Injuries to the central nervous system most often lead to irreversible damage in humans. Brittle stars are marine animals related to sea stars and sea urchins, and are one of our closest evolutionary relatives among invertebrates. Extraordinarily, they can perfectly regenerate their nerves even after completely severing the nerve cord after arm amputation. Understanding what genes and cellular mechanisms are used for this natural repair process in the brittle star might lead to new insights to guide strategies for therapeutics to improve outcomes for central nervous system injuries in humans.Amphiura filiformis can regenerate both components of its central nervous system as well as the peripheral system, and understanding the molecular mechanisms underlying this ability is key for evolutionary comparisons not only within the echinoderm group, but also wider within deuterostomes. Here we characterise the neural regeneration of this brittle star using a combination of immunohistochemistry, in situ hybridization and Nanostring nCounter to determine the spatial and temporal expression of evolutionary conserved neural genes. We find that key genes crucial for the embryonic development of the nervous system in sea urchins and other animals are also expressed in the regenerating nervous system of the adult brittle star in a hierarchic and spatio-temporally restricted manner.Neural regeneration is very limited in humans but extremely efficient in echinoderms. The brittle star  Regeneration of the nervous system is a question that fascinated scientists for decades, especially considering that axons of the central nervous system (CNS) notoriously fail to regenerate in humans compared to peripheral nerves. This is due to a variety of limiting factors such as the lack of attractive or trophic factors in the adult brain and spinal cord, or in fact the presence of inhibitory factors of neurite growth in the CNS . In contAmphiura filiformis, which showed that the regeneration of the adult skeleton re-uses parts of the established embryonic skeletogenesis gene regulatory network [Amphipholis kochii [Amphiura filiformis [Amphipholis kochii and Ophioderma brevispinum [Brittle stars are members of the echinoderm phylum of marine invertebrates characterised by their pentaradial body plan organisation. Echinoderms belong to the ambulacrarians, which together form a phylogenetic sister group with chordates, and remarkably can regenerate most adult structures including their central nervous system. Both the generation of new neurons under normal physiological conditions as well as neurogenesis during post-traumatic regeneration has been extensively studied in related echinoderms—sea cucumbers ,13,14,15 network ,23,24. P network ,26,27. Bs kochii  and Amphliformis  revealinvispinum ,31. Remavispinum ,33.soxB genes) have been shown to first regulate the formation of the embryonic neurectoderm and also to maintain neurectodermal cells in a progenitor state in a variety of animals from Nematostella, through sea urchins to vertebrates [atonal and neuroD are involved in the specification of these cells and differentiation into subsets of neurons [musashi, and embryonic lethal abnormal vision (elav) are involved in the final differentiation stages of neurogenesis [six3 has been shown to be required for the development of all neurons in the embryo [pax6 expression has not been extensively studied in the sea urchin embryo but it is expressed in adult sea urchin tube feet where it is presumed to have a role in photoreception [Amphiura filiformis.Here, we elucidate the potential molecular drivers of neuronal growth and repatterning during arm regeneration in the brittle star by looking at the expression of key transcription factors regulating neurogenesis across the animal kingdom. Group B SRY-related HMG box genes  in London in filtered artificial seawater . During all manipulations live Amphiura were anaesthetised in a solution of 3.5% MgCl2·6H2O in a 50:50 solution of filtered sea water (FSW) to milliQ H2O. For 50% differentiation index (DI) and 95% DI regenerated stages arms were amputated at the intersegmental boundary at a fixed distance from the disc dependent on its size in accordance with standardised tables composed previously [eviously  or at 1 eviously . A maximeviously  or earlyeviously . Followi2O gradient  at room temperature (RT). Following 3 PBST  washes (RT), a permeabilization step involving PBSTX (PBS with a 1:100 Triton-X100) was used for 1hr at RT before washing with a blocking buffer (PBST + 4% Goat serum) for 30 mins at RT. The primary antibodies used in this work are listed in Regenerating arm samples that were fixed for WMISH as above in 4% PFA were used for the majority of antibody experiments with the exception of the samples for anti-synaptotagmin B staining, which were fixed in PFA for 15 min at room temperature and postfixed in 100% methanol for 1 min. Arms were first rehydrated using a descending Ethanol: HWMISH was carried out according to the protocol outlined previously , with noSupplementary Materials (Table S2)The NanoString nCounter dataset used in this study was created, validated and described in detail in a previous publication . BrieflyAmphipholis kochii and Ophioderma brevispinum, suggesting that the architecture of the brittle star arm central and peripheral nerves is conserved within ophiuroids [Amphiura filiformis [A. filiformis is similar to that in other brittle stars. The central nervous system composed mainly of the radial nerve cord is positioned in the oral part of the segments and extends throughout the arm  staining highlights not only the radial nerve cord running through the middle oral side of the proximal differentiating segments of the regenerating arm , while Afi-pax6 and Afi-soxC are strongly expressed in the developing distal structure. Interestingly, their expression precedes the morphological differentiation of the distal structures including the terminal podium surrounded by the terminal ossicle [Afi-atonal and Afi-nkx2.1 have no detectable expression at this stage  of  ossicle . Afi-atois stage . In addior cells , is exprNext, we wanted to study in depth the quantitative changes in expression of the various neuronal genes throughout regeneration using a high-throughput gene expression analysis system—the nanostring nCounter. We used probes from a previously published codeset  and lookAfi-elav, Afi-soxB2, Afi-soxB1, Afi-musashi, Afi-neuroD, and Afi-six3 , the transcription factors Afi-pcna, Afi-mycb and Afi-piwi, they are also strongly downregulated right after amputation and begin to be turned on between 48 and 72 hpa, and are very strongly upregulated in blastemal stages and late regeneration stages  is free from neurogenic gene expression until clear segments and/or podia appear. It seems then that there is a growth zone just proximal to the terminal tip that adds new metameric units in which the new radial nerve cord and its axonal extensions begin to differentiate and mature, reminiscent of the recently proposed mechanism of indeterminate homeostatic growth for adult brittle star arms [Afi-mycb, Afi-piwi and Afi-vasa compared to the neuronal genes Afi-elav, Afi-neuroD and Afi-pax6 (Afi-six3) can dedifferentiate to give rise to new neurons. It would also be very interesting to understand whether the early regeneration of the nerve cord drives the formation of the whole arm, such as the nerve dependent regeneration of many animals including salamander limbs [Afi-soxB2 expression suggests its role in the laying out of the future metameric units as well as the formation of the peripheral neuroectodermal territories such as the ciliated epidermis and podia. This is in line with the role of soxB2 in posterior growth in annelids [The ability to regenerate a complete central nervous system is extraordinary among deuterostomes, and so understanding how it occurs in brittle stars may yield important information to focus efforts for regenerative medicine. We show here that the brittle star neration , neurogetar arms . This apAfi-pax6 . Togetheer limbs ,48 and eer limbs . Afi-soxneration % distal annelids . Perhapsannelids  and Deltannelids .We provided a first glimpse into the anatomy and regulatory genes involved in brittle star neuronal regeneration, which will provide a platform for understanding adult neurogenesis in deuterostomes using an experimental model which so readily regrows and repatterns components of both the central and peripheral nervous system.


Thrombotic thrombocytopenia purpura is characterised by microangiopathic haemolytic anaemia and red cell fragmentation on the peripheral smear, neurological involvement and thrombocytopenia. Diagnosis in the context of sickle cell disease can be challenging due to the inherent haemolytic state and the multitude of other associated complications of the latter. Specifically, fat embolism syndrome characterised by respiratory failure, neurological impairment and thrombocytopenia can be misdiagnosed this way. Confirmation of a diagnosis of thrombotic thrombocytopenic purpura requires demonstration of very low levels (<10%) of the metalloproteinase ADAMTS13 which in fat embolism syndrome is normal. Existing scoring systems used to estimate the pre-test probability for thrombotic thrombocytopenic purpura cannot be applied in patients with sickle cell disease due to the chronic underlying haemolysis. Here, we analyse the diagnostic approach in published cases of thrombotic thrombocytopenic purpura affecting patients with sickle-cell disease. The vast majority of cases were characterised by severe respiratory failure before any other manifestation, a feature of fat embolism syndrome but not of thrombotic thrombocytopenic purpura, and all received red cell transfusion prior to receiving therapeutic plasma exchange. Despite the potential overestimation of the pre-test probability using the existing scoring systems, a large number of cases still scored low. There were no cases with documented low ADAMTS13. In the majority this was not tested, while in the 3 cases that ADAMTS13 was tested, levels were normal. Our review suggests that due to many overlapping clinical and laboratory features thrombotic thrombocytopenic purpura may be erroneously diagnosed in sickle cell disease instead of other complications such as fat embolism syndrome and confirmation with ADAMTS13 testing is essential. Thrombotic microangiopathies (TMA) are a group of conditions defined by thrombocytopenia and microangiopathic haemolytic anaemia with resultant end organ damage. Thrombotic thrombocytopenic purpura (TTP) is a type of TMA defined by a deficiency of the metalloproteinase ADAMTS13, and can be immune-mediated (iTTP) or congenital (cTPP) ,3. Howev− [There is significant overlap between the clinical presentation of iTTP and complications of sickle cell disease (SCD). Like iTTP, complications of SCD include haemolysis, often with abnormal peripheral blood morphology. Fat embolism syndrome (FES) is a catastrophic complication of SCD that can cause neurological impairment, multiorgan failure, elevated lactate dehydrogenase (LDH) and thrombocytopenia similar to iTTP, making the two diagnoses challenging to distinguish ,7,8. FES− ,14,15. SA PubMed literature search was performed in order to identify published cases of TTP in patients with SCD and review the diagnostic approach. The terms “sickle cell” and “thrombotic thrombocytopenic purpura”/“TTP” were used. The references of identified articles as well as articles citing those selected were also searched and included herein.Nineteen cases of iTTP in patients with SCD were identified: 9 were individual case reports ,22,23,249/L. LDH was markedly elevated in all cases with a mean of 3613 iU/L (range 280–7689). Peripheral blood morphology was variable: schistocytes were identified in all cases but, in the majority, at relatively low levels; at the same time, all had large numbers of nRBCs.All patients had thrombocytopenia but only 7/19 (37%) had a platelet count <30 × 10Brain imaging was documented in 7 cases: 3 computed tomography (CT) and 4 combined CT and magnetic resonance imaging. In 1 patient, intracranial haemorrhage was noted, while the remainder were normal. Chest imaging, plain radiographs and/or CT were performed in 16 cases: 2 were normal whereas 14 showed extensive infiltrates with an ARDS picture.As mentioned in the introduction, existing scoring systems to estimate the pre-test probability of iTTP are not suitable for application in patients with SCD due to the chronic haemolysis characteristic of the latter. In addition, several required parameters were not included in the case reports. Despite those significant limitations, at least 4/19 (21%) patients still had a score of 0 as per the French criteria and at least 11/19 (58%) had a PLASMIC score of ≤5 . No publEighteen patients received TPE and 1 received an infusion of donor plasma. However, prior to TPE, all patients had already received red cell transfusion (exchange or simple) with an aim to achieve Hb S levels of ≤30%. There were 2 (11%) deaths while the remainder of patients achieved complete recovery. One patient died of an intracranial haemorrhage and the other, sepsis, in the context of immunosuppression for prevention of iTTP recurrence.SCD is often associated with severe acute complications that share features with TTP: Neurological signs may be due to sickle-related stroke whereas overwhelming hypo splenic sepsis, a well-known complication of SCD, can cause fever, altered sensorium and thrombocytopenia if coexistent disseminated intravascular coagulation. In addition, haemolysis which is one of the hallmarks of TTP is an innate characteristic of SCD and the peripheral smear of SCD patients can be grossly abnormal thus masking or mimicking the morphology characteristic of TTP.More than that, none of the described cases was characterised by severe renal failure requiring replacement as seen in other TMAs, e.g., haemolytic uraemic syndrome. The combination of fever, neurological signs, thrombocytopenia, and renal failure is characteristic of FES but also of TTP and can lead to confusion as the two conditions share many characteristics but also differ distinctly . This coCollectively, reported cases demonstrated an almost universal presence of severe respiratory failure prior to the development of other symptoms or signs (including thrombocytopenia), a feature of FES but not TTP. In addition, the inconclusive morphological findings in the peripheral smear, low pre-test probability of iTTP and, most importantly, complete absence of low ADAMTS13 activity required for definitive iTTP diagnosis also bring the diagnosis to question. It should also be noted here that the markedly elevated levels of LDH observed in these reported cases is also a universal feature of FES .Therefore, we suspect that many of these patients, in fact, had FES. Further supporting this possibility, we have identified 3 case reports where an original diagnosis of iTTP was revised to FES ,8. The pIn conclusion, the diagnosis of iTTP in patients with SCD is challenging due to many overlapping clinical and laboratory features with FES. Established scoring systems to estimate the pre-test diagnostic probability of iTTP are not relevant in SCD due to chronic hemolysis, and likely inflate the risk due to significant clinical overlap. A diagnosis of iTTP in patients with SCD, therefore, should not be made without confirmatory ADAMTS13 results. Furthermore, the possibility of alternative diagnoses should be explored swiftly.


Here, we therefore discuss associations and relationships between HDL, DOX and cancer and discuss whether assessing HDL subclass/composition/function may be considered as a possible early biomarker to detect DOX-induced cardiotoxicity.Doxorubicin (DOX) is an anthracycline antibiotic frequently used against a wide range of cancers, including breast cancer. Although the drug is effective as a treatment against cancer, many patients develop heart failure (HF) months to years following their last treatment with DOX. The challenge in preventing DOX-induced cardiotoxicity is that symptoms present after damage has already occurred in the myocardium. Therefore, early biomarkers to assess DOX-induced cardiotoxicity are urgently needed. A better understanding of the mechanisms involved in the toxicity is important as this may facilitate the development of novel early biomarkers or therapeutic approaches. In this review, we discuss the role of high-density lipoprotein (HDL) particles and its components as possible key players in the early development of DOX-induced cardiotoxicity. HDL particles exist in different subclasses which vary in composition and biological functionality. Multiple cardiovascular risk factors are associated with a change in HDL subclasses, resulting in modifications of their composition and physiological functions. There is growing evidence in the literature suggesting that cancer affects HDL subclasses and that healthy HDL particles enriched with sphingosine-1-phosphate (S1P) and apolipoprotein A1 (ApoA1) protect against DOX-induced cardiotoxicity Noncommunicable diseases are the main cause of death worldwide, with cancer predicted to be the leading cause and an important deterrent in increasing life expectancy in all countries in the twenty-first century . In 2020Previously, the diagnosis of a cancer had a poor outcome for many patients. Fortunately, the discovery of chemotherapy has allowed patients to survive and live longer . The antAlthough the primary function of HDL particles is to facilitate the reverse cholesterol transport (RCT) by exporting cholesterol from the periphery back to the liver for degradation, their functionality is diverse and includes anti-oxidative, anti-inflammatory, anti-apoptotic and anti-thrombotic properties (see review ). Most iWhile DOX is widely used as a therapy for cancer, many patients suffer from cardiac dysfunction consecutive to the treatment, with a decline in left ventricular ejection fraction (LVEF) that will progress into HF . The AmeA meta-analysis reported that after a median follow up of 9 years of 22 815 cancer patients of whom the majority was breast cancer patients, 17.9% developed subclinical cardiotoxicity, 6.3% developed clinical overt cardiotoxicity and 10.9% of patients had overall cardiac events . The latThe main mechanism of antitumour activity of DOX is to target the deoxy-ribonucleic acid (DNA) (see review ). Once DThe main mechanism of how DOX induces toxicity to healthy tissues is believed to be mediated by oxidative stress. However, in preclinical studies the use of anti-oxidants are not able to protect against DOX-induced cardiotoxicity doubting the role of generating reactive oxygen species (ROS) as the main mechanism for toxicity , 20. Sev2• ®) and hydrogen peroxide (H2O2). Both enter the Harber-Weiss and Fenton reaction, catalysed by the presence of ferrous iron (Fe2+) to form hydroxyl radicals (•OH) . Th reviews . Nitric  reviews . DOX exp reviews , 25. If  reviews . The cel reviews . DOX fur2+) by interacting with ryanodine receptors found on sarcoplasmic reticulum (SR) resulting in the release of Ca2+ into the cytosol. The mitochondria are located near SR within cardiomyocytes and aim to balance the cytosolic Ca2+ levels by retaining it. Accumulation of Ca2+ above the mitochondrial threshold, triggers opening of the mPTP, resulting in the loss of mitochondrial membrane permeability and ultimately the release of cytochrome c activating the intrinsic pathway of apoptosis [The mitochondria are one of the major targets for DOX-induced cardiotoxicity and it involves several mechanisms of injury . Thpoptosis . As mentpoptosis . Howeverpoptosis , 33. DOXpoptosis –36. DOX poptosis , 38. Iropoptosis , 22). Thpoptosis ).Maintaining mitochondrial function by preserving mitochondrial topoisomerase 1 (Top1mt) may offer protection against DOX-induced mitochondrial dysfunction. Top1mt maintained mtDNA stability and biochemical functions in wildtype mice on DOX treatment while Top1mt knockout mice were sensitive to DOX-induced mechanisms of injury .Inflammation plays a key role in the development of HF with inflammatory cytokines contributing to progressive HF (see review ). DOX trAs mentioned previously, DOX induces cancer cell death by exploiting the activity of the topoisomerase enzyme , 17. TopThe ubiquitin proteasome system (UPS) and autophagy are protein degradative pathways which are active at basal conditions and during cellular stress to promote cell survival and development, however their dysregulation could lead to cell death (see review ). The UPDOX treatment is also associated with changes in the use of metabolites as an energy source, and the disruption of iron homeostasis within cardiomyocytes , 55. FatAccumulation of free intracellular iron contributes to the generation of ROS and consequently mitochondrial dysfunction as outlined above . ReDOX-induced damage leads to cardiomyocyte death which may occur in a regulated or unregulated manner. Regulated cell death is under the control of biomolecules involved in signalling pathways while unregulated cell death occurs suddenly without the control of signalling pathways (see review ). ApoptoEndogenous cardiac progenitor cells (CPCs), c-kit positive and multipotent cells in the heart contribute to tissue repair and homeostasis in several diseases . In an iIt is important to highlight that the severity of DOX-induced cardiotoxicity may be influenced by multiple factors including age and sex (see review ). AnimalAlthough DOX is an effective and widely used chemotherapeutic agent, it is detrimental to the myocardium. Monitoring left ventricular function with the best possible imaging tool and myocardial deformation with speckle tracking to measure GLS during therapy is recommended to detect early changes in the myocardium . UnfortuInterestingly, HDL particles are traditionally associated with cardiovascular health and preclinical research suggests that healthy HDL particles are protective against myocardial damage induced with DOX . There iLipoproteins are particles responsible for the transport of dietary fats, including triglycerides, cholesterol and fatty acids within the blood . There aHDL particles are spherical shaped with an outer monolayer consisting of lipids and proteins surrounding an inside core of dietary lipids, that includes triglycerides and cholesterol esters. They are the smallest of the lipoproteins (5–12 nm) and have the highest density (1.063–1.21 g/ml) due to the high ratio of proteins to lipids , 78.HDL particles are well known to be anti-atherogenic due to their RCT functionality . It is lThe anti-oxidative function of HDL particles is mediated by multiple components present in the particles and these include the apolipoproteins, the lipid transfer proteins and different enzymes such as the paraoxonase (PON) , the CET−/− mice, PAF-AH was also found to display anti-atherogenic properties through anti-oxidative activity where it reduced endothelial cell adhesiveness and monocyte recruitment [PAF-AH also contributes to the anti-inflammatory and anti-thrombotic activities of HDL particles. Whilst the majority of plasma PAF-AH is bound to LDL, only 20–30% is present in HDL particles . Its proruitment .The sphingolipid, sphingosine-1-phosphate (S1P), also present in HDL particles, contributes significantly to the cardioprotective properties of HDL particles by promoting cell survival . In modeMultiple proteins and lipids have been identified as key components to contribute to the different physiological functions of HDL. Interestingly, the composition of these proteins and lipids found in HDL particles varies function to the type of HDL subclass (see review ).The subclasses of HDL can be separated with ultracentrifugation based on their protein to lipid ratio, predominantly HDL2 and HDL3 (see review ). HDL2 iAlthough scant information on sex differences is available in the literature, there seems to be a sex difference in HDL subclass distribution, with females having a higher value of large HDL subclass compared to men . HoweverThere is strong evidence in the literature suggesting that a shift in HDL subclasses and functionality occurs in the presence of existing CVD and other conditions that increase the risk for developing CVD. In HF patients with reduced EF (HFrEF) or preserved EF (HFpEF), the small HDL subclass was decreased, and the large HDL subclass was increased compared to the healthy controls . This ef, while adding S1P to the diabetic HDL increased its S1P content and restored its cardioprotective capacity [The HDL particle profile is also altered in other pathophysiological conditions such as diabetes, obesity, hypertension and renal disease, which are known to be associated with an increased risk for cardiovascular related mortality , 108–110capacity . HyperglAs ApoA1 is responsible for the maintenance of HDL structure and for controlling the activity of LCAT, alterations to the structure of ApoA1 consequently affects HDL –116. ApoObesity and hypertension were associated with an increase in the smaller HDL subclass , 121. AlSurprisingly, HDL particles in end stage renal disease had greater cardioprotective functionality due to its increase in S1P concentration compared to healthy patients . HoweverDespite some discrepancies reported in the literature with regards to HDL subclass shifts associated with different pathophysiological conditions , it is Many breast cancer patients present with an alteration in their lipid profile . Cancer Mechanisms have been proposed on how cancer cells interact with HDL particles . In briIn addition, the interaction with cancer cells may not only decrease HDL-c levels but it affects its composition and functionality (see review ). The oxA different aspect of the relationship between HDL and cancer, is the anti-tumour activity of ApoA1 . In vitrIn addition to the cancer itself, chemotherapy further affects plasma lipids , 152. BrConsidering that the cancer itself impacts on the plasma lipid levels and that cancer patients most often receive a combination of chemotherapeutic agents, exploring the effects of an individual agent on the lipid profile is not easy to pinpoint in the clinical setting. Additionally, the cancer itself could further have an impact on the plasma lipids. Sharma, Tuaine  investigWe speculate that the mechanisms involved in DOX-induced cardiotoxicity could involve the formation of dysfunctional HDL that become unable to protect cardiomyocytes against the toxic effect of DOX. As treatment with DOX will result in ROS generation, disruption of intracellular iron homeostasis and mitochondrial dysfunction which all promote oxidative stress and inflammation. Dysfunctional HDL will lose their anti-inflammatory and antioxidant functions as characterized by a reduction in PON1 anti-oxidative functionality, structural and functional changes of its composition such as ApoA1 and S1P . The usIn conclusion, both the cancer cells and chemotherapy alter the plasma lipid profile in breast cancer patients with a decrease in HDL-c commonly observed. Few studies have explored HDL subfractions and HDL composition and all of them converge to the conclusion that the cancer environment leads to a shift in HDL subclasses and a change in HDL composition favouring dysfunctional HDL particles. Preclinical studies suggest that healthy HDL and rHDL enriched with S1P may protect against DOX-induced cardiotoxicity. Of note, most preclinical studies have been conducted in the absence of the cancer environment which may affect the HDL particle dynamic. In addition, it is important to take into consideration that multiple confounders including age, sex, ethnicity and multiple cardiovascular risk factors  may also influence the change in HDL subclass, functionality, and composition in cancer patients , 163–165


Rationale: Hepatocellular carcinoma (HCC) is a highly heterogeneous and malignant disease with the complex immune microenvironment, which ultimately influence clinic outcomes of patients. However, the spatial expression patterns of diverse immune cells among tumor microenvironment remain to be further deciphered.Methods: Spatial transcriptomics sequencing (ST) was implemented on two portions of HCC specimens. Differentially expressed genes, cell cycle phases, epithelial-mesenchymal features, pseudo-time and immune infiltration analysis were applied to demonstrate the intratumor heterogeneity and define the specific immune-related regions, and the results were further validated by a second analysis on another ST study. In vitro and in vivo experiments were conducted to confirm the functional mechanisms of key molecules such as CCL15, CCL19 and CCL21. Clinical tissue samples were used to assess their potential prognostic and therapeutic values.Results: Totally, 7553 spots were categorized into 15 subsets by hierarchical clustering, and malignant subsets with intratumor heterogeneity phenotypes were identified. Spatial heterogeneity from distinct sectors highlights specific chemokines: CCL15 is remarkable in the core region of the carcinoma sector and facilitates the immunosuppressive microenvironment by recruiting and polarizing M2-like macrophages in vitro and in vivo; High expression of CCL15 and CD163 respectively predicts poor prognosis of HCC patients, and the combined application of them has better predictive value. CCL19 and CCL21, sharing similar spatial expression patterns, are highly-correlated and prominent in the immune infiltration enrichment and recruit CD3+ T cells and CD20+ B cells to inhibit the growth of HCC, indicating a good prognosis of HCC patients.Conclusions: Taken together, our studies preliminarily reveal intratumor heterogeneity of HCC based on ST techniques and unravel the previously unexplored spatial expression patterns in the immune microenvironment. We also highlight the clinical significance and spatial discrepancy of key molecules, providing novel insight for further developing therapeutic strategies in HCC. The latest cancer statistics for 2020 indicate that primary liver cancer (PLC) ranks sixth in morbidity and third in mortality worldwide, with approximately 906,000 new cases and 830,000 deaths yearly, and that hepatocellular carcinoma (HCC) accounts for approximately 80% of PLC cases Tumor heterogeneity in HCC can be roughly classified into phenotypic heterogeneity and molecular heterogeneity In the present study, we performed ST sequencing for two portions of HCC specimens with clearly demarcated and continuous cores, peripheral regions, and boundary regions and investigated the potential makers and regulatory mechanisms of immunosuppressive or immune enrichment regions with distinct prognoses based on analyzing spatial gene expression patterns. Accordingly, our studies highlighted the spatial heterogeneity of gene expression patterns as well as their clinical significance in the tumor immune microenvironment and provided novel insight for the further prognostic and therapeutic strategies for HCC.Totally, two fresh continuous tissues  of one HCC case were acquired from surgical resections without preoperative treatment at Eastern Hepatobiliary Surgery Hospital . Another 89 HCC tissues were obtained from the Department of Pathology at Changhai Hospital . Diagnoses of HCC were evaluated by two certificated pathologists. Collections of tissue specimens were approved by the Ethics Committee of EHBH and CH, respectively. Written informed consent was provided by individuals donating HCC tissues. Fresh tissues for ST sequencing were promptly filled with optimal cutting temperature (OCT) compound and then snap frozen in isopentane and liquid nitrogen. The samples used for Western blotting were freshly frozen in -80 °C until use. Tissues utilized for immunohistochemistry analysis were formalin fixed and paraffin embedded (FFPE). Clinical characteristics from CH cohort were listed in The two fresh HCC tissues were surgically resected, washed with pre-cooled PBS solution and drained by gauzes. The tissues were then transferred to isopentane for soaking and freezing, and moved to a cryopreservation tube with tweezers for subsequent embedding with the OCT mixture. The procedures are as follows: (1) Hematoxylin-eosin staining (HE) was used for histological imaging; (2) Tissues were then fixed, stained and permeabilized to release mRNA, which can bind to the probes that contained a 16 bp spot barcode and a 12 bp UMI sequence. The capturing probe with poly (dT) sequence acquired gene expression information by binding to mRNA 3'-poly(A) tail; (3) cDNA synthesis and sequencing libraries were prepared using the captured RNA as templates; (4) This sequencing was based on 10× Genomics Visium and the paired-end sequencing mode of the Illumina sequencing platform, which was previously described before After sequencing, the data were visualized and analyzed via Space Ranger (version1.1.0). The Space Ranger was used to ensemble the reference genome database and FastQC software was utilized for data quality control. Further sequencing and application of R software and other applications were used for data visualization.R software version 4.0.3 and “Seurat” software package version 3.1.1 were used for analysis. After excluding low-quality units, we used the “SCTransform” function to normalize data, find variable features and scale data.Next, principal component analysis (PCA) was performed on the first 2000 highly variable genes using the “RunPCA” function. Then, the number of PCs corresponding to ElbowPlot was selected and the “RunUMAP” function with a default perplexity value of 30 was executed to obtain the bidimensional coordinates for single-spot. At the same time, we used the “FindClusters” function to cluster the unsupervised units at 0.6 resolution on the same PC as for the “RunUMAP” function. Therefore, the datasets were visualized through UMAP plots. The closer the spot distance was, the closer the expression trend of spot was.The “FinAllMarkers” function is used to identify the differentially expressed genes (DEGs) in different clusters. Bonferroni correction method was used to adjust p value, and DEG with p value larger than 0.05 after correction was eliminated. The nonparametric Wilcoxon rank sum test in the “Seurat” software package was used to analyze the differential expression between subsets.Cells in each spot were divided into specific cell cycle and differentiation stage based on G2/M and S related gene expression  and cellThe “Monocle2” software is used to identify differential genes that change between clusters or spots for developmental trajectory and pseudo-time analysis. The “Monocle2” package for trajectory and pseudo-time analysis contained 400 marker genes from the “differentialGeneTest” function that is designed to infer potential pedigree differentiation trajectories. A generalized additive model (GAM) is constructed to generate the average expression of each isotype. RNA counts of all spots in the cluster were selected as the input of “Monocle2” for downstream analysis. Additionally, we also used “AddModuleScore” function to evaluate the feature scores for immune infiltration enrichment based on the specific markers for T cells. B cells, NK cells and myeloid cells .To systematically explore the relations between spatial expression patterns and tumor heterogeneity in hepatocellular carcinoma, we randomly sampled tissues from two regions of surgically resected and pathologically confirmed HCC HCC-R1, R2) of the same case with clearly demarcated and continuous cores, peripheral regions, and boundary regions. The fresh-frozen samples were then subjected to spatial transcriptomics sequencing via the 10× Genomics Visium platform with histological staining, cDNA sequencing library preparation, and further sequencing procedures. Hematoxylin-eosin staining revealed histological locations and boundaries; thus, we annotated them as sectors of carcinoma, para-carcinoma and fiber cord based on spatial locations  were in the carcinoma sector, while clusters  were in the para-carcinoma sector and cluster HC-11 was in the fiber cord sector. It was also obvious that the number of nCounts in the carcinoma sector was larger than that in the para-carcinoma region and fiber cord sector, revealing a high abundance of gene expression in the tumor region  PTPRC (leukocyte marker) CD2 (T cell and NK cell marker) LYZ (myeloid cell marker) To further characterize the identified clusters, we performed differentially expressed gene (DEG) analysis and profiled the featured DEGs in each cluster at the set of log-fold change (log FC) thresholds of 0.25 A. We exaIn the multi-step process of tumorigenesis, the basic biological functions of tumor cells have altered, such as the acquisition of unlimited replication and colonization ability and the activation of metastasis and invasion capacity Taken together, our recent analysis of spatial transcriptomics on two sections of HCC samples preliminarily reveals the discrepancies of gene expression patterns within the different regions of HCC microenvironment, providing new insights and novel strategies for exploring the relations between spatial gene expression and tumor heterogeneity of HCC.Next, we intended to elaborate the spatial expression pattern and underlying mechanisms in distinct sectors. We set out to assess the differentiation and development trajectory of spots in the carcinoma sector using pseudo-time analysis and observed the developmental trajectory among clusters in HCC-R1/R2  were notably downregulated in core regions, possibly indicating an immunosuppressive microenvironment (ISME). Among the upregulated genes presented, most of them  have been documented previously to be elevated in HCC and play oncogenic roles in various aspects , which increases the expression of macrophage markers  and median survival time  for OS and the five-year survival rate (2.85%) and median survival time  for RFS of the CCL15hiCD163hi group were the worst among the four groups , and dendritic cells (DCs) in HCC IGHG1, IGHG3, IGHG4, IGLC2, IGKC, IGHA1, IGHM and other molecules were dramatically elevated  and control AAV were then intratumorally injected when the largest diameter of the xenograft reached 5 mm. The results showed that the growth of tumors from the oe-AAV group was slowed and the tumor volume was gradually reduced, whereas the tumor growth and tumor volume of the control AAV group continued to increase Figure B. The we+ T cells, CD8+ T cells and B lymphocytes  in subcutaneous xenografts, and the results showed that Ccl19 correlated positively with Cd3e, Cd19, Cd20, and Ifng  High expression of CCL15 and CD163 respectively predicts poor prognosis of HCC patients, and the combined application has better predictive value; (6) Highly-correlated CCL19 and CCL21 were synergistically upregulated in the IIE and inhibited the growth of HCC by increasing the infiltration degree of T cells and B cells.In our study, we conducted ST sequencing on two pieces of HCC specimens from one patient. Each piece of tissues comprised distinct but continuous regions from carcinoma tissue and the tumor boundary region to para-carcinoma tissue. We first described overall landscapes of spatial gene expression patterns among two samples and intratumor heterogeneity. Next, we defined the tumor core region and the IIE via bioinformatics analysis, which was also confirmed by our secondary data analysis on previous study focusing on tumor heterogeneity of primary liver cancers in vitro and in vivo. Clinical relevance analysis also indicated that high expression of CCL15 or the M2-like macrophage marker CD163 predict a poor prognosis of HCC and the combined predictive value of CCL15 and CD163 in the prognosis is superior to that using either marker alone. Currently, macrophage polarization involves a variety of molecular mechanisms, including TLR4/NF-κB, JAK/STATS, TGF-β/SMADS, PPARγ, NOTCH and microRNA signaling pathways Concretely speaking, we profiled the overall landscapes of hepatocellular carcinoma with ST sequencing and all the 7553 spots were classified into 15 clusters. We then determined the features of 15 clusters belonging to distinct sectors by virtue of the histological boundaries, marker genes and differential gene expression, cell cycle phases and differentiation origins. Intratumor heterogeneity, especially immune-ITH, has been documented to impact the clinical outcomes of HCC, which is also characterized by an increased immunosuppressive or exhaustive TME; However, spatial distribution characteristics and expression of other cell markers in the tissue structure of HCC still need further consideration, and the origins and contributing factors for ITH are still largely unknown Furthermore, we also evaluated the infiltration scores and spatial distributions of various immune cells, including T cells, B cells, NK cells and myeloid cells among 15 clusters. Clustering results showed that two clusters are rich in immune cells especially T cells and B cells, and are annotated as immune infiltration enrichment. Contrary to the conditions of tumor core region, upregulated DEGs in the IIE are characterized by immunoglobin families and two prominent chemokines, CCL19 and CCL21, and the results are verified by our second data analysis on a previous study In vivo experiments of AAV showed that high expression of CCL19 and CCL21 inhibits the growth of HCC by influencing the infiltration of CD3+ T cells and CD20+ B cells in the subcutaneous xenograft model, while there was a slight significance in the DEN/CCl4-induced tumor models when compared with tumor sizes in two groups. The reason may well be that CCL19 and CCL21 function better in the tumor progression rather than the tumorigenesis process of HCC, and DEN/CCl4-induced tumor models are not strongly consistent due to individual differences among mice and the sample sizes. Anyway, the above conclusions indicate good prognosis and may serve as a novel therapeutic target for HCC immunotherapy. Taken together, we determined the specific chemokines in either the tumor core region or IIE, which have remarkably different roles in clinical applications due to spatial heterogeneity in gene expression. Notably, the recruiting mechanism of these chemokines to immune cells still needs further exploration.Further studies elucidated their strong correlations and similarities in spatial expression pattern, biological process and clinical significance of HCC. In conclusion, our study comprehensively reveals intratumor heterogeneity, especially immune-ITH, in HCC based on spatial transcriptomics technology. We further reveal the spatial expression patterns in specific regions of some key molecules such as CCL15, CCL19, and CCL21, which affect the infiltration and recruitment of various immune cells and collectively promote intratumor heterogeneity in the HCC microenvironment, thus influencing the prognosis of HCC patients. Our studies also highlight the clinical significance of the spatial heterogeneity and gene expression patterns, laying the foundation for developing new prognostic markers and therapeutic strategies for HCC.Supplementary methods, figures and tables.Click here for additional data file.


Suppurative labyrinthitis continues to result in significant hearing impairment, despite scientific efforts to improve not only its diagnosis but also its treatment. The definitive diagnosis depends on imaging of the inner ear, but it is usually clinically presumed.To analyze the clinical factors and hearing outcomes in patients with labyrinthitis secondary to middle ear infections and to discuss findings based on imaging test results.Retrospective cohort study, based on the charts of patients admitted with middle ear infection-associated labyrinthitis.We identified 14 patients, eight (57%) of whom were females and six (43%) males. Mean age was 40 years. Cholesteatomatous chronic otitis media was diagnosed in six patients (43%), acute suppurative otitis media in six (43%), and chronic otitis media without cholesteatoma was diagnosed in two patients (14%). Besides labyrinthitis, 24 concomitant complications were identified: six cases (25%) of labyrinthine fistula, five cases (21%) of meningitis, five cases (21%) of facial paralysis, five cases (21%) of mastoiditis, two cases (8%) of cerebellar abscess, and one case (4%) of temporal abscess. There was one death. Eight (57%) individuals became deaf, while six (43%) acquired mixed hearing loss.Suppurative labyrinthitis was often associated with other complications; MRI played a role in the definitive diagnosis in the acute phase; the hearing sequel of labyrinthitis was significant. The advent of antibiotics and immunizations in the last century led to a considerable decline in the incidence of complications from otitis media, and therefore a discussion on suppurative labyrinthitis associated with middle-ear infection may seem, at first glance, an outdated issue. However, complications still occur, particularly in developing countries, with significant morbidity .The diagnosis of suppurative labyrinthitis secondary to otitis media is essentially a clinical one, through the observation of vertigo, nystagmus, tinnitus and hearing impairment in the presence of a middle ear infection. In many diseases of the inner ear, the inflammatory process that ensues is usually presumed, rather than effectively diagnosed, and corticosteroids are empirically prescribed as the treatment of choice.in vivo.The complex location of inner ear structures in the temporal bone, housed in the dense bone of the otic capsule, represents a significant barrier for the access and identification of any alterations in this region. Considering that the current knowledge about inner ear physiopathology is mainly derived from animal studies involving the collection of tissues and histological, molecular and inflammatory marker analyses, little is known about the mechanisms involved in diseases of the human inner ear in vivo. The use of gadolinium as a contrast for the study of the inner ear adds sensitivity to the NMR, particularly for diseases such as labyrinthitis.11Imaging tests are important tools in an attempt to better understand the dynamics of inner ear inflammation. Currently the high-resolution computed tomography (HR-CT) best evaluates diseases affecting the bony labyrinth and nuclear magnetic resonance (NMR) imaging defines diseases that affect the inner ear and retrocochlear pathways. Recent advances in NMR techniques offer interesting opportunities for the study of cochlear structure, function and metabolism The aim of this research is to analyze clinical factors and hearing outcomes of patients with labyrinthitis secondary to middle ear infections and discuss the results of imaging tests.A retrospective cohort study was carried out based on medical records of patients treated at the Otology/Otoneurology department of a tertiary university hospital between 1987 and 2013. The study included patients whose medical records contained the diagnosis of labyrinthitis secondary to otitis media. Medical records with incomplete information were excluded, as well as those in which the diagnosis of suppurative labyrinthitis was not well established. The following data were collected from medical records: age, gender, medical history, type of middle ear infection, associated complications, audiometry and imaging test results. All patients received antibiotic therapy and systemic corticosteroids. The study was approved by the Ethics Committee of the institution under Protocol number 0081/10.A total of 14 patients diagnosed with labyrinthitis associated with otitis media were identified. Eight were female (57%) and six (43%) were male. The mean age was 40, with an age range from 9 to 67 years .Table 1Dn = 24); meningitis, facial paralysis and mastoiditis in five patients each ; cerebellar abscess (n = 24), and temporal lobe abscess in one patient . There was one death in this study (case 7) (All patients had otitis media before the diagnosis of labyrinthitis. Otitis media was classified as follows: cholesteatomatous chronic otitis media (CCOM) in six (43%) patients, acute otitis media (AOM) in six (43%) patients, and non-cholesteatomatous chronic otitis media (NCCOM) in two (14%) patients. Thirteen (93%) patients had one or more of the following associated complications : labyrinthine fistula  in six p abscess  in two p(case 7) .Figure 1Tinnitus was reported in 14 patients (100%), vertigo in 10 patients (71%), and nystagmus in five (36%). Eight (57%) patients developed anacusis, and six (43%) progressed to mixed hearing loss (moderate to severe). In nine patients (64%), the diagnosis of labyrinthitis was confirmed with the aid of NMR: with contrast  and withIt is known that the sequence of events that follow an episode of suppurative labyrinthitis typically occurs in three stages. In the acute phase, the bacteria and leukocytes appear in the perilymphatic space; in the fibrous phase, granulation tissue consisting of fibroblasts associated with neovascularization result in fibrosis, and finally, the ossification phase is characterized by metaplastic bone formation.n = 14), similar patterns were reported in studies with a larger number of patients.12The predominant otologic diagnoses were CCOM and AOM; although the sample was small n = 1, similarThere is little direct evidence of inflammation occurring during labyrinthitis, and the knowledge of the inflammatory response dynamics within the human inner ear is still limited.Some animal studies have investigated the mechanisms of inflammation-induced inner ear disorders; however, studies in humans are limited. The reasons could be: the intralabyrinthine fluids and tissues are housed in the temporal bone complex and are difficult to collect for culture; acute hearing loss or vertigo are rarely fatal, making inner ear tissues rarely available for autopsies; the temporal bone is not usually removed in routine autopsies, thus limiting histological analysis.4Imaging tests help to overcome these obstacles and currently play an important role in diagnosis. Few medical areas have benefited from advances in diagnostic imaging as much as otology.Previous studies13In the radiological literature, three phases of labyrinthitis are usually described: acute, fibrous stage, and labyrinthitis ossificansCT studies are of little help in this phase, as well as in the fibrous stage of labyrinthitis. With the replacement of intralabyrinthine fluids by fibrous tissue septa, which are characteristic of the fibrous phase, there may still be inner ear enhancement by gadolinium, although less pronounced. At the T2-weighted NMR and in the FIESTA sequence  it is poWe emphasize that all patients (100%) complained of tinnitus and all of them developed some degree of sensorineural hearing loss (with or without an air-bone gap), and 71% complained of vertigo, usually correlated with infection severity. In a more comprehensive series of cases,It is pertinent to observe that labyrinthitis occurred in association with other complications in all except one patient 93%); these complications usually receive greater attention due to their more urgent clinical presentation, resulting in a delay in the labyrinthitis diagnosis. When it is finally identified, the hearing loss is already established. With the current available treatment, even if it were possible to attain an early diagnosis, once the inflammatory process starts in the inner ear, it develops into an inevitable cell damage process. Unfortunately, the early diagnosis of labyrinthitis still does not change the hearing prognosis, except in cases of immune-mediated diseases, such as Wegener's granulomatosis, in which it is possible to improve hearing thresholds with specific treatment.%; these Suppurative labyrinthitis was often associated with other complications and MRI helped to establish the definitive diagnosis in its acute phase. Hearing sequel of labyrinthitis was significant.The authors declare no conflicts of interest.


The glymphatic system is a unique pathway that utilises end-feet Aquaporin 4 (AQP4) channels within perivascular astrocytes, which is believed to cause cerebrospinal fluid (CSF) inflow into perivascular space (PVS), providing nutrients and waste disposal of the brain parenchyma. It is theorised that the bulk flow of CSF within the PVS removes waste products, soluble proteins, and products of metabolic activity, such as amyloid-β (Aβ). In the experimental model, the glymphatic system is selectively active during slow-wave sleep, and its activity is affected by both sleep dysfunction and deprivation. Dysfunction of the glymphatic system has been proposed as a potential key driver of neurodegeneration. This hypothesis is indirectly supported by the close relationship between neurodegenerative diseases and sleep alterations, frequently occurring years before the clinical diagnosis. Therefore, a detailed characterisation of the function of the glymphatic system in human physiology and disease would shed light on its early stage pathophysiology. The study of the glymphatic system is also critical to identifying means for its pharmacological modulation, which may have the potential for disease modification. This review will critically outline the primary evidence from literature about the dysfunction of the glymphatic system in neurodegeneration and discuss the rationale and current knowledge about pharmacological modulation of the glymphatic system in the animal model and its potential clinical applications in human clinical trials. The glymphatic system is a waste clearance system that utilises perivascular channels formed by astroglial cells surrounding penetrating arterioles ,2,3. ThrThe comprehension of the role of the glymphatic system and its associations with age, sleep and protein clearance could lead to a deeper understanding of a common pathophysiological pathway underpinning several neurodegenerative conditions . MoreoveAlzheimer’s disease (AD) is a progressive neurodegenerative condition  neuropatExciting possible associations between AD, glymphatic function and sleep have been a contemporary topic of interest . Clinica11C]-PiB, an Aβ-sensitive tracer proposed to be also sensitive to CSF clearance alterations in AD, has shown defective CSF clearage in AD patients Florbetaben in a large cohort of PD subjects , increasing age was associated with increased cortical [18F]Florbetaben retention in the PD group. However, after adjusting for age, no correlation was found between cortical [18F]Florbetaben uptake and global cognitive ability within the PD cohort H2O JJ vicinal coupling proton exchange MRI, they found increased turnover of labeled water within the PVS in TGN-073 treated mice , w, w166], 2A receptors [Adenosine is a somnogenic neuromodulator that plays a role in sleep-wake regulation , shown teceptors . Substaneceptors ; therefoeceptors . Such fieceptors .Another upcoming approach of potential utility in glymphatic modulation is photobiomodulation (PBM) therapy, which is defined as the utilisation of light (red or near-infrared) to elicit advantageous changes in cellular biochemistry . ContempIn conclusion, the dysfunction of the glymphatic system plays an important, multifaceted, yet largely mysterious role in several neurodegenerative diseases. For example, it is uncertain if the glymphatic system changes play a primary role in the pathogenesis of disease onset and propagation or a secondary response to changes associated with neurodegenerative conditions. In such diseases, exploring the available evidence on the modulation of the glymphatic system presents a highly fascinating, promising and novel avenue for future research and potential clinical applications in human clinical trials . It rema


Environmental exposures to endocrine disrupting compounds (EDCs) such as the organochlorines have been linked with various diseases including neurodevelopmental disorders. Autism spectrum disorder (ASD) is a highly complex neurodevelopmental disorder that is considered strongly genetic in origin due to its high heritability. However, the rapidly rising prevalence of ASD suggests that environmental factors may also influence risk for ASD. In the present study, whole genome bisulfite sequencing was used to identify genome-wide differentially methylated regions (DMRs) in a total of 52 sperm samples from a cohort of men from the Faroe Islands (Denmark) who were equally divided into high and low exposure groups based on their serum levels of the long-lived organochlorine 1,1-dichloro-2,2-bis(p-chlorophenyl)ethylene (DDE), a primary breakdown product of the now banned insecticide dichlorodiphenyltrichloroethane (DDT). Aside from being considered a genetic isolate, inhabitants of the Faroe Islands have a native diet that potentially exposes them to a wide range of seafood neurotoxicants in the form of persistent organic pollutants (POPs). The DMRs were mapped to the human genome using Bismark, a 3-letter aligner used for methyl-seq analyses. Gene ontology, functional, and pathway analyses of the DMR-associated genes showed significant enrichment for genes involved in neurological functions and neurodevelopmental processes frequently impacted by ASD. Notably, these genes also significantly overlap with autism risk genes as well as those previously identified in sperm from fathers of children with ASD in comparison to that of fathers of neurotypical children. These results collectively suggest a possible mechanism involving altered methylation of a significant number of neurologically relevant ASD risk genes for introducing epigenetic changes associated with environmental exposures into the sperm methylome. Such changes may provide the potential for transgenerational inheritance of ASD as well as other disorders. Environmental exposures to endocrine disrupting compounds (EDCs) have been linked with various diseases including neurodevelopmental disorders . Autism Experimental and epidemiological studies have reported harmful effects of environmental exposures to both short- and long-lived EDCs, such as the short-lived bisphenol A (BPA) and phthalates, and persistent organic pollutants (POPs) like polychlorinated biphenyl (PCB) and polybrominated diphenyl ether (PBDE) compounds, on social behavior and neurodevelopment. Rodent studies have shown associations between EDC exposure and disruption of social hormones, social recognition, locomotion, and excitatory-inhibitory synapse pathways . ExposurWith respect to mechanisms of action, many studies have investigated epigenetics as a potential mediator of EDC-induced changes in phenotype and disease state. Epigenetic mechanisms involve modification of gene expression or regulation that do not involve changes in DNA sequence. These changes may be due to DNA methylation, histone modification and microRNA induction or suppression. For example, In addition to having an impact on neurodevelopment, EDCs have also been implicated in many other diseases or human conditions, such as cancer, diabetes, obesity, metabolic disorders, and infertility . With reIn recent years, the sperm methylome  has come under increasing scrutiny as a source of epigenetic changes that may have an impact on a wide variety of human disorders, from infertility to neurodevelopmental and psychiatric disorders . DNA metBased on the above studies, we postulated that elevated exposures to EDCs is associated with methylomic changes in sperm DNA in regions that may influence risk for ASD. The primary objectives of this study are to: 1) determine if high versus low exposures to the persistent organochlorine 1,1-dichloro-2,2-bis(p-chlorophenyl)ethylene (DDE) as determined by serum levels are associated with DMRs in sperm from a Faroese cohort whose native diet, often rich in pilot whale meat and blubber, may expose a fraction of the individuals to higher than average amounts of EDCs; 2) determine the nature of genes associated with such DDE DMRs; 3) identify pathways and functions over-represented among DMR-associated genes.n = 26) and lowest (n = 26) exposures to DDE, a stable breakdown product of the insecticide DDT. The highest and lowest exposures were determined by the levels of DDE that were quantitated in the serum of semen donors. The serum levels were divided into tertiles, with the highest tertile representing the high exposure group, and with the lowest tertile representing the low exposure group which served as the “control” group for this study. Serum levels of DDE within the first tertile ranged from 1 to 249 ng/g lipid while those within the third tertile ranged from 578 to 3,328 ng/g lipid. For comparison, a recent meta-analysis of blood levels of DDE across the globe after the year 2000 shows an average blood level of 207 ng/g lipid (n = 32) and validation sets (n = 20) for WGBS analyses of the methyl sequencing data. The purpose of using discovery and validation sample sets in this study was to assess the reproducibility of the DMRs identified by the methyl-seq analyses. Gene ontology and pathway analyses were employed to determine enrichment in pathways and functions among the DMR-associated genes. Differential methylation of selected genes relevant to ASD and other neurodevelopmental disorders was further validated by pyrosequencing analyses.The overall workflow for this study is summarized in /g lipid  which is6 per ml were divided by Dr. Melissa Perry  into three exposure groups based on the recorded levels of DDE in serum, and 26 samples each from the first (lowest exposure) and third (highest exposure) tertiles were provided for this methylation study without assignment of the samples to exposure tertiles. Thus, the semen samples were processed through bisulfite sequencing analysis without knowledge of their respective exposure levels.The semen donors whose de-identified samples were included in this study were among several hundred young men recruited from the general population register of individuals born in the Faroe Islands (Denmark) between 1981 and 1984. Samples were collected throughout 2007 and 2008 and de-identified by the Faroese Hospital System. Information related to height, weight, age, smoking status, sample collection date, length of abstinence at the time of collection, and the serum concentrations of 13 persistent organic pollutants (POPs) including the four most prevalent PCB congeners,  as well as DDT and DDE were recorded for each sample as part of the Faroese General Population research cohort. This cohort was part of a broader parent study on Faroese health and fertility that was approved by the local Science Ethics Committee for the Faroe Islands. Thus, the sample data also included information on sperm concentration and motility levels, both of which have relevance to male fertility. Raw ejaculate  from 52 young men in this cohort was kindly provided for this specific study through a Data Processor Agreement between Dr. Pál Weihe, Director of the Department of Occupational Medicine and Public Health of the Faroese Hospital System in Tórshavn, Faroe Islands and Dr. Valerie Hu at The George Washington University. The advantage of using the Faroese cohort for this study on the sperm methylome in the context of environmental exposures to EDCs is that inhabitants of the Faroe Islands are considered genetic isolates and therefore are expected to have similar genetic background and polymorphisms that would be considered major confounding variables in genomics studies. In addition to the de-identified semen samples, Dr. Weihe also provided the demographic data and serum levels of the POPs that had been determined for each donor as previously described . FaroeseSperm was isolated from semen (which contains various cell types) using a published discontinuous gradient protocol . BrieflyDNA was isolated from the purified sperm cells using a Qiagen AllPrep DNA/RNA mini kit following the manufacturer’s protocol. Sperm pellets were first lysed in 450 μl RLT buffer with 50 μl added tris(2-carboxyethyl)phosphine solution  by vortexing with 0.1 gm RNase/DNase-free stainless steel microbeads for 5 min at RT in a Disruptor Genie . Lysates were immediately stored at −80°C until DNA extraction which was usually completed the next day using the standard protocol.The sperm DNA samples were sent to Admera Health , a CLIA-certified laboratory, for WGBS analyses. The directional Illumina TruSeq DNA Methylation library kit was used for sample preparation. WGBS was then performed on an Illumina HiSeq X sequencer resulting in 150 bp PE reads achieving roughly 4x coverage genome wide per sample. Raw FASTQ files were received from Admera Health for further analyses.https://github.com/ben-laufer). CpG_Me builds on previously published bioinformatic tools and pipelines  ranged from ±7 to ±38 for all DMRs, as shown in DMRs were identified using dmrseq and bsseq Bioconductor packages in the wrapped pipeline of DMRichR. Default parameters for the DMRichR executable script were used which included coverage set to 1x, per Group set to 100%, minCpGs set to 5, and maxPerms set to 10. Covariables adjusted for in DMR analysis included BMI, age, days in storage, batch effects of processor and date of processing, percent motile sperm, and smoking status. DMRs with permutation p-value analysis was performed using the sum of logs of the p-values of GO terms to integrate the data from the discovery and validation sets, and then terms with a meta p-value < 0.05 were slimmed using REVIGO  between the first and third DDE exposure tertiles, while subsequent analyses of the validation set of 20 samples resulted in a total of 865 DMRs  revealed a total of 894 DMRs .The overall distribution profiles of annotated gene and CpG regions were similar for both analyses . AnalysiGene ontology analyses of the genes within the DMRs in the discovery and validation samples were performed using two different gene mapping and ontology approaches. GOfuncR analysis provides an overview of the top biological processes, cellular components, and molecular functions associated with the DMR-associated genes from each of the study cohorts , while SThe enrichment in neuronal processes was further confirmed by independent pathway and functional analyses of the DMR-associated genes from the discovery and validation cohorts using Ingenuity Pathway Analysis (IPA). CREB signaling in neurons, the endocannabinoid developing neuron pathway, netrin signaling, calcium signaling, GABA signaling, and axon guidance signaling are among the canonical pathways significantly enriched in both data sets , while r−58). Moreover, IPA analysis of the overlapping genes revealed significant over-representation of genes involved in nervous system development and function  which are located in an imprinted region identified as significantly hypermethylated on a genome-wide level in this study as well as in a previous study on paternal sperm from men with an autistic child  reached genome-wide significance (FAM19A5 and BRD1) overlap with the DDE DMR-associated genes from sperm. Furthermore, this region contains a copy number variant for ASD  methods to treat infertility) were enriched for genes involved in ASD and neurogenesis, especially in regions with bivalent chromatin structure  analyses of brain tissues from individuals with Dup15q syndrome but not from that of individuals with idiopathic ASD (CSMD1 and 6 SNORD115 variants) from this brain study  that participate in the modification of other non-coding RNAs and site-specific 2′-O-methylation of substrate RNAs  to allow for isolation of sufficient amounts of DNA for the bisulfite sequencing analyses followed by pyrosequencing validation of selected gene regions. Despite the limitation in sample size, by dividing the samples into discovery and validation sets for the WGBS analyses, we were able to show a significant overlap of 138 DMRs between the two experimental cohorts. In addition, there were no completely unexposed samples for comparisons. Because EDCs are known to exhibit non-monotonic dose-response curves  years old, thus eliminating confounding results based on age which has been shown previously to influence methylation patterns . Another advantage of this cohort is the natural Faroese diet, rich in pilot whale meat and blubber, that exposes a fraction of the population to higher than average levels of fat-soluble long-lived organochlorines and other POPs. While we show differences in methylation patterns in sperm from men with high versus low exposures to DDE that were highly correlated with the sum of the four most prevalent PCB congeners , an obvie curves , we canne curves , it has e curves . Thus, iAside from investigating the possible relationships between organochlorine exposures, sperm DNA methylation profiles, and neurodevelopmental disorders, the availability of this publicly accessible methylation data on DDE/organochlorine-associated changes in the sperm methylome of exposed individuals will provide a valuable resource for studies on other diseases and conditions, such as cancer, obesity, diabetes, and infertility, which have also been linked to environmental exposures. Indeed, the data in Future studies should include additional cohorts sampled longitudinally to monitor temporal changes in individual sperm methylation levels as a function of cumulative EDC exposures as well as expanded concentration levels of DDE and other POPs. Moreover, it is unknown whether the DNA methylation differences noted in this study arose from direct exposure of the individual sperm donors to EDCs or from ancestral exposures that led to the inheritance of specific DNA methylation patterns in the donors’ sperm. Thus, future studies would also benefit from the analyses of sperm DNA samples coupled with measured EDC exposure levels within families  to determine potential heritability of the sperm methylome in the context of environmental exposures. Finally, given the association between exposure levels of DDE and altered methylation of many neurodevelopmental genes, it will also be of interest to investigate the relationships between DDE exposures of the semen donors, epigenetic changes in sperm, and the health outcomes of their children.per se, this study supports a link between environmental EDC exposures and epigenetic changes in germ cells that may impact the regulation of many genes associated with disease phenotypes, including ASD. Studies involving animal models have shown that DNA methylation patterns as well as associated phenotypes or diseases can be stably and heritably transmitted through the germline in several generations of offspring, specifically in relation to EDC exposure. If transgenerational epigenetic transmission can be demonstrated in humans, our results would suggest that heritable methylomic patterns in sperm may contribute to disease phenotypes, including ASD, in human progeny.This study shows that elevated exposure to DDE, one of a class of persistent organochlorines, is associated with genome-wide differential DNA methylation patterns in sperm when compared against the lowest exposure levels. The DMRs are enriched for genes involved in many biological processes, including neurological functions and pathways impacted by neurodevelopmental disorders. While not establishing a causal relationship between EDC exposures and the disorders


During the last decades, the utilization of imaging modalities such as single photon emission computed tomography (SPECT), positron emission tomography (PET), and magnetic resonance imaging (MRI) in every day clinical practice has enabled clinicians to diagnose diseases accurately at early stages. Radiolabeled iron oxide nanoparticles (RIONs) combine their intrinsic magnetic behavior with the extrinsic character of the radionuclide additive, so that they constitute a platform of multifaceted physical properties. Thus, at a practical level, RIONs serve as the physical parent of the so-called dual-modality contrast agents (DMCAs) utilized in SPECT/MRI and PET/MRI applications due to their ability to combine, at real time, the high sensitivity of SPECT or PET together with the high spatial resolution of MRI. This review focuses on the synthesis and in vivo investigation of both biodistribution and imaging efficacy of RIONs as potential SPECT/MRI or PET/MRI DMCAs. The NOTA ligand forms thermodynamically stable complexes even at low pH (logKML = 23.33); however, the Cu(II) complex exhibits low kinetic inertness [ML = 22.3) and adequate in vivo stability, but DOTA requires more time (~1 s) and heating for complex formation. All in all, NOTA appears to be the better ligand for complexing Cu2+. Gallium3+ is hexacoordinated to DOTA, resulting in an N4O2 distorted octahedron. It has been shown that the large size of the macrocycle is not ideal for Ga3+ complexation (logK1 = 26.05) [3+ complexes with DOTA exhibit high thermodynamic stability (logKML = 21.3) and have excellent in vivo stability [68Ga-based radiopharmaceuticals in clinical practice are based on the DOTA chelator. Finally, another important issue refers to the comparison between passive and selective accumulation onto the tumor in both qualitative and quantitative terms. Upon administration in blood circulation, passive and selective DMCAs should possess specific physicochemical characteristics  ; howevertability . DespitelogKML = .3 and haDue to their multifaceted physical properties, RIONs serve as the parent platform for the development of powerful DMCAs utilized in SPECT/MRI and PET/MRI applications. Their main advantage originates from the fact that they can be employed in the above mentioned complementary, minimally invasive imaging modalities simultaneously, ultimately providing the optimal combination of the highest possible sensitivity with the maximum achievable spatial resolution. We seek out that in the near future, research goals should focus on the production of RIONs with ideal characteristics that affect their in vivo fate and, consequently, their targeting and imaging potential. With the continuous development of new RIONs, the future of DMCAs is definitely promising in the clinical diagnosis of diseases.


Sclerocormus from the Early Triassic of China that clearly demonstrates an omphalosaurid affinity, allowing for the synonymy of the recently erected Nasorostra with Omphalosauridae. The skull also reveals the anatomy of the unique feeding apparatus of omphalosaurids, likely an adaptation for feeding on hard-shelled pelagic invertebrates, especially ammonoids. Morphofunctional analysis of jaws shows that omphalosaurids occupy the morphospace of marine turtles. Our discovery adds another piece of evidence for an explosive radiation of marine reptiles into the ocean in the Early Triassic and the rapid recovery of pelagic ecosystems after the PTME.Marine ecosystem recovery after the Permo-Triassic mass extinction (PTME) has been extensively studied in the shallow sea, but little is known about the nature of this process in pelagic ecosystems. Omphalosauridae, an enigmatic clade of open-water durophagous marine reptiles, potentially played an important role in the recovery, but their fragmentary fossils and uncertain phylogenetic position have hindered our understanding of their role in the process. Here we report the large basal ichthyosauriform  Sclerocormus specimen.Further evidence for an explosive marine reptile diversification event in the Early Triassic is presented via morphological analysis of a new  However, this view has been challenged by an increasing number of recent discoveries suggesting the rapid emergence and evolution of Mesozoic marine reptiles in the aftermath of the PTME8. Marine reptiles attained high taxonomic and ecomorphological diversity as early as the late Early Triassic, and included generalist predators9, piscivores10, specialized forms using non-visual prey detection11, lunge feeders6 and durophages12. Durophagy evolved independently in several clades of Triassic marine reptiles, including ichthyosauriforms 14, placodonts15 and thalattosaurs16, but these durophagous ecomorphs were mostly limited to shallow-water environments and likely fed on abundant, sessile and benthic hard-shelled invertebrates. In fact, our understanding of the ecosystem recovery after the PTME is in general heavily biased towards data from shallow-water environments17 and relatively little is known about the nature of the recovery in open-water ecosystems3.Marine ecosystem recovery after the Permo-Triassic mass extinction (PTME), the most severe extinction in the history of the Earth, has been traditionally regarded as delayed and gradual18. Immediately after the mass extinction, however, ammonoids diversified explosively in the first few million years, and continued to play a significant ecological role for the rest of the Mesozoic due to their abundance, wide distribution, and high evolutionary rates18. Despite their high abundance in Mesozoic marine ecosystems, little is known about predation on ammonoids19. While sharks, mosasaurs, and cephalopods have been hypothesized as major groups that preyed upon ammonoids in the Jurassic and Cretaceous oceans, information regarding predators on Triassic ammonoids is scanty19.Ammonoids, a now extinct group of open marine cephalopods, had a successful ecological and evolutionary history for >300 million years during the Palaeozoic and Mesozoic, but their evolution experienced a severe bottleneck, with only three genera surviving the PTMEOmphalosaurus, is an enigmatic group of durophagous marine reptiles known hitherto from Early–Middle Triassic pelagic sediments of Western North America21, Svalbard22, and the Bavarian Alps23. There is one exception where a single jaw fragment was found in the Middle Triassic shallow marine carbonates of Southern Poland, but this occurence is likely a stray carcass24. Omphalosaurids are characterised by the presence of durophagous dentition arranged in unique dental batteries both in the upper and lower jaws, in which functional teeth underwent extreme tooth wear leading to almost complete tooth loss before shedding23. Even though omphalosaurids were first reported over a century ago20, they are represented in the fossil record mostly by jaw fragments and only a few specimens preserving partial cranial and postcranial remains have been discovered to date23. As a consequence, the taxonomic affinity of omphalosaurids has remained elusive, with several groups of Mesozoic reptiles, including rhynchosaurs, placodonts and ichthyosaurs, proposed as their closest relatives23. The incomplete nature of omphalosaurid fossil material, as well as some stark anatomical differences between them and other groups of Mesozoic reptiles, have hindered their unambiguous placement in a phylogenetic context and their role in pelagic ecosystem recovery after the PTME.Omphalosauridae, comprising several species in the genus Sclerocormus25 from the Early Triassic of China. Its skull and dentition share numerous synapomorphies with those present in Omphalosaurus and another basal ichthyosauriform –  Cartorhynchus13. Our phylogenetic analysis unambiguously establishes omphalosaurids as early diverging ichthyosauriforms and necessitates the synonymy of the recently erected Nasorostra (comprising Sclerocormus and Cartorhynchus) with Omphalosauridae. The skull of the new specimen also reveals the anatomy of the bizarre feeding apparatus of omphalosaurids, suggesting the presence of a grinding mechanism unique among Triassic marine reptiles, that probably evolved as an adaptation for feeding on hard-shelled pelagic invertebrates, especially ammonoids. The peculiar feeding ecology of omphalosaurids likely enabled these reptiles to become geographically widespread rapidly following the PTME event, suggesting the early establishment of complex and functional trophic webs in pelagic waters.Here, we describe a new specimen of the basal ichthyosauriform Diapsida Osborn, 1903Ichthyosauromorpha Motani et al., 2015Ichthyosauriformes Motani et al., 2015Omphalosauridae Merriam, 1906 Omphalosaurus nevadanus, Cartorhynchus lenticarpus and Sclerocormus parviceps and all of its descendants.The last common ancestor of Omphalosauridae is differentiated from all other Ichthyosauriformes by the presence of the following synapomorphies: elongate nasal reaching the snout tip; maxilla excluded from the border of the external naris; crushing dentition forming irregular maxillary and dentary batteries concentrated along cranial midline; dome-shaped tooth crown; first maxillary and dentary teeth rounded and blunt; premaxilla edentulous; convex occlusal surface of maxilla and corresponding concave occlusal surface of dentary; deep posterior mandible with slanting end and low jaw joint; wing-like process on posterior dentary.Sclerocormus Jiang et al., 2016Skull small; tail long; trunk short and deep; preorbital snout constricted and short; preorbital and postorbital parts of skull subequal in length; postorbital extending backward to the posterior edge of the upper temporal fenestra; parietal posteriorly bifurcating into two prongs; coracoid larger than scapula; humerus and femur straight; radius and ulna subequal in length; gastralia robust and comprising three sets, each gradually tapering towards their lateral ends.Sclerocormus cf. parviceps Jiang et al., 2016Omphalosaurus cf. O. nevadanus . The new specimen was collected from Majiashan quarry in Chaohu, Hefei, Anhui Province, China Fig. . MeasureSclerocormus on the basis of its large body size compared to the co-occurring Cartorhynchus13, as well as a well-developed gastral basket and ossified centralia that clearly differentiate it from Cartorhynchus13. The skull length of HFUT MJS-16-012 cannot be accurately measured since the tip of the snout is damaged. However, the upper and lower jaws are well articulated with each other in HFUT MJS-16-012 25. Using the simple skull-body length proportion of the holotype specimen, the total length of HFUT MJS-16-012 is estimated to have reached as much as 3.6 m. However, HFUT MJS-16-012 might still not be fully mature as the carpal region is poorly ossified. Whether the stark difference in body size between HFUT MJS-16-012 and the holotype of S. parviceps is due to taxonomic or intraspecific variation remains uncertain. HFUT MJS-16-012 also possesses a proportionally smaller orbit than the holotype specimen , a frontal contribution to the orbit , three rows of gastralia , and one centralium and one distal carpal preserved in the forefin . The decrease in the relative size of the orbit throughout ontogeny has been demonstrated in ichthyopterygians and other reptiles26, so a similar phenomenon in Sclerocormus seems likely. Furthermore, the skull of the holotype specimen is dorsolaterally compressed. This is likely to have an effect on the perceived differences in the relative contributions of the frontals to the orbital margin between both specimens. Because the gastral basket in the holotype specimen is largely disarticulated and scattered, it cannot be excluded that when articulated, the gastralia would have also formed three rows of elements. Finally, the numbers of ossified forefin elements have been demonstrated to vary in the forefin of the basal ichthyosauriform Chaohusaurus9, so the differences in the number of centralia and distal carpals in both specimens of Sclerocormus likely represent intraspecific variation. Because HFUT MJS-16-012 differs notably from the holotype only in its larger body size, we refrain from erecting a new species and tentatively refer the new specimen to S. cf. S. parviceps, pending a detailed comparative study between HFUT MJS-16-012 and the holotype.HFUT MJS-16-012 is referred to Cartorhynchus13 is also of relevance. The exposed area of the quadrate is much smaller than the squamosal in HFUT MJS-16-012, although it is partially covered by the squamosal  in Cartorhynchus14. The splenials of HFUT MJS-16-012 meet within the symphysis and extend to the tip of the jaw, while they do not in Cartorhynchus14. Cartorhynchus has three functional tooth rows on the right dentary14, and no dental battery23, while HFUT MJS-16-012 only has a single functional tooth row on the lower jaw but has a dental battery in both the upper and lower jaws . The gastralia are arranged into three sets in HFUT MJS-16-012 and are robust, curved, and flat with grooves on the surface , we included these taxa into a modified phylogenetic matrix focusing on the relationships among diapsid reptiles .To test the phylogenetic position of 7. PCO 1 and 2 explain nearly 50% of the craniodental morphological variation among Mesozoic marine reptiles. Morphospaces of durophagous forms such as placodonts, the durophagous mosasaur Globidens, and the omphalosaurid Cartorhynchus and Sclerocormus are convergently shifted towards higher PCO 1 and 2 values within each subclade . However, Cartorhynchus and Sclerocormus do not occupy the morphospace of shallow-marine durophagous marine reptiles, but instead plot beyond the edge of the ichthyosauromorph space among that of marine turtles , seems to indicate that throughout their evolution, the teeth of omphalosaurids underwent root reduction. An efficient grinding surface formed by numerous teeth dispersed along the occlusal surfaces of the jaw bones likely allowed omphalosaurids to process their food even more efficiently23. Given the lack of modern analogues of the feeding apparatus of omphalosaurids and modern open-water ecosystems dominated by hard-shelled invertebrates, an analysis of feeding and lifestyle in omphalosaurids inherently involves much speculation.Our phylogenetic analysis, which demonstrates that 12. Therefore, we argue that omphalosaurids represented a unique lineage of pelagic predators capable of feeding on hard-shelled invertebrates. Even though the body plan of omphalosaurids lacked anatomical features associated with efficient pelagic cruising, they were likely well adapted to a pelagic lifestyle as evidenced by their highly cancellous bone histology, since the disappearance of compact bone in tetrapods is generally a manifestation of more aquatic adaptation23. Furthermore, omphalosaurids do not need to be efficient pursuit predators, as they could have fed on high-density but low-speed pelagic invertebrates such as ammonoids39. The fact that omphalosaurids are known almost exclusively from pelagic deposits in which they co-occur with ammonoids also supports our hypothesis.The dentition and feeding apparatus of omphalosaurids differ strikingly from those of other contemporaneous marine reptiles with blunt teeth that were restricted largely to more shallow-marine environments and likely fed on sessile and benthic invertebratesCartorhynchus was previously determined to be at least semi-terrestrial on the basis of its highly unossified flippers, low dorsal vertebral count, and relatively weak visual capacity13. However, strongly unossified flippers are also present in the likely fully aquatic basal ichthyosauriform Chaohusaurus9 and the vertebral count of Cartorhynchus also overlaps with the range for some other marine reptiles13. In addition, the seemingly weakly developed underwater vision of Cartorhynchus could still have been useful for detecting pelagic invertebrates, especially in the photic zone13. Therefore, Cartorhynchus is probably a pelagic animal like other omphalosaurids, as also supported by the deep water environment of Majiashan quarry . The individual teeth were manually segmented using the Lasso tool.The new specimen (HFUT MJS-16-012) was scanned with a micro X-ray computed tomography scanner  at the X-Ray Computed Tomography and Multi-Scale Simulation Laboratory, School of Civil Engineering and Architecture, Zhejiang University, Hangzhou, China. A total of 3142 slices were generated. The original slices were read into ImageJ 1.52a25. The matirx was prepared using Mesquite. Two new characters were added to the original data matrix. Character scores for eight taxa  were modified and Pachypleurosauria was replaced with an Anarosaurus-Dactylosaurus operational taxonomic unit (see Supplementary Data 1 for details). The new specimen (HFUT MJS-16-012) was included in the matrix independently of the holotype of Sclerocormus, and two more taxa, Eusaurosphargis and Omphalosaurus, were included as well. The coding of the holotypes of Cartorhynchus and Sclerocormus was updated based on the recent restudy14, and that of Eusaurosphargis was based on ref. 45. The coding of Omphalosaurus was based on the literature and direct specimen observations. See Supplementary Table 46. All multistate characters were treated as unordered. A bootstrap sampling of 1000 replicates of the dataset was conducted to measure the nodal support. The NEXUS file is available in Supplementary Data We modified the recent taxon-character matrix for diapsids with a particular focus on marine reptiles7 was modified for the morphospace analysis. Characters 4–6 were removed due to the subjectivity of assessing the extent of temporal musculature in extinct taxa. Measurements and coding for C. lenticarpus and Sclerocormus (HFUT MJS-16-012) were modified or newly added to the original dataset from published photographs and personal examination. Following the method described in ref. 47 was generated from the combined continuous + discrete data, using the R package StatMatch48. The distance matrix was subjected to principal coordinate analysis (Supplementary Data 49.A previous data matrix of 18 morphofunctional characters and 207 taxa (Supplementary Data Further information on research design is available in the Supplementary InformationDescription of Additional Supplementary FilesSupplementary Data 1Supplementary Data 2Supplementary Data 3Supplementary Data 4Reporting Summary


Neurofibromatosis type 1 is a genetic disorder that follows an autosomal dominant pattern of inheritance. Ocular involvement is not uncommon, but spontaneous dialysis of the retina in the absence of a history of trauma is a rare clinical entity. Rare cases of retinal involvement such as retinal detachment or dialysis of ora serrata could be linked with the abnormal cell-matrix formation in neurofibromatosis type 1. Here, we present a case of a 36-year-old man having Neurofibromatosis Type 1 with spontaneous dialysis of ora serrata without prior history of ocular trauma. A routine fundoscopic examination should be done in addition to the examination of the anterior chamber in patients with neurofibromatosis type 1 despite the absence of ocular complaints. SOB was gradually progressive and of modified Medical Research Council (mMRC) grade 3 at the time of presentation. It was relieved while lying on the left side but aggravated on lying on the right side. The cough was productive, whitish, and non-blood mixed. He also had a history of night sweats and weight loss. On examination, 7-9 dark brown coloured cafe au lait spots of approximately 3x2 cm size each were present along with multiple neurofibromas over the trunk .There were several non-tender cervical lymph nodes palpated of which the largest lymph node measured approximately 20 mm. Following clinical examination, a Chest X-ray (CXR) was done which showed obliteration of left costophrenic and cardiophrenic angles with a homogenous opacity in the left middle and lower lung zones without tracheal or mediastinal shift, suggestive of left-sided pleural effusion .To confirm the diagnosis, a Contrast Enhanced Computed Tomography (CECT) chest was done which showed left-sided moderate pleural effusion with normal lung parenchyma along with features suggestive of plexiform neurofibromatosis. Pleural fluid analysis was done which showed lymphocytic effusion with high pleural fluid adenosine deaminase (55 IU/l). The patient was treated with anti-tubercular treatment with a diagnosis of neurofibromatosis with left-sided tubercular pleural effusion. He had no significant family history related to neurofibromatosis or tuberculosis. He was referred to the Department of Ophthalmology for ocular evaluation for NF 1.On further inquiry in the Department of Ophthalmology, he gave no history of ocular symptoms. There was no history of trauma to the eye and preceding ocular surgeries. On ophthalmological evaluation, the patient had a best-corrected visual acuity of 6/9 on the right eye Oculus Dexter (OD), and 1/60 on the left eye Oculus Sinister (OS) with no error noted. Further evaluation showed left exotropia of an eight-degree prism dioptre. Pupillary examination showed no anisocoria; however, a positive Relative Afferent Pupillary Defect (RAPD) of grade 2 was present on the left eye (OS). There was no evidence of angle recession, iridodialysis, phacodonesis, or choroidal ruptures. Slit-lamp examination revealed no iris Lisch nodules in either eye.Fundoscopy following pupil dilation revealed inferiorretinal detachment with the macula off in the left eye. The right eye was normal. The dialysis of the ora serrata was noted from the 4 o'clock to the 7 o'clock positions .The detailed ocular examination did not yield clinical evidence suggesting ocular trauma, and the patient repeatedly denied the same. Rhegmatogenous retinal detachment with spontaneous dialysis of the ora serrata in the left eye was diagnosed. On follow-up after 3 weeks, he was admitted with a lung abscess, for which chest tube insertion and drainage of the fluid were done. However, he had no ocular complaints, and the ophthalmic evaluation revealed no deterioration of vision. Even so, the ocular findings of Rhegmatogenous Retinal Detachment (RRD) with spontaneous retinal dialysis persisted , raising6 The cutaneous features include cafe-au-lait macules, axillary and groin freckling (Crowe sign), dermal tumors, etc.1 It can also affect the gastrointestinal tract, eye, musculoskeletal and neurovascular systems.1 The ocular manifestations can range from benign growths like iris hamartoma (Lisch nodules) and neurofibromas to malignant tumors like Optic Pathway Glioma (OPG).3Neurofibromatosis type 1 (NF 1) is characterized by abnormal growths of epithelial, mesothelial, and endothelial components.7 Likewise, retinal dialysis also occurs secondary to trauma; however, a few inherited spontaneous retinal dialysis cases have also been reported.8 Retinal detachment with spontaneous dialysis of ora serrata in the absence of a history of ocular injury or tumor has rarely been reported in patients with neurofibromatosis type 1.3Retinal detachment in patients with NF 1 usually occurs following mechanical trauma to the eye or with retinal tumors like hamartomas and capillary hemangioma.3 Fibroblasts migrate and proliferate in this region following vitreous haemorrhage and penetrating ocular trauma and are thought to be responsible for the vitreous base avulsion.9 In NF 1, vitreoretinal pathologies are linked with abnormal cellular proliferation in the vitreous base even in the absence of previous ocular pathology.10 One article hypothesized that there is Matrix Metalloproteinase 1 (MMP1) downregulation in stromal cells in NF 1 leading to disturbance in the collagenolytic process and thereby accumulation of abnormal fibroblast and neo-collagen in the ocular region in NF 1.10 Another study showed that there is decreased receptor binding of Epidermal Growth Factor (EGF) in NF 1, which might lead to membrane defect in the retina.6 These factors alter the cellular matrix of the vitreous base resulting in reduced production of collagen fibers that keep the peripheral neurosensory retina intact.10The vitreous base contains numerous fibroblasts in its cortex, which form collagen fibrils that insert through the basal lamina of the pars plana and peripheral retina.3 Unlike in their case, our patient had diminished visual acuity in the left eye noted after examination along with exotropia and grade 2 RAPD therefore, we performed an MRI of the brain instead of an exploration of the retina to rule out tumors. In their case, the underlying defect was corrected by ophthalmological intervention but this approach was not done in our case since the patient had asymptomatic chronic RRD. We opted to observe the patient with routine follow-up as the risk of progression of the RRD outweighed the benefit of surgical repair.In a pediatric case study, the ocular examination revealed retinal detachment with dialysis of the ora serrata in the left eye, similar to our case, the patient had no ocular symptoms and no history of ocular trauma.Although retinal dialysis is a rare entity in cases with NF 1, with this case, we would like to emphasize performing routine funduscopic examination under mydriatics in all the patients with NF 1 even in the absence of ocular complaints in addition to evaluating the anterior segment. Furthermore, imaging modalities like MRI of the brain should be employed to rule out underlying lesions. This will not only aid in ruling out the presence of ocular lesions but also help in diagnosing any obscured asymptomatic retinal pathologies like spontaneous retinal dialysis, as in our case thereby, preventing vision-threatening complications.