Glucagon-like peptide-1 (GLP-1) receptor agonists are important treatment options in obese patients with type 2 diabetes. To date, few immediate allergic reactions due to GLP-1 receptor agonists were reported. One report revealed that a patient with a level 1 anaphylaxis according to Brighton Criteria due to an exendin based GLP-1 receptor agonist was able to tolerate liraglutide , the alternative GLP-1 receptor agonist. Since exenatide is the only available GLP-1 receptor agonist covered by insurance in Turkey, a drug desensitization protocol, the only therapeutic method in hypersensitivity reactions used in case of absence of an alternative drug, was considered. Here, we report a successful desensitization protocol for the first time in two obese diabetic patients with an immediate hypersensitivity to exenatide.The first patient was a 47 year-old female. She was referred to our outpatient allergy clinic because of a generalized urticaria developed within minutes after the last dose, following a week of an exenatide BID 5 mcg/20 mcl treatment. Although the reaction was sudden onset, it did not meet the Brighton Criteria of anaphylaxis. The second patient was a 46 year-old female. She had a large local immediate injection site reaction that appeared 15 min following an exenatide BID 5 mcg/20 mcl injection. The injection site reaction was not accompanied by a systemic allergic reaction. We performed desensitization with exenatide to two patients who need GLP-1 receptor agonist treatment. Protocol was completed in 7 steps in approximately 3 h, with the aim of reaching the daily dosage of exenatide. Throughout this process, we observed that both cases tolerated the protocol without any complaints or complications. Following the protocol, the patients safely tolerated the treatment for 3 months.We present the first successful desensitization protocol to exenatide in both local and/or systemic immediate hypersensitivity reactions and indicate the importance of desensitization in patients who do not have alternative therapies. Glucagon-like peptide-1 (GLP-1) receptor agonists, exenatide, lixisenatide, liraglutide, dulaglutide and semaglutide, are among the recent subcutaneous treatment options that provide better glycemic improvement and weight loss in patients with type 2 diabetes according to the current guidelines .To date, few case reports have revealed that GLP-1 analogues can cause allergic reactions –6. One oExenatide and lixisenatide are exendin based GLP-1 receptor agonists. They are structurally similar to exendin-4, a peptide found in the saliva of the Gila monster lizard. In contrast, liraglutide, dulaglutide and semaglutide are structurally analogous to the human GLP-1 molecule . In termDesensitization is a process that aims to administer essential medications while protecting patients from severe reactions induced by anaphylactic and anaphylactoid mechanisms . AlthougA 47 year-old female was referred to our outpatient allergy clinic because of a generalized urticaria developed within minutes in the first week of an exenatide BID 5 mcg/20 mcl treatment. The urticaria developed within minutes after the last dose of injection on the 7th day of treatment, presumably after a 1 week sensitization period. This was the patient's first episode of urticaria. There was no suspected any other allergens such as drug or food exposure as well as the patients did not have any signs or symptoms related to any concomitant infections. Urticaria was not accompanied by respiratory, cardiovascular or gastrointestinal symptoms and disappeared spontaneously after the drug administration had stopped. Although the reaction was of sudden onset, it did not meet the Brighton Criteria of anaphylaxis [To confirm the diagnosis of exenatide allergy, we performed skin tests with a stepwise approach depending on the previously published report . The skiWe suggested to treat the patient with an alternative treatment. However, it was stated by her endocrinologist that an alternative GLP-1 analogue was not available. Since there was no previously published desensitization protocol with exenatide, we created a new protocol inspired by a previous protocol made for a subcutaneous medication . No premA 46 year-old female, diagnosed with type 2 diabetes and obesity, was referred to us because of having a large local immediate injection site reaction appeared in 15 min following an exenatide BID 5 mcg/20 mcl injection for a month. After the fourth injection, an itchy, edematous and hyperemic lesion had developed on the injection site , the usage of a human GLP-1 analogue  may provide a solution since no cross-reactivity has been reported so far , 8. HoweWe assumed that both reactions were immediate IgE-mediated hypersensitivity reactions based on the timing of the reaction and the test results. The test results of the case report of Shamirz O. et al. and the results of our control group showed no false positivity . HoweverAlthough we could not find any data in the literature, mast cell activation with a non-IgE-mediated way, for example MRGPRX2, is also possible. Besides, there is also the possibility of the allergic reactions may occur not with the active drug, but because of metacresol, an antimicrobial additive. However, both patients were able to use insulin glargine containing metacresol after exenatide allergy.Our desensitization protocol was completed in 7 steps in approximately 3 h, with the aim of reaching the daily therapeutic dosage of exenatide. Throughout this process, we observed that the patients tolerated the protocol without any complaints or complications during injections. Besides, the daily regular administration of exenatide without dose interruptions enabled us to achieve success in suppressing the immediate hypersensitivity reaction with a single desensitization period. 3 months after the desensitization protocol, our patients were still applying exenatide injections without any allergic reactions. Besides, the skin tests with exenatide of case 2 unexpectedly turned to negative after desensitization. This finding suggested us the possibility of cessation of a local IgE-mediated response to the drug after the desensitization protocol which is uncommon in most of the desensitization procedures.Temporary toleration is achieved in hours and can be maintained if drug antigens are administered at regular intervals, depending on pharmacokinetic parameters . If the As a limitation, the fact that both patients successfully desensitized with exenatide had non-anaphylactic immediate allergic reactions, leading to insufficient evidence for the application of this protocol in anaphylaxis. However, we think that the use of this protocol in non-anaphylactic immediate reactions in the presence of appropriate indications will provide the expected benefit.In conclusion, this first report demonstrated a successful desensitization protocol to exenatide in patients with isolated immediate skin reaction and local reactions and indicated the importance of desensitization in patients who do not have alternative therapies.


Structured and harmonized implementation of molecular tumor boards (MTB) for the clinical interpretation of molecular data presents a current challenge for precision oncology. Heterogeneity in the interpretation of molecular data was shown for patients even with a limited number of molecular alterations. Integration of high-dimensional molecular data, including RNA- (RNA-Seq) and whole-exome sequencing (WES), is expected to further complicate clinical application. To analyze challenges for MTB harmonization based on complex molecular datasets, we retrospectively compared clinical interpretation of WES and RNA-Seq data by two independent molecular tumor boards.High-dimensional molecular cancer profiling including WES and RNA-Seq was performed for patients with advanced solid tumors, no available standard therapy, ECOG performance status of 0–1, and available fresh-frozen tissue within the DKTK-MASTER Program from 2016 to 2018. Identical molecular profiling data of 40 patients were independently discussed by two molecular tumor boards (MTB) after prior annotation by specialized physicians, following independent, but similar workflows. Identified biomarkers and resulting treatment options were compared between the MTBs and patients were followed up clinically.n = 38) and 82 molecular aberrations from WES only (n = 3) were considered for clinical interpretation for 40 patients (one patient sequenced twice). A median of 3 and 2 targeted treatment options were identified per patient, respectively. Most treatment options were identified for receptor tyrosine kinase, PARP, and mTOR inhibitors, as well as immunotherapy. The mean overlap coefficient between both MTB was 66%. Highest agreement rates were observed with the interpretation of single nucleotide variants, clinical evidence levels 1 and 2, and monotherapy whereas the interpretation of gene expression changes, preclinical evidence levels 3 and 4, and combination therapy yielded lower agreement rates. Patients receiving treatment following concordant MTB recommendations had significantly longer overall survival than patients receiving treatment following discrepant recommendations or physician’s choice.A median of 309 molecular aberrations from WES and RNA-Seq (Reproducible clinical interpretation of high-dimensional molecular data is feasible and agreement rates are encouraging, when compared to previous reports. The interpretation of molecular aberrations beyond single nucleotide variants and preclinically validated biomarkers as well as combination therapies were identified as additional difficulties for ongoing harmonization efforts.The online version contains supplementary material available at 10.1186/s12916-022-02560-5. Precision oncology is expected to improve cancer treatment by taking into account molecular alterations . TargeteThe workflows of both MTB were compared  and 82 (WES) aberrations per patient were reported to the MTB, of which the majority were gene expression outliers in the WES/RNA-Seq group and SNV in the WES group  were reported per concordant treatment option than per discordant treatment option. Recommended combination therapies were assessed individually and only one of 25 recommended combination regimens was identically recommended by both MTB.To identify challenges for future harmonization efforts, treatment options were analyzed with regard to the type of biomarker, the evidence level (EvL), and therapeutic basket Fig. . More coAfter communication of identified treatment options, 22 of 40 55 %) patients started at least one recommended therapy. Eighteen out of these 22 patients received treatment following concordant treatment recommendations Figs.  and 5.Fi5 % patieP = 0.005, Fig. Patients receiving therapy as recommended by both MTBs (Matched MTB therapy) displayed a significantly longer overall survival P = 0.00, Fig. 5,Specific attention and additional research is required to improve the clinical annotation of molecular data, which is still unstandardized and inconsistent between molecular tumor boards . The intIn this work, we retrospectively analyzed the clinical interpretation of identical and high-dimensional molecular alterations of 40 patients by two molecular tumor boards that were prospectively sequenced within the DKTK-MASTER-program. This analysis yielded a mean overlap coefficient of 66%. Previous studies of recommendation heterogeneity yielded overall agreement rates between 40%  and 86% This analysis allows for a detailed look at challenges with the interpretation of complex molecular data for these efforts. Generally, more heterogeneous recommendations were found in the setting of biomarkers with low evidence levels and combination therapy, probably due to the wealth and heterogeneity of preclinical studies , differeThe agreement rate in our study was lower for identified predictive biomarkers than the corresponding treatment option. This can be explained by the frequent identification of several alterations that point towards identical treatment recommendations  but were not always all named by both tumor boards. Yet, agreement rates of predictive biomarkers were significantly associated with agreement rates of treatment options. Therefore, the structured identification of potential (predictive) biomarkers from molecular data remains key to the identification of treatment options. Efforts for a harmonization of databases is expected to greatly aid with this . InterdiSome limitations of this study should be considered: Patients were discussed in parallel by the molecular tumor boards in Heidelberg and Berlin only in the beginning of the MASTER program, thus limiting the number of patients. Additionally, the recommendations reflect MTB practices of the inclusion years 2016–2018, which have evolved significantly since then, thus possibly underestimating current concordance rates. This limitation is important, since great efforts have been put into biomarker annotation and database development, and since efforts are ongoing . A folloThe availability of new data and treatment options will eventually bring established targets into clinical routine and away from a more experimental MTB setting. An analysis of unclear and complex data can therefore be expected to remain a problem for precision oncology. Therefore, this analysis also has several strengths: Identical molecular data were available, limiting the effect of potential confounders. The retrospective, real-world design of this study reduced the risk of bias. Furthermore, the interpreted molecular data are highly complex, incorporating WES and RNA-Seq data, and thus allow for an analysis of a wealth of biomarkers and treatment recommendations even in the setting of two participating MTBs. High-dimensional molecular data beyond targeted sequencing are increasingly incorporated into precision oncology –21. The An improved outcome could be shown in the I-PREDICT and WINTHER trials, as well as a real-world data analysis, for patients that received treatment that was better matched to their tumor’s molecular profiles , 23, 29.Reproducible and evidence-based interpretation of complex molecular data is feasible with the use of structured workflows. Additional attention is required for the interpretation of data beyond genomic analyses and biomarkers with preclinical evidence levels as well as for the introduction of rational combination therapies.Patients with advanced solid tumors of a rare histology or younger age (< 50y), no available standard therapy, and available fresh-frozen tumor tissue were included in the DKTK-MASTER precision oncology program of the German Cancer Consortium (DKTK) . Patienthttp://varianttools.sourceforge.net/Annotation/dbNSFP), using functional impact tools as well as several gene lists of interest, including the Cancer Gene Census (https://cancer.sanger.ac.uk/census). One excel file was generated per patient including single nucleotide variations/indels, gene fusions and structural variations, mutational signatures, RNA information, and germline findings, if available. RNA expression ranking was based on the first 148 RNA samples of the MASTER program. These information were provided to the molecular tumor boards.Fresh-frozen tumor tissue was obtained from all participating patients and shipped to the central laboratory in Heidelberg. Specimen handling, DNA and RNA extraction, next-generation sequencing, and bioinformatic analyses were performed as published . BrieflyMolecular tumor boards (MTB) were established independently in Heidelberg and Berlin. In Berlin, a weekly meeting comprising at least medical oncologists, bioinformaticians, pathologists, and molecular biologists was established. In Heidelberg, a MTB was held in parallel, with medical oncologists, bioinformaticians, pathologists, and molecular biologists in regular weekly attendance. Prior to the MTB, specialized physicians analyzed the identified alterations  using at least one database as well as a structured PubMed search to identify potential biomarkers. After establishment of a list of potential biomarkers, predictive information were collected and annotated using predefined evidence levels , 20. Prehttp://www.R-project.org/).Patients with treatment recommendations from an independent discussion of identical molecular results in both MTBs were identified from the internal MTB database. Number and type of molecular alterations were collected from the initial report as provided to both molecular tumor boards. Identified biomarkers and treatment options were retrieved from the final MTB recommendations and supporting documents used in the MTB discussion. Survival data were retrieved from follow-up documents. Data were then structured in a harmonized format and analyzed using R (R version 4.0.0 (2020-04-24) -- “Arbor Day”. R: A language and environment for statistical computing. R Foundation for Statistical Computing, Vienna, Austria. URL Treatment recommendations were defined as treatment options that were identified and communicated after multidisciplinary discussion in the respective MTB . Predictive biomarkers were defined as genetic aberrations that were used for the identification of treatment recommendations after annotation and multidisciplinary discussion in the MTB, thus allowing for multiple predictive biomarkers as a rationale for one identified treatment option .Treatment recommendations and predictive biomarkers were matched for each individual patient. The overlap coefficient was calculated using the Szymkiewicz–Simpson formula ∩YminX,Y .Additional file 1: Figure S1. Shows the respective molecular tumor board workflows of Heidelberg (HDB) and Berlin (BLN). Corresponding steps are indicated by their respective colors.Additional file 2: Table S1. Reports all gene names and corresponding number of alterations, including SNV, gene expression outliers, indels and gene fusions that were identified in the cohort. Table S2. Lists the genetic alterations that were identified as predictive biomarkers and corresponding treatment options per patient by Heidelberg (HD) and Berlin (BLN) MTBs. Results from a second sequencing for the same patient are indicated by a comma. Table S3. Provides information on treatment options and respective biomarkers for the analyzed cohort. Results from a second sequencing for the same patient are indicated by a comma.


In recent years, many different biosensors are being used to monitor physical health. Electrospun nanofiber materials have the advantages of high specific surface area, large porosity and simple operation. These properties play a vital role in biosensors. However, the mechanical properties of electrospun nanofibers are poor relative to other techniques of nanofiber production. At the same time, the organic solvents used in electrospinning are generally toxic and expensive. Meanwhile, the excellent performance of electrospun nanofibers brings about higher levels of sensitivity and detection range of biosensors. This paper summarizes the principle and application of electrospinning technology in biosensors and its comparison with other technologies. Globally, it has been observed that improvement in standards of living has also caused the rise of diseases. Biosensors that can monitor our physical health in real time are particularly important. Electrospinning technology has developed rapidly in recent years, and there are several nanofiber membranes that can be used in biosensors. The biosensor has high biocompatibility, good flexibility, remarkable mechanical properties and high sensitivity. For example, biosensors can assess the activity of enzymes in the body to determine the health of stem cells . In receWith the continuous advancement of current technology, there are many manufacturing methods for nanofiber membranes, with electrospinning being the easiest and relatively low-cost method. The nanofiber membrane made by electrospinning can form a typical network structure with high surface area and high porosity . The proElectrospinning is a high-throughput technology that can produce nanofiber membranes with controllable fiber orientation . In the The influencing factors of electrospinning mainly include: solution concentration and viscosity, voltage intensity, receiving distance, air humidity and temperature. The viscosity and concentration of the solution play an important role in the morphology and performance of the fiber . VoltageDifferent parameters and methods of electrospinning lead to different morphology of nanofibers. For example, core-shell nanofibers can be obtained by coaxial electrospinning. Different kinds of nanofibers have different characteristics such as oriented nanofibers with high axial mechanical properties and good dimensional stability. High porosity and large specific surface area are key properties that contribute to a successful biosensor, hence the wide use of electrospun nanofibers . MeanwhiThe types of nanofibers are described above and the preparation techniques used for different nanofibers are also different. The preparation technology and characteristics of different fibers are described in detail below.Randomly distributed nanofibers: The preparation of such fibers is the simplest form by direct drawing of the instabilities of the jet. This results in a relatively small range of applications and poor mechanical properties for such fibers. However, the advantage is that the preparation is simple and there is no complicated process.Aligned nanofibers: This fiber is obtained by suppressing the instability of the jet on the basis of randomly distributed nanofibers. This makes the fibers have the advantages of high axial mechanical strength, good dimensional stability and high application value in tissue engineering, composite reinforcement, electrical and optical fields. However, due to the different methods used, the collection speed may be slow and the amount of fibers is relatively small.Core-shell nanofibers: Fibers of this structure are mostly obtained by coaxial electrospinning devices. Core-shell structured nanofibers solve the problem that some materials are not spinnable. Core-shell structured nanofibers solve the problem that some materials are not spinnable. This also makes this fiber widely used in biomedical fields such as drug release systems, tissue engineering scaffolds, drug-loaded medical dressings and sutures.Multispace nanofibers: The preparation of such fibers is mostly used to induce phase separation. For example, solvent evaporation and heating can induce phase separation to form multispace nanofibers. This fiber is characterized by a substantial increase in the specific surface area of the fiber.Pine-needle nanofibers: Such fibers are formed based on randomly distributed nanofibers. Then, other materials are generally grown on randomly distributed fibers by hydrothermal method.Patterned nanofibers: This fiber is obtained by changing the shape, movement mode and material of the collecting device. Its physical and chemical properties are basically the same as those of disordered fibers.Cobweb nanofibers: This fiber is a two-dimensional mesh fiber membrane material with ultrafine electrospun fibers as a scaffold. It has the advantages of large specific surface area, good adsorption and stable mechanical properties.Hollow nanofibers: This fiber is obtained on the basis of the core-shell structure fiber. Such fibers are generally obtained by coaxial electrospinning with a soluble or volatile substance as the core layer and a polymer solution as the shell layer and then removing the core layer by dissolving or heating. The disadvantage is that the production efficiency is relatively low.A biosensor is mainly composed of a biosensor and signal transducer. The main function of the biosensor is to select the substance to be measured. The main function of signal transducers is to convert the chemical effects generated by the interaction between the biological components and measured substances into electrical signals that can be considered as output . Thus, iElectrospinning is also widely used in the field of biosensors. This is because the nanofiber film obtained by electrospinning has a high specific surface area. This enables more biological elements to be loaded on the fiber surface, which greatly improves its sensitivity. This is why electrospinning technology has been widely used in the field of biosensors in recent years. There are four main characteristics of biosensors  . SelectiIn recent years, the development of electrospinning technology has been very rapid and the fields of application are expanding. Compared with other technologies, electrospinning is relatively simple and versatile . TherefoPiezoelectric biosensors are often used to test whether the heart rate, pulse and limb movements are normal. The detection principle is based on the deformation of the sensor to drive the resistance to change to reflect the health of the body. For example, when detecting a pulse, the resistance response is normally stable and fluctuating. However, if the resistance response suddenly appears abnormal, it means that the pulse beat is abnormal. The same is true when detecting body movement.Heart rate is a very important indicator of the human body and its abnormality can threaten life. Therefore, it is particularly important to invent a sensor that can monitor heart rate in real time. There are many sports bracelets on the market that can monitor heart rate in real time. However, the stability and sensitivity of this equipment are generally not high enough to reflect abnormal conditions in time . This grIn recent years, the development of sensors for monitoring heart rate has advanced rapidly. Mainly manifested in increased functions, increased sensitivity and ease of production . StudiesThe movement changes of human joints are also one of the important indicators to reflect the physical condition. Under normal circumstances, the changes of human joints are regular and do not undergo long-term mutations. However, when the changes in the joints are so abrupt that they do not stick to the same pattern, it is important to consider the health of the body to avoid getting worse. Such real-time surveillance is crucial for the early detection and treatment of disease . In rece–1), high precision (0.1 °C) and fast response time (2.46 s).Compared with traditional sensors, today’s sensors are more convenient and provide a more timely feedback. As time progresses, the requirements for sensors are get higher. Not only should the sensitivity and stability be high, but also the cost should be as low as possible . Chen etThe sensors described above are based on electrospinning. It can be seen from the data that the sensor has excellent performance and it is versatile. Electrochemical biosensors are composed of various biomolecules  and electrochemical converters . Its main principle is to convert chemical signals into electrical signals through chemical reactions between the biological materials on the sensitive components and the biological information in the measured object. Among them, there are many sensors using enzymes as biological materials, but the disadvantage is that the activity of enzymes is difficult to guarantee. There are many sensors based on other biological materials, such as DNA, cells, nucleic acids, and microorganisms. However, due to the peculiarity of these materials, they are not widely used in the field of electrospinning. The more commonly used enzymatic biosensors and non-enzymatic biosensors are introduced here.In recent years, the rapid development of non-invasive biosensors brings great convenience to sensor detection . Human s−1) and low detection limit (0.01 mM).In recent years, the main reason for the slow development of enzymatic sensors is that the inference from biological information carried in these liquids is not comprehensive enough . In addi−1 cm−2) and a low detection limit (0.08 μM).The disadvantage of enzyme sensors is that enzymes are affected by various environmental factors, such as temperature, humidity, oxygen concentration and pH value. This limits the application of enzymatic sensors in continuous detection . The advIt can be seen from the above that both the enzymatic and non-enzymatic sensors have good resolution for glucose. This is because the electrospun nanofibers have a higher specific surface area and can adsorb more biological elements to fully react with the analyte. Body temperature is one of the important signs for detecting human health . The traHowever, the measurement results may be inaccurate compared with mercury thermometers. In recent years, temperature sensors have developed rapidly. Sensors that can feedback body temperature information in time and measure it accurately have emerged.As a wearable sensor, it must have a certain degree of biocompatibility and the material should be non-toxic . The curThermosensitive biosensors are becoming more sensitive to temperature, which results in higher requirements for sensors. Electrospun nanofibers have attracted much attention due to their excellent properties. Humans are exposed to excessive including ultraviolet rays, which are more harmful to the human body. Long-term exposure to ultraviolet light may cause abnormalities such as skin cancer and skin aging . This br−1. As a strain sensor, the detection range is 7–74% and GF (gauge factor) can reach 34. Therefore, this sensor is feature-rich and has excellent performance.Today’s wearable UV sensors are sticker-shaped, which can change color according to the intensity of UV rays . They alBiosensors based on electrospinning to monitor light intensity are still relatively few. Electrospinning improves the performance of the sensor for light intensity detection to a certain extent. It can be seen from the above comparison that electrospinning technology has great advantages over other technologies. Not only does it have a better sensitivity and stability, but also its functions are broad. This is attributed to the high specific surface area of electrospun nanofibers. This also enables electrospinning technology to be widely used in the field of biosensors.In recent years, electrospinning technology has developed rapidly and its application in the field of biosensors has become extensive . ElectroAlthough the development of electrospinning technology in the field of biosensors is very extensive, biosensors themselves have certain limitations that prevent them from being widely used. Because biological elements are the most important components in biosensors, biological materials on the surface of biological components are easy to inactivate and have poor reproducibility, making it difficult to guarantee their service life and storage. The biological element is closely related to the performance of the sensor. The high specific surface area of the electrospun nanofibers enables the attachment of a large number of biomaterials and contributes to the enhanced sensor performance. At the same time, electrospinning also has disadvantages, such as relatively unstable product consistency and many control factors. I believe that with the continuous advancement of electrospinning technology in future, more breakthroughs will be made in the field of biosensors.


Highly effective systemic treatments have globally improved outcomes in metastatic clear-cell renal cell carcinoma (m-ccRCC). However, despite many efforts, reliable biomarkers predicting individual responses are currently lacking. Moreover, mixed responses are commonly observed. We hypothesized that molecular heterogeneity between primary tumors and their metastases could flaw biomarker research based on features of the primary tumor and explain mixed responses. Therefore, we studied the heterogeneity of the ccrcc1–4 molecular subtypes across patient-matched primary and metastatic lesions over time in 62 patients with m-ccRCC who underwent both nephrectomy and metastasectomy. These subtypes characterize underlying disease biology and are associated with outcomes in both the primary and metastatic settings. We observed a concordance rate of 58% . This concordance was not affected by the interval between nephrectomy and resection of the metastatic lesion. Across discordant pairs, the metastatic lesions mostly exhibited a less favorable molecular subtype. Moreover, primary tumors with the favorable ccrcc2 molecular subtype were characterized by favorable prognosis and a long interval between nephrectomy and metastasectomy. Conversely, tumors with the unfavorable ccrcc4 molecular subtype relapsed quickly and had poor prognosis. Thus, the considerable molecular heterogeneity between patient-matched m-ccRCC primary and metastatic lesions provides an explanation for mixed responses to systemic therapy and could impact the development of biomarker studies in which the primary tumor is often considered a surrogate for metastatic disease.We studied primary tumors and metastases from patients with kidney cancer and found considerable heterogeneity in their molecular features. This heterogeneity explains mixed responses to systemic therapy and is important to take into account in future biomarker studies for this disease. In recent years, novel and highly effective systemic treatments for metastatic clear-cell renal cell carcinoma (m-ccRCC) have revolutionized the treatment landscape and globally improved outcomes for patients Molecular subtypes have emerged as a way to characterize underlying disease biology and potentially guide therapeutic decision-making. We previously described four molecular subtypes of ccRCC that are associated with outcomes in both the localized and metastatic settings for patients receiving systemic therapy and patients undergoing surgical metastasectomy Brannon et al. We collected archived formalin-fixed, paraffin-embedded (FFPE) tissue specimens from 62 patients with m-ccRCC who underwent nephrectomy at our institution between 1998 and 2018 and who had tissue available from both the primary tumor and at least one metastasis. We performed whole-transcriptome RNA sequencing to determine the ccrcc molecular subtypes for the 62 primary tumors and 121 metastases as previously described The baseline characteristics of our cohort are shown in We calculated the ccrcc molecular subtype concordance between the primary tumor and the first resected metastasis. If multiple metastases resected at the same time point had discrepant molecular subtypes, the observation was considered discordant. The 95% confidence intervals (CIs) were calculated using the Clopper-Pearson exact method.Out of the 62 pairs, 36 (58%) were concordant (95% CI 45–71%). Out of the 26 discordant pairs, 18 (69.2%) switched to a more unfavorable subtype in at least one of the resected metastases, while six (23.1%) switched to a more favorable subtype and two (7.7%) switched between the ccrcc2 and ccrcc3 subtypes, which both have favorable prognosis. The fact that discordant subtypes more often switch to a more aggressive subtype is in line with the Serie study which reported a switch from the favorable ccA to the unfavorable ccB subtype in 80% of discordant cases.p = 0.79).However, concordance analysis does not consider the effect of time. To assess whether the concordance rate depends on the time between resection of the primary tumor and the metastasis, we performed logistic regression with molecular subtype concordance between the primary tumor and metastasis as the dependent variable and time as the independent variable. This showed no significant impact of the time to the first metastasectomy on the concordance , are the following: None.Funding/Support and role of the sponsor: This research was funded by Bristol-Myers Squibb. The sponsor played a role in the design and conduct of the study and approval of the manuscript.


The correlation between ReDS values and CSIs became stronger in the heart failure cohort . In patients with mild congestion (ReDS < 35%), ReDS values, instead of CSI, stratified the degree of congestion. In patients with severe congestion (ReDS > 35%), both modalities stratified the degree of congestion. Conclusions: Both chest X-ray and ReDS are useful for assessing severe pulmonary congestion, whereas ReDS would be preferred to chest X-ray in stratifying the severity of mild pulmonary congestion.Background: Chest X-ray is a practical tool to semi-qualify pulmonary congestion. Remote dielectric sensing (ReDS) is a recently introduced, non-invasive, electromagnetic energy-based technology to quantify pulmonary congestion without expert technique. We compared these two modalities to clarify appropriate clinical situations for each modality. Methods: ReDS and chest X-ray measurements were prospectively performed on admission in consecutive hospitalized patients with cardiovascular diseases. In the chest X-ray, the congestive score index (CSI) was calculated blindly by two independent experts and averaged. CSIs were correlated with ReDS values. Results: A total of 458 patients  years old, 267 men, and 130 heart failure) were included. Median ReDS value was 28% . There was a mild correlation between ReDS values and CSIs (r = 0.329,  Chest X-ray (CXR) is a practical and established tool to assess pulmonary congestion during daily clinical practice . The exiRecently, congestion score index (CSI), which is a semi-quantitative approach to evaluate pulmonary congestion based on a 6-segment evaluation of CXR, scoring each zone from 0 (no congestion) to 3 , has been proposed to overcome the above challenges. Several studies have demonstrated its utility for assessing pulmonary congestion ,5.Remote dielectric sensing (ReDS) has been established as a noninvasive methodology to quantify the degree of pulmonary congestion without expert technique . Its utiHowever, the clinical utility of these two modalities in each clinical scenario remains uncertain: i.e., none, mild, or severe pulmonary congestion with/without heart failure [HF]). We aimed to correlate ReDS and CSI in patients with and without HF and discuss the appropriate clinical situations for each modality.From August 2021 to July 2022, consecutive patients who were hospitalized in our institute to examine and treat cardiovascular diseases were included in our prospective study. Successive measurements of ReDS values and CXR were performed on admission in all participants. Patients who could not wear ReDS vests or had obvious lung diseases, such as acute pneumoniae, lung cancer, interstitial lung disease, asthma, and chronic obstructive pulmonary disease, were excluded. HF was defined according to the Framingham’s criteria. All patients provided written informed consent before inclusion. The study protocol complied with the Declaration of Helsinki and was approved by the Ethic Committee of the University of Toyama (MTK2020007).ReDS is a noninvasive system to evaluate lung fluid content by analyzing the dielectric properties of the lung portion using sonar-released, low-power electromagnetic signals. The ReDS value was taken by wearing the vest and maintaining a resting and sitting position for one minute . The manThe severity of the radiographic pulmonary congestion was evaluated using a previously reported CSI  3,4,5].,4,5.3,4,p-values < 0.05 were considered statistically significant.Continuous variables were presented as median and interquartile, and categorical variables were presented as numbers and percentages. Pearson’s correlation coefficient was used to evaluate correlations between ReDS values and CSIs. The correlations were reevaluated among HF patients. Statistical analyses were performed using SPSS Statistics 26 . Two-sided 2. The median ReDS value was 28% , and the median CSI was 0.08 . Our study included 130 HF patients (28%). The κ value to assess inter-observer reliability between the two readers was 0.88  for the scoring of CSI.A total of 500 consecutive patients were considered to be included in this prospective study. Of them, 42 patients were excluded due to inappropriate physical health or lung diseases. Ultimately, 458 patients were included  . The medp < 0.001; p = 0.891, n = 391). In patients with significant congestion , ReDS had a strong linear correlation with CSI  .There was a mild correlation between ReDS values and CSI among all 458 patients  B. In HF We investigated the correlations between ReDS values, which was measured by novel ReDS technology, and CSI, which was measured by experienced cardiologists from CXR, in the assessment of pulmonary congestion. There was a mild correlation between ReDS values and CSIs in all participants. In patients with normal ReDS values , CSI remained at relatively low levels irrespective of a variety of ReDS values. In patients with significant pulmonary congestion with ReDS > 35%, ReDS values had a linear and strong correlation with CSI.The accurate assessment of pulmonary congestion is a key to risk stratification and constructing tailor-made therapeutic strategies in patients with congestive HF ,9. HowevReDS technology is a recently introduced, novel modality to quantify the percentage of lung fluid noninvasively and without expert technique. ReDS values have a moderate correlation with high-resolution computed tomography, a mild correlation with invasively measured pulmonary artery wedge pressure, and weak correlations with plasma B-type natriuretic peptide levels and lung ultrasound ,11,12. CThe correlation between ReDS values and CSIs was mild. Of note, CSI remained at low levels when pulmonary congestion was nonexistent or mild . Nevertheless, ReDS values presented a variety of levels. In other words, ReDS system may stratify even mild pulmonary congestion, which CXR cannot distinguish. This may not be surprising, because ReDS can detect only a slight degree of pulmonary congestion. On the other hand, it would be challenging to distinguish mild pulmonary congestion from the physiological consolidation of lung vasculatures in CXR.In patients with significant pulmonary congestion , ReDS values and CSIs showed a stronger linear correlation. As experienced in daily clinical practice, the apparent and broad lung shadow can be easily diagnosed with severe pulmonary congestion in CXR. We do not deny the usefulness of CXR in daily practice to assess pulmonary congestion or any other cardio-pulmonary abnormalities . Of noteReDS values showed good agreement with CSIs which were scored by expert cardiologists, particularly among those with severe pulmonary congestion and/or HF. ReDS may be a good alternative to CXR, particularly when repeated CXR is hesitated or unavailable in this cohort.Additionally, ReDS technology can stratify those with no or mild pulmonary congestion. Such mild pulmonary congestion may also be caused by non-HF etiologies, including chronic kidney diseases. ReDS technology may have advantage over CXR in accurately quantifying the degree of mild pulmonary congestion and adjusting the doses of diuretics. Of note, only a slight residual pulmonary congestion at the index discharge can worsen clinical outcomes . ReDS teThere are several concerns and limitations. Since our study was a single center study, we require further studies to evaluate the scientific rigor or external validity. We included only in-hospital patients. The applicability of our findings in remote monitoring and home care settings is the next concern . We exclBoth CXR and ReDS are useful to assess severe pulmonary congestion, whereas ReDS is preferred to CXR in stratifying the severity of mild pulmonary congestion.


Indeed, publications concerning radiomic technique for oncology keep increasing over the past decade. In their valuable article, Ding et al.  conducteIn the study , they usNatural language processing (NLP), a popular research field where human language is decoded by machine learning, has been deployed to analyze medical information recently . Latent It seemed that one third of publications were concerned about the reliability of radiomics (Topic 2: standardization), and it remained as the most productive topic between 2016 and 2019. This might not be a surprise, because every novel technique would undergo a standardization process during the early stage. Researchers focused on the standardization of radiomics, as various procedures like segmentation of region of interest, scan protocol, preprocessing details would compromise the consistency of extracted features and the reproducibility of the findings. After the standardization of radiomics, more and more publications focused on the utility of radiomics (Topic 1: utility), and it remained as the most productive topic after 2019. These publications were simply identifying radiomic signature or building nomogram tools for certain clinical issues, but they surged up and contributed to nearly 40% of this field. It seemed that survival prognostication was the major prediction target of developing nomogram based on radiomic features. Interestingly, it should be noticed that publications concerning artificial intelligence and multi-source also grew up quickly (Topic 3: miscellaneous), and they caught up the productivity of Topic 2 after 2020. As radiomics was basically a technique of feature engineering, it seemed that comparisons with other techniques of like deep learning-based feature extraction were often in applications. Additionally, multi-source data like genomics was frequently combined with radiomic features, which theoretically could realize better predictions like higher precisions.In our perspective, publications of Topic 3 would become a future growing point, as multi-disciplinary researchers were likely to flock into this field and multi-source data would enable more ambitious and achievable goals. First, image segmentation of the region of interest (ROI), a prior step in radiomic workflows, needs the implementation of deep learning technique to improve the applicability of radiomic techniques . Manual We selected the searching formula [2] instead of the formula [1] in their study to extract data for our LDA analysis, because the formula [2] they proposed seemed to be more directly pertinent to radiomics. Nevertheless, it was not always easy to create a perfect searching strategy, and we admitted that it was also possible that the formula [2] miss some relevant publications. Additionally, other relevant publications not included by the WoSCC database remained as another limitation of our results.In conclusion, we appreciated their efforts in informing beginners and encouraging more researchers to participate in practicing radiomics by a bibliometric study. Meanwhile, we believed our LDA analysis also provided more in-depth analysis of radiomics in oncology, which should be beneficial to beginners who were eager to quickly grasp the major topics in this field and how they evolved. We hope the previous study  and our GF: writing and data interpreting; JQ: drafting and data analysis; HL: data extraction and revision; XL: study design and critical comments. All authors contributed to the article and approved the submitted version.Guangdong Basic and Applied Basic Research Foundation (2019A1515111171) and National Natural Science Foundation of China (82102640) were received in support of this work. The funders had no role in study design, data collection, data analysis, interpretation, writing of this report and in the decision to submit the paper for publication.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Pharmacists play an important role in infection control and antimicrobial stewardship teams in hospitals and the community. The need for education on antimicrobial resistance (AMR) and clinical infectious diseases (CID) is increasing. However, the status of Japanese pharmaceutical education on CID and AMR is unclear.We conducted a nationwide cross-sectional survey of pharmaceutical department in Japanese universities between February and March 2022. Data on the following were collected using questionnaires: 1) basic information; 2) number of faculty members with experience in CID; 3) lecture times for CID and AMR; 4) self-evaluation of CID and AMR education; and 5) the most important issues in CID and AMR education. Descriptive analyses were conducted.Among the 74 participating universities, 44 (59.5%) universities responded. The median number [IQR] of faculties of infectious disease education was 7 [4–12], and clinical faculties was 3 [1–6]. Twenty-seven (62.8%) of the universities had faculties with clinical experience in infectious diseases. Lectures time for CID and AMR varied among the universities.Respondents indicated that CID and AMR lectures were insufficient, compared to microbiology basic lectures. Additionally, insufficient lecture time and specialists were the most common responses as the most important issues in CID and AMR education.University background and questions regarding CID and AMR lectures (N=44)Boxplot of lectures time for clinical infectious disease and antimicrobial resistance educationSelf-evaluation of clinical infectious disease and antimicrobial resistance educationThis study revealed that educational status and resources for CID and AMR education varied widely, and that practical lectures on CID and AMR may be insufficient. We suggest that the entire core curriculum and resources, including the number of faculties, needs to be examined and improved.The most important issues in clinical infectious disease and antimicrobial resistance education.All Authors: No reported disclosures.


V. vulnificus-infected patients suffer from hemolytic anemia and circulatory lesions, often accompanied by venous thrombosis. However, the pathophysiological mechanism of venous thrombosis associated with V. vulnificus infection remains largely unknown. Herein, V. vulnificus infection at the sub-hemolytic level induced shape change of human red blood cells (RBCs) accompanied by phosphatidylserine exposure, and microvesicle generation, leading to the procoagulant activation of RBCs and ultimately, acquisition of prothrombotic activity. Of note, V. vulnificus exposed to RBCs substantially upregulated the rtxA gene encoding multifunctional autoprocessing repeats-in-toxin (MARTX) toxin. Mutant studies showed that V. vulnificus-induced RBC procoagulant activity was due to the pore forming region of the MARTX toxin causing intracellular Ca2+ influx in RBCs. In a rat venous thrombosis model triggered by tissue factor and stasis, the V. vulnificus wild type increased thrombosis while the ΔrtxA mutant failed to increase thrombosis, confirming that V. vulnificus induces thrombosis through the procoagulant activation of RBCs via the mediation of the MARTX toxin. Vibrio vulnificus infection remains largely unknown. In this work, the authors investigate this association, focusing on effects of the pore-forming MARTX toxin of V. vulnificus on red blood cells, and the utilisation of a rat venous thrombosis model.The pathophysiological mechanism of venous thrombosis associated with  V. vulnificus, an opportunistic human pathogen and a causative agent of foodborne disease, is characterized by rapid disease progression leading to hemolysis and fatal septicemia with high mortality rates within a few days2. The pathogenesis of V. vulnificus infection is also often accompanied by circulatory disorders such as venous thrombosis, which brings about tissue destruction and limb amputation5. These pathological features imply that V. vulnificus infection may induce intravascular coagulation6, which causes blood clotting and blockade of small blood vessels. Also, the pathological features suggest that the prevention of venous thrombosis is an attractive strategy to alleviate the symptoms of V. vulnificus infection. However, little is known about the virulence factor(s) of V. vulnificus responsible for the development of venous thrombosis and its molecular mechanisms.Infection of 7. However, recent studies demonstrated that thrombosis, especially in the vein, also occurs through the increased procoagulant activity of human RBCs under low blood flow9. Procoagulant activity is often increased along with the elevation of the intracellular calcium [Ca2+]i level, and caspase-3 and scramblase activities by various external stimuli such as mechanical and chemical stress, resulting in the externalization of an anionic phospholipid, phosphatidylserine (PS) of RBCs10. Along with PS exposure, RBCs often shed the PS-bearing microvesicles  by exocytosis11 and undergo progressive morphological changes from normal discocytes to echinocytes12. The PS exposure of RBCs provides sites for the assembly of prothrombinase and tenase complexes, which facilitates thrombin generation and enhances prothrombotic activity to prompt blood clotting and, ultimately, venous thrombosis13.Normally, thrombosis, such as arterial thrombosis, occurs via platelet activation under high blood flowV. vulnificus, the multifunctional autoprocessing repeats-in-toxin (MARTX) toxin, which is encoded by the rtxA gene, is the most crucial exotoxin responsible for the pathogenicity of V. vulnificus15. As a very large protein toxin, the MARTX toxin forms a pore on the host cell membrane using its repeats-containing regions and then delivers its own multiple cytotoxic or cytopathic effector domains into the cytosol of epithelial cells16. A mutant V. vulnificus defective in the MARTX toxin production was less cytotoxic and lethal when compared with the isogenic wild type (WT)15. However, it remains unelucidated whether the MARTX toxin also plays a role in venous thrombosis caused by V. vulnificus infection.Among multiple virulence factors of V. vulnificus induces shape changes, PS exposure, and MV generation in RBCs, along with the elevation of the [Ca2+]i level, ultimately leading to the procoagulant and prothrombotic activity of RBCs. Especially, V. vulnificus infecting RBCs substantially upregulated rtxA, illuminating that the pore-forming activity of the MARTX toxin induced the cytopathic changes of RBCs. Finally, we conducted rat model studies in vivo to confirm the role of the MARTX toxin in V. vulnificus-associated thrombosis.Here, we show that V. vulnificus were screened. The RBCs freshly isolated from healthy volunteers were infected with V. vulnificus MO6-24/O  at an MOI lower than 10, but severe hemolysis (up to 80%) was observed at an MOI of 25  before hemolysis, an optimal titer and incubation time for sub-hemolytic infection of  25 Fig. . Similar1 h Fig. . TherefoV. vulnificus (red arrowheads) infection at the sub-hemolytic level induced the morphological changes of the RBCs (discocyte) as analyzed with scanning electron microscopy (SEM)  Fig.  and tranEM) Fig. . As the EM) Fig. . The moropy Fig. . The mortes Fig. , as ofteV. vulnificus at the sub-hemolytic level were further investigated. Flow cytometry analysis demonstrated that the RBCs uninfected and infected with V. vulnificus at an MOI of 5 are observed as a uniform size (mostly RBCs) and two clearly different sizes (RBCs and MVs), respectively  were analyzed by fluorescence microscopy. Notably, the RBCs pre-infected with more V. vulnificus (MOI of 5 to 10) revealed more adherence (white arrows) and aggregation (yellow arrowheads) to ECs  responsible for procoagulant activity could be specifically induced upon exposure to RBCs. Indeed, the sequencing analyses of the whole transcriptome of V. vulnificus unexposed or exposed to RBCs identified a total of 121 differentially expressed genes   encoding the MARTX toxin. To examine the effects of the MARTX toxin on procoagulant activity, effects of the rtxA deletion in V. vulnificus on the changes of PS exposure and MV generation of the RBCs were primarily characterized. The ΔrtxA mutant showed significantly reduced PS exposure and MV generation , of which the rtxA gene was inactivated by insertional rather than deletional mutation, revealed similarly attenuated procoagulant activity of RBCs  was constructed  ted Fig. . Of noteion Fig. . AltogetV. vulnificus infection in vitro were examined as bridge studies. The rat RBCs infected with the sub-hemolytic level of V. vulnificus  in vitro increased PS exposure and thrombin generation in an MOI-dependent manner  as well as the insertion mutant (rtxA::nptI) did not show any significant increase in thrombus formation when compared with the uninfected control. Furthermore, the revertant of the insertion mutant (rtxA::nptI) rescued the ability of V. vulnificus WT to increase thrombus formation  rescued the ability of V. vulnificus WT to increase thrombus formation  and alpha-hemolysin (HlyA) were also observed to induce the elevation of the [Caion Fig. . Notablyion Fig. . PreviourtxA encoding MARTX toxin by directly binding to its promoter28. Interestingly, this study revealed that the expression of rtxA was significantly increased following the exposure of V. vulnificus to RBCs  (LBS) and appropriate antibiotics at 30 °C and monitored by measuring A600 of the cultures.The strains and plasmids used in this study are listed in Table S1g for 15 min. Packed human RBCs were washed two times with phosphate-buffered saline  and once more with Ringer’s solution . The washed RBCs were resuspended in Ringer’s solution to a concentration of 1 × 108 cells/ml, and CaCl2 was added to a final concentration of 1 mM prior to use for further experiments.With the approval from the Ethics Committee of the Health Service Center at Seoul National University (IRB No. 1909/001-007), all participants provided written informed consent before enrollment for the study. Human blood was obtained from healthy male donors (20–30 years old) using a vacutainer with acid citrate dextrose (ACD) and a 21-gauge needle (Becton Dickinson) on the day of each experiment. Platelet-rich plasma and buffy coat were removed by aspiration after centrifugation at 200×V. vulnificus at various MOIs and incubated for different incubation times was centrifuged at 10,000×g for 2 min. The extent of hemolysis of the RBCs was determined by measuring released hemoglobin spectrophotometrically at 540 nm (A540). The complete lysis of RBCs by 1% Triton X-100 (Sigma-Aldrich) was expressed as 100% hemolysis.The RBCs resuspension infected with V. vulnificus for 1 h and then fixed with 2% glutaraldehyde solution in the refrigerator overnight. On the second day, the fixed RBCs were washed three times with PBS and then post-fixed with 1% osmium tetroxide for 30 min. The fixed RBCs were washed briefly with distilled water twice and serially dehydrated with 30, 50, 70, 80, and 90% ethanol, and finally with 100% ethanol three times. Next, the dehydrated RBCs were treated twice with propylene oxide for 10 min each, infiltrated once with the 1:1 mixture of propylene oxide and Spurr’s resin for 2 h, and then finally left with only Spurr’s resin in a desiccator overnight. On the third day, the RBCs were infiltrated again with fresh Spurr’s resin for 2 h in a desiccator and then kept in a 70 °C oven overnight for polymerization of the resin. Finally, the RBCs were analyzed by TEM . For scanning electron microscopy (SEM) analysis, the RBCs were infected with V. vulnificus for 1 h and then fixed with 2% glutaraldehyde solution (Sigma-Aldrich) for 1 h at 4 °C. The fixed RBCs were then centrifuged, washed three times with PBS, and then post-fixed with 1% osmium tetroxide (Sigma-Aldrich) for 30 min at room temperature. After washing with PBS twice, the RBCs were dehydrated serially with 50, 70, 80, 90, and 100% ethanol, coated with gold and then analyzed by SEM . For confocal observation, the RBCs were diluted to 3 × 106 cells/ml, added to a 4-well-chambered coverslip (Thermo Fisher), and then incubated at RT for 1 h for complete attachment. Unattached RBCs were removed by washing with Ringer’s solution containing 2% BSA, and then infected with V. vulnificus. After infection, the RBCs were stained with the phycoerythrin-labeled monoclonal mouse anti-human CD235a antibody ) for 30 min, washed with Ringer’s solution once, and then analyzed by confocal microscopy equipped with an argon laser . Excitation and emission filters were set at 488 and 550–600 nm, respectively. To observe PS exposure, the RBCs were stained with fluorescein-isothiocyanate (FITC)-labeled Annexin V  instead of glycophorin-A-PE.For transmission electron microscopy (TEM) analysis, the RBCs were infected with V. vulnificus were analyzed using the FACSCalibur flow cytometer (Becton Dickinson) equipped with an argon-ion laser emitting at 488 nm. The Annexin V-FITC  was used to detect PS exposure, whereas the anti-glycophorin-A-PE  was used to determine RBCs and MV generation. RBCs and MV were identified by the relative size of cells  and granularity . Fluorescence data from 5,000 events were collected and analyzed using Cell Quest Pro software v.6.0. When required, the RBCs were pretreated with caspase-3 inhibitors ) before being infected with V. vulnificus. A gate for PS exposure-positive events was set using the analyzed data from RBCs treated with 2.5 mM of CaCl2 instead of 2.5 mM EDTA before the flow cytometry analysis. Data from 5000 events were collected and analyzed using Cell Quest Pro software v.6.0. For the detection of the intracellular Ca2+ level, the RBCs were pretreated with 3 μM of Fluo-4 acetoxymethyl ester (fluo-4 AM) (Thermo Fisher Scientific) for 1 h at 37 °C in the dark were infected with V. vulnificus and then the fluorescence of Fluo-4 AM was analyzed by flow cytometry. For the evaluation of caspase-3 activity, three hundred microliters of the RBCs infected with V. vulnificus were mixed with 1 μL of the in situ fluorescent marker for caspase-3 ) and then incubated for 30 min in a thermomixer . Then, the RBCs were centrifuged (1000×g for 5 min), washed twice with Ringer’s solution, and resuspended in 500 μL of Ringer’s solution. The fluorescence from the RBC suspension was analyzed by flow cytometry. Data from 10,000 events were collected and analyzed. For the assessment of scramblase activity, 500 μL of the RBCs were infected with V. vulnificus in the presence of 0.5 μL of fluorescent phosphatidylcholine (PC) (C6-NBD-PC (Avanti Polar Lipids)) for 0, 10, and 30 min at 37 °C. Then, 50 μL of aliquots were transferred to tubes containing 450 μL of ice-cold Ringer’s solution either with or without 1% BSA and further incubated for 10 min. Because the PCs associated with the outer leaflet of the RBC membrane would be back-extracted by the BSA, the degree of PC translocation into the inner leaflet of the RBC membrane was measured by comparing the fluorescence intensity of the cells before (without 1% BSA) and after (with 1% BSA) back-extraction. Data from 5000 events were collected and analyzed using Cell Quest Pro software v.6.0. Percentage of PC translocation was calculated by the following: PC translocation (%) = (fluorescence intensity after back-extraction/fluorescence intensity before back-extraction) × 100.PS exposure and MV generation of the RBCs infected with V. vulnificus, washed with Ringer’s solution with 1 mM of CaCl2, and then incubated with 5 nM of factor Xa (Hematologic Technologies) and 10 nM of factor Va (Hematologic Technologies) in Tyrode buffer , 5 mM glucose, 2.9 mM KCl, 1 mM MgCl2, 12 mM NaHCO3, 0.34 mM Na2HPO4, 0.3% BSA, and 2 mM CaCl2 at pH 7.4) for 3 min at 37 °C. After the addition of 2 μM of purified human prothrombin (factor II) (Hematologic Technologies) to the suspension for 3 min, a 10 μl aliquot was transferred to a tube containing 490 μl of stop buffer . Thrombin generation was determined by measuring A405 and 1 μmol thrombin generation per min is defined as a unit (U) per 106 RBCs in vitro and per μL whole blood cells ex vivo11. In experiments using purified Annexin V 51, human RBCs were pre-incubated with the Annexin V for 10 min, and then infected with V. vulnificus at 5, and 10 MOIs for 30 min. Thrombin generation was determined using the prothrombinase assay as mentioned above.The RBCs were infected with V. vulnificus for 30 min were moved to human umbilical vein endothelial cells  pre-stained with Calcein green (Invitrogen) and then incubated at 37 °C for 30 min. The RBCs pre-infected but not adhered to ECs were washed out using the EC growth medium (EBM-2 (Lonza)). The RBCs pre-infected and adhered to ECs were stained with the anti-glycophorin-A-PE  and then analyzed by fluorescence microscopy . To observe self-aggregation, the RBCs were infected with V. vulnificus, stained with the anti-glycophorin-A-PE , and then analyzed by fluorescence microscopy.Human umbilical vein endothelial cells were originally purchased from Lonza (C2517A). The RBCs pre-infected with V. vulnificus MO6-24/O WT at an MOI of 10. After 30 min incubation at 37 °C, total RNAs from the V. vulnificus were isolated52. Libraries of total RNAs were prepared with the TruSeq stranded mRNA library prep kit (Illumina) according to the manufacturer’s protocol and then sequenced on the Illumina NovaSeq6000 platform. The sequencing reads were mapped to the complete reference genome of V. vulnificus MO6-24/O , and the expression level of each gene was calculated as a relative log expression (RLE) value. The RLE values were normalized and analyzed statistically using DESeq2 v.1.26.0 to identify differentially expressed genes (P < 0.05) when exposed to the RBCs.For transcriptome analysis, the freshly prepared RBCs were infected with V. vulnificus and then, thrombin generation was determined using the prothrombinase assay as mentioned above. For in vivo RBC morphological studies, rats were infected IV with either V. vulnificus WT or ΔrtxA mutant for 1 h, and then whole blood cells were collected. RBCs were prepared after centrifugation at 5800×g for 1 min followed by removal of platelet-rich plasma and buffy coat. RBC morphology was analyzed using SEM as mentioned above. For in vivo thrombus formation studies, a venous thrombosis rat model was used, based upon the previous report that thrombus formed in this model was due to RBCs and fibrin clots with only a few platelets53. The rat abdomen was surgically opened, and the inferior vena cava (IVC) was exposed after 1 h IV infection with V. vulnificus. Both ends of the IVC, about 16 mm apart from each other, were tied loosely. However, all side branches were tied tightly with cotton threads. Thromboplastin (Instrumentation Laboratory) was infused into the IVC for 1 min to initiate thrombus formation. Upon tightening the cotton threads at the ends of the IVC, the abdominal cavity was provisionally closed to maintain the blood stasis for 15 min. The tied IVC segments were excised and opened longitudinally to isolate the thrombus, of which weights were immediately determined. The thrombus was photographed at a 1× magnification using a Stemi 305 stereomicroscope (Zeiss) equipped with an Axiocam 105 color camera (Zeiss).All the protocols used for animal experiments were approved by the Ethics Committee of the Animal Service Center at Seoul National University (SNU-170417-27-5). For bridge studies, the rat RBCs were prepared from the whole blood (citrate-dextrose treated) withdrawn from the abdominal aorta of male Sprague-Dawley (SD) rats  anesthetized with urethane  following the method used for the human RBC preparation, and then, PS exposure and thrombin generation were determined as mentioned above. For ex vivo studies, whole blood cells were collected from the abdominal aorta of the rats after 1 h IV infection with t-test to determine statistical significance when compared with the control using SigmaPlot v.12.The means and standard errors (SE) of means were calculated from at least five independent experiments. The data were subjected to a two-way analysis of variance (ANOVA) followed by Duncan’s multiple range test or two-tailed Student’s Further information on research design is available in the Supplementary InformationPeer review fileReporting Summary


Bioinformatics applies computer science approaches to the analysis of biological data. It is widely known for its genomics-based analysis approaches that have supported, for example, the 1000 Genomes Project. In addition, bioinformatics relates to many other areas, such as analysis of microscopic images , molecular modelling , and visualization of biological networks . Design is a highly interdisciplinary field that incorporates aspects such as aesthetic, economic, functional, philosophical, and/or socio-political considerations into the creative process and is usually determined by context. While visualization plays a critical role in bioinformatics, as reflected in a number of conferences and workshops in the field, design in bioinformatics-related research contexts in particular is not as well studied. With this special issue in conjunction with an international workshop, we aim to bring together bioinformaticians from different fields with designers, design researchers, and medical and scientific illustrators to discuss future challenges in the context of bioinformatics and design. It is widely known for its genome-based analysis approaches that have supported, for example, the 1000 Genomes Project. However, it also relates to many other areas, such as analysis of microscopic images , molecular modelling , and visualisation of biological networks .Since design is a highly interdisciplinary field, aspects such as aesthetic, economic, functional, philosophical, and/or socio-political considerations enter into the creative process and are usually determined by context. While visualisation and visual approaches play a crucial role in bioinformatics, as reflected in a number of conferences and workshops in the field, this is not yet the case for design, especially in bioinformatics-related research contexts.http://desxbioinf.i2d.uk/.With this special issue, in conjunction with an international workshop initiative, we aim to bring together bioinformaticians from different fields with designers, design researchers, and medical and scientific illustrators to discuss future challenges in the context of bioinformatics and design: Because bioinformatics is a field that often combines abstract and spatial data and frequently considers standardization approaches, it offers a number of opportunities for future designers.In addition, we would like to place Design X Bioinformatics in the context of the emerging field of Biodesign, which uses the biological capabilities of organisms to meet specific user needs, for example, in terms of materials development, synthetic biology applications, or the creation of bio-digital hybrid systems. In such systems, both the digital and biological components are engineered to work together.1.1–Challenges in Design X Bioinformatics–Contextualization of previous work to the design field–Investigating different areas of design  in the context of Bioinformatics–Bio-related 3D Modelling and Design–Generation of medical and scientific illustrations–Visualization of biological phenomena  and their abstractionFor this initiative, and thus for this special issue, contributions were solicited from a variety of areas that combine bioinformatics and design approaches. A non-exhaustive list of such potential topics includes:As the summary of research contributions below shows, a variety of these topics are covered, providing a snapshot of current research directions in the field.1.2Biodesign is an emerging topic for which a clear definition has not yet been found. Several challenges and workshops on Biodesign have been held in recent years –3. The oHowever, previous workshops have not yet addressed the relationship between design and bioinformatics in depth, often focusing on more general design issues related to biological systems.–The “Visualizing Biological Data (VIZBI)” conference has attracted – besides computer scientists and Bioinformaticians – in numbers of artists and designers in recent years . This co–The “Workshop on Molecular Graphics and Visual Analysis of Molecular Data” has promoted visual analysis of molecular data at the EuroVis conference  annually since 2018 and also attracts papers with a minor design components .–The workshop “From Virtual Reality to Immersive Bioinformatics” was organised in 2018 as part of the Stereoscopic Displays and Applications conference . It explThere are already a number of workshops and conferences that are active in related areas and have a strong focus on visualization but are not yet closely related to design. In a non-exhaustive list, we briefly describe three such initiatives related to this special issue:1.3in vitro method to study and visualize the physical principle underlying the phenomenon of microtubule self-assembly by designing the structure of microtubule assemblies in a simplified and ideal form.Microtubules are critical for supporting cellular architecture, intracellular transport, and chromosome segregation. Microtubule self-assembly is an essential physical process underlying these cellular functions. The micro-patterning technique is an in vitro systems such as microtubule micropatterning are already powerful tools for studying cellular phenomena observed under the microscope. However, microscopy has limitations in terms of the scale at which it can observe, and it is a challenging task to visualize cellular structures and phenomena at the molecular level. Recent computational approaches can visualize such phenomena at the molecular scale and break the limitations of wet lab experiments.Designed From another point of view, the various geometric patterns created by the micro-patterning technique using biomaterials  are a kiin vitro approach.Therefore, we chose the final image as the logo to promote the Design X Bioinformatics workshop, created by this 2Of the nine manuscripts submitted for this special issue on design in the context of bioinformatics, five papers have been accepted for publication after peer review. We provide here an overview of the accepted submissions:2.1In his keynote contribution, David Goodsell presents an approach to building a cross-sectional model of an entire JCVI-syn3.0 minimal cell . To this2.2This manuscript provides an overview of the design principles to consider when creating visualizations of systems biology information, using the Systems Biology Graphical Notation (SBGN) as an example. A review of the literature on the design of graphical representations in biology using the SBGN recapitulates previous work. The paper provides a detailed overview of the theoretical background, critically discusses the use and design of glyphs, criteria for good network layouts, and building blocks for SBGN PD maps . The pri2.3In this manuscript, the authors outline the importance of using design in structural biology and bioinformatics. First, the authors present practical examples of the benefits of researchers working with designers/illustrators and the collaborative process between the two. The fusion of art and science also new designs for visualising, representing, and understanding the invisible molecular world of proteins. Such designs go beyond molecular visualization and redefine the way we see the molecular world. Inspired by a panoramic camera, the authors propose a method to flatten the three-dimensional structure of proteins and convert it into a two-dimensional projection, which is then used to design information visualization for ion channels . This pr2.4At the molecular level, structures such as proteins, sugars, nucleic acids, etc., have no color of their own. But to illustrate and explain the diversity and functionality of these cellular components, colors are often used. In this article, the author proposes a new initiative to help bring together biologists, artists, computer graphic designers, and perception experts to reconcile these two situations. The concept can be developed in a series of iterations that engage the community, including discussion panels, technical challenges, experimental studies, and outreach activities. Examples discuss, e.g., how color ranges can be used to encode intensities of molecular structures . A uniqu2.5Garrison and Bruckner discuss the use of color in molecular visualizations, focusing on the reasons and problems associated with the broad and often arbitrary use of color. They provide background information and considerations that underscore the need for a color system for molecular visualizations. The guidelines offer the added benefit of simplifying the creation process for those unfamiliar with color theory. Best practise considerations relate to three key concepts – aesthetics, interpretability, and effectiveness. The challenges and perspectives described highlight the importance of involving multiple stakeholders and diverse perspectives in the development of such guidelines. The overall goal is to link colors better and more consistently to the functional or structural semantics of molecules . These e3This special issue offers several examples of how the worlds of design and bioinformatics can be brought together to open new avenues of research and promote cross-fertilization between these disciplines. We can already see that there are some recurring themes, such as that of color, and that it is desirable to develop generally applicable design principles for the particular area where design and bioinformatics meet. There may currently be a lack of places where these communities naturally intersect, and the organization of workshops specifically focused on this area may be necessary to further advance such studies.We have also begun to explore the relationship between Biodesign and Bioinformatics here. As mentioned earlier, the field of Biodesign is not yet clearly defined, but obviously Design X Bioinformatics Design shares a large cross-section with Biodesign. The flourishing of Biodesign offers great potential for the future development of the Design X Bioinformatics Design field.


Clathria reinwardti, Rhabdastrella globostellata, and Spheciospongia sp. The results reveal that the three metagenomes contain a high number of predicted BGCs, ranging from 282 to 463 BGCs per metagenome. The types of BGCs were diverse and represented 12 different cluster types. Clusters predicted to encode fatty acid synthases and polyketide synthases (PKS) were the most dominant BGC types, followed by clusters encoding synthesis of terpenes and bacteriocins. Based on BGC sequence similarity analysis, 363 gene cluster families (GCFs) were identified. Interestingly, no GCFs were assigned to pathways responsible for the production of known compounds, implying that the clusters detected might be responsible for production of several novel compounds. The KS gene sequences from PKS clusters were used to predict the taxonomic origin of the clusters involved. The KS sequences were related to 12 bacterial phyla with Actinobacteria, Proteobacteria, and Firmicutes as the most predominant. At the genus level, the KSs were most related to those found in the genera Mycolicibacterium, Mycobacterium, Burkholderia, and Streptomyces. Phylogenetic analysis of KS sequences resulted in detection of two known ‘sponge-specific’ BGCs, i.e., SupA and SwfA, as well as a new ‘sponge-specific’ cluster related to fatty acid synthesis in the phylum Candidatus Poribacteria and composed only by KS sequences of the three sponge-associated bacterial communities assessed here.Recent reviews have reinforced sponge-associated bacteria as a valuable source of structurally diverse secondary metabolites with potent biological properties, which makes these microbial communities promising sources of new drug candidates. However, the overall diversity of secondary metabolite biosynthetic potential present in bacteria is difficult to access due to the fact that the majority of bacteria are not readily cultured in the laboratory. Thus, use of cultivation-independent approaches may allow accessing “silent” and “cryptic” secondary metabolite biosynthetic gene clusters present in bacteria that cannot yet be cultured. In the present study, we investigated the diversity of secondary metabolite biosynthetic gene clusters (BGCs) in metagenomes of bacterial communities associated with three sponge species:  Bacterial secondary metabolites, also referred to as natural products or specialized metabolites, are an important source of biologically-active compounds for healthcare and agriculture applications ,2,3,4. T–carbon bond formation between the downstream product chain and the upstream extender unit [In bacteria, non-ribosomal peptide synthase (NRPS) and polyketide synthase (PKS) gene clusters are the two main clusters responsible for the biosynthesis of secondary metabolites . Polyketder unit . Non-ribder unit . Interesder unit ,13,14,15der unit ,17,18,19Dysidea herbacea are actually produced by the endosymbiotic cyanobacterium Oscillatoria spongeliae [Theonella swinhoei has been found to be contained in a δ-proteobacterial symbiont [Marine sponges are well known to be a particularly rich source of structurally unique secondary metabolites with a broad spectrum of biological properties ,21,22, mongeliae . In addisymbiont . Subsequsymbiont ,31,32,33symbiont ,34,35,36symbiont ,38,39,40symbiont . Thus, tSpheciospongia sp., Rhabdastrella globostellata and Clathria reinwardti harbored diverse communities of bacteria [C. reinwardti [R. globostellata [Spheciospongia spp. [We have previously investigated the bacterial and archaeal diversity of the 18 marine sponge species and found that three species in particular bacteria . Furtherinwardti ,44,45, Rstellata ,51,52,53gia spp. ,55,56,57Spheciospongia sp., Rhabdastrella globostellata, and Clathria reinwardti were sequenced. The numbers of paired-end reads obtained from the samples were in a range of 30,414,932 to 66,835,559 with average G+C content between 61% and 62 %  from the metagenomes from sponge-associated bacteria, only contigs longer than 1000 bp were analyzed. These yielded a total of 1116 BGCs as identified by antiSMASH . The numTo identify orthologous clusters among BGCs across the different sponge metagenomes, we reconstructed a BGC sequence similarity network . The anaIn general, approximately one-third (103) of GCFs were found in all specimens, 89 GCFs in 2 specimens, and 171 GCFs only in individual specimens (singletons) A. It appThe ketosynthase (KS) domain is the most conserved catalytic domain of the PKS gene cluster and involved in the tailoring the polyketide by catalyzing chain condensation. Therefore, the KS sequences extracted from the identified BGCs were used for phylogenetic analysis . The KS Candidatus Poribacteria metagenome-assembled genomes as best hit and formed a phylogenetically distant clade from other KS sequences from fatty acid BGCs derived from other sources , followed by the classes of Proteobacteria (3.5–11.7%) and the phylum Firmicutes (9.1%). Each of remaining phyla accounted for less than 5%. At the genus level, the KS sequences of the identified BGCs from the metagenomes were linked to 142 genera , particularly the genera Mycolicibacterium (37%), Mycobacterium (28%), Burkholderia (12%), Streptomyces (7%), and Sciscionella (5%) (Proteobacteria (> 80% of the sequences), including Betaproteobacteria (45%), Gammaproteobacteria (26%), and Deltaproteobacteria (11%) with Verminephrobacter, Cellvibrio, Syntrophus, Cupriavidus, Lechevalieria, and Thiobacillus as main genera (Proteobacteria (Alpha- and Delta-) and three genera Tropicibacter, Sandaracinus, and Pseudovibrio  . On the n genera . In casedovibrio .Candidatus Poribacteria, a sponge-specific bacterial candidate phylum. Members of the phylum Candidatus Poribacteria were not detected in the same sponge specimens by 16S rRNA amplicon sequencing [Candidatus Poribacteria in the PCR amplicon-based study may be attributed to a bias of the specificity of primers [Gene clusters related to putative fatty acid synthesis is the most predominant BGC type identified from the metagenomes of sponge-associated bacteria in our study. This is expected because fatty acids are essential components in cell membranes of bacteria. Of particular interest is the detection of a separate cluster of KS genes related to fatty acid synthase from quencing . However primers . Except  primers , many fa primers ,62,63. S primers ,66,67,68trans-AT PKSs were also detected in the sponge-associated bacteria metagenomes. Trans-AT PKSs lack integrated AT domains. Instead, a free-standing AT domain acts in trans to load acyl building blocks into the assembly line [trans-AT type, suggesting that the polyketides of these enzymes constitute a major natural product class [trans-AT PKS products  [trans-AT PKS pathways  [trans-AT PKS gene clusters detected in the sponge-associated bacteria metagenomes in our study had a best BLAST hit to gene clusters involved in the production of puwainaphycins and epothilones  ,79. Manyamide A) ,81,82. Tthilones . HoweverType II PKSs are found exclusively in bacteria and are responsible for producing aromatic polyketides such as the antibiotics actinorhodin, tetracycline or doxorubicin . AlthougTerpene BGCs were the third most predominant among BGCs predicted from the three metagenomes. Originally, terpenes were considered to be plant and fungal products. However, extensive bacterial genome analysis has shown that terpene synthase genes are also widespread in bacteria ,89,90. R–16 bacteriocin BGCs and 1–3 lantipeptide BGCs per metagenome. Bacteriocins are ribosomally synthetized peptides or proteins that act as antimicrobial compounds against other Gram-positive and Gram-negative bacteria [Bacillus subtilis MMA7 associated with the sponge Haliclona simulans and found that the subtilomycin biosynthetic cluster is widespread among B. subtilis strains isolated from different shallow and deep water marine sponges. However, the generally large discrepancy between bacteria isolated from sponges and the dominant bacteria present in sponges [Clostridium difficile infections [Several RiPP BGC types were detected in the metagenome of sponge-associated bacteria in our study, including 12bacteria ,97. Genobacteria ,93,98. Ibacteria  isolated sponges  is also  sponges ,101,102  sponges ,105,106.fections ,108,109 fections ,111,112.Discodermia dissoluta from three metagenomic libraries  revealed that NRPS sequences were only detected in the fraction enriched by filamentous bacteria, where no NRPS sequences were found in whole sponge tissue and the unicellular-enriched fraction [Interestingly, no NRPS-like BGCs were detected in three sponge bacterial metagenomes in the present study. This may be attributed to several reasons. Firstly, an investigation of PKS and NRPS from microbes associated with fraction . During fraction ,117,118.C. reinwardti [R. globostellata [Spheciospongia spp. [Previous chemical investigations have reported diverse natural products isolated from the three sponges inwardti ,44,45, Rstellata ,50,51,52gia spp. ,55,56,57Actinobacteria, Proteobacteria, Bacteroidetes, Firmicutes, and Cyanobacteria [Actinobacteria, such as Mycolicibacterium, Mycobacterium, and Streptomyces, were most dominant as predicted hosts of the KS sequences. Apart from Streptomyces and Bacillus, which are rich sources of natural products [Pseudomonas, Clostridium, Burkholderia, Pseudonocardia, Photorhabdus, Xenorhabdus, Chitinophaga, Herpetosiphon, and Planctomyces [Candidatus Entotheonella” has been recognized for its unusually rich and varied arsenal of BGCs [Streptomyces, Pseudomonas, and Burkholderia were among the prominent genera to which KS sequences were assigned, but “Candidatus Enthotheonella” was not identified, which corroborates with its absence in these three sponge species based on 16S rRNA gene analysis [Pseudovibrio and Bacillus, that are frequently isolated from sponges and linked to a high prevalence of antimicrobial activity [Previous extensive investigations of BGCs in bacterial genomes revealed non-uniform distribution of BGCs in bacterial taxa. The phyla known to possess a high number of BGCs in their genomes include bacteria . Since tproducts , other bctomyces ,122. Par of BGCs . In our analysis . In addiactivity  were notGenerally, the predicted taxa of bacteria harboring the BGCs in the present study corresponded to 16S rRNA gene analysis in a previous study at the phylum level, but their relative abundances are different . This isSup). In another study, Sala et al. [Swf), in several high-microbial-abundance sponges. Notably, both SupA and SwfA are involved in the synthesis of methyl branched fatty acids [SupA and SwfA.It is well known that marine sponges harbor specific microbial communities . Interesa et al.  detectedty acids ,126. PhyCandidatus Poribacteria. This sponge-specific KS group includes KS sequences from our study and KS sequences retrieved from Candidatus Poribacteria metagenome-assembled genomes [Inflatella pellicula, Poecillastra compressa, and Stelletta normani through PCR screening of the KS domain and also found several sponge-specific KS groups. Additionally, other recent studies have identified other ‘sponge-specific’ gene clusters related to the biosynthesis of RIPP proteusins, bromotyrosine alkaloids, and ether lipids [The third sponge-specific KS group is involved in fatty acids synthesis in  genomes . Borcher genomes  investigr lipids ,32,128. Spheciospongia sp., Rhabdastrella globostellata, and Clathria reinwardti were collected by scuba diving from May to September 2015 from the central coastal region of Vietnam at 5–10 m depth and identified using molecular markers (18S rRNA and COI genes) in a previous study [Sponge specimens of us study .g for 5 min at 4 °C. The supernatant was then transferred to another tube and centrifuged at 1000× g for 15 min at 4 °C. Next, the supernatant was transferred to another tube and centrifuged at 3000× g for 15 min at 4 °C. Finally, the supernatant was then transferred to another tube and centrifuged at 8000× g for 15 min at 4 °C. The pellet was washed twice with TEN buffer and centrifuged at 8000× g for 20 min at 4 °C. This cell pellet was used for metagenomic DNA extraction.The specimens were rinsed three times with sterile artificial seawater to remove any debris attached to the sponge. Then the specimens were further cleaned with a sterile scalpel in order to remove sediment and other organisms more strongly attached to the sponges. Sponge-associated bacterial cells were enriched by stepwise centrifugation as described by . In briew/v) agarose gel. Metagenomic DNA was sequenced on an Illumina HiSeq2500 platform at BaseClear BV . Quality control of FASTQ files was done with FastQC v.0.11.7 . The raw reads were checked and the adapters, low-quality reads, artifacts, and PhiX contamination were removed using the command bbduk.sh of the BBmap v.34 . Reads with a low Phred quality score (< 30) and reads shorter than 70 base pairs were filtered out. Next, reads were coverage-normalized with the command bbnorm.sh of BBMap v.34 at default settings. Reads were then assembled with SPAdes v.3.5.0 [Metagenomic DNA of sponge-associated bacteria was extracted using the ZymoBeadTM Genomic DNA Kit  according to the manufacturer’s protocol. The concentration of the extracted DNA was determined with a Nanodrop 1000 spectrophotometer , and its integrity was examined by gel electrophoresis on a 1% . Biosynthetic gene cluster similarity networks and gene cluster families were generated using BiG-SCAPE with default settings. The networks were visualized using Cytoscape v. 3.6.0  [The predicted protein-coding genes were subjected to a search for secondary metabolite biosynthetic gene clusters (BGCs) using antiSMASH v.4.1.0 . The KnownClusterBlast function in antiSMASH was enabled to identify BGCs that are responsible for the biosynthesis of known compounds in the metagenomes of the sponge-associated bacteria. The predicted biosynthetic gene clusters were also searched for known biosynthetic pathways using the natural product domain seeker (NaPdoS) (st 2018) .http://napdos.ucsd.edu/, accessed on 15 August 2018) [blastp  against the nr database. KS sequences with a short length < 200 amino acids were excluded from the phylogenetic analysis. The KS sequences  were aligned using the MAFFT (v.7.504) program with the FFT-NS-i strategy [https://itol.embl.de/, accessed on 23 April 2021). Taxonomy of the bacteria harboring the KS sequences were predicted based on the most homologous KS sequences (the first hit) by a BLAST search of the KS sequences against the refseq_protein database (v.2018) at NCBI using blastp .For ketosynthase (KS) phylogeny, KS domain fragments of the biosynthetic gene clusters were extracted and trimmed using NaPdoS (st 2018) . The KS strategy . Poorly strategy . The ketstrategy  with thestrategy  via the Actinobacteria, Proteobacteria, and Firmicutes were the most predominant. At the genus level, the KS were related to 142 genera, of which Mycolicibacterium, Mycobacterium, Burkholderia, and Streptomyces were the most predominant. Notably, a large number of KS sequences retrieved from PKS BGCs was present in two known ‘sponge-specific’ clusters, i.e., SupA and SwfA, whereas a part of KS sequences retrieved from fatty acid BGCs formed a new sponge-specific KS cluster related to the fatty acid synthesis in Candidatus Poribacteria. Our study reinforces that sponge-associated bacteria are a rich source of novel BGCs and as such most likely, bioactive compounds.The metagenomes of associated bacteria of three sponges contained a high number of predicted BGCs, ranging from 282 to 463 BGCs per metagenome, with 12 different BGC types. After cf_fatty_acid, PKSs were the most dominant BGCs, followed by terpene BGCs and bacteriocin BGCs. The BGCs were grouped into 363 gene cluster families (GCFs) based on sequence similarity. Interestingly, not a single GCF from the metagenomes was assigned to pathways responsible for the production of known compounds, implying that they might be responsible for production of novel compounds. Based on taxa of the closest reference KS sequences retrieved from the NCBI database, the KS sequences of the identified BGCs from the metagenomes related to 12 bacterial phyla, of which