LRRTM4. Duplication of the terminal exon of LRRTM4 was found in two females with TS from the same family (mother and daughter) in association with autistic traits and ASD.Tourette syndrome (TS) is a neurodevelopmental disorder characterised by motor and vocal tics and strong association with autistic deficits, obsessive–compulsive disorder (OCD) and attention-deficit/hyperactivity disorder (ADHD). The genetic overlap between TS and autism spectrum disorder (ASD) includes those genes that encode the neurexin trans-synaptic connexus (NTSC) inclusive of the presynaptic neurexins (NRXNs) and postsynaptic neuroligins (NLGNs), cerebellin precursors (CBLNs in complex with the glutamate ionotropic receptor deltas (GRIDs)) and the leucine-rich repeat transmembrane proteins (LRRTMs). In this study, we report the first evidence of a TS and ASD association with yet another NTSC gene family member, namely  Tourette syndrome (TS) is a neurodevelopmental disorder characterised by motor and vocal tics, a prepubertal age of onset, a waxing and waning course and improvement of symptoms in adulthood ,2. AutisPhenomenological, epidemiological and pathogenetic evidence all indicate a strong overlap between TS and autism spectrum disorder (ASD) ,5. TS anNRXN1-3) and their postsynaptic ligands the neuroligins (NLGN1-3), cerebellin precursors (CBLNs with their GRID1 complexes) and leucine-rich repeat transmembrane proteins (LRRTMs), all of which have been implicated in TS and ASD through multiple associations , which regulates neuronal cell adhesion and neurotransmission, has been proposed to link TS and ASD . The NTSciations  7]. The. TheNRXN sharing , as haveulations ,11,12,13Copy number variation (CNV) analyses were performed on blood samples from the proband and her family using two different microarray CNV detection platforms: the Agilent SurePrint G3 Human Whole Genome Microarray 2 × 400 K  and the Illumina CYTOSNP12 vs2.1.HPLC was used for amino acid profiling and gas chromatography and mass spectrophotometry for organic acid profiling of a blood sample from the proband. The study has been approved by the Human Research Ethics Committee of the Sydney South West Area Health Service (Project id. 08/CRGH/233).The proband was a 9-year-old female referred by her mother following a history of behavioural, emotional and sensory concerns. A comprehensive assessment revealed other concerns, including restricted interests, repetitive behaviours, social difficulties and anxiety. Further assessment confirmed a diagnosis of autism spectrum disorder (ASD). It also became evident that in addition to the restricted repetitive behaviours that are characteristic of ASD, the patient also exhibited finger and body movements as well as animal sounds such as barking, meowing, etc. The frequency and intensity of these behaviours were noted to increase following the commencement of stimulant medication for her ADHD symptoms. This prompted further assessment by a child psychiatrist when it became evident that the patient had tics as early as her preschool years, including a nose twitch at 2 years of age. Since then, over the years, the proband has had a variety of motor tics, including blinking; eye staring; nasal twitching; flaring of nostrils; kissing self; gripping lips and other lip movements; facial grimacing; head nodding forward, backward and sideways; moving hair out of eye tic; fingers drumming, tapping, flexing and touching; abdominal contractions; body wiggling; leg flexing, extending and kicking; adjusting, picking and chewing clothes; picking skin and nails; poking self; chin on chest posture; stroking, especially her legs; complex movements such as stomping and skipping; abnormal gait in the form of raising the leg high with each step, etc. She has also exhibited a number of vocal tics, including sniffing; throat clearing; coughing; “ugh” and other baby sounds; t-t-t sounds with coordinated hand movements; squeaking, shrieking, screaming and other high-pitched sounds; barking, growling and other animal sounds such as chickens, cats, gorillas, etc. Other tic-related behaviours include echolalia (repeating words such as “pineapple” or “monkey” or phrases such as “Rum... Rum... I am in Mummy’s car”), echopraxia  and non-obscene socially inappropriate (NOSI) behaviours, such as saying “you are ugly” and then laughing inappropriately. She also reported having a number of obsessive–compulsive behaviours that fall into the tic spectrum of behaviours. For example, she had to do things in a particular way to get it “just right”, with pencils facing one way, colours not to be mixed and shoes and other things needing to be in a particular place. She also had an obsession with LEGO® and miniature items. She also exhibited self-injurious behaviours such as picking her nail or skin until they bled. She also had emotion regulation issues and rage attacks, with aggression mainly directed towards her brother, which have been a significant issue in the past but are reported to be better now. She has always been a poor sleeper. Anxiety is reported to make her tics worse. She reported to be able to suppress her tics voluntarily for a period of time, such as generally staying “under the radar” at school and coming home to “release” the tics. On certain days when she was not able to suppress the tics or cope effectively, her mum would get a call asking for her to be picked up from school. Her early developmental history included myoclonic and febrile seizures at 18 months. She had a stutter, her speech was delayed and there was also some slight delay in other developmental milestones. It is worth mentioning here that there is a family history of TS, autism, stuttering and ADHD . It appeRegarding her family history, her mum reported having had nasal flare movements and other tics, and the maternal uncle’s son has been diagnosed with Tourette syndrome. Another maternal uncle is reported to have had ADHD, and he has one son with ADHD and autism and a daughter with ADHD . The matIn summary, the female proband was diagnosed with Tourette syndrome with multiple motor tics and one or more vocal tics that have been present for more than a year and characterised by a waxing and waning course and one type of tic being replaced by another. The proband also has tic-related obsessive–compulsive behaviours and self-injurious behaviours, but she did not meet the diagnostic criteria for ADHD. The mixed features of autism spectrum disorder and the tic spectrum of behaviours is consistent with a family history of Tourette syndrome in association with autism. LRRTM4 in the female proband and her affected mother . Both platforms detected a heterozygous duplication of the terminal exon of d mother  which waAmino acid profiling by HPLC indicated no abnormalities. Organic acid profiling by gas chromatography and mass spectrophotometry indicated no abnormalities and no increase in lactate or methylmalonate. The glycosaminoglycan amount was 9.2 mg/mM cr.LRRTM4 is a component of the neurexin trans-synaptic connexus (NTSC) involved in neuronal cell adhesion, synaptogenesis and neurotransmission (LRRTM4 is part of a larger LRRTM family (LRRTM1-4) of postsynaptic ligands for the neurexins (NRXNs). Rare mutations of NRXN1 are strongly associated with Tourette syndrome and autism. The other neurexins (NRXN2-4) are also associated with TS and ASD (LRRTM4 in association with TS and ASD.smission  7]. LRR. LRRLRRT and ASD . PostsynNRXN1 mutations in TS or ASD. In this study, we report two affected females from the same family in association with the duplication of the terminal exon of LRRTM4, suggesting that the male bias more generally associated with TS/ASD may not apply as strongly with the NTSC or LRRTM4 association with TS/ASD.TS and ASD more often affect males compared to females at a ratio of ~4:1. Notwithstanding, there is little evidence of strong male bias associated with LRRTM4 is a paralog of LRRTM3 which is also associated with TS and ASD [LRRTM3 overlaps the CTNNA3 gene, which has recurrent partial deletions in TS and ASD [LRRTM3 is nested within and overlaps the CTNNA3 gene, where both genes are transcribed from opposing strands of the same DNA in convergent fashion [LRRTM3 is discordantly regulated by the convergent transcription of CTNNA3, thus extending the CTNNA3 association with TS/ASD to include LRRTM3 [LRRTM1 overlaps and is nested within an intron of the CTNNA2 gene and overlapping deletions have been identified near the 3′ end of CTNNA2, in a ~1Mb overlap region enriched with conserved DNA noncoding elements that may regulate the expression of CTNNA2 and LRRTM1 [ and ASD . LRRTM3  and ASD . LRRTM3  fashion . We preve LRRTM3 . Likewisd LRRTM1 .LRRTM4 (LRRTM4 gene expression in neuronal tissues; however, it is highly plausible that the partial duplication of LRRTM4 may have affected the expression of LRRTM4 in this family.The duplication breakpoints identified in the Tourette syndrome family reported in this study were also localised near conserved non-coding regions on either side of the terminal exon of LRRTM4 . DeletioLRRTM3 and LRRTM4 are prominently expressed in the dentate gyrus [LRRTM3 and LRRTM4 to induce synaptogenesis. LRRTM4 is the postsynaptic ligand for the potent synapse organiser NRXN1δ, and Roppongi et al. demonstrated that mice expressing a mutant form of LRRTM4 that fails to bind the heparan sulfate modification of NRXN exhibit structural and functional deficits at dentate gyrus excitatory synapses [LRRTM4, in specifying the molecular algorithm for neuronal circuitry formation [te gyrus . Presynasynapses . Togetheormation .


A bond mechanism at the reinforcement-concrete interface is one of the key sources of the comprehensive functioning of reinforced concrete (RC) structures. In order to apprehend the bond mechanism, the study on bond stress and slip relation (henceforth referred as bond-slip) is necessary. On this subject, experimental and numerical investigations were performed on short RC tensile specimens. A double pull-out test with pre-installed electrical strain gauge sensors inside the modified embedded rebar was performed in the experimental part. Numerically, a three dimensional rib scale model was designed and finite element analysis was performed. The compatibility and reliability of the numerical model was verified by comparing its strain result with an experimentally obtained one. Afterwards, based on stress transfer approach, the bond-slip relations were calculated from the extracted strain results. The maximum disparity between experimental and numerical investigation was found as 19.5% in case of strain data and 7% for the bond-slip relation at the highest load level (110 kN). Moreover, the bond-slip curves at different load levels were compared with the bond-slip model established in CEB-fib Model Code 2010 (MC2010). Overall, in the present study, strain monitoring through the experimental tool and finite element modelling have accomplished a broader picture of the bond mechanism at the reinforcement-concrete interface through their bond-slip relationship. Reinforced concrete (RC) is a composite material designed from different types of concrete and reinforcing bars. The reinforcing bars are strategically placed in the tensile areas of RC structures, resisting tension forces after cracking of concrete. Consequently, the interaction between concrete and reinforcement bars, often referred to as bond, is essential in developing the required performance of the structure.Modern non-destructive tests can be a possible option for the assessment of the conditions of concrete structures . HoweverAs an alternative to traditional force displacement based experimental arrangements, a direct strain measurement of reinforcement strain inside the concrete element may be applied to study the bond-slip behavior ,10. In fExperimentally measured reinforcement strain distribution in a tensile RC element gives valuable data on the force transfer mechanism from reinforcement to concrete, as well as the development of slip along the element. However, the diffusion of tensile stresses in the concrete, micro-cracking, and internal damage of concrete can hardly be measured experimentally . In thisAlthough several well-documented experimental campaigns have been carried out measuring the strain distribution in the tensile RC prisms ,10,18,19The experiment was performed by preparing a RC prism of cross-section 200 mm × 200 mm and the length of 390 mm with 1–20 mm ϕ reinforcement placed concentrically along its longitudinal axis. These dimensions were chosen considering one primary factor to avoid the risk of crack (primary) appearance during the load application. Recognizing the issue, the specimen length was kept shorter than the predicted mean crack spacing, calculated on the basis of the reinforcement ratio and the bar diameter . FurtherHigh strength concrete was prepared in the laboratory through the combination of Ordinary Portland Cement (OPC) of grade 42.5R, natural fine aggregate of grade 0/4 mm, coarse aggregate of 5/8 mm grade, and concrete superplasticizer (polycarboxylic ether polymer) to achieve desired workability. It should be noted that the water-cement ratio of the concrete was 0.35. The quantity, specific, and bulk density of the chemical compositions of concrete are mentioned in After 28 days of curing, the mechanical properties of the concrete were evaluated. Standard concrete cylinders (ϕ 150 × h 300), cubes (150 × 150 × 150), and prisms (100 × 100 × 400) were tested to determine the compressive strength, split tensile strength, and flexural strength of concrete respectively. Reinforcing bars of ϕ-20 mm and S500 grade were also tested to find out the yield strength and modulus of the elasticity of steel. Fixing the strain gauge sensors inside the reinforcement bar was undoubtedly the most delicate and time consuming part of this experimental campaign. However, on the positive side, this particular arrangement creates no interruption in the bond performance at the reinforcement-concrete interface, as the devices were placed inside the reinforcement core, not on its surface. At first, a reinforcement bar was cut in half longitudinally, and a groove 2 mm in depth and 10 mm in height was milled on its inner surface, as illustrated in The specimen was tested using a Universal Testing Machine  with the application of monotonic tensile load in a displacement controlled method. This double pull-out test was well-equipped with LFM100 , including DION STAT control  and digital controls along with a data procuring system, as displayed in FE analysis was frequently performed as a trustworthy tool in the study on reinforcement-concrete interface ,27, but Rf), defined as the ratio of the rib surface area’s projection to the surface area between the ribs [While designing the model, the authors put special attention on the rib geometry, which has a significant impact on the load transfer mechanism of RC structures . The maithe ribs . For theFor ease and simplicity, 1/8th of the FEM200×200×390 model was considered for further analysis by creating symmetric boundary conditions on relevant surfaces. During the mesh design, the tetrahedral shape of solid isoparametric finite elements with ten nodes were chosen for both the concrete and steel reinforcement. The concrete was modelled as 3D nonlinear cementitious material with the considerations as Young’s modulus 84.3 N/m . In the 8 MN/m3 were assumed. It was earlier shown by the authors that, for adequate bond analysis, the interface failure must occur so as to allow the slip between the reinforcement and concrete elements [The following are the notations of essential parameters of the interaction surface: material . In the elements . The ribThe load was applied in equally distributed loading steps of 5 kN (1.25 kN for a 1/4th of specimen), until the final step while it was reaching the yield stress of the reinforcement . Results from the FE analysis of the model FEM200×200×390 are illustrated in The authors have demonstrated the laboratory experiment of a double pull-out test and the FE analysis of a numerical model in earlier chapters. The current section presents the strain profiles as the key result from the above mentioned experiments followed by the further analysis. It is seen in In order to determine the bond-slip relation, the obtained strain data were further analyzed based on the stress transfer approach. This method provides the edge to directly model the cracks in accordance with the strain variation within the RC element. It also adopts the theory that the bond stress at the reinforcement-concrete interface directly controls the strains in the reinforcement and concrete through the relative slip . InherenIt is observed from It is important to note that the curves in In Here, The reinforcement strain profiles recorded at different load levels had a smooth and regular shape, and also kept symmetry in respect to the mid-section of the members representing zero slip. This implies that the employed reinforcement strain measurement technique based on the strain gauge sensors glued inside a groove of the bar may assure consistent and reliable test results.The bond-slip relations derived from the experimental reinforcement strain profiles of short RC ties had an ascending and a descending branch. The ascending branches of bond–slip curves were of a parabolic shape, and were in close agreement with each other at different load levels. This confirms that the same bond–slip relationship may be used for the whole loading analysis. Moreover, the ascending bond–slip branch may be considered to represent the inherent material feature of the reinforcement–concrete interaction, whereas the descending branch signifies the structural effect of cracking on the bond stresses in the close proximity of the cracks.In the case of strain monitoring, the numerical model performed extremely good correlation with the experimental one at lower load levels, though a maximum 19.5% disparity in strain data was noticed at the highest load level 110 kN.In the criteria of predicting bond-slip behavior, the numerical model has shown a thorough consistency in accordance with the experimental result. Only at higher loads has minor disparity (max. 7%) has been noticed.The current investigation demonstrated much stiffer bond response by fast reaching specific bond stress values at a significantly smaller slip compared to the MC2010.The paper reports the experimental results of the double pull-out tests of a singly reinforced concrete specimen produced from high strength concrete. Reinforcement strains were measured along the reinforcement bar using strain gauges spaced at 20 mm. Alongside a three-dimensional rib-scale, a finite element model was designed and analyzed in ATENA software. Later, its effectiveness was successfully verified with the laboratory test on the basis of the strains and bond-slip results. Subsequently, the bond-slip behavior of the specimens were obtained through stress transfer approach and compared with one prescribed in the MC2010 . Based oThe experimental-numerical study of strain monitoring techniques, followed by bond-slip investigation were successful. It creates a vast possibilities for further study related to reinforcement–concrete interaction, such as prediction of cracks, deformation, tension, stiffening, and so on.


Research is needed to delineate the relative and combined effects of different antibiotic administration and manure management practices in either amplifying or attenuating the potential for antibiotic resistance to spread. Here, we carried out a comprehensive parallel examination of the effects of small-scale (> 55 °C × 3 days) static and turned composting of manures from dairy and beef cattle collected during standard antibiotic administration , versus from untreated cattle, on “resistomes”  determined via shotgun metagenomic sequencing), bacterial microbiota, and indicator ARGs enumerated via quantitative polymerase chain reaction. To gain insight into the role of the thermophilic phase, compost was also externally heated to > 55 °C × 15 days.R = 0.424, p = 0.001, respectively) in all composts at the small-scale. Reduction in relative abundance  of total ARGs in finished compost (day 42) versus day 0 was noted across all conditions , except when externally heated. Sul1, intI1, beta-lactam ARGs, and plasmid-associated genes increased in all finished composts as compared with the initial condition. External heating more effectively reduced certain clinically relevant ARGs , fecal coliforms, and resistome risk scores, which take into account putative pathogen annotations. When manure was collected during antibiotic administration, taxonomic composition of the compost was distinct according to nonmetric multidimensional analysis and tet(W) decayed faster in the dairy manure with antibiotic condition and slower in the beef manure with antibiotic condition.Progression of composting with time and succession of the corresponding bacterial microbiota was the overarching driver of the resistome composition , and resistome risk suggested that composting reduced some potential for antibiotic resistance to spread, but the increase and persistence of other indicators of antibiotic resistance were concerning. Results indicate that composting guidelines intended for pathogen reduction do not necessarily provide a comprehensive barrier to ARGs or their mobility prior to land application and additional mitigation measures should be considered.This comprehensive, integrated study revealed that composting had a dominant effect on corresponding resistome composition, while little difference was noted as a function of collecting manure during antibiotic administration. Reduction in total ARGs, Video Abstract.The online version contains supplementary material available at 10.1186/s40168-021-01006-z. Antibiotic resistance is increasing in prevalence and economic costs, contributing substantially to human morbidity and mortality , 2. GlobUp to 100% of dosed antibiotics can be excreted as parent compounds, with the portion that is metabolized often remaining bioactive or converting back to the parent compound –11. ExcrEscherichia coli carrying ampicillin, kanamycin, and tetracycline resistance, as well as Acinetobacter and Enterobacteriaceae carrying resistance to erythromycin and tetracycline in antibiotic-dosed swine manure, cattle manure and poultry waste [Composting is often used to prepare manure for use as a soil amendment because it is beneficial for attenuating pathogen loads, reducing odors, and improving the quality of the soil . Variousry waste –43. Indiry waste , 44–46. sul1, tet(W), and the class 1 integron integrase gene, intI1. 16S rRNA gene amplicon sequencing was also performed on every sample to characterize succession of the bacterial microbiota as they related to antibiotic administration, progression of composting, the extent of thermophilic phase, and the composition of corresponding resistomes. The findings here have key implications for both livestock and manure management strategies for limiting the potential for antibiotic resistance to spread.The objectives of this study were (1) to provide a comprehensive, parallel comparison of how composting affects the composition and mobility of ARGs in manure derived from cattle undergoing antibiotic administration, versus from control cattle, and (2) to determine the relative roles of microbial ecological succession versus the duration of the thermophilic phase in reducing the potential for antibiotic resistance to spread. Here we examined manures of dairy and beef cattle, which have relatively similar physiologies, but vary in antibiotics used and routes of administration. Dairy and beef manures were generated for composting following intramammary infusion of cephapirin and pirlimycin or in-feed sulfamethazine, chlortetracycline, and tylosin, respectively, versus from untreated cattle. Collection of manure during peak antibiotic excretion following standard administration practices and in a natural metabolized state, rather than exogenously spiking antibiotics, was a key aspect of achieving realistic conditions. Composting was performed in small-scale composters to enable multiple replicated and parallel comparisons of static versus turned composting methods. A trial of the static dairy compost conditions externally heated to 55 °C for 15 days further evaluated the effects of an extended thermophilic phase. Shotgun metagenomic sequencing was performed on a cross section of 60 samples to profile resistomes while quantitative polymerase chain reaction (qPCR) provided a sensitive measure of specific indicators of anthropogenic sources of antibiotic resistance across all 270 samples collected, including The methods for cattle selection, manure collection, and small-scale composting were described in a prior study focused on the fate of antibiotics . In summNine Hereford steers were fed a basal diet of corn silage and medicated or non-medicated grain mix for seven days. Three steers were fed 350 mg of chlorotetracycline and sulfamethazine per day, three were fed 11 mg tylosin per kg feed, and three were fed a non-medicated diet. Feces and urine were collected from days 3 to 7 post-treatment. Nine dairy cows were selected for this study. Three peak lactation cows received no antibiotics, three peak lactation cows were treated with two intermammary doses of 50 mg pirlimycin; and three cows at the end of lactation received a single intermammary dose of 300 mg cephapirin per quarter (i.e. 300 × 4 = 1200 mg per cow), according to standard veterinary practice. Feces and urine were composited to obtain a homogenous mixture of “antibiotic” and “control” manure for both beef and dairy cattle  and to simulate the possibility of segregating antibiotic-containing manures as a management practice.The experimental setup and fate of antibiotics during composting were described previously . In summAn externally heated composting trial was employed to artificially impose a 15-day thermophilic phase using segregated aliquots of the same dairy manures used in the small-scale experiment. Composters were set up in 5-gallon barrels in triplicate for a total of 6 composters using the same compost feedstock recipe as the small-scale trial (stored at 4 °C prior to use). The compost was allowed to self-heat for the first 72 h, after which external heat tape was applied to maintain the thermophilic stage (>55°C) for 15 days. A mesophilic temperature range (35–45 °C) was then maintained for 3 weeks before allowing the compost to cool to room temperature. Samples were collected on days 0, 1, 3, 7, 14, 21, 28, 35, and 42 and raw manure samples were collected on day 0 for comparison with finished composts.E. coli quantification at higher specificity and sensitivity, the Colilert Quanti-Tray 2000 method  was applied to 0.1% peptone solution serial dilutions weekly after day 21 to determine if the FSMA guideline for E. coli reduction had been achieved.Ten grams of compost or manure were added to a sterile blender bag with 90 mL of 0.1% peptone solution to make a 1:10 dilution and mixed in a bag mixer for 2 min. A serial dilution was performed in 0.1% peptone solution prior to plating onto MacConkey agar and incubating for 24 h at 37 °C to enumerate total fecal coliforms . For The FastDNA Spin Kit for Soil  served as the primary extraction kit applied to all samples. Following blending, 500 mg of compost or manure was aseptically transferred to an extraction tube. Extraction followed the manufacturer’s instructions, except that a 2-h incubation period was added to both protocols immediately following the bead-beating step to optimize lysis of microbial cells and the final centrifugation step was extended to 3 min to maximize capture of DNA. The OneStep PCR Inhibitor Removal Kit  was applied to all DNA extracts.e-value cutoff (1e−10) utilized to ensure lower quality matches were filtered out prior to assessment [r = 0.8, p <0.01). ARG richness was determined by enumerating each unique ARG detected by CARD and normalizing to the total million of reads for the sample.Sixty representative DNA extracts were selected to provide a cross section among all manure types and the small-scale and externally heated compost conditions for metagenomic sequencing. DNA extracts were sequenced by the Biocomplexity Institute of Virginia Tech, Blacksburg, VA, on an Illumina HiSeq 2500 in High Output mode with 2 × 100 paired-end reads, with 12 samples pooled per lane across 5 lanes. Paired-end reads were annotated in MetaStorm using default parameters, with the amino acid identity (80%) aimed at preventing false positive annotations  and the sessment . ARGs wesessment  and plassessment , resistosessment . Relative-value < 1e−10 and amino acid identity > 60%. To assess the potential influence of the assembly method on the observed resistome risk trends, all small-scale samples were also assembled using MEGAHIT [p = 0.5). On average, 48% of metagenomic reads per sample were successfully assembled into contigs using IDBA-UD. Contigs that occurred more than 3 times across all samples were further examined. Relative abundances were calculated by normalizing gene counts to abundance of 16S rRNA genes, annotated against the Greengenes database [Contigs were assembled in MetaStorm using IDBA-UD . “Resistdatabase , factoridatabase .Two hundred and seventy DNA extracts from each composting trial were amplified via PCR targeting the V4 and V5 regions of the 16S rRNA gene following the online Earth Microbiome Project protocol using barcoded primers (515F/926R) . Triplictet(W) [intI1 [sul1 [qPCR was performed in triplicate on all DNA extracts using the CFX96 Touch Real-Time PCR Detection System  to quantify initial concentrations of 16S rRNA genes , tet(W) tet(W) , intI1 [) [intI1 , and sulI1 [sul1  using SsI1 [sul1 .α = 0.05 was applied. Statistical differences among gene abundances determined by qPCR were calculated using the Kruskal-Wallis nonparametric rank test and two-sided Wilcoxon rank sum tests. Nonmetric multidimensional scaling analysis (NMDS) and analysis of similarities (ANOSIM) were performed on unweighted UniFrac distance matrices for microbial communities and on Bray Curtis similarities using Primer 6 software [Statistical analyses were performed using R (3.4.1) . Graphicsoftware  and the software  to compahttps://dataview.ncbi.nlm.nih.gov/object/PRJNA506850, , rather than by cattle type, whether manure had been collected during antibiotic administration, or by the composting method employed. Presence/absence of antibiotic use for the feedstock raw manure significantly affected the composition of the initial microbiota in the beef and dairy conditions as well as the progression throughout composting across all samples and within the beef condition, though these did not play as large of a driving force . Throughout static and turned composting from day 0 to 4, in both the with and without antibiotic conditions, Bacilli significantly increased , while the Clostridia (p = 0.002) and Methylacidiphilae (p ≤ 0.03) classes significantly decreased from their starting abundances  clustered based on how many days the composting had progressed , indicating that the composting process generally imposed negative selection pressure on bacteria carrying ARGs. There was no significant difference in total ARG relative abundance among the four raw manure types , among the different initial day 0 compost mixtures , or among the different finished compost types .Shotgun metagenomic sequencing was performed on 60 compost samples to characterize the resistome of the initial compost mixture (day 0), thermophilic phase (day 4), and the finished compost day 42) p = 0.004; Fig. SThe range of ARG alpha diversity in terms of unique ARGs identified  in all samples was 1.76–12.8 ARGs/million reads, with an average of 6.4 ARGs/million reads. Significant decreases in diversity were observed in the dairy with antibiotic condition for both composting methods with time . There was also no significant difference in initial ARG profiles among the four uncomposted manures used in this study  or between these manures and their corresponding day 0 compost mixtures . Composting, on the other hand, significantly shaped ARG profiles , while manure type did not have an effect . The shift in ARG profile in the compost as a function of time was significant , but the effect was not as strong as that observed for the succession of the microbiota.NMDS analysis of ARG profiles  provided a broad comparison of the resistomes among the compost conditions Fig. . Contrarles Fig. . While tp = 0.03, dairy: p = 0.02).Plasmid-associated genes were also analyzed as a key indicator of the mobility of the resistome and, in general, were found to increase in relative abundance with time regardless of cattle type or antibiotic dosing condition . Notably, there were 39 pathogen/ARG/MGE contigs that persisted through composting. These were predicted to originate from Staphylococcus, Streptococcus, and Vibrio, encoding resistance to a wide array of antibiotics  and being carried on a diverse range of transposases, recombinases, and plasmids , with the Firmicutes phyla and the Erysipelotrichia class, respectively. These are both gut commensal-associated microbiota and thus expected to decrease during composting due to the elevated temperatures and microbial community succession.In terms of individual classes and mechanisms of resistance, the abundance of ARGs corresponding to trimethoprim, tetracycline, and macrolide-lincosamide-streptogramin (MLS) resistance classes decreased during composting to a striking extent Fig. . Only twp = 0.002), with beta-lactam ARGs being particularly dominant. Interestingly, although this class of antibiotics was only administered to the dairy cows, there was no difference in detection of the corresponding ARGs in terms of a time-paired comparison between beef and dairy experiments .A “clinically relevant” subset of ARGs, defined as genes that convey resistance to antibiotics currently used in clinical settings, was compiled and subject to separate comparison Fig. . Notablysul1, as measured by qPCR, absolute abundance  increased during composting across all experimental conditions . Similarly, intI1 absolute abundance also increased across all conditions . Further, sul1 and intI1 correlated strongly and positively , as would be expected based on the common insertion of sul1 in the class 1 integron [Notably, integron .Fig. 7atet(W) decreased across all compost conditions from day 0 to day 42 . Notably, tet(W) was the only of the three indicator ARGs for which the reduction pattern was influenced by whether the manure was collected from antibiotic-treated cattle. Counterintuitively, tet(W) decreased faster in the dairy with antibiotic than the dairy without antibiotic condition  decreased faster in the control than in the antibiotic manure.Absolute and relative abundances of ion Fig. . HoweverE. coli were successfully reduced below detection by the extended thermophilic period  , as well as on the final day of composting . The final compost from the externally heated condition was also more taxonomically diverse than the final compost at the small-scale .As was observed at the small-scale, NMDS analysis revealed duration of composting to be the overarching driver of the composition of the bacterial microbiota in the externally heated composts  Fig. . Similarp = 0.055), although NMDS analysis did not reveal a difference in resistome composition between the finished small-scale and externally heated composts. Consistent with the small-scale composting, ARG profiles did not vary as a function of antibiotic treatment , but did shift with time . The richness of ARGs did not vary with time, nor did plasmid-associated genes or clinically relevant ARGs  decreased during externally heated composting  and for tet(W) for all conditions from time 0 to day 42 (p = 0.01). Relative sul1, tet(W), and intI1 abundance , with intI1 and sul1 increasing and tet(W) decreasing.Consistent with the small-scale composting, absolute abundances of ing Fig. . This wace Fig. S all wereGiven that the majority of antibiotics sold in the USA and many parts of the world are administered to livestock, it is important to understand how this affects the carriage of ARGs in corresponding manures and if agricultural management practices, such as composting, act to amplify or attenuate their potential to spread downstream to environmental, food, and human receptors. Here, we carried out a comprehensive integrated study to assess effects of different composting approaches on manure collected during standard antibiotic administration, versus without antibiotic treatment, for both beef and dairy cattle. We utilized a variety of measurements to gain a mechanistic understanding into how the composting process affects the resistome and to evaluate the potential for antibiotic resistance in the finished compost to spread and pose a human health risk through association with pathogens.In general, composting had a strong effect in shaping the microbiomes of finished composts and both resistome and microbiota profiles tended to converge across conditions, regardless of whether the manure was collected from beef or dairy cattle or if antibiotics had been administered.The overall results indicate that the natural microbial ecological succession that occurs during composting likely plays a major role in dictating the composition of the resulting resistomes. The specific microbial taxa that shifted during composting was logical, with Clostridia being strict anaerobes and highly characteristic of cattle manure, thus their reduction with (aerobic) composting is as expected –76, whilTet(W) was reduced universally in all compost treatments, including both small-scale and externally heated trials. The decrease in tet(W) in response to composting is consistent with the observations of Selvam et al. [sul1 and intI1 both increased in relative and absolute abundance across all composting conditions. sul1 and intI1 are becoming widely applied as sensitive indicators of anthropogenic sources of antibiotic resistance and potential to spread [m et al.  and Storm et al. , suggesto spread . In parto spread , 80, preE. coli. These results are consistent with the known benefits of an extended thermophilic phase for pathogen reduction, but this study suggests that the benefits of time × temperature guidelines intended for diminishing fecal pathogen risk do not translate to reducing the total ARG load or their potential to mobilize.Metagenomic analysis of resistomes further revealed increases in several clinically relevant ARGs during composting, especially those encoding resistance to beta-lactams. While pirlimycin and tylosin, two of the antibiotics administered to the cattle, belong to the MLS drug class, ARGs encoding resistance to this class markedly decreased throughout composting, suggesting that there was little selection pressure. However, there were indications that the resistome may become more mobile during composting, enabling the spread of ARGs to new host organisms. In particular, plasmid-associated genes increased measurably in all composting conditions, including the externally heated trial. Resistome risk scores, which provided a relative comparison across samples of associations of ARGs with MGEs and taxonomic groups containing pathogens, increased initially during the thermophilic phase of small-scale composting, but then decreased back to levels similar to the initial condition by day 42. This suggests that the shock of the thermophilic phase might induce horizontal gene transfer of ARGs. However, with the extended thermophilic phase of 14 days, resistome risk scores attained the lowest levels observed throughout the study, as was indicated by substantially fewer contigs annotated as containing ARGs, MGEs, and pathogen-like sequences . Similarly, no differences were found in the finished composts in terms of overall resistome composition, relative abundance of total ARGs, clinically relevant ARGs, plasmid-associated ARGs, or resistome risk scores as a function of whether the manure had been collected during antibiotic administration. The only discernable differences based on antibiotic administration were with respect to tet(W), ARG alpha diversity, and the composition of the bacterial microbiota. Specifically, tet(W) decayed faster in the dairy antibiotic compost and slower in the beef antibiotic compost than in their respective controls. This could relate to the fact that chlortetracycline was administered to the beef cattle and thus continued to exert some selection pressure for bacteria carrying tet(W). In the case of the dairy manure, tet(W) reduction may have been enhanced by pirlimycin killing bacteria carrying tet(W), assuming that tet(W) did not co-occur with ARGs conferring resistance to pirlimycin. ARG alpha diversity was the only other antibiotic resistance indicator that varied by antibiotic administration, significantly decreasing during composting only in the dairy with antibiotic condition. According to NMDS analysis of bacterial 16S rRNA gene amplicons, taxonomic composition of both beef and dairy composts was influenced by whether antibiotics were administered across all timepoints. This brings to light an interesting point that the taxonomic composition of the compost was actually more sensitive to antibiotic administration than the composition of the resistome. Overall, the results indicated that antibiotic administration was a minor factor shaping the composition of the resistome compared with composting itself.Whether the manure was collected during antibiotic administration had surprisingly little effect on indicators of antibiotic resistance. Notably, there was no significant difference in initial resistome composition among the four manures  prior to composting , others persisted or even increased. sul1 and intI1 are widely being considered monitoring targets for assessing anthropogenic sources of antibiotic resistance and efficacy of mitigation measures [sul1, intI1, and indicators of mobility were not. Further, little to no difference was observed between turned and static composting conditions, suggesting that, from an antibiotic resistance standpoint, there may be little value in the extra effort required to turn and maintain compost heated for extended periods. Composting is well known and effective at reducing microbial pathogens and produces a consistent product, but additional mitigation measures are advisable to minimize the potential for antibiotic resistance to spread along the farm-to-fork continuum. Additional barriers beyond composting, such as imposed distances from water bodies for land application and wait periods before harvest would be wise to consider.The comprehensive comparative nature of this study provides new insight into the relative benefits of various on-farm management approaches in terms of their potential to control the spread of antibiotic resistance. The study was designed in such a manner to inform with respect to whether typical antibiotic use in cattle poses special concerns for the control of antibiotic resistance in resulting manure, and if such manures merit segregation and specialized treatment relative to antibiotic-free manures. Surprisingly, there were few discernable effects as a function of antibiotic administration, suggesting that the cattle used in this study already carried a robust gut resistome that was not substantially affected by antibiotic use. Lack of difference in antibiotic-treated and control livestock in manure-borne ARGs has been noted in other studies , 84. Commeasures . NotablyAdditional file 1.


Immunoglobulin G4-related disease (IgG4-RD) is an immune-mediated fibroinflammatory condition that can affect multiple organs. IgG4-RD may show a variety of initial symptoms. In the oral mucosa, lesions present as inflammatory fibrosis with a large number of IgG4-positive plasma cells. Evaluating treatment is a well-known problem in IgG4-RD due to the absence of an established assessment system. There are difficulties in defining the severity of the disease, which is why treatment is primarily based on its clinical manifestations.We present a case report of localized IgG4-RD with ulcerative and proliferative manifestations on the tongue, which clinically mimicked oral squamous cell carcinoma. A tumor-like lesion on the tongue can indicate something else other than the malignant or reactive changes commonly found in the oral mucosa. Multiple differential diagnoses of these atypical oral lesions, including localized IgG4-RD, should be considered. Immunoglobulin G4-related disease (IgG4-RD) is an immune-mediated fibroinflammatory condition affecting multiple organs, which may be difficult to distinguish from other malignant, infectious, and inflammatory diseases. IgG4-RD is characterized by organ enlargement, elevation of serum IgG4 levels, tissue infiltration with IgG4-positive plasma cells and various degrees of fibrosis –4. ManifIn this report, we present a case report of localized IgG4-RD with ulcerative and proliferative manifestations on the tongue, which clinically mimicked oral squamous cell carcinoma. After excluding multiple diagnoses, we considered localized IgG4-RD to be the likely diagnosis. We present the protracted clinical manifestations lasting for 5 years, diagnostic work-up, pathology, and management of this case.9 cells/L). There were no signs of dysplasia, and malignancy could be ruled out. She received treatment with Clobetasol and Chlortetracycline with no improvement of the symptoms.In December 2014, a 71-year-old woman was referred to the Department of Plastic and Oral and Maxillofacial surgery at Uppsala University from the Ear, Nose, and Throat Department (ENT). The reason stated for referral was a painful ulcer on the right side of the tongue, which had manifested 6 months earlier. The ulcer had a fibrinous base and was firm, but not indurated . There wIn January 2015, the lesion had increased in size, and an excisional biopsy was performed. The initial diagnostic hypothesis was a drug-induced side effect initiated by the Alendronate treatment , 20 for In June 2015, the patient reported increased pain from the enlarged ulcer without improvement of symptoms. A new excision was performed , where H/L (2.3–16 µg/L) during the examination, and the possible diagnosis of hyper eosinophilic syndrome  associated with inflammatory bowel disease was considered. As the patient had not displayed any characteristic pustules or a confirmed diagnosis of ulcerative colitis or Crohn’s disease, and had not shown any improvement when treated with Prednisolone, PV was excluded as a possible diagnosis.During the period 2016 and 2017, due to several recurring exophytic growths of the lesion, the patient underwent multiple excisions . We concIn February 2018, our patient was investigated at the Department of Rheumatology. Churg–Strauss syndrome (CSS), now better known as eosinophilic granulomatosis with polyangiitis (EGPA), was discussed as a possible differential diagnosis. EGPA is a form of vasculitis, affecting small- and medium-sized arteries. It causes granulomatous, eosinophilic inflammations, mostly found in patients with severe asthma . OverallFurthermore, in February 2018, another excision biopsy was retrieved from the lesion on the tongue, with an inquiry regarding IgG4-positive plasma cell levels. Histopathological analysis revealed that the tumor-like lesion on the tongue was caused by a candidiasis-associated ulceration. The ulceration formed an extruding polyploid lesion with granulation tissue rich in neutrophil granulocytes and lymphoplasmacytic cells . The preAn IgG-immunostaining was performed, a method that can be unreliable and difficult to interpret due to high-background staining . As a reMedical databases and literature revealed only one description of IgG4-RD involving the tongue . TherefoA more conclusive diagnosis of IgG4-RD would have validated treatment with Rituximab, a monoclonal antibody that targets CD20 presented by B cells . With thIn August 2018, 6 months after the latest excision biopsy of the lesion, the patient underwent fluoro-deoxy-glucose positron emission tomography with computerized tomography (FDG PET/CT) for evaluation and diagnosis of IgG4-RD. This method enabled an individually tailored treatment and provided a better understanding of the disease . FDG PETIn September 2018, a follow-up showed a progressive exophytic growth on the tongue. New, firm, soft tissue changes in the mucous membrane on the right side of the buccal cheek, much like the previous changes seen on the tongue, were observed . This neApproximately 1 year later, in 2019, the patient reported that she was completely pain-free. The lesions remained but were significantly less firm in consistency. Neither color change nor progression of the lesions were observed .In 2019, the American College of Rheumatology (ACR) and the European League Against Rheumatism (EULAR) developed new international classification criteria for IgG4-RD. The entry criteria require involvement of IgG4-RD in at least one organ. The exclusion criteria consist of 32 items, all of which must be unfulfilled to reach IgG4-RD classification. Finally, eight weighted inclusion criteria domains are applied, creating a point system that must meet a specific threshold . In the Mikulicz’s disease (MD) was first reported in 1888. It is characterized by painless, symmetrical, bilateral, enlarged salivary and lacrimal glands with lymphocytic infiltration. The histological findings of the disease were identical to Sjogren’s syndrome (SS), and MD was, therefore, considered to be a subtype of SS. However, the clinical picture and the histopathology of the two diseases vary, and in MD, one can find elevated levels of serum IgG4, as well as IgG4-positive cell infiltration. Hence, the disease was suggested to represent an systemic IgG4-RD , 14, 18.Küttners tumor (KT), a disorder in the salivary glands associated with IgG4-RD, is an inflammatory disease previously known as chronic sclerosing sialadenitis. It was first described in 1896, and it primarily affects the submandibular glands. Histologically, KT shows fibrosclerotic lesions containing IgG4-positive plasma cells. The histopathological and serological findings in KT are similar to those of MD , 39.There are reports of mucosal manifestations of IgG4-RD in the head and neck region as well. Most of these lesions have occurred on the mucosa of the gingiva, the alveolar mucosa, hard palate, and floor of the mouth. Manifestations on the laryngeal mucosa, nasal and paranasal sinus involvements have also been described . These lWe report a case of localized IgG4-RD manifesting as a progressive tumoral lesion on the lateral border of the tongue. Before reaching the final diagnosis of IgG4-RD and administering adequate treatment, many possible diagnoses were investigated and correspondingly treated without success. Throughout treatment, the mucosal lesion changed in character from a small ulcer to a painful, exophytic tumor-like lesion. With this case, we show that a tumor-like lesion on the tongue can suggest something else other than the malignant or reactive changes commonly found in the oral mucosa. Our case emphasizes the importance of working in a multidisciplinary manner in the absence of expected treatment results. Multiple differential diagnoses of these atypical oral lesions, including localized IgG4-RD, should be considered. More importantly, our case underlines the importance of being attentive when symptoms do not match histopathological findings, and why one should never exclude possible connections to the patient’s underlying conditions.Evaluating treatment in IgG4-RD is a well-known problem due to the absence of an established assessment system. Difficulties remain in outlining the severity of the disease. Treatment is, therefore, based mostly on the clinical manifestations and not on the severity of the disease. Retrospectively, treatment with Rituximab was identified as successful due to the lack of progression in size and relief of pain from the lesion. Further studies are needed to determine the pathophysiology and epidemiology to efficiently diagnose and treat cases of localized or general IgG4-RD.Based on the ACR/EURLAR classification criteria for IgG4-RD, our patient met the entry criteria. No exclusion criteria were detected. A threshold of 20 points is needed for a patient to be classified as having IgG4-RD. As obliterative phlebitis and storiform fibrosis were not observed in the excision biopsy of the tongue, 18 of a possible 29 points were obtained from the histopathology criteria.Furthermore, for the classification criteria for IgG4-RD, no additional points could be obtained for our patient to achieve the threshold of 20 out of a possible 96 points. However, based on the histopathological findings, ruling out other possible diagnoses and the fact that Rituximab was the only successful treatment leading to improvement of the patient’s symptoms, it was concluded that a localized form of IgG4-RD of the tongue was the most likely diagnosis.


EWSR1 rearrangement and subsequent MET upregulation. The European Organisation for Research and Treatment of Cancer 90101 phase II trial evaluated the MET inhibitor crizotinib in CCSA but resulted in only sporadic responses. The aim of this exploratory study was to identify the molecular alterations potentially relevant for the treatment outcome by using archival CCSA samples and trial-related clinical data. We characterized MET signaling and revealed an infrequent activation of MET, which may explain the lack of response to crizotinib in the disease cohort. Based on sequencing analyses, we discovered copy number alterations, mutations and dysregulated pathways with potentially predictive or prognostic values for patients’ outcomes. This work describes the molecular heterogeneity in CCSA and provides deep insight into the biology of this ultra-rare malignancy, which may potentially lead to better therapeutic approaches.Clear cell sarcoma (CCSA) is a rare subtype of soft tissue sarcoma characterized by EWSR1 rearrangement, resulting in aberrant transcription of multiple genes, including MET. The EORTC 90101 phase II trial evaluated the MET inhibitor crizotinib in CCSA but resulted in only sporadic responses. We performed an in-depth histopathological and molecular analysis of archival CCSA samples to identify alterations potentially relevant for the treatment outcome. Immunohistochemical characterization of MET signaling was performed using a tissue microarray constructed from 32 CCSA cases. The DNA from 24 available tumor specimens was analyzed by low-coverage whole-genome sequencing and whole-exome sequencing for the detection of recurrent copy number alterations (CNAs) and mutations. A pathway enrichment analysis was performed to identify the pathways relevant for CCSA tumorigenesis. Kaplan–Meier estimates and Fisher’s exact test were used to correlate the molecular findings with the clinical features related to crizotinib treatment, aiming to assess a potential association with the outcomes. The histopathological analysis showed the absence of a MET ligand and MET activation, with the presence of MET itself in most of cases. However, the expression/activation of MET downstream molecules was frequently observed, suggesting the role of other receptors in CCSA signal transduction. Using sequencing, we detected a number of CNAs at the chromosomal arm and region levels. The most common alteration was a gain of 8q24.21, observed in 83% of the cases. The loss of chromosomes 9q and 12q24 was associated with shorter survival. Based on exome sequencing, 40 cancer-associated genes were found to be mutated in more than one sample, with SRGAP3 and KMT2D as the most common alterations (each in four cases). The mutated genes encoded proteins were mainly involved in receptor tyrosine kinase signaling, polymerase-II transcription, DNA damage repair, SUMOylation and chromatin organization. Disruption in chromatin organization was correlated with longer progression-free survival in patients receiving crizotinib. Conclusions: The infrequent activation of MET may explain the lack of response to crizotinib observed in the majority of cases in the clinical trial. Our work describes the molecular heterogeneity in CCSA and provides further insight into the biology of this ultra-rare malignancy, which may potentially lead to better therapeutic approaches for CCSA.Clear cell sarcoma (CCSA) is characterized by a chromosomal translocation leading to  EWSR1/ATF1) rearrangement, resulting in the aberrant activation of microphthalmia-associated transcription factor (MITF) [MET, and the subsequent overexpression of the receptor tyrosine kinase (RTK) MET, which is suggested as a potential target for CCSA treatment [Clear cell sarcoma (CCSA) is an ultra-rare subtype of soft tissue sarcoma (STS) that accounts for approximately 1% of all STS cases . This dir (MITF) ,8. MITF reatment ,10.EWSR1 rearrangements in at least 15% of the tumor cells. This trial demonstrated that crizotinib provided some clinical benefits in patients with locally advanced or metastatic CCSA but did not meet its primary endpoint , as only one partial response was observed among 26 evaluable patients. This suggests that other factors may contribute to the oncogenesis and progression of CCSA in addition to MET upregulation.A prospective phase II trial, European Organization for Research Treatment of Cancer (EORTC) 90101 “CREATE”, evaluated the oral MET/ALK/ROS1 inhibitor crizotinib in patients with CCSA . The actEWSR1 rearrangement is required for MET expression and that MET inhibition could decrease CCSA cell growth in vitro [MET amplification or mutations, and bypass the signaling pathways [MET amplification and MET exon 14 alterations is known to be sensitive to treatment with crizotinib, while the presence of secondary MET mutations is described as a potential resistance mechanism [It has been reported that in vitro . The molpathways . MET-addechanism ,14. SimiWe performed an in-depth histopathological and molecular analysis of archival CCSA tumor materials from patients who participated in the CREATE trial, based on tissue microarrays (TMA) constructed from leftover tissue. Using immunohistochemistry and high-throughput sequencing, we characterized the relevant molecules in the MET-signaling pathway and assessed the copy number changes and mutational profiles, with the aim to identify potentially predictive/prognostic biomarkers and novel therapeutic targets for this rare cancer. A total of 36 formalin-fixed and paraffin-embedded (FFPE) tissue samples from patients with CCSA were available and had been archived in a central biorepository . The diagnosis of CCSA was centrally confirmed as part of the clinical trial. Thirty-two of the tumor blocks were used to construct a TMA with 1.5-mm triplicate cores per case . A serie® HiSeq4000  was used for sequencing at a low coverage (±0.1×). Raw sequencing reads (50 bp) were mapped to the human reference genome (GRCh37/hg19 version) using Burrows-Wheeler Aligner  and sorted with SAMtools . Picard tools were used to remove duplicates. QDNASeq and ASCAT were used to count and segment the aligned reads in bins of 50 kb [q-value < 0.25 were selected [DNA was extracted from 24 archived FFPE tumor samples having a quality acceptable for libraries preparation. Illuminaof 50 kb ,18. The GRCh37/hg version Libraries prepared for low-coverage whole-genome sequencing were enriched for exomic sequences using the SeqCapV3 exome enrichment kit  following the manufacturer’s instructions. They were sequenced on HiSeq4000 using a flow cell, resulting in 2 × 150-bp end reads that were further mapped and sorted, and the duplicates were removed as described above. Next, base recalibration, local realignment and single-nucleotide variant calling were performed with the Genome Analysis Tool Kit . Dindel was used for calling small insertions and deletions (indels). Mutations with a coverage <10× or a quality score <30 for substitutions or <50 for indels were discarded. Since no germline samples were available, a strict filtering strategy was applied based on publicly available databases to exclude the common single-nucleotide polymorphisms. Mutations occurring in large databases  with an allelic frequency >0.001, as well as mutations occurring in smaller, appropriate databases , were removed if they occurred in more than one individual. The Cancer Gene Consensus (CGC) set, developed by the Catalogue of Somatic Mutations in Cancer databases , was applied for a further analysis to include genes that have been implied in cancer . We alsoq-values < 0.01 were considered significant. Visualization was done by using EnrichmentMAP, AutoAnnotate and the Markov Cluster algorithm  in a Cytoscape v3.7.2  environment [To identify the pathways that were significantly dysregulated in CCSA, we conducted a pathway enrichment analysis using g:Profiler  to identify disrupted pathways enriched in the list of mutated CGC genes that were detected by WES. The g:Profiler is a tool combining bioinformatic and statistic methods to pick up pathways where the genes present in the dataset are significantly enriched or overrepresented, as compared to all the genes in the genome . All patironment ,28.p values < 0.05 were considered significant.The response to crizotinib was evaluated using Response Evaluation Criteria in Solid Tumors (RECIST v1.1), as previously reported . A totalArchival tumor material was available from 34 out of 36 CCSA patients enrolled in EORTC 90101. These samples included 30 MET-positive tumors according to the protocols, three MET-negative and one unevaluable for MET status. Among 26 out of the 34 eligible patients in the trial, only one achieved a partial response (PR), and 17 had SD as the best response to crizotinib by RECIST 1.1. PD was the best response in 8 cases. Six patients were treated but did not reach the primary end point, and two cases were not treated . The bioTo explore the expression and activation of MET and related molecules involved in RTK signaling, we performed IHC using TMA constructed from 32 cases. A series of TMA slides were successfully stained with antibodies against MITF, HGF, (p)MET, (p)GAB1, (p)MAPK, (p)AKT and (p)S6, with evaluable rates from 88% to 100% per staining. MITF expression was observed in 78% of the samples, including three MET-negative cases, while expression for HGF and MET was found in 16% and 82% of the samples, respectively. To assess the activation of the MET receptor, two phosphorylated MET sites (Tyr1234 to 1235 and Tyr1349) were evaluated, showing immunopositivity in 4% and 50% of the cases, respectively. The downstream-signaling molecules and their phosphorylated forms were positive in most of the cases, apart from phosphorylated AKT i.e., GAB1 (100%), pGAB1 (100%), MAPK (100%), pMAPK (79%), AKT (97%), pAKT (53%), S6 (97%) and pS6 (74%). Notably, 20%, 43% and 24% of the cases expressing MAPK, AKT and S6 were absent for the phosphorylated form, suggesting a dominance of MAPK and S6 activation in CCSA. The immunoreactivity for each molecule is summarized in To reveal the CNA profile in CCSA, we performed low-coverage whole-genome sequencing and a GISTIC analysis allowing the detection of recurrent regions affected by CNAs in 24 assessable samples. A number of the chromosomal arm-level (broad events) and regional-level  regions affected by CNAs were detected. The most frequent arm-level CNA was a gain of chromosome 8q, which was present in 16 out of 24 (67%) cases. Other broad events detected were the gain of 7q (38%), 7p (33%), 1q (29%), 6p (25%) and 8p (25%), with frequent losses of 9p (54%), 19q (38%), 9q (33%), 17q (33%), 19q (33%), 10p (29%) and 10p (25%) A. FurtheSRGAP3, KMT2D, NIN, TSC2, CACNA1D, WRN, AFF1, CREBBP, CYLD, DAXX, DICER1, FBXW7, GNA11, MAP2K1, MAP3K13, ITK, MN1, NOTCH1, RBM15, SH2B3, TBX3 and UBR5) displayed damaging mutations (PolyPhen-2 or VarSome) in more than one case. The proportion of mutations, including those with damaging phenotypes, is presented in SRGAP3 and KMT2D were the most common alterations (in four cases). SRGAP3 encodes a product inhibiting Rho GTPase Rac1 and interacts with actin remodeling proteins to regulate the cytoskeleton [KMT2D plays a critical role in epigenetic regulation and may have an impact on development, differentiation, metabolism and tumor suppression [MET mutations (p.S548L) located in the plexin–semaphorin–integrin (PSI) domain. However, this mutation was predicted to be non-pathognomonic. SRGAP3, KMT2D and MET. Among the genes mutated in more than one case, we identified 16 potentially druggable genes  using DGIdb.To assess the mutational landscape of CCSA, the DNA used for the CNA analysis was also subjected to whole-exome sequencing. We sequenced them at an average coverage of 77.2× and detected a total of 6181 mutations with an average of 258 mutations per sample (range 156–473). Among all the alterations detected, 4201 were no-synonymous mutations, including 3548 missense, 134 nonsense mutations and 519 insertions and deletions (indels). For further analysis, we focused on the genes that were previously documented in the CGC set. A total of 105 CGC genes affected by 211 mutations, with the average of nine (range 3–22) per case, were identified, and 40 of them were found to be mutated in more than one case. Only 22 altered genes  (p = 0.02) and PFS (p < 0.01), respectively (p = 0.02), 7p  and 7q  in metastatic lesions compared to the primary tumors, though they were not associated with patient survival. p = 0.025). We tried to perform a more advanced statistical analysis to support our oberservation. However, we were not able to obtain conclusive results due to the disease cohort being of a small sample size and with limited clinical data. To identify the potential prognostic factors, we correlated the molecular findings with the clinical parameters, including the disease status, OS, PFS and response to crizotinib. In the immunohistochemical analysis, the Kaplan–Meier estimates showed that the activation of MAPK was associated with longer OS (= 0.046) A. In theectively B. We alsEWSR1-rearrangements, leading to aberrant MET expression [n = 34) collected in the context of the clinical trial. The recurrent alterations were further analyzed and correlated with the treatment outcome, with the aim of identifying the therapeutic targets and potential predictive/prognostic biomarkers. The results of this work were a comprehensive analysis of the genomic landscape of CCSA including an in-depth analysis of MET signaling in this rare malignancy.CCSA is known as a translocation-associated sarcoma that is associated with pression . Crizotipression . Little EWSR1–ATF1 fusion has been proven to upregulate its transcriptional target of the MITF melanocytic transcription factor, leading to subsequent MET overexpression in CCSA [EWSR1 rearrangement showed MITF expression, while no MITF expression was detected by IHC in seven specimens with altered EWSR1, suggesting that not all EWSR1-rearanged cases induce MITF overexpression. As previously reported, CCSA with EWSR1-CREB1 rearrangement had little or no melanocytic signatures (absence of MITF) compared to those with common fusion involving ATF1 [ in CCSA . Since aing ATF1 . It mighing ATF1 . Under physiological conditions, MET activation requires the presence of its ligand HGF. In our study, a relatively low frequency of HGF expression  and MET phosphorylation at Tyr1234–35  was observed, while 50% of the cases were positive for MET phosphorylation at Tyr1349 . At the same time, the downstream signaling pathway was found activated in a majority of the cases, as illustrated by frequent and strong positivity for pGAB1 (97%) and pMAPK (79%). These findings suggested the presence of other activating mechanisms in CCSA. Several RTKs  engaged with their ligands could transactivate MET and the subsequent signaling in an HGF-independent manner , which mMYC and CDKN2A are located, respectively. Ozenberger et al. recently reported MYC amplifications and CDKN2A deletions as recurrent CNA in 13 CCSA cases and demonstrated that MYC overexpression contributes to tumorigenesis in this disease [MYC (gain) and CDKN2A (loss or mutation) have been frequently detected in various human cancers, including melanoma [MYC should be further investigated in the evolution of this disease, which may refine the therapeutic strategies for CCSA. Using low-coverage whole-genome sequencing, we detected recurrent CNAs in 24 CCSA samples, where a gain of chromosome 8q 67%) was the most common broad alteration. Similarly, a number of individual cases harboring gains of chromosome 8q were found in previous studies % was the, indicat disease . Aberranmelanoma ,42,43. Imelanoma ,45. It imelanoma . For theSRGAP3  and KMT2D  in 17% of the analyzed cases as the most common alterations. These mutations, apart from KMT2D p.Q791E, were bioinformatically predicted as damaging events. The product of SRGAP3 has been identified as having a tumor-suppressive role through the negative regulation of RAC [KMT2D transcript plays an important role in histone methyltransferase [KMT2D mutations may implicate epigenetic dysregulation in the disease biology of CCSA. Although the functionality of these mutations has not been fully clarified, mutations in SRGAP3 and KMT2D have been detected and considered relevant in a variety of sarcomas, including angiosarcoma, endometrial stromal sarcoma and alveolar rhabdomyosarcoma [MET mutations located in the tyrosine kinase domain [MET mutations (p.S548L) detected in our cohort occurred at the PSI domain that is responsible for positioning the binding site between the ligand and receptor [MET mutation we detected is unlikely to be an activating mutation and irrelevant for the response to crizotinib.For the first time, we detected recurrent alterations using WES in CCSA, discovering mutations in n of RAC , suggestosarcoma ,51,52,53e domain , the METreceptor . The bioTSC2 mutations that were detected in three cases in our cohort were known to result in mTOR activation [To broaden the scope of molecular findings, we applied a pathway enrichment analysis to systematically explore mutated CGCs, hopefully identifying overrepresented pathways that are disrupted in CCSA. Our findings indicated that CCSA-associated alterations might be actively involved in transcriptional regulation, SUMOylation, RTK signaling and chromatin organization and modification, as well as MMR. It is not surprising that some of these pathways have already been related to CCSA, such as transcriptional regulation and RTK signaling ,10. Furttivation . HoweverGTF2IRD1 and PEG10) associated with progression in other cancer types [KMT2D deficiency in chromatin organization have been identified as a favorable prognostic marker in small cell lung cancer, and its potential role to enhance the activity of antitumor treatment in pancreatic adenocarcinoma has been shown [In the correlative analysis, we identified a number of associations between the molecular findings and clinical features . The histopathological correlations revealed that a high-level expression of activated MAPK was significantly associated with a better OS, suggesting that MAPK is a favorable predictive marker for crizotinib treatment. However, we should not overestimate the significance of this finding because of the very small sample size. Next, the copy number gains of chromosomes 1q, 7p and 7q (arm level) were seen more frequently in metastatic CCSAs as compared to the primary tumors, suggesting their potential roles in disease progression, which is also consistent with literature, revealing the association between copy number changes and disease recurrence in various human cancers ,60. Receer types ,62. The er types . Alteraten shown ,65. To tThere are clear limitations in our study. Due to the lack of germline samples, the genetic analysis was performed based on computational strategies and applications of public databases , which required further experimental validation to confirm the functional consequences of the described alterations. Secondly, these databases have been validated and extensively used, but molecular findings and interactive pathways are still being accumulated. Updates should be considered if more comprehensive genome-wide analysis data are available in the future. Furthermore, for the identification of relevant alterations, we focused on cancer-related genes (CGCs) and involved pathways that were affected by recurrent alterations, which might have missed the importance of rare or noninterpreted alterations. Finally, because the number of assessable cases was too small to perform more advanced statistical analyses, we should not overestimate the statistical power of the described putative biomarkers. Ideally, the observed correlations should be confirmed by treating and analyzing a validation cohort of additional patients, but due to the orphan characteristics of CCSA, this is difficult to achieve. Nevertheless, our study presents one of the biggest subtype-specific series in this orphan disease. The compilation of existing and newly acquired datasets, that is currently planned, will likely broaden our knowledge of CCSA biology.We performed a comprehensive histopathological evaluation, characterized the MET-signaling pathway and described the genomic alteration landscape in CCSA and were able to illustrate the molecular heterogeneity of this rare malignancy. We correlated the molecular findings with the outcomes of patients receiving an experimental therapy and identified potential biomarkers with predictive values. Our study provides an insight into the biology of this ultra-rare cancer and will hopefully lead to the identification of novel targets to improve the clinical management of this commonly fatal malignancy.


Tursiops truncatus) towards conspecifics is widely described, but they have also often been reported attacking and killing harbour porpoises (Phocoena phocoena) around the world. However, very few reports exist of aggressive interactions between bottlenose dolphins and other cetacean species. Here, we provide the first evidence that bottlenose dolphins in the western Mediterranean exhibit aggressive behavior towards both striped dolphins  and Risso’s dolphins (Grampus griseus). Necropsies and visual examination of stranded striped (14) and Risso’s (2) dolphins showed numerous lesions . Indicatively, these lessons matched the inter-tooth distance and features of bottlenose dolphins. In all instances, these traumatic interactions were presumed to be the leading cause of the death. We discuss how habitat changes, dietary shifts, and/or human colonization of marine areas may be promoting these interactions.Aggressive behavior of bottlenose dolphins ( However, close encounters between species groups can also lead to aggressive interactions between individuals10. An often-reported example of aggressive interactions between cetaceans are the attacks on harbour porpoises (Phocoena phocoena) by bottlenose dolphins (Tursiops truncatus). Such incidents of aggression are reported relatively frequently from UK and western USA waters and occasionally Northwest Spain, and may often lead to mortalities17. Barnet et al.39 also reported that aggression from bottlenose dolphins might have led to the death of at least one Risso’s dolphin (Grampus griseus), one long-finned pilot whale , one striped dolphin , and four short-beaked common dolphins (Delphinus delphis) on the southwest coast of England. More recently, an interspecific kill of a common dolphin by bottlenose dolphins was filmed and analyzed in detail in Northwest Spain18.Many cetaceans form inter-species groups that could benefit in foraging strategies22. However, many hypotheses have been proposed to explain the causal factors behind the aggressive interactions between bottlenose dolphins and other cetacean species, which have been reviewed by several authors23. Aberrant behavior has been proposed, referred as the pattern that is outside the usual behavior for the species. Alternatively, it has been suggested that these interactions may result from bottlenose dolphins practice-fighting with the small porpoises23, or as a practice oriented to the acquisition of skills used in infanticidal attacks23. Other options such as high testosterone levels or sexual frustration have been considered when assessing the possible reasons driving interspecific traumatic interactions23. One usually discarded hypothesis is that dolphins are attempting to predate on the smaller cetaceans, although in most cases there is no evidence of attempted feeding23. However, perhaps the most feasible explanation is that it may be influenced by competition for resources23.Intraspecific aggression is commonly observed in bottlenose dolphin behavior repertories in many different contexts such as male competition, dominance, or female access for copulation, supported by wide field data24. In the open ocean, it is generally accepted that cetaceans rarely display territoriality due to the lack of spatially-defined environmental features that may promote individuals or groups to demarcate their territories and patrol or defend them26. However, territoriality has been hypothesized in resident bottlenose dolphins from Scotland excluding visitors from productive deep waters during winter26. On that way, a potential enabler of predictable resources could be fish farms. These facilities often lead to increases fish biomass in the vicinity of the cages27. In fact, this increase in prey availability induces a greater presence of fish predators, including dolphins. Multiple reports, relate bottlenose dolphins aggregating around aquaculture facilities33, including the present study area where bottlenose presence around fish farms has been reported in greater numbers and all year around compared to other areas in the same study34. If food patches created by aquaculture increase site-fidelity in coastal dolphins, then some individuals or groups could aggressively defend these spatially-fixed resources27. Although the striped dolphin is mainly pelagic, it has also been suggested that, in the Mediterranean, the species is shifting from oceanic to neritic habitats due to prey increases in coastal waters35.In this regard, many cetacean species adjust their distributions patterns in response to variability in food availabilityHere, we report on necropsies from fourteen striped dolphins and two Risso’s dolphins that stranded at different spots along the Valencian Community shoreline. In all instances, aggressive interactions with bottlenose dolphins were proposed as the leading cause of death.37 and state of gonadal development on internal examination, we estimated that of the affected dolphins, 6 were calves (considering until weaning), 3 were juveniles and 5 were sexually mature. Similar lesions were also present on two live stranded female Risso’s dolphins that died within 10 days of rehabilitation associated to infection, traumatic injuries and severe hypovolemic shock. One dolphin stranded in Valencia and the other in Alacant. One of these two dolphins was a calf and the other was a juvenile close to sexual maturity38.We conducted necropsies on 136 dolphins along the 11 years of study, including 13 bottlenose dolphins, 7 short-beaked common dolphins, 2 long-finned pilot whales, 9 Risso’s dolphins and 105 striped dolphins. Of the 105 striped dolphins stranded in the study area, 17 (16%) stranded in the province of Castelló, 29 (28%) in València and 59 (56%) in Alacant. We observed on 14 of the striped dolphins, including eight males and six females, both macroscopic and microscopic compatible lesions, external examination (14/14) and internal (13/14), with bottlenose dolphins’ traumatic interactions that were likely the leading cause of death. Seven striped dolphins stranded in València province and the other 7 in Alacant (24% and 12% of performed necropsies in the area). No cases with similar injuries were found in the province of Castelló. On the basis of growth rates and total lengths published for striped dolphins in the western MediterraneanAll external lesions on both striped and Risso’s dolphins consisted of three to fifteen parallel fresh lacerations (rake marks) that were spaced 1 to 1.2 cm apart  but, according to histopathology of tonsils and lung tissues, it was at an initial infection stage. No individuals tested positive for More details on the size, gender, stranding date, stranding location and main pathological findings are presented in Tables 39. Wounds are considered to be shark-inflicted if they are crescent-shaped, consistent with a shark's jaw. Lesions inflicted by sharks are usually deep, wide-spaced lacerations, which may be coupled with punctures of individual teeth40. Furthermore, many individuals also exhibited evidence of blunt-force trauma to the internal organs that may be attributable to ramming and tail slapping. Such aggressive behavior in bottlenose dolphins towards other cetacean species has been previously reported in other regions 39; however, this behavior is commonly focused towards conspecifics and harbor porpoises44. Other possible causes of traumatic death were ruled out in the striped dolphins in the absence of other external and internal lesions compatible with fisheries interactions, vessel collisions or live stranding47. Here, we report on the first evidence of bottlenose dolphins aggressing striped and Risso’s dolphins in the Mediterranean Sea.We provide evidence from necropsies that suggests that aggressive interactions from bottlenose dolphins led to the death and/or stranding of 14 striped dolphins and two Risso’s dolphins in the western Mediterranean. Supporting this claim, rake marks and lesions that consisted of a series of parallel lacerations that were spaced 1 to 1.2 cm apart were present. These distinctive lesions were likely to have been caused by the teeth of an odontocete and moreover, spacing between the lacerations only matches the inter-dental distance for a single odontocete species in the western Mediterranean—the bottlenose dolphin48. Each species generally inhabits different habitats based on depth50. Specifically, bottlenose dolphin primarily inhabits waters between 0 and 1000 m, striped dolphins between 900 and 1900 m, and Risso’s dolphins between 1500 and 2500 m. Nevertheless, the ranges of these species do still periodically overlap. For example, striped dolphins in the western Mediterranean regularly move over the continental shelf to hunt for neritic prey35 and similar behavior for this species has also been reported in the Ligurian Sea51. Bottlenose dolphins have also been reported to occasionally move away from the continental shelf and into deep-waters to forage49. Such overlap in the habitats of these species opens the possibility for inter-specific interactions. It should also be noted that the strandings associated with inter-specific interactions where often recorded in areas where the continental shelf is narrower  and southern short fin squid (Illex coindetii)35. This is also supported by evidence from boat surveys, which suggest that striped dolphin sightings over the continental shelf have increased in recent years54. Occasionally, sightings of striped dolphins have even been reported in the vicinity of aquaculture facilities48. This could likely bring the striped dolphins into direct competition with the bottlenose dolphins as their primary prey in the Mediterranean is hake55. As for Risso’s dolphins, this species predominantly feeds on squid at depth56.If there is overlap in the ranges of these two species, there is also potential for competition for food resources. While most diet studies of striped dolphins suggest that these species primarily prey on pelagic and/or bathypelagic cephalopods57. Indeed, Bonizzoni et al.29 found a higher occurrence of bottlenose dolphin occurrence within 20 km around fish cages. Furthermore, while territoriality is not a commonly-observed behavior in cetaceans, it has been suggested to be possible in situations concerning localized and predictable food sources26 such as fish farms. Thus, the presence of fish farms could trigger territoriality behavior displayed in bottlenose dolphin against striped dolphin in coastal areas close to aquaculture facilities. Such behavior has already been proposed to be occurring based on a 5-year study of dolphin presence around fish farms in the western Mediterranean27. In support of this hypothesis, the present study revealed that 12 of the 16 strandings reported here occurred within a range of 25 km of the fish farms  of the causes of death in striped dolphins necropsied during 2015. Stranding numbers likely represent only a small fraction of animals that actually die at sea, being strandings estimated to be one order of magnitude below the number of individuals dying at sea61 and CeMV and Brucella ceti are commonly-diagnosed infectious diseases for striped dolphins in the study area63. In fact, one of the animals in this study tested positive for CeMV.Finally, it is worth considering how inter-specific cetacean interactions could affect the risk of infectious disease transmission. Close contact from aggressive interactions would likely increase the possibility for infection via body fluids or tissues exposition . Some infectious diseases could also simply be transmitted by close contact between individualsWe provide evidence suggesting that aggressive interactions from bottlenose dolphins were the primary cause of death for 14 striped dolphins as well as involved in the stranding and death of two Risso’s dolphins. We suggest that a combination of shifting ranges and ecological overlap could have made interspecies interaction more likely. Territoriality around fish-farms has been hypothesized as a possible contributing factor. Nevertheless, it is necessary to further study this phenomenon to determine unequivocally which factors are driving such interactions. Specifically, additional research into how human-induced changes in marine ecosystems could affect cetacean behavior and their interactions with other species will improve our understanding of potential ecosystem effects.Conselleria d'Agricultura, Desenvolupament Rural, Emergència Climàtica i Transició Ecològica of the Valencian Government. Dolphins were encountered stranded along the coastline of the Valencian Community (518 km) in eastern coast of Spain (40°31′N–0°30′E to 37°50′N–0°45′W).Post-mortems of all dolphins were conducted under a collaborative official agreement between the 64, on any well preserved (Code 1 to 3)65 cetacean carcass encountered within the study area between January 2010 and October 2020. The external inspection included examination of eyes, mouth, blowhole, umbilicus, genital opening, anus, and skin. Scars, abscesses, ulcerations, erosions, and wounds were documented, making note of the size , shape, color, texture, location, and distribution. Internal tissues were systematically examined documenting any abnormality as detailed for external examination66. Photographic record was taken from normal external and internal structures and abnormal findings. External lesions compatible with rake marks from conspecifics or interspecific individuals were specifically registered and compared with reports from patterns of lesions reported on the literature67. Fresh tissue samples of brain, spinal cord, heart, lung, kidneys, liver, selected lymph nodes, tonsil, thymus, gastrointestinal tract, pancreas, spleen, urinary bladder, gonads, blubber, skeletal muscle, and skin were fixed in 10% neutral buffered formalin for histopathology, refrigerated for microbiology, and frozen for molecular diagnosis. Molecular diagnosis was also used to test for CeMV68 and herpesvirus69. Finally, serum samples and/or cerebrospinal fluid were collected from live and dead strandings respectively to test for brucellosis using the standard Rose Bengal agglutination test70.Post-mortems were performed, following standard protocols as outlined in Kuiken and Hartmannhttps://qgis.org/es/site/forusers/download.html). Fish farm data was obtained from public repositories on request to local authorities (https://agroambient.gva.es/es/web/pesca/acuicultura).Map was generated with QGIS 3.10.4-A Coruña (