Diabetic kidney disease (DKD) has become a key cause of end-stage renal disease worldwide. Inflammation and fibrosis have been shown to play important roles in the pathogenesis of DKD. MID1, also known as TRIM18, is an E3 ubiquitin ligase of the tripartite motif (TRIM) subfamily of RING-containing proteins and increased in renal tubule in patients with DKD. However, the function and molecular mechanism of TRIM18 in DKD remain unexplored. Herein we report that TRIM18 expression levels were increased in patients with DKD. An animal study confirms that TRIM18 is involved in kidney injury and fibrosis in diabetic mice. TRIM18 knockdown inhibits high glucose (HG)-induced epithelial–mesenchymal transition (EMT), inflammation, and fibrosis of HK-2 cells. This is accompanied by decreased levels of tumor necrosis factor alpha, interleukin-6, hydroxyproline (Hyp), connective tissue growth factor, and α-smooth muscle actin. Additionally, TRIM18 knockdown inhibits HG-induced increase in the phosphorylated-/total signal transducer and activator of transcription (STAT3). Treatment with niclosamide (STAT3 inhibitor) or protein tyrosine phosphatase-1B (PTP1B) overexpression blocked the TRIM18 induced EMT, inflammation and fibrosis. Co-immunoprecipitation and Western blot assays showed that TRIM18 promoted the ubiquitination of PTP1B. These findings highlight the importance of the TRIM18/PTP1B/STAT3 signaling pathway in DKD and can help in the development of new therapeutics for DKD treatment. Diabetes mellitus is characterized by elevated blood glucose levels , while tIncreased secretion of proinflammatory factors and profibrotic factors caused by hemodynamic changes and metabolic disorders has been implicated in DKD . EpithelThe ubiquitin-proteasome system, catalyzed by E1, E2, and E3 enzymes, is a method for getting rid of proteins , 2005. THerein, we aimed to explore the role of the TRIM18/PTP1B/STAT3 pathway in the pathogenesis of DKD and investigate its underlying mechanisms.RNA-seq data about TRIM18 expression in patients with DKD were acquired from the GSE20122 dataset, which included 10 cases of DKD renal tubule tissues and 37 control tissues. Gene set enrichment analysis (GSEA) was performed to determine the pathways of significant enrichment in high and low TRIM18 expression.n = 45) with type 2 DKD were obtained from the hospital, and normal kidney tissues from nephrec–tomies performed for renal hamartoma (n = 10) served as the control, between January 2017 and August 2019 were enrolled in this study. Clinical characteristics of patients with type 2 DKD and control subjects are presented in This study was approved by the Ethics Committee of the Affiliated Hangzhou First People’s Hospital, Zhejiang University School of Medicine and is in accordance with the 1995 Declaration of Helsinki. All participating patients gave consent before the investigation. Human kidney biopsy tissues of patients , constructed by Novobio Biotechnology . Serum and urine samples were collected at 8 and 12 weeks, and kidney tissues were collected for H&E, Masson’s trichrome staining, and immunohistochemistry staining with anti-TRIM18 antibody  as previously described (n = 6 per group). Serum creatinine, blood urea nitrogen, and urine protein were analyzed using enzyme-linked immunosorbent assay (ELISA) kits . The study was approved by the Animal Experimentation Ethics Committee of the Affiliated Hangzhou First People’s Hospital, Zhejiang University School of Medicine.This study used 6-week-old male C57BL/KsJ mice, both non-diabetic (db/m) and diabetic (db/db). The db/db mice exhibited diabetic symptoms as reported  and wereHK-2 cells from the American Type Culture Collection  were cultured in Dulbecco’s modified Eagle’s medium  with 10% fetal bovine serum  and 1× Pen/strep (Beyotime). HK-2 cells were treated with high glucose  for 48 h in the absence or presence of 10 μM niclosamide (STAT3 inhibitor) or 10 μM MG132 (proteasome inhibitor). For osmotic control, 5.5 mM glucose and 24.5 mM mannitol (NG) were used.Genes were silenced with shRNAs. Three TRIM18-specific shRNAs  were inserted into the pLKO.1 plasmid . The coding sequence of TRIM18 and PTP1B was ligated in pLVX-Puro plasmid (OriGene). The plasmids were transfected into 293T cells using lipo2000 to produce viruses.3 cell/well) were cultured in 96-well plates and incubated at 37°C overnight. After treatment for 0, 12, 24, and 48 h, 10 μl of the Cell Counting Kit-8 solution was added into each well and incubated for an extra 1 h. Cell viability was subsequently determined using a microplate reader at OD 450 nm.HK-2 cells (3 × 10Hydroxyproline (Hyp) levels were determined using the A030 Hyp assay kit .CT−ΔΔ method.RNAs were extracted using TRIzol (Beyotime). cDNAs were generated using Superscript II . Quantitative Real-time PCR was performed using SYBR Green master mix . The primers used are: TRIM18-F 5'-AGAGTGCGTGTAGCAACAG-3', TRIM18-R 5'-CAGACAAATAGGGCAGGTCAG-3', GAPDH-F 5'-AATAATATCACCATCTTC-3', and GAPDH-R 5'-AGGCTGTTGTCATACTTC-3'. GAPDH was used as a control. Fold changes were calculated using the 2Total proteins were prepared in the RIPA lysis buffer. Sodium dodecyl sulfate polyacrylamide gel electrophoresis was conducted to separate the protein samples. Then, proteins were electroblotted onto polyvinylidene difluoride membranes, blocked, and incubated with antibodies against TRIM18 , connective tissue growth factor , α-SMA , STAT3 , p-STAT3 , protein tyrosine phosphatase non-receptor type 6 , suppressor of cytokine signaling 1 , PTP1B , protein inhibitor of activated STAT 1 , and GAPDH  at 4°C for 12 h, followed by secondary antibodies (Beyotime). Bands were determined using the Quantity One software.For the co-immunoprecipitation (co-IP) assay, 2 mg of cell proteins was pre-cleared and then immunoprecipitated with anti-TRIM18 , anti-SOCS1 , and anti-PTP1B  for 12 h at 4°C, followed by immunoblotting. Proteins were immunoprecipitated with anti-PTP1B antibody or control IgG and then probed with anti-ubiquitin antibody to test ubiquitination.The levels of tumor necrosis factor alpha (TNF-α) and interleukin-6 (IL-6) in HK-2 cell supernatant and serum of mice were analyzed using ELISA kits. OD 450 was recorded.p < 0.05 was considered statistically significant.All data in this study were processed using the GraphPad Prism 8.0.2 and expressed as mean ± standard deviation (SD). All the experiments were performed in triplicates. To calculate statistical significance between groups, Mann–Whitney test was used for two groups, whereas an analysis of variance followed by Bonferroni’s method was used for multiple groups. Comparisons between categorical and non-parametric variables were conducted using a Chi square test, and a Mann–Whitney test, as appropriate. To evaluate the function of TRIM18 in DKD, microarrays of 10 patients with DKD and 37 controls from the GSE20122 dataset were analyzed. TRIM18 was significantly increased at the mRNA level in the tubules compared with controls . The GSEvia Hyp analysis and fibrotic gene expression. It revealed that db/db+shNC mice had significantly increased Hyp, p-STAT3, TRIM18, CTGF, and α-SMA levels at both 8 and 12 weeks  can be found in the article/The studies involving human participants were reviewed and approved by Affiliated Hangzhou First People’s Hospital, Zhejiang University School of Medicine. The patients/participants provided their written informed consent to participate in this study. The animal study was reviewed and approved by Affiliated Hangzhou First People’s Hospital, Zhejiang University School of Medicine.QC and CG supervised all the experiments. CG designed this study. MW, XF, and NZ performed the practical work and completed the experiments. QC provided help during the experiments. NZ helped in revising and improving language expression. All authors contributed to the article and approved the submitted version.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.


Treponema pallidum (syphilis), herpes simplex virus-2  and herpes simplex virus-1 , Haemophilus ducreyi (chancroid), Chlamydia trachomatis serotypes L1, L2 and L3 (lymphogranuloma venereum), and Klebsiella granulomatis (donovanosis). Epidemiological and clinical aspects of these infections and guidelines for their diagnosis and treatment are presented, including strategies for surveillance, prevention, and control actions to support health managers and professionals in the qualification of care.Infections that cause genital ulcers are one of the themes comprising the Clinical Protocol and Therapeutic Guidelines for Comprehensive Care for People with Sexually Transmitted Infections, published by the Brazilian Ministry of Health in 2020. The Protocol and Guidelines have been developed based on scientific evidence and validated in discussions with specialists. This article addresses clinical genital ulcer syndrome caused by sexually transmitted infections and its most common etiological agents:  Approximately 70% of the genital ulcers attended in specialized clinics are due to sexually transmitted infections, particularly in adolescents and young adults. This article addresses the infections that cause genital ulcers, a subject that comprises the Clinical Protocol and Therapeutic Guidelines (PCDT) for Comprehensive Care for People with Sexually Transmitted Infections (STI), published by the Health Surveillance Secretariat of the Brazilian Ministry of Health in 2020Approximately 70% of the genital ulcers attended in specialized clinics are due to STI, particularly in adolescents and young adults,,Establishing the etiologic agent on a clinical basis is difficult due to polymorphism in presentationsTreponema pallidum, which causes syphilis. These agents can also be found in association,The most common agent found in genital ulcers is the herpes simplex virus (HSV), type 1 (HSV-1) and type 2 (HSV-2). They are DNA viruses that belong to the Herpesviridae familyHaemophilus ducreyi, a sexually transmitted Gram-negative bacterium. The condition, which occurs most frequently in men and in tropical regions,Chancroid is caused by Chlamydia trachomatis, the highly invasive serotypes L1, L2, and L3. Transmission is also attributed to asymptomatic people,Lymphogranuloma venereum is caused by Klebsiella granulomatis, a sporadic condition most commonly found in countries with tropical and subtropical climates. Associated with sexual transmission, it has low transmissibility, and its transmission mechanisms are not well knownDonovanosis is caused by The clinical aspects of genital ulcers are very varied and have low sensitivity and specificity regarding the etiological agent,,Infection caused by HSV-1 and HSV-2 sometimes presents a symptomatic primoinfection, which occurs with incubation of six days on average and a duration of approximately 20 days,Once the infection occurs, the virus is transported along the peripheral nerves' axons to sensitive ganglia. It enters latency, a state in which it can persist for life or suffer reactivations. Reactivated, the virus migrates to the mucous and cutaneous surfaces, again through the sensory nerves, and can be eliminated asymptomatically or cause episodes of recurrent lesions. The outbreaks, which may be the first manifestation, are spontaneous or induced by several factors . Recurrent manifestations are more frequent by HSV-2 than HSV-1,,T. pallidum,,,Chronic affection transmitted by sexual contact, whether genital, anal, or oral, or even by vertical transmissionVery painful genital or perianal ulcers typically characterize soft chancre or Ducrey's chancre. It rarely occurs in the oral cavity or other regions of the tegument. It has an irregular border and unclean bottom, covered with a yellowish, necrotic, and fetid coating. When removed, it reveals friable granulation tissue and a non-infiltrated base. The most frequent locations in men are the frenulum and the balanoprepucial groove, and in women, the furcula and the inner face of the inner and outer lipsIt presents ulcers that usually go unnoticed by the infected person or the health professional. Its evolution occurs in three phases: inoculation, regional lymphatic dissemination, and sequelae.,,,The inoculation phase starts with a papule, a pustule, or a small painless ulcer, which disappears without leaving sequelae. It may occur in the rectum and rarely in the urethra or cervix. Depending on its location, it may present mucopurulent exudate,,The original lesion may vary in appearance. It can start with a flat edge ulcer that evolves to hypertrophic or vegetating ulcer, with granular bottom, single or multiple, well delimited, that grows slowly and progressively. It has a bright red aspect and easy bleeding to manipulation. There is a predilection for skin or mucous folds, with frequent "mirror configuration",,The assistance to people with lesions presents particularities. The interview must be conducted in an environment of privacy, in an empathetic manner, and without value judgments, either through speech or non-verbal language. Inquiries about sexual practices and identification of STI risk factors, such as age under 30, history of new or multiple sexual partnerships , sexual partnerships with STI, prior or concurrent STI, and irregular condom use are required,,The etiological diagnosis of genital ulcers based on anamnesis and physical examination can be imprecise; therefore, it is important to use diagnostic tests whenever they are available,Serological tests for HSV-1 and HSV-2 detections are available. Although they are of little help in diagnosis, identifying the viral type can help evaluate prognosis and counseling. It is estimated that HSV-2 has greater transmissibility, including transmission from the pregnant woman to the newborn Viral culture, reserved for research environments and considered a gold standard exam, is being replaced by tests based on nucleic acid amplification test (NAAT), which have greater sensitivity and practicalityT. pallidum particle agglutination assay (TPHA), electrochemiluminescence, and the enzyme-linked immunosorbent assay (ELISA). Nontreponemal tests, such as the venereal disease research laboratory (VDRL) and rapid plasma reagin (RPR), have particular importance in the serological follow-up,,Dark-field microscopy can allow the identification of spirochetes with characteristic shapes and movements. Lymph obtained by the expression is used, ideally without bleeding, which must be analyzed immediately. The use of material obtained from lesions of the oral cavity is inappropriate due to the presence of saprophytic spirochaetes unrelated to syphilis. Direct immunofluorescence or special stains require longer processing and will hardly be useful in clinical context. Identification of genital lesions indicates serological investigation for diagnostic clarification and screening of concomitant STI. There are two types of serological tests, treponemal and nontreponemal. Among the treponemal tests, the following can be mentioned: rapid tests by immunochromatography, treponemal fluorescent antibody-absorption (FTA-Abs), H. ducreyi, with sensitivity up to 98.4%. Some kits for polymerase chain reaction (PCR) have the advantage of simultaneously testing other agents, such as T. pallidum and HSV,,,Smear microscopy of lesions with abundant exudate may allow identification of organized streptobacilli two by two, a configuration compared to "train tracks". This examination has much lower sensitivity and specificity than culture, a procedure with many demands and challenging performance. DNA amplification techniques, such as NAAT, are now considered the gold standard for detecting C. trachomatis in material collected from ulcer specimens, rectum, bubo aspirates, pharynx, biopsy specimens, and urine samples, which are little available in the routine.,,C. trachomatis,.NAAT tests, which include PCR and ligase chain reaction, are the recommended methods for detecting ,,,The diagnosis is established by identifying Donovan bodies in the microscopy of tissue fragment smears or their anatomopathological study, stained by Giemsa, Leishman's, Wright's, or Rapi-Diff methods. It is a microorganism of difficult culture, and NAATs are available, especially in research institutionsSTIs are the main cause of genital ulcers and should invariably compose the diagnostic hypotheses, especially in the presence of epidemiological elements. Longer developing ulcers of more than four weeks in duration require a more careful evaluation since they may be related to neoplastic processes, chronic infectious diseases, or immunosuppression. Most of the time, these processes require specialized care, biopsy, and histopathological studies.,,,,,STIs' importance as a public health issue lies in their high frequency of occurrence and the need to interrupt transmission. The use of laboratory tests, although desirable, has limitations. In genital ulcers with STI suspicion, it is not acceptable to delay the treatment depending on laboratory test results. The use of combinations of therapeutic options to treat one or more agents is desirable in some scenariosT. pallidum, and H. ducreyi) using molecular tests in specific services. This project's findings may modify the current conduct flowchart regarding genital ulcers, as was the case for urethral discharge syndrome,per os, three times a day, for seven to ten days, and, in the case of recurrence, acyclovir 200mg, two tablets, per os, three times a day, for five days. In cases of need of genital herpes suppression (six or more episodes per year), acyclovir 200mg, two pills, per os, twice a day, for up to six months, with the possibility of prolonging the treatment for up to two years. During the evaluation of genital ulcer complaints, given evidence or history of characteristic vesicular lesions, the treatment of genital herpes is indicated. In case of first episode, acyclovir 200mg, two pills, per os. In cases of lesions with more than four weeks of evolution, a biopsy must be performed. The institution of treatment for syphilis, chancroid, lymphogranuloma venereum, and donovanosis must be assessed. For lymphogranuloma venereum and donovanosis, more prolonged treatments are necessary. In the case of lymphogranuloma, doxycycline 100mg must be taken twice a day orally for 21 days as the first option, and azithromycin as an alternative. In the case of donovanosis, it is recommended to take azithromycin 500mg, two pills, per os, once a week, for at least three weeks or until the lesions heal, and, as an alternative treatment, the use of doxycycline, ciprofloxacin, or sulfamethoxazole-trimethoprim may be assessed,For the treatment of other STI with genital ulcers and clinical pictures with less than four weeks of evolution, syphilis (hard chancre) must be treated with benzathine penicillin, a single dose of 2.4 million international units (IU), deep intramuscular (1.2 million IU in each gluteus), and chancroid with azithromycin 1g, single-dose, ,,,,,per os, single dose,,,,,,per os, single-dose, or doxycycline 100mg, one tablet, per os, twice a day for seven days . In the case of donovanosis, presumptive treatment of asymptomatic sexual partners is not recommended because of low infectivity,The sexual partners of the last three months must be oriented about exposure risks and evaluated and treated according to clinical, laboratory, or epidemiological findings,Counseling centered on the person and their sexual practices aim at recognizing riskier practices and establishing a risk reduction plan in light of combined prevention recommendations for STI, HIV, and viral hepatitis,,C. trachomatis and Neisseria gonorrhoeae infections in urine samples, urethral or cervical exudate is recommended, despite its unavailability in most servicesRapid testing for HIV, syphilis, and hepatitis B and C infection must invariably be recommended. Post-therapeutic serological follow-up tests are indicated for people with syphilis. Vaccination for HPV and hepatitis A and B prophylaxis should follow the recommendationsSyphilis cases with ulcers that present a reagent test  must be compulsorily reported as acquired syphilisT. pallidum. The suspicion of syphilis in pregnant woman indicates treatment according to the clinical stage, in a mandatory way, independently of laboratory results. Post-treatment nontreponemal tests must be performed every 30 days,,,,-Genital ulcers caused by STI deserve careful attention due to the potential for vertical transmission of some etiological agents, especially ,,,,,,STI treatment regimens are the same as those recommended for people without HIV infection. Genital ulcers facilitate HIV transmission, increasing the importance of early treatment, and the higher risk is especially significant for genital herpes, syphilis, and chancroid


Second, two sets of potential biomarkers were identified, being descriptors for the metabolomic changes in the bee’s hemolymph following viral infection. Third, the biomarker sets were evaluated in a new dataset only containing wild bees and successfully discriminated virus infection versus non-virus infection with an AUC of 0.985. We concluded that screening hemolymph metabolite markers can underpin physiological changes linked to virus infection dynamics, opening promising avenues to identify, monitor, and predict the effects of virus infection in a bee community within a specific environment.State-of-the-art virus detection technology has advanced a lot, yet technology to evaluate the impacts of viruses on bee physiology and health is basically lacking. However, such technology is sorely needed to understand how multi-host viruses can impact the composition of the bee community. Here, we evaluated the potential of hemolymph metabolites as biomarkers to identify the viral infection status in bees. A metabolomics strategy based on ultra-high-performance liquid chromatography coupled to high-resolution mass spectrometry was implemented. First, we constructed a predictive model for standardized bumble bees, in which non-infected bees were metabolically differentiated from an overt Israeli acute paralysis virus (IAPV) infection (R Viruses are etiological agents of diseases and also infect bee species . UnderstBombus terrestris, a metabolic biomarker-based approach showed significant potential to predict artificial food stress in worker bees [Different “omics” technologies exist that describe a specimen’s condition by generating a massive amount of data. Big data has been suggested to develop predictive models, for instance, to develop a diagnostic algorithm to identify specimens suffering from some disease . The metker bees . With thB. terrestris and performed a biomarker discovery study for the Israeli acute paralysis virus (IAPV) and the slow bee paralysis virus (SBPV). Injection of IAPV in bumble bees leads to high bee mortality within the first week after injection, while SBPV-infected bees survive, although viral titers increase [We used standardized commercial increase . An untaincrease ,22,23, wB. terrestris . Wild B. terrestris worker bees were caught in urban areas of Ghent, Belgium during June 2019. Upon arrival in the lab, five random worker bees  were placed in one microcolony. In total, we started 48 microcolonies . These microcolonies were placed in an incubator at 30 °C, 60% relative humidity, and continuous darkness, and were all fed with gamma-irradiated pollen ; 20 + 4 microcolonies  received a standardized sugar syrup , while the other 20 + 4 microcolonies  received a 25% sugar syrup to mimic a low carbohydrate nutritional stress [5 virus particles), or 5 µL of filter-sterilized PBS was injected through the membrane between the second and third segment in the abdomen. The IAPV and SBPV stocks were provided by Joachim de Miranda . Both virus stocks were stored in aliquots at –80 °C and had an estimated number of 1 × 106 particles per mL (measured by transmission electron microscope). In addition, virus stocks had < 0.1% contamination of other common honeybee viruses, as determined by RT-qPCR [All bees had a random age. Commercial bumblebees were randomly picked from indoor mass-reared queen-right colonies of l stress . Bees we RT-qPCR . w/v; Sigma Aldrich, Overijse, Belgium) in 1 mL of 50% methanol/PBS to prevent melanization. The hemolymph sample was collected on ice and immediately put on dry ice afterwards. All hemolymph collection was performed under binocular microscope, and three rules were strictly followed to guarantee the quality of sampling: (i) the hemolymph should be pure and transparent; (ii) no other tissues were perforated; (iii) sampling time (incision and extraction) per bee is less than 35 s. All samples were stored at −80 °C until chemical analysis. Bee hemolymph was collected by making a small incision in the dorsal thorax and extracting 10 μL per bee using Wiretrol II Capillary micropipettes (VWR) into an Eppendorf tube that contained 2 μL of 5% N-phenylthiourea  vial with glass insert. Solvents used for extraction of hemolymph metabolites were of LC-MS grade, obtained from Fisher Scientific  and VWR International . Ultrapure water was obtained by usage of a purified-water system .Polar and medium polar metabolites were extracted from bee hemolymph as previously described . In brien = 150) was used to make quality control (QC) samples for instrument conditioning and data normalization. Experimental samples were run in a randomized order, except for QC samples, which were analyzed in duplicate after every nine experimental samples.For ultra-high-performance LC hyphenated to Quadrupole-Orbitrap high-resolution MS (UHPLC-Q-Orbitrap-HRMS) analysis, the methodology used was as previously described . The chrWe randomly assigned 200 commercial bees to 40 microcolonies consisting of four treatment groups, including PBS-injected bees, IAPV-injected bees, SBPV-injected bees, and mock bees (without any treatment). For each group, we also introduced the stressor malnutrition . In total, we sampled 110 bees for hemolymph extraction .The choice of our sampling timepoint was based on previously established infection dynamics for IAPV and SBPV in commercial bumble bees. IAPV has a quick replication, resulting in high mortality in the first week, while SBPV has a steep increase in the first week, with a more moderate growth later and no mortality . We opte® 14.1 multivariate statistics software . Model validation was performed through various quality parameters such as CV-ANOVA , permutation testing (n = 100), Q2 (>0.5), and R2Y (>0.5) [Untargeted data analysis was performed as previously described by Wang et al. 2019 ,28. To cY (>0.5) . S-plotsm/z-value and 13C isotope profile) and retention time. Hereby, the identification process was based on an in-house library of analytical standards (±300 compounds), which were injected through various mixtures at the beginning of the analytical sequence. Statistical analysis was performed using SPSS 22.0 software, applying two-way ANOVA and Tukey HSD test for post-hoc comparisons, whereby a p-value < 0.05 was considered as statistically significant. Further filtering of candidate biomarkers was based on biological qualification, performing literature survey and metabolic pathway analysis ). To judge the overall diagnostic efficacy of the selected metabolites in the standardized bee dataset, we calculated the AUC value using ROC curve analysis in MetaboAnalyst.Targeted data analysis was performed using XcaliburTM 2.1 (Thermo Fisher Scientific), allowing quantitative analysis (peak integration) of metabolites that were identified based on the mass spectrum  [The 40 wild caught n 3.8.4) . The selUpon analysis of 150 hemolymph samples, obtained from B. terrestris hemolymph following different virus infections, nutritional stresses, and sample resources , a total of 1149 (+ionization mode) and 911 (−ionization mode) components were defined as constituents of the metabolic fingerprint. The PCA-X score plots revealed good clustering of the internal QC samples , confirmn = 110). We performed multiple pairwise comparisons of various treatment groups.In We first analyzed if the viral infection status can be discriminated in standardized commercial bees, without any additional stressor, as stressors could potentially obscure clear virus related metabolome fingerprints. In bees receiving 50% sugar water, models had a predictive power to (1) find an overt (IAPV) and covert (SBPV) infection  of model 13 is lower, meaning that including stressed bees indeed make the classification harder. This selection strategy resulted in five potential biomarkers for IAPV infection  bees, our results confirmed that UHPLC-Orbitrap-MS-based metabolomics can clearly distinguish bees with or without virus infection in many situations (from single virus-infected bees to a mixture of single- and double-stressed bees). To select robust biomarkers, we defined components with discriminative power  are further on named as the “targeted biomarker set”. In the commercial bees, the AUC of this biomarker set to identify a viral infection was 0.876 (95% CI: 0.783–0.949).In B. terrestris. Twenty-nine metabolites  were finally selected as potential biomarkers, descriptive for virus infection. There is growing evidence that the impacts of viruses on their bee hosts can be exacerbated by poor nutrition [In summary, this study provided the first evidence of biomarker discovery for predicting virus infection in bees. A dual targeted and untargeted metabolomics approach was established to analyze both covert and overt virus infection in utrition ,41; henc


A multimodality approach is often used in the management of high-grade soft tissue sarcoma of the extremity (eSTS). Limb-sparing surgery is the keystone of eSTS management, often accompanied with radiotherapy (RTX) and/or chemotherapy (CTX). The National Comprehensive Cancer Network (NCCN) and European Society for Medical Oncology (ESMO) guideline recommend RTX and CTX in selected groups of high-risk patients.Our cross-sectional study aimed to acquire insight into the variation of eSTS management across the world and to assess the importance of several clinicopathologic risk factors for the indication of multimodality therapy.The optimal management of patients with eSTS remains an important field of research. Recent developments of prediction tools and advances in genomics and proteomics may allow for better patient selection for multimodality therapy. Because in recent years some retrospective studies have been performed to assess the use of these tools for the indication of multimodality therapy, further studies are needed to improve patient selection for the indication of multimodality therapy, with a special focus on perioperative CTX and new perioperative modalities. Also, the cost-effectiveness of chest CT scan versus chest X-ray and the use of prognostic tools for patient-tailored follow-up regimen should be explored. Given the rarity and heterogeneity of eSTS, large collaborative efforts of international sarcoma centers are crucial to further improve patient-tailored management of eSTS.


Klebsiella pneumoniae carbapenemase (KPC)-producing Klebsiella pneumoniae. Our hospital, Rambam Health Care Campus (RHCC), was one of the medical centers affected by this outbreak. We aimed to investigate the changing epidemiology of CPE at RHCC since 2006.Israeli hospitals were confronted with a major national outbreak of carbapenemase-producing Enterobacterales (CPE) starting in 2006, caused predominantly by monoclonal This was a retrospective observational cohort study performed in Northern Israel (Haifa) at RHCC, which is a primary tertiary acute care academic hospital. The study included all patients who had acquired CPE at RHCC between January 2005 and December 2020.K. pneumoniae dropped from 100% of all CPE in the first years to 28% (37/134) in 2020. In 2014, the carbapenemase in 94% of all CPE patients (89/95) was KPC. This decreased to 56% in 2020, while New Delhi metallo-β-lactamase (NDM) and OXA-48 carbapenemases increased from 4% and 2% to 29% (39/134) and 12.7% (17/134) of CPE, respectively.The proportion of patients infected with K. pneumoniae to involve different Enterobacterales and carbapenemases. Our results are a microcosm of the current global epidemiology attesting to globalization in bacteriology. The results have implications for infection control and antibiotic treatment of CPE infections.The CPE epidemic evolved from KPC-producing  Klebsiella pneumoniae carbapenemase (KPC), New Delhi metallo-β-lactamase (NDM), imipenemase (IMI), oxacillinases (OXA)-48, and Verona integron-encoded metallo-β-lactamase (VIM). Israeli hospitals confronted a major national outbreak starting in 2006, caused predominantly by monoclonal KPC-producing Klebsiella pneumoniae.2Carbapenem-resistant Enterobacterales (CRE) are amongst the major challenges that have been facing healthcare institutions throughout the world in the last two decades.K. pneumoniae; from January 2006 to April 2007, all 88 carbapenem-resistant K. pneumoniae patient isolates carried KPC.3Our medical center, Rambam Health Care Campus (RHCC), located in Northern Israel, was among the affected hospitals. The prevalence of CRE at RHCC increased from 2006, reaching a peak of 186.6 new acquisitions per 100,000 hospital-days in 2008.Some of the carbapenemases are chromosomal, but most often, and notably, the KPCs are found on plasmids, hence they are able to move between bacterial species. Indeed, with time, KPC spread to different Enterobacterales, and the introduction of diverse carbapenemases was noted. The aim of this study was to describe the introduction of new carbapenemases at RHCC and the spread of carbapenemases among Enterobacterales.The study was conducted at RHCC, a 1,000-bed primary and tertiary university hospital in Northern Israel. All patients who acquired CPE at RHCC between January 2005 and December 2020 were included in the study. At RHCC, CPE was considered as acquired if the positive sample was taken ≥72 hours after admission, within 72 hours after any discharge, or within one month after discharge, if the patient was not hospitalized in another healthcare facility. Patients who acquired CRE before admission to RHCC were excluded.blaKPC/blaNDM/blaOXA-48/blaVIM, have been detected routinely using polymerase chain reaction (PCR)-based multiplexed assays specific for these genes.5Rectal swab screening samples were cultured prior to 2019 on PD420 CHROMagar KPC plates; since 2019, samples were cultured on PD517 MSUPERCARBA plates . The latter medium was used because of its higher sensitivity to non-KPC CPE. Carbapenemase-producing Enterobacterales was defined as Enterobacterales of any type resistant to all tested carbapenems using the Clinical and Laboratory Standards Institute (CLSI) M100S guidelines definition of MIC>1. We extracted DNA from suspected CRE colonies using the Qiamp DNA mini kit  in accordance with the manufacturer’s instructions. Since 2014, β-lactamase (bla) carbapenemase genes, i.e. K. pneumoniae. Starting in 2009, the proportion of patients carrying CRE with other strains increased gradually, reaching 72% (97/134) in 2020, while the proportion of K. pneumoniae decreased to 28% (37/134) in 2020  of all CPE acquired at RHCC possessed the KPC gene. Only 6% (6/95) of isolates possessed other resistance genes. This proportion decreased to 56% (75/134) in 2020 . The proK. pneumoniae, spread of carbapenemases to other Enterobacterales strains was reported in RHCC as early as 2010.K. pneumoniae, and less than half possessed the KPC gene. Carbapenemases spread to many different Enterobacterales, and new carbapenemases were introduced.We examined the changing epidemiology of CPE in terms of pathogens and resistance mechanisms in one Israeli institution (RHCC) endemic for CPE. Starting from an outbreak confined to KPC-producing K. pneumoniae in North Carolina, USA, in 1996,K. pneumoniae has spread to many locations worldwide.8Since the first description of KPC-producing Our findings are consistent with this spread, which has a major impact on infection control. The diversity of isolates and resistant genes may indicate that, besides clonal transmission of CRE in the hospital, alternative transmission occurs through horizontal spread of mobile genetic elements and plasmids. Modalities to prevent transfer of carbapenemases between Enterobacterales are necessary, possibly avoiding certain antibiotics, such as carbapenems which are direct drivers of carbapenem resistance. While previously all CRE carriers could be cohorted, separate isolation according to the type of carbapenemase is currently necessary. The diverse resistance mechanisms also affect our choice of empirical and definite antibiotic treatment, since the new β-lactamases are carbapenemase-specific.Our study has some limitations. First, it was conducted in one medical center that was endemic for CPE. Second, we included only CPE acquired in RHCC due to lack of microbiological data and molecular studies on isolates acquired elsewhere. However, 95% of CPE carriers hospitalized in RHCC during the study period had acquired CPE there. This study does not analyze the mechanisms leading to this spread of carbapenemases among bacteria and the reasons leading to the introduction of new carbapenemases to RHCC; only the CPE epidemiology at RHCC over time is described. Similar changes are most likely occurring in other Israeli hospitals. Data published in the 2019 national report for all CPE found in Israeli hospitals reported that only 47% were KPC, 33% were NDM, 12% OXA, 5% VIM, and 3% other; it is likely that these CPE circulate within and among hospitals.12In conclusion, we demonstrate the change in carbapenem resistance mechanisms in one Israeli hospital, the introduction of new carbapenemases, and the spread of carbapenemases across Enterobacterales. These findings, if representative of global epidemiology, are of great concern. Irrespective of location, infection control efforts are essential to prevent the further spread of carbapenemases and CPE.


Brevipalpus are members of the genera Cilevirus, family Kitaviridae, or Dichorhavirus, family Rhabdoviridae. They produce non-systemic infections that typically display necrotic and/or chlorotic lesions around the inoculation loci. The cilevirus citrus leprosis virus C (CiLV-C) causes citrus leprosis, rated as one of the most destructive diseases affecting this crop in the Americas. CiLV-C is vectored in a persistent manner by the flat mite Brevipalpus yothersi. Upon the ingestion of viral particles with the content of the infected plant cell, virions must pass through the midgut epithelium and the anterior podocephalic gland of the mites. Following the duct from this gland, virions reach the salivary canal before their inoculation into a new plant cell through the stylet canal. It is still unclear whether CiLV-C multiplies in mite cells and what mechanisms contribute to its movement through mite tissues. In this study, based on direct observation of histological sections from viruliferous mites using the transmission electron microscope, we posit the hypothesis of the paracellular movement of CiLV-C in mites which may involve the manipulation of septate junctions. We detail the presence of viral particles aligned in the intercellular spaces between cells and the gastrovascular system of Brevipalpus mites. Accordingly, we propose putative genes that could control either active or passive paracellular circulation of viral particles inside the mites.Plant viruses transmitted by mites of the genus  Brevipalpus-transmitted viruses (BTV) result in non-systemic infections that produce local necrotic and chlorotic lesions on leaves, stems, and fruits  that accumulate both in the nucleus and the cytoplasm of plant and mite cells . The genoviridae .Cilevirus, family Kitaviridae  in the infected plant cell cytoplasm . BTV-C gaviridae . Citrus aviridae .Cilevirus, the family Kitaviridae comprises other two plant-infecting virus genera: Higrevirus and Blunervirus -B. californicus  as indicator plant . Viral particles are consistently found between adjacent cells at the basal part of the caeca and in the anterior podocephalic gland of mites  RNA of the cileviruses CiLV-C and CiLV-C2 in viruliferous e vector . HoweverLuteoviridae, Geminiviridae, and Nanoviridae occurs without replication of the viral genome. Virus particles are transported across cells into membrane vesicles, preventing any contact between viruses and the insect cell cytoplasm in the epithelia of the gut and salivary gland  and the anterior podocephalic gland. Ultrastructural observations of viruliferous B. yothersi mites feeding on CiLV-C-infected plants reveal the presence of viral particles between cells , present in ectodermal-derived epithelium, i.e., epidermis, fore- and hindgut, salivary gland, etc., and the smooth septate junctions (sSJ), which occurs in the endodermal-derived epithelium, i.e., midgut, gastric caeca  act as a barrier that separates distinct compartments, limiting the paracellular passage of fluids . Unlike ic caeca . MutatioIn vertebrates, TJs could act as physical barriers from the innate immune defense system, especially on the respiratory tract. The coordination of TJ opening is mediated by chemical signals and membrane receptors, as happens during the paracellular passage of lymphocytes through the walls of capillary vessels . In humaBrevipalpus mites, composed of the highly branched caeca that fill all spaces left by other tissues on the hemolymph, may serve as a pathway to the circulation of viruses inside the mites.The gastrovascular system of Brevipalpus mites.Based on our transmission electron microscopy studies following the methodology proposed by Paramyxoviridae, spreads laterally into the epithelium via AJs , are found associated with the membrane by a glycosylphosphatidylinositol anchor and play essential roles in cell adhesion, signaling, and lipid metabolism . TFPs di insects . In flie insects .1, we were able to identify putative homologs of proteins that regulate SJ and sSJ in Drosophila encoded by the genomes of T. urticae (the most studied mite in the superfamily Tetranychoidea) and B. yothersi (pyd) gene, which is a recognized homolog of zonulin ssRNA viruses, a common virus-vector mode of transmission is circulative non-replicative . In manyvia RNA silencing mechanisms that protect plant germ cells against viral infection  required for the virus entry into different groups of tissues by interactions with SJ. Studies on the molecular interactions between mite vectors and plant viruses are scarce  and Aceria tosichella (Eriophyidae) showed the upregulation of two gene families that participate in the SJ formation  interaction has to be assumed. Accordingly, the transmissibility or not and efficiency of this process for a given pair of virus- e scarce . Global ormation . Recent i genome  and invei genome  may provBrevipalpus mites is confirmed, it will be the first example of a plant virus using this unconventional route of cell-to-cell movement in its arthropod vector. The unusual type of interaction with its vector, as also happens during the interaction of CiLV-C with their plant hosts, suggest that this virus, and probably other members of the family Kitaviridae, represent unique chimeric genetic systems that are likely reshaping features inherited from their ancestors to adapt to new ecological challenges.In case the paracellular route of CiLV-C movement in The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author/s.AT, EK, PR-G, and JF-A: conceptualization and methodology. PR-G, AT, EK, and TS: formal analysis. JF-A and EK: funding acquisition. AT, TS, PR-G, EK, and JF-A: investigation. PR-G, EK, and JF-A: supervision. AT: writing—original draft. AT, PR-G, EK, TS, and JF-A: writing—review and editing. All authors contributed to the article and approved the submitted version.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.


Boiga irregularis and American bullfrog Lithobates catesbeianus), with 10.3 and 6.0 billion US$ in costs, respectively. Costs for the remaining 19 reported species were relatively minor (< 0.6 billion US$), and they were entirely unavailable for over 94% of known invasive herpetofauna worldwide. Also, costs were positively correlated with research effort, suggesting research biases towards well-known taxa. So far, costs have been dominated by predictions and extrapolations (79%), and thus empirical observations for impact were relatively scarce. The activity sector most affected by amphibians was authorities-stakeholders through management (> 99%), while for reptiles, impacts were reported mostly through damages to mixed sectors (65%). Geographically, Oceania and Pacific Islands recorded 63% of total costs, followed by Europe (35%) and North America (2%). Cost reports have generally increased over time but peaked between 2011 and 2015 for amphibians and 2006 to 2010 for reptiles. A greater effort in studying the costs of invasive herpetofauna is necessary for a more complete understanding of invasion impacts of these species. We emphasise the need for greater control and prevention policies concerning the spread of current and future invasive herpetofauna.Biological invasions by amphibian and reptile species (i.e. herpetofauna) are numerous and widespread, having caused severe impacts on ecosystems, the economy and human health. However, there remains no synthesised assessment of the economic costs of these invasions. Therefore, using the most comprehensive database on the economic costs of invasive alien species worldwide (InvaCost), we analyse the costs caused by invasive alien herpetofauna according to taxonomic, geographic, sectoral and temporal dimensions, as well as the types of these costs. The cost of invasive herpetofauna totaled at 17.0 billion US$ between 1986 and 2020, divided split into 6.3 billion US$ for amphibians, 10.4 billion US$ for reptiles and 334 million US$ for mixed classes. However, these costs were associated predominantly with only two species (brown tree snake  These often lead to invasions, with ecological impacts that include extinctions of native species and disruptions of ecosystem functioning2 through direct and indirect effects4. They also have major impacts on economic activities and human society—with high monetary costs to multiple economic sectors5, disruptions of livelihoods and loss of human health7 and welfare8. Synthesised assessments of the impacts of major taxonomic groups are sparse10 inhibiting management actions, assessment of biases, and the filling of knowledge gaps11.The transportation rate of alien species into new regions is unprecedented and accelerating with globalising trade and transport networks12. These impacts are present in different forms, such as ecological , evolutionary , epidemiological  impacts. For example, the yellow-bellied slider Trachemys scripta elegans has degraded the functioning of ecosystems by altering abiotic characteristics and through the displacement of native species17; the cane toad Rhinella marina has led to morphological changes in the heads of native ranivorous snakes18, declines in native predators due to toxicity19, and caused substantial management expenditures20; the common coqui frog Eleutherodactylus coqui has led to declines in property values in areas infested due to its extremely loud mating song21; the brown tree snake Boiga irregularis has generated significant economic costs through island-wide power outages, resulting in a loss of US$ 4.5 million US$ per year22, as well as ecological impacts through the extirpation of most endemic birds via depredation23.In recent decades, invasive herpetofauna (i.e. amphibians and reptiles), have gained great interest on various platforms  due to the numerous impacts they cause and the interest they generate from a wide range of sectors of society24 and the number of alien herpetofauna species has continued to increase in recent decades25, primarily due to the globalisation of human activity26 and other cultural or social factors28. Thus far, the primary introduction pathways for alien herpetofauna arise from unintentional and intentional transportation by humans, for example, via stowaways in cargo shipments or the nursery trade, and via the pet trade, religious releases, aquaculture or biocontrol programmes25. Furthermore, climate change is expected to increase the invasibility of regions such as Central and South America and central Africa to alien herpetofauna29.Yet, there are now more than 1000 known occurrences of alien reptile and amphibian species worldwide30. The filling of these knowledge gaps is crucial to incentivise a better development of invasion pathway regulatory policy and investment in control or biosecurity measures . In this context, the monetisation of the effects of invaders is a valuable option to raise societal awareness as well as motivate and justify policy actions by decision-makers5. The use of monetary values also allows highlighting of knowledge gaps among taxa using a common metric. The recent publication of the InvaCost database, where literature on the costs of alien species is compiled, standardised and described across a large number of indicators in a public form31, has increased interest in the synthesised study of invasive alien species monetary costs35.Knowledge gaps regarding invasive herpetofauna are pervasive due to the lack of adequate data and the presence of geographic and taxonomic biases38; (ii) costs will be biased towards particular regions, as shown previously by other studies36; (iii) most economic costs will come from damage to primary sectors such as agriculture, due to these being commonly impacted by invasions39; and (iv) temporal trends will be characterised by substantially increasing costs over time, due to the increase in invasion rates worldwide1 and a growing number of pathways introducing herpetofauna.Here, we quantify and synthesise, for the first time, the economic costs of alien herpetofauna worldwide, based on the current state-of-the-art in the published literature. We expect our results to reveal high costs, but also patterns and possible spatial biases that will inform the direction of future research: (i) we expect a minority of species will occupy the majority of the research effort and costs40, 263 species did not have any economic impacts recorded in the InvaCost database . Similarly, for the richest families of reptiles, Gekkonidae or Scincidae, no costs were reported.The total reported costs of the invasive herpetofauna exceeded 16.98 billion US$ between 1986 and 2020. We found that out of 280 invasive herpetofauna species recorded in the herpetofauna databaseWhile amphibians contributed a total of 6.27 billion US$, nearly all costs originated from high reliability entries, amounting to more than 6.24 billion US$ (99.58%), compared to 26.08 million US$ (0.42%) in low reliability entries. In terms of implementation, only 70.56 million US$ came from observed costs (1.28% of costs), and 6.19 billion US$ from potential costs (98.72% of costs). For reptiles, 8.12 billion US$ (78.10%) of cost entries came from high reliability sources and 2.25 billion US$ (21.89%) from low reliability sources  with a total cost of 6.04 billion US$ . The rest of the amphibian species accumulated less than 0.23 billion US$ (3.66%. n = 293).The overall cost of invasive herpetofauna was due to a handful of species, which contributed the bulk of total costs Fig. . For ampB. irregularis registering a total of 10.34 billion US$ , while the rest of reptilian species accumulated less than 0.03 billion US$ (0.70%). The species with the highest number of entries were T. scripta (n = 109), B. irregularis (n = 78) and the Green anole Anolis carolinensis (n = 60); the remaining species accounted for 91 entries in total.For the reptiles, the total cost of 10.37 billion US$ was divided among 15 species, with the R. marina , followed by E. coqui with 18.30 million US$ . The remaining amphibian species (n = 5) recorded less than 8.3 million US$ .For the amphibians, most of the observed costs belonged to B. irregularis dominated with 3.46 billion US$ , while the remaining species (n = 14) cumulated less than 28 million US$ .For the reptiles, observed species-level costs totalled 3.49 billion US$, and W = 4051.5, p-value ≤ 0.001) numbers of publications for the species with recorded costs in the InvaCost database compared to those that did not have any record of economic costs. Further, for those species with costs, we found a significant positive correlation between the total cost of the recorded species and the number of respective publications   followed by Public and social welfare  and Agriculture , while the remaining impacted sectors recorded less than 728.88 thousand US$ together . Focusing on observed costs only, we found that the Authorities-stakeholders sector was still the most affected  by invasive amphibians, followed by Public and social welfare  and Agriculture . The remaining impacted sectors reported less than 397.12 thousand US$, < 0.56%).For the amphibians, we found that the Diverse  followed by Public and social welfare  and Authorities-stakeholders . The remaining impacted sectors recorded less than 415.38 thousand US$ together . This pattern was not repeated for observed costs considering invasive reptiles, with Public and social welfare , followed by Authorities-stakeholders  and then the Diverse  sectors impacted most. The remaining impacted sectors reported less than 412.20 thousand US$ (< 0.01%).For the reptiles, we found that the most impacted sector was Management , followed by Damage  and Mixed  types. Focusing on Observed costs only, we found again that the highest costs were for Management  followed by Damage  and Mixed  types , followed by Management  and Mixed  types. This pattern was repeated for Observed costs, with first Damage , followed by Management  and Mixed  types  followed by Oceania and Pacific Islands  and diverse regions . Of these costs, the majority was Potential , being mostly in Europe (6.04 billion US$) and relatively little for Oceania and Pacific Islands (0.15 billion US$), while Observed costs were less than 72 million US$. The regions with the highest Observed costs were Oceania and Pacific Islands , followed by Europe  and Diverse . The remaining geographic areas recorded only a total of < 2 million US$ , split among Asia , North America  and South America . Lastly, no economic costs of amphibians were recorded in Africa , followed by Asia  and North America . Here, 66.24% came from Potential costs, with 6.86 billion US$ of the total, and 3.49 billion US$ (33.76%) came from Observed costs. The regions with the most costs observed were, again, Oceania and Pacific Islands  followed by Asia  and North America . The remaining geographic areas recorded only a total of < 2 million US$ , split among Africa , Europe  and South America  . There could thus be a positive feedback loop between research effort and cost reporting—with most research focused so far on a few high-profile species. Indeed, we found significantly greater numbers of papers concerning species with reported costs than those without reported costs. While InvaCost synthesises available cost information and allows it to be distinguished using various descriptors, we note the potential variability in underlying data that reflects methodological differences across the underlying literature. In addition, we acknowledge that the differences in research effort based on the number of papers for certain species, paired with a conceivable time lag in invasion and monetised impact reporting, may alter the total economic costs found in this study. These factors could make the total cost a conservative estimate, especially considering the gaps for those known invasive species that have few or no costs reported. Government policies may further influence research effort, resulting in certain widely distributed species receiving more attention from the scientific community. This corroborates earlier identifications of bias in studies of invasive alien herpetofauna36. While we acknowledge that not all invasive alien herpetofauna will have tangible monetary impacts, greater research effort is required to distinguish the true absence of economic cost from gaps in cost detection, especially for those taxa with the worst known ecological impacts41.The present study found a total reported cost of global herpetofauna invasions of 16.98 billion US$ between 1986 and 2020. However, reported costs were principally due to just a few species, namely L. catesbeianus potential costs in Europe, which represent more than 95% of the total amphibian’s cost. Additionally, whereas the potential costs were higher, the number of entries was lower than the observed costs, indicating that potential cost entries tend to report much greater costs, which is to be expected since they often extrapolate costs from smaller to larger scales . It is also possible that potential invasion costs were projected from one spatiotemporal unit to a much larger scale, but that the species then failed to spread, went bust or was eradicated, leading to an initial overestimation. For potential management costs, interventions can also become more efficient and less cost intensive as new protocols are developed, reducing the level of future costs than those predicted. However, in each of these scenarios, the reverse could occur, leading to an alternative underestimation of potential costs.44. This could indicate a massive underestimation of this cost type, although this pattern is not found in reptiles. Especially surprising is the low reported cost from Burmese python Python bivittatus considering the large number of impacts generated in the USA by the high predation capacity of this species, which has caused the decline of native species45, as well as great effort in the country to control its populations46. Yet, many of these ecological impacts are not monetised or available as they are not in the InvaCost database, and all recorded economic costs generated by P. bivittatus thus far are from a single eradication program targeting this species in Florida47. In addition, there is a possible cost bias as our study range is limited to 1986–2020, while some of the most notorious species were introduced long before that [e.g. R. marina were introduced in 1936 in Australia48].We found that most of the reported costs for amphibians are from management, while most other studies consistently show damage as the main source of costs, with values of 10–100 times higher than management37, Singapore and Southeast Asia35, fishes49, and ants50, among others. Concerning sectors of economic activity for amphibians, most costs impacted authorities-stakeholders. The vast majority of these costs were recorded from post-invasion management actions (e.g. control or eradication), and so the improved investment in and development of prevention measures could reduce future economic costs from invasions by reducing needs for long-term management and the scale of incurred damage. A more diverse pattern was found for reptiles among sectors, emphasising a large amount of damage from these species across various sectors, which could again be mitigated by more timely management.The lack of reported costs found for the vast majority of reported alien species corroborates other studies at various spatial and taxonomic scales, including for the United Kingdom5, freshwater crayfish  and concomitant “Massive” effects on recipient communities, such as the tiger salamander (Ambystoma tigrinum) and Italian pool frog (Pelophylax bergeri)41. However, the impacts of these species appear to be characterised by hybridization53, and therefore these impacts are difficult to quantify economically due to their ecological nature. Regarding reptiles, we observed that three of the five most notorious aliens  had reported economic costs, but we did not find economic costs for the remaining two: the curious skink  and common house gecko (Hemidactylus frenatus) in the InvaCost database. To date, there is no assessment of the effects of reptiles in recipient ecosystems based on EICAT, but we hope that addressing for the first time the economic costs for these taxa will motivate their ecological assessment in the future.Costs of herpetofauna invasions were of a similar or greater magnitude to other taxa, despite knowledge gaps, such as rodents  studies pioneered early works in economic costs estimations for invasive alien species59, promoting further research in the last few decades that resulted in burgeoning reported economic impacts from invasions there60.While taxonomic gaps were pervasive across socio-economic sectors, these were coupled with a similar lack of data for geographic regions . Island ecosystems are regarded as more vulnerable to invasion by alien species and their impacts62. They can incur economic costs through a different suite of sectors and species than mainland areas64. Islands are substantially more biodiverse than mainlands65. While ecological impacts can be challenging to monetise, alien species there generate high rates of extinction of native species in groups such as vertebrates and plants66, which can have high intrinsic or cultural value67. Nonetheless, our study may help develop more effective policies to reduce these future economic costs.In addition, costs from 5, as well as the possibility that there is a lag phase in the impacts generated by these species, which can take decades68. Indeed, the range in which the greatest costs of amphibians was detected coincides with the years with the highest number of entries, with a total of 105 entries between 2006 to 2010, while recent years' entries record a much lower amount, with only 46 entries between 2016 to 2020. This pattern was not seen in reptiles, where in the period with the highest number of costs from 2011 to 2015, only 40 entries were reported. On the contrary, 149 entries were reported from 2016 to 2020. We, therefore, suggest that the presented costs over time for herpetofauna invasions have been heavily influenced by the evolution of the costs of the three costliest species  and the timing of underlying studies.Analysis of the cost trend between 1986 and 2020 revealed similar patterns for both classes. For amphibians, the cost increased until 2006, when it declined steeply. For the reptiles, the cost increased until 2006–2010, when it declined steeply until the final years of this study. Primarily, these trends likely relate to the delay in reporting impacts32. Furthermore, because invasion rates are expected to increase in the future69, we expect this increase to be followed by a rise in economic costs. Yet, it could be reduced by investing in management, especially in the prevention of the arrival of alien species and early detection measures71. While research interest and economic impacts of biological invasions are globally increasing, their costs have been shown to grow faster than the relative number of reports of costs72. For herpetofauna, because these trends were driven by specific species or taxonomic groups, it can be expected that these will be prioritised, if neglecting wider data gaps. However, future research priorities should also be based on the recognition of data gaps, as that would result in improved research efforts to address the lack of data (e.g. on damages) and focus not only on those sites with the highest records of economic costs by invasive herpetofauna  but also on the monetisation of the costs generated by the ecological degradation by other herpetofauna species (e.g. A. tigrinum and P. bergeri).This study presents the first global synthesis of the costs of invasive herpetofauna based on the current state-of-the-art in the InvaCost database. While costs are high, they are likely severely underestimated due to knowledge gaps, with costs focused on only a few taxa, regions and limited to recent decades73. For amphibians, it is expected that future trade will not differ from the current patterning, where some of the species that are expected to still be traded widely are the yellow cururu toad Rhinella icterica, Müller's platanna Xenopus muelleri or Woodhouse's toad Anaxyrus woodhousii74. In contrast, the reptile pet trade is expected to change its most traded species in the future, expectedly resulting in new economic impacts to potentially different sectors. While I. iguana, P. bivittatus and T. scripta have declined the most in popularity, other species such as blue-tongued lizards Tiliqua spp., crested geckos Correlophus ciliatus, and ball pythons Python regius are expected to increase with a growing economic cost36. For this reason, the continuous updating of lists of invasive  species found in the pet trade could result in large economic savings for governments and stakeholders75.Further, the main pathway of invasion of alien herpetofauna is the pet trade76. Given the high context-dependence in invasion science, whereby the impacts generated may vary depending on the area invaded, habitat type and socio-economic sector, we also recommend conducting assessments at more granular spatial and taxonomic scales77. We emphasise the importance of the development of policies by the responsible authorities and governments to prevent the arrival of alien species since effective prevention will decrease future expenses70.Because of a high reliance so far on predictions and extrapolations, we urge further work to quantify costs actually incurred by invasive herpetofauna, which could in turn inform more robust predictive efforts and cost–benefit analyses of these animals for managers. In addition, pre-invasion management techniques could save a large economic burden for governments and stakeholders from invasive species damages and long-term managementhttps://doi.org/10.6084/m9.figshare.12668570) at the time of writing, per the “getInvaCostVersion” function of the invacost R package78. This version of the database contains a total of 13,123 cost entries, extracted primarily from four different sources: Web of Science (https://webofknowledge.com/), Google Scholar (https://scholar.google.com/), Google search engine (https://www.google.com/) and biological invasion experts, that were contacted as part of this collection and identification process, to gather additional data. The collection and collation of data from these sources were based upon systematic search strings used to identify the costs of biological invasions5. The InvaCost database contains entries reported in 15 languages in a sufficiently detailed manner to analyse the cost of invasive alien species55.To estimate the costs of invasive alien reptiles and amphibians worldwide, we used the most recent version  of the InvaCost database  or by adding new data. The reliability of the data is therefore reliant on the underlying reporting material. InvaCost, however, does not include benefits quantitatively. While these data were also screened according to their method reliability in an objective manner, we could not capture the full spectrum of underlying approaches to cost estimation in a holistic way. Each entry was converted into 2017 US dollars to standardise the cost values, making costs directly comparable. In addition, each entry has 65 descriptor variables, including, for example, information on species’ taxonomy, the type of ecosystem invaded and the type of economic impact caused. The data in InvaCost were carefully screened to minimise spatial overlaps, e.g. at local and national scales over time.78. This function summarises the cumulative costs and average annual costs of invasive alien species and splits the total costs evenly into annual cost estimates reflecting the respective period over which the cost was incurred, without inflating the cost estimates78.We performed a series of steps before proceeding with the analysis of the data Fig. . First, 78. This function expands the estimated costs over the years they occurred or were estimated to have occurred. This means that one cost entry that reports a total cost of 100 million over 10 years, for example, will be transformed into ten cost entries with an annual cost of 10 million dollars each. The duration of the study was determined from the columns “Probable_starting_year_adjusted” for the start of the impact and “Probable_ending_year_adjusted” for the end of the impact. All analyses were carried out on this expanded database to reflect the duration of the costs according to each entry. Our final, expanded dataset had 710 entries from 1986 to 2020, thus covering 35 years.This final dataset contained 353 entries which were all homogenised and converted on an annual basis, whereby the total cost for each estimate and each species was divided by the duration time of each cost entry. This process of ‘expanding’ the dataset was performed by the function “expandYearlyCosts'' of the invacost R packageI.Method reliability Parameter used to indicate the degree of reliability of the estimation of costs. A cost is classified as High reliability if it is reported from official reports or peer-reviewed articles, and if not, whether the estimation method is reproducible; if these criteria are not met, the estimate will be categorised as being of Low reliability.II.Implementation Indicates whether the cost estimate has been produced directly in the invaded study area , or whether it is instead a predicted or extrapolated cost derived from the distribution area of alien species elsewhere, i.e., a cost not incurred but is predicted to occur over time and/or space expected . Examples of potential costs are the extrapolation of economic impacts due to the loss of tourist revenue from the presence of R. marina in Australia19, or the cost predicted for the removal of B. irregularis in Guam57. Further, to avoid the overestimation of extrapolated costs, the InvaCost database followed a strict selection and classification of costs, notably excluding previous potential costs that were later incurred and listed as observed costs.III.pecies This descriptor shows the name of the species responsible for the incurred costscost totals per species. In addition, entries that contained more than one species that could not be distinguished (column: “Species”) were added to the “Species” category “Diverse” (n = 44).SIV.Impacted sector market and/or activity sector affected by the costs of invasive herpetofauna, . All entries with more than one category were pooled into “Diverse”.V.Type of costs Type of cost generated by the invasion: Damage , Management  and Mixed (indistinguishable damage and management components).VI.Geographic region Refers to the continental region where the cost of alien species invasion was recorded. We also made several changes to the descriptor variable “Geographic_region” to homogenise the sub-group. These changes were the joining of “Pacific Islands” and “Pacific Islands/Oceania” into “Oceania and Pacific Islands”, and “Central America” and “South America” into “Central/South America” and “Oceania/South America'' into ''Diverse”.The cumulative costs of invasive alien species were aggregated according to the following descriptor variables:40. To our knowledge, this is the most complete database published on invasive herpetofauna records, whereby each entry represents the occurrence of an alien established species in a country, federal state or biogeographically-separate island or archipelago.We also examined the proportion of invasive alien species for which costs were reported using a database reporting alien herpetofauna worldwide, separately by order and familyFurthermore, we examined whether species with costs were more studied than those without costs, and correlated the magnitude of costs with the number of studies (Supplementary 75 to estimate the total costs of alien species per year and the mean for each 5-year period, considering amphibians and reptiles separately.To assess the trend of alien reptilian and amphibian species costs, we considered 5-year intervals ranging from 1986 to 2020 due to data availability. We used the “summarizeCosts” function of the invacost packageSupplementary Information.


Rupture of HCC (rHCC) is a life-threatening complication of hepatocellular carcinoma (HCC), and rHCC may lead to a high rate of peritoneal dissemination and affect survival negatively. Treatment for rHCC mainly includes emergency surgery, interventional therapies, and palliative treatment. However, the management of rHCC should be carefully evaluated. For patients with severe bleeding, who are not tolerant to open surgery, quick hemostatic methods such as rupture tissue ablation and TAE/TACE can be performed. We described clinical presentation, prognosis, complication, interventional management, and current evidence of rHCC from the perspective of interventional radiologists. Overall, our review summarized that interventional therapies are necessary for most patients with rHCC to achieve hemostasis, even in some patients with Child–Pugh C. Moreover, TAE/TACE followed by staged hepatectomy is a beneficial treatment for rHCC according to current clinical evidence. TAE/TACE is the first choice for most patients with rHCC, and appropriate interventional treatment may provide staged surgery opportunities for those who are not tolerant to emergency surgery to reach an ideal prognosis. Hepatocellular carcinoma (HCC) is the sixth most diagnosed cancer and the fourth leading cause of cancer-related death worldwide . Hepatitvia the hepatic artery and other extrahepatic arteries. The application of interventional therapy is still broader than open surgery, as interventional therapy can be applied in some cases with Child–Pugh C , transarterial embolization (TAE), transarterial radioembolization (TARE), microwave ablation (MWA), and radiofrequency ablation (RFA), remains a minimally invasive treatment for HCC, even suitable for some patients who have extrahepatic metastases or recurrence . TACE/TAd–Pugh C . Furtherd–Pugh C . This reThe mechanism and prognosis of rHCC have not been completely investigated. Studies have also reported that tumor location, tumor size, hypertension, hepatic parenchyma, and liver cirrhosis were associated with rHCC.The overlying liver parenchyma plays an important role in rHCC. As tumor progresses, when the tumor invades the hepatic arteries or portal vein, the pressure inside the liver may increase and lead to rHCC –26. MoreThe relationship between tumor size and HCC rupture is controversial. Although the size of tumor is associated with the pressure inside the tumor and vessel, Li et al.  reportedPrevious studies have identified that vascular injury may be associated with rHCC. A widely accepted mechanism of rHCC is that hepatic arteries and veins are invaded or occluded by tumor cells, leading to high pressure within the tumor mass . As HCC In addition, rHCC is associated with factors involving liver function, portal vein thrombus, location, and so on. Monroe et al.  reportedAlthough rHCC can present with various symptoms, acute abdominal pain can be observed in most cases (66%–100%) of rHCC . Shock iUltrasonography (US) and computed tomography (CT) are recommended for detecting rHCC, the location of ruptured tumor, and changes in hematoma density. The most direct evidence of rHCC from CT scans is hemoperitoneum , but hemPatients with rHCC usually presented with acute abdominal pain, and with the utilization of medical imaging, laboratory analyses, and history of patients, a diagnosis of rHCC can usually be made. However, in some cases of rHCC, the diagnosis of rHCC is still a challenge, as some cases are noncirrhotic and there is an absence of risk factors, indicating that other potential factors may also play an important role in carcinogenesis and rHCC , 52.Abdominal paracentesis can be a choice when patients are suspected with rHCC . If the Although the prognosis of rHCC is not ideal, survival benefits for interventional radiology and hepatectomy have been reported in patients with rHCC –57. ActuThe aim of interventional therapy is not only to achieve successful hemostasis, but also to prolong the survival of patients with rHCC. Lee et al.  and HsueChen et al.  and KiriInterventional radiologists should also note that extrahepatic rupture of metastatic HCC has been reported as a rare complication of TAE . Suoh etKodama et al.  reportedHCC can recruit extrahepatic collateral vessels from a wide range of vessels such as gastroduodenal artery, superior mesenteric artery, and suprarenal arteries –90; thusIt is worth noting that TAE/TACE-led injuries of portal vein or hepatic vein may lead to recurrent bleeding and an unsatisfactory hemostatic effect , 96, as Various embolization materials can influence the patient prognosis, and the effect of different embolic agents is not completely known. Koçyiğit et al.  reportedDiscussing the level of embolization, Lee et al.  reportedAnother issue that should be noted is that aforementioned observational studies, non-randomized studies, case reports, and cluster-randomized trials have a known limitation as these studies will force us to focus more on benefits than harms .Furthermore, the overall survival rate of patients with rHCC who underwent TAE/TACE is lower than those who underwent radical resection; hence, the long-term effect of TAE/TACE is still questioned .Ablation is another interventional therapy for rHCC, but the efficacy and safety are only reported in a few studies Table 2 Gao et al.  reportedAmong these studies, image-guided percutaneous ablation is commonly used, as it can be minimally invasive, and most cases can achieve hemostasis. Because only limited cases were reported, the benefit of ablation is not completely understood. Three studies , 110 repFurthermore, another issue of ablation that should be noted is the “heat sink” effect, which is defined as a phenomenon whereby flowing blood adjacent to or within tissues being targeted for ablation results in relative tissue cooling due to heat transfer by convection , leadingThe application of interventional combination therapies is used in some settings. However, interventional combination therapies for rHCC are only reported in several studies Table 3 Recently, the interventional combination therapies for HCC include TACE plus brachytherapy and transarterial ethanol ablation combined with TACE –125. MorIn conclusion, the clinical management of rHCC remains a challenge. Our review summarized that interventional therapies are necessary for most patients with rHCC to achieve hemostasis, even in some patients with Child–Pugh C. Furthermore, TAE/TACE followed by staged hepatectomy is the most beneficial treatment for rHCC according to current clinical evidence. However, the efficacy and safety of ablation are still questioned because only a few well-documented studies were reported. Interventional radiologists should also keep in mind that TAE/TACE is the first choice for most patients with rHCC, and appropriate interventional treatment may provide staged surgery opportunities for those who are not tolerant to emergency surgery to reach an ideal prognosis.All authors listed have made a substantial, direct, and intellectual contribution to the work and approved it for publication.This study was supported by Project of Science and Technology Department of Qinghai Province (2020-ZJ-Y01).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. 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Consequently, sCPD9 can ease and accelerate SARS-CoV-2 research under BSL-2 conditions, particularly in experiments requiring replicating virus, such as diagnostics and development of antiviral drugs.Research with infectious SARS-CoV-2 is complicated because it must be conducted under biosafety level 3 (BSL-3) conditions. Recently, we constructed a live attenuated SARS-CoV-2 virus by rational design through partial recoding of the SARS-CoV-2 genome and showed that the attenuated virus, designated sCPD9, was highly attenuated in preclinical animal models. The recoded sequence was designed by codon pair deoptimization and is located at the distal end of gene ORF1ab. Codon pair deoptimization involves recoding of the viral sequence with underrepresented codon pairs but without altering the amino acid sequence of the encoded proteins. Thus, parental and attenuated viruses produce exactly the same proteins. In Germany, the live attenuated SARS-CoV-2 mutant sCPD9 was recently classified as a BSL-2 pathogen based on its genetic stability and strong attenuation in preclinical animal models. Despite its high attenuation  The goal of recoding by CPD is to increase the occurrence of codon pairs that are naturally underrepresented in host protein coding sequences. The recoding algorithm must therefore be able to assess which codon swaps reduce the overall codon pair bias of the recoded sequence and which do not. Codon pair bias of each codon pair can be calculated as codon pair score (CPS) To generate SARS-CoV-2 mutants that would be attenuated in humans, we recoded the SARS-CoV-2 genome using the CPS calculated for each of the 3,721 codon-pair combinations  based on all representative human protein-coding genes Saccharomyces cerevisiaeIn vitro growth kinetics demonstrated stable replication of sCPD9 to titers about 10-fold lower compared to parental wild type The sCPD9 virus was constructed using a reverse genetics system of SARS-CoV-2 2One of the most important criteria for live attenuated virus vaccines is that they are genetically stable and thus unable to revert to their pathogenic phenotype and cause disease. This risk particularly exists when only a few nucleotide changes are responsible for the attenuation, as was the case with the oral poliovirus vaccine To determine the genetic stability of sCPD9 virus in cell culture, we passaged sCPD9 ten times in African green monkey Vero, Vero E6 or human intestinal epithelial Caco-2 cells. Sequencing showed that limited serial passage did not significantly alter the structure of the sCPD9 virus genome, as no single nucleotide polymorphisms (SNP), insertions or deletions were detected within the recoded sequence after 10 passages on Vero, Vero E6 or Caco-2 cells , Table 1N-terminal domain of the spike protein (Most sCPD9 virus variants that emerged from the propagation on Vero E6 cells contained a 10 amino acids deletion (NSPRRARSVA) in the spike protein that encompasses the entire furin cleavage site. Similarly, sCPD9 virus populations that originated from the passage on Vero cells contained a 15 nt deletion encoding 5 amino acids ‘QTQTN’ located directly upstream of the furin cleavage site . These r protein . BecauseWe applied to the German Central Committee on Biological Safety  for reclassification of the attenuated SARS-CoV-2 mutant sCPD9 from BSL-3 to BSL-2 based on its biological properties, in particular its near-complete attenuation in preclinical animal models and its genetic stability under limited passage in cultured cells. Our application was positively evaluated, and the SARS-CoV-2 sCPD9 with and without the furin cleavage site is now classified as a BSL-2 organism in Germany.3We contend that downgrading of the attenuated virus sCPD9 sets an important precedent. We anticipate that reclassification of attenuated SARS-CoV-2 viruses to BSL-2, as in the case of sCPD9, will democratize, simplify, and expedite SARS-CoV-2 research that requires the use of replicating virus, especially but not only at institutions that only operate BSL-2 laboratories. In addition to the recent ZKBS recommendation for BSL-2 classification of sCPD9 in Germany, sCPD9 was recently downgraded to BSL-2 level in the Netherlands and a similar application is currently under evaluation by the Swiss Expert Committee for Biosafety (SECB).The sCPD9 virus, and its future derivatives, could prove to be particularly valuable tools in virus diagnostics, for example for determining virus neutralization titers. The sCPD9 virus could also be used for (high-throughput) screening of neutralizing antibodies or antiviral compounds. In addition, we envision that the sCPD9 virus could be also very useful in basic research to study biological questions that do not need to be addressed with a pathogenic virus. Moreover, the ability to produce virus under BSL-2 conditions greatly facilitates vaccine production, both in the case of a live attenuated vaccine and vaccines based on inactivated virus particles.The major limitation of sCPD9 virus for many potential applications, such as viral diagnostics or screening of neutralizing antibodies, is that its genome is that of the ancestral viral variant Wuhan-01 Alternatively, for applications that require an intact antigenic repertoire, SARS-CoV-2 variants can become attenuated through insertion of the sCPD9 mutations into their genome. We are currently generating SARS-CoV-2 variants Delta and Omicron BA.2 that would carry the same attenuating mutation, and we expect to expand the number of SARS-CoV-2 mutants that can be studied under BSL-2 conditions soon.44.12.African green monkey Vero (WHO RCB 10–87), Vero E6 cells (ATCC CRL-1586) or human intestinal epithelial Caco-2 (ATCC HTB-37) cells were grown in minimal essential medium (MEM) containing 10% fetal bovine serum (PAN Biotech), 100 IU/ml penicillin G and 100 µg/ml streptomycin at 37 °C and 5% CO4.2To recover infectious sCPD9 virus, Vero E6 cells were grown to 70% confluence in a T25 cell culture flask and transfected with 4 µg of BAC DNA and 1 µg of helper plasmid pVITRO2-EGFP-N using the Xfect single shots transfection reagent (Takara Bio). The transfected cells were incubated for 72 h, then the cell culture medium containing the recovered virus was transferred to fresh Vero E6 cells to create passage 1 stocks of recovered virus.4.3Upon development of visible cytopathic effects, virus stocks were frozen and titrated by plaque assay. To initiate passaging, Vero, Vero E6, or Caco-2 cells were infected with an MOI of 0.05. After 48 h, infected cells were lysed by a freeze–thaw cycle and 1% of the supernatant and cell lysate was used as inoculum for the next passage, this procedure was repeated 10 times in total.4.4The genomic sequence of sCPD9 virus populations after serial passage was determined by next-generation sequencing on the Illumina MiSeq platform.To sequence viral genomes, total RNA was isolated from infected cells using Trizol reagent (Thermo Fisher Scientific) according to the manufacturer’s instructions. Sequencing libraries were constructed using the NEBNext Ultra II RNA kit (New England Biolabs) with NEBNext Multiplex Oligos for Illumina (New England Biolabs).The obtained sequencing reads were processed with Trimmonatic v.0.36 and mapped against the genomic sequence of sCPD9 virus using the Burrows-Wheeler aligner, version 0.7.15. Single nucleotide polymorphisms (SNP) as well as insertions or deletions (indel) were evaluated using FreeBayes version 1.1.0–333. Data were merged by position and mutation using R v.3.2.3. The SNPs were additionally assessed using Geneious R11 software (Biomatters).Dusan Kunec: Conceptualization, Methodology, Software, Investigation, Writing – review & editing, Visualization. Nikolaus Osterrieder: Conceptualization, Writing – original draft, Writing – review & editing, Funding acquisition. Jakob Trimpert: Conceptualization, Methodology, Investigation, Writing – original draft, Writing – review & editing, Supervision, Project administration, Funding acquisition.Related to this work, Freie Universität Berlin has filed a patent application for the use of sCPD9 as vaccine. In this application, JT, NO and DK are named as inventors of sCPD9. Freie Universität Berlin is collaborating with RocketVax Inc. for further development of sCPD9 as vaccine and receives funding for research.