Structure-selective endonucleases cleave branched DNA substrates. Slx1 is unique among structure-selective nucleases because it can cleave all branched DNA structures at multiple sites near the branch point. The mechanism behind this broad range of activity is unknown. The present study structurally and biochemically investigated fungal Slx1 to define a new protein interface that binds the non-cleaved arm of branched DNAs. The DNA arm bound at this new site was positioned at a sharp angle relative to the arm that was modeled to interact with the active site, implying that Slx1 uses DNA bending to localize the branch point as a flexible discontinuity in DNA. DNA binding at the new interface promoted a disorder-order transition in a region of the protein that was located in the vicinity of the active site, potentially participating in its formation. This appears to be a safety mechanism that ensures that DNA cleavage occurs only when the new interface is occupied by the non-cleaved DNA arm. Models of Slx1 that interacted with various branched DNA substrates were prepared. These models explain the way in which Slx1 cuts DNA toward the 3′ end away from the branch point and elucidate the unique ability of Slx1 to cleave various DNA structures. DNA repair is an important biological process that ensures genome stability. Cells rely on an intricate network of DNA repair pathways to counteract various types of DNA damage. One of the most deleterious types of damage is the DNA double-strand break. Double-strand breaks can form when two single-strand breaks are in close proximity, after the attempted replication of nicked DNA or upon replication fork collapse. If double-strand breaks are not repaired, then this can lead to the loss of entire segments of genetic information. Homologous recombination is generally considered an error-free pathway of double-strand break repair because it utilizes a homologous sequence as the template for DNA repair. Homologous recombination invariably involves the generation of joint (branched) DNA intermediates  . The strStructure-selective endonucleases belong to various nuclease families, including the FEN1/XPG, XPF/ERCC4 and GIY-YIG families. As the name implies, these enzymes cleave specific types of branched DNA structures, as opposed to specific DNA sequences. Most of these enzymes have defined substrate specificities and thus play roles in distinct pathways of DNA repair . For exaInterestingly, some SSEs function in a coordinated manner to utilize their distinct substrate specificities. One prominent example is cooperation between XPF-ERCC1, MUS81-EME1 and SLX1, which are brought together via their interactions with the SLX4 platform protein ,20. NuclWe previously reported the mechanism by which the Slx4 scaffold activates Slx1 nuclease. In the absence of Slx4, Slx1 adopts a homodimeric architecture that occludes the active site. Conversely, Slx4 disrupts the inhibitory Slx1 homodimer and forms a catalytically active Slx1–Slx4 heterodimer. Guided by structural similarities between the active site of Slx1 and GIY-YIG type II restriction endonucleases, we proposed a possible mode of DNA binding by Slx1 . In contThe present study investigated the mechanism of DNA binding by Slx1 and defined a new DNA-binding interface. The positioning of this interface explains the way in which the enzyme utilizes helical discontinuity in branched DNA substrates as the main determinant for incision point selection. We propose a mechanism that explains the ability of Slx1–Slx4 to cleave various DNA joint molecules that are intermediates during DNA repair.Candida glabarata Slx1 (Cg-Slx1) and the conserved C-terminal domain of Slx4 (Cg-Slx4CCD) (residues 557–726) were codon-optimized for expression in Escherichia coli and obtained from Bio Basic Inc., Canada. Synthetic genes for Thielavia terrestris Slx1 (Tt-Slx1) and a fragment of Slx4CCD (Tt-Slx4CCD3) (residues 834–936) were codon-optimized for expression in E. coli and obtained from GenScript®. Cg-Slx1 and Cg-Slx4CCD were subcloned into a pET28a vector (Novagen) with an N-terminal 6xHis-SUMO tag. Tt-Slx1 and Tt-Slx4CCD3 were subcloned into a pRSFduet-1 vector (Novagen) using multiple cloning site I with an N-terminal 6XHis tag and multiple cloning site II without a tag, respectively. All point substitutions were introduced by QuikChange Site-Directed Mutagenesis (Agilent Technologies). All proteins (wild type and mutants) were expressed in E. coli BL21 (DE3) Rosetta (Novagen). For protein expression, bacteria were grown in LB medium at 37°C, induced with 0.4 mM isopropyl β-d-1-thiogalactopyranoside at OD600 = 0.7–0.9, and grown overnight at 18°C. The cells were harvested by centrifugation, and pellets were washed with phosphate-buffered saline (PBS) prior to purification.Synthetic genes for Cg-Slx1 and Cg-Slx4CCD (wild type and point substitution variants) was purified as described earlier  and lysed by sonication. The lysate was centrifuged at 186 000 × g at 4°C for 40 min, and the supernatant was loaded onto a His-Trap HP column  that was equilibrated with lysis buffer. Protein was eluted using a linear gradient of imidazole from 5 to 500 mM. The fractions were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Fractions that contained a complex of Tt-Slx1 and Tt-Slx4CCD3 were further purified using a Superdex-200 size exclusion column  in a buffer that contained 20 mM Tris–HCl (pH 8.5), 10% (v/v) glycerol, and 5 mM 2-mercaptoethanol. Fractions that contained the Tt-Slx1–Slx4CCD3 complex were pooled and concentrated using a 30k MWCO Amicon Ultra Centrifugal Filter Device (Millipore).A complex of  earlier . A complTt-Slx1E79Q-Slx4CCD3 at a concentration of 13 mg/ml was crystallized in the presence of either HJs or self-annealing splayed-arm substrate in a buffer that contained 20 mM Tris–HCl (pH 8.5), 100 mM NaCl and 5 mM ethylenediaminetetraacetic acid. The oligonucleotide sequences are presented in The active-site mutant of Tt-Slx1E79Q-Slx4CCD3 was used as the starting model for molecular replacement for dataset 2. Coot was used for iterative model building  were collected at Diamond Light Source at a wavelength of 1.2828 Å. These crystals diffracted to a maximum resolution of 3.2 Å. Diffraction data for crystals with splayed-arm substrate (dataset 2) were collected at beamline 14.1 at Berliner Elektronenspeicherring-Gesellschaft für Synchrotronstrahlung (BESSY)  at a wavbuilding ,27. The lProbity . The dif were col2, 1 mM DTT and 0.5% glycerol. The substrates comprised 200 nM unlabeled DNA substrate and 50 nM DNA that was labeled with fluorescein on X0-1 and Cy5 on X0-4. The samples were then taken out at various time points and stopped by adding equal amounts of 100% formamide that contained Orange G dye and boiling the samples for 5 min at 95°C. The samples were run on a 20% TBE-Urea polyacrylamide gel at a constant power of 18 W for 40 min. The cleavage sites were mapped using fluorescein- or Cy5-labeled DNA marker oligonucleotides of various lengths , 2.5 mM CaCl2, 100 mM NaCl and 0.1 mg/ml bovine serum albumin at 25°C. Binding reactions were set in Corning 96 flat bottom black polystyrene plates. Anisotropy was measured using a Tecan Infinite M1000 microplate reader at an excitation wavelength of 635 nm and emission wavelength of 670 nm.The binding of 5′-flap DNA substrate to Secondary structure analysis was performed using a Bruker Tensor 27 spectrometer. Protein samples were used at a concentration of 0.5–1.0 mg/ml in 20 mM HEPES (pH 7.5) and 150 mM NaCl. The results were analyzed using OPUS-PRO software.CCD) from several fungal species in the presence of various DNA substrates . We obtained two crystal forms of Slx1–Slx4CCD3 from Thielavia terrestris   of 2.3 Å over 142 C-α atoms . The main difference between them was the connection between the GIY-YIG and RING domains. This region formed a long α-helix in Cg-Slx1–Slx4CCD3, whereas it lacked a secondary structure and was partly disordered in Tt-Slx1–Slx4CCD3  . One for Å Table . The ove earlier . The strSlx4CCD3 . The strTt-Slx1–Slx4CCD3 that were grown in the presence of splayed-arm substrate belonged to the P21 space group. These crystals diffracted to a resolution of 2.7 Å. Phases were determined by molecular replacement using the apo structure as the search model. Clear electron densities were observed for DNA that was bound by the protein  and site II . In the new Tt-Slx1–Slx4CCD3-DNA structure, the DNA was located away from the active site and occupied a markedly different position from the position that we predicted earlier. Thus, our Tt-Slx1–Slx4CCD3-DNA structure revealed an additional and novel DNA-binding interface. We designated this newly identified DNA-binding surface as site III.We previously used structural similarities between Slx1 and GIY-YIG type II restriction endonucleases to predict the interface for catalytic DNA binding by Cg-Slx1 . The preapo and Tt-Slx1–Slx4CCD3-DNA structures revealed intriguing structural differences. Two residues that formed important DNA contacts, R111 and R114, were located in a loop that could be visualized in the protein-DNA complex . Further studies will be necessary to confirm this potential mechanism. We also note that the region that comprised residues 97–107 was still disordered in the DNA-bound structure. This region may become ordered when DNA binds to site I around the active site of the nuclease, potentially forming another safety mechanism.Comparisons of the x Figure . In the Tt-Slx1–Slx4CCD3. The DNA molecules from GIY-YIG complexes were used to model nucleic acid binding at and around the active center and site I. This analysis revealed that the two interfaces did not overlap, and the two DNA duplexes were at an angle of ∼50°  is related to the previously postulated interface around the Slx1 active site. We superimposed the catalytic structures of GIY-YIG type II restriction endonuclease on our new structure of DNA-bound ° Figure  and B. S° Figure .Our models explain the way in which Slx1 is able to accommodate various substrates and cleave them in multiple positions. The key feature of Slx1 is that it introduces cuts near the branch point. Our models suggest that this is achieved by bending the DNA substrate, such that Slx1 recognizes the branch point as a flexible discontinuity in the DNA. By interacting with two arms of the DNA substrate, Slx1 is able to bind and cleave various branched substrates.CCD. These experiments were performed using C. glabrata protein, for which substantial structural and biochemical information is available .Structure-selective endonucleases are thought to require tight regulation in cells because the unrestrained DNA cleavage of branched DNA structures would destabilize the genome. This regulation can be achieved by protein-protein interactions, post-translational modifications, and cell cycle-dependent activation, among others ,31. AddiCg-Slx1–Slx4CCD3 and Tt-Slx1–Slx4CCD3 did not reveal any features that could be related to the specific binding of particular DNA structures. We could only identify patches of positively charged residues binding DNA substrate in a specific orientation around the catalytic site to facilitate catalysis. Therefore, an intriguing issue is the way in which Slx1 interacts with various DNA substrates.Slx1 is quite different, in which it cleaves many diverse types of branched DNA structures and at different positions near the branch point ,20,21. IArabidopsis thaliana has been shown to bind branched DNA structures that represent homologous recombination intermediates. No nuclease activity has been observed for the GIY-YIG domain of Arabidopsis MSH1 (Our structural and biochemical work identified three positively charged patches on the surface of Slx1, designated sites I, II and III ( and pressis MSH1 , but thesis MSH1 .Cg-Slx1, which otherwise exists as an inactive homodimer (Slx1 is active only in the presence of Slx4. We previously reported the role of Slx4 in the activation of omodimer . Our stromodimer ,38.Slx1 is a very promiscuous endonuclease and needs to be tightly regulated. Our present and previous  work ideTt-Slx1E79Q-Slx4CCD3 alone and in complex with the DNA were deposited in the PDB under the accession codes 6SEH and 6SEI, respectively.The structures of gkz842_Supplemental_FileClick here for additional data file.


Retroperitoneal sarcomas (RPS) include a heterogeneous group of rare malignant tumours, and various treatment algorithms are still controversially discussed until today. The present study aimed to examine postoperative and long-term outcomes after resection of primary RPS.Clinicopathological data of patients who underwent resection of primary RPS between 2005 and 2015 were assessed, and predictors for overall survival (OS) and disease-free survival (DFS) were identified.P = 0.030) was significantly associated with diminished OS in univariate and multivariate analyses. When assessing DFS, histologic high grade , positive surgical resection margins , and vascular involvement , were significantly associated with inferior DFS in univariate and multivariate analyses.Sixty-one patients underwent resection for primary RPS. Postoperative morbidity and mortality rates were 31 and 3%, respectively. After a median follow-up time of 74 months, 5-year OS and DFS rates were 58 and 34%, respectively. Histologic high grade (5-year OS: G1: 92% vs. G2: 54% vs. G3: 43%, High-grade tumours indicated poor OS, while vascular involvement, positive surgical resection margins, and histologic grade are the most important predictors of DFS. Although multimodal treatment strategies are progressively established, surgical resection remains the mainstay in the majority of patients with RPS, even in cases with vascular involvement. Soft tissue sarcomas (STS) are a heterogeneous group of rare malignant tumours that can occur in almost any anatomic region . WhereasThe retroperitoneum represents a sophisticated anatomical space with multiple vital structures, and therefore RPS is associated with several therapeutic challenges . EspeciaThe objective of this study was to review our recent experience with RPS and analyse postoperative and long-term oncological outcomes. Besides, we aimed to evaluate factors associated with overall survival (OS) and DFS in this cohort and thus identify patients who may derive the most benefit from a multimodal approach including radical surgery.Retrospective single-centre analysis conducted at the Charité – Universitätsmedizin Berlin, Campus Virchow-Klinikum in Berlin, Germany. Following permission from the local institutional review board (EA1/361/14) clinicopathological data of patients who underwent resection for primary RPS between 2005 and 2015 were collected in a prospective database and further reviewed. We excluded recurrent RPS from this analysis, due to divergent management approaches and outcomes. An interdisciplinary tumour board indicated all resections and all patients obtained written informed consent.Standard preoperative clinical assessment included physical examination, serum laboratory testing, imaging studies, and an anaesthesia evaluation. Multiphase computed tomography (CT) with contrast agents or magnetic resonance imaging (MRI) were computed to define the dimension and location of the sarcoma and to assess the involvement of adherent structures. A multimodal therapeutic approach was individually formulated and scheduled by a multidisciplinary tumour board, which consisted of surgeons, medical oncologists, specialised radiation therapists, and radiologists, for every patient. Due to the high recurrence rate and aggressive nature of dedifferentiated liposarcoma, neoadjuvant chemotherapy with/without radiation therapy was individually considered for treatment, depending on the individual patient presentation. Therefore, preoperative tissue diagnosis (89%) of retroperitoneal liposarcoma included percutaneous biopsy (image-guided core needle biopsy (CNB) or fine-needle aspiration (FNA)) in order to facilitate an accurate subtype-specific consideration for neoadjuvant therapy.Perioperative antibiotics [Metronidazole 500 mg (i.v.) and Cefuroxime 1 g intravenously (i.v.)] were routinely given. All procedures were performed in an open surgical approach according to international standards at that time . A midliAll patients were administered and monitored at a specialised surgical intensive care unit for at least 1 day. The Clavien-Dindo classification was used to grade postoperative complications . ComplicP < 0.05 in univariate analysis. A P value below 0.05 was considered significant, and all statistical analyses were conducted using the SPSS software package, version 24.0 .Unless otherwise specified, qualitative and quantitative variables are constituted as medians (range) and numbers (frequencies). Postoperative morbidity, mortality, OS, and DFS were defined as primary outcomes. Survival analysis was determined using the Kaplan-Meier method, calculating OS from the date of resection to the date of death or the last follow-up. DFS contained the period from the date of resection to the date of first recurrence or last follow-up. Log-rank tests estimated the significance of univariate analyses. To identify factors associated with survival after resection of RPS, the following clinicopathological characteristics were recorded and analyzed: patient sex, BMI, patient age at resection, tobacco use, tumor entity, histologic grade, staging according to the AJCC 8th Ed., chemotherapy, radiotherapy, surgical resection margin, vascular involvement, ASA physical status, and need for intraoperative transfusion. Furthermore, a Cox multivariate regression model was executed, including all variables associated with survival with 2 (16–42), and 15 (25%) patients acknowledged consistent tobacco usage. Leiomyosarcoma and dedifferentiated liposarcoma had the highest incidence with 19 (31%) and 14 (23%) patients, respectively. Twelve patients (20%) suffered from well-differentiated liposarcoma, while undifferentiated sarcoma, not otherwise specified, were identified in 13% of all resected tumours. Pleomorphic liposarcoma, malignant peripheral nerve sheath tumours, were detected in 5% of patients, respectively. Histologic grading showed low-grade tumour tissue in 21 (34%) patients, while intermediate grades and high grades were verified in 11 (18%) and 29 (48%) cases, respectively. In terms of the AJCC 8th Ed., the majority of patients (41%) were classified to be Stage IIIB, while Stage IA, Stage IB, Stage II, Stage IIIA and Stage IV were discovered in 3, 28, 7, 13, and 8%, respectively. Forty-nine patients (80%) received no chemotherapy, while ten patients (16%) obtained adjuvant chemotherapy. Two patients (3%) received neoadjuvant chemotherapy. Based on the recommendations of an interdisciplinary tumour board, 44 patients (72%) did not require additional radiotherapy. Four patients (7%) received neoadjuvant radiotherapy, while 13 patients (21%) received adjuvant radiotherapy. In 33 patients (54%) complete tumour removal was achieved. The histological examination discovered persistent tumour cells on the surgical resection margin in 18 patients (30%). In two patients (3%) only an R2 resection could be achieved, while in 8 patients (13%) histological examination did not state sufficient information whether complete tumour resection with surgical margins microscopically negative for tumour cells was successful. Seventeen patients (28%) revealed vascular involvement. In this context, we planned vascular resection in 12 cases already preoperatively. In five cases, preoperative imaging studies underestimated the dimension of RPS and diagnosis of vascular involvement, appeared intraoperatively. The vena cava inferior was the most common vessel involved. In eight cases, segmental resection required a reconstruction by PTFE prosthesis. Within the remaining four cases, direct suture or patch plastic occluded the area of resection. Five patients revealed infiltration of the right renal vein. In all cases, nephrectomy facilitated radical tumour removal. The local recurrence rate of RPS was 41%. ASA physical status included ASA I in 20% of patients, ASA II in 52% of patients, ASA III in 23% of patients, and ASA IV in 5% of patients. In 32 patients (52%) there was no necessity for intraoperative red blood cell concentrate (RBCC) transfusion during the surgical procedure.Between 2005 and 2015, 61 patients with primary RPS underwent open resection at our institution. Table Thirteen patients (21%) required up to two RBCCs during the operation, while 26% of patients demanded more than three RBCCs. Postoperative morbidity, according to the Clavien-Dindo classification ≥3, was 31%, and the 90-day mortality rate was 3%.After a median follow-up time of 74 months 2–131), the median survival of patients who underwent resection for RPS was 38 months. The 5-year OS rate was 58% , and histologic tumour grade (P = 0.015). In multivariate analysis, only histologic tumor grade  independently associated OS (Table Table P = 0.006), histologic tumor grade (P = 0.018), classification according to the AJCC 8th Ed. (P = 0.004), surgical resection margin (P = 0.047) and vascular involvement (P = 0.044). In multivariate analysis, histologic grade , positive surgical resection margins  and vascular involvement , were independently associated with DFS (Table P = 0.044)  program of patients undergoing resection for primary RPS from 1988 to 2005 and reported similar patient characteristics to those reported in our study . GiulianP = 0.015) independently influenced OS. Equivalent to previous findings it might reflect the more advanced nature of tumours with high-grade transformation [Tumour entity and histologic tumour grade of RPS significantly impact on OS in our univariate analysis. However, in multivariate analysis only histologic tumour grade  compared to survival in patients having biopsy only (17%) . It seemTo date, data regarding vascular involvement and oncological outcomes after resection of RPS is still limited. Previous studies established the feasibility and safety of en-bloc vascular resection for RPS and recommended vascular resection and reconstruction to achieve radical tumour removal when needed , 31. HowChemotherapy and radiotherapy did not demonstrate a significant impact on survival. While neoadjuvant therapy is an established treatment for a variety of other tumour entities, evidence on preoperative treatment for RPS is still undetermined, and high-quality trials are needed , 34. TheThe present study is limited by common biases that are mainly due to the retrospective character of this analysis. Furthermore, treatment regimens are still rather heterogeneous. While extensive surgery remains the mainstay of treatment in RPS, conflicting data on the benefit of neoadjuvant and adjuvant therapies exist, and thus interdisciplinary treatment plans are mainly based on individual preference and expertise of the treating institution .In conclusion, in our analysis of 61 patients, we found that patient characteristics of RPS is similar to those of other extensive studies, with liposarcoma and leiomyosarcoma being the most common histologies. High-grade tumours indicated poor OS, while histologic grade, positive resection margins, and vascular involvement are the most important predictors of DFS. Although preoperative and postoperative radiotherapy and chemotherapy did not significantly affect survival, procedures tailored to the individual needs of our patients are the current advancement of choice. While effective adjuvant treatment regimens are continuously developed, surgical resection, even in cases with vascular involvement should be individually attempted in selected patients with RPS. Furthermore, international collaborations are mandatory in order to enhance the management of RPS and guide clinicians in their daily decision-making.


Pharmaceuticals and personal care products (PPCPs) are abundantly used by people, and some of them are excreted unaltered or as metabolites through urine, with the sewage being the most important source to their release to the environment. These compounds are in almost all types of water  at concentrations ranging from ng/L to µg/L. The isolation and concentration of the PPCPs from water achieves the appropriate sensitivity. This step is mostly based on solid-phase extraction (SPE) but also includes other approaches (dispersive liquid-liquid microextraction (DLLME), buckypaper, SPE using multicartridges, etc.). In this review article, we aim to discuss the procedures employed to extract PPCPs from any type of water sample prior to their determination via an instrumental analytical technique. Furthermore, we put forward not only the merits of the different methods available but also a number of inconsistencies, divergences, weaknesses and disadvantages of the procedures found in literature, as well as the systems proposed to overcome them and to improve the methodology. Environmental applications of the developed techniques are also discussed. The pressing need for new analytical innovations, emerging trends and future prospects was also considered. The production and consumption of pharmaceutical and personal care products (PPCPs) is considered an important environmental risk ,2,3,4. IThese considerations on the sources of PPCPs shows the key role that the analysis of water plays to fight against contamination. In fact, water—the most affected environmental compartment—may be considered as a mirror of the pollution status of an area, and also a scarce resource that must be preserved with optimal quality and zero pollution. In addition, water contaminants, depending on their physicochemical properties, may also (bio)accumulate in sediments and biota, consequently harming human health . TherefoDetermining and quantifying PPCPs in different types of water provides considerable interesting information not only related to pollution status. For example, the analysis of wastewater samples, divided into influent and effluent waters, could offer information on the PPCPs consumption of a community, estimate the wastewater treatment plant efficiency and establish the most recalcitrant compounds difficult to eliminate. River, lake, and seawater samples could give us an idea of the more persistence substances. The detection in irrigation channels could identify food quality issues. In addition, drinking water is certainly another matrix that should be monitored to assess the potential risks on human and animal health for long-term use ,19.For an accurate analysis of PPCPs in different types of water, it is fundamental to consider all water characteristics that could influence recovery of the contaminants. The pH could affect the structure of molecule, promote ionization according to pKa, or activate a prodrug with a change of structure. Many substances were thermolabile and photosensitive, for this reason, sample temperature must always be considered. The salinity of water could increase or decrease extraction efficiency due to different ionic strengths of the media, or the formation of molecular complexes between PPCPs and multivalent metal cations present in the samples that are soluble in water . Other wIn addition to the matrix, it is also important to consider the structural variability of PPCPs, designed to interact with specific targets. The presence of distinct functional groups  or the existence of nucleophile/electrophile substituents contribute to all chemical-physical characteristics of each active substance. Influencing stability, reactivity, and solubility in water are all parameters that need to be considered before a sample’s preparation for analysis. Despite the variability in the PPCPs’ chemical structures, for most laboratories specialized in the analysis of these compounds, the use of multiresidue methods is very attractive because not only attains a reduction of cost and time, but also offers global patterns of contamination with only one analysis. Moreover, these methods easily facilitate an eco-friendly analysis with decreases in waste. In these multiresidue methods, the sample preparation becomes the heart of analysis that influences any other procedural steps from sample collection and storage to the specific instruments selected for final quantification (high performance liquid chromatography-mass spectrometry (HPLC-MS), gas chromatography-mass spectrometry (GC-MS), etc.). The choice of a liquid or gas chromatography (LC or GC), to analyze the final extract, is guided by the analytes’ polarity. Generally, compounds with polar characteristics are more suitable for LC, and those with non-polar properties are more amenable to GC; most PPCPs are polar or moderately polar . SurprisTherefore, the goal of this review was to critically analyze the status of sample preparation to determine PPCPs in an all-water matrix. Each step of sample preparation, including all analytical variants  used to detect these contaminants were considered. Furthermore, each sample preparation method was critically analyzed, highlighting advantages and disadvantages. This review performs an examination of all studies published from January 2018 to May 2020. The search was conducted on the database Scopus (Elsevier), with two different inputs: “extraction pharmaceutical environmental”/“extraction personal care products environmental” and “extraction pharmaceutical water”/“extraction personal care products water”. More than one thousand one hundred works have been viewed. The selection criteria to choose the studies were based on (i) the presence at least of 10 PPCPs to include attractive multiresidue methods; and (ii) water compartmentation, in all variants , was chosen. In addition, some reviews outside the interval of time were also chosen.PPCPs are organic compounds, and traditionally this type of compound has been extracted by solid-phase extraction (SPE). This technique was commercialized in the late 1970s and rapidly replaced the liquid-liquid extraction that was previously used . Table 1Only 16% of the studies use other types of methods, such as direct injection, dispersive liquid-liquid extraction (DLLME) (based on liquid-liquid extraction), polyether sulfone microextraction (PES) or buckypaper devices. It is important to note here that many of the methods classified as “other” are based on the basic principles of SPE, but using new phases or formats.This technique involved the use of a small amount of sorbent (commonly hundreds of mg) in a cartridge or syringe barrel. After activation of the sorbent, a water sample of hundreds of mL was passed through the sorbent, which retained the analytes of interest (in this case PPCPs) whereas the water was discarded. Then, the analytes retained in the sorbent were eluted using a few mL of organic solvent. This technique has some advantages, such as the minor investment in reagent and materials, and rapidity.® HLB, Strata®X and Cleanert® PEP). Another polymeric sorbent applied was marked by the presence of polystyrene-divinylbenzene  that was applied to polar analytes, or the presence of octadecyl endcapped silica RP (Supelclean™ LC-18 SPE) that was used for nonpolar to moderately polar analytes from aqueous samples.There are different marketed sorbents that work principally with two distinct separation mechanisms: classical reversed phase chromatography (RP) or ion exchange chromatography (IC). Characteristics of these sorbents are summarized in ® MCX, Strata®X-CW or X-Drug B and UCT XTRACT® XRDAH) were the most prevalent.The IC sorbents were used in the so-called mixed-mode cartridges that combine the polymeric sorbent with an ionic exchanger that could be weak or strong. The IC can be of cations or anions, and this affects the target specificity. Cationic exchange sorbents (weak or strong) were designed to extract basic PPCPs, and anionic exchange sorbent (weak or strong) were to extract the acidic ones. However, weak polymeric cationic-exchangers (OasisN-vinyl pyrrolidone-co-divinylbenzene) (NVP-co-DVB) that improved the average absolute recovery for 44 PPCPs, with respect to the use of HLB sorbents. Alternatively, Caban et al. [Different studies applied attractive modifications to these traditional cartridges, to obtain the best recoveries for a large spectrum of compounds. Zhu et al.  developen et al.  studied n et al.  mixed fon et al.  combinedThe format of the cartridge and the volume of samples that pass through the cartridge were other elements to take into consideration. The sorbent weight (mg), capacity (mL) and pore size (µm) can influence the efficiency of the columns. These parameters have a key role on the surface area, on which analytes interacted. The amount of sorbent ranged from 60 to 600 mg, and the most used was a quantity of 200 mg. The capacities most used were 3 and 6 mL. There is a global consensus on this point.In SPE, the volume of water processed generally involves hundreds of milliliters. The capacity to detect lower amounts is one of the advantages of SPE. The matrix effect (ME) could be an element to take in consideration in the choice of volume because it is directly proportional to volume as well as the organic matter content of the water; in this case, clogging of the cartridge slows the process too much. For this reason, some studies chose different volumes of water (depending on its characteristics) with the same method. For example, influent wastewater samples were generally analyzed with smaller volumes compared to effluent samples ,48.In SPE, the first step is the conditioning of the sorbent in order to favor the interaction of the sorbent with the analytes by a mechanism namely “solvation”. RF and IC sorbents are usually activated by filling the column two or three times with a solvent miscible with water  followed by the solvent in which the analyte is dissolved . Methanol and water are most frequent solvents to activate cartridges. Water used for activation can be pH-adjusted or spiked with salt, counter ions or metal sequestrators . The wash was followed by cartridges air-drying by vacuum or pressure to remove the remaining water, although in few cases, the cartridges can be dried with nitrogen instead of air [Once the sample passed through the sorbent, another important step is to remove the impurities retained on the SPE packing. For this reason, a wash solution strong enough to remove these impurities, but weak enough to leave the analytes of interest, is passed through the cartridge. These solutions are water, pH-adjusted water, or in a few cases methanol-water , PTFE, RC, GHP, PP, etc. The size used were 0.20 and 0.22 μm. This procedure was an ulterior clean-up to remove the particles with a large size to optimize analysis, for example by reducing the presence of obstruction phenomena in column. For this step, it was fundamental to remember which combinations (solvent-filter) are safe and which are corrosive.Regarding the characteristics of the water sample, the preservation of its integrity from the sampling point to the moment of the analysis was vital. To this end, in many cases, the water samples were spiked by preservative compounds or solutions. As described in the U.S. Library of Medicine (2017), a pharmaceutical preservative is referred to as “substances added to pharmaceutical preparations to protect them from chemical change or microbial action” . In the To ensure the proper quantification of the analytes is always very important. Therefore, samples were spiked by a solution of internal standard (IS) in more than 83% of the studies to obtain more reliable results, taking matrix effects into consideration. It was not always possible to use an isotopic reference for every compound, because the cost of the ISs are high, and are not available for some of the target analytes .Another pretreatment widely used in water sample preparation was filtration, the role of which was to remove suspended substances, such as suspended particles, colloids and microorganisms from samples to prevent obstruction of the SPE cartridges or significant interferences in subsequent treatment processes . In variSPE can be used offline  or online (directly connected to the chromatographic system). However, there are few difference in the components of the techniques between the two formats, with the exception of the valves system used to connect SPE online with the determination technique (commonly any type of HPLC-MS). In both, the main factors that affected the results of these technique were the formats and sorbents of stationary phase (cartridge) and the solvent(s) used for activation, washing and elution. Online SPE can be coupled to both, LC or GC. However, the preferred technique is online SPE liquid chromatography tandem mass spectrometry (SPE-LC-MS/MS), PPCPs and the SPE eluents are much more compatible with the mobile phase of LC than with that of GC.To be functional, online systems require the use of 6 or 10 port valves to automate extraction and connection to the instrument. A schematic illustration of the analytical system is presented in The columns for online-SPE mode were characterized by similar polymeric RP sorbents. The main differences, with offline-SPE, were the length of columns . OASIS HLB  ,50,78, MThe online SPE systems present the advantage to provide automatic and efficient sample loading, clean-up, desorption, separation, and detection at the same time, to reduce the sample volume, save time and solvents, prevent sample contamination and PPCP loss, and improve the method performance. The reduction in sample volume that could decrease sensitivity is normally compensated for by the increase in sensitivity as all the analyte retained in the sorbent passes to the chromatographic column.The disadvantage of these methods is that they are not very versatile to be adapted to different types and conditions of analysis, because sample pH, injection volume, and valve-switching time needs to be very carefully optimized to ensure appropriate method performance for target PPCPs . Once esThe disks are a variant of cartridges that follow the same principle to retain an elute PPCPs. The disks have a higher diameter (commonly ca. 45 mm) and low height (a few millimeters). This format attempts to address several disadvantages of the cartridges, such as plugging due to the suspended particulate matter, high back-pressure that reduces flow rates, and improve retention kinetics of the analytes by using lower particle size. Generally, it was used with higher volumes of samples than SPE. The passage of the sample was much quicker (up to 100 mL/min). Moreover, the use of disks was advantageous for handling dirty samples. Only four studies used this approach . There aHydrophilic divinylbenzene (DVB) disks have been compared with Oasis HLB cartridges. Although the most apolar analytes (LogP ≥ 4) attained higher process efficiencies following Speedisk extraction, it could be noticed that in general, process efficiency was lower than for Oasis HLB extraction: 16 versus 59 analytes having a process efficiency >60% for Speedisk and Oasis HLB, respectively . HoweverThe combination of different disks in order to improve recoveries has also been tested for GC-MS amenable analytes using, in sequence, a glass microfiber disk , a styrene-divinylbenzene disk , and an active carbon disk  . HoweverThe main disadvantages, as evidenced in the studies, are related with highest waste of samples and reagents used to active, wash and elute the sorbent.Other approaches have been reported to extract the PPCPs from water, and even though they are not as used as SPE, can be advantageous for some applications. These approaches are commonly focused on more environmentally friendly alternatives that reduce the use of materials, organic solvents and reagents; the so-called green chemistry. The simplest process is direct injection without any pre-concentration steps ,83. ThisThe microextractions, both solid and liquid, are an attractive alternative to SPE. The Dispersive Liquid-liquid Microextraction (DLLME) ,47 has bOther approaches are based on testing new phases consisted of nanomaterials as more efficient sorbents. Tomai et al.  performeThe last approach was the use of a PASSIL sampler . The acrSeventy-six studies have been selected . Nine st−1), TDS , DO , TSS  and BOD . Influent and effluent wastewater samples from hospitals or wastewater treatment plants (WWTPs) were the most analyzed (46% of studies). This matrix was marked by complexity due to the presence of numerous interferents. Some studies investigated the presence of PPCPs in raw wastewater at the treatment plant, some just the effluents, and some investigated both [Different types of water were collected. The principal parameters monitored were temperature, pH, EC . The 40% of works analyzed and studied in this group investigated a large spectrum of water sources: streams, rivers, estuaries, lakes, seas, ground water, and urban and agricultural storm waters. Drinking and tap water (DW) constituted the third group, with an occurrence of 12% in the studies selected. DW was regulated by “The Drinking Water Directive 98/83/EC” that supervises the quality of water (for human consumption). It provided a general framework and a minimum value of 48 specific parameters that must be monitored regularly .Lastly, two works included other two aqueous matrices: thermal and swimming pool water (SP). Chemicals in these matrices can come from different sources, such as bathers, who continuously release organic matter mainly through sweat and urine .The review article focused on the extraction methods for PPCPs. Although these extraction methods are clearly dominated by offline solid-phase extraction using cartridges, there are significant knowledge gaps in accurately understanding the extraction mechanisms for PPCPs, including some metabolite and/or degradation products. Many of the most recent and innovative methods are based on the combination of sorbents with different chemical-physical properties either in the same cartridge, in parallel, or even in series. These modifications are considered to be small steps, but nevertheless, they represent a great advance by improving the extraction of a group of compounds with very different polarities. Multiresidue methods able to cover more than 100 compounds are already a reality. Rapid, lower cost, and eco-friendly sample preparation techniques are urgently needed. Automated, online preconcentration, and clean-up steps prior to the instrument analysis would be the future of the technique. Solvent-free microextraction methods are the trend of extractions such as DLLME. However, these techniques are rarely used, as they work especially well with non-polar PPCPs, but most are polar.Most of the environmental studies carried out so far have two aspects, (i) analytical including validation of the methods that mainly improves the accuracy in the quantification and the elimination of the matrix effects, and (ii) the environmental aspect in which the whole cycle of the water is covered, including the identification of the sources of these compounds to the environment, the efficiency in the elimination, and the influence of environmental factors such as seasonality. All these studies have contributed to an important advance of knowledge about the distribution and hazards of PPCPs. A gap detected in these studies is the lack of knowledge about the mixtures of PPCPs found in the environment and on the different metabolites and/or degradation products that can be present. It is expected that, in the near future, there will be an increase in knowledge in these fields.


Drosophila suzukii Matsumura has become one of the most important invasive agricultural pest insects across climate zones in much of Asia, Europe, North America, and South America. Populations of D. suzukii have demonstrated notable behavioral and physiological plasticity, adapting to diverse environmental and climatic conditions, interspecific competition, novel food sources, and potential predators. This adaptability and plasticity have enabled rapid range expansion and diversified niche use by D. suzukii, making it a species particularly suited to changing habitats and conditions. This article reviews factors and evidence that influence plasticity in D. suzukii and promotes this species’ invasiveness.After its initial discovery in California in 2008,  Plasticity is a driving force behind the spread of numerous invasive species. Plasticity refers to the degree to which traits of individuals or populations can rapidly adapt to new or changing environmental conditions . AdaptatResistance to or tolerance of stressful conditions and a short generation time increase the risk of unintentional transportation and facilitate introduction . Newly iGrapholita molesta (Busck) (Lepidoptera: Tortricidae), which is able to use many apple varieties as oviposition substrates, or the ability to outcompete local species, as in the Asian ladybird, Harmonia axyridis  (Coleoptera: Coccinellidae), which develop more quickly than the North American native ladybird, Adalia bipunctata L. (Coleoptera: Coccinellidae) , available diet, or pressure from predators or competitors . This plellidae) .Drosophila suzukii Matsumura was first described in Japan , acclimation (long-term exposure in a laboratory setting), or acclimatization   . Adult Ds −7.5°C . Acclimas −7.5°C . Acclimas −7.5°C . Thus, es −7.5°C . Five dae levels .D. suzukii to survive temperatures below 0°C for longer periods, withstand chill coma symptoms at lower temperatures, and recover from cold exposure more quickly  to volatiles produced by ripening fruit, there is evidence to suggest D. suzukii also uses differences in leaf tissue volatiles produced during fruit development as a supplemental means to locate potential feeding and oviposition sites  had no oviposition preference as adults between blackberry and American pokeweed , flies reared on American pokeweed preferred to oviposit on blackberry , skin toughness, and acidity (pH) have been investigated most commonly (Drosophila suzukii are able to use a wide variety of host fruits by selecting for characteristics which signal health and ripeness of host fruits and a lack of potential competitors, pathogens, or predators. Other plant characters, such as leaf odors, leaf color, and overall health of plant, have received less attention, but are important supplemental cues used to locate host fruits of healthy plants (Characters associated fruit species have been correlated with suitability for feeding and oviposition behavior of commonly . Other fcommonly . Drosophy plants . Foraginy plants .D. suzukii larvae with critical nutrients, including protein, vitamins, and minerals (Drosophila species, yeast quality affects development, fecundity, and lifespan of D. suzukii (Drosophila species, D. suzukii larvae develop most successfully on foods containing lower ratios of protein to carbohydrates, developing more quickly into larger adults with greater potential fecundity (Larval development time varies depending on larval diet. Larvae feeing on fruits such as cherry, blueberry, or raspberry, develop more quickly than those that feed on standard diet media . Larvae minerals . As with suzukii . Howeverecundity .D. suzukii have been documented feeding on floral nectar, tree sap, and honeydew when other food sources were unavailable (D. suzukii will also oviposit and larvae can successfully develop on less ideal materials, including mushrooms and chicken manure (D. suzukii to novel dietary choices, for both the adult and larval stages, contributes to its invasion success and interact with other aspects of its plasticity.In temperate regions, fruit can be of limited availability or quality as a food source for adult flies and as an oviposition site. In laboratory studies, adult vailable . In the n manure . Thus, tD. suzukii and in behavior of larvae. Chemical cues or signs of previous oviposition by heterospecifics, such as D. melanogaster, can deter female D. suzukii from ovipositing in the same fruit (D. melanogaster sex pheromone, cis-vaccenyl acetate, transferred to fruit during oviposition. In direct interspecific competition situations, D. melanogaster larvae have greater survival than D. suzukii larvae (D. melanogaster larvae than fruit containing D. suzukii larvae (D. suzukii females prefer to oviposit in ripe fruit, they are able to shift ecological niches and use ripening fruit to avoid competitive pressures and reduce potential ethanol exposure to their larvae.Interspecific and intraspecific competition can induce changes in oviposition behavior of female adult me fruit . This mai larvae . This isi larvae . AlthougD. suzukii were not deterred from ovipositing in fruit containing eggs from other D. suzukii females (D. suzukii larvae are less likely to remain within their original host fruits throughout their development. In the absence of competition, larvae remain on or in host fruits throughout pupation. However, as the number of intraspecific competitors within a fruit host increases, larvae move greater distances away from the fruit to pupate (In contrast, female  females . In respo pupate .D. suzukii select fruit for oviposition that contain atropine compounds to infer prophylactic protection to the next generation (D. melanogaster’s use of ethanol laden oviposition sites following detection of parasites and parasitoids (In the presence of parasitoid populations, neration . This isasitoids .Success of an introduced species in a novel environment depends primarily on its ability to adapt and explore its surroundings . SpeciesD. suzukii makes it easily overlooked or misidentified in field settings. Without close inspection, it is easily mistaken for native Drosophilids in each of its invasive regions. Although no voucher specimens exist in mainland American entomological collections prior 2008, it is highly likely that D. suzukii was introduced to North America or Europe long before its first recognized detection in California, but it was not present in sufficient numbers to cause significant crop damage or invite notice (D. suzukii invasion (The relatively small size and inconspicuous nature of e notice . Effectsinvasion .Drosophila suzukii has demonstrated a notable ability to adapt behaviorally, physiologically, and morphologically to new environments. Although each of these responses is of themselves modest in scope, the nature of its adaptive responses is arguably more important to its success as an invasive species than is the extent of any one variable response (Drosophila suzukii has shown itself to be highly opportunistic and it has been able to adapt to a wide range of host plant fruit for rearing its larvae and for adult feeding. As a result of its willingness to explore and test novel fruit species, it has become extremely polyphagous. In addition, D. suzukii has adapted to a wide range of temperature and humidity conditions, through its behavior and through physiological, developmental, and morphological plasticity. As D. suzukii has expanded its range into temperate regions, its ability to adapt to changing environmental conditions and then reverse those adaptations as conditions change through successive seasons as been vital to its success. Plasticity within multiple aspects of behavior, physiology, and morphology has allowed D. suzukii to move from a localized introduced species to an established invasive species over a global range.response . Drosophieaa034_suppl_Supplementary_TablesClick here for additional data file.


Drosophila suzukii, and the anthracnose pathogen Colletotrichum fioriniae are an important insect pest and fungal disease of highbush blueberries, respectively, in the United States. However, whether C. fioriniae infection affects D. suzukii preference and performance remains unknown. Here, we conducted choice and no-choice studies to determine the repellent, oviposition-deterrent, and insecticidal effects of C. fioriniae on D. suzukii. In choice tests, blueberry fruit treated with anthracnose solutions containing spores from either field-collected infected fruit (‘fruit’) or a laboratory C. fioriniae culture (‘colony’) were less attractive to sexually mature D. suzukii females, but not males, than untreated fruit. The plant tissue (fruit or leaves) did not influence C. fioriniae repellency effects on D. suzukii. In no-choice tests, 55% fewer numbers of eggs were laid on, and 65% fewer adults emerged from, blueberry fruit treated with either the ‘fruit’ or ‘colony’ anthracnose solution than untreated fruit. Egg-to-adult D. suzukii survival was also 12% lower on C. fioriniae-infected fruit. No repellency or negative effects on survival were observed when C. fioriniae spores were filtered out of the solution. These findings will help efforts towards the discovery of microbial-derived repellent/oviposition-deterrent compounds that could be used in behavior-based management strategies for D. suzukii.Spotted-wing drosophila,  Drosophila suzukii (Matsumura) (Diptera: Drosophilidae), an insect native to Southeast Asia, has become a major pest of soft- and thin-skinned fruit crops, including raspberries, strawberries, blueberries, blackberries, and cherries1. This frugivorous pest has rapidly expanded its geographic range across multiple continents, which now includes many Asian, North and South American, and European countries4. In the United States, this insect was first detected in the Western states  in 2008 and quickly spread to other states; by 2011, it was found in most Northeastern states3. Several behavioral and morphological features in D. suzukii, such as its attraction to odors from ripening fruit6 and an enlarged, serrated ovipositor, enable gravid females to be attracted to, and oviposit on, fresh fruit in addition to overripe fruit7. These features, among others, have contributed to its worldwide pest status compared with most Drosophila species that prefer rotten fruits and are, thus, not considered pests.Spotted-wing drosophila, 8, understanding complex plant-pathogen-insect interactions could help in the discovery of novel pest management strategies in agroecosystems. Drosophila spp., including D. suzukii, have associations with microbes  that can be mutualistic (where both species benefit)9, commensal (where only one of the species benefits)10, or antagonistic (where one species has a negative effect on the other)11. For example, D. suzukii has a strong relationship with mutualistic yeasts that improve larval survival and development and increase oviposition on fruits12. One example is Hanseniaspora uvarum, the most abundant yeast present in the alimentary canal of wild D. suzukii populations13. This may partially explain the greater attraction of D. suzukii to H. uvarum odors than to odors from other yeasts9; this yeast when present on fruits also increases feeding by gravid females14. Additionally, Drosophila suzukii can facilitate infection by pathogens that often results in fruit rotting, reduction of yield, and loss of marketability15. Indeed, a variety of yeasts and bacteria that cause sour rot in grapes gain access to the fruit’s carbohydrate-rich mesocarp through the feeding and oviposition activities of Drosophila spp.16. However, fruit infection by fungal pathogens can also negatively affect Drosophila spp. For example, D. suzukii larval survival and adult size were significantly reduced when they were reared on raspberries infected by Botrytis cinerea Pers., and fruit infection by this fungus also reduced fly attraction and oviposition11. Therefore, odors from microbes associated with D. suzukii could serve as sources of attractive and repellent compounds to manipulate the fly’s behavior in agroecosystems18.Because plant–insect and plant-pathogen interactions share many features including adaptations and counter-adaptions and negative effects of invasive species on humansVaccinium corymbosum L.) agroecosystem, anthracnose fruit rot is an important disease mainly caused by the fungal pathogen Colletotrichum fioriniae (Sordariomycetes: Glomerellaceae)20, which can result in pre- and postharvest fruit losses23. The primary anthracnose symptoms are rotting of ripe fruit, both in the field before harvest and during storage after harvest, and the appearance of distinctive salmon-colored  droplets on the surface of rotting fruit24. If left untreated during bloom and if favorable  conditions persist, C. fioriniae infestation can result in significant crop losses26. Colletotrichum spp. infection is thought to speed the conversion of acids to sugars as a defense response in blueberries27 because there is a positive correlation between sugar concentration in fruit and fruit rot resistance28. This “overripe” effect of the pathogen on blueberry fruit could stimulate avian and mammalian herbivory31 and also have a positive effect on D. suzukii by increasing adult survival, longevity, and oviposition34. In fact, D. suzukii adults have a strong bias towards carbohydrate‐rich foods35. However, the Chinese chestnut anthracnose pathogen can cause high mortality of the Asian chestnut gall wasp Dryocosmus kuriphilus Yasumatsu36, indicating that interactions with anthracnose might not be beneficial to insects.In the highbush blueberry . Alternatively, if C. fioriniae competes with D. suzukii for resources, we hypothesized that females are repelled by pathogen-fruit-associated odors and avoid ovipositing on infected fruits (hypothesis 2). To address this, we conducted choice and no-choice experiments to investigate the effects of C. fioriniae-infected fruit on D. suzukii attraction, oviposition, and immature performance. Specifically, we asked the following: (i) Are D. suzukii female and male responses to an attractive odor (blueberry fruit) affected by odors emitted from anthracnose (C. fioriniae)? (ii) Do anthracnose solutions from either infected fruit or a laboratory-grown C. fioriniae culture differentially affect D. suzukii adult attraction, oviposition preference, and offspring performance? (iii) Is the effect of C. fioriniae odors on D. suzukii influenced by the plant tissue it infects (fruit or leaf)? In other words, is the effect tissue-specific? (iv) Is there a relationship between D. suzukii adult preference and offspring performance on C. fioriniae-infested fruit? An understanding of this blueberry-C. fioriniae-D. suzukii association constitutes the first step towards the development of behavioral control strategies for D. suzukii based on odors from plant pathogens.In this study, we tested two opposing hypotheses. First, given that D. suzukii adults  were likely driven mainly by chemical rather than visual cues. Symptoms of fruit infection were seen after 7.23 ± 0.28 and 7.05 ± 0.26 days in the 'fruit’ and ‘colony' treatments ; no symptoms were observed in the 'filtered' and 'control' treatments.No symptoms, such as the distinct salmon-colored droplets on the surface of fruits, were observed prior to experiments; thus, any behavioral effects of anthracnose on D. suzukii females and males to anthracnose-infected blueberry fruit odors. When given a choice between blueberry fruit  versus an empty vial (without fruit), 100% of the responding D. suzukii females and males were attracted to the fruit odors   versus untreated blueberry fruit, sexually mature D. suzukii females were 3 times more attracted to the untreated fruit than fruit treated with ‘fruit’ and ‘colony’ solutions . In contrast, no differences in D. suzukii female attraction were found when they were given a choice between fruits treated with a ‘filtered’ C. fioriniae solution (without conidia) and untreated fruit  , ‘fruit’ , or ‘filtered’  solutions compared with the untreated fruit  plus leaves (nonfood tissue) treated with C. fioriniae and fruit plus untreated leaves. Drosophila suzukii females were 2 times more attracted to the fruits with the untreated leaves than to fruits with the leaves treated with C. fioriniae      C. fioriniae odors reduce the attraction of sexually mature D. suzukii females  to attractive blueberry fruit odors, showing that these females are repelled by odors from anthracnose-infected fruit; (ii) the ‘fruit’ and ‘colony’ anthracnose solutions had similar repellent, oviposition-deterrent, and insecticidal activity on D. suzukii females, indicating that the fungal pathogen C. fioriniae is mainly responsible for these effects; (iii) removing the fungal spores (conidia) from the anthracnose solution (‘filtered’ solution) eliminated the repellent effects of C. fioriniae on female D. suzukii; and (iv) the effect of C. fioriniae on D. suzukii was independent of the plant tissue (fruit or leaf).The current study documents four important features of the interactions between the invasive insect pest D. suzukii females were less attracted to odors from blueberry fruits when treated with the anthracnose solutions (‘fruit’ and colony’). There are three possible, not mutually exclusive, mechanisms for these inhibitory effects. First, volatiles emitted from C. fioriniae could directly be responsible for the observed repellency effects. In fact, fungal odors such as geosmin and 1-octen-3-ol38 have been shown to negatively affect D. suzukii host-seeking and acceptance behaviors. Using video observations, Wallingford and collaborators38 found that 1-octen-3-ol prevents D. suzukii flies from coming into contact with the fruit , whereas geosmin inhibits egg-laying behaviors . Second, anthracnose infection could indirectly induce changes in volatile emissions from blueberry fruit, making them less attractive to D. suzukii. Many pathogens successfully exploit their host plants by inducing chemical changes to avoid, circumvent, or silence the hosts’ detection and/or defense systems41. These pathogen-induced chemical changes could affect volatile emissions from the host plant43. Although it needs verification, the fact that we observed repellent effects of C. fioriniae on D. suzukii females regardless of the plant tissue (fruit or leaf) suggests that these effects might not be due to changes in fruit odors but to odors emitted from C. fioriniae itself. Third, C. fioriniae odors could camouflage the blueberry odors, i.e., masking effect, thus preventing D. suzukii flies from detecting  and/or responding to these otherwise attractive odors44. Although testing these mechanisms was beyond the scope of this study, work is underway to isolate and identify the behaviorally active compounds from C. fioriniae-infected blueberry fruit on D. suzukii. Despite that both D. suzukii males and sexually mature females were attracted to noninfected fruit odors, only females were repelled by C. fioriniae odors, indicating that these odors provide information to host-seeking females on the suitability of oviposition sites. Although not tested in this study, we expect these fungal odors to affect (repel) mated females more strongly than virgin females, since the former likely utilize fruit odors for oviposition17. According to Revadi et al.45, D. suzukii females start producing offspring 2.5 days after emergence when kept together with males of the same age. We kept female and male flies together for at least 3 days prior to bioassays, which means that most females were likely ready to start egg laying. Future studies are needed to investigate whether physiological status  of D. suzukii females influences their response to anthracnose odors.Sexually mature C. fioriniae on D. suzukii females, about 20% of the tested flies were not affected by the fungal odors. In our studies, two variables that were constant included the time of anthracnose incubation and its concentration. We used a C. fioriniae incubation period of 48 h because this time of exposure was previously used to test the susceptibility of blueberry species (Vaccinium spp.) to anthracnose46. Previous studies have also shown that fruit rot pathogens, Colletotrichum spp., can successfully infect blueberry fruit after only 10 to 12 h postinoculation47 and induce chemical changes in fruit as early as 24 h after incubation27. Moreover, in preliminary studies, we showed that field-collected anthracnose-infected fruit had levels of spore concentrations similar to those used in our experiment (1.0 × 107 per mL). Although further studies are needed to determine if increasing the time of anthracnose incubation or reducing the spore concentration affects D. suzukii behavior, our study documents for the first time the repellent effects of C. fioriniae under realistic conditions.Despite our choice assays showing clear repellent effects of D. suzukii oviposition was reduced in fruits infected by C. fioriniae. Anthracnose infection is known to increase sugar concentrations in fruits, possibly as a defense mechanism27 because there is a positive correlation between fruit sugar concentration and resistance to Colletotrichum spp.28. Given that D. suzukii females prefer to oviposit on fruit with high sugar concentrations due to their nutritional benefit7, we expected female oviposition preference for anthracnose-infected fruits (hypothesis 1). However, D. suzukii also prefers ovipositing on ripe fruit in which sugar concentrations are typically lower than in rotten fruit48. It is, thus, likely that D. suzukii oviposition preference is not determined solely by the sugar content in fruits49. Infection by many fungal pathogens, including Colletotrichum spp., typically leads to significant changes in the phytochemical composition of not just sugars but also alcohols, pH, and antimicrobial compounds50. These changes may be collectively perceived by D. suzukii females when assessing the quality of fruits during oviposition. It is also possible that females used visual cues to avoid oviposition on anthracnose-infected fruits; however, fruits did not show any signs of spoilage 48 h after infection.Under a no-choice scenario, C. fioriniae that were of lower quality for offspring survival. Certain fungi, such as the yeast H. uvarum, have a mutualistic association with D. suzukii13 and have positive effects on D. suzukii larval development and survival12. In contrast, our study showed an antagonistic  effect of the fungal pathogen C. fioriniae on D. suzukii. Previously, Graziosi and Rieske36 had also shown that infection by Colletotrichum acutatum increases the mortality of the Asian chestnut gall wasp D. kuriphilus. Raspberries infected by the fungus B. cinerea also repelled D. suzukii and reduced larval survival and adult oviposition and size11. Interestingly, our results showed that both anthracnose solutions (‘field’ and ‘colony’), but not the ‘filtered’ solution, have repellent and insecticidal effects on D. suzukii, indicating that removing C. fioriniae conidia eliminates these negative effects. However, some yet unknown components of the ‘filtered’ solution also had some negative effects on D. suzukii oviposition. Compared to the ‘colony’ solution that was simply C. fioriniae, the ‘fruit’ solution was composed of more than one fungal species, although C. fioriniae was by far the most abundant of all species . In fact, C. fioriniae is highly competitive and typically reduces the abundance of other fungal species51. Therefore, it is likely that C. fioriniae competes with D. suzukii larvae for resource when co-exiting in blueberry fruits, which supports our hypothesis 2. The fact that disease symptoms were visible in fruit treated with the ‘fruit’ and ‘colony’ solutions, but not with the ‘filtered’ solution, about a week after infection suggests that fruit became a less suitable host for D. suzukii, which takes ~ 10 days to develop from egg-to-adult at 25 °C52, as the infection progressed.There was a positive correlation between adult preference and immature performance: females avoided fruit infected by C. fioriniae and the insect pest D. suzukii. We showed that C. fioriniae odors, likely emitted from the spores (conidia), elicit a repellent response in sexually mature D. suzukii females and that C. fioriniae infection of blueberry fruits reduces both oviposition and immature survival in this fly. Altogether, these findings open new opportunities for the potential use of odors from the fungus C. fioriniae to manage D. suzukii in fruit crops once the behaviorally active compounds are identified.In conclusion, this study is the first to demonstrate the antagonistic association between the fungal pathogen V. corymbosum; cv. ‘Bluecrop’) from a field located at the Rutgers P.E. Marucci Center, Chatsworth, New Jersey (USA), were used for laboratory experiments. Three fruit clusters per bush were bagged at fruit set  to prevent pesticide exposure and natural insect infestation and considering that fruits are susceptible to D. suzukii once they start to color7. Fruit was harvested during maturation  from mid-June through mid-July, placed in clear polyethylene bags, transported to the laboratory, and used for the experiments. For experiments with leaves, green, healthy, mature highbush blueberry leaves  were collected from greenhouse-grown potted plants. Prior to experiments, the surface of fruits and leaves were thoroughly cleaned with sterile deionized water and then dried at room temperature (~ 25 °C).Highbush blueberry fruit  with ~ 15 mL of diet and plugged with BuzzPlugs (Fisher Scientific) and were kept in an incubator  set at 25 ± 1 °C, 65 ± 5% relative humidity (RH), and 16:8 light:dark cycle (L:D). Male and female flies used in the experiments were 3–7 days old and, thus, were sexually mature45. Flies were removed from the colony ~ 5 h before the start of each experiment.C. fioriniae culture consisted of a single spore isolate (isolate BB#10)19 collected in 2005 from infected blueberry fruit at the Rutgers P.E. Marucci Center and was stored on corn meal agar . High-density sporulation of the isolate was induced by streaking conidia onto clarified V8 juice agar54 with no calcium carbonate and a higher concentration of agar (28 g/L)19. Cultures were incubated at 25 °C in the dark for at least 7 days before spore suspension preparation.The laboratory C. fioriniae culture . For the ‘fruit’ treatment, blueberry fruit showing symptoms of anthracnose, i.e., salmon-colored  droplets on the surface of rotting fruit, were collected at the Rutgers P. E. Marucci Center and incubated in plastic containers (15 × 15 × 5 cm) at room temperature (~ 25 °C) to promote sporulation. Once conidia were abundant , fruit were placed into a plastic screen mesh filter and repeatedly submerged into sterile deionized water. Once conidia were liberated, the spore concentration was estimated using a hemocytometer and adjusted to 1.0 × 107 spores per mL of deionized water immediately before the beginning of experiments. This concentration was chosen because it was similar to the concentration of spores from the field-collected infected fruit samples. For the ‘colony’ treatment, C. fioriniae spore suspensions were prepared by washing conidia from the high-density cultures into deionized water and adjusting the spore concentration to 1.0 × 107 spores per mL of deionized water, as described for the ‘fruit’ treatment, immediately before their use in experiments. In addition, to remove the conidia from any extracellular enzymatic compounds, a ‘filtered’ solution was prepared by sterile filtering  the ‘colony’ spore suspension described above immediately before use . Treated fruit were left to incubate at room temperature for 48 h before use in choice and no-choice assays (see below) to allow the solutions to settle on the fruit surface; this anthracnose incubation period is sufficient to activate defensive genes and induce secondary metabolite production in blueberries27. A group of 5 fruit from each treatment was set aside to confirm C. fioriniae infection by checking for symptoms (only fruit treated with the C. fioriniae ‘fruit’ and ‘colony’ solutions were symptomatic).Anthracnose spore suspension treatments were prepared using two sources: field-collected infected fruit (referred to as ‘fruit’) and a laboratory C. fioriniae-infected fruit on D. suzukii female and male behavior. Flies were given a choice between untreated fruit (control) and fruit treated either with the ‘fruit’ solution, ‘colony’ solution, or ‘filtered’ solution. In addition, flies were given a choice between untreated fruit and a blank control to determine their attraction to fruit in the absence of other odors. The experimental setup followed similar methods used by Feng and collaborators55. Choice arenas consisted of clear plastic cups . The lid of each cup had an 80-mm diameter circular hole cut in it, which was covered with a nylon mesh  to provide ventilation while retaining flies. Polystyrene tubes  were labeled for the treated and untreated fruit. Equal quantities (~ 5 g) of blueberry fruit (N = 5) were used for all treatments, and fruits were placed inside of each tube. Fruits were sprayed with their corresponding treatment until run-off (approx. 1 mL of solution per fruit); untreated fruits were sprayed with deionized water only. Each pair of tubes in each choice test was sealed with Parafilm® , with a 4-mm-diameter hole in the center of the Parafilm to provide an entrance for the flies, and placed vertically on opposite sides of a cup. A cotton wick moistened with deionized water was placed on the bottom of each cup as a water source for the flies during the experiment. Ten D. suzukii  were released inside each cup (choice arena), and each choice test was replicated 10 times (N = 100 flies tested per choice test and sex). Testing flies in groups does not appear to affect their behavior in previous studies using similar choice tests55. The number of D. suzukii flies inside and outside the tubes in each choice arena was counted after 24 h. The experiment was carried out under a fume hood at 25 ± 1 °C, 60 ± 5% RH, and 16:8 h L:D.Choice assays were conducted to investigate the effects of odors from D. suzukii to C. fioriniae-infected fruit odors was mediated by changes in fruit odors. To address this, females were given a choice between blueberry fruit (food tissue) plus leaves (nonfood tissue) treated with the ‘colony’ solution versus fruit plus untreated leaves. Note that C. fioriniae primarily affects fruits but can also attack all other aboveground parts, including leaves. Only females were used in these experiments because males did not respond to C. fioriniae odors (see “N = 5) plus leaves  treated with “colony” solution and (ii) a tube containing the same amount of fruit and leaves but the leaves were treated with deionized water only. Leaves were sprayed with their respective treatment until run-off. Ten D. suzukii females were placed inside each choice arena, and the experiment was replicated 10 times (N = 100 flies tested). The experiment was conducted under a fume hood under the conditions described above. The number of D. suzukii females inside and outside the tubes within the arenas was counted after 24 h.We conducted choice tests to determine if the response of ors see “” sectionC. fioriniae infection on D. suzukii oviposition and adult emergence. Blueberry fruit were placed individually in 118-mL (4 oz) translucent plastic cups  and simultaneously exposed to one D. suzukii sexually mature female. Each fruit was treated with one of the four treatments described above: fruit’ solution, ‘colony’ solution, ‘filtered’ solution, or untreated control. A cotton wick moistened with deionized water was placed on the bottom of the plastic container as a water source. After 24 h of exposure, D. suzukii females were removed and all eggs laid in the fruit were counted under a stereomicroscope. Note that D. suzukii eggs are visible on the surface of fruit skins by observing the oviposition hole and two white breathing filaments protruding out of the egg7. All fruit containing eggs were subsequently stored in the cups with filter paper at room temperature (~ 25 °C). The number of emerged adults was recorded after14 days, which allowed enough time to complete egg-adult development56. As a measure of immature performance, for each fruit, we calculated the percent egg-to-adult survival by dividing the number of emerged adults by the number of eggs laid multiplied by 100. Each treatment was replicated 25 times.No-choice studies were conducted to determine the effects of D. suzukii flies inside each tube were recorded and the differences in D. suzukii response between treatments compared using paired t-tests. Flies that did not make a choice were considered as nonresponding and were not included in the statistical analysis. Choice data are shown as percentages in Figures.In choice experiments, for each cup (arena), data on the total number of D. suzukii oviposition, adult emergence, and percent egg-to-adult survival (immature performance) was analyzed using generalized linear models (GLMs), followed by Bonferroni post-hoc tests. For oviposition and adult emergence, we used a Poisson distribution with a logit-link function. To analyze the percentage egg-to-adult survival, data were first arcsine transformed, and then analyzed using a normal distribution with identity link function. All statistical analyses were performed using SPSS Statistics 23.0 .In no-choice experiments, the effect of treatments on


In vitro, adhesion depended on fibrinogen and coagulation factor XIII (FXIII), and supraphysiological FXIII improved adhesive strength. Furthermore, when exogenous FXIII was topically applied into the wound pocket of rats, eleven adhesive failures occurred between eight rats, compared to seventeen adhesive failures between eight untreated rats, whereas the number of cohesive failures remained the same at sixteen in both groups. In conclusion, rebleeding from both adhesive and cohesive failure of clots decreases survival from hemorrhage in vivo. Both endogenous and exogenous FXIII improves the adhesive strength of clots.The adhesion of blood clots to wounds is necessary to seal injured vasculature and achieve hemostasis. However, it has not been specifically tested if adhesive failure of clots is a major contributor to rebleeding and what mechanisms prevent clot delamination. Here, we quantified the contribution of adhesive and cohesive failure to rebleeding in a rat model of femoral artery injury, and identified mechanisms that contribute to the adhesive strength of bulk clots in a lap-shear test in vitro. In the rat bleeding model, the frequency of clot failures correlated positively with blood loss (R = 0.81,  During cohesive failure, the sealant fractures internally from forces that disrupt structural integrity within the clot. Whereas during adhesive failure, the sealant remains mechanically intact, but its substrate interface is disrupted. It is crucial to understand the mechanisms behind the failure of blood clots as it may inform approaches to mitigate rebleeding during severe hemorrhage, but it is not known if adhesive failure contributes to hemostasis and rebleeding in vivo.Blood clots, like all sealants, fail either by cohesive failure or adhesive failure2. Rebleeding typically occurs when blood pressure exceeds the mechanical stability of the clot. This can occur when blood pressure increases during fluid resuscitation2. The failure of clots and volume of blood loss from rebleeds can be exacerbated by trauma-induced coagulopathies, including hyperfibrinolysis and the depletion of coagulation factors3, as well as physical perturbation of the clot during patient transport4. The destabilization of blood clots and subsequent rebleeding correlates with poor survival in trauma patients2. The critical stress a clot can withstand before internal fracture, its cohesive strength, is positively correlated with its elastic modulus, a measure of its stiffness, as measured by a clinically-utilized technique, thrombelastography (TEG)5. While the factors that contribute to clot stiffness have been extensively characterized, such as by using TEG7, the adhesive properties and mechanisms of clots, examined in bulk rather than examining individual components in isolation, are less characterized.Blood coagulation is typically effective at sealing damaged blood vessels to achieve hemostasis, but blood clots can fail during severe hemorrhage, leading to rebleeding and secondary hemorrhage. Rebleeding is common during severe hemorrhage, normally occurring many times before definitive hemostasis is reached8. Fibrin is formed by proteolytic cleavage of fibrinopeptides of its precursor, fibrinogen, during activation of the coagulation cascade. The fibrin monomers then self-assemble to protofibrils, which aggregate into a mesh-like structure10. This fibrin polymer can both directly crosslink and mechanically interlock with extracellular matrix (ECM) proteins in tissue. FXIII is present in plasma as a heterotetrameric proenzyme (FXIII-A2B2) and is converted to the activated transglutaminase  by thrombin in the presence of Ca2+. FXIIIa mediates the covalent crosslinking of fibrin to ECM proteins. FXIIIa also crosslinks fibrin to antifibrinolytic proteins, such as α2-antiplasmin11, as well as fibrin subunits to each other, which increases the density of the fibrin network12. Both of these processes stabilize the blood clot against premature fibrinolysis, and clinical observations indicate that a low FXIIIa concentration in blood is correlated with hyperfibrinolysis in trauma patients13. Platelets are adhesive to materials in the subendothelial space, including collagen and von Willebrand Factor (vWF), through receptors such as glycoprotein (GP) VI and the GPIb-V-IX complex15. While fibrin, FXIIIa and platelets all have adhesive properties, it is not known which of these components contribute to the adhesive strength of the bulk clot. We hypothesized that rebleeding can occur due to adhesive failure, and that supplemental FXIII could increase the adhesiveness of clots during bleeding. To confirm whether the delamination of clots from wound tissue leads to rebleeding, we analyzed the modes of clot failure in a rat femoral artery bleeding model. To test if depletion of FXIII and other clot components would decrease clot adhesiveness, we measured adhesive clot failure of depleted plasma ex vivo, and also tested if reintroducing exogenous FXIII would recover adhesion.Several components of blood are known to have adhesive properties. Fibrin and coagulation factor XIII (FXIII) have been used as surgical sealantsExperiments involving rat models were approved by the University of Washington Institutional Animal Care and Use Committee (IACUC). All experiments were performed in accordance with IACUC guidelines and regulations.Experiments involving human participants were approved by the Human Research Ethics boards of the University of British Columbia (UBC) and the Canadian Blood Services (CBS). All experiments were performed in accordance with UBC and CBS guidelines and regulations. Informed consent was obtained from all healthy human volunteers prior to whole blood donation.All donors were deidentified. The recruitment criteria were inclusive of both sexes.16. Wild-type male Sprague–Dawley rats weighing between 300–450 g were used. Rats of only one sex were used to reduce sex-related variability in this pilot study, but should be extended in the future to involve both sexes. The rats were anesthetized with ketamine and xylazine and maintained under isoflurane for the duration of the protocol . Before injury, the carotid artery and jugular vein were catheterized to monitor blood pressure (BP) and to infuse saline, respectively. A tracheotomy was performed and a small polyethylene tube was inserted to aid respiration using a mechanical ventilator. The left femoral artery was isolated and clamped proximally and distally. A wound pocket of roughly 2 mL in volume was made at the incision site to access the femoral artery prior to isolation and incision of the artery to induce bleeding. A 3-mm longitudinal incision was then made in the anterior wall of the femoral artery segment between the clamps to allow for free bleeding. Prior to releasing the clamps, blood was drawn from the carotid artery through the inserted catheter to standardize starting blood pressure to as close to 50 mmHg as possible  to prevent exsanguination and standardize the initial bleed volume. Following catheter hemorrhage, clamps were removed to initiate bleeding from the wound, and this time was designated t = 0 min. Solutions containing the test reagents  were applied immediately into the wound pocket and allowed to mix with the forming blood clot while still in its liquid phase. This FXIII concentration was chosen to correspond with a concentration that improved clot adhesion in vitro, accounting for the volume of the wound pocket and potential loss of the exogenous FXIII from the wound pocket during the initial bleeding. After 5 min of free bleeding to allow clot formation, fluid resuscitation was initiated. Saline (0.9%) was infused intravenously at 3 mL/kg/min to raise mean arterial pressure to 60 mmHg as needed. The wound pocket was video recorded continuously starting at t = 0 min. Each video was viewed by two blinded observers to review each rebleeding event to determine whether the clot failed by adhesion (from the edges) or cohesion . Pre-weighed cotton gauze was used to collect blood spilling over the wound to track blood loss every 5 min. The blood loss attributed to each clot failure event was estimated as the blood loss within the 5-min interval in which the failure occurred. When more than one failure occurred in the 5-min interval, blood loss was divided equally among the failures. Survivors were euthanized under anesthesia if surviving up to 80 min or if arterial wave form was lost and mean arterial pressure confirmed to be < 20 mmHg, whichever occurred first. Animals that did not survive the initial catheter hemorrhage were excluded from the analysis.Test reagents were applied to a rat model of femoral artery injury to evaluate their effects on clot failure mode and hemostatic efficacy. The procedure for the rat model was modified from a previous studyg for 20 min to separate the RBC from platelet-rich plasma (PRP). The RBC fraction was centrifuged at a higher speed of 500 g for 5 min to separate residual plasma from the RBC. The RBC fraction was then washed twice with phosphate buffered saline . In experiments that investigated the effect of RBCs on clot adhesion  (BD Biosciences). The blood was centrifuged at 100×2, 1.1 mM MgCl2, 2.7 mM KCl, 137 mM NaCl, 0.4 mM NaH2PO4, 10 mM HEPES, 5.6 mM D-glucose, pH 6.5) to prevent platelet activation prior to the start of the experiment. The platelets were centrifuged at 250 g for 10 min for a final wash. The buffer was removed and the platelets resuspended in fresh Tyrode’s HEPES and counted by a XN-550 Automated Hematology Analyzer (Sysmex). Platelet concentration was adjusted by the addition of more Tyrode’s HEPES as specified in the protocol for each experiment in Supplementary Table Platelets were isolated from platelet-rich plasma (PRP) that was processed by the CBS from whole blood pooled from four deidentified female and male donors. The PRP was stored at 22˚C with mild shaking on a small platelet incubator and agitator  and used within five days of the blood draw. To extract platelets, the PRP was centrifuged at 250 g for 20 min to pellet the platelets. The platelet-poor plasma (PPP) was removed from the pellet and the pellet was resuspended in citrate glucose saline buffer . The platelets were again centrifuged at 250 g for 10 min, the buffer removed, and the platelets resuspended in Tyrode’s HEPES buffer  for 103 min. The A405 increased with increased turbidity as the clot became insoluble through fibrin formation and then decreased as the clot was lysed17. Clots were measured until complete lysis, defined by when the absorbance of all reactions had reached the baseline level as indicated by the no-calcium negative control.Lysis was measured by transmission spectrometry. Plasma samples (100 µL) with or without tissue plasminogen activator (tPA)  were added to a 96-well microplate using the recipes listed in Supplementary Table t test was used to calculate the p values in specific comparisons between two groups of interest. SEM denotes standard error of mean, and is defined as the standard deviation of the data set divided by the square root of the number of replicate values recorded.Normality was assumed for all data sets but could not be verified due to low sample sizes. A Pearson (R) calculator was used to calculate the Pearson’s correlation coefficient and p value for correlation analyses. One- and two-way analyses of variance (ANOVA) and the two-stage Benjamini, Krieger, and Yekutieli procedure, with adjusted p values for multiple comparisons, were used to calculate the p values between multiple groups. A Student’s To determine if rebleeding occurs in a rat model of lethal hemorrhage, intravenous fluids were administered following a femoral artery incision to challenge the hemostatic capacity of the formed clot. Blood loss was quantified every 5 min by weighing the bleed volume soaked by pre-weighed gauze Fig. a,b. Fail20. To test if platelets contribute to the adhesive strength of bulk clots to collagen in vitro, PRP clots were prepared by supplementing platelet-poor plasma  with washed platelets for final concentrations that mimic the low (170 × 109 cells/L), normal (340 × 109 cells/L) and high (690 × 109 cells/L) physiological concentrations in blood. Adhesive strengths of PPP clots and PRP clots at low and normal platelet concentrations were not significantly different. At high platelet concentrations, PRP clots were 52% more adhesive than PPP clots  and FXIII on clot adhesion to collagen in vitro, clots were formed between collagen coverslips for 1 h and subjected to a lap-shear test until adhesive failure occurred. The concentrations of Fg were varied by supplementing purified Fg to Fg-poor PPP (PPP with less than 0.1 g/L fibrinogen). Supplementing with Fg increased the adhesive strength in a concentration-dependent manner Fig. a. This rmin Fig. c. Lap shmin Fig. . To testmin Fig. d. Clot smin Fig. e. Whole min Fig. f.Figure 2B2 was chosen for this study, rather than FXIIIa, to ensure FXIIIa was only generated at the site of coagulation and minimize off-target crosslinking of proteins by purified FXIIIa. This treatment was compared to the vehicle control of 0.9% saline, using groups of eight rats. Blood in the systemic circulation of the rats was diluted by intravenous administration of saline, reaching approximately 53% and 55% of the original concentration after 20 min in the control and FXIII groups respectively  was added directly to the wound pocket of rats subjected to femoral artery puncture prior to fluid resuscitation Fig. a,b. FXIIBlood clots seal injured vasculature, and thus clots must adhere to the wound site to achieve hemostasis. However, no studies had previously quantified adhesive failure in wounds and correlated them to survival, and it was not clear what mechanisms contribute to clot adhesive strength. This work shows for the first time that adhesive failures are common. We found that adhesive failures accounted for 51 ± 14% of rebleeds in untreated control animals. The in vitro results also highlight the important contribution that FXIII plays in preventing adhesive failure of the clot, presumably by enhancing the attachment of fibrin to ECM proteins.5. Our studies that measured clot stiffness and lysis were specifically designed and performed in order to have direct comparisons to clot adhesion. Comparing the results from the lap-shear adhesion assay and the TEG assay showed that the stiffness of a clot was not directly correlated to its adhesive strength. For example, while platelets dramatically increased clot stiffness by approximately 16-fold, consistent with previous reports on the contribution of platelets to stiffness21, the clot adhesive strength to collagen was unchanged by platelets at the same concentration, and only increased by 1.5-fold at twice this concentration. This reflects the importance of platelets in clot cohesion through aggregation and binding of cells and proteins in blood22. While adhesives require cohesion to function, adhesive strength is also affected by the interactions between the adhesive and its substrate23. While platelets had only a mild effect on the adhesive strength of a formed clot 60 min after initiating clotting, they did contribute to adhesion at a very early stage of coagulation; PRP had higher adhesion than PPP at 2 min. This may have been due to platelets quickly binding to the adherend and enabling the initial attachment of blood components to collagen, adhering through receptors such as glycoprotein VI and Ia/IIa25. It is important to note that the adhesion test used here was a static system. In microfluidic studies, platelets are important in mediating the initial attachment and subsequent clot formation on collagen surfaces under flow26, and platelets may have a greater impact on mature clots under flow. RBC count did not affect the clot adhesive strength. This was similar to the effect of RBC on clot stiffness. It is known that hematocrit levels do not improve clot stiffness as measured by thromboelastrography20. This could be due to the deformability of RBC27. In conditions where RBC become less deformable, such as sickle cell disease, clot stiffness increases with RBC concentration28.The stiffness of blood clots as measured by thrombelastography correlates with clot cohesive strength and is used often to effectively predict the severity of bleeding30. Weak clot adhesion occurred in PPP deficient in FXIII, but supplementing FXIII increased the clot’s adhesive strength. In particular, a high dose of FXIII (30 µg/mL) increased clot adhesive strength by roughly 3-times compared to a lower physiological concentration (10 µg/mL). Consistent with previous reports31, clot stiffness also increased by adding FXIII, but the high dose of FXIII only increased clot stiffness moderately (27%) compared to the lower physiological concentration tested. We found that concentrations of FXIII that achieved only modest increases in clot stiffness led to large increases in adhesive strength. This highlights that in addition to increasing clot cohesion through interchain crosslinking of fibrin strands, FXIIIa plays an important role in enhancing the attachment of fibrin to wound tissue. Whole blood clots were more adhesive to extracellular matrix proteins, collagen, fibronectin, and laminin, compared to plain glass, indicating that both the blood composition and the substrate to which the clot is adhered affect clot adhesion.We observed that clot adhesive strength can be affected by both its intrinsic composition and the interactions between the clot and the substrate to which it is adhered. Both fibrin and FXIII improved clot adhesion to collagen, in the absence of platelets, in a dose dependent manner. This suggests that perhaps the primary contributions of fibrin and FXIII on enhancing clot attachment to the collagen surface was independent from the known role of platelets in mediating fibrin-collagen interactions. Unsurprisingly, fibrin was essential for clot stiffness32.After showing in vitro that supplementing FXIII enhanced clot adhesion, we tested if supplementing FXIII directly into the wound site would prevent adhesive failure in our rat hemorrhage model. Of rats that received FXIII, more survived, fewer adhesive failures and lower blood loss occurred compared to control animals. While these comparisons between the control and FXIII-treated groups of rats were not statistically significant with this sample size of eight rats per group, these in vivo results warrant further investigations into the effects of FXIII on clot adhesive strength in future studies with a larger sample size. Models of hemorrhage typically have high variability in survival rate and blood loss. The model used in this study also showed high variability in the mode of clot failures. A larger sample size would be needed to better distinguish the effects of exogenous treatments from intrinsic differences between animals and injuries. Future studies using rats with a genetic knockout of FXIII would also be useful for testing the physiological contribution of FXIIIIt is probable that clot contraction influences adhesion of clots, but this was not specifically tested in this study. Inhibitors of clot contraction, eptifibatide and blebbistatin, did not influence adhesion in the specific in vitro assay used here. In vivo, some shrinkage of the clots occurred even though the wounds were held open, although we did not detect differences in clot contraction between the treatment groups.In conclusion, the bulk adhesion of clots contributes to hemostasis in vivo. Clot failures were associated with blood loss and poor survival, and adhesive and cohesive failures were equally common. Bulk clot adhesion is controlled by fibrin and FXIII, but only minimally by platelets in vitro, demonstrating that clot stiffness and clot adhesion do not have a linear relationship. This highlights the importance of mechanisms of clot adhesion to hemostasis and encourages the development of therapeutics for bleeding disorders or hemorrhage that enhance these innate mechanisms.Supplementary Information.