Personalized treatment of metastatic non-squamous non-small cell lung cancer (NSCLC) requires detailed molecular characterization of the tumour including detection of predictive driver mutations and programmed death ligand 1 (PD-L1) expression. Complete detection is influenced by the amount of tumour cells sampled as well as their quality. Different sampling techniques may be necessary to provide sufficient tumour material for comprehensive molecular characterization. Missing the detection of targetable molecular genetic aberrations would have a serious impact on the quality of life and prognosis of a patient. This case report highlights the importance of biopsy technique in a patient with NSCLC. Several procedures—pleural puncture, transthoracic lung biopsy and endobronchial ultrasound-guided transbronchial needle aspiration (EBUS-TBNA)—could not provide sufficient tumour material for precise tumour characterization. Only the addition of EBUS-guided transbronchial lymph node cryobiopsy (EBUS-TBLNC) enabled complete immunohistochemical and genetic tumour characterization, demonstrating PD-L1 expression in 100% of the tumour cells in the absence of actionable genetic alterations. Based on these results, immunotherapy was initiated. Mutational analysis and programmed death ligand 1 (PD-L1) expression are required for personalized treatment of metastatic non-small cell lung cancer (NSCLC). The detection of a targetable driver mutation has prognostic and predictive relevance, giving the patient the opportunity to receive highly effective first line targeted treatment with a kinase inhibitor ,3. The bTissue sampling is affected by size and/or localization of tumour lesions, which may result in insufficient biopsy samples, both in terms of quantity and quality. In cases of mediastinal or hilar lymph node involvement, cytological specimens obtained by EBUS-TBNA can often confirm malignancy , but cytEndobronchial and transbronchial cryobiopsy can yield large tissue samples allowing exact tumour characterization and may be superior to other biopsy techniques . EBUS-TBWe describe the case of a patient in whom several attempts of characterization of a lung tumour by different sampling techniques  were unsuccessful due to low cellularity of the samples. Only EBUS-TBLNC yielded sufficient tumour material for detailed immunohistochemical and molecular tumour characterization and allowed to determine the optimal systemic therapy for this patient’s non-squamous NSCLC.In an 83-year-old male former smoker (30 pack-years) with multiple comorbidities but with a good performance status (ECOG 1), the work-up of a symptomatic left pleural effusion led to the detection of a lung lesion in the left lower lobe accompanied by lymphangiosis, as well as discrete hilar and mediastinal lymphadenopathy. Drainage of the pleural effusion revealed single cells of a thyroid transcription factor 1 (TTF-1)-positive pulmonary adenocarcinoma. Further molecular analysis was not possible due to the scarcity of tumour cells in the cyto spin. For further classification of the non-squamous NSCLC, cytological material was obtained by EBUS-EBNA from mediastinal lymph nodes (LN) in position 4L, the best accessible site according to the staging CT A. DespitTherefore, we suggested tissue sampling with the aim of extracting solid tissue from the mediastinal LN by EBUS-TBNLC. The patient was informed about potential side effects and the limited experience with this technique. He agreed to undergo the procedure.Under EBUS guidance, tunnelling through the bronchial wall in the direction towards LN 4L was made using a monopolar electric needle knife A. A 1.1 Extensive infiltrates of TTF1 positive adenocarcinoma cells were seen C. MolecuBased on these findings and the positive risk-benefit ratio, first-line PD-1-directed therapy was initiated with Pembrolizumab as monotherapy ,16,17. UWe present a case in which, after several unsuccessful sampling attempts for NSCLC characterization, EBUS-TBLNC succeeded in adequate tumour sampling for molecular characterization, thereby allowing personalized treatment of the patient. EBUS-TBNLC may contribute to optimized molecular workup of NSCLC in the future, thereby providing important information for patient treatment.


Drosophila model to gain insight into the role of these extracellular matrix receptors in tumoural progression. We find that the role of integrins as tumour suppressors is conserved. The depletion of integrins enhances the growth and invasive behaviour of tumours induced by a gain of function of an oncogenic form of Ras, by regulating cell shape and cycle progression. Furthermore, our results show that integrin loss enhances the ability of Ras tumour cells to affect the tumour microenvironment through the activation of the JNK pathway.Studies over the last few years have revealed that integrin function in cancer is controversial. Accumulating evidence has shown that integrins can act as both tumour promoters or tumour suppressors. The targeting of integrins has shown enormous potential in both the diagnosis and treatment of cancer. Thus, further mechanistic insights into the roles of integrins as regulators of cancer progression are required. In this work, we aim to use the Drosophila, offer great potential to unravel underlying conceptual principles. Here, we find that in the Drosophila wing disc epithelium the βPS integrins act as suppressors of tumours induced by a gain of function of the oncogenic form of Ras, VRas12. We show that βPS integrin depletion enhances the growth, delamination and invasive behaviour of VRas12 tumour cells, as well as their ability to affect the tumour microenvironment. These results strongly suggest that integrin function as tumour suppressors might be evolutionarily conserved. Drosophila can be used to understand the complex tumour modulating activities conferred by integrins, thus facilitating drug development.Cancer is the second leading cause of death worldwide. Key to cancer initiation and progression is the crosstalk between cancer cells and their microenvironment. The extracellular matrix (ECM) is a major component of the tumour microenvironment and integrins, main cell-ECM adhesion receptors, are involved in every step of cancer progression. However, accumulating evidence has shown that integrins can act as tumour promoters but also as tumour suppressor factors, revealing that the biological roles of integrins in cancer are complex. This incites a better understating of integrin function in cancer progression. To achieve this goal, simple model organisms, such as  Cancer is the second leading cause of death worldwide. In 2020, there were 19.3 million new cancer cases, and by 2040, this number is expected to rise to 29.5 million [Drosophila, with fewer genes compared to the human genome and lower genetic redundancy, have great potential to unravel underlying conceptual principles.The crosstalk between cancer cells and their microenvironment plays a key role in cancer initiation and progression. The extracellular matrix (ECM) is a major component of the tumour microenvironment and integrins, main cell adhesion receptors for components of the ECM, are involved in almost every step of cancer progression, including cancer initiation, proliferation, metastasis and survival of circulating tumour cells (reviewed in ). IntegrDrosophila genome encodes only two β subunits, βPS and βν, and five α subunits, αPS1 to αPS5. The βPS subunit, encoded by the gene myospheroid (mys), the orthologue of vertebrate β1, is widely expressed and forms heterodimers with all α-subunits  in the wing disc results in hyperplastic growth. This has been used in genetic screens to isolate additional genes, which can act as either suppressors or enhancers of the growth of VRas12 overexpressing cells [VRas12 wing disc epithelial cells stimulates their basal extrusion [In contrast to vertebrates, the iewed in ,9). The iewed in ,12,13,14iewed in ). Integriewed in ,17,18. Tiewed in ). More s in [Ras , mutated in [Ras . The indng cells . In thisVRas12 epithelial tumour cells. We find that downregulation of the βPS integrins enhances VRas12-mediated tissue hyperplasia. The elimination of integrins promotes the growth of VRas12 tumour cells. In addition, using live imaging and confocal microscopy, we find that the reduction of βPS integrin levels induces the delamination and invasion of tumour cells to neighbouring wild-type regions. These results demonstrate that, as is the case in some mouse models and human cancer cell lines, in Drosophila, integrins inhibit the metastatic capacity of tumour cells. These results also strongly suggest that the role of the β1 integrin family as suppressors of tumour metastasis might be evolutionarily conserved.In this work, we use the wing disc to analyse the role of integrins on the progression of VUAS-Ras12 [Ap>myrT/Cyo, Ap>GFP/Cyo, Ap-Gal4-UAS-flyfucci/Cyo (gifts from Dr. Marco Milán’s laboratory); RNAiUAS-mys and UAS-RNAihid . Flies were raised at 25 °C.The following stocks were used: S-RasV12 ; Ap>myrTThird instar larvae wing imaginal discs were dissected and stained following standard procedures and mounted in Vectashield . The primary antibodies used were: goat anti-GFPFICT , rabbit anti-caspase Dcp1 , rabbit anti-pJNK , mouse anti-βPS , rabbit anti-PH3 , rabbit anti-Perlecan , rabbit anti-aPKC , Rat-anti-RFP . The secondary antibodies employed were Alexa fluor 488,  and Cy3 and Cy5  at 1: 200. DNA was labelled using Hoechst . F-actin was visualized using Rhodamine Phalloidin .Confocal images were obtained using a Leica SPE microscope, equipped with a 20× oil objective (NA 0.7) and a Leica Stellaris microscope equipped with 20×, 40× and 63× oil objectives with NA 0.75, 1.4 and 1.4, respectively.For live imaging, wing disc samples were prepared as described in  and filmTo measure the fluorescent intensity of all markers, fluorescent signalling was quantified on several confocal images per genotype, using the square tool FIJI-Image J version 2.0.0-rc-59/1.51n. The microscope settings, such as the laser parameters and z-confocal interval, were maintained between imaging sessions in each experiment. Measurements of whole fluorescence intensity were carried out using the “mean grey value” tool in FIJI. This tool divides the mean of all included pixel intensities by the outlined cell area. The different areas of the wing disc, apterous  and control  domains were selected using the wand tool.To quantify cell proliferation, dots of the fluorescent intensity of wing discs stained with an anti-PH3 antibody were quantified using the FIJI-Image J tool to analyse particles. Images were previously processed using the tool threshold, which transforms 8-bit images into a binary system.Cell areas were calculated by manually delineating the apical and basal surfaces of the cells and measuring the resulting surfaces using FIJI-ImageJ software. To quantify cell height, a vertical line from the apical to the basal surface was drawn in a region of interest of a wing disc stained for F-actin, and the length of this line was measured using FIJI.Flyfucci analyses were performed using the “merge channels” tool in FIJI to generate an image containing the green and red channels together. The “TrackMate” tool was used to measure the fluorescence intensity of both channels at the same 3D coordinates. Spots showing similar levels of the fluorescence intensity of both channels correspond to G2 and those with higher levels, at least three times greater, in the red channel with respect to the green one and vice versa correspond to S and G1, respectively.p-value was calculated using the Welch test and assuming that all distributions were normal with unequal variances. Multiple comparison p-values were adjusted following the BH procedure .All graphics and mathematical analysis were generated using R software and the ggplot2 package. For statistical comparisons, the VRas12 in the dorsal domain of wing discs, by means of an apterous-Gal4 line , caused tissue overgrowth and the formation of ectopic folds [VRas12-mediated oncogenesis, we decided to reduce integrin levels in tumour cells by expressing a mys-specific RNAi, a method that was shown to cause a strong reduction in βPS protein levels in wing discs [mys RNAi in the dorsal compartment of wing discs  induced the formation of ectopic folds and a reduction in its size  enhanced the formation of folds and overgrowth due to VRas12 overexpression alone . We found that, although the overexpression of a RNAihid in Ap>GFP; RNAimyswing discs led to an increase in fold formation and overgrowth . Furthehenotype . Thus, w was not D,D’. In 12 cells E,E’.VRas12 on its own does not grossly affect the BM, loss of cell polarity in these tumour cells induces BM degradation and the formation of aggressive, malignant tumours . In addiewed in ). Defectiewed in . Thus, tVRas12 resulted in a reduction in the number of PH3+ cells  showed that, in agreement with previous results ,25,33,343+ cells . This wa3+ cells . To anal3+ cells . We founVRas12 promotes G1-S phase transition, as a consequence of E2F activation and cyclin E induction [VRas12 in inducing transition from G1 to S phase and also showed that VRas12 expressing tissues exhibited a reduction of the number of cells in S phase and an accumulation in G2, compared to control tissues [VRas12 in promoting cellular growth and senescence in Drosophila imaginal epithelia [VRas12 overexpression [VRas12 tumour cells in G2 . This iDrosophila [β1 family of integrins as tumour enhancers or suppressors. Deletion of β1 integrins blocks and delays cancer initiation in the Polyoma middle T (PyMT) and ErbB2 oncogene models, respectively [β1 integrins leads to impaired tumour growth and metastasis [β1 integrins appear to play a tumour suppressor-like role. Thus, the deletion of β1 integrins in the TRAMP prostate adenocarcinoma model led to an increase in the expansion of the tumour cell population, an enhancement of the rate of tumour progression and a reduction in overall animal survival [Drosophila models to analyse this issue. The zebrafish has become a promising model for vertebrates in recent years, as it is small, transparent, low cost and reproduces fast. At present, zebrafish xenografts have been used to analyse the role of integrins in metastasis. These studies have shown that disrupting the expression of some integrins, such as β1, α3 or α6 in cancer cells reduces their ability to extravasate or metastasize when transplanted in zebrafish, suggesting a role for integrins as tumour promoters in this context [Drosophila, despite the fact that it has been extensively used to study cancer, there is little evidence for its use as a platform to analyse the role of integrins in tumour development. Using Drosophila Intestinal Stem Cell tumours in the adult midgut, it was shown that integrins were required for the initiation of hyperplasia upon loss of adenomatous polyposis coli and for the growth of tumours induced by the gain of an oncogenic form of the transcription co-factor Yki [Drosophila wing imaginal disc epithelium, suggesting that, the dual role of integrins as tumour modulators is conserved. In the future, Drosophila could be used to understand the opposing tumour modulating activities conferred by integrins. This will facilitate further development of drugs targeting integrin signalling pathways.To better understand the genetic bases and the development of cancer, researchers have used small animal models, such as mice, zebrafish and osophila . In partectively ,42. Liketastasis ,44. In csurvival . In cont context ,47,48. Ictor Yki ,50. Thes2β1 integrin does not affect tumour growth in vivo or in vitro, the α2-null/Neu tumour cells demonstrate enhanced anchorage-independent growth [Drosophila, loss of integrin function inhibits the proliferation of Yki-driven intestinal stem cell tumours in the adult [VRas12 in the Drosophila wing disc produces hyperplasia, as a consequence of increased cell growth, accelerated G1−S transition and cell shape changes [VRas12 in wing disc cells induces a cell shape change from columnar to cuboidal [VRas12 overexpression, by enhancing cell shape changes and cellular growth. We have previously shown that the removal of integrins on its own induces a change in cell shape similar to that caused by VRas12 overexpression [The role of integrins in tumour growth is also complex. Thus, while loss of the αt growth . In Droshe adult . As ment changes . In agrecuboidal . Here, wpression . In addipression . VRas12 wing discs when downregulating integrin expression could just be an additive effect of simultaneously disrupting two pathways independently regulating cell shape. Alternatively, integrins could modulate oncogenic Ras signalling. In fact, many crosstalk between integrins and oncogenic Ras have been proposed, including the regulation of RTK activity by integrins [Ras signalling , ratherVRas12 wing disc cells does not seem to clearly affect the adjacent basement membrane, it increases the ability of the tumour cells to cause activation of the JNK pathway and non-autonomous death in neighbouring wild-type tissue. This effect has been proposed to increase the proliferation of tumour cells through the secretion of growth factors and Eiger (tumour necrosis factor homolog) [Integrins not only regulate the properties of the cancer cells but also their ability to alter the tumour microenvironment, which includes the extracellular matrix and surrounding cells, stimulating non-autonomous tumour progression  ,37. Here3β1 and α2β1 integrins have been shown to suppress metastasis of prostate and breast cancer [9 reduces tumour metastasis in triple-negative breast cancer [VRas12 alone is not sufficient to create invasive tumours in wing disc epithelial cells, the elimination of integrins in these tumour cells triggers an invasive behaviour. As integrins are the main receptor implicated in cell migration, how can their elimination induce invasion? Even though collective cell movement of primary melanoma explants requires β1-integrin mediated cell-matrix adhesion, impairment of cluster cohesion resulted in detachment and dissemination of amoeboid single tumour cells [1 integrin function swapped the migratory behaviour of human and mouse E-cadherin–positive triple-negative breast cancer (TNBC) cells from an integrin-dependent collective movement to an integrin-independent “amoeboid” crawling and dissemination [VRas12 wing disc epithelial cells induces their detachment [1 family of integrins as promoters of epithelial phenotype and suppressors of dissemination. Understanding the apparent metastatic suppression activity conferred by integrins in certain cancer contexts is crucial to developing proper treatment strategies targeting integrins.Most studies have shown that the upregulation or overexpression of integrins associates with cancer metastasis, conferring integrins a pro-metastatic activity (reviewed in ). Furthet cancer ,57,58,59t cancer , while it cancer . Here, wur cells . Similartachment . Here, wDrosophila cancer models has been established in several tissues besides the wing imaginal disc, such as the gut, the brain and the eye imaginal disc, by means of transgenesis, genome editing, transplantation and drug-induced toxic damage. These tumour models have provided new concepts and findings in cancer biology that have significant parallels and relevance to human cancer. Our findings establish the Drosophila wing imaginal disc as a platform to address the complex role of integrins in cancer. We can now extend these studies to the other Drosophila cancer models to further explore the diverse biological functions integrins perform in cancers. As targeting integrins has shown huge potential in the diagnosis and treatment of cancer, deeper mechanistic insights are necessary to better comprehend the mechanisms of the integrin-mediated biological behaviour of cancer cells.A variety of Drosophila wing disc epithelium, we show that the βPS integrin, the orthologue of vertebrate β1, can act as a suppressor of tumours induced by a gain of function of the oncogenic form of Ras, VRas12. We show that the βPS integrin restrains the growth, delamination and invasiveness of VRas12 tumour cells, as well as their ability to affect the tumour microenvironment. These results strongly suggest that the role of the integrin of the β1 family as tumour suppressors might be evolutionarily conserved. In the future, it will be interesting to study the contribution of the βPS integrin to other Drosophila cancer models to further explore the multiple biological functions mediated by integrins in cancers. Drosophila can be used to understand the mechanisms underlying the complex tumour modulating activities conferred by integrins, thus facilitating the design of cancer drugs targeting integrins.Using the


Dear Editor,2. Immune cells, mostly neutrophils and epithelial cells form the cellular components of saliva. Since neutrophils rapidly react to the changes in the niche, they play a significant role in oral cancer immunology. A novel mechanism of neutrophil-mediated host response called NETosis was reported in 20043. Neutrophils form neutrophil extracellular trap (NET) that protect the host from an array of infectious diseases have been reported to play a role in malignancy as well. This NET-mediated trapping mechanism of circulating tumour cells has been detected in a number of diverse types of cancers, including head and neck cancers. It has been reported that the tumour infiltrating neutrophils produces NETs and also lead to tumour progression and subsequently promote metastasis in many cancers including head and neck cancer4. In addition, they are known to contribute to the progression as well as regression of tumours in many oral cancers through multiple molecular mechanisms, such as the suppression of cytotoxic and helper T-cell responses. Also, these NETs are known to induce tumour angiogenesis contributing to the metastatic risk of tumours. Neutrophilia and a high neutrophil-to-lymphocyte ratio have been linked with poor prognosis in several head and neck cancers5. These changes may be due to the spread of the tumour to the lymph nodes. Thus, understanding their molecular basis would help in figuring out the tumour immune escape mechanisms, as well as their diagnostics and therapy.Neutrophils are the most abundant type of leucocytes (white blood cells), which play an important role in the innate immune response by providing defence against a number of bacterial and other microbial infectionset al.6 in a mouse model of chronic myelogenous leukaemia. The role of NETs in tumours varies according to the status of the immune system. On one hand, NETs induce apoptosis in head and neck squamous cell carcinomas7. On the other hand, NETs have been found to enhance the proliferation of tumours by elevating the expression of activation markers and also by inhibiting the apoptosis of tumour cells8. NETs also play a role in the protection of tumour cells from cytotoxicity by the suppression of CD8 + and natural killer (NK) cells in the tumour niche or microenvironment, thus promoting tumour survival and growth9. Analysing and understanding the molecular mechanisms underlying NET formation has revealed that there are some key molecules, receptors, and signalling pathways, such as the upregulation of cytokines and chemokines, impairment of immune cell cytotoxicity, and induction of angiogenesis during metastasis which also participate in both NETosis and the biological behaviour of tumours.How NETosis promotes tumour progression in cancers?: NETs in cancer were first identified by Demers 10. NET inhibitors can also be used in conjunction with immunotherapies or as adjuvants to other cancer drugs to enhance their effectiveness9. In addition, platelets developing from NETosis might protect circulating tumour cells from immune cells through their trapping properties. Metastasis can be minimized or stopped by depleting or blocking platelets by disrupting this interaction of platelets, tumour cells, and NETs. Hence, NETs might serve as an effective target for cancer treatment12. Observing the role of NETs in upgrading the metastatic potential of tumour cells, inhibiting their formation, or reducing their activity in cancers could be advantageous for better prognosis in the treatment of cancer. However, only limited studies are currently available, and the ongoing clinical trials also have not yet provided any clarification or clear inferences to target NETs. Currently, the challenge in targeting NETs is a lack of precise biomarkers that can predict an adequate response to NET-interfering therapy in patients. Further studies may reveal detailed information about the connections between NETosis, cancers, and other diseases and help in identifying appropriate strategies to effectively modulate the dysregulated NETosis and associated mechanisms.Significance of NETosis in cancer therapy: NETosis can be considered a double-edged sword. Circulating levels of NET components, such as cell-free DNA (cfDNA), neutrophil elastase (NE)–DNA, myeloperoxidase (MPO)–DNA, and citrullinated histone H3 (CitH3) can serve as beneficial biomarkers for several kinds of cancers. Blocking the formation of NETs via small-molecule drugs against DNase I, NE inhibitors, MPO inhibitors, and PAD4 inhibitors might also have great potential, and studies have revealed that targeting NETs has resulted in a significant reduction of tumour proliferation and metastasis as well as decreased resistance to radiotherapy, chemotherapy, and immunotherapyNot applicable.Not funded by any agency and received funding in any manner/form.D.G., V.P.V., K.M.S., L.P., and U.M.: conceptualization, data curation, and writing – original draft preparation; K.M.S.: conceptualization, study design, data curation, writing – original draft preparation, reviewing and editing, and visualization and supervision.The authors declare no conflicts of interest.Dr Krishna Mohan Surapaneni.This correspondence is based exclusively on resources that are publicly available on the internet and duly cited in the ‘References’ section. No primary data were generated and reported in this manuscript. Therefore, data have not become available to any academic repository.


We have identified the cell guidance receptor EphA3 on distinct stromal/fibroblast-like cell types in the tumour microenvironment (TME) that promote growth and angiogenesis, both in human cancers and in mouse tumour models. To investigate its role in the TME, we developed novel transgenic mice with inducible shRNA-mediated knockdown of EphA3 expression. Knockdown of EphA3 expression in the TME in syngeneic mouse tumours reduced mesenchymal stromal/fibroblast-like cells in tumours, with corresponding reductions in tumour vasculature and tumour growth, and increased immune infiltrate, indicating an important role in tumour progression.Tumour progression relies on interactions with untransformed cells in the tumour microenvironment (TME), including cancer-associated fibroblasts (CAFs), which promote blood supply, tumour progression, and immune evasion. Eph receptor tyrosine kinases are cell guidance receptors that are most active during development but re-emerge in cancer and are recognised drug targets. EphA3 is overexpressed in a wide range of tumour types, and we previously found expression particularly in stromal and vascular tissues of the TME. To investigate its role in the TME, we generated transgenic mice with inducible shRNA-mediated knockdown of EphA3 expression. EphA3 knockdown was confirmed in aortic mesenchymal stem cells (MSCs), which displayed reduced angiogenic capacity. In mice with syngeneic lung tumours, EphA3 knockdown reduced vasculature and CAF/MSC-like cells in tumours, and inhibited tumour growth, which was confirmed also in a melanoma model. Single cell RNA sequencing analysis of multiple human tumour types confirmed EphA3 expression in CAFs, including in breast cancer, where EphA3 was particularly prominent in perivascular- and myofibroblast-like CAFs. Our results thus indicate expression of the cell guidance receptor EphA3 in distinct CAF subpopulations is important in supporting tumour angiogenesis and tumour growth, highlighting its potential as a therapeutic target. It is now widely recognised that tumour development depends not only on genetic alterations and ensuing protein expression changes in tumour cells, but also on complex interplay with the tumour microenvironment (TME). Thus, cells from surrounding tissues, or recruited from the bone marrow, contribute to the tumour stroma and vasculature, and provide structural and nutrient support that promotes tumour growth . ReciproEph receptors make up the largest family of receptor tyrosine kinases (RTKs) and are distinguished from other RTKs by binding to cell-bound ligands (ephrins), interacting across cell-cell junctions and controlling cell adhesion and segregation, cell fate, and survival. They are expressed most prominently during development, where they control cell guidance and differentiation during diverse processes including tissue boundary formation, organogenesis, and development of the neural, vascular, and immune systems ,9,10. WhWe previously found that EphA3, known to be overexpressed in haematopoietic malignancies and many solid tumour types , is most2) to maintain EphA3 expression. 5B3 ES cells were cultured on a mouse embryonic fibroblast (MEF) feeder layer as previously described [Lewis lung carcinoma (LLC), B16F10 melanoma, and fibroblast cell lines were grown in DMEM/10% foetal calf serum. Primary mesenchymal stromal cells were isolated and maintained, as previously described , under hescribed .g for 1 h at 32 °C. Transduced cells were selected using puromycin resistance and analysed for GFP fluorescence and EphA3 expression via flow cytometry and quantitative RT-PCR.shRNA sequences targeting mouse EphA3 were designed using DSIR  and SensshRNA EphA3.3466 was inserted into the pColTGM (Col1A TREtight-eGFP-miR30) plasmid (EcoR1/XhoI restriction sites), containing a tetracycline (Tet)-responsive TRE-tight promoter element  driving RNA was extracted using Isolate II RNA kit (Bioline). Samples were analysed via SYBR Green qRT-PCR, using a Rotorgene 6000 (Corbett Research). Relative gene expression was determined using tubulin β4a as a reference gene.qRT-PCR primers: EphA3 2721For CAAGTTCGAGCAGATCGTCA, 2879 Rev CCGTTAAGCCAATCACCAGT; tubβ4a For CCAGATCTTTCGGCCAGACAAC, Rev CCCTCGGTGTAGTGACCCTTG.PCR primers for genotyping: EphA3 sh 3466For CCACAGATGTATTAAAAGAAAC, RBG Rev GAAAGAACAATCAAGGGTCC; Ren 713For ATGTATAGATAAGCATTATAATTCC, RBG Rev GAAAGAACAATCAAGGGTCC; rtTA For CGGTATCGAAGGCCTGACGACAAGG, Rev AGAAGCCTTGCTGACACAGGAACGC.n ≥ 5), and tumour growth was monitored by calipers until ethical endpoint or at earlier time points for analysis, as specified in the figure legends. The results show representative data from at least three replicate experiments. All animals were handled in strict accordance with good animal practice, as defined by the National Health and Medical Research Council  Code of Practice for the Care and Use of Animals for Experimental Purposes, and approved by the Austin Hospital and Monash University Animal Ethics Committees.Aged-matched controls and EphA3 shRNA mice  were fed on mouse food pellets containing Doxycycline  for 2 weeks prior to experiments. For syngeneic tumour models, we used LLC cells and B16F10 melanoma cells, which we previously characterized as EphA3-negative and EphA3-positive, respectively. 5 million LLC or 2 million B16F10 cells were injected subcutaneously in the flank of the control and EphA3 shRNA mice  was from Millipore; rat anti-mouse CD90-PB (30-H12), CD45-PB (30F-11) and Sca1-PE/Cy7 (D7) monoclonal antibodies were from BioLegend, San Diego, CA, USA; rat anti-mouse CD8 (4SM15) and TER119 (ef450 conjugated 48-5921-80) monoclonal antibodies were from eBioscience ; CD31 rat anti-mouse monoclonal antibody (Clone MEC 13.3) was from Becton Dickinson; rabbit transgelin (155272) polyclonal and alpha smooth muscle actin (SMA) (ab124964) monoclonal antibodies were from Abcam ; α-EphA3 mouse monoclonal IIIA4 and sheep polyclonal antibodies have been previously described ,33. AlexMouse tumours were embedded in optimal cutting temperature (OCT) medium. When relevant, to preserve GFP, tumours were fixed in 4% PFA overnight, and transferred to 30% sucrose for 24 h prior to embedding and freezing in OCT. Formalin-fixed, paraffin-embedded human tumour samples were sectioned and stained with the primary antibodies listed above. For the IHC antibody, binding was detected using the IHC Vector Labs ABC staining kit, before haemotoxylin counterstaining. Stained sections were scanned using the Aperio Scanscope XT , and biomarker analysis was conducted using the Aperio image analysis Positive Pixel Count v9 algorithm. For immunofluorescence staining of human tumours, Alexa Fluor-labelled secondary antibodies  were used, nuclei were stained with DAPI, and imaging was carried out using a Zeiss LSM 980 confocal microscope (displayed as a maximum projection of a 6 micron z-stack), using Zeiss imaging software. Human tissue samples were collected following approval from the Austin Health Human Ethics Committee.Cells stained with the indicated primary antibodies and Alexa Fluor-conjugated secondary antibodies were analysed via flow cytometry using LSRII and FACS Canto II flow cytometers (Becton Dickinson). Dead cells detected with propidium iodide were excluded, and FLOWJO software version 10.5.3  was used for raw data analysis and multivariate compensation.p value range to indicate statistical significance, as determined by unpaired two-tailed student’s t test, or as otherwise specified.Graphs show individual data points, with mean and standard error, and We first investigated EphA3 expression in MSC-supported angiogenesis using the aortic ring model of sprouting angiogenesis, where a small piece of mouse aorta explant is embedded in type I collagen and incubated in the presence of vascular endothelial growth factor-A (VEGF-A) to encourage vessel sprouting . After 1To investigate if EphA3 functionally supports neo-angiogenesis, we developed shRNA transgenic mice, allowing doxycycline-induced expression of shRNA, linked with green fluorescent protein (GFP) as a reporter ,35. ThisTo test and quantify inducible EphA3 knockdown in these mice, we again isolated primary cells from the aortae of EphA3 shRNA or control shRNA mice, and cultured them in vitro with or without the tetracycline analogue doxycycline. FACS analysis of total cell populations showed robust induction of GFP expression upon doxycycline stimulation, accompanied by a marked decrease in EphA3 expression, compared to aortic cell cultures from control shRNA mice A. As aboAs a functional readout in vitro, we compared the angiogenic sprouting of aortic rings recovered from EphA3 and control shRNA mice. These were cultured in the presence of doxycycline to induce shRNA-GFP expression, and stained with fluorescently labelled lectin to mark vessel sprouts. Immunofluorescence staining confirmed expression of EphA3 in cells with a mesenchymal, migratory appearance, around lectin-stained sprouts, which was notably reduced in explants from EphA3 shRNA mice A. ImaginTo investigate a functional role for EphA3 in cells contributing to the tumour microenvironment, we used a Lewis lung carcinoma (LLC) syngeneic mouse tumour model, chosen because we showed previously that EphA3 was expressed in the TME, including MSCs recruited from the bone marrow, but not in the tumour cells . Mice weWe next compared LLC primary tumours grown subcutaneously in mice with doxycycline-induced shRNA expression. Analysis of early-stage tumours showed EphA3 was almost exclusively expressed in GFP+, CD90+/Sca1-high MSCs , and thihttps://melanoma.cellgeni.sanger.ac.uk, accessed on 7 September 2023), showing EphA3 expression in CAFs .Finally, we sought to characterise EphA3 expression in the TME of human tumours. We used a single-cell RNA sequencing analysis of 26 early-stage breast tumours  to analyle_cell, , and colInterestingly, we also tested EphA3 expression in a human breast fibroblast cell line that had been ‘tumour-educated’ by co-implantation with human breast tumour cells in a mouse xenograft model, inducing a myofibroblast-like CAF phenotype . Flow cyThe tumour microenvironment is of increasing interest for therapeutic intervention, with anti-angiogenic and immune modulatory agents already used in the clinic. Eph receptors have long been recognised as potential therapeutic targets due to their deregulated expression in tumour cells. More recently, their detection in cells of the TME suggests that they may act more broadly in tumour development. We determined previously that EphA3 was frequently expressed in the stroma in a range of different human tumours, and in tumour cell xenografts in mice, even when not expressed in the tumour cells . Bone maHere, we have developed novel transgenic mice in which EphA3 expression can be specifically and temporally suppressed by shRNA interference in adult mice prior to tumour inoculation, thus avoiding the developmental and possible compensatory effects of germline gene knockout. Using syngeneic mouse tumour allografts of EphA3-negative LLC cells implanted in these mice, we show that EphA3 expression is detectable in the MSC/CAF-like cells of the tumour microenvironment, and that knockdown of EphA3 expression indeed has impacts on tumour growth. EphA3 suppression reduced levels of MSCs in tumours, including cells resembling perivascular and myofibroblast-like CAFs, and was associated with decreased blood vessel density and cell proliferation in tumours. Accordingly, EphA3 knockdown decreased vessel sprouting in tissue explants from mice, suggesting an important role for EphA3+ MSCs in supporting angiogenesis, as previously described for MSCs more generally . Bone maOther Eph receptors have previously been shown to be important for both normal and oncogenic angiogenesis, particularly EphB receptors and EphA2, along with their cognate ephrin ligands  ,18,20. EImportantly, we also confirm EphA3 expression in similar cell subtypes in the TME of human cancers, including breast, colon and melanoma, by analysis of single-cell RNA sequencing data. Our data from early-stage breast tumours  shows thIn conclusion, we identify the cell guidance receptor EphA3 as a novel marker in distinct myofibroblast-like perivascular/smooth muscle cells in the microenvironment of human tumours and in analogous mesenchymal/fibroblast cells in mouse tumour models. Our novel transgenic mice, with specific knockdown of EphA3 expression in the TME of implanted syngeneic tumours, showed reduced tumour growth and angiogenesis, and associated defects in stem-like and angiogenic properties in mesenchymal stromal progenitor cells. Our results thus indicate an important functional role of EphA3 in mesenchymal CAF-like cells recruited to tumours that promote angiogenesis and tumour growth, and suggest the potential of EphA3 as a therapeutic target in the TME of solid tumours.


Tuberculous pleural effusion (TPE) and malignant pleural effusion (MPE) may occasionally show similar cytological and biochemical picture including ADA. In such cases, differentiating TPE and MPE is challenging and needs histopathology of pleural tissue which may involve invasive procedures. The present study aims to evaluate the diagnostic accuracy of pleural fluid ADA to serum CRP (ADA/CRP) ratio to discriminate between tuberculous and malignant pleural effusion. In addition, we investigated whether the ratio ADA/CRP adds diagnostic value to ADA.p < 0.05 was considered statistically significant for all tests.This cross-sectional study was conducted in the National Institute of Diseases of the Chest and Hospital (NIDCH), Mohakhali, Dhaka, from July 2021 to February 2022 on diagnosed patients of TPE and malignant pleural effusion MPE. A receiver operating characteristic curve (ROC) was constructed for identifying TPE. The added value of the ADA/CRP ratio to ADA was evaluated using the net reclassification improvement (NRI) and integrated discrimination improvement (IDI). A value of Fifty-nine patients were enrolled in this study, of which 31 had TPE, and 28 had MPE. Pleural fluid ADA to serum CRP ratio and pleural fluid ADA level was significantly higher in patients with TPE, but there was no significant difference in serum CRP levels between patients with TPE and MPE. At cut off value of > 1.25, pleural fluid ADA to serum CRP ratio had a sensitivity of 93.8%, specificity of 85.2%, and positive and negative predictive values were 88.2% and 92% respectively, in the diagnosis of TPE and area under ROC curve (AUC) was 0.94. The NRI and IDI analyses revealed added diagnostic value of ADA/CRP to ADA.This study shows that the ADA/CRP ratio improves the diagnostic usefulness of ADA for TPE. Pleural effusion, an abnormal accumulation of fluid within the pleural space, is not a disease itself but rather an important clinical manifestation of systemic and pleural diseases . InternaTuberculosis occurs in every part of the world, but most cases and deaths are registered in developing countries. Nearly 10.6 million people around the world developed TB in 2021, with an estimated 1.6 million deaths . One of Due to their low cost, and short turn-around time, PF biomarkers serve as complementary diagnostic tools. Adenosine deaminase (ADA) is one such commonly explored investigation tool in the case of tuberculous pleural effusion (TPE)with 90% sensitivity and a 92% specificity . But ADAPleural fluid ADA to serum CRP ratio (ADA/CRP) can be a novel, cost-effective tool in differentiating malignant from tuberculous pleural effusion; however, there is insufficient and contradictory evidence regarding its effectiveness , 15. So,This cross-sectional study was conducted from July 2021 to February 2022. Department of Respiratory Medicine of the National Institute of Diseases of the Chest and Hospital (NIDCH), Mohakhali, Dhaka, Bangladesh, was selected as the study site as it is the highest referral center for chest diseases and receives patients from all over the country.Patients with tuberculous pleural effusion diagnosed based on histopathology of pleural biopsy or patients with malignant pleural effusion diagnosed based on pleural fluid cytology for malignant cells or histopathology of pleural biopsy and providing consent were included in the study. Patients with transudative pleural effusion, congestive heart failure, chronic liver disease, chronic kidney disease, and connective tissue diseases were excluded from the study.Sample size was calculated using Buderer’s formula  where \dRP = 79% , specifiRP = 79% , prevaleRP = 79% , and absAll the newly admitted patients with unilateral pleural effusion  during the study period were consecutively approached for enrolment in the study. Among whom, 272 were excluded for meeting one or more of the exclusion criteria, and 57 were excluded as they/their guardian didn't provide consent to enter the study, leaving a final sample size of 59 Fig. .Fig. 1A Abnormal fluid accumulation between the parietal and visceral pleura is defined as pleural effusion . If pleuData were collected through face-to-face interviews of patients/guardians using a pretested structured questionnaire. Background information, previous medical records, physical findings, and laboratory reports were recorded. After receiving informed consent, diagnostic thoracocentesis was performed on all patients. Once the thoracocentesis site has been identified, the skin encompassing the site is thoroughly rinsed with an antiseptic solution. Then, 2% xylocaine is used to administer local anesthesia to the skin, subcutaneous tissue, muscles, and parietal pleura. Then, a 20-cc syringe with a 22 G needle is inserted into the intercostal space at the upper border of the lower rib, and 10 to 20 cc of pleural fluid is aspirated. Pleural fluid was then sent for pleural fluid study, including physical appearance, biochemistry (protein and glucose), cytology, exfoliative cytology for malignant cell, Gene Xpert for MTB and ADA. Pleural biopsy was performed using Abrams pleural biopsy needle with all aseptic precautions followed by histopathological examination. Blood was collected and analyzed for serum CRP. Echocardiography was performed to rule out heart failure, ascitic fluid analysis was performed to determine the cause of ascites when associated with pleural effusion, blood urea, and serum creatinine were measured to rule out renal failure as a cause of pleural effusion, and a thyroid function test was performed to rule out hypothyroidism as a cause of pleural effusion.p-value of less than 0.05 was deemed statistically significant. All the reporting was done according to the Standards for Reporting of Diagnostic Accuracy Studies (STARD) guideline [Using Microsoft Excel, collected data were cleansed, validated, and encoded. For data analysis, we used Stata . Using a histogram, a normal Q-Q plot, and the Kolmogorov–Smirnov test, the normality of continuous data was determined. As a measure of the center of quantitative data, the arithmetic means and the median was used, while the standard deviation and interquartile range were used as a measure of dispersion. We summarized qualitative data using frequency and relative frequency. When applicable, the chi-square test and t-test were used to examine associations between predictor and outcome variables. A receiver operating characteristic (ROC) curve was plotted to evaluate the sensitivity, specificity, and area under the curves (AUC) with a 95% confidence interval (CI) to measure the efficacy level of the ratio of pleural fluid ADA to serum CRP, pleural fluid ADA and serum CRP for the diagnosis of TPE. We determined the added value of the pleural fluid ADA to serum CRP ratio to ADA using the net reclassification improvement (NRI) and integrated discrimination improvement (IDI) . A two-tuideline .A total of 59 patients participated in the study, among which the majority (52.5%) had tuberculous pleural effusion, and the rest (47.5%) of the respondents had malignant pleural effusion. Table p-value was highly significant (< 0.001). Both the lower and upper bound area was also above the area of 0.5, indicating that both ADA/CRP and ADA could accurately predict TPE; among them, ADA/CRP is better. The AUC of CRP was 0.32 , and the upper and lower bound of the 95% confidence interval was below 0.5. Therefore, serum CRP is a poor predictor of TPE.The receiver operating characteristic (ROC) curve for pleural fluid ADA to serum CRP ratio, pleural fluid ADA and serum CRP is depicted in Fig. Table p < 0.05). These findings suggest that the ADA/CRP ratio adds diagnostic value to ADA.Table This study was carried out to assess the validity of pleural fluid ADA to serum CRP ratio for differentiation of tuberculous from malignant pleural effusion. While prior research investigated the diagnostic accuracy of the pleural fluid ADA to serum CRP ratio for TPE , 15, 22,p < 0.001) which is consistent with Ernam et al. (2005)  [In this study, a significantly higher level of pleural fluid ADA was found in TPE in comparison to MPE  . A retro< 0.001)  In our s< 0.001) .p < 0.001). A concordant finding was also found in Swetha et al. [p < 0.001) and a cut-off ≥ 1.25 showed 93.8% sensitivity and 85.2% specificity. These findings are corroborated by the findings of Swetha et al. [Patients with TPE have significantly higher levels of ADA/CRP ratio compared to patients with MPE . We employed the NRI and IDI to determine whether the ADA/CRP ratio offers additional diagnostic value beyond ADA because the AUC of ROC has certain limitations in determining the overall diagnostic accuracy of a given test . These tTherefore, ADA/CRP ration can be an efficient and cost-effective tool to differentiate TPE from MPE in clinically perplexed situations as these two diseases often present with similar clinical pictures. Before opting for a more expensive and invasive procedure which is also often difficult to obtain in resource-limited healthcare settings, this tool can bolster the clinical impression of primary care physicians and in difficult-to-refer cases give them the confidence to initiate anti-TB medication. However, it is also important not to forget the role of gold standard tests for the diagnosis of TPE and MPE.Even though this is one of the first studies to look at the additional diagnostic value of ADA/CRP over ADA, it has some limitations. Our sample size was relatively smaller and further comprehensive studies with larger sample sizes are required to validate our findings. Moreover, we only included tuberculous and malignant pleural effusion cases. The inclusion of other causes of exudative effusion in the study would have provided a more comprehensive finding. Tissue culture was not done in the study due to the long turn-around time, which would have delayed the treatment of the patient and complicate the case. As our country is one of the top TB burden countries, if we have a patient suggestive of signs and symptoms of TB who demonstrated caseating granuloma in pleural biopsy, we can make a diagnosis of TPE as per our national guideline .Although both pleural fluid ADA to serum CRP ratio and pleural fluid ADA are useful tools in differentiating between tuberculous and malignant pleural effusion, ADA/CRP ratio has added diagnostic value over ADA. A cut-off value of ≥ 1.25 is identified as the optimal cut-off value for ADA/CRP ratio, with 93.8% sensitivity and 85.2% specificity. ADA/CRP can therefore help to differentiate TPE from MPE in clinically puzzling scenarios, especially in resource-limited settings.


CHST14) or in the dermatan sulfate epimerase gene (mcEDS-DSE). As gastrointestinal complications of mcEDS-CHST14, diverticula in the colon, small intestine, or stomach have been reported, which may lead to gastrointestinal perforation, here, we describe sisters with mcEDS-CHST14, who developed colonic perforation with no evidence of diverticula and were successfully treated through surgery (a resection of perforation site and colostomy) and careful postoperative care. A pathological investigation did not show specific abnormalities of the colon at the perforation site. Patients with mcEDS-CHST14 aged from the teens to the 30s should undergo not only abdominal X-ray photography but also abdominal computed tomography when they experience abdominal pain.Musculocontractural Ehlers–Danlos syndrome (mcEDS) is a heritable connective tissue disorder characterized by multiple congenital malformations and progressive connective-tissue-fragility-related manifestations in the cutaneous, skeletal, cardiovascular, visceral, ocular, and gastrointestinal systems. It is caused by pathogenic variants in the carbohydrate sulfotransferase 14 gene (mcEDS- CHST14) encoding dermatan 4-O-sulfotransferase 1  or in the dermatan sulfate epimerase gene (DSE)  [CHST14 and 14 patients (eight families) with mcEDS-DSE have been reported [CHST14, the identified gastrointestinal features included constipation (most commonly), diverticula, abdominal pain, diarrhea, large bowel sounds, and gastric ulcers [Musculocontractural Ehlers–Danlos syndrome (mcEDS) is a rare subtype of EDS, caused by biallelic pathogenic variants in the carbohydrate sulfotransferase 14 gene #615539) ,3. To da9 ,3. To reported . Patientreported ,3. In a CHST14, both of whom developed colonic perforation without any diverticula in their 20s. This report could have implications for the pathomechanism of colonic perforation as well as for the diagnostic and therapeutic strategy for this lesion in patients with mcEDS.Here, we describe detailed clinical and pathological findings of two sisters with mcEDS-CHST14 were recruited in this study. Clinical and radiological information was obtained from their medical records at Tohoku University Hospital. Their past histories were included in a recently reported large international collaborative study on mcEDS-CHST14, in which no individual clinical information or history was described [Two sisters with mcEDS-CHST14 (NM_130468.3:c.842C>T:p.Pro281Leu) homozygously through Sanger sequencing.Patient 1, the older sister, is currently 29 years old. In early childhood, she underwent orthopedic surgical treatment for bilateral clubfeet and a left hip dislocation. During her schooldays, she developed recurrent dislocations in bilateral patellae and the right shoulder, requiring manual reduction. She also repeatedly developed large subcutaneous hematomas on her extremities from minor traumas, which recovered spontaneously after about 3 weeks. The clinical course of the disease was complicated by pyelonephritis at age 15 years, gastritis at age 19, and left ureteral stones at age 20, all of which were treated with pharmacological therapy. She had severe constipation, with defecation only once every 7 days, and her stools varied from hard to soft. After graduating from college, she worked in an office for a company. At age 16 years, she was suspected of having mcEDS clinically, and was found to have a recurrent variant in CHST14 as her sister through Sanger sequencing.Patient 2, the younger sister, is currently 27 years old. In childhood, she underwent correction of bilateral flat valgus feet with braces. Deformity and rigidity of the ankle joint persisted, and she sprained her left ankle three times, complicated by subcutaneous hematomas and skin laceration requiring surgical suture. She repeatedly developed large hematomas on her extremities from minor traumas, which recovered spontaneously after about 3 weeks. She developed five dislocations of the left shoulder and one dislocation of the right shoulder during her schooldays. At age 13 years, she underwent corrective surgery for progressive scoliosis. At age 17 years, she fell and dislocated her left hip, which was reduced manually. Bilateral retinal detachments occurred at age 18 years, which were treated with photocoagulation. She had moderate constipation, with defecation once every 3 to 4 days, and her stools varied from hard to soft. She suffered from acute gastritis at age 24 years and gastroesophageal reflux disease at age 26, both of which were treated pharmacologically. After graduating from university, she worked in an office. At age 14 years, she was suspected of having mcEDS clinically, and was found to have the same homozygous variant in Paraffin-embedded samples of a perforated tissue and a normal tissue adjacent to it in the transverse colon of Patient 2 were obtained. Hematoxylin–eosin staining, immunochemical staining for desmin, and elastica-Masson staining were performed in accordance with the standard protocols. Written informed consent for the publication of this case report was obtained from the patients.Patient 1 visited our emergency department due to abdominal pain after 2 weeks of constipation at age 25 years. After administering an enema, the abdominal pain progressed and her blood pressure decreased. Abdominal X-ray photography (Xp) failed to determine the cause of the abdominal pain, while contrast-enhanced computed tomography (CT) showed free air in the abdominal cavity and ascite accumulation A. EmergePatient 2 had mild pain in her upper left abdomen from the morning at age 24 years. She had a bowel movement after eating lunch, which relieved her abdominal pain. Because her abdominal pain became worse in the evening, she left work early and visited our hospital. Abdominal Xp showed no abnormal findings, but the possibility of gastrointestinal perforation was considered based on her past history with acute gastritis as well as Patient 1’s episode. Contrast-enhanced CT showed a small amount of intra-abdominal free air B, so sheHistopathologically , the musCHST14 as described in this paper developed spontaneous colonic perforation with no evidence of diverticula or of no apparent macroscopic or pathological abnormalities of the colon and were treated successfully with no sequelae. We cannot exclude the possibility of additional gene mutations that could contribute to the severity of findings in these women because they were diagnosed not through comprehensive genetic screening such as whole exome sequencing but through targeted sequencing.The sisters aged 25 and 24 years with mcEDS-CHST14 who developed colonic perforation were complicated by diverticula [CHST14, developed colonic perforation at the splenic flexure 11 days after corrective surgery for talipes equinovarus, and underwent an emergency laparotomy plus partial colectomy with proximal fistulotomy [Previously reported patients with mcEDS-erticula ,8. A rectulotomy . Recovertulotomy . Divertitulotomy . It was tulotomy . The sisO-sulfotransferase 1 or dermatan sulfate epimerase [To the best of our knowledge, no systematic investigation has been performed on histopathological abnormalities of the gastrointestinal system in patients with mcEDS. Microscopic and ultrastructural abnormalities of the skin have been systematically investigated: light microscopy—fine collagen fibers predominantly present in the reticular to papillary dermis and a marked reduction of normally thick collagen bundles; transmission electron microscopy—dispersed collagen fibrils in the papillary to reticular dermis ; and transmission electron microscopy with cupromeronic blue staining to visualize glycosaminoglycan (GAG) side chains of decorin—linear, stretched GAG chains from the outer surface of collagen fibrils to adjacent fibrils  . The skipimerase ,10. Becapimerase , it mighIncluding the data for the sisters reported here, the age of onset of gastrointestinal perforation in mcEDS tends to range from the teens to the 30s . AbdominFurther reports on gastrointestinal perforation in patients with mcEDS are needed to uncover the pathomechanisms of this critical complication as well as to establish the management strategy, including prevention  and treatment .CHST14, who developed spontaneous colonic perforation with no evidence of diverticula or of no apparent macroscopic or pathological abnormalities of the colon and who were treated successfully with no sequelae. The threshold for abdominal computed tomography in patients with mcEDS-CHST14 aged from the teens to the 30s presenting with otherwise unexplained acute abdominal pain should be low so that spontaneous perforation is diagnosed early and immediately followed by antibiotic therapy and laparotomy.We have presented two sisters aged 25 and 24 years with mcEDS-


The nano-cleaning fluid enables the deconstruction and carrying of the sludge attached to the well wall, which accomplishes the purpose of downhole cleaning.In drilling and completion projects, sludge is formed as a byproduct when barite and oil are mixed, and later sticks to the casing. This phenomenon has caused a delay in drilling progress, and increased exploration and development costs. Since nano-emulsions have low interfacial surface tension, wetting, and reversal capabilities, this study used nano-emulsions with a particle size of about 14 nm to prepare a cleaning fluid system. This system enhances stability through the network structure in the fiber-reinforced system, and prepares a set of nano-cleaning fluids with adjustable density for ultra-deep wells. The effective viscosity of the nano-cleaning fluid reaches 11 mPa·s, and the system is stable for up to 8 h. In addition, this research independently developed an indoor evaluation instrument. Based on on-site parameters, the performance of the nano-cleaning fluid was evaluated from multiple angles by heating to 150 °C and pressurizing to 3.0 Mpa to simulate downhole temperature and pressure. The evaluation results show that the viscosity and shear value of the nano-cleaning fluid system is greatly affected by the fiber content, and the cleaning efficiency is greatly affected by the concentration of the nano-emulsion. Curve fitting shows that the average processing efficiency could reach 60–85% within 25 min and the cleaning efficiency has a linear relationship with time. The cleaning efficiency has a linear relationship with time, where R While the Tarim Basin is rich in oil and natural gas reserves ,2,3, itsAt present, there are various cleaning methods for underground oily sludge. From a technological point of view, some scholars use ultrasonic waves to decompose oily sludge ,18,19. HNano-emulsions are fluids consisting of tiny emulsion structures containing an average radius of about 10–100 nm. During the emulsion formation process, surface active substances are adsorbed on the oil–water interface to form a stable interfacial film, which reduces the interfacial tension between oil and water. Nano-emulsions with ultra-low surface interfacial tension and strong cleaning ability have been widely used in oil repelling as well as oil washing in recent years. Ye Yan  applied This article analyzes the components of the obstruction taken from an existing ultra-deep well  using thChemicals used in nano-cleaning fluid preparation include Tween 80 , n-pentane (AR. 99.99%), octane number , fiber , and superfine calcium carbonate The downhole sludge used in the experiment was provided by Tarim Oilfield, and was used in oil–solid separation of oil sludge by Soxhlet extraction. As shown in The Varia EL Ⅲ type elemental analyzer produced by Elementar was used to analyze the element content of the inorganic solid phase substances in the oil sludge. The results are shown in Nano-emulsions have the advantages of low surface interfacial tension, small particle size, good stability, and strong biodegradability ,28,31,32Use Tween 80 (or Tween 60) and n-butanol as the surfactant (S) and auxiliary surfactant (A) to construct the “S+A” system. Control the ratio of “S+A” and n-octane to 9:1, 8:2, 7:3, 6:4, 1:1, 4:6, 3:7, 2:8, and 1:9, followed by dropwise introduction of deionized water into a nano-emulsion system . The pse3 for weighting, and weigh zeta the nano-cleaning fluid system to 1.5–1.8g/cm3, after weighting and high stirring for 30 min to form a uniform nano-cleaning fluid. The nano-cleaning fluid formula is shown in As shown in A clear and transparent nano-emulsion liquid is prepared according to the three-phase diagram. Select nano-emulsion samples, using Manern’s Zetasizer 4 particle size analyzer and zeta potential measuring instrument to determine the nano-emulsion’s particle size and zeta potential.A nano-cleaning fluid with a nano-emulsion concentration of 0.06–1.2% was prepared, and the surface tension of the cleaning fluid was tested by using an interfacial tensiometer (BZY-2).Contaminate the 5 cm × 1 cm P110 steel sheet with sludge , and then use the nano-cleaning fluid to soak the steel sheet, and use the contact angle analyzer (AY-PHb) to test the contact angle of the steel sheet before and after the nano-cleaning fluid soaking, to analyze the wetting and flipping effect of the nano-cleaning fluid on the casing.The development of ultra-deep wells is faced with the problem of high downhole temperatures. The current cleaning fluid fails due to its poor temperature resistance. A vertical electric test bench (MIRCOE-300) is used to test the high-temperature stability of the nano-cleaning fluid kept at 180 °C for 8 h.By changing the nano-emulsion concentration in the formula, the amount of fiber added, and the density of the cleaning fluid, different nano-cleaning fluid formulations were formed, as shown in The cleaning efficiency of the cleaning fluid was simulated by the cleaning efficiency evaluation instrument developed by the China University of Petroleum (Beijing). The device diagram is shown in The cleaning efficiency evaluation instrument is composed of a cleaning liquid storage tank, a heatable simulated casing, a circulating pump, and a particle size analyzer. The sludge is heated at 150 °C and adsorbed on the simulated casing, and a circulating pump is used to circulate the cleaning liquid to flush the simulated casing. There is a screen at the bottom of the casing. When the sludge is dispersed into the cleaning liquid, it can pass through the screen to communicate with the simulated casing. The cleaning efficiency of the nano-cleaning fluid was calculated by measuring the quality difference before and after the simulated casing was cleaned. The particle size change of the nano-cleaning fluid before and after the cleaning was measured by a particle size analyzer, and the cleaning efficiency of the nano-cleaning fluid was evaluated in combination with the color change of the nano-cleaning fluid during the cleaning process. The evaluation system model established by the evaluation device is shown in According to the steps shown in 50 value is about 14.8 nm.The nano-emulsion is the core component of the cleaning fluid, and its stability and particle size are the guarantees for efficient cleaning by the nano-cleaning fluid. The zeta potential of the nano-emulsion was measured to be −31.4, indicating that the emulsion system has good stability. The particle size test result of the nano-emulsion is shown in The lipophilic-treated P110 steel sheet was immersed in a nano-cleaning fluid with a concentration of 0.8%, and then the contact angle was measured at different time intervals. After 30 min, the contact angle between the P110 steel sheet and the water droplet changes from 101° to 27°, the wettability changes from hydrophobic to hydrophilic, and the wetting reversal occurs. It is confirmed that the nano-cleaning fluid has efficient wetting and turning ability, and good cleaning ability.Aiming at the problem that the nano-cleaning fluid often fails in the high-temperature downhole environment of ultra-deep wells, the high-temperature stability of the nano-cleaning fluid measured by the vertical motorized test stand (MIRCOE-300) is shown in In the cleaning process, the rheological properties of the cleaning fluid have a certain influence on the treatment effect and fluid circulation, and the rheological parameters of the cleaning fluid are studied by using the measurement and release method of drilling fluid. Using nano-cleaning fluids of different formulas to clean the sludge, the viscosity and shear performance of the nano-cleaning fluid after the measurement is shown in According to the method in As can be seen from 0 and m1 are weighed according to the evaluation method in The different formulas of the nano-cleaning fluids are shown in As shown in The cleaning mechanism of nano-cleaning fluid was studied by fitting the experimental data, and its action mechanism was described. The fitting results are shown in 3, a concentration of 1% nano-emulsion, and a fiber addition of 0.8 g was simulated indoors. The cleaning efficiency was tested for different periods within a 25 min range and then curve fitting was performed. The cleaning efficiency changes linearly with time, the fitting curve is y = 3.66 + 1.6x, and the fitting height is R2 = 0.98335. According to the analysis based on the principle, the nano-cleaning fluid mainly has a physical dispersion effect in the auction process, so it does not cause secondary pollution and can meet the requirements of efficient treatment. After the cleaning, the sludge can be taken out of circulation.2 = 0.98335, which confirms that the treatment mechanism is mainly “deconstruction-carry.“ The nano-cleaning solution with adjustable density developed in this study has high treatment efficiency and can be used for ultra-deep well cleaning. In addition, the indoor simulation method also guides field application, and provides a guarantee for the realization of clean and environmentally friendly production.The percentages of oil, water, and barium sulfate in the downhole oil sludge of the Tarim deep block are 15.67%, 6.2%, and 75%, respectively. The sludge is well-cemented and not easily soluble in acid, which seriously affects the development progress. The nano-emulsion prepared in this study has an interfacial tension of about 4.5 mN/m and a surface tension of 35 mN/m. It can change the wetting angle of the oil sludge surface from 101° to 27° and has a strong wetting reversal ability. A set of nano-cleaning solutions with adjustable density were developed based on nano-emulsion and through a fiber-reinforced system network structure. The apparent viscosity of the nano-cleaning fluid is 11mPa-s and the stabilization time can reach 8 h, which can be used for the cleaning treatment of downhole oil mud in the field. During the treatment of oil mud, the particle size changes from a single peak to a bimodal peak, and deconstruction and dispersion of sludge exists, and the average treatment efficiency could reach 74.885%. The nano-emulsion in the nano-cleaning fluid disperses the oil sludge solid phase to realize oil–solid separation, and the separated solid phase is wound by the fiber to hinder its settlement, and carried out of the ground through the circulation system. The change in treatment volume with time is obtained by curve fitting, and the curve y = 3.66 + 1.6x is highly fitted with R


All other patients (n = 706) were considered as a control group and 1 mg protamine for each 100 units of heparin was administered. Results: The combined intra-hospital endpoint of death, life-threatening major bleeding, and major vascular complications were significantly more frequent in patients receiving low-dose protamine . After propensity matching (n = 130 for each group) for relevant clinical characteristics including anti-platelet therapy , low-dose protamine predicted the combined endpoint , and even in multivariable analysis, low-dose protamine continued to be a predictor of the combined endpoint in the matched model  alongside baseline hemoglobin. Conclusions: In this propensity-matched retrospective analysis, a low-dose protamine regime is associated with a higher rate of major adverse events compared to a full-dose protamine regime following transfemoral TAVI.Objectives: We aimed to evaluate the efficacy and safety of low-dose protamine in reducing access site-related complications during Transcatheter Aortic Valve Implantation (TAVI) as compared to full-dose protamine. Background: Access site-related complications represent an independent predictor of poor outcomes of TAVI. Data regarding heparin reversal with protamine and the dosage needed to prevent bleeding complications are scarce among patients undergoing TAVI. Methods: A total of 897 patients were retrospectively included in the study. Patients who underwent percutaneous coronary intervention within 4 weeks before or concomitantly with TAVI ( The prevalence of aortic valve disease and particularly calcific aortic valve disease is increasing due to the aging population . Since iCompared with surgical access such as the transapical approach, transfemoral access was associated with mortality and morbidity benefits that justify its use as a mainstream strategy ,6. HowevTherefore, our retrospective analysis aimed to evaluate the efficacy and safety of low-dose protamine compared to full-dose protamine in preventing bleeding and vascular complications in patients undergoing transfemoral TAVI.n = 60, transaortic n = 29, transsubclavian n = 2, transaxillary n = 1). All patients undergoing TAVI via the femoral artery (n = 897) were included in this retrospective study. Further details including patient disposition are demonstrated in Between January 2015 and December 2020, patients admitted to the Department of Cardiology of the University Hospital of the RWTH Aachen due to severe aortic valve stenosis or severe aortic valve regurgitation who met the criteria for treatment based on the European Society of Cardiology guidelines on valvular heart disease  were disThe protocol for this study as a retrospective analysis of routinely collected data was approved by the local ethics committee (EK 481/21). The study was performed in accordance with the ethical standards defined by the latest version of the Declaration of Helsinki.The procedure was carried out by an interventional cardiologist, a cardiothoracic surgeon, and an anesthesiologist. TAVI procedures in our center are carried out under analgesic sedation. Femoral arterial access was guided by using a preoperative computed tomography angiogram of the pelvic vasculature. According to our standard operating procedure, weight-adapted unfractionated heparin was administered before placement of the TAVI introducer sheath. ACT was measured every 20 min and additional heparin was given to maintain ACT above 300. Closure of the access site was performed using either Prostar, Manta, or ProGlide devices. An additional pressure bandage was applied for 12 h following the intervention. Dual anti-platelet therapy was prescribed for 6 months.Heparin reversal took place after vascular closure in all patients. The standard dose was 1 mg protamine for every 100 units of unfractionated heparin that were given in the last 30–60 min. In long procedures, 0.75 of protamine was additionally administered per 100 IU of heparin that were given earlier than 60 min. ACT measurements were done both before and after protamine administration. Indications for low-dose protamine included planned PCI during the index procedure or in the 4 weeks prior to the TAVI. ‘Low dose’ protamine was half the usual calculated dose.After the procedure, patients were monitored for 24 h in the intermediate care unit. Transthoracic echocardiography was performed immediately after TAVI and before discharge from our center. All patients underwent a duplex Doppler sonography of the access site of the femoral artery.The classifications of the vascular access site and access-related complications were based on the standardized definitions for important clinical endpoints in TAVI as proposed by the updated consensus document of the Valve Academic Research Consortium from 2012 (VARC-2 criteria) . Primaryt-test for normally distributed variables and with a Mann–Whitney test for non-normally distributed variables; distributions of binary variables were compared with a chi-squared test. In order to assess the association of two different protamine doses with interventional outcomes and complications, we performed a univariable logistic analysis; results are expressed as an odds ratio (OR) with a 95%-confidence interval. In order to adjust for possible confounders, we performed multivariable logistic analysis for the prediction of the combined endpoint, including in the model all variables with p < 0.10 in the univariable analysis  and then performing backward selection.Continuous variables are reported as mean ± standard deviation if normally distributed and median (interquartile ratio) if not normally distributed. Normal distribution was assessed by visual histogram inspection as well as with the Kolgomorov–Smirnov test. Dichotomic variables are reported as a proportion (percentage). The comparison of baseline continuous variables was performed with a To exclude the effects of different baseline characteristics between the two study groups, we performed propensity matching based on sex, age (with a tolerance of ±5 years), and dual antiplatelet therapy. We then repeated our analyses in the matched population.p < 0.05.Analysis was performed with SPSS software. Statistical significance was awarded for p = 0.005). The average logistic Euroscore, as a predictor for cardiac operative risk and mortality, was significantly higher in patients receiving the low-dose protamine scheme. These patients also had a higher prevalence of coronary artery disease (CAD)  and dual anti-platelet inhibition , while other clinical characteristics were comparable between both groups.A total of 897 patients were included. The median age was 82 (IQR 78-85) years. Men were represented significantly more in the low-dose protamine group at 58.1% (p = 0.88). In the majority of the patients, A. femoralis was used as an additional vascular access site with no significant difference between both groups . There was no difference between the groups in terms of closure device used. (p = 0.574). Further procedural characteristics are presented in Propensity matching was performed on 130 patients from the low-dose protamine group and 130 patients from the full-dose group. After matching, both groups presented similar characteristics regarding the above-mentioned differences, except for CAD which remained higher in the low-dose protamine group. Importantly, no in-between group difference was present regarding anti-coagulatory and anti-platelet therapy. The baseline characteristics of matched patients are presented in p < 0.001]. In particular, the low-dose protamine group presented a higher incidence of life-threatening  and major bleeding events . No significant difference between both groups was observed for stroke and  and myocardial infarction , respectively.The combined primary intra-hospital endpoint of death, life-threatening major bleeding, and major vascular complications occurred significantly more often in patients receiving low-dose protamine compared to patients treated with full-dose protamine [29 (15.2%) vs. 50 (7.1%), p < 0.001).Additional outcomes for both groups are presented in p < 0.001) and life-threatening  and major bleeding events  in p = 0.001) and pre-procedural hemoglobin  were able to predict the combined endpoint; details of the univariable analysis are reported in p = 0.006) alongside with EuroScore and pre-procedural hemoglobin, as reported in Univariable logistic regression analysis was performed to assess the association between low-dose protamine treatment and adverse clinical outcomes. Low-dose protamine treatment significantly predicted the combined endpoint . Details are reported in p = 0.004); details of this analysis are shown in p = 0.023) together with pre-procedural hemoglobin . Details are shown in Following propensity matching, low-dose protamine was still significantly associated with a higher incidence of the composite endpoint  following transfemoral TAVI was associated with a lower incidence of the combined intra-hospital endpoint of all-cause mortality, major and life-threatening bleeding, as well as major vascular complications when compared with a low-dose protamine scheme (0.5:1 protamine/heparin ratio), mainly due to a significantly lower incidence of major and life-threatening bleeding. First, our data show an overall complication rate mostly comparable to major cohorts , especian = 100), provided no evidence for a significant decrease in major and life-threatening bleeding complications among patients who routinely received protamine sulfate as compared to those in the placebo group [Although the only existing single-center randomized clinical trial (PS TAVI) on protamine sulfate during TAVI, which was limited by its small sample size  with the intention of balancing bleeding complications with stent thrombosis offered the opportunity to compare two doses of protamine in a real-world scenario. Patients receiving low-dose protamine experienced a significantly higher rate of the combined intra-hospital endpoint of death, life-threatening major bleeding, and major vascular complications compared with patients receiving full-dose protamine. This was driven by a significantly higher rate of both life-threatening and major bleeding events in the low-dose group, and translated into a higher blood transfusion volume as well as a longer hospital stay.Whilst it is tempting to speculate that incomplete heparin reversal using the low-dose protamine scheme is insufficient in preventing access site bleeding in patients after TAVI, the conclusion should be tempered by the higher risk profile, as demonstrated by EuroScore and more frequent medication of anti-platelet and anticoagulant agents in those allocated low-dose protamine. In order to control for these baseline differences, we performed propensity matching between both groups, although this was only partially successful given that the primary indication for low-dose protamine was a recent or concurrent coronary intervention. Nevertheless, despite propensity matching, the primary combined endpoint as well as secondary endpoints such as minor bleeding, blood transfusion volume, and the length of hospital stay were still significantly greater in the low-dose group. A multivariable analysis determined that low-dose protamine alongside with EuroScore and baseline hemoglobin predicted the combined endpoint. Even after repeating the same analysis in the matched model, the combined endpoint was predicted only by low-dose protamine and baseline hemoglobin.With respect to thrombosis, the overall rates of intervention-related stroke and myocardial infarction were extremely low in both study groups. We found no association between stroke rates and the dosage of protamine used. Stent thrombosis was confirmed only in one patient who received low-dose protamine after recent elective PCI, a further two cases of myocardial infarction due to coronary embolism were reported in patients in the full-dose protamine group. Overall, the extremely low rate of thromboembolic complications demonstrates that protamine application, independently of the suggested dosing scheme, seems not to be associated with thrombotic events.Despite being the first study assessing two different dosing schemes of protamine following TAVI, the main limitation is the retrospective nature of our analysis and the possible resultant selection bias which partially persists despite propensity matching for relevant clinical characteristics including anti-platelet therapy. Taking this into consideration highlights the role of conducting large randomized clinical trials to validate our results. Furthermore, in spite of the very low incidence of thromboembolic events following protamine administration in our cohort as well as in previous studies, the safety of a high-dose regime in patients who underwent a recent PCI still has to be assessed.In this retrospective propensity-matched study of a large and unselected patient cohort, a low-dose protamine regime (0.5:1 protamine/heparin ratio) was associated with a higher rate of intra-hospital major adverse events compared to a full-dose protamine (1:1 protamine/heparin ratio) after transfemoral TAVI. This may suggest that a complete reversal of the heparin effect is needed to reduce bleeding complications after TAVI. These findings need to be confirmed in a prospective randomized trial.