrafish in which all CD63-positive EVs were fluorescently labelled. This model expressed CD63-pHluorin (emits green fluorescence) and therefore, endogenous EVs were labelled and could be tracked in real time in vivo by confocal microscopy [Several applications of confocal imaging of EV include the observation of vesicles inside the Golgi apparatus with a size of approximately 50–80 nm , and in croscopy . What isAmong the advantages of fluorescence microscopy are the direct localization of the labelled target within the system of interest , the ability to track the EVs in real time and the fact that image analysis software can nowadays give accurate quantitative results on colocalization of fluorophores, tracking speed and directionality, etc. In addition, in contrast to other microscopy methods such as STORM, PALM and FRET, there is a wide variety of fluorophores which can be used for confocal imaging. The main limitations of this imaging strategy mainly pertain to the resolution limit, allowing only larger EV to be accurately imaged, and the sample preparation procedure, which can sometimes involve long protocols including fixation. Another issue always appearing in microscopy is the selection of the appropriate target for labelling, and the suitable fluorophore which needs to be stable and resistant to photobleaching from prolonged exposure .An excellent example of the most advanced applications of fluorescence is super-resolution microscopy, where different techniques use distinct properties of fluorescence to break through the light diffraction limit . Among the most popular techniques are Structured Illumination Microscopy (SIM) which is based on the illumination of the specimen by a striped pattern  and Stimet al., as well [EVs have been imaged with PALM and dSTORM to study the interactions of exosomes with neurons in Alzheimer’s disease. CD9-labelled vesicles were found in the cytoplasm and endosomes of dendrites isolated from mouse brain tissue with a precision of 25 nm . In anot as well . Labelli as well . EVs fro as well . STED is as well . A more  as well .The benefits of super-resolution microscopy and SMLM are by definition the ability to break the resolution limit and image particles non-detectable by conventional microscopy. It is therefore an approach very suitable for EV analysis regarding size, concentration and content, although this does not come without limitations, mainly from the perspective of sample preparation and the variety of imaging modes which are possible. In more depth, although the protocols for fixation and staining of the biological materials are considered well established for confocal microscopy, when it comes to SMLM, the precision of imaging reveals insufficient labelling of all target molecules which can be due to inadequate staining with affinity reagents or inappropriate labelling strategy. In other words, for imaging intended for quantitative analysis, the location of the target epitope on the particle under examination is a point which needs to be carefully considered when choosing the appropriate affinity probe and labelling conditions. Another limitation which was only discovered with the advent of SMLM approaches is the fixation method, as it has been shown that the structure and clustering of the target can vary significantly with different fixation protocols . MoreoveFluorescence lifetime imaging microscopy (FLIM) differs from steady-state fluorescence microscopy as in the former, the contrast of the image is generated by the difference in the excited state lifetime of the fluorophores rather than the total fluorescence intensity in case of the latter. In addition to fluorescence localization and intensity information provided by steady-state microscopy, FLIM is especially useful for studying the spatial arrangement of biological membranes, as fluorescence lifetime of a dye depends on its microenvironment ,113.et al. where the incorporation of the anti-fungal drug amphotericin B in membranes was investigated by treating cell lines with the drug and imaging the secreted EVs, recording both the lifetime and anisotropy. The images reveal the different microdomains on the EV membrane and show that amphotericin replaces cholesterol in the membrane [Imaging membrane microdomains of EVs can be useful in revealing potential mechanisms of interaction with other membranes and especially cells, and has been used by several groups for cellular uptake imaging. EVs from osteosarcoma cells and their interactions with lung tissue have been imaged with FLIM using the dye PKH67, providing high-quality images of the membrane . A detaimembrane .Overall, as FLIM is based on the lifetime of the fluorophores, it is independent of probe concentration, excitation intensity or photobleaching and can be used even with spectrally similar fluorophores, given that their decay times are different. Moreover, it can directly detect differences in the probe’s microenvironment, without the need for special treatment of the specimen. On the other hand, the main drawbacks are the need for expertise in lifetime spectroscopy in order to understand the data and the special equipment required . AnotherFörster Resonance Energy Transfer (FRET) is the phenomenon whereby energy from one fluorophore is transferred non-radiatively to another via dipole–dipole interaction when the two molecules show some spectral overlap (resonance) and are practically less than 10 nm apart in space. Thus, these measurements are used to determine molecular distance and interactions between fluorescently labelled targets.et al., showing potential for loading and targeting vesicles from patients by the attachment of a special peptide on the CD63 surface molecules [The combination of FRET/FLIM has enabled quantification of the FRET effect using the advantages of time-resolved lifetime spectroscopy and has been useful in the case of HER receptor in breast cancer since the status of dimerization of HER provides more valuable prognostic/diagnostic information than the levels of expression. HER monomers on circulating EVs were immunostained with the FRET pair AlexaFluor 546 and Cy5 and imaged with the above combination of techniques to quantify the different forms of HER . Besidesolecules . An indiolecules .Although FRET offers unique imaging information over other microscopy techniques, the principles of Förster resonance bring disadvantages, as the appropriate combination of emitters needs to be carefully selected. It is also observed that the signal-to-noise ratio (SNR) can be low and emission is sensitive to changes of the microenvironment of the fluorescent molecule; however, recent advances in the field are tackling these issues .Quantitative Polymerase Chain Reaction is a technique based on the well-established PCR method, which is enhanced with numeric quantitative data in contrast to the traditional PCR which is semi-quantitative. This method assesses EV populations in total, rather than single EVs, as it requires vesicle lysis. The basis for its inclusion here is the use of fluorescence to detect the amount of nucleic acid produced in every cycle of the reaction. As the majority of EV studies are conducted on mRNA and miRNAs, qPCR is a common method of choice .Microfluidics-based technologies continue to evolve for biological and analytical applications and there have also been recent innovations in the field of EV detection. Most often, these involve the capture and separation of EVs on a solid substrate surface localized with a microfluidic compartment. There are several strategies for EV immobilization and these can be distinguished as passive  and active . The analytes can be detected by a variety of different methods depending on the desired readout signal. For instance, nucleic acids can be amplified on-chip with PCR-based methods, proteins can be detected with principles similar to enzyme-linked immunosorbent assay (ELISA) assays, or the EVs can be lysed, releasing their contents which can be subsequently processed off-chip for the detection of various components such as Western blots or mass spectrometry ,125.et al. using a catalysed hairpin DNA circuit (CHDC) on special lipid-polymer nanoparticles. When EVs are captured on the surface of the nanoparticles, EV RNA interacts with the hairpin DNA complex which emits an amplified signal upon hybridization, enabling detection of low levels of mRNA expression [et al. where individual EVs were transported in a flow stream and visualized on a microscope. The results on synthetic lipid particles showed potential applications in measurement of concentration and size distribution, as well as dual colour imaging for different markers [EV secretion from single cells has been presented on a microfluidic setup, where the EVs secreted by each cell are captured on CD63 beads and visualized by fluorescence imaging. Antibodies labelled with PE were attached on the EVs captured on microbeads and the values of fluorescence intensity of the beads could indicate the rate of cell secretion, concentration and phenotype of the vesicles . A biochpression . A platfpression . A minia markers .Other applications of microfluidic systems have been developed for the capturing and concentration of EVs from biopsies, where the amount of available vesicles is limited . Taking advantage of the natural surface charge of EVs, an ion-selective membrane can be used as a trap for vesicles flowing onto this surface, resulting in maximized concentrations and a surface saturated with EVs for further studies . EV enriet al. [The miniaturization of EV capture and detection processes on microfluidic chips has promoted research towards the development of hand-held analysis devices, such as the system reported by Ko et al. . The baset al. . The reset al. .et al. to capture the total EV population from blood plasma on array spots, followed by visualization using fluorescent detection antibodies. This has enabled screening for 21 different EV surface antigens with the use of less than 10 μl of sample [The development of antibody arrays on solid surfaces offers high-throughput screening for various EV markers often by detection of fluorescence signal. A cocktail of antibodies against CD9, CD63 and CD81 were used by Jørgensen f sample . Later of sample . Anotherf sample .In this section, we discuss some non-conventional tools with potential for EV analysis, such as fluorescence anisotropy and other optical methods.Fluorescence anisotropy takes advantage of the polarization properties of fluorescence that can be used to describe rotational diffusion and energy migration. More specifically, when fluorescent molecules are exposed to a polarized excitation source , only the molecules found in the same plane as the polarized light will get excited. Due to Brownian motion and other energy migration phenomena, the excited polarized molecules will fluoresce while rotating back to randomized planes (depolarization) [ization) . The depization) . The addThe literature shows an extended interest in anisotropic properties of lipid bilayers over several decades, and these approaches have revealed the behaviour of the membranes under different conditions, usually altering the temperature, pH and lipid composition, using liposomes as artificial membrane models . The reset al., who studied the membrane order and phase transition of the particles for a range of temperatures and conclude the lamellarity of the vesicles, evident by the broad phase transition points around 37°C [There has been extensive use of this technique in studying lipid rafts of cell membranes even from the 70s ,145 to tund 37°C . Howeverund 37°C .In this review, we have focused on the phenotypic analysis of EVs highlighting the versatility of fluorescence. It is also important to acknowledge techniques which use principles different from fluorescence, but which are of great significance in EV analysis , micronuclear magnetic resonance (microNMR), electron microscopy (EM), cryoEM-tomography and numerous others).in vivo, as well as their lifetime in the blood circulation. More advanced tools are required to address these properties of EVs and one of the future directions could be the combination of fluorescence with other optical techniques, as well as multiplexed analysis by adding modalities which use different and non-overlapping principles.There are currently many outstanding questions about the biology of EVs including their routes of biogenesis, release and uptake mechanisms by recipient tissues For example, in NTA, advances in the method would tackle limitations such as the speed of sample analysis and allow fluorescence screening for multiple surface markers. Currently, instruments with two different fluorescence channels are under development, so that samples labelled with two fluorescent probes can be studied simultaneously, and in addition to this, it is being converted to high-throughput versions, such as with the use of an autosampler . AnotherAlso, a recent and promising development for FLIM is the introduction of single-photon avalanche diode (SPAD) array cameras with on-chip pixelated timing capabilities ,154. Thiet al., as mentioned earlier). Finally, the need for clinically applicable devices has started with the development of microfluidics and arrays, which promise rapid, point-of-care analysis of multiple markers simultaneously. Fluorescence-based methodologies for phenotyping EVs in healthcare applications will undoubtedly continue to develop apace and future EV-based diagnostics are highly likely to increase in efficacy and popularity.Looking into the current approaches for EV phenotyping, we conclude that the heterogeneity of size and composition of EVs dictates the use of multiple techniques in order to eliminate the errors resulting from the differences in size. Moreover, the chemistry of fluorescent probes has seen rapid development. There is currently a wide range of options and the selection of the appropriate probe depends on the imaging strategy, as some methods have specific requirements . Because of the growing interest in the use of EVs in clinical analysis, standardization of the phenotyping guidelines is required, as there is significant variability in the techniques in current use. As one of the key questions about extracellular vesicles still remaining is the mechanism of interaction with tissues in vivo, future research could be directed towards the development of live EV imaging and tracking in living models (similar to Verweij


Cervical component of thymus is noted more in children and young adults than in older age group. CT texture (lobules of soft tissue interspersed with fat), similarity with CT density of mediastinal thymus and continuity with mediastinal thymus on sagittal/coronal images, are given as the criteria for diagnosis of the cervical thymus. But CT densities of cervical and mediastinal components of the thymus may vary. The purpose of our study was to compare CT densities of cervical and mediastinal parts of the thymus, in cases where ultrasonography correlation was available.We retrospectively identified 22 patients who had undergone CT between May 2015 and May 2017 and in whom ultrasonography (USG) correlation was available. CT densities of cervical and mediastinal components of thymus were measured.CT density of cervical thymus is lower than the CT density of mediastinal thymus by ~ 25 HU.There is a moderate positive correlation between CT densities of cervical and mediastinal parts of the thymus.CT densities of both cervical and mediastinal thymus were found to reduce with age, but the reduction was statistically significant only in the cervical thymus in this study.CT densities of cervical and mediastinal components of the thymus may vary, with CT density of cervical thymus being lower. There is a positive correlation between CT densities of cervical and mediastinal parts of the thymus.CT density of cervical thymus reduces with age.Understanding these may help avoid confusion on CT and avoid the need for correlative USG, saving time and effort.The online version of this article (10.1186/s13244-019-0781-z) contains supplementary material, which is available to authorized users. CT densities of cervical and mediastinal components of the thymus may vary, with CT density of cervical thymus being lower.CT density of cervical thymus reduces with age.Understanding these may help avoid confusion on CT and avoid the need for correlative USG, saving time and effort.Although the thymus is predominantly a mediastinal organ, part of it can often be seen in lower neck normally , 2. WhenIt is a common practice at our center to do USG correlation when there is any ambiguity in the neck  on CT scan. We encountered some cases where the cervical component of thymus had a density different from the mediastinal thymus, but ultrasound  confirmed that the cervical lesion was indeed a normal variant and not some small lymph node/other pathology. We did a retrospective study of cases of the cervical thymus in which USG correlation was available, to compare CT densities of cervical and mediastinal parts of thymus.The institutional review board approved this retrospective study of existing imaging data, and written consent was waived. We retrospectively searched our USG and CT (PACS) records to identify patients in whom USG correlation Fig.  had beenCT was performed on a 16-channel scanner  with variable milliampere and kilovolt (peak) of 120. Display FOV varied with patient size but was approximately 25 mm; coronal and sagittal reformats were used in this evaluation.Cervical thymic tissue was defined as an oval focus of soft tissue above the level of sternal notch that had continuity with mediastinal thymus on axial/sagittal/coronal images. Each CT examination was evaluated by two radiologists  to determine densities of mediastinal and cervical components of thymus Fig.  and the t test. The comparison of density with age (groups) was tested using ANOVA test and the comparison of density with sex was tested using Student’s t test. A p value of .05 was chosen as the threshold for statistical significance. SPSS, Version 11  was used for all statistical calculations.The difference in density of cervical and mediastinal parts of thymus was tested using paired p 0.001). Mean value of CT density of cervical thymus is lower than CT density of mediastinal thymus by ~ 25 units . That is, if the CT density of mediastinal part of thymus is high, CT density of cervical thymus is also likely to be high; and vice versa.CT densities of both cervical and mediastinal thymus were found to reduce with age, but the reduction was statistically significant only in the cervical thymus in this study Table .After adjusting for age, there is no difference demonstrated in densities  between males and females.The 22 studied patients included 10 males (45.5%) and 12 female patients (54.5%); age varied from 1 to 34 years (mean age 14); 19 patients had contrast-enhanced CTs and 3 patients had non-contrast CTs Additional file Embryological pathway of thymic descent is from the angle of mandible to the superior mediastinum ; hence, thymus can be found anywhere along this path. Continuity with mediastinal thymus distinguishes cervical component of thymus from ectopic thymic tissue. Cervical component of thymus may be present at all ages , though On ultrasound, thymus of infants may be homogeneous, with echogenicity slightly less than liver and spleen  or have Thymic size and density are affected by a number of factors. Thymic size decreases with age; acute size reduction may occur during stress ; true hyperplasia occurs in recovery from recent stress ; when reOur study demonstrates that CT density of cervical part of thymus may vary from that of mediastinal part of the thymus, with density of cervical thymus being lower than the density of mediastinal component. But densities of cervical and mediastinal parts of thymus show positive correlation. We did not study the inter-observer variation in CT density, based on the excellent intra- and inter-observer agreements noted previously , 13, 14.The difference in CT density between cervical and mediastinal parts of the thymus may be partially due to the non-involvement of the cervical thymus in thymic hyperplasia. Most of our oncology patients would have had stress  and reactive thymic hyperplasia after recovery from stress.Limitations of our study include the retrospective nature of the study and absence of histological correlation. All patients in the study were imaged for non-uniform clinical reasons. Sick patients may have a higher rate of thymic hyperplasia and hence increased the difference in densities between cervical and mediastinal components of the thymus, but this is acceptable because it mirrors the actual incidence that would be seen on medical imaging.CT densities of cervical and mediastinal components of thymus may vary, with density of cervical thymus being lower than the density of mediastinal component. When continuity with mediastinal thymus is seen on CT, diagnosis of the cervical thymus can be made even in the presence of different CT densities. Densities of cervical and mediastinal parts of thymus show positive correlation. Decrease in CT density of thymus with age, noted previously in the mediastinal thymus, has been demonstrated in cervical thymus also. Understanding these may help avoid confusion on CT and avoid the need for correlative USG, saving time and effort.Additional file 1:Table S1. Patient data. (DOCX 20 kb)


Meralgia paresthetica is a neuropathy of the lateral femoral cutaneous nerve. Traditionally, the diagnosis is based on classical symptoms and signs. In cases where there is a diagnostic dilemma, the role of magnetic resonance imaging has been to exclude other causes for the patient’s presentation, as the small extraspinal peripheral nerves were not well visualised at imaging. The development of 3-Tesla magnetic resonance neurography, however, has made pathology of these nerves more conspicuous. The diagnosis is based on the classical symptoms and clinical findings of pain, an unpleasant sensation, numbness and paraesthesia on the anterolateral aspect of the thigh.4 It emerges from the lateral aspect of the psoas muscle and crosses the anterior surface of the iliacus muscle across the ilium towards the anterior superior iliac spine (ASIS). It enters the thigh by passing above, below or through the inguinal ligament  or proton density fat saturation (PDFS) are useful. Short Tau Inversion recovery (STIR), diffusion weighted imaging or diffusion-weighted whole-body imaging with background body signal suppression (DWIBS) is also good for identifying signal abnormalities but usually has lower resolution.While the diagnosis is based predominantly on history and examination of the patient, the condition may mimic other pathologies of the pelvis and lumbar spine, such as lumbar disc herniation and metastases to the iliac crest.3 Motor neuropathies may also be supported by the findings of muscle oedema in a specific nerve distribution, in the acute phase of denervation. This finding will not be present in the purely sensory nerve neuropathy such as in the LFCN in meralgia paresthetica. The diagnosis then depends on findings of signal alteration in the LFCN (3Magnetic resonance neurography allows the detection of changes in perifascicular and endoneurial signal intensity, which may indicate nerve injury. An increase in nerve size, loss of the normal appearance of nerve fascicles and surrounding fat stranding support the findings of nerve injury, but these morphological changes may be more difficult to detect in smaller, more peripheral nerves such as the LFCN.the LFCN  and b. Ithe LFCN  to adequ2A study published by Chhabra et al in 2013 investigated the diagnostic accuracy and observer performance of 3T MRN in the investigation of meralgia paresthetica. The study found a moderate inter-observer agreement between two readers for detecting signal alterations of the LFCN with a sensitivity and specificity of ≥71% and ≥94% for both readers, respectively. The authors concluded that 3T MRN was reliable and accurate in the diagnosis of meralgia paresthetica.Meralgia paresthetica, a neuropathy of the LFCN, may be spontaneous or iatrogenic. The highly variable anatomy puts it at risk of compression, as well as injury during various surgical procedures. The diagnosis has traditionally been based on clinical findings, but occasionally a diagnostic dilemma may arise. The development of 3T MRN is now proving useful in assisting with the diagnosis of neuropathies of the smaller peripheral nerves.


Immediate breast reconstruction and postmastectomy radiation therapy (PMRT) each play an integral part in multimodal breast cancer treatment. With the increasing use of neoadjuvant systemic therapy (NST) in early-stage breast cancer, the indications for PMRT are affected, and this contributes to the complexity of immediate breast reconstruction planning. The choice between immediate or delayed reconstruction is dependent on the preoperative need for PMRT. Residual axillary lymph node involvement after NST is the main determinant for PMRT since these patients have an increased risk of locoregional recurrence.This study reports on the risk of positive sentinel lymph node (SLN) after NST in clinically node-negative breast cancer patients, and therefore the likelihood of PMRT.Both breast reconstruction and PMRT are increasingly used, and the integration of these two important treatments requires a multidisciplinary approach and careful patient counseling. Our study provides data on low- and high-risk PMRT patients and can be included in the decision-making process. In high-risk SLN positive patients with a desire for immediate breast reconstruction, an SLN biopsy can be performed after NST prior to breast surgery in order to determine the pathologic axillary outcome and, consequently, the need for PMRT. If PMRT is required, the patient should be well-informed on the pros and cons of the combination of immediate breast reconstruction and PMRT, as well as the possibility of a delayed reconstruction. Finally, the decision to perform immediate or delayed reconstruction in the setting of PMRT must be individualized and must take into account the experience of the (plastic) surgeon and the expectations of the patient.


In recent years, dielectric elastomer actuators (DEAs) have been widely used in soft robots and artificial bio-medical applications. Most DEAs are composed of a thin dielectric elastomer layer sandwiched between two compliant electrodes. DEAs vary in their design to provide bending, torsional, and stretch/contraction motions under the application of high external voltages. Most compliant electrodes are made of carbon powders or thin metallic films. In situations involving large deformations or improper fabrication, the electrodes are susceptible to breakage and increased resistivity. The worst cases result in a loss of conductivity and functional failure. In this study, we developed a method by which to exploit stretchable metallic springs as compliant electrodes for cylindrical DEAs. This design was inspired by the extensibility of mechanical springs. The main advantage of this approach is the fact that the metallic spring-like compliant electrodes remain conductive and do not increase the stiffness as the tube-like DEAs elongate in the axial direction. This can be attributed to a reduction in thickness in the radial direction. The proposed cylindrical structure is composed of highly-stretchable VHB 4905 film folded within a hollow tube and then sandwiched between copper springs (inside and outside) to allow for stretching and contraction in the axial direction under the application of high DC voltages. We fabricated a prototype and evaluated the mechanical and electromechanical properties of the device experimentally using a high-voltage source of 9.9 kV. This device demonstrated a non-linear increase in axial stretching with an increase in applied voltage, reaching a maximum extension of 0.63 mm  at applied voltage of 9.9 kV. Further miniaturization and the incorporation of compressive springs are expected to allow the implementation of the proposed method in soft micro-robots and bio-mimetic applications. Recent developments in soft robots and artificial biomimetic structures have greatly increased interest in dielectric bio-compatible elastomers capable of producing large deformations. These devices have been widely used in actuators and sensors. Recent applications include tunable lenses , micro-pA variety of polymers and compositions have been studied to produce DEAs of higher performance. It has been demonstrated that compliant electrodes can affect the deformation of DEAs. Most compliant electrodes are made of carbon powder (graphene or carbon black) mixed with conductive glue ,22,23,24In this research, we adopted metallic springs as compliant electrodes for cylindrical DEAs to provide low resistance and stiffness under the effects of deformation. This design was inspired by the extensibility of mechanical springs. The proposed cylindrical DEA comprises a VHB 4905  thin film folded into a hollow tube and sandwiched between two individual copper springs inside and outside. VHBs are acrylic polymers widely used as dielectric elastomers with large expansion exceeding 100% strain ,36 and hThis paper is organized as follows. In the next section, we outline the configuration and theoretical model of the proposed cylindrical DEA and then describe the fabrication process. We then examine the mechanical properties of the fabricated device as well as electromechanical characteristics using a variable high-voltage source. Important results are summarized at the end of the paper.As mentioned in the previous section, we used copper springs as stretchable electrodes for their ability to remain conductive and retain low stiffness even as the elastomer is stretched. Without external electrical stimuli, the proposed cylindrical DEA retains its original shape, i.e., without deformation, as shown in L. Under application of external voltage, V, the tube actuator deforms, such that the length is increased to Y is the Young’s modulus. The analytical simulations of the proposed spring-based DEA based on the electromechanical model of the tube DEA will be presented in the later section. Note that this model is not necessarily applicable to large deformations.Under microscopic stretching and contraction, the distance between adjacent turns of the spring is relatively small when the cylindrical DEA is extended in the axial direction. The application of external electrical stimuli can result in an electric field uniformly distributed across the entire surface of the device. This is similar to the behavior of the tube actuator with thin compliant electrodes as shown in uator in , the innThe inside of the barrel was filled with Gypsum after being smeared with Vaseline for later mode-stripping. The proportion of Gypsum to water was optimized to enable a short curing time while ensuring strength sufficient for the fabrication of the actuator. After curing, the axle was demolded with Gypsum from the barrel, as shown in We obtained stress-strain relation of the fabricated cylindrical DEA by measuring the axial strain under axial loads, as shown in The proposed cylindrical DEA demonstrated a non-linear stress-strain relation similar to that of the VHB film. The individual copper spring presented a linear stress-strain relation with little overall impact on the actuator. In other words, the non-linear behavior of the proposed DEA was due mostly to the nonlinear properties of VHB film. The Young’s modulus of the proposed metallic springs was lower than that of the electrodes coated with a metallic film, falling within the GPa range suitable for actuation applications.Based on the aforementioned theoretical model, we determined that the axial deformations in the cylindrical DEA were proportional to the square of the applied external voltage. We repeated the experiments in In the previous sections, we demonstrate that the proposed cylindrical DEA undergoes deformation under external electrical stimuli, resulting in a maximum axial strain of 2.35% under an external electric field of 20 V/Overall, the proposed cylindrical DEA demonstrates good electromechanical performance with linear motion at the micro-scale. The proposed device could be scaled down using precision microelectromechanical system (MEMS) fabrication techniques. Miniaturized cylindrical DEAs could be used as cylindrical legs or arms for soft micro-robots. The proposed approach could also be incorporated with compressive springs to provide additional bending motions for use as swing legs. In addition, small cylindrical DEAs could be used to achieve linear movements in bio-mimetic structures .This paper describes the use of copper springs as compliant electrodes for cylindrical dielectric elastomer actuators. Our objective was to preserve the conductivity and low stiffness of the compliant electrode under deformation. We fabricated a prototype and evaluated the mechanical and electromechanical properties of the device experimentally using a high-voltage source of 9.9 kV. Our results demonstrate that the proposed cylindrical DEA stretches under external electric stimuli and contracts following removal of the stimuli. Further miniaturization and the incorporation of compressive springs is expected to allow the implementation of the proposed method in soft micro-robots and bio-mimetic applications.


Fractures of the coracoid process are uncommon and when they do occur, are oftenmistaken for injuries to the acromi oclavicular joint. We report a case of a15-year-old boy who sustained a Salter-Harris Type 1 fracture through hiscoracoid process alongside strain of the acromioclavicular and coracoclavicularligaments. Additional imaging, specifically MRI, was critical in both correctlyidentifying this injury as a coracoid process fracture and also in determiningthat conservative management was the best course of action. Optimum managementof such injuries remains controversial, specifically with regards to skeletallyimmature patients. In our case, the injury was identified clearly on MRI andmanaged conservatively, with the patient making a full recovery and a return tocontact rugby after 3 months. The case presented here is of an isolated coracoid processfracture managed conservatively.Fractures of the coracoid process are uncommon.A 15-year-old right-hand dominant schoolboy tackled an opposition player whilstplaying rugby. He tackled head-on and, in-so-doing, sustained an impact to the rightshoulder. Upon completing the tackle, he was knocked to the ground. There wasimmediate pain, which he localized to the anterosuperior aspect of the shoulder;this was sufficiently severe to prevent him from finishing the match.An initial clinical diagnosis of AC injury was made. There was no clinical deformitybut tenderness in the region and increased pain when the joint was stressed. Theother positive finding of note was that subscapularis testing demonstratedsignificant pain with preservation of power.On the anteroposterior (AP) radiograph, there is apparent widening of the AC jointwith superior subluxation of the distal clavicle in relationship to the acromion;with an unfused distal acromial apophysis. The axial radiograph showed normalalignment of the AC joint and marked widening of the physis at the base of thecoracoid process . NormalRoutine MR shoulder sequences, including a sagittal and axial proton density sequenceto give adequate evaluation of hyaline cartilage, were performed 2 days after theaforementioned radiographs.Subsequent MRI confirmed a Salter-Harris Type 1 fracture through the base of thecoracoid process with widening of the physis, extensive surrounding haematoma andcircumferential soft tissue oedema at the site of the coracoid physeal fracture,extending deep to the supraspinatus and subscapularis muscle bellies, bestappreciated on the sagittal view . This paThe AC joint demonstrated normal alignment on MR, with a mild strain of the ACligaments and intermediate strain of the coracoclavicular ligaments.The injury was managed non-operatively with rest in a sling, physiotherapy and aphased return to play. An AP and axial radiograph were taken 10 weeks after theinjury with the AP radiograph showing progressive callus formation at the coracoidphyseal injury; whilst the lucency of the physis persists on the axial radiograph. The patCoracoid process fractures are rare, and as such our understanding and knowledgeregarding management of these fractures is limited.4 The corachobrachialis and short head of biceps tendons make up the conjointtendon, which inserts onto the tip of the coracoid process, with the pectoralisminor tendon being the only other muscular attachment to the coracoid process,inserting along the medial border  was obtained from the patient(s) for publication of this case report,including accompanying images.


Xenotransplantation using pig cells, tissues and organs may help to overcome the shortage of human tissues and organs for the treatment of tissue and organ failure. Progress in the prevention of immunological rejection using genetically modified pigs and new, more effective, immunosuppression regimens will allow clinical application of xenotransplantation in near future. However, xenotransplantation may be associated with the transmission of potentially zoonotic porcine microorganisms. Until now the only xenotransplantation-associated transmission was the transmission of the porcine cytomegalovirus (PCMV) into non-human primates. PCMV caused a significant reduction of the survival time of the pig transplant.Here the available publications were analysed in order to establish the mechanism how PCMV shortened the survival time of xenotransplants. PCMV is a herpesvirus related to the human cytomegalovirus and the human herpesviruses 6 and 7. These three human herpesviruses can cause serious disease among immunocompromised human individuals, including transplant recipients. It was shown that PCMV predominantly contributes to the reduction of transplant survival in non-human primates by disruption of the coagulation system and by suppression and exhaustion of the immune system.Although it is still unknown whether PCMV infects primate cells including human cells, indirect mechanism of the virus infection may cause reduction of the xenotransplant survival in future clinical trials and therefore PCMV has to be eliminated from donor pigs. In the last years, a significant progress was achieved in the development of xenotransplantation –4. The mViruses affecting the health of the donor pigs will harm the transplant and have to be eliminated. For some of these viruses effective vaccines exist, which prevent the infection or at least the outbreak of the disease. More difficult is the situation with viruses not affecting the health of the donor pigs. With two exceptions we do not know whether transmission of these viruses will harm the transplant recipient. On exception is HEV. It was shown that HEV can be transmitted to humans by undercooked meat or contact and that infection of immune-compromised humans leads to a chronic HEV infection, whereas infection of individuals with pre-existing liver disease may be fatal , 21. TheThe risk posed by PERV is difficult to evaluate. Retroviruses are known to induce tumours and / or immunodeficiency in the infected individuals. Until now, no transmission of PERV was observed in numerous pig to non-human primate xenotransplantations and in first clinical trials in humans , 27. If Herpesviridae, subfamily Betaherpesvirinae, genus Roseolovirus .–60.54–60Transplanting pig kidneys into baboons, the first kidney coming from a PCMV-positive animal failed after 13 days and a second kidney from a PCMV-negative animal was transplanted after excision of the first kidney. The second transplant survived for 60 days without hemorrhagic changes, clearly demonstrating the PCMV infection was the reason of the short survival time of the first transplant .Table 2XTransmission of PCMV was also observed in orthotopic pig heart transplantations in baboons , 62. PCMAt present it is still unclear, whether PCMV can infect human and non-human primate cells. There is one report showing infection of human cells  and anotAnother example for transspecies transmission of a herpesvirus is the baboon cytomegalovirus (BaCMV). This virus was shown to infect human cell in vitro . FurtherSince several reviews concerning the role of PCMV in xenotransplantation have been published , 22, 72,Consumptive coagulopathy (CC) defined by thrombocytopenia, decreasing fibrinogen, and haemorrhage, has been observed in pig to non-human primate xenotransplantations –75. InfePCMV is an immunosuppressive virus modulating the expression of immune-related genes . A PCMV No increased expression of PERV in transplanted pig kidneys from PCMV-positive animals compared with kidneys from uninfected animals was reported . The intLike many of the potentially zoonotic microorganisms, PCMV can be eliminated by selection of PCMV-negative animals or by early weaning. Since PCMV can be transmitted via the placenta, Caesarean section, colostrum deprivation and embryo transfer may be useful. There were some efforts to eliminate PCMV by early weaning , 87. OnlAntiviral drugs such as ganciclovir, cidofovir and to a lesser extent the more toxic compounds foscarnet and acyclovir have been shown to inhibit replication of PCMV . HoweverPCMV is the first virus with proven pathogenicity in xenotranplantation. In numerous preclinical trials transplanting pig organs into non-human primates, a significant reduction of the survival time was observed when the organs were PCMV-infected. The possible mechanisms of reduction of survival time are based on the disruption of the coagulation system and by suppression and exhaustion of the immune system. Although PCMV is resistant against antivirals which are effective against other herpesviruses and although vaccines against PCMV do not exist, PCMV can easily eliminated by early weaning and isolation of the negative animals.


The association between biofilm-forming microorganisms and prosthetic joint infection influences all aspect of management including approaches to diagnosis, management and prevention. This article will provide an overview of new anti-biofilm strategies for management of prosthetic joint infection. Staphylococcus aureus and Staphylococcus epidermidis, and some fungal species, such as Candida albicans  is a reducing agent that has been compared to sonication with similar sensitivity between the two techniques . SonicatBoth techniques, however, are performed on a resected prosthesis and may not be available if debridement and implant retention approaches are undertaken . Other rClinicalTrials.gov Identifier: NCT02868736) comparing the ELISA and lateral flow test in 3000 patients has recently completed recruitment and the results will provide important data on the test performance.Despite these advances, the diagnosis may still be missed in up to 30% of cases of prosthetic joint infection . Non-culThe goals of prosthetic joint treatment are 2-fold: to remove the biofilm-associated pathogens whilst maintaining a functional, pain-free joint . As withStaphylococcus species and Streptococcus species both in in vitro and clinically , a Phase 2b trial, is comparing the safety and efficacy of the SA4Ag Vaccine (Pfizer) for the prevention of S. aureus blood stream infections and/or deep surgical site infections in participants undergoing spinal implantation surgery. This trial aims to recruit 6000 participants with the planned completion data of August 2019. If successful, this trial may have implications for preventative strategies for other surgical site infections, including prosthetic joint surgery.The final prevention strategy that has been extensively investigated is vaccination strategies targeting practice . The chapractice . The STRThe inclusion of biofilm-focused strategies for arthroplasty surgery in the Centers for Disease Control and Prevention Surgical Site Infection guidelines highlight the prominence of this clinical entity. Understanding the link between biofilm and arthroplasty infections has driven research into novel strategies to diagnose, treat and prevent these infections, with the promise of significant advancements in this field in the coming years.TP was involved in all aspects of the manuscript.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.


X inactivation represents a complex multi-layer epigenetic mechanism that profoundly modifies chromatin composition and structure of one X chromosome in females. The heterochromatic inactive X chromosome adopts a unique 3D bipartite structure and a location close to the nuclear periphery or the nucleolus. X-linked lncRNA loci and their transcripts play important roles in the recruitment of proteins that catalyze chromatin and DNA modifications for silencing, as well as in the control of chromatin condensation and location of the inactive X chromosome. A subset of genes escapes X inactivation, raising questions about mechanisms that preserve their expression despite being embedded within heterochromatin. Escape gene expression differs between males and females, which can lead to physiological sex differences. We review recent studies that emphasize challenges in understanding the role of lncRNAs in the control of epigenetic modifications, structural features and nuclear positioning of the inactive X chromosome. Second, we highlight new findings about the distribution of genes that escape X inactivation based on single cell studies, and discuss the roles of escape genes in eliciting sex differences in health and disease. Evolution of the mammalian sex chromosomes from a pair of autosomes resulted in the emergence of distinct heteromorphic chromosomes that govern sex determination . The Y ccis-coating of the future inactive X chromosome (Xi) by the long-non-coding RNA (lncRNA) Xist , a mechanism that results in silencing of a randomly chosen X chromosome in early female embryogenesis . XCI is NA) Xist . Layers n the Xi . These mNA Firre .Despite the multiple layers of gene repression that stabilize XCI, a subset of developmentally critical genes remains expressed, albeit at a lower level, from the Xi . These eThis review focuses first on mechanisms that govern X chromosome structure and nuclear location in relation to XCI, with a specific emphasis on the role of X-linked lncRNAs in these processes. We next discuss mechanisms that allow a select subset of genes to escape silencing in the context of the repressed Xi environment and how single-cell RNA sequencing has been used to identify novel escape genes. Lastly, we review new data on the role of escape gene dosage in sex differences in health and disease.Xist, Dxz4, and Firre, which have been implicated in various aspects of the onset and maintenance of XCI.The importance of lncRNAs in controlling nuclear structure and gene expression has become increasingly clear . Here weXist in great detail   . Interesn of XCI .Xist RNA   . Most of “Dxz4”) . Xist RNilencing . Indeed,granules .Xist RNA-mediated gene silencing is accomplished through recruitment of proteins that establish epigenetic and structural modifications on the Xi  that act as waystations to enhance Xist RNA coating throughout the Xi 3D space  with different functions in XCI. The utmost importance of the A-repeat in X silencing but not in Xist RNA coating was recognized early on, and has been further strengthened by demonstrating that it recruits the transcriptional repressor SPEN  emanating from Dxz4 in the contact map  with incomplete XCI in a subset of cells, and also confirmed heterogeneity of XCI for TIMP1  and Klinefelter syndrome  directly implicate escape genes in abnormal phenotypes, since such genes would be haplo-insufficient and overexpressed, respectively, in these conditions. One of the hallmark of Turner syndrome is premature ovarian failure and infertility. The X chromosome is three-fold more enriched for genes expressed in female reproductive organs when compared to autosomes, indicating a role in female fertility . Defininectively . Recent ectively . Of courectively . The infectively . Furtherectively . TogetheWhile a great deal has been learned about the various lncRNAs and proteins that control structural and epigenetic features of the Xi and its silencing, their exact modes of action remain to be further studied. In terms of the 3D structure of the Xi it will be of great interest to define factors involved in nuclear compartmentalization and phase separation. Additional experiments are needed in order to link the specific 3D structure and nuclear location of the Xi with its distinct epigenetic landscape. Moreover, little is known about contacts between each X chromosome with the rest of the genome in female cells and tissues, and about such contacts with the heterochromatic Y chromosome in male cells and tissues. Tissue-specific differences in the arrangement of chromosomes within the nucleus are poorly understood, and few functional studies have examined the consequences of manipulating chromosomal location. The epigenetic controls of escape from XCI also warrant further functional studies. Unfortunately, current analyses toward identifying escape genes in specific cell types and tissues are limited due to the relatively small number of informative polymorphisms in human. However, single-cell analyses are progressing at a fast rate, with some methods allowing analyses in thousands of cells in tissues to establish maps of gene expression or accessibility in a whole organism, as shown for example in a recently published atlas of mouse tissues/cell types . While tJB, HF, and CD outlined and wrote the review. All authors approved the final 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.


One of the major components of the functional process in the nasal cavity is taken up by the respiratory epithelium covering the posterior two-thirds of the nasal cavity. Disruption in the cytoarchitectonics and subcellular changes in this epithelium results in a number of functional changes in the nasal cavity. One of the rare and usually iatrogenic disturbances of this type is described in 1996, although noticed and discussed significantly earlier, by Kern and Stenkvist empty nose syndrome (ENS) or secondary atrophic rhinitis. The clinical hallmarks of ENS are described as paradoxical feeling for nasal obstruction in the presence of actually widened nasal airways. This phenomenon is attributed to the efferent neuronal signal dissociation accompanying the changes in the nasal mucosa. Herein we report the findings in a 50-year-old male. The patient presented with chronic right-sided headache, foul discharge and complaints of a stuffed nose in 2011. Endoscopy and computed tomography (CT) showed complete destruction of the hard plane, nasal septum, and right maxillary septum, leading to a formation of a huge neocavity. Due to the past medical history and the severity of the case biopsy specimens were obtained under general anesthesia. The sections showed severe but unspecific changes of the nasal epithelium with areas of minimal remaining preserved respiratory epithelium. Based on the clinical data and endoscopic, CT and histomorphologic data, despite the case is not applicable to the current classification of ENS, the diagnosis of ENS was accepted based on the combined extensive but unspecific findings. A seven-year follow-up period included multiple hospital admissions for monitoring of the condition and extensive sinus lavage. No advancement was noticed. One of the major components of the functional process in the nasal cavity is taken up by the respiratory epithelium covering the posterior two-thirds of the nasal cavity . DisruptOne of the rare and usually iatrogenic disturbances of this type is described in 1996, although noticed and discussed significantly earlier, by Kern and Stenkvist empty nose syndrome (ENS) or secondary atrophic rhinitis . ENS is The clinical hallmarks of ENS are described as paradoxical feeling for nasal obstruction in the presence of actually widened nasal airways . This phThe most common reasons for ENS as already mentioned are repeated endonasal procedures, based on the locations of which ENS can be classified into four categories: ENS due to inferior turbinate, middle turbinate, and combined inferior and middle turbinate resections, and recurrent turbinate sparing procedures .Herein we report the findings in a 50-year-old male. The patient presented with chronic right-sided headache, foul discharge and complaints of a stuffed nose in 2011. Previous medical history included maxillary osteomyelitis in 2009 with multiple interventions resulting in a complex mediofacial resection, with a subtotal defect of the hard plate, maxillary sinus, and nasal septum. Concomitant diseases included depression and type two diabetes mellitus on peroral therapy.A nasal endoscopy and computed tomography (CT) showed a huge pathologic cavity resulting from communication between the oral cavity, right maxillary sinus, and nasal cavity, with a minor communication with the right orbit Figures -2.Due to the past medical history and the severity of the case biopsy specimens were obtained under general anesthesia. The tissues were fixated in 10% buffered formaldehyde solution and later embedded in paraffin (FFPE) for staining with hematoxylin and eosin (H&E) and Azan.The sections showed severe but unspecific changes of the nasal epithelium with areas of minimal remaining preserved respiratory epithelium Figure . These c Based on the clinical data and the endoscopic, radiologic and histomorphologic data, despite the case is not applicable to the current classification of ENS, the diagnosis of ENS was accepted based on the combined extensive but unspecific findings. Due to the current classification criteria, however, the case was concluded to be ENS not elsewhere classified (NEC) as the currently used classification does not include such an entity.The patient was discharged following training for self-lavage of the neocavity, due to refusal for obturator placement to compartmentalize the neocavity and restore physiological relation of its sections. A seven-year follow-up period included multiple hospital admissions for monitoring of the condition and extensive sinus lavage. No advancement was noticed on endoscopy and CT. No further histological evaluations were performed due to the steady clinical course and lack of evidence for malignant transformation of the now squamous epithelial lining of the neocavity.Many current revisions of classifications are now expanding to include NEC types, due to the wide variety of clinical, endoscopic, radiological and histological criteria . The curThe current case, despite its severity, shows that continuous monitoring and patient education can lead to a relatively stable condition, without progression over a seven-year follow-up period, despite initial presentation.The current report illustrates the wide range of histological changes found in a single ENS case. Despite being a secondary iatrogenic entry, ENS classifications should widen the scope to include histological criteria, as the severe changes illustrated by this case, can be considered as a precancerosis.


Newcastle disease virus (NDV) causes morbidities and mortalities in wild and domestic birds globally. For humans, exposure to infected birds can cause conjunctivitis and influenza-like symptoms. NDV infections in mammals are rarely reported. In this study, using next-generation sequencing, an NDV was identified and isolated from Vero cells inoculated with the nasal swab of an aborted dromedary fetus in Dubai, during the time when an NDV outbreak occurred in a pigeon farm located in close proximity to the dairy camel farm where the mother of the aborted dromedary fetus resided, and there were a lot of pigeons in the camel farm. Genome analysis revealed that the structurally and functionally important features of other NDVs were also present in this dromedary NDV genome. Phylogenetic analysis based on the nucleotide sequences of fusion protein (F), hemagglutinin-neuraminidase protein (HN) and complete polyprotein showed that the virus belonged to sub-genotype VIg of class II NDV and is most closely related to pigeon NDVs in Egypt in the same year. The present study is the first that demonstrated isolation of NDV in dromedaries. Further study is warranted to investigate the relationship between NDV infection and abortion. Avian avulavirus 1, is a negative-sense single-stranded RNA virus that belongs to the genus Avulavirus of the Paramyxoviridae family. It is an avian virus causing high morbidities and mortalities in most species of wild and domestic birds globally and is associated with major economic loss in poultry farms worldwide .,29.N-gly strains  .d 96–110  28,30]..N-glycosd 96–110  [30]. Ovd 96–110 .To determine the phylogenetic relationship between the DcNDV and other NDV strains reported, phylogenetic trees based on the nucleotide sequences of F, HN, and complete polyprotein of DcNDV and those of other NDVs were constructed a–c. PhylThe nucleotide sequence of the genome of the DcNDV in this study has been lodged within the GenBank sequence database under accession no. MK673997. Raw data of Illumina HiSeq have been submitted to Sequence Read Archive (SRA) under accession no. SRR8569164.112KRQKRF117 in the F protein, conserved amino acid residues at neutralizing epitopes on the HN protein, and the size  of the HN protein (We report the first isolation of NDV in a dromedary. NDV is basically an avian virus which has caused numerous outbreaks of devastating disease in poultries. In the present study, we isolated an NDV from the nasal swab of an aborted dromedary fetus in Dubai using Vero cells, and the virus isolate also produced the typical CPE caused by NDV on chicken embryo fibroblasts. During that period, an NDV outbreak occurred in the pigeon farm located in close proximity to the dairy camel farm where the mother of the aborted dromedary fetus resided and there were a lot of pigeons in the camel farm. We speculate that the mother dromedary had probably acquired the NDV from the pigeons and subsequently transmitted to the fetus. Whole genome sequencing and comparative genome analysis revealed that the structurally and functionally important features in the F and HN proteins of the NDV genome observed in previous studies were also present in the genome of this NDV in dromedary. In particular, it also possessed genomic features similar to other velogenic NDVs, including cleavage site motif  protein  30,31].,31.112KRThe present dromedary NDV was probably resulted from spillover from NDVs in birds of the Middle East. NDVs of class II genotype VI were first isolated from pigeons in the Middle East in the 1960s and spread rapidly throughout Northern Africa to Europe and other parts of the world ,32. So fThe use of next generation sequencing technologies has led to the discovery of many novel viruses as well as identification of known viruses that were previously not reported in dromedaries. The MERS epidemic and discovery of dromedaries as the reservoir of MERS-CoV and our discovery and isolation of a novel dromedary camel CoV UAE-HKU23 have boosted interest in search of more novel viruses in dromedaries ,36,37. I


N,N-Dimethyl-N-dodecylglycine betaine). A combination of absorbance, fluorescence, and circular dichroism spectroscopic measurements elucidated the protein denaturation and heme dissociation from myoglobin. The results indicated that Empigen BB was not able to fully denature the myoglobin structure, but apparently can induce the dissociation of the heme group from the protein. This provides a way to estimate the heme binding free energy, ΔGdissociation. As ionic liquids (ILs) have been shown to perturb the myoglobin protein, we have investigated the effects of the ILs 1-butyl-3-methylimidazolium chloride (BMICl), 1-ethyl-3-methylimidazolium acetate (EMIAc), and 1-butyl-3-methylimidazolium tetrafluoroborate (BMIBF4) in aqueous solution on the ΔGdissociation values. Absorbance experiments show the ILs had minimal effect on ΔGdissociation values when compared to controls. Fluorescence and circular dichroism data confirm the ILs have no effect on heme dissociation, demonstrating that low concentrations ILs do not impact the heme dissociation from the protein and do not significantly denature myoglobin on their own or in combination with EBB. These results provide important data for future studies of the mechanism of IL-mediated protein stabilization/destabilization and biocompatibility studies.We have investigated myoglobin protein denaturation using the zwitterionic detergent Empigen BB (EBB,  It has proven very difficult to directly investigate the thermodynamics and kinetics of native protein folding due to the fact that proteins fold in the cell, often co-translationally. Many natural proteins also require the action of chaperones to properly fold into the functional three-dimensional 3D) conformation. The folding process in cells also is directly impacted by the incorporation of non-protein cofactors and prosthetic groups such as heme. However, only very elegant experimental designs using whole cell extracts and non-native amino acids can begin to address folding in a cellular context . As suchD conformThe use of surfactants or detergents to denature proteins is widely applied in life sciences research, most ubiquitously in the use of SDS-PAGE (Sodium Dodecyl Sulfate—PolyAcrylamide Gel Electrophoresis) ,11. DeteProtein unfolding studies in the presence of ionic liquids (ILs) have recently gained attention in order to understand the effects of ILs on protein structures and stabilities for their potential biomedical applications. The tunable properties of the cation-anion pairs in ILs allow for selection of compatibility and optimal functionality for a variety of desired applications. There have been a variety of reports describing the application of ILs for enzyme inhibition, drug delivery, and biomedical devices ,21,22,234) was shown to enhance the thermal stability of bovine serum albumin (BSA)  in each ionic liquid environment was calculated byR is the ideal gas constant (8.314 J·mol−1), T is the temperature in kelvin , and OD409 is the peak absorbance of myoglobin solution at 409 nm. Data were analyzed in Microsoft Excel  and fit to a linear trendline with an R2 cutoff of 0.95. The y-intercept of each trendline was the estimated Gibbs free energy of dissociation (ΔGdissociation). This value reflects the energy of dissociation of the heme group from the myoglobin protein. The ΔGdissociation values were tested for statistical significance using an analysis of variance (ANOVA) and post-hoc testing for each variable using MS Excel with the XL-toolbox add-in [The equilibrium constant of dissociation [x add-in .Interventionary studies involving animals or humans, and other studies require ethical approval must list the authority that provided approval and the corresponding ethical approval code.Empigen BB is not a typical denaturant for proteins, and therefore it is important to characterize the interaction of this zwitterionic detergent with myoglobin and heme. As absorbance spectroscopy in the heme region is typically used to quantify myoglobin unfolding ,42, the In light of these findings, we compared heme fluorescence dequenching when myoglobin was denatured by EBB with denaturation by the chaotrope guanidinium chloride (GuHCl). It has been well-demonstrated in the literature that as the distance between the native Trp residues and the heme increases, Trp fluorescence emission intensity increases. Our fluorescence results D show thConsidering EBB does not cause full denaturation of the protein but is sufficient to remove the heme, the effect of low concentrations of ILs on this process was monitored using absorbance spectroscopy, specifically using the heme absorption, or Soret band, at 409 nm. As the heme is lost from the binding pocket there is a concomitant loss of absorptivity and a slight red shift in the Soret band ,43,47. C4 did not significantly affect the dissociation profile with EBB. This is an important finding as it was previously shown that high concentrations of BMIBF4 can denature myoglobin in the absence of other chaotropes [4 exhibited a lower absorbance than the samples treated with detergent alone .Myoglobin was exposed to increasing concentrations of Empigen BB in the presence of a fixed concentration of IL or salt. The results of these experiments are shown in aotropes ,43. Howent alone F. This sThe EBB-induced heme dissociation was next investigated by changes in emission intensity of the intrinsic tryptophan. In the folded state, the iron at the center of the heme group efficiently quenches Trp fluorescence emission through a heavy atom quenching mechanism ,50. As tThe results of the dissociation experiments monitored by Trp fluorescence are shown in Circular dichroism spectroscopy is a widely utilized method for evaluating protein folding because of the distinctive spectral shapes associated with protein secondary structures in the far UV spectral region. However, due to the strong absorption of the imidazolium-based ILs in the far UV region, traditional protein structural analysis by CD is unreliable. For this reason, the signal produced by heme was used, as the restricted chiral environment provided by myoglobin generates a positive CD band to protein-associated heme . This apHeme dissociation was also monitored by the loss of the CD signal at 409 nm as a function of EBB in the presence of varying concentrations of ILs or salts. Similar to the results from the absorbance measurements, the CD results showed the sigmoidal dissociation profiles . Consist4 which has been shown to destabilize myoglobin, enhancing the denaturing effects of GuHCl. The results presented in 4 (or the other ILs) on heme dissociation, indicating there is likely no additional protein denaturation caused by the inclusion of the ILs. In aggregate, the spectroscopic results indicate that the dissociation of heme from myoglobin by EBB is generally insensitive to the addition of a variety of ILs and salts, and to modest changes in the ionic strength of the solution environment. The lack of sensitivity to ionic strength is not surprising considering the hydrophobicity-driven mechanism of detergent mediated unfolding that is also the likely driver of heme displacement from the binding pocket. The range of ionic strengths tested in this work is relatively modest considering the typical ionic strengths in biological environments, so the protein stability is not likely to be significantly affected. More interestingly, the heme dissociation profile was not significantly affected by the inclusion of ILs, especially BMIBFH-values (Equations (1) and (3)) to compute heme-dissociation free energies. Using the data collected, the ΔGdissociation was calculated for each of the IL/salt conditions tested. As reported previously for denaturation of myoglobin, the A409 values from the absorbance spectroscopy experiments can be used to calculate the equilibrium constant (eqK) from the transition region of unfolding curve and then the ΔGdissociation can be directly calculated [unfolding for the complete denaturation of the protein, but in this case, the transition more accurately reflects the ΔGdissociation of the heme from the protein. The values for ΔGdissociation under all conditions are shown in dissociation were ~8000 kJ/mol in most cases, regardless of the IL identity or concentration. The values were compared using an ANOVA method which showed no statistical significance based on IL identity or IL concentration.The spectroscopic experiments described allows for further quantification of the dissociation of heme from myoglobin by EBB. Assuming that the loss in heme absorbance and CD signal is attributable primarily to heme dissociation and not protein unfolding, we can use the lculated ,43. PrevMany reports have focused on ILs ability to form micelles and the investigation of the denaturing mechanism in relation to a micelle- or detergent-like mechanism . ConsideThe CMCs for EBB in the presence of varying concentrations of IL or control salts are shown in The interactions of ionic liquids with biomolecules is an area of great interest due to the proliferation of ILs in various electrochemical and industrial applications. The role of the employed chemical species, concentration, and mechanism of unfolding have all been areas of significant investigation. Nonetheless, there are still questions that surround the interactions, making predictive modeling difficult at this point.N-dodecyltrimethylammonium chloride (DTAC) demonstrated that the detergents destabilize certain regions of the myoglobin protein, rather than causing total denaturation [Myoglobin has been a well-studied model protein over several decades. The high abundance in biological samples, commercial availability, and favorable spectroscopic properties make it an ideal system for studying protein folding and stability. Additionally, the fact that myoglobin is exclusively composed of α-helical secondary structure allows for straightforward and unambiguous interpretation of CD spectra. These studies provide a well-established baseline of behavior for myoglobin in the presence of ionic species and detergents. The work of Tang et al. investigated the role of ionic strength on myoglobin denaturing, demonstrating that ionic strength stabilized both the native conformation and the transition (T) state of myoglobin on the unfolding pathway when thermally denatured . Similarturation ,63. Thisturation . There wturation ,65,66. TThe denaturation profile of myoglobin with and without ILs present show similar behavior, with only partial loss of helical content. However, this loss of helical content is concomitant with a dramatic loss of heme absorbance. These results can be interpreted in the detergent denaturing secondary structural elements near the heme binding pocket, thus disrupting the molecular contacts that allow the heme to be positioned within the protein. The preponderance of hydrophobic amino acids in the heme binding pocket provide an ideal binding site for detergent molecules, and thus provide a likely point of local structural disruption . The EBB4 on myoglobin denatured by GuHCl. The same work showed a much weaker destabilizing effect of EMIAc, pointing to the role of the anion in the mechanism of protein destabilization. Similarly, Constantinescu et al. investigated the role of EMI-based ILs and the Hofmeister series on the stability of RNase, demonstrating that the anion (Tf2N)—was the most destabilizing anion when coupled with the EMI cation, [4 (300 mM) will denature myoglobin over time thus the effects of low concentrations of IL are relevant and important to understand. The results presented here indicate that lower concentrations of the disruptive IL BMIBF4 do not have significant effects on the dissociation of heme from myoglobin. There have been many previous reports of how ILs affect stability of proteins, including myoglobin, and the role of both the cation and anion in these interactions. Previous work by Fiebig et al. demonstrated the significant destabilizing effect of 150 mM and 300 mM BMIBF cation, . These s cation, . Notably4−, which was attributed to either a hydrophobic or H-bonding driven mode of action [4 alone do not induce myoglobin unfolding under these conditions, indicating that the enhancement of unfolding was likely driven by an additive or synergistic effect between GuHCl or urea and BMIBF4, which is clearly not present for the EBB interactions. However, it is important to recognize that the behavior of ILs at the lower concentrations tested here may differ when concentrations are increased as in previous reports where higher concentrations of BMIBF4 will denature myoglobin over longer timescales, well beyond those presented above. Nonetheless, the lack of enhancement of denaturation or heme dissociation indicates the BMIBF4 and the EBB may destabilize the protein and heme through different mechanisms, or perhaps acting at different sites on the myoglobin. The spectroscopic results indicate that the destabilizing effects of ionic liquids on myoglobin are more complex than a simple ion-protein interaction and are likely linked to the chaotrope and the underlying molecular mechanism by which they act.The mechanism of IL-based protein denaturation is also still an open question in the field. Many reports have focused on the surfactant-like properties of imidazolium-based ILs, linking the surfactant properties to the length of the alkyl chain attached to the imidazole ring ,71,72. Mf action . Thus, tTaken together and in the context of the known mechanisms of interactions between myoglobin and detergent, the EBB provides an attractive alternative to traditional cationic and anionic surfactants for protein denaturation studies. The incomplete unfolding of the protein does not prevent complete loss of the heme from the binding pocket, setting up the potential for incorporation of EBB in expedited purification of heme or apo-forms of the protein. Additionally, this work shows that ionic liquid enhanced protein denaturation is dependent on the denaturing agent used. This lack of interaction between the IL and the EBB denaturing agent provides insight into related studies of proteins and ILs. Further, it suggests that ILs may be useful in applications that require partial unfolding of proteins, selective folding/unfolding conditions, and lead to more fundamental understanding of the nature of protein-IL interactions as well as the mechanisms of IL mediated stabilization and destabilization of proteins.


A definitive diagnosis of extensive suture line exostoses affecting the nasofrontal, nasolacrimal, nasomaxillary, frontolacrimal, lacrimozygomatic and lacrimomaxillary suture lines in a 7-year-old Thoroughbred mare with chronic bilateral epiphora and facial deformation was achieved using standing computed tomography (CT) examinations. Positive contrast dacryocystorhinography using CT revealed partial bilateral obstruction of the nasolacrimal ducts. Minimally displaced depression fractures of the right nasal bone, the right maxillary bone and right frontal bone were also demonstrated. The cosmetic appearance of the periosteal reaction associated with the suture line exostosis and epiphora significantly improved within 3 months of diagnosis and treatment. The equine skull is composed of many flat bones bound together by collagen fibres and overlying periosteum Dixon . CraniofInflammation and suture line exostosis of the equine craniofacial suture lines result in regional swellings and is a common cause of firm facial swellings in the horse Carslake . Suture This case report describes the clinical, radiographic and computed tomographic findings of suture line exostoses of the nasofrontal, nasolacrimal, frontolacrimal, lacromaxillary and lacrimozygomatic craniofacial suture lines in a skeletally mature Thoroughbred mare who suffered severe facial trauma. The use of computed tomography (CT) with dacryocystorhinography has not previously been reported in a standing horse.Ethical clearance was not needed or required for the study.A 7-year-old Thoroughbred mare was presented to the Onderstepoort Veterinary Academic Hospital (OVAH) for the evaluation of a severely painful facial swelling of approximately 6 weeks’ duration. The owners reported that she had suffered severe violent blunt trauma directly to the facial region that resulted in extensive facial swelling over the following 48-hour period. Intermittent treatment with non-steroidal anti-inflammatory medicines and stable confinement in the 6 weeks prior to referral had failed to resolve the facial swelling, and bilateral epiphora had developed. Additionally, bilateral conjunctivitis developed 5 days prior to presentation at the OVAH.Clinical examination at presentation showed normal clinical parameters and mentation. Bilateral epiphora with a purulent discharge from the medial canthi and three discrete firm prominent swellings situated between the eyes in the areas of the frontal, nasal and lacrimal bones  were obsRadiographic and CT examinations with retrograde dacryocystorhinography, under a standing sedation protocol, were performed. An intravenous catheter was aseptically placed in the left jugular vein , and the mare was sedated using intravenous detomidine hydrochloride  and intravenous butorphanol tartrate . Radiographic evaluation of the head was performed using a ceiling-mounted computed radiography unit . Latero-lateral and the oblique (RtDLeVO and LeDRtVO) radiographic projections  and c reBilateral retrograde dacryocystorhinography was performed using 15 mL of Iohexol , with a 15-cm 6-FR Foley catheter (Foley catheter) inserted into the nasal puncta. Motion artefact was present in all the scans because of the standing sedation method employed but did not significantly affect the diagnostic quality of the images. The examination revealed mild soft tissue swelling associated with underlying marked bilateral irregular periosteal proliferation of the caudal aspects of the nasal bones, lacrimal bone, rostral aspect of the frontal bone and the nasofrontal-, nasolacrimal- frontolacrimal-, lacrimozygomatic and lacrimomaxillary suture areas  and e. TMinimally displaced depression fractures of the right nasal bone, the right maxillary bone and right frontal bone were demonstrated, despite the presence of motion artefact. Surgical intervention was not deemed necessary based on the mild displacement, good stability of the fracture and callus formation.At the completion of the CT examination and with confirmation of the patency of both nasolacrimal ducts, retrograde flushing of both ducts was performed successfully, although significant pressure had to be applied to the right nasolacrimal duct. The mare was discharged with instructions of stable confinement for 6 weeks, and a therapeutic regimen of ocular chloramphenicol  twice daily for 5 days and oral meloxicam  once daily for 14 days.A re-examination 6 weeks post-presentation and 12 weeks after the initial traumatic episode revealed that the mare had normal clinical parameters and mentation. A firm area of swelling was evident extending between the medial canthi of both eyes. Palpation of this swelling revealed a moderate pain response, but this was markedly reduced compared to 6 weeks previously. There was no evidence of epiphora or conjunctivitis, and the owners reported the mare to be showing significantly less resentment to general palpation of the facial region.A second radiographic evaluation was performed under a standing sedation protocol, which revealed no significant differences compared to the initial radiographs.The owners were requested to maintain the mare in stable confinement and to commence hand walking and trot work on the lunge over the next 6 weeks. A telephone update at this point confirmed that the mare had made excellent progress, and small paddock turnout was commenced.Eight months after the initial injury, the mare had returned to her normal routine, although a firm swelling was still present at the nasofrontal suture line area. The lacrimal and maxillary bones appeared normal . The marLocal facial trauma has been suggested as the likely aetiology of the inflammatory response seen in suture line exostosis Carslake . This aeAlthough, like the mare in this case, some affected horses have a definite history of a single episode of head trauma, present with acute-onset epistaxis or have a history of recent facial bone surgery, the majority of cases of suture line exostosis do not. Two studies reporting a series of suture exostosis cases revealed 0/16 cases (Gibbs & Lane Clinical signs of suture line exostosis of the nasolacrimal, maxillolacrimal and frontolacrimal suture lines commonly include bilateral diffuse swellings situated rostrally to the medial canthus of the eye with or without accompanying epiphora Carslake . These sRadiographic evaluation is the most common method of diagnosing suture exostosis (Butler et al. In this case, the resolution afforded by CT with the use of dacryocystorhinography confirmed partial obstruction of both nasolacrimal ducts and allowed the severity and location of the nasolacrimal duct obstructions to be determined.Dacryocystorhinography has been frequently performed under standing sedation Carslake , and theDacryocystorhinography is a moderately technically demanding procedure and can be irritating for the horse because of the pressure within the duct Carslake . It has After accurate identification, craniofacial suture line exostoses are normally treated successfully with stable confinement and anti-inflammatory treatment (Bonilla et al. The significant improvement in this case after 8 months has been previously recorded in studies assessing the long-term outcome of these cases Carslake . NasofroReassessment and surgical excision or stabilisation has been recommended in cases with persistent epiphora, large lesions unresponsive to conservative therapy, or in cases where the cosmetic appearance and epiphora are unacceptable to the owners (Bonilla et al. It is the opinion of the authors that CT examination with positive contrast dacryocystorhinography is a very useful technique for the diagnosis of suture line exostosis and evaluation of the patency of the nasolacrimal ducts when the nasofrontal or lacrimomaxillary suture lines are affected and epiphora is present. There is minimal risk to the horse as both diagnostic techniques can be performed in the standing, sedated horse. However, a sliding gantry design CT is required for standing equine CT to be performed. Further studies utilising CT will be of benefit to assess the complexities and relationships of suture line exostosis, as well as to define the relationship between fractures and exostosis.