Based on the GRS CT-based grading, 97.7% (340/348) were graded A or B, 1.7% (6/348) screws were graded C, and only 0.6% (2/348) of screws were graded D. Two complications, explantation of interbody and vacuum-assisted wound closure, were reported as requiring a return to the OR, but these were not related to robotic guidance or pedicle screws. This study demonstrated a high level of accuracy (97.7%) in the first 56 cases using navigated, robot-assisted surgery based on the GRS. There were two non-screw-related complications requiring return to the operating room.In the emerging field of robot-assisted spine surgery, the radiographic evaluation of pedicle screw accuracy in clinical application is an area of high interest. This study describes the pedicle screw accuracy of the first 56 consecutive cases in which navigated robotic assistance was used in a private practice clinical setting. A retrospective, Institutional Review Board-exempt review of the first 56 navigated robot-assisted spine surgery cases was performed. Pedicle screw malposition, reposition, and return to operating room (OR) rates were collected. A CT-based Gertzbein and Robbins system (GRS) was used to classify pedicle screw accuracy. In the first 56 robotic cases, 356 total pedicle screws were placed. Eight screws were placed without the robot due to surgeon discretion. Of the 348 pedicle screws inserted by navigated robotic guidance, only 2.6% (9/348) were repositioned, resulting in a 97.4% (339/348) successful screw placement rate. The average age was 64, and 48% were female. Average body mass index was 31 kg/m Pedicle screw constructs have become the standard for stabilization and fusion in spinal surgery. HoweverSince the launch of pedicle screws for spinal stabilization, numerous techniques have been used to direct and confirm screw placement. ExampleAn Institutional Review Board-exempt retrospective chart review was conducted from January to August 2018 on the first 56 minimally invasive navigated robotic-assisted spine surgeries. Demographic, intraoperative, perioperative data and radiographs of patients who underwent lumbosacral pedicle screw placement with minimally invasive navigated robotic guidance using intraoperative CT were analyzed. Pedicle screw accuracy, malposition, and reposition rates were collected.®; Globus Medical, Inc. Audubon, PA, USA)  Fig.  uses radIn this study, the robotic system operated on one functional modality, intraoperative CT. In this mode, the image coordinate system was obtained from a portable intraoperative CT  or a standard CT scan taken at the time of surgery with the patient already in surgical position (prone). The spinal levels were identified and a CT scan was taken. Pedicle screw trajectories were planned and saved. Reference frames were installed and fixated to the pelvis, and instruments and arrays with reflective markers were registered. A surgeon-controlled foot pedal activated and positioned the robot arm to the planned pedicle trajectory. Stab incisions were made on the skin using a scalpel. Pedicle screws were inserted using navigated instruments guided by the robotic arm. This sequence was repeated until all pedicle screws were placed. Rods were then placed and locking caps were set once the rods were in the proper position. Intraoperative CT images were taken to verify screw and rod position. In cases where interbody spacers were placed, they were placed manually. Surgical incisions were cleaned and closed in the standard fashion.A CT-based Gertzbein and Robbins system (GRS) was used to classify pedicle screw accuracy. According to the GRS, screws completely within the pedicle are considered grade A; a breach of < 2 mm is grade B; a breach of 2 to < 4 mm is grade C; a breach of 4 to < 6 mm is grade D; and a breach of > 6 mm is grade E Table . Accordip < 0.05 for all statistical analysis. Statistical analysis was performed using SPSS Statistics Version 25 software .Frequency tables and measures of central tendencies were used for descriptive statistics. Parametric and nonparametric tests were used for continuous quantitative variables and qualitative variables, respectively. The level of statistical significance was set to 2 (range 21–44 kg/m2). There were 6 mean number of screws per case and 3.2 mean number of vertebrae. The most common number of levels with screws placed is 3, with a total of 22 patients (39.3%). The most common disposition was home (33.9%) or rehabilitation (32.1%). The most common levels instrumented were L4 (26.4%) and L5 (27.0%)  were repositioned resulting in a 97.4% (339/348) successful screw placement rate. The average age was 64 ± 11 years and 48% (27/56) were female. Average body mass index was 31 kg/mRobotic-assisted spine procedures are in early development. This stTechnological advances including navigation have improved the safety and accuracy of pedicle screw fixation. A meta-analysis performed by Meng et al. includedThe robotic guided navigation technique used in this study is a viable approach to pedicle screw placement, with a lower malposition rate (2.3%) compared to 4.2% reported by Sarwahi et al.. OverallData from this study demonstrated a 97.7% accuracy rate in the clinical use of navigated, robot-assisted surgery. Navigated robotic guidance of pedicle screw placement is a safe and useful tool for assisting spine surgeons with pedicle screw placement.


An appropriate screw trajectory and insertional depth were confirmed using X-ray imaging prior to pullout testing in both groups. The pullout forces of larger-diameter screws (6.5 mm × 45 mm) and longer screws (6.0 mm × 50 mm) were significantly higher (p < 0.05) in the semi- and non-pedicle modalities in the normal-density group, whereas only longer screws (6.0 mm × 50 mm) had a significantly higher (p < 0.05) pullout force in the non-pedicle modalities in the osteoporosis group. The pedicle plays an important role in both the normal bone density group and the osteoporosis group, as revealed by analyzing the pullout force percentage contributed by the pedicle. Use of a longer screw would be a way to salvage a broken pedicle of osteoporotic vertebra.Screw loosening due to broken pedicles is a common complication resulting from the insertion of screws either with inadequate diameters or into an osteoporotic pedicle. In this novel in vitro study, we tried to clarify the contribution of the pedicle to screw fixation and subsequent salvage strategies using longer or larger-diameter screws in broken pedicles. Sixty L4 fresh-frozen lumbar vertebrae harvested from mature pigs were designed as the normal-density group (n = 30) and decalcified as the osteoporosis group (n = 30). Three modalities were randomly assigned as intact pedicle (n = 30), semi-pedicle (n = 15), and non-pedicle (n = 15) in each group. Three sizes of polyaxial screws  over five trials were used in each modality. The associations between bone density, pedicle modality and screw pullout strength were analyzed. After decalcification for 4 weeks, the area bone mineral density decreased to approximately 56% ( Screw loosening due to broken pedicles is a common complication resulting from the insertion of screws either with inadequate diameters or into an osteoporotic pedicle7. In a well-designed L4 sawbone model4, the pedicle contributes to the screw pullout strength, and only 29–39% of the pullout strength is retained after the pedicle breaks. However, there is a lack of biomechanical studies addressing the pullout strength of broken pedicles under normal bone density and osteoporosis conditions that are more clinically practical. Biomechanical testing has shown a decrease of 80.7% in elastic modulus, 74.7% in yield stress and 74.5% in ultimate stress in decalcified vertebral bodies compared with non-decalcified bodies8, but the strength of the pedicle cannot be evaluated only by the vertebral body strength. Varghese et al. used polyurethane foam blocks to investigate the effects of bone density, insertion depth and insertion angle on pedicle screw pull out strength and insertion torque. The contribution of bone density was found to be the most influential factor for the pullout strength and insertion torque10; however, their result was hardly convincing, as polyurethane foam blocks were used instead of an anatomical vertebral model.Pedicle screw fixation during spinal surgery has been a common procedure for several decadesTo the best of the authors’ knowledge, no existing research has addressed the effect of the absence of pedicles on screw anchoring power using different-density porcine vertebrae rather than test blocks or simulated sawbones. The present study is the first to determine any difference in pullout strength on the fixation stability of pedicle screws in different broken pedicle modalities and under different bone densities.In this novel in vitro experimental study, we compared the pullout strength of screws between the intact pedicle, semi-pedicle and non-pedicle modalities in normal and osteoporotic porcine vertebral models and then examined subsequent salvage strategies.We hypothesized that the pedicle contributes to the screw pullout strength in both the normal bone density group and the osteoporosis group, and both longer and larger-diameter screws could be used for salvage methods.2 in the normal group, 0.9 ± 0.07 g/cm2 in the first week, 0.81 ± 0.05 g/cm2 in the second week, 0.68 ± 0.07 g/cm2 in the third week and 0.57 ± 0.02 g/cm2 in the fourth week. After treatment with EDTA for 4 weeks, the area BMD decreased to approximately 56% (p < 0.05) of that of the normal vertebrae and reached the osteoporotic stage8. Specimens that had been treated with EDTA for 4 weeks were assigned to the osteoporosis group, and those without EDTA treatment were assigned to the normal-density group.The serial changes in area BMD from normal to decalcified states are shown in Fig. An appropriate screw trajectory and insertional depth were confirmed using X-ray imaging prior to pullout testing in the normal-density group Fig.  and osteIn the normal-density groups, 6.0 mm × 45 mm, 6.0 mm × 50 mm, and 6.5 mm × 45 mm screws had mean pullout strength values of 1,710.1 ± 387.6 N, 2039.1 ± 443.1 N, and 2,493.1 ± 383.2 N, respectively, in the intact pedicle modality; 762.6 ± 341.8 N, 1,325.1 ± 282.6 N, and 1686.1 ± 327.4 N, respectively, in the semi-pedicle modality; and 565.2 ± 226.9 N, 1,019.4 ± 324.8 N, and 901.3 ± 127.6 N, respectively, in the non-pedicle modality Fig. . CompareIn the osteoporosis groups, the 6.0 mm × 45 mm, 6.0 mm × 50 mm, and 6.5 mm × 45 mm screws had mean pullout strength values of 162.2 ± 34.7 N, 189.1 ± 36.0 N, and 217.3 ± 15.3 N, respectively, in the intact pedicle modality; 89.5 ± 42.6 N, 124.7 ± 49.1 N, and 145.4 ± 41.4 N, respectively, in the semi-pedicle modality; and 43.1 ± 22.0 N, 88.2 ± 17.1 N, and 101.4 ± 62.1 N, respectively, in the non-pedicle modality Fig. . CompareA comparison of the pullout force between the normal-density group and the osteoporosis group for various screw sizes and pedicle modalities is shown in Fig. The pullout force percentage contributed by the pedicle for the normal and osteoporotic groups is shown in Fig. 4, the results revealed that pullout strengths of only 41–45% in the semipedicle group and 29–39% in the nonpedicle group were retained for all three screw sizes. These results indicate that the pullout force percentage contributed by the pedicle reaches 59–71%. The pullout force contributed by the pedicle seems to be higher in standard sawbone than in porcine vertebrae.The pullout force percentage of the pedicle was 60 ± 11% in the normal-density group and 57 ± 19% in the osteoporosis group, but the difference was not significant  and longer screws (6.0 mm × 50 mm) were significantly higher than those of the 6.0 mm × 45 mm screws in the semi- and non-pedicle modalities, which means that switching to larger-diameter screws or longer screws could be a salvage method when the pedicle is broken during surgery in individuals with normal bone density. However, in the osteoporosis group, the significantly higher pullout force in the non-pedicle modality belonged only to the longer screw, which implies that the use of only a longer screw can be considered a means of salvaging. Reasons for the difference in salvage conditions between the normal bone density and osteoporosis groups are the following: (1) a result of the bone density decreasing from the surroundings to the center of the vertebral body during the decalcification process, which implies that a longer screw rather than a larger-diameter screw would be suitable for the inner undecalcified bone Fig. ; and (2)21 reported that the 503 N (279/731) pullout force in the osteoporotic human spine, which is significantly larger than that found in our study in the osteoporosis group, could be explained using different pedicle contributions and different pedicle anatomical sizes between humans and swine22. Lee et al.8 stated that approximately 16.02% of the mean compression ultimate strain in similar osteoporotic vertebrae is conducted in a biomechanical axial compression system instead of a pullout test. The similar decrease in BMD did not imply the corresponding reduction in strength due to the use of different anatomical sites for measurement and testing methods. Lai et al.23 reported that larger diameter screws (5.0 mm × 35 mm) achieved a higher pullout strength than that of smaller (4.35 mm × 35 mm) screws in an osteoporotic model. The osteoporotic density was not uniform, and tests were performed in human thoracic vertebrae, yielding different results than those of this study.In our study, the pullout force decreased to 9–12% of that of the osteoporotic group Fig. , which i4 because of the different pedicle sizes between humans and swine22. A higher pullout force contributed by the pedicle in sawbones is predictable because the pedicle width and height are significantly larger in humans than in pigs11.In our normal-density porcine vertebrae, the average pullout strength after the complete percentage of the pedicle contributed to the pullout force reached 40% Fig. , which i29 or methods32 should be applied clinically, especially when pedicles are broken in osteoporotic spines.To the best of the authors’ knowledge, no biomechanical reports have addressed the pullout strength of the pedicle in a normal BMD and osteoporotic porcine vertebral model or the subsequent salvage strategies to prevent screw loosening. The percentage of pullout force contributed by the pedicle reached 60% in the normal-density group and 57% in the osteoporosis group, without a significant difference, which indicates the important role of the pedicle in either normal bone density or osteoporotic vertebrae through the whole screw pullout performance. Due to the preserved force of only 9–12% after decalcification, longer screws with supplementary strengthening materials33. Second, the lack of blindness of practitioners, data collectors and analysts could result in potential selection bias. Third, limited number of specimens might increase variability of data and reduce the statistical reliability. Finally, data from standard experimental semi-pedicles and non-pedicles may not completely reflect clinical biomechanical data from irregular-shaped broken pedicles.The present study had some limitations. First, inherent limitations, such as different geometries or density distributions of porcine vertebra compared with those of humans may limit clinical practiceThe pedicle plays an important role in both the normal bone density group and the osteoporosis group on the basis of the whole screw pullout performance. In the normal-density group, both longer and larger-diameter screws could be used for salvaging; however, in the osteoporosis group, only the use of a longer screw would be a salvage method.This study was approved by the review board of Chang Gung Memorial Hospital (CRRPG3H0082). All experiments conformed to the regulations for the care and use of animals. The usage of the porcine spines was in accordance with the guidelines of replacement, reduction and refinement. A flowchart of the study design is presented in Fig.  every week after embedding in a 10% formalin solution for 24 h.This study was performed using sixty L4 fresh-frozen lumbar vertebrae harvested from mature pigs . All animals were healthy before harvesting and never exposed to any drugs or procedures that could affect the bone density. All specimens were separated into individual vertebrae after the surrounding musculature, ligaments, and periosteum were stripped off. Thirty of these 60 vertebrae were designed as the normal-density group and embedded in 10% formalin solution  for 24 h before the biomechanical and imaging studies. The other 30 specimens were designed as the osteoporosis group and stored in a glass tank at room temperature for four weeks of decalcification. The glass tanks were filled with 500 ml, 0.5 Mof EDTA decalcification solution , with renewal of the EDTA X-ray images  were obtained every week during the decalcification process Fig. . The pos4. A pilot hole was drilled using a 2.5 mm “twist” metric drill bit attached to a Dremel 4000 rotary tool that was mounted on a Dremel WorkStation Model 220-01. This trajectory was selected based on previously reported morphometric characteristic data33. The pilot track was followed with a standard straight pedicle probe to a depth of 45 mm. Three sizes of polyaxial screws   were chosen and randomly implanted into each pedicle of the vertebrae by an experienced surgeon. These chosen screw sizes were based on the most commonly used clinically. Then, the specimens were randomly distributed into the intact pedicle modality (n = 30), the semipedicle modality (n = 15), or the nonpedicle modality (n = 15). These experiments were conducted over five trials for each screw size . Failure was defined as the maximum load or the load peak prior to a decrease in load associated with increasing displacement36. After the pullout test was completed, the specimen and the screws were closely examined for signs of fracture and damage and any findings were carefully recorded.Each of the 60 specimens was potted in metal boxes using specific epoxy resins . Judicious potting was performed to ensure that the cement did not come into contact with any portion of the pedicle screw. The prepared specimens were mounted onto a material testing machine  to conduct axial pullout tests with the screws Fig. . The scrp < 0.05.Statistical software  is used to analyze the pullout force of all specimens. All of the measurements were collected from 60 vertebrae and expressed as the mean ± standard deviation. Normal distribution of the sample was examined using Shapiro–Wilk test; whereas Levene's test was used to test the Homogeneity of Variances. ANOVA test with post hoc LSD analysis was performed to evaluate the differences between groups if the data is normally distributed and homogeneity. Differences were considered significant at Supplementary information.


Online biological databases housing genomics, genetic and breeding data can be constructed using the Tripal toolkit. Tripal is an open-source, internationally developed framework that implements FAIR data principles and is meant to ease the burden of constructing such websites for research communities. Use of a common, open framework improves the sustainability and manageability of such as site. Site developers can create extensions for their site and in turn share those extensions with others. One challenge that community databases often face is the need to provide tools for their users that analyze increasingly larger datasets using multiple software tools strung together in a scientific workflow on complicated computational resources. The Tripal Galaxy module, a ‘plug-in’ for Tripal, meets this need through integration of Tripal with the Galaxy Project workflow management system. Site developers can create workflows appropriate to the needs of their community using Galaxy and then share those for execution on their Tripal sites via automatically constructed, but configurable, web forms or using an application programming interface to power web-based analytical applications. The Tripal Galaxy module helps reduce duplication of effort by allowing site developers to spend time constructing workflows and building their applications rather than rebuilding infrastructure for job management of multi-step applications. The execution of scientific workflows is increasingly important in biological research, especially as the costs for data acquisition are decreasing and the quantity and complexity of data is increasing. The fields of genomics, genetics and phenomics are important generators of large-scale data in biology as evidenced, in genomics, by more than 31 petabytes of DNA sequence deposited into the NCBI Sequence Read Archive (SRA)  of resulAdditionally, scientists have varying access to computational resources which may include stand-alone workstations, institutional HPC clusters, national-level infrastructure such as XSEDE , JetStrehttps://usegalaxy.org/), powered by the Texas Advanced Computing Center (TACC) and CyVerse  analysis , genomicDespite the benefits of Galaxy and other scientific workflow tools, there is a need for the online data repository (or database) to provide analytical tools for their users. When online genome databases first appeared in the 1990s, they often included computational tools that directly used the data they provided. The most common tool offered was an online BLAST , 15 toolTo this end, the Tripal  platformhttps://github.com/tripal/tripal_galaxy with full online documentation at https://tripal-galaxy.readthedocs.io/en/latest/. It is a Gold rated module (the highest level) according to the current Tripal Module Rating System.As with all online data repositories, Tripal-based sites may want to provide analytical tools to their users, yet due to the complexity of modern large-data workflows, this becomes challenging, especially for the smaller teams that typically manage a Tripal-based site. To this end, the Tripal Galaxy extension module was created to support ‘wrapping’ of workflows that are available in a remote Galaxy instance. Tripal-based sites can integrate with Galaxy workflows in two different ways: first, via auto-generated web forms for commonly executed workflows, education and training or clinical applications, and second via the Tripal Galaxy API to power online web applications. Here we describe these two approaches with brief usage examples. The Tripal Galaxy module source code is available on GitHub at The Tripal Galaxy module is an extension module for Tripal. Thus, any Tripal-based site can download, install and enable the module and quickly add support for Galaxy workflows with minimal effort. The Tripal Galaxy module uses the Blend4php library , also wrThe first method for integration with Galaxy is through automatic generation of web forms for a specific Galaxy workflow. With this method, a Tripal-site developer can specify an existing workflow on a remote Galaxy server and the module will automatically create a step-by-step web form that an end user can use to upload or specify input data, set parameters and launch the workflow. There are several reasons why a site developer may want to provide a web form within their site rather than redirecting the user to a Galaxy instance. First, using a web form within the site’s visual theme maintains the end user experience. Second, the site developer can control the workflows the site is willing to support. This can be helpful for sites that receive data from their users for inclusion in the site. By ensuring that workflows follow proper community-accepted standards and protocols, the site can more easily verify the quality of input data and reduce the time required by site curators. Results that already reside on the site are more easily integrated. Third, by providing commonly used workflows, the site can easily provide site-specific data for use in workflows for their users, thus easing the data management and computational burden that some of their users may have difficulties with. Fourth, workflows can be used for educational purposes. Site visitors can be trained in analytical concepts without the burden of learning Galaxy or HPC systems. Fifth, web form workflows might be more appropriate for a clinical setting, where clinicians need not be distracted by the wealth of functionality in Galaxy.To demonstrate creation and use of web forms, a small Galaxy-created NCBI BLAST workflow is shown in After a workflow is selected, a step-by-step web form is created. As an example, When a workflow begins or terminates (either successful or failure) the user receives an email informing of the status. Site administrators can customize the text of these emails for each workflow, if desired, using the Tripal Galaxy administrative interface. During execution, Galaxy houses results within a storage concept referred to as a ‘history’, and each invocation of a workflow via Tripal uses a unique history. Therefore, results for all workflows are kept separate from one another, and the entire set of files  for a single workflow execution are available to the end user. Upon successful completion, the user can view the status of their workflow from their site profile pages and click a link to view the results page. If an error occurs, the end user (and site administrator) can view the error on the results page. The Tripal Galaxy module will provide all final and intermediate result files that are present in the workflow history. Unfortunately, the end results of a workflow are not always best understood via a list of files that the end user must peruse. Often, summary statistics, coupled with results, housed in a descriptive report will best serve the end user. The Tripal Galaxy module does not provide any visualization or summarization of results into reports. For this purpose, the Aurora Galaxy Tools toolkit  exists. https://tripal-galaxy.readthedocs.io/en/latest/API/api.html.In some cases, a Tripal-based site developer may wish to create a PHP-based web application that is meant to use a scientific workflow ‘behind-the-scenes’. The end user, in this case, does not interact with a web form as previously shown, but rather interacts with the web application. The web application integrates with the Tripal site and allows the end user to execute tasks through some custom interface. Those tasks are then executed on behalf of the user using pre-built Galaxy workflows that are also created by the site developer. The web application can communicate with the Galaxy server using the Tripal Galaxy API function set—a set of PHP functions. The API enables the programmer to upload the necessary files, initialize the workflow with appropriate parameters, launch the workflow, check its status and retrieve results once completed. The programmer can therefore customize the experience the web application provides to the user without exposing the Galaxy or Tripal Galaxy interfaces. https://treegenesdb.org/ct)  , 36 on tFor either approach, the Tripal Galaxy module provides a variety of administrative features. First, as previously mentioned, site administrators can add remote Galaxy servers such that Tripal knows about them. This in turn makes remote workflows discoverable for integration on the site. Galaxy servers and workflows that are added via the Tripal Galaxy API will also be visible via the administrative pages.Second, a job queue is available that provides to site administrators a listing of active, completed, and deleted workflows. This will include jobs added by users via the web form or by web applications using the API. From this job queue, the site administrator can view results from any executed workflow, view error logs (if necessary) and view the parameter settings for each submission.Third, a report page is provided that includes usage graphs of the top workflow submitters (users) on the site, and the top workflows submitted. These reports will include both web form submitted workflows and those submitted by the API. They can help site administrators gauge usage and popularity of workflows.Fourth, a form is provided to specify site-specific files that should be made available to all users. Site-specific files will appear in the data steps of each workflow as shown in Finally, the site administrator can specify the number of days a history will remain on a remote Galaxy server. Once the set number of days has passed, the Tripal Galaxy module will automatically delete the history and results will no longer be available for viewing or download. This helps ensure that storage space on the remote Galaxy server does not fill with older workflow results. Similarly, Tripal itself provides a quota system for user uploaded files. The Galaxy module utilizes this system to ensure that end users do not overload the server with unused older files. Site administrators must set quotas and expiration days based on expected demand and the size of the storage available.To help support adoption by site administrators, A Tripal site can communicate with any number of remote Galaxy instances. To do this, the site administrator must add the API key of the Galaxy user account for which the workflows will be executed. This API key enables anyone with it to interact with the remote Galaxy server on behalf of the user. Therefore, to secure this API key, the Tripal Galaxy module provides a security role, compatible with Drupal, that allows a site administrator to limit the set of users that have access to view the key. Ideally, this should only be site administrators. End users will not see the API key and are restricted from viewing administrative pages where the API key is visible. Workflow results are protected such that only the site administrator and the user who submitted the job request can view workflow results.In general, Tripal depends on Drupal to provide overall site security from attacks such as SQL injection and others. Drupal developers post security updates when new vulnerabilities are found. Also, Drupal coding standards provide mechanisms that, if followed, ensure that Drupal sites are secure. Both the Tripal core package and the Tripal Galaxy module follow these coding standards. Therefore, any Tripal-based site should remain secure if the site administrator maintains and patches the software when security updates are released.However, any site, no matter how secure, may be compromised, potentially exposing the API key and other sensitive data. Moreover, it is recommended that the Galaxy account provided to the Tripal Galaxy module is a ‘service’ account, pre-created specifically for Tripal to submit jobs, and should never be the API key of an actual user. The Tripal Galaxy module automatically cleans old results, so if a site is compromised results from the most recent job submissions could be accessed. We recommend sites inform users of such vulnerabilities.Finally, neither Tripal nor Galaxy can ensure security for sensitive data, such as human identifying information or data that might fall, for example, within the regulations such as the Health Insurance Portability and Accountability Act (HIPAA) of the USA. Tripal sites that provide analytical tools to end users should discourage submission of such data to their workflows.While the Tripal Galaxy module does provide important functionality to support execution of scientific workflows from a Tripal website, several challenges remain. The most significant is that of scale related to demands on hardware. Galaxy can scale well when enough resources are available, but with large data, CPU, RAM and storage can be overrun if site administrators do not set limitations. Determining these limitations can be difficult. To help with storage challenges, Galaxy workflow developers who intend to share their workflows for repeated use should take care to build in cleanup steps to remove unwanted intermediate files, and perhaps input files. Additionally, network bandwidth limitations may create bottlenecks, when a user desires to analyze large amounts of data, such as hundreds of RNA-seq datasets, for example. A Tripal-site perhaps would not be able to support such an extremely large dataset. In cases where a Tripal site could support larger data, the network bandwidth required may be prohibitive.UseGalaxy.org service. However, public services may not perform well when very busy, may not have all the tools needed or may not want to support a community of users behind a single Tripal-site user account.Access to a responsive Galaxy instance with sufficient resources can also be problematic. Site developers can install their own Galaxy instances provided they have enough computational resources. They may also take advantage of public Galaxy services such as the The Tripal Galaxy module strives to meet the need for Tripal-based sites that wish to provide complex analytical workflows for larger data sets. The module provides cyberinfrastructure for scientific workflow execution to perhaps less-funded communities that use the Tripal framework. It helps support sustainability of these sites by adding functionality their site developers need without having to create it themselves. It can also help improve the quality of data submissions for a site, support technical and analytical training and serve as a toolkit to power online research web applications.


CD40L is a member of the TNF superfamily that participates in immune cell activation. It binds to and signals through several integrins, including αvβ3 and α5β1, which bind to the trimeric interface of CD40L. We previously showed that several integrin ligands can bind to the allosteric site (site 2), which is distinct from the classical ligand-binding site (site 1), raising the question of if CD40L activates integrins. In our explorations of this question, we determined that integrin α4β1, which is prevalently expressed on the same CD4+ T cells as CD40L, is another receptor for CD40L. Soluble (s)CD40L activated soluble integrins αvβ3, α5β1, and α4β1 in cell-free conditions, indicating that this activation does not require inside-out signaling. Moreover, sCD40L activated cell-surface integrins in CHO cells that do not express CD40. To learn more about the mechanism of binding, we determined that sCD40L bound to a cyclic peptide from site 2. Docking simulations predicted that the residues of CD40L that bind to site 2 are located outside of the CD40L trimer interface, at a site where four HIGM1 (hyper-IgM syndrome type 1) mutations are clustered. We tested the effect of these mutations, finding that the K143T and G144E mutants were the most defective in integrin activation, providing support that this region interacts with site 2. We propose that allosteric integrin activation by CD40L also plays a role in CD40L signaling, and defective site 2 binding may be related to the impaired CD40L signaling functions of these HIGM1 mutants. CD40L is a type II protein ligand member of the tumor necrosis factor (TNF) superfamily expressed by activated T cells. CD40L is a costimulatory molecule critical in a variety of T cell–antigen presenting cell (APC) interactions, including activating APCs to provide help to cytotoxic T cells . It alsoCD40L functions through its interactions with cell surface proteins CD40, and α5β1 and αIIbβ3 integrins. CD40 belongs to the tumor necrosis factor receptor (TNF-R) family and was first identified and functionally characterized on B lymphocytes . CD40–CDe.g., CX3CL1, CXCL12, and secreted phospholipase A2 type IIA (sPLA-IIA)) activated integrins by binding to a second ligand-binding site (site 2) in an allosteric manner in addition to binding to a primary site (site 1) (We recently identified vascular integrin αvβ3 as a new receptor for CD40L . We loca(site 1) , 15, 16.(site 1)  indicateHerein, we now present data that integrin α4β1 is a new receptor for CD40L. Notably, we showed that CD40L is an allosteric activator of integrins αvβ3, α5β1, and α4β1. Specifically, we demonstrate that CD40L can bind to an allosteric ligand-binding site (Site 2) , 15, 16,2+ to keep αvβ3 inactive (A). sCD40L enhanced binding of soluble αvβ3 to immobilized γC399tr in a dose-dependent manner. Activation is defined by the increase in binding of soluble integrin αvβ3 to immobilized ligand (γC399tr) by soluble activators (sCD40L). These findings suggest that CD40L activates integrin αvβ3 in an allosteric manner.Previous studies showed that several proinflammatory integrin ligands activated integrins in an allosteric manner (see Introductions). We hypothesized that another proinflammatory cytokine CD40L activates integrins. Previous studies showed that soluble integrin αvβ3 bound to many ligands including the fibrinogen γ-chain C-terminal domain with truncation at the C terminus (γC399tr) (See Introduction). γC399tr specifically binds to site 1 of αvβ3, but not to site 2 . We studinactive A. sCD40L2+ to keep αvβ3 inactive and bound FITC-labeled γC399tr was measured in flow cytometry. sCD40L markedly enhanced the binding of γC399tr, indicating that sCD40L activated αvβ3 on the cell surface (B). The activation of αvβ3 by sCD40L was dose-dependent (up to 50 μg/ml) and can be detected at 6 μg/ml. The binding of FITC-labeled γC399tr shown by mean fluorescent intensity (MFI) correlated well with the sCD40L concentrations (D). Although high concentrations of sCD40L were required to activate soluble integrins, it is likely that integrin activation by sCD40L is biologically relevant, since CD40L is a membrane-bound protein and is highly concentrated on the cell surface.We next studied if sCD40L activates integrin αvβ3 on the cell surface using CHO cells (CD40-negative) that express recombinant αvβ3 (β3-CHO cells). β3-CHO cells were incubated with sCD40L and FITC-labeled γC399tr in the assays medium with 1 mM Ca surface B. The actrations D. Althoue.g., CX3CL1, CXCL12, and sPLA2-IIA) bound to an allosteric site (site 2) and activated several integrins (D).We previously showed that several integrin ligands . The position of cyclic site 2 peptide is shown in Figure 2B. These findings suggest that sCD40L activates integrins by binding to site 2.We previously reported that the peptide from site 2 of β3  directly bound to CX3CL1, sPLA2-IIA, and CXCL12 and the peptide suppressed integrin activation by these activators, suggesting that they directly bind to site 2 and mediate integrin activation , 15, 16.Fig. 2B). Amino acid residues involved in the predicted CD40L-αvβ3 (closed form) interaction are shown in Fig. 2C) and were defective in activating cell surface αvβ3 . These finding are consistent with the docking model that these mutations (K143T and G144E) are located in the predicted site-2-binding site of CD40L .The docking simulation predicts that four HIGM1 mutations  are clustered in the predicted site-2-binding interface of CD40L B. Amino E). This finding is consistent with the model that site-1-binding and site-2-binding sites are distinct.Previous studies identified amino acid residues in the trimeric interface that are involved in binding to the classical ligand-binding site (site 1) of activated integrins . ConsistFig. 2Previous studies showed that integrin α5β1 binds to CD40L and induces signals independent of CD40 . We prev+2 (A). FN8-11 binds to site 1, but does not bind to site 2 (2+. sCD40L enhanced the binding of soluble α5β1 to immobilized FN8-11 in a dose-dependent manner (B). These findings indicate that sCD40L activates soluble integrin α5β1 in cell-free conditions.We confirmed that biotinylated soluble α5β1 binds to the fibronectin fragment (FN8-11), a specific ligand to α5β1, in ELISA-type activation assays in the presence of 1 mM Mn+2 A. FN8-11o site 2 . We studt manner B. These B). The levels of α5β1 activation by sCD40L in MFI and sCD40L concentrations correlated well (C and D). We studied if four HIGM1 mutants can activate cell-surface α5β1 in CHO cells. They were defective in activating α5β1 and the K143T was the most defective (E). These findings suggest that sCD40L binds to site 2 of α5β1 in a manner similar to that of αvβ3 and activates α5β1.We showed that sCD40L activated cell-surface α5β1 in CHO cells  using FITC-labeled FN8-11 in a dose-dependent manner B. The leted well , C and Defective E. These 2+ bound to immobilized WT sCD40L in a dose-dependent manner (A), indicating that CD40L is a ligand for α4β1 that binds to site 1.CD40L is primarily expressed in activated CD4+ T cells, but αvβ3 and α5β1 are not major integrins in T cells. sCD40L binds to site 1 of activated αvβ3 and α5β1 . It is ut manner A, indica2+ (B).We then studied if sCD40L activates α4β1 by binding to site 2. A fibronectin fragment H120, a ligand specific to α4β1, binds to site 1, but does not bind to site 2 . Activat2+ B.2+ (C). The α4β1 activation (MFI) correlated well with the sCD40L concentrations, indicating that sCD40L activated cell-surface α4β1 (D). We studied if the four HIGM1 mutants that are clustered in the predicted site-2-binding site of CD40L can activate α4β1 on α4-CHO cells. The HIGM1 mutants were defective in activating cell surface α4β1. The K143T and G144E mutants were the most defective in activating α4β1 among four HIGM1 mutants (E), indicating that site-2-binding interface in α4β1 is similar to those of αvβ3 and α5β1. These findings suggest that α4β1 is a new receptor for CD40L. CD40L binds to site 2 and activates α4β1 in a manner similar to those of αvβ3 and α5β1.Also, sCD40L enhanced the binding of FITC-labeled H120 to CHO cells that express recombinant α4β1 (α4-CHO cells) in the presence of 1 mM Ca2+ C. The α4ace α4β1 D. We stu mutants E, indicaA). We tested their ability to bind to activated soluble αvβ3 in ELISA in the presence of 1 mM Mn2+, which reflects their ability to bind to site 1. S128R/E129G and L155P were defective and K143T and G144E were partially defective in binding to activated integrin αvβ3, suggesting that K143T and G144E are still able to bind to site 1 (B). S128R/E129G and L155P did not bind to activated αvβ3 (to site 1) although these mutations are not in the site-1-binding site in the trimeric interface. It is possible that these mutations induced conformational changes in the trimeric interface.We further characterized the four HIGM1 mutants to determine the role of site 2 in CD40L signaling. They all bound to CD40 in ELISA A. We teso site 1 B. S128R/C). K143T at high concentration (5 μg/ml) induced substantial NF-kB activation (D). We tested if excess (20-fold) mutants can suppress NF-kB activation by WT sCD40L. We found that all mutants tested suppressed NF-kB activation by WT sCD40L (D), indicating that they act as antagonists. We previously showed that eight other HIGM1 mutants, which are clustered in the trimeric interface, were defective in integrin binding (to site 1). Their defect in site 1 binding is likely related to their defect in CD40L signaling . We showed that the HIGM1 mutants (particularly K143T and G144E) are defective in activating integrins, consistent with the prediction. The present study showed that sCD40L is a new allosteric activator of integrins by binding to site 2. The four HIGM1 mutants, including K143T and G144E, were defective in activating integrins αvβ3, α5β1, and α4β1 because they are defective in site 2 binding. This suggests that the defective binding of CD40L to site 2 is related to defective CD40L/CD40 signaling. Since integrins on normal blood cells  are not activated, it is possible that CD40L-mediated integrin activation by binding to site 2 is required for CD40L/CD40 signaling, in addition to site 1 binding. Previous study showed that eight HIGM1 mutants clustered in the trimeric interface are defective in integrin binding and in signaling because they are defective in site 1 binding . We thus propose that integrin activation is primarily induced by membrane-bound CD40L in activated T cells or platelets, in which CD40L is brought to the cell surface upon activation of T cells or platelets, and CD40L is highly concentrated on the cell surface. It is, however, possible that sCD40L can be concentrated by binding to cell surface CD40. If so, sCD40L at levels in autoimmune disease patients may sufficiently trigger integrin activation and subsequent CD40/CD40L signaling for inflammatory responses. The present results provide the first evidence that CD40L can induce integrin activation, regardless of whether it is soluble or membrane-bound form.It is well known that high concentrations of sCD40L (>1000-fold) are required to induce CD40L/CD40 signaling compared with membrane-bound CD40L. In our previous study, sCD40L at >1 μg/ml was needed to achieve maximal NF-kB activation in HEK293 reporter cells . We propB) is required for their signaling functions. This includes FGF-1  K1 cells using the soluble αv and β3 expression constructs and purified by Ni-NTA affinity chromatography, as described . BiotinyWe synthesized recombinant sCD40L  with no N-terminal KGD motif as described . sCD40L The cDNA fragment encoding the CD40 fragment (residues 21–144) was amplified by PCR and subcloned into the BamHI/EcoRI site of PGEX2T. We synthesized the proteins in BL21 cells and purified using glutathione-Sepharose affinity chromatography.The CD40 fragment fused to GST was coated to wells of a 96-well microtiter plate (100 μg/ml in PBS) for 1 h, and the remaining protein-binding sites were blocked by BSA (0.1%).We then incubated the wells with sCD40L and incubated for 1 h, and bound sCD40L was measured using HRP-conjugated anti-His.http://cptweb.cpt.wayne.edu/DbD2/) (We introduced 6His tag to the BamHI site of pGEX-2T (resulting vector is designated pGEX-2T6His). We synthesized GST fusion protein of site 2 peptide  and a scrambled site 2 peptide (VHDSHYSGQGAMSDNTNSPQT) by subcloning oligonucleotides that encodes these sequences into the BamHI/EcoRI site of pGEX-2T6His as described , 15. We u/DbD2/) . It pred3, 0.4 mM NaH2PO4, 2.5 mM KCl, 0.1% glucose, 0.1% BSA) with 1 mM CaCl2 for 1 h at room temperature. After unbound αvβ3 was removed by rinsing the wells with binding buffer, bound αvβ3 was measured using anti-integrin β3 mAb (AV-10) followed by HRP-conjugated goat anti-mouse IgG and peroxidase substrates. For activation assays with α5β1, biotinylated soluble α5β1 and α4β1(AcroBio) and their specific ligands  were used, and bound biotinylated soluble integrins were measured by using streptavidin conjugated to HRP.ELISA-type binding assays were performed as described previously . BrieflyCHO cells were cultured in Dulbecco's modified Eagle's medium (DMEM)/10% fetal calf serum. The cells were resuspended with HEPES-Tyrodes buffer/0.02% BSA (heat-treated at 80 C for 20 min to remove contaminating cell adhesion molecules). The β3-CHO or CHO cells were then incubated with WT or mutant CD40L for 30 min on ice and then incubated with FITC-labeled integrin ligands  for 30 min at room temperature. The cells were washed with PBS/0.02% BSA and analyzed by BD Accuri flow cytometer . The data were analyzed using FlowJo 7.6.5.5 cells in 500 μl DMEM without fetal calf serum) and stimulated with CD40L for 4 h. We measured the levels of alkaline phosphatase activity in the medium, as described  reporter (Invivogen). Cells were maintained in DMEM/10% fetal calf serum. For reporter assays, cells were plated in wells of a 24-well culture plate (2.5 × 10escribed .Docking simulation of interaction between CD40L (Protein Data Bank code 1ALY), which does not contain the N-terminal KGD motif, and integrin αvβ3 was performed using AutoDock3, as described . In the Treatment differences were tested using ANOVA using Prism 7 (GraphPad Software).Coordinate of the docking model presented in this paper is available upon request. All remaining data are contained within the article.The authors declare that they have no conflicts of interest with the contents of this article.


Controlling the activation of the NLRP3 inflammasome by post-translational modifications (PTMs) of critical protein subunits has emerged as a key determinant in inflammatory processes as well as in pathophysiology. In this review, we put into context the kinases, ubiquitin processing and other PTM enzymes that modify NLRP3, ASC/PYCARD and caspase-1, leading to inflammasome regulation, activation and signal termination. Potential target therapeutic entry points for a number of inflammatory diseases focussed on PTM enzyme readers, writers and erasers, leading to the regulation of inflammasome function, are discussed.Video Abstract Inflammasomes are multi-protein complexes that serve as important sensors to fend off pathogens and to resolve aberrant cellular physiology. Among the four types of inflammasomes, the NACHT, LRR and PYD domains-containing protein 3 (NLRP3) inflammasome subtype is the best characterised one. It can be activated by a variety of pathogens and sterile stimuli, which are termed pathogen-associated molecular patterns (PAMPs) or damage-associated molecular patterns (DAMPs). Inflammasome activation triggers caspase-1 dependent activation of pro-inflammatory cytokines, including IL-1β, as well as induction of an immunogenic type of cell death referred to as pyroptosis . Molecul. reported that 10 min treatment with lipopolysaccharides (LPS) was sufficient to prime the NLRP3 inflammasome in mouse bone marrow derived macrophages (BMDMs) without an increase in NLRP3 protein expression, suggesting that the effect of LPS does rely on post-translational processes other than transcriptional upregulation of NLRP3 .–28.26–28Conversely, Jun N-terminal kinase-1 (JNK1) mediated S194  phosphorylation positively regulate NLRP3 activation. It was reported to be an essential priming step and required for NLRP3 self-association. Mechanistically, S194 phosphorylation might provide a receptor site for BRCC3 binding, which induces deubiquitination and promotes subsequent activation by the second stimulus. Notably, even though S194 phosphorylation is essential for priming, a NLRP3-S194D phosphomimetic mutant does not seem sufficient as a second signal to induce complex assembly .In addition, phosphorylation of S295  was shown to be regulated by two kinases PKA and PKD. During the cAMP-induced NLRP3 inhibition, activated PKA directly phosphorylates NLRP3 at S295 and subsequently suppresses ASC oligomer formation . AnotherRecently, it was shown that phosphorylation of Y132 (in mouse NLRP3) in airway epithelial cells (AECs) interfered with ASC oligomerization and precluded IL-1β production. Mechanistically, Y132 phosphorylation is induced by Ephrin type-A receptor 2 (EphA2), which is selectively expressed in AECs and contributes to inhibiting the NLRP3 inflammasome in an ovalbumin‐induced asthma model [ASC/PYCARD has been shown to be subjected to K-63-linked poly-ubiquitination  and lineIn resting cells, ASC is usually hypo-ubiquitinated and remains in an inactive state. Upon stimulation by LPS, ASC is ubiquitinated by the linear ubiquitin chain assembly complex (LUBAC), consisting of HOIL-1, HOIP, and SHARPIN. It has been reported that ASC is a direct substrate of HOIL-1, and its linear ubiquitination is required for the ASC oligomer formation  Table 1. Notably. showed that, ubiquitin specific peptidase 50 (USP50) directly binds to ASC and positively regulate ASC oligomerization by promoting deubiquitination of K-63-linked ubiquitin chains on the ASC protein [In addition to ubiquitination, the deubiquitination process of ASC remains poorly understood, with only one DUB reported. Lee et al protein  , S46, S58 , Y60 4141, Y137 In resting cells, active IKKα consecutively phosphorylates ASC at S16 and S193 (in mouse ASC), which negatively regulates the activation via its interaction with ASC. Upon stimulation by LPS/ATP, IKKα dissociates from ASC. IKK-related kinase, IKKi, subsequently induces S58 phosphorylation on ASC and facilitates its redistribution from the nucleus to the cytoplasm, which in turn activates downstream signalling pathway   have been identified in human caspase-1 , althoug3 kinase .Fig. 4Ca. found that Helicobacter pylori LPS alone was able to trigger the maturation and release of IL-1β via Rac1/PAK1 signalling pathway. Upon stimulation, PAK1 interacts with and directly phosphorylates caspase-1 at S376, which in turn leads to its cleavage and subsequent IL-1β secretion [Although there are 10 phosphorylation sites  , 48 idenIn resting cells, NLRP3 is Sumoylated by SUMO E3-ligase MAPL. Upon stimulation by nigericin, MAPL dissociates from NLRP3, and subsequently, Sumoylation level of NLRP3 is markedly reduced. Furthermore, knockdown of SUMO de-conjugating enzymes SENP6 and SENP7 selectively repress ASC oligomerization, caspase-1 cleavage and IL-1β secretion in a NLRP3-dependent manner, strongly indicating Sumoylation involvement in NLRP3 inflammasome activation . In addiRecently, NLRP3 was shown to be acetylated at K21, K22 and K24  promoting the assembly and activation of NLRP3 inflammasome, controlled by the NAD-dependent protein deacetylase sirtuin-2 (SIRT2). LPS and ATP were shown to promote the acetylation level on NLRP3, and in turn increase the production of IL-1β, which was completely repressed by K21/22/24R mutations . The SIRMycoplasma pneumonia-derived toxin CARDS interacts with and activates NLRP3 inflammasome, dependent on its ADP-ribosyltransferase activity. Upon stimulation by CARDS, NLRP3 was found to be ADP-ribosylated, suggesting ADP-ribosylation of NLRP3 is a functional PTM for its positive regulation. Further studies are needed to understand the detailed molecular mechanism [Several pathogen-derived proteins have been reported to act as activators for NLRP3 inflammasome assembly. It was shown that a echanism .. found that NLRP3 S194 phosphorylation promotes its interaction with the deubiquitinase BRCC3, which in turn induces the deubiquitination of NLRP3 and its subsequent activation. Deubiquitination can be prevented by either S194A mutation or using a JNK agonist (anisomycin) [While single PTM regulation of NLRP3 inflammasome has been studied, the interplay between different PTMs is just beginning to be evaluated, largely expanding the complexity of NLRP3 regulation. Crosstalk between multiple PTMs can be either positive or negative, and occur at different levels , 51. In somycin) , which sAnother example for the crosstalk between phosphorylation and ubiquitination is the Src-family kinases (SFKs)-Cbl-Pyk2 axis in the regulation of ASC phosphorylation. Phosphorylated Pyk2 was shown to co-localize with ASC upon activation and promote ASC phosphorylation that is required for subsequent oligomerization . The tyrAdditionally, in the case of NLRP3, K689 can be modified by both ubiquitination and sumoylation. It was reported that, poly-ubiquitination of K689 represses the formation of NLRP3 inflammasome , 23, andIn summary, the combinatorial control of NLRP3 complex by multiple PTMs is emerging as an essential early event for the priming process, while more studies are needed to decipher the PTM codes and explain the crosstalk between them.PTMs are able to affect protein biological functions in multiple ways, including regulation of protein–protein interaction, functional activities, stability , folding and subcellular localization. An important focus on NLRP3 inflammasome research is the translocation of inflammasome components between different subcellular compartments during the priming and activation process .PTM regulation of either NRLP3 or ASC was reported to affect their localisation. For example, upon stimulation, the increase of diacylglycerol (DAG) in Golgi induces recruitment of PKD, which in turn phosphorylates NLRP3 (at S295) in MAMs. This leads to the release of phosphorylated NLRP3 from MAMs and subsequent assembly of the inflammasome complex in the cytosol .Another example is the translocation of ASC induced by IKKi-mediated phosphorylation. Mechanistically, under resting condition, IKKα constitutively interacts with ASC in the nucleus and prevents the complex from hyper-activation. Upon stimulation with LPS and ATP, ASC is released from IKKα and is phosphorylated at S58 by IKKi, which facilitates its translocation from the nuclear to the perinuclear area resulting in complex assembly . HoweverAs NLRP3 inflammasome is linked to multiple diseases, there is growing interest in exploring the therapeutic potential of direct or indirect targeting this complex. A small number of selective and potent NLRP3 inhibitors  [The Syk-Pyk2 pathway has been shown to be involved in the phosphorylation regulation of NLRP3 inflammasome , 57. R40lbicans) .As a key modulator downstream of B cell receptor signalling pathway, Bruton’s tyrosine kinase (BTK) is emerging as a promising drug target in B cell malignancies and autoimmune disorders . The FDAG5 methylene)-1,1-dioxide,tetrahydro-4H-thiopyran-4-one), a pan DUB inhibitor, was initially identified in a screening for compounds that are able to trigger caspase and induce apoptosis . It was In addition to the DUB inhibitor G5, another study found that P22077, an inhibitor of USP7/USP47, precludes the formation of ASC oligomers, which subsequently inhibits caspase-1 and IL-1β maturation . Two othThe uncontrolled activation of NLRP3 inflammasome has huge impact on multiple inflammatory and autoinflammatory diseases, making NLRP3 a promising therapeutic target. The increasing complexity of NLRP3 inflammasome regulation is partly ascribed to the post-translational regulation of its components. Although the discoveries of multiple PTMs on NLRP3/ASC/capase-1 and the enzymes responsible for these PTM regulation have advanced our knowledge of single PTM function in controlling NLRP3 inflammasome activation, a few important questions remain unanswered, including the interplay of multiple PTM regulation in sequential orders, and the identity of PTM enzyme readers, writers and erasers. In addition to NLRP3, ASC and caspase-1, other proteins, such as NIMA- related kinase 7 (NEK7)  and BTK The direct NLRP3 inhibitor MCC950  has not progressed towards the clinic. Therefore, there is still a great need to identify therapeutic intervention points in the pathway to modulate the NLRP3 inflammasome . With th


Here, we report that knock-out of Rab29 does not influence endogenous LRRK2 activity, based on the assessment of Rab10 and Rab12 phosphorylation, in wild-type LRRK2, LRRK2[R1441C] or VPS35[D620N] knock-in mouse tissues and primary cell lines, including brain extracts and embryonic fibroblasts. We find that in brain extracts, Rab12 phosphorylation is more robustly impacted by LRRK2 inhibitors and pathogenic mutations than Rab10 phosphorylation. Transgenic overexpression of Rab29 in a mouse model was also insufficient to stimulate basal LRRK2 activity. We observed that stimulation of Rab10 and Rab12 phosphorylation induced by agents that stress the endolysosomal system  is suppressed by LRRK2 inhibitors but not blocked in Rab29 deficient cells. From the agents tested, nigericin induced the greatest increase in Rab10 and Rab12 phosphorylation (5 to 9-fold). Our findings indicate that basal, pathogenic, as well as nigericin and monensin stimulated LRRK2 pathway activity is not controlled by Rab29. Further work is required to establish how LRRK2 activity is regulated, and whether other Rab proteins can control LRRK2 by targeting it to diverse membranes.Mutations that enhance LRRK2 protein kinase activity cause inherited Parkinson's disease. LRRK2 phosphorylates a group of Rab GTPase proteins, including Rab10 and Rab12, within the effector-binding switch-II motif. Previous work has indicated that the PARK16 locus, which harbors the gene encoding for Rab29, is involved in Parkinson's, and that Rab29 operates in a common pathway with LRRK2. Co-expression of Rab29 and LRRK2 stimulates LRRK2 activity by recruiting LRRK2 to the surface of the  The VPS35[D620N] autosomal dominant mutation that causes PD markedly elevates Rab protein phosphorylation by LRRK2 through an unknown mechanism  pathogenic mutant. Our data demonstrate that knock-out or moderate transgenic overexpression of Rab29 in a new mouse model we created, does not significantly impact the ability of LRRK2 to phosphorylate Rab10 or Rab12. Furthermore, we show that knock-out of Rab29 does not impact the ability of the VPS35[D620N] mutation or monovalent cation ionophore antibiotics nigericin and monensin to promote Rab10 and Rab12 protein phosphorylation. Our data indicate that Rab29 is not a major regulator of basal wildtype or LRRK2[R1441C] activity as measured in whole cell or tissue extracts that we have analyzed. Further work is, therefore, required to clarify how LRRK2 activity is regulated.MLi-2 LRRK2 inhibitor was synthesized by Natalia Shpiro (University of Dundee) and was first described to be a selective LRRK2 inhibitor in previous work . MicrocyRabbit immunization and rabbit antibody generation was performed by Abcam Inc. . To generate the Rab29 total antibodies, full length recombinant proteins as well as N-terminal and C-terminal peptides were used. Abcam performed three subcutaneous injections using the immunogens conjugated with keyhole limpet hemocyanin (KLH), followed by two subcutaneous injections using the immunogens conjugated with ovalbumin. Target immunogen is described in The MJFF rabbit monoclonal antibody Rab10 pThr73 was previously characterized  and purcCell or tissue extracts were mixed with a quarter of a volume of 4× SDS–PAGE loading buffer  and heated at 95°C for 5 min. Samples ranging from 15 to 40 μg were loaded onto a NuPAGE 4–12% Bis–Tris Midi Gel  or self-cast 10% Bis-Tris gel and electrophoresed at 130 V for 2 h with NuPAGE MOPS SDS running buffer . At the end of electrophoresis, proteins were electrophoretically transferred onto a nitrocellulose membrane  at 90 V for 90 min on ice in transfer buffer (48 mM Tris–HCl and 39 mM glycine supplemented with 20% methanol). The transferred membrane was blocked with 5% (w/v) skim milk powder dissolved in TBS-T , 0.1% (v/v) Tween 20) at room temperature for 1 h. Membranes were washed three times with TBS-T and were incubated in primary antibody overnight at 4°C. Prior to secondary antibody incubation, membranes were washed three times for 15 min with TBS-T. The membranes were incubated with secondary antibody for 1 h at room temperature. Thereafter, membranes were washed with TBS-T three times with a 15 min incubation for each wash, and protein bands were acquired via near-infrared fluorescent detection using the Odyssey CLx imaging system and quantified using the Image Studio software.Mice selected for this study were maintained under specific pathogen-free conditions at the University of Dundee (U.K.). All animal studies were ethically reviewed and carried out in accordance with the Animals (Scientific Procedures) Act 1986 and regulations set by the University of Dundee and the U.K. Home Office. Animal studies and breeding were approved by the University of Dundee ethical committee and performed under a U.K. Home Office project license. Mice were housed at an ambient temperature (20–24°C) and humidity (45–55%) and were maintained on a 12 h light/12 h dark cycle, with free access to food (SDS RM No. 3 autoclavable) and water. For the experiments described in tm1a(EUCOMM)Wtsi mice were bred with Taconic Total Body Cre mice expressing Cre recombinase (Model 12524), which recognizes the loxP sites that flank the inserted promoter-driven neomycin cassette and exon 4 of Rab29, which is critical for expression. Following the deletion of exon 4, the mice were then bred and maintained on a C57Bl/6j background to remove the Cre recombinase allele, and to produce the experimental animals used in this study. The genotypes of the Rab29 knock-out mice were confirmed by PCR using genomic DNA isolated from ear biopsies and primers that amplify the entire Rab29 gene, as well as by immunoblotting.Rab29 knock-out mice are made available through the Wellcome Trust Sanger Institute, distributed by Infrafrontier: EMMA mouse repository (EM: 05517), and were characterized previously . LacZ-knThe generation of LRRK2[R1441C] knock-in mice and VPS35[D620N] knock-in mice were described previously ,26. Rab2in situ hybridization with probes for all murine chromosomes (mFISH) to confirm that the cell line meets the quality standard for parental ES cell lines used for targeting experiments. To generate chimeras, superovulated BALB/c females were mated with BALB/c males following hormone administration. Blastocysts were isolated from the uterus at dpc 3.5. For microinjection, blastocysts were placed in a drop of DMEM with 15% FCS under mineral oil. A flat tip, piezo actuated microinjection-pipette with an internal diameter of 12–15 micrometer was used to inject 10–15 targeted C57BL/6NTac ES cells into each blastocyst. After recovery, eight injected blastocysts were transferred to each uterine horn of 2.5 days post coitum, pseudopregnant NMRI females. Chimerism was measured in chimeras (G0) by coat color contribution of ES cells to the BALB/c host (black/white). Germline transmission occurred during an IVF expansion using chimeric males and C57BL/6NTac oocyte donors. The colony is maintained by mating wildtype C57BL/6NTac females to heterozygous males and heterozygous females to wildtype C57BL/6NTac males.The Michael J. Fox Foundation for Parkinson's Research generated the transgenic Rab29-overexpressing mouse model (C57BL/6NTac-Gt(ROSA)26Sortm1(Pgk-Rab29)Tac), which is made available through Taconic (Model 16552). The constitutive knock-in of Pgk-Rab29 in the ROSA26 locus via targeted transgenesis was undertaken by Taconic. The Rab29 sequence was synthesized according to the NCBI transcript NM_144875.2. The following elements were inserted into the ROSA26 locus (NCBI gene ID: 14910) using recombination-mediated cassette exchange (RMCE): a Pgk promoter, the Rab29 open reading frame together with a Kozak sequence (GCCACC), the human growth hormone (hGH) polyadenylation signal and an additional polyadenylation signal. The RMCE vector was transfected into the Taconic Biosciences C57Bl/6 embryonic stem (ES) cell line equipped with RMCE docking sites in the ROSA26 locus. The ES cell line pre-equipped with F3/FRT — RMCE docking sites was grown on a mitotically inactivated feeder layer comprised of MEFs in ES cell culture medium containing Leukemia inhibitory factor and Fetal Bovine Serum. The cells were co-transfected with the circular exchange vector containing the transgene and the recombinase pCAG-Flpe pA. The transfection was performed via lipofection with a commercially available kit. From day 2 onwards, the medium was replaced daily with medium containing the appropriate selection antibiotics. The recombinant clones were isolated using positive neomycin resistance selection. On day 7 after transfection, resistant ES cell colonies (ES clones) with a distinct morphology were isolated. The clones were expanded and frozen in liquid nitrogen after extensive molecular validation by Southern Blotting and/or PCR. Quality control of the ES cell line used for transfection was performed at Chrombios GmbH (Germany). Karyotype analysis was undertaken by multicolor fluorescence Genotyping of mice was performed by the MRC genotyping team at the MRC-PPU, University of Dundee, by PCR using genomic DNA isolated from ear biopsies. For this purpose, Primer 1 (5′ CACACACATGGTACACAGATATACATGTAGG 3′) Primer 2 (5′ ACATCCATGACACGACTCTACTATAGAGAT 3′) and Primer 3 (5′ CTATCCCGACCGCCTTACTGC 3′) were used to distinguish between wildtype and Rab29 knock-out alleles . The VPS35[D620N] knock-in mouse strain required Primer 1 (5′ TCATTCTGTGGTTAGTTCAGTTGAG 3′), Primer 2 (5′ CCTCTAACAACCAAGAGGAACC 3′) and Primer 3 (5′ ATTGCATCGCATTGTCTGAG 3′) to distinguish wildtype from D620N knock-in alleles . The LRRK2[R1441C] knock-in mouse strain required Primer 1 (5′ CTGCAGGCTACTAGATGGTCAAGGT 3′) and Primer 2 (5′ CTAGATAGGACCGAGTGTCGCAGAG 3′) to identify wildtype and R1441C knock-in alleles . To detect the constitutive KI allele in the transgenic Rab29 overexpression mouse, Primer 1 (5′ TTGGGTCCACTCAGTAGATGC 3′) and Primer 2 (5′ CATGTCTTTAATCTACCTCGATGG 3′) as well as internal PCR control Primer 1 (5′ GTGGCACGGAACTTCTAGTC 3′) and Primer 2 (5′ CTTGTCAAGTAGCAGGAAGA 3′) were used . To detect the wildtype allele in the transgenic Rab29 overexpression model, Primer 1 (5′ CTCTTCCCTCGTGATCTGCAACTCC 3′) and Primer 2 (5′ CATGTCTTTAATCTACCTCGATGG 3′) and internal PCR control Primer 1 (5′ GAGACTCTGGCTACTCATCC 3′) and Primer 2 (5′ CCTTCAGCAAGAGCTGGGGAC 3′) were used . All genotyping primers were used at a final concentration of 10 pmol/μl. PCR reactions were set up and run using KOD Hot Start Polymerase standard protocol. PCR bands were visualized on Qiaexcel (Qiagen) using the standard DNA screening kit cartridge.g for 30 min at 4°C and supernatant was collected for subsequent Bradford assay and immunoblot analysis.Mouse tissues were collected and snap frozen in liquid nitrogen. Snap frozen tissues were weighed and quickly thawed on ice in a 10-fold volume excess of ice-cold lysis buffer containing 50 mM Tris–HCl pH 7.4, 1 mM EGTA, 10 mM 2-glycerophosphate, 50 mM sodium fluoride, 5 mM sodium pyrophosphate, 270 mM sucrose, supplemented with 1 μg/ml microcystin-LR, 1 mM sodium orthovanadate, complete EDTA-free protease inhibitor cocktail (Roche), and 1% (v/v) Triton X-100. Tissue was homogenized using a POLYTRON homogenizer (KINEMATICA), employing three rounds of 10 s homogenization with 10 s intervals on ice. Lysates were centrifuged at 20 800(−ΔΔCt) , and the difference between means of the four biological replicates for each genotype revealed that >99% power was achieved to reject the null hypothesis at α = 0.05 for Rab12 phosphorylation in post hoc power calculations derived from the standard deviations of wildtype and pathogenic mutant VPS35[D620N], and the difference between means of the four biological replicates for each genotype revealed that >99% power was achieved to reject the null hypothesis at α = 0.05 for Rab12 phosphorylation in Graphs were made using Graphpad Prism 7 and 8 software. Error bars indicate SD. Two-tailed unpaired tration, ,49,51,54http://www.immprot.org/) as well as tissues (https://www.proteinatlas.org/).We raised two novel Rab29 monoclonal antibodies termed MJF-30-Clone-124 and MJF-30-Clone-104 that detect endogenous Rab29 in wildtype but not in Rab29 knock-out human A549 cells . MJF-30-To investigate the role of endogenous Rab29 in regulating LRRK2 pathway activity, we obtained Rab29 knock-out mice made available through the Wellcome Trust Sanger Institute, Infrafrontier EMMA mouse repository and the Michael J. Fox Foundation . These mice have also been deployed in other studies mentioned above ,44,46. AAs the human genetic data point toward variants within the PARK16 locus enhancing expression of Rab29 ,38,39, wTo demonstrate that the overexpressed Rab29 was functional, we undertook immunofluorescence studies of Rab29 knock-out and littermate-matched wildtype and transgenic MEFs . This reWe examined LRRK2-mediated Rab10 and Rab12 phosphorylation in wildtype and homozygous transgenic Rab29 mouse brain , large iTo study whether endogenous Rab29 was necessary for the previously reported elevated Rab10 phosphorylation observed in LRRK2[R1441C] knock-in MEFs and mouse tissues ,51, we gWe also immunoblotted for the pRab specific phosphatase PPM1H , and fouWe also attempted to measure endogenous phosphorylation of LRRK2 at Ser1292, an autophosphorylation site, using a commercially available antibody ,54. ThisTo investigate whether endogenous Rab29 was necessary for the elevated Rab10 phosphorylation observed in VPS35[D620N] MEFs and mouse tissues , we geneStreptomyces hygroscopicus  mutants are more readily activated by Rab29, which could explain why this mutation elevates LRRK2 activity. However, our results suggest that knock-out of endogenous Rab29 has no significant impact on endogenous LRRK2 activity, assessed by monitoring pRab10 and pRab12 levels in six mouse tissues as well as MEFs and lung derived fibroblasts. Moreover, we also found that knock-out of Rab29 does not impact elevated Rab10 or Rab12 phosphorylation observed in the LRRK2[R1441C] knock-in MEFs or mouse tissues. This would suggest that endogenous Rab29 is not sufficient to explain the elevated activity of the LRRK2[R1441C] pathogenic mutant. We also found that Rab29 knock-out had no effect on the elevated LRRK2-mediated Rab10 or Rab12 phosphorylation observed in VPS35[D620N] knock-in MEFs and mouse tissues. To our knowledge, there is no evidence implicating Rab29 in mediating the effects of the VPS35[D620N] mutation. We also find that in brain extracts, Rab12 phosphorylation appears to be more robustly impacted by LRRK2 inhibitors and pathogenic mutations than Rab10 phosphorylation, consistent with a recent study .In this study, we also report that agents that induce endolysosomal pathway stress including nigericin, monensin, chloroquine and LLOMe significantly enhance Rab10 and Rab12 phosphorylation in MEFs and A549 cells, in a manner that is blocked by LRRK2 inhibitors. Out of the 4 agents tested, nigericin was the most potent in enhancing Rab10 and Rab12 phosphorylation and increases the basal phosphorylation of these substrates 5 to 9-fold . NigericIt is possible that other Rab proteins, or even other regulators that have not yet been characterized, could also interact with and activate LRRK2 in a similar manner to Rab29 by recruiting LRRK2 to a variety of cellular membranes. In the absence of Rab29 overexpression, LRRK2 is widely distributed in cells with ∼90% cytosolic localization and ∼10% localization on a variety of cellular membranes ,57,71,72The ability of exogenous Rab29 to recruit LRRK2 to the Golgi (or other compartments to which it is targeted) confirms the ability of Rab29 to bind LRRK2 in cells. Yet our knock-in and knock-out models failed to reveal clues regarding the functional significance of this interaction. Rab29 is a relatively poorly abundant Rab and may play a specific role in macrophages and dendritic cells where it is most abundant. Indeed, LRRK2 activation triggered by Rab29 could occur in a specific cell type or tissue, or following a physiological stimulus, stress or infection that we have not investigated. Our data do not rule out the possibility that Rab29 knock-out is compensated by another cellular protein(s) other than Rab32. However, our findings with transgenic mice that overexpress Rab29 from 1.5 to 25-fold, without enhancing LRRK2-mediated Rab10 or Rab12 phosphorylation, reveal that increasing Rab29 expression is not sufficient to stimulate the activity of endogenous LRRK2.We have previously shown that Rab3, Rab8A, Rab10 Rab12, Rab35 and Rab43 are the main substrates of LRRK2 ,26,74, a


Elevated %C37:4 is widely interpreted as an indicator of low sea surface salinity from polar water masses, but its biological source has thus far remained elusive. Here we identify a lineage of Isochrysidales that is responsible for elevated C37:4 methyl alkenone in the northern high latitude oceans through next-generation sequencing and lab-culture experiments. This Isochrysidales lineage co-occurs widely with sea ice in marine environments and is distinct from other known marine alkenone-producers, namely Emiliania huxleyi and Gephyrocapsa oceanica. More importantly, the %C37:4 in seawater filtered particulate organic matter and surface sediments is significantly correlated with annual mean sea ice concentrations. In sediment cores from the Svalbard region, the %C37:4 concentration aligns with the Greenland temperature record and other qualitative regional sea ice records spanning the past 14 kyrs, reflecting sea ice concentrations quantitatively. Our findings imply that %C37:4 is a powerful proxy for reconstructing sea ice conditions in the high latitude oceans on thousand- and, potentially, on million-year timescales.Alkenones are biomarkers produced solely by algae in the order Isochrysidales that have been used to reconstruct sea surface temperature (SST) since the 1980s. However, alkenone-based SST reconstructions in the northern high latitude oceans show significant bias towards warmer temperatures in core-tops, diverge from other SST proxies in down core records, and are often accompanied by anomalously high relative abundance of the C Some algae produce compounds called alkenones that can reconstruct sea surface temperature through geological time, but in high latitudes unknown species complicate use of this proxy. Here the authors find a lineage of sea ice algae that produces alkenones and can be used as a paleo-sensor for sea ice abundance. They are a class of C35–42 methyl and ethyl ketones with two to four double bonds produced exclusively by the algae in the order Isochrysidales8: in ocean settings Emiliania huxleyi and the closely related Gephyrocapsa oceanica have been considered as the exclusive producers of di-unsaturated (C37:2) and tri-unsaturated (C37:3) methyl alkenones5. The unsaturation index of alkenones (37:2/(C37:2 + C37:3), is positively correlated with temperature, as validated both by algal culture experiments and global core-top calibrations13.Alkenones are among the best proxies for paleo sea surface temperature (SST) reconstructions, and their exceptional diagenetic stability has enabled the generation of palaeotemperature records spanning tens of millions of years of Earth’s history37:4), which is usually absent in mid-to-low latitude oceans and E. huxleyi cultures even under exceedingly low growth temperature8. This pattern is reported in surface sediment and seawater filtered particulate organic matter (POM) samples from the Nordic Seas and the Bering Sea16, and downcore sediment records in Okhotsk Sea during glacial periods19. As a result, Rosell-Melé14 concluded that 37:4 among the total C37 methyl alkenones (%C37:4) is >5%. Yet, %C37:4 greater than 5% is widely reported in sediment records in the northern high latitude oceans, thereby complicating SST reconstructions from these regions  on a regional scale were observed20, with elevated %C37:4 often associated with fresher and colder water mass22. The mechanisms for these observations, however, are poorly defined, with previous studies suggesting unknown oceanographic parameters14 and/or alkenone producers22 as the possible explanations. The occurrence of miscellaneous compounds that co-elute with C37:4 alkenone in sediment samples extracts adds an additional level of complication when interpreting %C37:4 data23. Regardless of these problems, elevated %C37:4 is widely applied as an indicator of fresher and colder polar water mass or decreased SSS  to identify Isochrysidales in water column, sea ice, and sediment samples in northern high latitude marine environments. We show that Isochrysidales communities in subarctic regions are composed of a lineage that is distinct from 37:4 , Group 2 , and Group 3 . Our phylogenetic analysis revealed close relationships between the majority of Isochrysidales sequences recovered from marine sites with perennial or extended seasonal sea ice and those of published sequences from lakes with seasonal or perennial ice cover 30 where the western part of the Fram Strait was ice-covered and eastern part was ice-free showed E. huxleyi was present in both ice-covered and ice-free sites. However, Group 2i was only detected in seawater samples collected under ice cover. The DNA data suggest that the presence of Group 2i is tightly associated with sea ice.We followed the Isochrysidales classification of Theroux et al.er Figs. . The seq37:4, we cultured the unclassified Isochrysidales strain RCC5486 at 3 and 6 °C in f/2 medium under salinity of 15, 21, 26, 31, and 38 ppt. The RCC5486 strain falls within the Group 2i in our phylogeny and it is the most phylogenetically similar strain to the Isochrysidales found in Canadian Arctic Archipelago surface sediments that is available in pure culture . The alkenones produced by RCC5486 showed exceptionally high %C37:4 (~80%) at both 3 and 6 °C , matching alkenone production by other Group 2 Isochrysidales and differing from E. huxleyi8. However, C38Me was present in the Canadian Arctic Archipelago surface sediments, even though Group 2i was the only Isochrysidales detected through NGS. The C38Me detected in Canadian Arctic Archipelago is likely produced by E. huxleyi even though their DNA was not detected. The discrepancy between DNA and alkenone preservation could result from differences in the life cycles of Group 2i compared to E. huxleyi: the former potentially forms cysts and enters a resting stage in surface sediment,36 while the latter does not form resting spores during winter37, after the bloom they die and disaggregate during downward transportation into sea floor38.To confirm that Group 2i produces high %C37:4 as a reliable sea ice proxy. Elevated %C37:4 from surface sediment and seawater POM22 is located at regions with seasonal sea ice in the Northern Hemisphere  or estuarine environments where Group 1 Isochrysidales are present and contribute to elevated %C37:440. Group 1 Isochrysidales are typically found in freshwater to oligosaline environments and predominantly produce C37:441. Alkenone contribution from Group 1 can be identified through the presence of C37:3b isomer, which is not produced by Groups 2 and 341. Our results show that both Group 1 and the Group 2i are present in brackish water underlying sea ice in the northwest of Gulf of Finland, Baltic Sea42  in the Svalbard area during the past ca. 14.5 kyr  is located close to the modern maximum winter sea ice extent in the northeastern Norwegian Sea where temporal variations of sea ice conditions have been identified64. Alkenones were not detected prior to 14.1 kyr BP in M23258-2, likely due to seasonally extended sea ice cover limiting productivity. The general trend of %C37:4 from M23258-263 is in direct agreement with inferred-temperature trends from the δ18O values of Renland ice core located in east Greenland65, with increasing %C37:4 corresponding to decreasing temperature, and is positively correlated with IP25 concentration and coarse fraction content in M23258-2  located in the glacial trough of Storfjordrenna during the Bølling–Allerød (13.95–12.8 kyr BP) may represent partial sea ice cover and a retreating ice edge; high coarse fraction content during this time in M23258-2 suggests rapid breakup of the Svalbard–Barents Sea ice sheet. The highest peak in %C37:4 in M23258-2 occurs at the beginning of the Younger Dryas , indicating an expansion of sea ice. This is in agreement with increases in the coarse grain-size fraction and IP25 from M23258-2 and increases in IP25 at nearby site JM09-KA11-GC66 . The gradual decrease in %C37:4 observed in M23258-2 and JM09-020-GC after the termination of YD (~11.7 kyr BP) is coincident with increasing Northern Hemisphere summer insolation and temperature. Low %C37:4 in M23258-2 and the closely adjacent core SV0467  suggests that sea ice extent retreated and was present in low concentrations at these sites between 10.8 and 4 kyr BP during the Holocene Thermal Maximum, while the fluctuating %C37:4 in JM09-020-GC suggests a variable ice-edge condition at Storfjordrenna during this period. %C37:4 in M23258-2 increased after ca. 4 kyr BP indicating a slight expansion of the sea ice margin during the neoglacial cooling period in the Northern Hemisphere, as the coarse fraction contents in this core also increased, reflecting sediment injections caused by cascades of dense brine water down the Barents shelf induced by seasonal sea ice56. IP25 in M23258-2 is, however, absent (or below detection limit) during this time period. The return to partial seasonal sea ice cover during neoglaciation is also evident in the rapid IP25 increase in the Fram Strait60  and northern Barents Sea records68 , as well as the re-emergence of IP25 in JM09-KA11-GC.To further evaluate %C37:4 is a reliable and sensitive indicator for paleo sea ice, and has the potential for quantitative sea ice reconstructions based on surface sediment calibrations. As alkenones are solely produced by Isochrysidales, %C37:4 directly reflects proportion of Group 2i Isochrysidales within Isochrysidales community induced by sea ice, and is not affected by variations in total productivity or sedimentation rates as IP25. %C37:4 is also less likely to be affected by diagenetic factors due to the strong diagenetic stability of alkenones, and does not suffer from dissolution like microfossil sea ice proxies such as diatoms, ostracods, and foraminifers. As an example of the durability of the %C37:4 proxy, a gradual increase of %C37:4 from 0 to 10% was observed in Bering Sea after the onset of MIS M2 glaciation (~3.3 Ma) in concordance with an increase in sea ice-related diatoms50. In addition, %C37:4 provides site-specific sea ice information which is different from proxies such as methanesulfonic acid and sea salt in ice from glaciers and ice sheets that records the averaged sea ice condition at a variable  sector of ocean. Future work combining molecular biology and organic geochemistry analyses in culture experiments and large-scale analyses of seawater, sea ice, and sediment samples will improve the %C37:4-sea ice calibration and shed light on other sea ice properties such as seasonality.In this study, we demonstrate that %CSurface sediment samples for both DNA sequencing and alkenone analyses were collected from Canadian Arctic Archipelago during Amundsen 2018. The samples were stored at −20 °C before DNA extraction. Other surface sediment samples for alkenone analyses were collected during several cruises including M86/1 , P435 , EMB046 , JR51 , JR106 , KN158-4 , POS175 , HLY0601 , HLY0702 , M13/2 , and M 21/2 . The locations of the surface sediment samples are shown in Supplementary Data https://jonahventures.com/). Genomic DNA was extracted from the samples using the DNeasy PowerSoil HTP 96, then a two-step PCR protocol was performed. First, a portion of 18S rRNA was amplified by an adapted version of primer pair 528Flong and PRYM01+7 . The first PCR step following the program described in Egge et al.69 was performed in 25-μl reactions containing Phusion GC Buffer, 0.4 mM of each primer, 0.2 mM dNTP, DMSO 3%, 0.5 U Phusion polymerase (Thermo Scientific), and 2 µl of DNA template. After cleanup of the amplicons, a second round of PCR was performed to give each sample a unique 12-nucleotide index sequence. The final indexed amplicons were then cleaned and normalized using SequalPrep Normalization Kit  and proceeded to sequencing on an Illumina MiSeq using V2 600-Cycle Kit . Sequencing success and read quality was verified using FastQC v0.11.8. The forward and reverse primers were removed from the sequences, and sequences with length below 100 bp were discarded. The ASVs were compiled for each sample after denoising by UNOISE3 algorithm with an alpha value of 5 to remove sequencing error70. Taxonomy of the ASVs was assigned using the SILVA reference database  and assign_taxonomy.py as implemented in QIIME/1.9.171.Sequencing of the surface sediment samples was performed by Jonah Ventrues, Boulder, CO, USA, . Sequences classified to the order Isochrysidales were selected to proceed to further analysis. Close relatives of the environmental sequences were identified through BLAST search against NCBI GenBank and compiled as reference sequences for phylogeny. All sequences were then aligned against a curated alignment from Gran-Stadniczeñko et al.76 using PyNAST77. In total, the alignment contains 105 sequences, including 6 Coccolithales, 16 Groups 2 and 3 Isochrysidales cultures, 46 published environmental sequences from GenBank, and 37 recovered or reanalyzed environmental sequences from this study. A maximum-likelihood phylogeny was inferred using the RAxML78 program with substitution model GTRCAT with 1000 bootstraps through CIPRES79.We searched for short-read studies including the term “sea ice” on the European Nucleotide Archive, and runs from studies targeting subfragments of 18S rRNA that included sea ice or seawater samples were downloaded. Primers were removed from the downloaded raw reads using cutadapt−2 · s−1. Culture experiments under each salinity and temperature condition were triplicated.We obtained the RCC5486 strain (GenBank accession: MH764930) from the Roscoff Culture from the National Institute for Environmental Studies. The strain was acclimatized to 3 and 6 °C for 10 days before the start of corresponding culture experiments. The f/2 medium was prepared from filtered and sterilized seawater collected from Vineyard Sound, Woods Hole, MA, USA, adjusted to 15, 21, 26, 31, and 38 ppt. Cultures were grown under a light:dark cycle set at 16:8 h. The light intensity was 140 μE · mCultures were harvested at early stationary phase ) by filtering onto 0.7 μm glass fiber filters . All filters were wrapped with aluminum foils and immediately frozen at −20 °C before further extraction and analysis. For alkenones analyses, all of the filters were freeze-dried overnight and then dissolved in dichloromethane (DCM) and sonicated . The total extracts were separated and analyzed for alkenones as described in the section “Alkenones analyses.”TM accelerated solvent extraction system with DCM:methanol mixture  to obtain a total lipid extract (TLE). The TLE was then separated into alkane, ketone, and polar fractions through silica gel columns by eluents with increasing polarity: hexane, DCM, and methanol. The ketone fraction containing alkenones was analyzed by gas chromatography–flame ionization detection Agilent 7890N Series instrument equipped with mid-polarity column RTX-200 (105 m × 250 μm × 0.25 μm) with the oven program described in Zheng et al.80. This method is able to fully eliminate the co-elution between alkenones and alkenoates, as well as co-elution between C38 methyl and ethyl alkenones80. Alkenone peaks were identified and quantified based on retention time compared with standard Group 1 alkenone samples extracted from BrayaSø Lake (Greenland) through Agilent ChemStation B.03.02. A gas chromatography–mass spectrometer  equipped with RTX-200 columns (105 m × 250 μm × 0.25 μm) was used to examine co-elution. Samples with compounds co-eluting with alkenones were purified using silver thiolate silica gel columns with hexane: DCM , DCM, and acetone23. Regressions of alkenone indices and environmental parameters were performed via MATLAB (R2018b).Surface sediment samples were analyzed for lipids by extracting around 10 g of freeze-dried sediment using a Dionex25 in core M23258-2 was carried out as described by Belt et al.81 with minor modification. An internal standard  was added to dried sediment material (ca. 2 g), which was extracted using DCM/methanol (MeOH)  and ultrasonication. Following evaporation of the total organic extract (TOE) to dryness , elemental S was removed according to Cabedo-Sanz and Belt82. Thus, the TOE was dissolved in hexane (1 ml) followed by addition of tetrabutylammonium sulfite (1 ml) and propan-2-ol (2 ml). After brief shaking by hand (1 min), ultrahigh purity water (3 ml) was added, reagitated (1 min), and centrifuged . The hexane layer was transferred to a clean vial and the procedure repeated twice more. Combined hexane extracts were dried using N2. Partially purified TOEs were resuspended in hexane and purified using column chromatography , with fractions containing IP25 collected using hexane (6 ml). Final purification was achieved using Ag-ion chromatography . After elution of saturated hydrocarbons , unsaturated hydrocarbons including IP25 were collected in acetone (2 ml).Analysis of IP25 in purified sediment extracts was carried out via GC–MS using established methods81 with an Agilent 7890 gas chromatograph equipped with a HP5MS fused-silica column  coupled to an Agilent 5975 series mass spectrometric detector. IP25 was identified by its characteristic retention index (RIHP5-MS = 2081) and mass spectrum. Quantification was carried out in single-ion monitoring mode. The resulting peak areas for IP25 were normalized to internal standard responses, instrumental response factors, and sediment mass.Identification and quantification of IPFurther information on research design is available in the Supplementary InformationPeer Review FileSupplementary Data 1Supplementary Data 2Description of Additional Supplementary FilesReporting Summary