All of them showed more potent inhibition than a commonly used positive control, d-saccharic acid 1,4-lactone, with the IC50 values ranging from 1.2 µM to 23.1 µM. Inhibition kinetics studies indicated that compound 1–3 were competitive type inhibitors for EcGUS. Molecular docking studies were performed and predicted the potential molecular determinants for their potent inhibitory effects towards EcGUS. Structure–inhibitory activity relationship study revealed that chloro substitution on the phenyl moiety was essential for EcGUS inhibition, which would help researchers to design and develop more effective thiazolidin-2-cyanamide type inhibitors against EcGUS.Gut microbial β-glucuronidases have the ability to deconjugate glucuronides of some drugs, thus have been considered as an important drug target to alleviate the drug metabolites-induced gastrointestinal toxicity. In this study, thiazolidin-2-cyanamide derivatives containing 5-phenyl-2-furan moiety ( Interestingly, all these types of compounds contain an extremely similar moiety, a phenyl group substituted with a heterocycle. Molecular docking studies further demonstrated that both the phenyl and heterocyclic groups interacted with the corresponding pocket residues via π–π stacking, and the heterocyclic nitrogen, sulphur and/or oxygen increased hydrogen bonding capability of these compounds for pocket bindingXanthomonas oryzae pv. oryzae on the rice cultivar IR24Interestingly, recent studies demonstrated that gut microbial β-glucuronidase inhibitors, such as p-Nitrophenyl-β-d-glucuronide acid (PNPG) was used as the probe substrate for EcGUS. Then the inhibitory behaviours of these compounds against EcGUS were characterised and their structure–inhibitory activity relationships were explored. Finally, molecular docking studies were performed to predict the molecular determinants of thiazolidin-2-cyanamide derivatives against EcGUS.Therefore, in this study, 13 thiazolidin-2-cyanamide derivatives containing 5-phenyl-2-furan moiety were selected and subjected to evaluate their inhibitory effects on EcGUS. 2.2.1.p-Nitrophenyl-β-d-glucuronide acid (PNPG), d-saccharic acid-1,4-lactone (DSL), and dimethyl sulfoxide (DMSO) were supplied by Sigma-Aldrich . Dulbecco’s phosphate-buffered saline (PBS) was provided by Life Technologies . Imidazole, kanamycin, and isopropyl β-d-1-thiogalactopyranoside (IPTG) were supplied by Biosharp . Recombinant E. coli BL21 (DE3) harbouring pET28a-EcGUS was generously provided by Prof. Ru Yan from the University of Macau . Deionised water was purified by a Milli-Q purification system . Purities were all >98%.Thirteen thiazolidin-2-cyanamide derivatives containing 5-phenyl-2-furan moiety were provided by Prof. Zi-Ning Cui from South China Agricultural University . 2.2.,Supplementary Materials). The structures were also further confirmed by X-ray single-crystal analysis and a perspective view of the compound 6 (CCDC No.: 1565820) was shown in The synthetic route of title compounds was shown in 2.3.EcGUS was prepared according to our previous report2.4.Thirteen thiazolidin-2-cyanamide derivatives were subjected to screening for potent EcGUS inhibitor. The inhibitory effects of these compounds were determined by measuring the PNP formation generated from PNPG by EcGUS. Briefly, the assays were conducted in 96-well flat-bottomed tissue culture plates  with a total volume of 100 µL which consisted of 10 µL EcGUS , 70 µL PBS buffer (pH 7.4), 10 µL test compound or DSL  and 10 µL PNPG . DSL was used as a positive control, and 1%DMSO was used as blank control. All reactions were performed in triplicate and the produced PNP was monitored by measuring absorbance at 405 nm in a plate reader  after incubating at 37 °C for 30 min. The PNP concentration was determined from a standard curve  in PBS buffer (pH 7.4). Relative activity was defined as a percent of PNP formation in the presence of test compound within 30 min compared with the blank control group2.5.50 values of the 13 thiazolidin-2-cyanamide derivatives against EcGUS were investigated using similar assays as described in 50 values were calculated by plotting the concentration–response curve using GraphPad Prism 6.0 .ICi values were calculated by nonlinear regression of different inhibition types using the GraphPad Prism 6.0  To further characterise the inhibition behaviour of discovered inhibitors, multiple concentrations of compounds (0.5–12 µM) or DSL (50–100 µM) and PNPG  were applied to determine the corresponding reaction rates. The inhibition type was evaluated through determining the intersection point in Lineweaver–Burk plots, and the K2.6.Molecular docking studies were conducted to explore the molecular determinants of discovered inhibitors against EcGUS using MOE . Docking studies were performed according to our previous report2.7.50 values were defined as the concentration of inhibitor that is required for 50% inhibition and evaluated by nonlinear regression using GraphPad Prism 6.0 software . The inhibition type was determined according to the intersection location of Lineweaver–Burk plot: competitive inhibition, the intersection at the y-axis; uncompetitive inhibition, parallel lines; non-competitive inhibition, the intersection at the x-axis; mixed inhibition, the intersection in first or second quadrantAll experiments were carried out in triplicate and repeated at least two times. All data were expressed as mean ± standard deviation (SD). The IC3.3.1.1–13) tested, all of them showed more potent inhibitory effects against EcGUS than DSL at 10 µM, a known positive control. Compound 2 showed the highest inhibition towards EcGUS with the inhibition rate of 95.2 ± 0.3%, followed by compounds 3 and 1, whose inhibition rates were 80.2 ± 1.8% and 77.2 ± 0.6%, respectively  showed more potent inhibition against EcGUS than compound 1 and 3 , and compound 13 , suggesting that the chloro substitution on the phenyl moiety was essential for the potent inhibition of EcGUS and meta-chloro-substitution was more beneficial than ortho-chloro-substitution or para-chloro-substitution for EcGUS inhibition.As shown in 3 with a para-chloro-substituted phenyl moiety (IC50 = 4.7 ± 0.6 µM) ranked the most potent inhibitor among compounds with a para-substituted phenyl moiety, including compound 5 (IC50 = 7.3 ± 0.7 µM), 9 (IC50 = 22.2 ± 2.5 µM), 10 (IC50 = 10.8 ± 0.2 µM), 11 (IC50 = 22.3 ± 2.6 µM), 12 (IC50 = 18.5 ± 2.1 µM), and 13 (IC50 = 10.7 ± 0.5 µM), indicating that effect of substitution group on EcGUS inhibition are as follows: Cl > F > Br ≈ H > OMe > NO2 ≈ Me. In other words, chloro and fluorine substitutions at the para position of the phenyl moiety are beneficial for EcGUS inhibition, while methoxy, methyl and nitro group substitutions are unbeneficial.It also can be seen from 1, 4, 8, and 13, it could be easily found that chloro substitution at the ortho position of the phenyl moiety is beneficial for EcGUS inhibition, while fluorine substitution is unfavourable. Interestingly, the IC50 values of compound 5 (para-fluorine substitution on the phenyl moiety), 7 , 4 (ortho-fluorine substitution on the phenyl moiety), and 6  were determined as 7.3 ± 0.7 µM, 9.3 ± 1.1 µM, 11.9 ± 1.5 µM and 23.1 ± 1.1 µM, respectively, demonstrating that para-fluorine substitution is more beneficial than ortho-fluorine substitution on the phenyl moiety for EcGUS inhibition, and fluorine substitution at the 2-position of the phenyl moiety of compound 5 is unfavourable for EcGUS inhibition, while fluorine substitution at the 6-position of the phenyl moiety of compound 4 is beneficial for EcGUS inhibition.Similarly, after comparing the difference in structure and inhibitory activity of compounds 3.4.1, 2, 3, and DSL were further investigated. As shown in 1, 2, 3 were competitive inhibitors for EcGUS as judged from the intersection point at the y positive axis, implying that the binding areas of these compounds on EcGUS may be overlapped with the binding area of the substrate, PNPG, while DSL was mixed inhibitors for EcGUS as judged from the intersection point at the second quadrant. Besides, the Ki values of compounds 1, 2, 3, and DSL also were determined as 3.5 µM, 0.7 µM, 2.0 µM, and 30.9 µM, respectively (1, 2 and 3 can be as potent competitive inhibitors against EcGUS with Ki values less than 3.5 µM.The inhibition behaviours of three potent EcGUS inhibitors, compounds ectively . In summ3.5.1–3 in inhibiting EcGUS, molecular docking studies were performed using MOE. As shown in 1, 2, and 3 could be well docked into the active site of EcGUS. PNPG formed hydrogen bond interactions with His330, Asn412, Tyr472, Glu505 and Arg562 (1 were found with residues His330, Tyr472, Asn566 and Lys568, and the binding interactions were formed with the phenolic or thiazolidine groups (2 and 3 with EcGUS were nearly the same to each other, and also very similar to that of compound 1, except that one more hydrogen bond interaction was observed in compounds 2 and 3. In detail, the binding area of compound 2 was highly overlapped with that of PNPG (1.To predict the molecular determinants of thiazolidin-2-cyanamide derivatives d Arg562 , while he groups . Ligand  of PNPG , and it  of PNPG , which m4.50 values ranging from 1.2 µM to 23.1 µM, which were much more potent than a positive control, d-saccharic acid 1,4-lactone. Structure–activity relationship analysis suggested that chloro and fluorine substitutions at the para position of the phenyl moiety in thiazolidin-2-cyanamide derivatives are beneficial for EcGUS inhibition, while methoxy, methyl and nitro group substitutions are unbeneficial. Inhibition behaviour analysis indicated that compounds 1, 2, and 3 were identified as competitive EcGUS inhibitors with Ki values less than 3.5 µM and the potential molecular determinants were predicted by molecule docking studies.Gut bacterial β-glucuronidases have been proved to play important roles in CPT-11-induced intestinal toxicity, and EcGUS inhibitors have shown great potential in alleviating the toxicity. Previously identified EcGUS inhibitors usually contain heterocyclic moiety and compounds with a furan ring frequently show various bioactivities. Therefore, we evaluated the inhibitory activities of our previous synthesised thiazolidin-2-cyanamide derivatives containing 5-phenyl-2-furan moiety towards EcGUS and revealed that 13 synthetic thiazolidin-2-cyanamide derivatives were potent EcGUS inhibitors with IC50 values ranging from 2.16 ± 0.01 µM to 58.06 ± 1.60 µM by Khan’s research group. Structure–activity relationship analysis also indicated that chloro substitution at the phenyl moiety in thiadiazole derivatives is beneficial for β-glucuronidase inhibitionRecently, 49 thiadiazole derivatives were synthesised and 38 of them were evaluated as potent bovine liver β-glucuronidase inhibitors with ICIn the present study, we focus on investigating the effects of different substitution groups on the phenyl moiety of the thiadiazole-2-cyanamide derivatives on EcGUS inhibition and revealed that different substitution groups greatly affect the inhibitory potency towards EcGUS, and meta-chloro-substitution was most beneficial for EcGUS inhibition among all substitution groups tested. The effects of substitution on furan or thiazolidin moiety worth further studies.,1, 2, and 3, and figured out that they were competitive EcGUS inhibitors. These findings demonstrated the complex interactions between EcGUS and the inhibitors, whose influence on the in vivo efficacy needs detailed analysis.When Wallace et al.In conclusion, the present study identified 13 synthetic thiazolidin-2-cyanamide derivatives as potent EcGUS inhibitors and found that chloro and fluorine substitutions at the C4-position of the phenyl moiety of the furan are beneficial for EcGUS inhibition. Our findings are beneficial for developing more potent EcGUS inhibitors from thiazolidin-2-cyanamide derivatives and the in vivo efficacy of discovered EcGUS inhibitors also warrant further study.Click here for additional data file.


To investigate in the conventional techniques of the pedicle screws using triggered screw electromyography (t-EMG), considering different threshold cutoffs: 10, 15, 20 25 mA, for predicting pedicle screw positioning during surgery of the adolescent with idiopathic scoliosis (AIS).Sixteen patients  were included, with an average curve magnitude of 50 degrees and placement of 226 pedicle screws. Each screw was classified as “at risk for nerve injury” (ARNI) or “no risk for nerve injury” (NRNI) using CT and the diagnostic accuracy of EMG considering different threshold cutoffs  in the axial and Sagittal planes for predicting screw positions ARNI was investigated.The EMG exam accuracy, in the axial plane, 90.3% screws were considered NRNI. In the sagittal plane, 81% pedicle screws were considered NRNI. A 1-mA decrease in the EMG threshold was associated with a 12% increase in the odds of the screw position ARNI. In the axial and sagittal planes, the ORs were 1.09 and 1.12, respectively. At every threshold cutoff evaluated, the PPV of EMG for predicting screws ARNI was very low in the different threshold cutoff (10 and 15); the highest PPV was 18% with a threshold cutoff of 25 mA. The PPV was always slightly higher for predicting screws ARNI in the sagittal plane than in the axial plane. In contrast, there was a moderate to high NPV (78–93%) for every cutoff analyzed.EMG had a moderate to high accuracy for positive predicting value screws ARNI with increase threshold cutoffs of 20 and 25 mA. In addition, showed to be effective for minimizing false-negative screws ARNI in the different threshold cutoffs of the EMG in adolescent with idiopathic scoliosis (AIS). Pedicle screw fixation plays an important role in spine surgery due to its firm three column control and thus provide superior reconstruction stability. As a common surgical procedure, the pedicle screw placement method has been widely employed to reconstruct local stability in spine surgery due to its great three-column control . The useVariety of techniques have been introduced to assist screw insertion and to reduce the prevalence of pedicle violation, given the clinical complications that may occur during and after the surgical procedure. A misplaced pedicle screw may result in neurovascular damage, dural tearing, or visceral involvement, and such complications are potential threats to life and limb . The conRecently, robot-assisted systems have been developed to address the issue of pedicle screw malposition. Many retrospective studies consistently reported that the clinically acceptable screw positioning accuracy under robotic guidance is near 99% . Two proDespite this controversy, several literature studies reveal that the instrumentation with pedicle screws allows for better correction of spinal deformities in the coronal, sagittal and rotational planes, less correction loss, shorter constructions and improved lung function without increasing neurological complications , 17. PedDue to peculiar vascular and neurologic anatomical features of the vertebrae and spinal canal, caution is needed during insertion of pedicle screws. For safety reasons, it is of utmost importance to ensure precise insertion and to confirm an intraosseous position . IntraopIn patients with scoliosis, rotational deformity increases the risk of perforating the pedicle cortical wall, which is not always easy to detect during surgical procedures. Intraoperative assessment of the position of the screws with conventional radiography or fluoroscopy can help detect misplaced implants, although computed tomography (CT) provides greater accuracy . EMG canThis was a prospective study  involving patients with AIS who underwent surgical treatment in the same hospital and in whom intraoperative EMG measurements were compared with the implant positions evaluated by CT. The study was reviewed and approved by the Departmental Research Committee of the Institute of Medical Assistance to the State Public Hospital Servant – IMASPS (registration number: 533.892), in accordance with relevant guidelines and regulations. All participants provided their informed consent by written underwent assessment and experimental procedure.Sixteen patients  who underwent surgery performed by the same surgical team in the same institution from March to December 2013 were included in the study, with an average curve magnitude of 62 degrees and placement of 226 pedicle screws to attain an average curve correction of 77.7%. Patients were excluded if they had scoliosis with a known etiology , if they were undergoing revision surgery, or if no postoperative CT was available for review.All patients were operated by a posterior approach, with insertion of pedicle screws from the same manufacturer  by the “free hand” technique  and undeAfter pedicle screw insertion, the stimulation was performed using EMG with monopolar electrode (cathode) and a subdermal needle electrode inserted into the paravertebral musculature (anode). Stimulation was performed with a frequency of 3 Hz, a duration of 0.1 ms and an increasing intensity until an EMG response could be observed. The maximum intensity used was 30 m-amperes (mA). For each screw, the lowest intensity able to generate a measurable response was recorded as the EMG threshold for that screw. If no response was observed, a value of 30 was assigned to that screw.Pedicle screw stimulation was performed using DS7A current stimulation  to generate and deliver square wave constant-current pulses to each pedicle screw following insertion. We used repetitive 4-pulse trains, applied with an interpulse interval of 2 msec between trains and an intertrain rate of 3 Hz. Maximal stimulus delivered varied between cases with maximums of 20, 30, or 40 mA. Minimum stimulus intensities were recorded for each level with a pedicle screw. All screws demonstrating a minimum intensity ≥30 mA was defined as a maximal intensity. Only one patient was classified as having a threshold ≤8 mA was defined as abnormal and underwent screw re-direction, but underwent screw removal and probing of the pedicle track using a ball-tip probe. This information has been clarified in the text for better understanding [In the EMG exam was considering different threshold cutoffs: 10, 15, 20 and 25 in the axial and sagittal planes to analysis the accuracy after AIS surgery. All patients performed the computed tomography (CT) exam, immediately after the surgery before the patient performs the gait, to evaluate the implant positioning and classification according to the criteria proposed by Abul-Kasim et al. . This grEach individual screw to the corresponding pedicle was assessed and classified in both the medial and sagittal planes as follows: normally placed in the medial plane; medial cortical perforation (MCP) grade 1, partially medialized; MCP grade 2, totally perforating the medial pedicular cortex; lateral cortical perforation (LCP) grade 1, partially lateralized but anchored in the vertebral body; LCP grade 2, abutting the outer cortex of the vertebral body and not anchored in the vertebral body; normally placed in the sagittal plane; perforating the inferior underlying neural foramen (INF); or perforating the superior underlying neural foramen (SUP). Additionally, screws classified as MCP grade 1 or MCP grade 2 in the axial plane and as perforating the INF or the SUP in the sagittal plane were considered “at risk for nerve injury” (ARNI), as these screws are closer to neural elements. Screws classified as normally placed and screws classified as LCP, although recognized as misplaced, were considered “no risk for nerve injury” (NRNI)  was used to induce anesthesia in the AIS surgeries, and medications that usually do not interfere with intraoperative neurophysiological monitoring (propofol and remifentanil) were administered. All AIS corrections included in this study were performed by the same surgical team, using the same techniques for surgery and electromyography evaluation. All CT scans were evaluated by the same observer (BMG).F = 0.25), a power of 80%, and a significance level of 5%”. Continuous variables are presented as the mean and standard deviation, and categorical data are presented as absolute and relative frequencies. A descriptive analysis of the positioning of all screws was performed. To evaluate the diagnostic accuracy of EMG for predicting screws ARNI, we excluded all screws inserted above T6, as those pedicles have lower reliability for EMG acquisition [The sample size calculation on 16 patients  was based on the mean age and Cobb angle preoperative, using the G-Power 3.0 software, and considered a moderate effect size (uisition . A singlp < .05 (two-sided) were considered significant.The diagnostic accuracy of EMG for predicting screw malposition was investigated using a receiver operating characteristic (ROC) curve. The curve represents a plot of the sensitivity and specificity at progressive cutoffs of a diagnostic test measured on a continuous scale. Therefore, the area under the curve (AUC) is a measure of the ability of EMG to discriminate between screws ARNI and screws NRNI. Estimates of diagnostic accuracy, including sensitivity (Sn), specificity (Sp), negative predictive value (NPV) and positive predictive value (PPV), were calculated for cutoffs at every 5 mA and are presented with their 95% confidence intervals (95%CI). All statistical analyses were performed using the statistical package STATA 14 . Associations with n = 12, 75% female and n = 4, 25% male) were female, and the average age was 16.6 years. No patient had neurological complaints, experienced irradiated pain to dermatomes or exhibited any observed change in the physical exam indicating nerve injury.In the study period, 16 patients underwent surgical treatment for AIS and were included in this study. Most . There was lateral cortical perforation in cases of which 25 (8.9%) were classified as LCP1, and 16 (5.7%), as LCP2. Medial cortical perforation was found in 45 cases  , while 48 (17.1%) violated the inferior foramen (FP1 INF), and 7 (2.5%), the superior foramen (FP1 SUP). Considering axial and sagittal planes together, 59.1% (166/281) of all screws had no cortical perforation , representing ideal positions, while 40.9% (115/281) of screws showed at least one degree of cortical perforation . In the axial and sagittal planes, the ORs were 1.09  and 1.12 , respectively. However, the ROC curves showed that EMG had moderate ability to discriminate between screws ARNI and NRNI.We observed a statistically significant association between EMG responses at lower intensities and the positioning of screws associated with risk for nerve injury. Overall, a 1-mA decrease in the EMG threshold was associated with a 12% increase in the odds of the screw position ARNI , .63 (95% CI = .51–.75) and .65 (95% CI = .55–.75), respectively. Table AIS is a complex three-dimensional deformity associated with rotation and structural abnormalities of the vertebrae, making treatment technically challenging. Implants for pedicle fixation have been widely used in surgical treatments of the thoracic and lumbar spine, with better results in arthrodesis rate, correction power and early mobilization of the patient compared with fixation systems that employ hooks or mixed systems , 30. PedScrews with LCP are associated with risk of vascular or visceral damage , 33, 34.Intraoperative neurophysiology evaluation can allow the early detection and correction of possible lesions during spinal surgery. Such techniques include the evaluation of motor evoked potentials, somatosensory evoked potentials and EMG , 24. TheIn evaluating the accuracy of EMG as an intraoperative diagnostic method to detect misplaced screws ARNI, there was a statistically significant association between EMG responses and the positioning of screws associated with risk for nerve injury. A decreased EMG threshold was associated with an increased odds of the screw position ARNI among thoracic and lumbar screws. The association between EMG threshold and screw misplacement in thoracic pedicles was recently shown using pulse-train stimulation . The preDespite the association found between EMG and the position of screws, the ability of EMG to intraoperatively discriminate between screws ARNI and NRNI was poor to moderate. NPV and PPV are the most meaningful measures of diagnostic accuracy in terms of making clinical decisions based on a test result. NPV expresses the probability of not having the condition under study given a negative test outcome, and PPV expresses the probability of having the condition given a positive test outcome. EMG showed a very low PPV (< 18%) at every threshold cutoff evaluated, meaning that less than 1 out of 5 screws that test positive  would actually be positioned ARNI.Considering the risk for neurological deficit or stenosis of the spinal canal if a screw breaches the medial wall during thoracic pedicle screw instrumentation in AIS surgery, the consequences of a false-negative result of a diagnostic test for screw malposition can be severe . TherefoThe main limitation of the present study is that despite the aim to analyze the ability of EMG to intraoperatively predict screws ARNI, the sample was composed exclusively of patients with no nerve injury, spinal cord injury, or nerve root injury. Some false-positive and false-negative cases were found among the EMG responses of the diagnostic test, but these cases did not result in any clinical consequence. Therefore, it is not possible to assume that EMG is not an accurate tool for predicting screws ARNI. Furthermore, we consider intraoperative neurophysiologic monitoring, particularly motor evoked potentials, as extremely important during thoracic screw insertion for the early detection and prevention of severe neurological complications.EMG had a moderate to high accuracy for positive predicting value screws ARNI with increase threshold cutoffs 20 and 25 mA. In addition, showed to be effective for minimizing false-negative screws ARNI.Additional file 1: Table 1. Summary of the positions of the pedicle screws in the axial and sagittal planesmfor each patient and the total pedicles analyzed. Table 2. Summary of the ideal screw positioning in the sagittal and axial planes and inadequate positioning in any of the planes.


Pasteurella multocida is a gram-negative opportunistic pathogen that causes various diseases in poultry, livestock, and humans, resulting in huge economic losses. Pasteurella multocida serotype A CQ6 (PmCQ6) is a naturally occurring attenuated strain, while P. multocida serotype A strain CQ2 (PmCQ2) is a highly virulent strain isolated from calves. Compared with PmCQ2, it was found that bacterial loads and tissue lesions of lung tissue significantly decreased and survival rates significantly improved in mice infected with PmCQ6 by intranasal infection. However, comparative genome analysis showed that the similarity between the two strains is more than 99%. To further explore the virulence difference mechanism of PmCQ2 and PmCQ6, transcriptome sequencing analysis of the two strains was performed. The RNA sequencing analysis of PmCQ2 and PmCQ6 showed a large number of virulence-related differentially expressed genes (DEGs) in vivo and in vitro. Among them, 38 virulence-related DGEs were significantly up-regulated due to PmCQ6 infection, while the number of PmCQ2 infection was 46, much more than PmCQ6. In addition, 18 virulence-related DEGs  were up-regulated in PmCQ2 infection compared to PmCQ6 infection, exhibiting a higher intensive expression level in vivo. Our findings indicate that these virulence-related DEGs  might be responsible for the virulence of PmCQ2 and PmCQ6, providing prospective candidates for further studies on pathogenesis. Pasteurella multocida is a gram-negative bacterium well-known for causing diseases in a wide range of birds and mammals   according to the specificity of capsular antigens (P. multocida mainly causes pneumonia and serious bovine respiratory diseases.species) , includispecies) –5, whichantigens , 7 and 1antigens . The capPasteurella multocida possesses a number of virulence factors, mainly including capsular polysaccharide, iron utilization-related genes, LPS, and outer membrane proteins (OMPs)  mutant for P. multocida LPS biosynthesis was completely attenuated in mice (s (OMPs) , 9, 10. ogenesis –13. For  in mice ; the gal in mice .P. multocida  of PmCQ2 in mice were significantly higher than that of PmCQ6  and highly virulent bovine P. multocida serotype A strain CQ2 (PmCQ2) were isolated from the lungs of calves with pneumonia in Chongqing, China  were purchased from Hunan SJA Laboratory Animal Co., Ltd. . The mice were housed in individually ventilated pathogen-free cages  with free access to food and water. The mice were acclimated for 4 days after arrival before starting the experiments. All animal experiments were carried out with approval from the Animal Ethics and Research Committee of Southwest University (Permit No. 11-1025), Chongqing, China.8 colony-forming units (CFU) in 20 μL, and the number of mice used for each strain detection was equal. The mice were monitored for 7 days to determine the survival curves, and mice showing severe clinical signs  were considered moribund and were humanely killed. Survival rates (n = 10/group) were measured in the two groups after injection. The mice were also euthanized for collection of lung tissues to measure the bacterial loads (n = 10/group) and histopathological examination  at 16 h post-infection.To determine the pathogenicity of PmCQ2 and PmCQ6, C57BL/6 mice were infected by intranasal exposure with PmCQ2 and PmCQ6 at a dose of 1 × 10n = 6/group) were immediately fixed in 4% paraformaldehyde for 24 h, dehydrated in graded ethanol, and then embedded in paraffin wax. The tissues were sliced at 3 μm thickness and then stained with hematoxylin and eosin (H&E)  . Accordiin (H&E) .n = 10 in each group) were collected at 16 h post-bacterial infection. The tissues were homogenized aseptically, and bacterial loads were quantified by 10-fold serial dilution in saline. These different dilutions were plated in triplicate on Martin's broth agar and were incubated at 37°C for 24 h to count the CFU.To measure the bacterial loads, lung tissues (n = 3) and PmCQ6 (n = 3) at a dose of 1 × 108 CFU in 20 μL. The mice were euthanized at 16 h post-infection; lung tissues were collected and quickly frozen in liquid nitrogen. Simultaneously, 1 mL each of fresh mid-logarithmic phase of PmCQ2 (n = 3) and PmCQ6 (n = 3) was seeded in 100 mL fresh Martin liquid medium and incubated at 37°C with shaking at 220 revolution/min for 12 h. The bacterial cultures were centrifuged at 3,000 revolution/min for 10 min at 4°C; the bacterial cell pellets were suspended in 10 mL ice-cold phosphate-buffered saline (PBS) and centrifuged again. The bacterial cells were quickly frozen in liquid nitrogen after washing three times. The lung tissues and bacteria samples were sent to Beijing Genomics Institute  for transcriptome sequencing.C57BL/6 mice were infected by intranasal inoculation with PmCQ2 , following the manufacturer's protocol. RNA integrity was confirmed by agarose gel electrophoresis and quantified by NanoDrop . Subsequently, biotin-labeled specific probes were used to remove rRNA, and then RNA is purified and fragmented. Then, 1 μg of each RNA sample was used to construct a strand-specific cDNA library according to the recommendations of Illumina TruSeq Stranded Kit. The cDNA library size was analyzed by using an Agilent 2100 Bioanalyzer, and the effective concentration was determined using qPCR . Finally, the samples were sequenced on Illumina sequencing platform (HiSeq 4000), and 150-bp paired-end reads were generated.via SOAP (HISAT software (Bowtie2 software (http://bowtie-bio.sourceforge.net/Bowtie2/index.shtml), and then RSEM software (http://deweylab.biostat.wisc.edu/RSEM) was used to calculate the gene expression levels. Differential expression analysis was performed using estimateSizeFactors in DESeq (version 1.18.0) R package; p-value and fold change value were identified using nbinomTest in DESeq with R package. P < 0.05 and |log2foldchange| ≥ 1 were set as the threshold for DEG identification. The hierarchical cluster analysis of DEGs was performed by clustering software cluster  involving a gDNA elimination step. cDNAs were synthesized with an iScript cDNA synthesis kit , and quantitative real-time RT-PCR was performed according to previous study using a CFX96 instrument  . Each ta−ΔΔCt . SpecifiAccording to the method described previously , bacterit-tests if the data were in Gaussian distribution and had equal variance or by unpaired t-test with Welch's correction if the data are in Gaussian distribution but show unequal variance or by non-parametric test (Mann–Whitney U-test) if the data were not normally distributed. The Gaussian distribution of data was analyzed by D'Agootino–Pearson omnibus normality test and Kolmogorov–Smirnov test. The variance of data was analyzed by Brown–Forsythe test. Significant differences were considered at P < 0.05 .All data were displayed as means ± standard deviation (SD). GraphPad Prism 6.0 software was applied for all statistical analysis. The survival rates of mice were evaluated using Kaplan–Meier analysis. Data between the two groups were analyzed using unpaired 8 CFU) by intranasal injection and observed for 1 week, 60% of them survived, while the PmCQ2 (1 × 108 CFU)-infected mice all died rapidly within 72 h (8-1 × 109 CFU) in mice lungs was significantly higher than that of PmCQ6 (1 × 107.5-1 × 108.5 CFU) at 16 h , a total of 960 DEGs were observed in PmCQ2-m (in vivo)/PmCQ2 (in vitro), including 448 up-regulated and 512 down-regulated genes; there were 841 DEGs (401 up-regulated genes and 440 down-regulated genes) in PmCQ6-m (in vivo)/PmCQ6 (in vitro); 433 DEGs, with 262 up-regulated genes and 171 down-regulated genes, were in PmQ2/PmCQ6; and 91 DEGs were in PmQ2-m/PmCQ6-m, of which 31 were up-regulated and 60 were down-regulated , three iron utilization-related genes , and two LPS-related genes  were chosen for qPCR analysis, which were similar to those obtained from the RNA-seq results  in PmCQ2-m/PmCQ2 , five iron utilization-related genes , three LPS-related genes , and one OMP-related gene (ompA) were used for qPCR analysis, which were similar to those obtained from the RNA-seq results in PmCQ2-m/PmCQ2 , which were mainly involved in ribosome, RNA degradation, biosynthesis of type II polyketide products, and bacterial chemotaxis in vivo , seven iron utilization-related genes , and one LPS-related gene (lpcA) were chosen for qPCR analysis, which were similar to those obtained from the RNA-seq results  and PmCQ6 (1 × 108 CFU) was established via intranasal infection. The mice died within 72 h post-infection with PmCQ2, while only 40% died after PmCQ6 infection. The bacterial burden of PmCQ2 in mice lung tissue was significantly higher than that of PmCQ6, and PmCQ2 causes serious lung lesions  than that of PmCQ6 in mice at 16 h post-infection. This result indicates that PmCQ2 is a virulent strain and PmCQ6 is an attenuated strain for mice as reported previously , hyaBCDE (capsule biosynthesis), and hexABCD (capsule transport) (hyaC and hexA/B/C/D) and ten predicted capsule-associated DEGs wza (putative polysaccharide export protein YccZ precursor), wzb (cytoplasmic phosphatase), wzc (tyrosine protein kinase), WbiI (epimerase/dehydratase WbiI), spsC (spore coat polysaccharide biosynthesis protein spsC), WeeH , wecB (UDP-N-acetylglucosamine 2-epimerase), WbjC , fnlA (UDP-glucose 4-epimerase), and VipA (Vi polysaccharide biosynthesis protein VipA/TviB). Moreover, the transcriptome data showed that there are 46 up-regulated virulence-related DEGs in PmCQ2-m/PmCQ2, including six reported capsule-related hyaB/C and hexA/B/C/D and two predicted capsule-associated genes hscA (capsule polysaccharide transporter) and hscB (capsule polysaccharide modification protein). Furthermore, 38 up-regulated virulence-related DEGs in PmCQ6-m/PmCQ6, including six reported capsule-related hyaB/C and hexA/B/C/D and two predicted capsule-associated genes wza (putative polysaccharide export protein YccZ precursor) and oppF (dipeptide transport system ATP-binding protein). In addition, there are 18 up-regulated virulence-related DEGs in PmCQ2-m/PmCQ6-m, including six reported capsule-related hyaB/C and hexA/B/C/D and capsular regulatory gene Hfq  . In thisgene Hfq . The aboCP033599.1) and over 2.1% (46 genes) (CP033600.1) are predicted to encode proteins for iron uptake or acquisition. We found that the expression of six iron utilization-related DEGs malK (iron ABC transporter ATP-binding protein), manB (phosphomannomutase), luxS (S-ribosylhomocysteine lyase), fbpC (Fe3+ ions import ATP-binding protein FbpC), pvuE (iron-dicitrate transporter ATP-binding subunit), and afuC (Fe3+ ions transport system ATP-binding protein) were remarkably increased in PmCQ2/PmCQ6. Four TonB-receptor-related DEGs , seven iron utilization-related DEGs  were significantly up-regulated in PmCQ2-m/PmCQ2. Two TonB-receptor-related DEGs (hhuA and hemR), six iron utilization-related DEGs , and two heme utilization-related DEGs  were obviously increased in PmCQ6-m/PmCQ6. Two TonB-receptor-related DEGs (hhuA and hemR), four iron utilization-related DEGs , and one heme utilization-related DEG (hutZ) were especially up-regulated in PmCQ2-m/PmCQ6-m. These results suggest that iron uptake played an important contribution/role in the virulence difference of the two strains.Iron is necessary for bacteria to survive ; genome-6 genes)  of the tP. multocida , rfaF (LPS heptosyltransferase II), and rfaC/opsX (LPS heptosyltransferase I) were significantly increased in PmCQ2/PmCQ6. Three LPS-related DEGs  were significantly increased in PmCQ2-m/PmCQ2; lptA (lipid A ethanolamine phosphotransferase) was sharply increased in PmCQ6-m/PmCQ6, and lpxC was sharply upregulated in PmCQ2-m/PmCQ6-m. This result indicates that lpxC plays an important role in the virulence of PmCQ2.LPS is one of the most important factors associated with the pathogenesis of ultocida , 40. TraP. multocida (OmpA and OmpP5 (outer membrane protein P5) were significantly upregulated in PmCQ2-m/PmCQ2 and PmCQ6-m/PmCQ6, and OmpP5 was increased in PmCQ2-m/PmCQ6-m. This result indicates that OmpP5 may be a key factor in the difference of virulence between the two strains.As the main structure of the outer membrane of gram-negative bacteria, OMPs  play an important role in bacterial infection and pathogenic processes, such as adhesion, invasion, and intracellular survival –43. For ultocida , 45. OmpP. multocida.In conclusion, the bacterial loads and lesions of lung tissue significantly decreased, and survival rates significantly improved in mice infected with PmCQ6 than that with PmCQ2. Notably, the differential transcription of virulence-related genes  is the main reason for the different pathogenicity of PmCQ2 and PmCQ6. This work provides new guidance for the mechanism study of the host–pathogen interactions of https://www.ncbi.nlm.nih.gov/, PRJNA597831; https://www.ncbi.nlm.nih.gov/, PRJNA629108; https://www.ncbi.nlm.nih.gov/, PRJNA664837.The datasets generated in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found below: The animal study was reviewed and approved by the Animal Ethics and Research Committee of Southwest University (Permit No.11-1025), Chongqing, China.YP and PL designed the experiment. FH, ZZ, PL, XW, and LD conducted the experiment. FH, PL, RF, and NL analyzed the data and prepared the figures. FH and PL drafted the manuscript. YP, RF, NL, and PL revised and approved the final manuscript. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Delineating the grounding line of marine-terminating glaciers—where ice starts to become afloat in ocean waters—is crucial for measuring and understanding ice sheet mass balance, glacier dynamics, and their contributions to sea level rise. This task has been previously done using time-consuming, mostly-manual digitizations of differential interferometric synthetic-aperture radar interferograms by human experts. This approach is no longer viable with a fast-growing set of satellite observations and the need to establish time series over entire continents with quantified uncertainties. We present a fully-convolutional neural network with parallel atrous convolutional layers and asymmetric encoder/decoder components that automatically delineates grounding lines at a large scale, efficiently, and accompanied by uncertainty estimates. Our procedure detects grounding lines within 232 m in 100-m posting interferograms, which is comparable to the performance achieved by human experts. We also find value in the machine learning approach in situations that even challenge human experts. We use this approach to map the tidal-induced variability in grounding line position around Antarctica in 22,935 interferograms from year 2018. Along the Getz Ice Shelf, in West Antarctica, we demonstrate that grounding zones are one order magnitude (13.3 ± 3.9) wider than expected from hydrostatic equilibrium, which justifies the need to map grounding lines repeatedly and comprehensively to inform numerical models. Upstream of the grounding line, ice rests on land and is resisted by bedrock and sediments and along valley walls. Downstream of the grounding line, ice is no longer grounded; it floats in the ocean waters and melts in contact with these waters. This boundary is fundamental to the study of ice sheets for many reasons. First, it is the boundary where we measure ice mass fluxes into the ocean3. Second, ice experiences a fundamental change in force balance at that boundary, transitioning from basal friction at the bed to frictionless motion on seawater at sea4, so it is essential to inform ice sheet numerical models about the location of the grounding line. At the grounding line, ice traverses hydrostatic equilibrium but bending stresses maintain ice below hydrostatic equilibrium over considerable distances seaward (10 km) until ice reaches full hydrostatic equilibrium in seawater5, which is important to consider when interpreting data from altimetry missions. Third, the surface slope is typically 1% at the grounding line, so a 1-m change in ice thickness translates into a 100-m horizontal migration of hydrostatic equilibrium1, therefore the grounding line position is a sensitive indicator of glacier thinning. Fourth, the grounding line is challenging to detect from above, it leaves little evidence in optical imagery6, it is elusive from the ground, and historically has been misplaced with errors ranging from kilometers to 100 km9.The grounding line is the transition boundary where land ice meets with ocean waters along the periphery of Greenland and Antarctica1, but the most efficient and precise method is satellite interferometric synthetic-aperture radar (InSAR). InSAR documents the motion of ice in the line of sight of the radar illumination with millimeter precision. Provided enough crossing tracks of InSAR data, this technique maps ice motion in vector form10. By differencing two consecutive radar interferograms spanning the same interval, it measures the short term variability in ice motion associated to processes such as glacier subsidence in response to subglacial lake drainage or tidal motion in response to changes in ocean tide. Because differential InSAR (DInSAR) combines four epochs to “image” the grounding line, it mixes four different tidal displacements, but the data provide a snapshot of grounding line positions over considerable distances, i.e. hundreds of km, anywhere around the continent, and repeatedly over time.Grounding lines are detected in several ways using remote sensing dataAt the grounding line, the DInSAR data reveals a step in vertical motion that is characteristic of the visco-elastic bending of ice afloat in ocean waters across the flexure zone Fig. . The inn11 fit the data with an elastic model, which was generalized in two dimensions12. The modeling identified the limit of tidal flexing, which is a proxy for the grounding line. While this approach worked well for smooth grounding line delineations, it failed in the presence of noise, required information on ice flow direction, and could not be automated or scaled to large areas. Instead, human experts have resorted to the practice of performing the work by hand, in part to resolve complex topological problems appearing in certain situations. For instance, interferometric fringes may be missing due to a loss of phase coherence, stretch out significantly along deep, narrow valleys, under-sampled, or not well resolved. While the manual approach has been acceptable for a few DInSAR pairs of Greenland and Antarctic glaciers, it has become cumbersome as more data become available from more SAR satellites. Furthermore, interpretation may vary among human experts and errors are not documented.To digitize the grounding lines, Rignot et al. (1998)14. This situation contrasts with the ERS-1/2 mission which provided only a handful of DInSAR data to analyze. Digitizing Sentinel-1a/b DInSAR data is impractical by hand on a large scale.The European Space Agency launched the Sentinel-1a/b constellation in 2014 and 2016, respectively, as part of the European Union’s Copernicus Earth Observation program to provide InSAR data over the ice sheets systematically for the first time and for decades to come with a 6-day repeat cycle (for the constellation) or a 12-day repeat cycle (for a single satellite)Here, we present a novel approach to the automated delineation of grounding line which uses a machine learning (ML) algorithm. After introducing the training and evaluation data sets, we discuss the ML architecture employed to capture the grounding line, the evaluation of the results and estimation of errors, and demonstrate its use at the large scale around Antarctica with thousands of interferograms.We employ DInSAR observations from the Sentinel-1a/b satellite captured in year 2018. The radar operates at the C-band (radar wavelength is 5.6 cm) and each interferometric fringe or 360 degree variation in phase corresponds to a 28 mm motion of the icy surface along the line of sight of the radar. Each interferogram is 250 km by 300 km in size, geocoded at 100 m spacing. Each sector of the grounding line is covered by several repeat tracks. The geocoded DInSAR data are first manually interpreted to serve as training and evaluation datasets, as outlined below. In total, we analyze 252 DInSAR-delineation pairs on the Getz Ice Shelf for training. To train the neural network, we use both the phase and coherence of the signal. Furthermore, we analyze 19,510 DInSAR interferograms with 6-day repeat between the pairs acquired from 22 Sentinel -1a/b tracks, and 3425 DInSAR interferograms with 12-day repeat between the pairs acquired from 23 Sentinel-1a/b tracks, totalling 22,935 interferograms over Antarctica in 2018. The 12-day tracks were trimmed to exclude in-land tiles far from the grounding zone to reduce the processing time. However, by including all the tiles from the 6-day tracks we demonstrate that the network is capable of dealing with areas without grounding lines. The detailed break-down of the number of interferograms and tiles is listed in Table We rasterize the manually-delineated grounding lines from the DInSAR data and split into 15. The neural network is trained on 5320 tiles. Each training epoch takes under 10 min on the Google Colab GPU accelerator, which is currently a Tesla P100-PCIe with 16 GB of memory. Using batch size of 30, the training is stopped after 9 epochs to avoid overfitting based on a minimum improvement threshold of 0.0001 in the value of the loss function applied to the randomly selected validation data in each epoch.We use a fully-convolutional architecture for the semantic segmentation of grounding lines. Given the noisy and varied nature of the DInSAR data, we aim to directly segment the grounding lines, as opposed to classifying the various surfaces in each tile. The goal is to derive the probability of each pixel belonging to a grounding line or no grounding line. We use a 40-layer neural network with 966,119 trainable parameters, to perform the segmentation see “”. The laThe fully-trained network is then tested on 500 new tiles. The neural network provides the probability of each pixel belonging to a grounding line. The original input data, the human-made training labels, and the output of the neural network are shown in Fig. 16. We use the length of the perimeter of polygons to filter out noisy outputs. We use a threshold of 6 km to eliminate small features introduced by the network. The 6-km threshold is chosen to minimize the detection of spurious grounding lines, while avoiding the exclusion of real features. Residual errors remain, e.g. on Carlson and Evans glaciers feeding into the Ronne Ice Shelf where segments of both sides of the flexure zone are included in the grounding line detection. In other areas, e.g. Pine Island Glacier, we find short spurious line segments downstream of the grounding line. These errors, which comprise a small fraction of the total, are manually removed from the final product. The uncertainty estimates allow us to pinpoint the grounding line by drawing a centerline through the polygons  of 101 m, and an interquartile range (IQR) of 131 m.16 which comprise narrow  and tide-dependent connections to the continent. In other areas, however, the ML interpretation is superior a posteriori to the human interpretation, for instance in areas of high noise where a conservative human interpreter would not identify a grounding line but would subsequently agree with the ML delineation . The activation maps in the second row correspond to the fifth layer of the network (Panels C–F). For each row, we divide the channels into quarters and showcase four activation maps at equally spaced intervals. At layer 5, the network picks up large scale features that are not necessarily relevant to the grounding line, and artifacts, e.g. faint diagonal feature across the interferogram on the floating ice tongue. This highlights the need for a deep network capable of encapsulating the many features of the data, hence the relatively large depth of the neural network. Panels G–J show the activation maps of layer 16, which is the convolutional layer with dimensions The network distinguishes two sides of the grounding zone, represented by asymmetric bands in the activation maps at the boundaries of the grounding zone Fig. . The net34. With slopes of the order of 1% and tides in the range of 1 m, we expect migrations of the order of 100 m. Instead, we detect migrations of several kilometers, well beyond the uncertainty in slopes. To quantify this discrepancy, we calculate the grounding zone width based on hydrostatic equilibrium as in Tsai and Gudmundsson35 along the Getz Ice Shelf, in West Antarctica, where we have continuous and high-quality delineations with dense coverage of 6-day interferograms  grounding zone, is calculated as35. Note that the downstream migration distance is 9 times smaller than the upstream distance. Here, we use the upper case as a conservative estimate of the grounding zone width. In addition, we assume a uniform 2 m tide height to get an upper bound of the HE grounding zone. We calculate 26 using the surface and bed topography from BedMachine v136, over a spatial scale of 3 km, which is several times the mean ice thickness. We compare the result with the observed width of the grounding zone measured along the same ice flow direction. A set of comparison points are shown in Fig. By analyzing delineations across the entire ice sheet, we find that the grounding zone, which is the area over which a grounding line moves back and forth with changes in oceanic tides, is several km in width. In the case of interaction of solid ice on a hard bed, the level of migration of the grounding line with tide is dictated by hydrostatic equilibrium, which depends on both surface and bed slopesWe find that the observed grounding zone width is one order of magnitude larger than the width expected from HE. Berry Glacier is an exception with a grounding zone width two orders of magnitudes wider than expected. The inset Fig. b shows t38 instead of a hard bed, or if un-grounding of ice is interpreted as the propagation of an elastic crack at the grounding line35. Our broader mapping exercise indicates that Getz Ice Shelf is not an isolated case but the norm in Antarctica, i.e. grounding zones are typically several km wide and are not well represented from hydrostatic equilibrium on a hard bed41. This result has implications for ice sheet models who treat grounding lines with hydrostatic equilibrium.Broader grounding zones are predicted when ice interacts with a deformable bed42. Conversely, in areas of steep slopes or abrupt transition boundary where the grounding zone is narrow and seawater intrusion is likely to be limited, a treatment with hydrostatic equilibrium and no melt at the grounding line is probably satisfactory.A consequence of wider grounding zones is that seawater must intrude over considerably longer distances beneath grounded ice at high tide than expected. Water intrusions in turn imply that ice probably melts at tidal frequencies in these broad regions. This information is relevant to ice sheet models because wider grounding zones and the possibility of high ice melt rates within the grounding zone will make the model more sensitive to ocean thermal forcing and the glaciers retreat faster in warming scenariosThe application of the ML method to the entire Antarctic Ice Sheet provides a unique and novel data set for the scientific community. The results confirm that wide grounding zones are the norm. Due to the uneven and sparse temporal sampling of the data, we cannot analyze changes within a tidal cycle, or from month to month, and only examine aggregated changes over an entire year at present. The ML approach detects the grounding line with a precision of 200 m and the width of the grounding zone over a tidal cycle. This precision is sufficient for large scale ice sheet numerical models. We also find that the approach is scalable to pan-Antarctica without retraining the network, at high speeds of computation compared to manual interventions. Visual inspection of the results in other areas, e.g. Pope, Kohler, and Smith Fig. , confirmThe complete mapping of grounding lines around Antarctica performed by the ML algorithm provides a new basis for the analysis of grounding line dynamics in Antarctica that was not available previously. We expect that the product from year 2018 to be of considerable interest for the scientific community, in particular numerical ice sheet modelers and ocean modelers, and will help improve projections of ice sheet evolution and contributions to sea level rise from models. We anticipate to perform this delineation routinely with Sentinel-1a/b and other InSAR data, e.g. the Italian CSK-2G, the Japanese ALOS PALSAR-2, -3 and -4 and the upcoming NASA/Indian Space Research Organization (ISRO) NISAR mission.43), inspired by the DeepLabv3+ architecture44. In order to minimize the number of trainable parameters, we use depthwise-separable convolutions32 for the parallel atrous layers, but we find that extending depthwise-separable convolutions to all layers degrades the performance of the network. We add skip connections between encoding and decoding layers to convey contextual information, as in45. All convolutional layers use the Expontential Linear Unit (ELU) activation function46, with the exception of the last convolutional layer with a sigmoid activation function producing the predicted probability of the class of each pixel. In total, we have 966,119 trainable parameters in the models. Descriptions of all layers are provided in Table N is the number of pixels, R is the penalization ratio which is 727 based on the average ratio of GL vs non-GL pixels, i between 0 and 1, and i from the human-made training dataset). The neural network provides raster images of the GLs, which are vectorized by drawing closed contours around GL pixels using a threshold of 0.3 (30% probability of pixel belonging to GL class). The width of these contours provide the uncertainty estimate for the neural network output. In order to draw the centerline through the contours, we use the label_centerlines Python package.Images are split into overlapping tiles that avoid edge effects. Partially overlapping tiles in four directions combined with a staggered grid produces 8 partial coverings of every area as a form of augmentation. We generate 5320 tile pairs for training. We use a 40-layer deep encoder-decoder convolutional neural network with parallel atrous convolutional layers (Atrous Spatial Pyramid PoolingSupplementary Information.


Chemoradiation, either alone or in combination with surgery or induction chemotherapy, is the current standard of care for most locally advanced solid tumors. Though chemoradiation is usually performed at the maximum tolerated doses of both chemotherapy and radiation, current cure rates are not satisfactory for many tumor entities, since tumor heterogeneity and plasticity result in chemo- and radioresistance. Advances in the understanding of tumor biology, a rapidly growing number of molecular targeting agents and novel technologies enabling the in-depth characterization of individual tumors, have fuelled the hope of entering an era of precision oncology, where each tumor will be treated according to its individual characteristics and weaknesses. At present though, molecular targeting approaches in combination with radiotherapy or chemoradiation have not yet proven to be beneficial over standard chemoradiation treatment in the clinical setting. A promising approach to improve efficacy is the combined usage of two targeting agents in order to inhibit backup pathways or achieve a more complete pathway inhibition. Here we review preclinical attempts to utilize such dual targeting strategies for future tumor radiosensitization. Chemoradiation is a current standard of care for the curative treatment of most locally advanced solid malignancies. Both modalities are generally administered at the maximum-tolerated doses to achieve best possible cure rates, which for many entities such as lung, brain, colorectal, bladder, or human Papillomavirus (HPV)-negative head and neck cancer, are still far from satisfactory. Due to the intense treatment regimes a considerable fraction of patients suffer from severe acute as well as late and partly irreversible side effects that can seriously impact quality of life. For example in head and neck squamous cell carcinoma (HNSCC) the addition of platin-based chemotherapy to radiotherapy increases 5-year overall survival by about 10%  at the cCombining radiotherapy with molecular targeting agents may offer an alternative to chemoradiation with potentially less severe side effects, provided the tumor cells are more dependent on the specific target than normal tissue. To be effective, the targeting agent needs to be directly toxic for the tumor and/or has to induce a meaningful radiosensitization. Despite a plethora of promising preclinical data, the results achieved in the clinic are so far exceedingly disappointing. The only currently approved molecular targeting agent for the combination with radiotherapy is the anti-epidermal growth factor receptor (EGFR)-antibody cetuximab in HNSCC. The combination was approved on the basis of the IMC 9815 phase III clinical trial, which demonstrated superiority over radiation alone in a range similar to the addition of cisplatin to radiotherapy . HoweverA general limitation for the effective use of molecular targeted agents is the current lack of biomarkers that could predict a possible oncogenic addiction to a given druggable target or a possible role of the target in radiation resistance. Also in the case of cetuximab in HNSCC, no predictive biomarker has been established. In order to fully exploit the potential of precision medicine, such biomarkers are mandatory to select the best agents for a given tumor. Sequencing individual tumors for druggable driver mutations is one way forward. However, to what extent the targeting of such potential oncogenic driver proteins will also result in an enhanced sensitivity toward radiotherapy is currently unknown.Another important concern is therapy resistance due to backup pathways or incomplete inhibition. In such cases, combined molecular targeting approaches may be an effective way to increase efficacy. Combined targeting often follows three main strategies:  blockingHere we review preclinical attempts to utilize such dual targeting strategies for future tumor radiosensitization.*, radiosensiti*, radiotherapy, molecular targeted therapy, combined molecular targeting” was conducted and the results were screened for use of combined molecular targeting for radiosensitization in the preclinical setting. In addition, because titles and abstracts do not follow any regular pattern, references from identified articles were further screened for suitable publications and PubMed was additionally screened for publications from the last/senior authors of identified articles  includes the members EGFR , Her2 (ErbB2), HER3 (ErbB3), and HER4 (ErbB4). These transmembrane receptors are located at the cell surface, harbor an intrinsic protein kinase domain and regulate proliferation, migration, cell fate determination and apoptosis via diverse downstream signaling pathways such as MAPK and AKT signaling . EGFR isCombined inhibition of different members of the HER sub-family indeed showed promising results in terms of radiosensitization. For example in first studies Fukutome et al. combined the EGFR inhibitor gefitinib (TKI) and the anti-HER2 antibody trastuzumab. Both inhibitors induced radiosensitization on their own and their combination resulted in a synergistic sensitization in vulvar squamous cell carcinoma cells expressing EGFR and HER2 .Also EGFR/HER-2 inhibition by the dual inhibitor lapatinib resulted in enhanced radiosensitivity in cancer cells of various entities, such as bladder cancer, peripheral nerve sheath tumors, pancreatic or breast cancer. This sensitization was shown to be partly dependent on the expression of the specific targets  and to be inhibited through the constitutive activation of downstream signaling factors, such as Ras & Raf mutations –27.To inhibit EGFR and HER3 Huang et al. used the dual inhibitor MEHD7945A. They demonstrated that MEHD7945A inhibits growth in cetuximab (EGFR mAb) and erlotinib (EGFR TKI) resistant cells with a significant PI3K and MAPK pathway inhibition. In a xenograft model, MEHD7945A reduced the growth of tumors resistant to mono-EGFR-targeting, and, in contrast to cetuximab, the combination with radiation resulted in a more pronounced growth inhibition than either modality alone. EGFR and HER3 are both activated upon radiation and the blockade of one receptor may be compensated by the other. Treatment with MEHD7945A but not with cetuximab reduced survival signaling and DNA repair . The samIn addition to the formation of heterodimers within the HER-family there is also a cross talk between EGFR and other receptor tyrosine kinases such as the insulin like growth factor 1 receptor (IGF-1R), which is also involved in tumor development and progression . In thisThe HER receptors transduce their signals through several downstream pathways including the Ras-Raf-MAPK, the PI3K-Akt and the JAK/STAT pathway , 34. AltIn this context Bonner et al. assessed the effect of combined treatment of head and neck cancer cells with cetuximab and the JAK inhibitor JAK1i. STAT3 is a downstream protein activated by JAK (among others) protecting cells from apoptosis. The authors observed enhanced anti-proliferative and apoptotic effects upon dual inhibition plus radiation. Dual inhibition was accompanied by a more complete inhibition of STAT3-phosphorylation and, in contrast to single inhibition, resulted in radiosensitization in colony formation assays .Eke et al. identified the activation of c-Jun N-terminal kinase 2 (JNK2) via the scaffold protein JNK-interacting protein 4 (JIP-4) as a possible signaling bypass after EGFR targeting. The authors knocked down JIP4 or JNK2 via siRNA and used the JNK2 inhibitor SP600125 in addition to cetuximab treatment and achieved enhanced tumor cell radiosensitization in an additive manner as compared to single inhibition .in vitro and in a xenograft model  and their specific receptors (VEGFRs) are frequently targeted in cancer therapy, e.g., in lung, breast, kidney, ovarian and cervix cancer. A fundamental difference in this therapeutic strategy is that, although the inhibition of tumor cell signaling is also of relevance, the main target of VEGF(R)-inhibition is tumor angiogenesis. VEGFs and VEGFRs are critical factors in the formation and maintenance of new vasculature in both normal tissues and solid tumors , 41. TheIn this context Bozec et al. demonstrated promising results using the VEGFR inhibitor cediranib (AZD2171) (targeting VEGFR1/2/3) concurrent with the EGFR inhibitor gefitinib and radiotherapy in a VEGF secreting HNSCC xenograft model. Combined treatment plus radiation clearly inhibited tumor growth more effectively than dual or single inhibition or radiotherapy alone. Dual inhibition was associated with decreased vessel density and dual inhibition plus irradiation showed the highest decrease in proliferation as assessed by Ki67 staining . The groIn vivo the combination with irradiation showed enhanced tumor growth inhibition as compared to single treatment , which often results in the up-regulation of the mTOR Complex 2. Therefore, combined inhibition of mTOR Complex 1 and 2 has been studied using dual inhibitors. Sapanisertib is an ATP-competitive mTORC1 and mTORC2 inhibitor. Miyahara et al. demonstrated an enhanced inhibition of proliferation and induction of apoptosis when combining the dual inhibitor and radiation in diffuse intrinsic pontine glioma cells . Liu et In vivo PP242 alone had no impact on tumor growth but enhanced the radiation-induced growth reduction  in patient-derived and in radioresistant oral squamous cell carcinoma cells on alone . Dactoli in vivo , 62.In vivo dactolisib alone had little effect on tumor growth but profoundly enhanced the effect of irradiation  and Wee1  in HPV-positive HNSCC cells because they had observed an activation of Chk1 upon Wee1 inhibition that may in part counteract the effects of sole Wee1 targeting. Analyzing proliferation, inhibition of G2 arrest and radiosensitization, they found dual targeting to be effective at profoundly reduced concentrations as compared to single agent usage. Additionally, they observed only minimal radiosensitization in p53 proficient normal human fibroblasts, thus demonstrating tumor specificity .Vance et al. combined the inhibition of PARP1 through olaparib and Chk1 through AZD7762 in p53 mutant pancreatic cancer cells and observed an additive radiosensitization. The authors observed G2 checkpoint abrogation, inhibition of HR and a persistent γH2AX signal after combined inhibition of the two targets. There was no significant radiosensitization in G1-checkpoint-proficient intestinal epithelial cells, backing up the hypothesis that tumor cells harboring aberrations in p53 or other DNA damage response pathways are more selectively sensitized . In lineIn vitro the combination of both inhibitors caused enhanced radiosensitization as compared to single inhibition. In vivo, there was no radiosensitization with olaparib alone and a moderate effect of adavosertib. Combined targeting, however, demonstrated highly significant radiosensitization  is a key DDR kinase acting directly upstream of Chk1. Through Chk1 activation but also partly independent from Chk1, ATR is critically involved in replication processes, such as the stabilization of stalled replication forks, and in DSB repair pathways , 103. TaOlaparib was further combined with the Rad51 inhibitor B02 and X- as well as proton-irradiation with the intention to induce HR deficiency that would synergize with PARP inhibition. Lung and pancreatic cancer cell lines were radiosensitized by the inhibitors, with the strongest effect for dual inhibition, similarly for both types of irradiation. Radiosensitization was found to be dependent on the proliferation rate, as serum deprivation reduced the effectiveness of dual targeting and in slowly proliferating PANC1 cells the combination was even less effective than sole PARP-inhibition .The addition of the Chk1/2 inhibitor prexasertib to cetuximab and irradiation was investigated by Zeng et al. in HPV-positive and HPV-negative HNSCC cell lines. Prexasertib caused an accumulation of cells in the S-phase, the triple combination partly resulted in decreased proliferation and increased apoptosis as compared to single or double treatment .DNA-dependent protein kinase, catalytic subunit (DNA-PKcs) is well known as an essential component of the classical NHEJ pathway but is further associated with genomic stability, hypoxia, inflammatory responses, metabolism and regulation of transcription , 109. HaCell matrix interaction by integrins was shown to be a modulator of tumor progression, invasion, metastasis and response to therapy. β1-integrin, a member of the integrin family of cell adhesion molecules is significantly involved in tumor survival and proliferation and is associated with radio- or chemotherapy resistance . β1-inteEke et al. investigated the effect of concurrent β1 integrin and EGFR targeting using the monoclonal inhibitory antibodies AIIB2 and cetuximab, respectively in head and neck cancer cells. They observed enhanced cytotoxicity and radiosensitization upon combined inhibition in 8 out of 10 cell lines and, in line with that, enhanced survival in a xenograft model of a responder cell line . FAK wasRecently, a screen for predictive biomarkers for the dual β1-integrin/EGFR targeting approach showed different mutational profiles of responding and non-responding cells and suggested some proteins as potential resistance factors. Using an RNAi screen and pharmacological inhibition  Kelch like ECH associated protein 1 (KEAP1) and mTOR were identified as druggable targets for radiosensitization in combination with β1-integrin/EGFR targeting .C-Abl is a tyrosine kinase found to be hyperphosphorylated upon β1-integrin inhibition. Therefore, dual β1-integrin (AIIB2) and c-Abl (imatinib) targeting was tested in a panel of tumor cell lines from various entities, where a cell line dependent cytotoxicity and enhancement or induction of radiosensitivity was observed as compared to single treatment in a subgroup of the panel. Radiosensitization was accompanied by altered expression of DSB repair proteins KU70 and NBS1 and was associated with reduced DSB repair .in vitro and in vivo resulted in a superior effect when combined with radiation as compared to single inhibition, i.e., increasing the median survival of orthotopic, radiochemotherapy-treated GBM mice. In vitro the authors observed defects in DNA repair associated with chromatin changes, enhanced ATM phosphorylation, and prolonged G2/M cell cycle arrest as the underlying mechanism of radiosensitization , a known stress mediator, to mediate bypass signaling after β1-integrin-inhibition in established glioblastoma cell lines, as well as stem-like and patient-derived glioblastoma cells. Dual β1-integrin/JNK inhibition through AIIB2 and the JNK inhibitor SP600125 tization .Heat shock proteins (HSPs) are a group of proteins with enhanced expression in response to various kinds of stresses, such as hyperthermia, infections, heavy metals, or oxidative stress . As moleIt was shown that the inhibition of Hsp90 compromises DNA repair after irradiation and enhances tumor cell radiosensitivity , 126. HoFollowing the same concept as dual HSP70/90-inhibition the PI3K/mTOR inhibitor PI-103, which had previously been shown to suppress the up-regulation of Hsp70 , was comIn vitro from a panel of four cell lines only U373MG showed a profound radiosensitization after dual targeting as compared to single inhibition. Still, in vivo, U87MG showed a more pronounced tumor growth delay compared to single inhibition with and without the combination with irradiation  with the HSP90 inhibitor HSP990, which resulted in downregulation of the AKT pathway and induction of apoptosis. adiation .Also targeting human glioma cells Dungey et al. combined the inhibition of PARP through olaparib with the Hsp90 inhibitor 17-AAG. The rationale behind is that Hsp90-inhibition decreases HR, which is needed to repair replication-associated DSB generated through PARP inhibition. They observed a downregulation of Rad51 and BRCA2 protein levels and inhibition of HR upon HSP90 inhibition. Combined treatment resulted in additive radiosensitization in proliferating cells. Since the authors did not observe radiosensitization through HSP90 inhibition in non-tumor control cells and had previously described olaparib-mediated radiosensitization to be replication-dependent, they expect an enhancement of the therapeutic ratio by taking advantage of the non-dividing state of normal brain tissue .Tao et al. observed that in Kras-mutant NSCLC cells, the inhibition of MEK through trametinib resulted in p16 expression and reduced phosphorylation and therefore activation of the tumorsuppressor RB in the cell line most sensitive toward both sole MEK-inhibition and MEK-inhibition induced radiosensitization. Likewise, activation of RB through CDK4/6-inhibition through palbociclib sensitized the more resistant cells to MEK-inhibition and resulted in enhanced radiation sensitivity as compared to single treatment. Dual targeting plus irradiation was also most effective in a xenograft model .Histone deacetylase inhibitors are a heterogeneous group of epigenetic therapeutics, which a.o. interfere with DNA damage signaling and repair . Moertl In vitro, the inhibitor sensitized subsets of HPV-negative and HPV-positive HNSCC cell lines to TNF family death ligands TNFα and TRAIL, which involved a reactivation of p53 in the HPV-positive strains. In HPV-positive and -negative human HNSCC xenografts the authors observed significantly delayed growth when the dual inhibitor was combined with radiation, which was attenuated by anti-TNFα pretreatment blockade radiation. However, when searching for trials with combined molecular targeting for radiosensitization in the narrow sense, we only found two running studies combining two inhibitors with sole radiotherapy, both in HNSCC. One trial is testing the combination of cetuximab and the CDK4/6 inhibitor palbociclib, a combination, for which we did not find a preceding preclinical evaluation but only a similar approach combining MEK-inhibition plus palbociclib  radiation. A common approach is the combination of chemoradiation, inhibition of signal transduction pathways and VEGFR-inhibition. However, while these combined inhibitor approaches clearly cover anticipated effectiveness through dual targeting, the purpose of radiosensitization is less in focus than the idea of achieving additive effects through repression of angiogenesis. Especially the combination of chemoradiation, EGFR-inhibition and the anti-VEGF antibody bevacizumab has been tested in different entities but efficacy so far appears limited: In HNSCC, the addition of bevacizumab to radiation, cetuximab and pemetrexed was reported to increase toxicity without an apparent improvement in efficacy . SimilarAt present, molecular targeting is often added to current chemoradiation regimes to increase efficacy and trials are mostly in early clinical development. It is therefore not surprising that combined targeting approaches are still quite rare and often based on dual specific inhibitors, which may be easier to implement than inhibitor combinations for which toxicity data may still be lacking, even without radiotherapy.+-, glucose- or mitochondrial metabolism . This should make them more sensitive to kinase inhibitors than normal tissue, providing some tumor specificity. What remains a major challenge is the choice of pathway inhibition for individual tumors, which requires reliable biomarkers. Unfortunately, the commonly analyzed kinase expression level is a poor surrogate for actual kinase activity, as we have recently demonstrated for EGFR activation in HNSCC . KeepingA major advantage for the targeting of DDR components is their direct involvement in radiation-induced DNA repair. On one hand, this makes it likely that a majority of tumors will be affected. On the other hand, specificity can be a major issue, as normal cells utilize the same pathways for DNA damage recognition, processing and repair. Nine of the 13 studies identified in this field combined PARP- and S/G2 phase checkpoint-inhibition. This approach is partly based on the model of synthetic lethality see , which hSimilar characteristics, i.e., high pathway activity and tumor cells‘ reliance also motivate the targeting of adhesion molecules  or the in vivo analyses of side effects other than weight loss and inspection of the skin/mucosa in the radiation field. Future approaches should therefore not only focus on the identification of the most efficacious radiosensitization but also more deeply on the safety of a possible clinical translation. Detailed in vivo studies on systemic as well as in-field toxicity may help design the most promising clinical trials and achieve better clinical outcomes.The tailored use of molecular targeting agents based on individual tumor characteristics is referred to as precision oncology. Apart from enhancing efficacy and thereby cure rates, molecular targeting is also expected to reduce toxicity, in case it can replace chemotherapy. It has to be noted, however, that the use of targeting agents can result in considerable side effects, which can of course be more severe and difficult to predict when agents are combined and added to (chemo-) radiotherapy. For example, EGFR inhibition frequently causes skin rash and diarrhea , which cin vitro mechanistic studies that should ideally include predictive biomarkers, with the results substantiated in a relevant number of adequate model systems, such as cell lines and (patient derived) xenografts, but possibly also tumor stem cell cultures as well as ex vivo cultured tumor tissues. The most promising combined targeting approaches should be thoroughly inspected for treatment efficacy and safety based on normal tissue toxicity in vivo. Such a concept should lead to the identification of effective targeting strategies for subsets of tumors, based on reliable predictive biomarkers to provide the best possible preclinical rationale to allow clinicians to implement the most appropriate combined targeting strategies in well-designed clinical trials.In conclusion, dual targeting for tumor radiosensitization has shown promising results in pre-clinical studies. The way to proceed toward a substantial future clinical benefit requires convincing KH and TR: idea, literature search, and preparation of tables and figures. All authors were involved in the preparation of the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Several broadly circular structures up to 16 m in diameter, into which higher strata have sagged and locally collapsed, are present in a tephra outcrop on southwest Öræfajökull, southern Iceland. The tephra was sourced in a nearby basaltic tuff cone at Varða. The structures have not previously been described in tuff cones, and they probably formed by the melting out of large buried blocks of ice emplaced during a preceding jökulhlaup that may have been triggered by a subglacial eruption within the Öræfajökull ice cap. They are named ice-melt subsidence structures, and they are analogous to kettle holes that are commonly found in proglacial sandurs and some lahars sourced in ice-clad volcanoes. The internal structure is better exposed in the Varða examples because of an absence of fluvial infilling and reworking, and erosion of the outcrop to reveal the deeper geometry. The ice-melt subsidence structures at Varða are a proxy for buried ice. They are the only known evidence for a subglacial eruption and associated jökulhlaup that created the ice blocks. The recognition of such structures elsewhere will be useful in reconstructing more complete regional volcanic histories as well as for identifying ice-proximal settings during palaeoenvironmental investigations.The online version of this article (doi:10.1007/s00445-016-1048-x) contains supplementary material, which is available to authorized users. Öræfajökull is the largest active stratovolcano in Iceland. It is situated on the southern margin of Vatnajökull and has a basal diameter of c. 23 km, rising to 2110 m a.s.l. at Hvannadalshnúkur a rhyolite lava dome, which is the highest peak in Iceland Fig. . ÖræfajöThis paper describes distinctive features in the tephra apron, comprising broadly circular structures up to 16 m in diameter in which higher strata have sagged and locally collapsed. The structures appear to have formed as a result of collapsing into voids. They show evidence for the soft-state deformation of tephras. Soft-state deformation features are commonly observed in pyroclastic deposits of tuff cones, tuff rings and tephra aprons  at the base of the southern flank of a small pyroclastic cone 900 m to the north Fig.  that is 5° at theThe discontinuous stratification, abundance of ash (including fine ash), variably vesicular juvenile lapilli with blocky shapes, abundant impact structures as well as clasts lacking impact structures, together with the presence of dune and antidune bedforms and impact structures filled by large lapilli are characteristic of deposits formed during phreatomagmatic eruptions  for the area to have been flooded by the sea. The tuff cone may have erupted either in a surface lake caused by melting through an extensive ice cover or else the supply of water was from a free-flowing aquifer  contains most of the unusual features that are the focus of this study. The tephra forming the ridge was deposited on a pre-existing broad topographical divide, or watershed, as exposed today Fig. . The ridTwo of the structures  faulted steep-dipping  margins that sharply crosscut external lapilli tuff strata; (2) downbending of some beds directly adjacent to the faulted margins; (3) local presence of a marginal zone of massive lapilli tuff with a semi-homogenised appearance, within which are indistinct blocks of stratified beds; (4) the higher beds simply bend down into the structure , defining symmetrical and asymmetrical concentric sag-like geometries; and (5) some structures contain breccia composed of stratified blocks with bedding in various dips. A lack of burial by younger tephra suggests that the structures formed after the eruption had ceased. Moreover, distinctive sedimentary structures would have formed in pyroclastic density current deposits passing over surface hollows, with variations in bed thickness and grain size.The surfaces between adjacent blocks in the breccias are ill-defined Fig.  and the Bed strength would have been greatly weakened by any coeval fractures caused by settling in the tephra pile as it accumulated, and they are perhaps represented by the intersecting tensile fractures, some with decimetre displacements, observed in the higher downsagged tephra beds. Collapsing beds that remained intact as stratified slabs may have been more cohesive or been deformed at lower strain rates, perhaps related to more gradual mass withdrawal due to slower melting or the local geometry of the melting ice. The ubiquitous evidence for sagging suggests that the overall circular structural features are collapse structures. Analogue models suggest that collapse rates during subsidence vary temporally and spatially, and are associated with incremental accelerations  a thick snow cover, (2) snow drifts (sastrugi), (3) dirt cones, (4) deep bedrock depressions filled by snow and (5) ice blocks. These are examined further below.How tephra subsides when it covers a thick layer of snow or firn is hard to predict. Tephra fall deposits with grain sizes comparable to those at Varða are completely cold on deposition  resting on a melting layer of snow but some localised circular collapse structures with steeply tilted beds and pits have developed below the 1875 tephra at Askja . However, there is evidence that thick snow (i.e. more than a few metres) was probably absent at Varða when the tephra was deposited (see above). If the melting layer was isotropic (i.e. had a constant thickness and uniform grain size), we speculate that subsidence of an overlying thick tephra layer might be relatively uniform, with pits restricted to anisotropic patches of snow, i.e. with a different crystal structure or hardness. If the snow layer was highly anisotropic  prior to tephra deposition), then melting might over time lead to differential subsidence. However, sastrugi are typically ridge-like with sharp, linear, wavy or otherwise sculpted crests and sloping often asymmetrical flanks, and occur in fields of closely spaced landforms. Sastrugi are also generally smaller, seldom more than a few metres high and wide. Whether their melting beneath a thick tephra layer might result in isolated concentric collapse pits with vertical to slightly overhanging margins  is unclear as there are no described examples.Pits might also form by the melting of the snow cores of buried dirt cones. Dirt cones in volcanic regions like Iceland commonly form by the differential melting of snow beneath a surface ash layer of variable thickness; they are particularly common on glacier ice. Although dirt cones are usually <1–3 m high, exceptionally they can be much larger, up to 85 m  and, even if such features are unexposed, there would be lithofacies evidence, such as bed thickening and preferential deposition of coarser clasts. They are thus unlikely to be involved.The collapse of sediment into voids created by the melting of buried ice is plausible and is our favoured option. Similar surface structures on glacial outwash plains (sandurs) are known as kettle holes. Kettle holes are hollows created in the sedimentary deposits when partially to completely buried blocks of glacier ice melt out (e.g. Maizels The dimensions of the Varða structures (c. 16 m in width) can be used to estimate the approximate dimensions of the former buried ice blocks, taking into account that the subsided volume will flare upwards at the angle of draw (θ), thus widening the diameter of the structure at the surface . Although relatively well preserved, apart from the post-glacial carving out of the Bæjargil gorge, the Varða tuff cone is also glacially eroded and it is draped by till from the last glaciation. The agglutinate cone that fed the underlying ‘a‘ā lava also shows signs of overriding ice . However, ice was not present at the site of either pyroclastic cone during their eruption. An interglacial Eemian age (i.e. c. 132–116 Ka) is thus possible for eruption of both the agglutinate cone and the Varða tuff cone . It could have occurred following the Allerød warm period, when the Icelandic ice sheet expanded greatly at least twice  are possible but less plausible and identification as former ice blocks is favoured. However, whatever their precise nature, the buried objects melted to create voids and were thus glacial in origin. The ice blocks were probably formed as a result of a preceding jökulhlaup that may have been triggered by a subglacial eruption. Called ice-melt subsidence structures, they are the sole evidence for that eruption which is preserved today. The location of the subglacial centre is unknown but presumably was within an ice cap situated higher upslope on Öræfajökull. Thus, the distribution of ice and snow-free ground implied by our study would have been broadly similar to present, precluding eruption within a glacial period. For such structures to be created relies on a tuff-forming eruption burying ice blocks generated by a preceding jökulhlaup since ice blocks do not survive long on the surface . The structures are analogous to kettle holes found much more commonly on proglacial sandurs and in some lahars sourced in ice-capped volcanoes. The ice-melt subsidence structures at Varða are another proxy for recognising an ice-proximal setting for volcanism and their recognition will enhance our ability to reconstruct palaeoenvironments more reliably, as well as for compiling more compete eruptive histories of regions experiencing glaciovolcanic eruptions.ESM 1View looking northwest across the mouth of Bæjargil showing steeply dipping Varða tephra draped across an older cliff face. (JPEG 1147 kb)ESM 2a–c Views showing numerous large ice blocks strewn across Skeidarársandur following the jökulhlaup associated with the 1996 eruption of Gjálp, Vatnajökull, photographed in 1997. Note the angular shapes of the individual blocks, their varied and often large sizes and steep sides. Although these ice blocks are exposed on the sandur, ice blocks also become completely buried during jökulhlaups (see d). d Kettle hole developing above an ice block completely buried in a lahar deposited by a jökulhlaup from Gígjökull during the 2010 eruption of Eyjafjallajökull (cf. ice-melt collapse pits of Branney and Gilbert 1995); photographed in July that year. The two steep-sided pits seen in the background are more fully developed kettle holes with vertical sides, also associated with completely buried ice. All images courtesy of Andy Russell. (JPEG 13163 kb)ESM 3Series of diagrams illustrating the evolution of subsidence in calderas (modified after Acocella 2007). Four empirical stages are recognised, with different surface expression and development of internal structures. The subsidence may stop at any stage depending on the local circumstances. Note the development of surface sagging and the progressive upward growth of reverse and normal ring fractures. In stage 3, normal faults are depicted migrating upward draped by downsagged beds, a relationship broadly similar to that observed in Varða structures 3 and 4 . The lack of a reverse ring fault cutting the surface at Varða may be due to a combination of a very slow strain rate and the ductility of the cohesive, mechanically weak lapilli tuffs, which reacted by downsagging. However, the presence of local breccias at Varða  indicates that strain rates were variable. (JPEG 399 kb)