Antibody production depends on B cell internalization and presentation of antigens to helper T cells. To acquire antigens displayed by antigen-presenting cells, B cells form immune synapses and extract antigens by the mechanical activity of the acto-myosin cytoskeleton. While cytoskeleton organization driving the initial formation of the B cell synapse has been studied, how the cytoskeleton supports antigen extraction remains poorly understood. Here we show that after initial cell spreading, F-actin in synapses of primary mouse B cells and human B cell lines forms a highly dynamic pattern composed of actin foci interspersed with linear filaments and myosin IIa. The foci are generated by Arp2/3-mediated branched-actin polymerization and stochastically associate with antigen clusters to mediate internalization. However, antigen extraction also requires the activity of formins, which reside near the foci and produce the interspersed filaments. Thus, a cooperation of branched-actin foci supported by linear filaments underlies B cell mechanics during antigen extraction. Generation of high-affinity, protective antibodies requires B cell uptake of antigens via their antigen-specific B cell receptors (BCRs). After antigen binding, the BCR undergoes endocytosis and delivers the antigen into intracellular processing compartments, where antigenic peptides are loaded onto major histocompatibility II (MHC II) proteins . PresentThe uptake of soluble antigen by the BCR occurs predominantly via clathrin-coated pits (CCPs) . In contNevertheless, the organization of the actin cytoskeleton in B cell synapses that leads to antigen extraction is incompletely understood. Synaptic actin polymerization is mediated by two mechanisms: the Arp2/3 complex producing branched actin networks, and formins producing linear actin filaments . It is tOne possibility is that myosin IIa causes contraction of formin-generated actin filaments connecting to, or sweeping through the actin foci to generate inward or lateral forces, respectively. This mechanism would be similar to transport of antigen clusters from the synapse periphery to the center by the contractile act-myosin arcs . AlternaHere we image the F-actin at the B cell synapse using super-resolution microscopy in 2D and 3D, and using live-cell imaging with high temporal resolution to resolve the structures that are relevant for antigen extraction. We show that the B cell immune synapse contains a highly stochastic and dynamic pattern of actin foci interspersed with actin fibers. The pattern originates from transient spots of Arp2/3 activity generating branched actin in the foci and from closely associated formin activity producing the surrounding linear filaments. The actin foci are more intimately associated with antigen endocytosis, however, manipulation of the actin-polymerization pathways shows that both Arp2/3 and formins are needed for antigen uptake. We propose that these two actin polymerization mechanisms create a functional foci-filament network which translates local bursts of actin polymerization into an inward force for antigen internalization.2. Total internal reflection fluorescence (TIRF) microscopy time-lapses showed that after initial cell spreading, between 5 and 20 min after synapse formation, the Lifeact-labeled F-actin was distributed throughout the synapse in a range of small structures including actin foci and short fibers  using phalloidin-AlexaFluor647 staining of fixed cells . Super-rDual color live-cell TIRF microscopy indicated that although the actin structures did not overtly colocalise with antigen, some actin foci did transiently coincide with antigen clusters , in lineTo determine the mechanisms by which the actin foci and fibers are generated, we blocked actin polymerization in PMS-attached B cells either with CK666, which specifically inhibits the activity of the Arp2/3 complex, or with SMIFH2, which inhibits formins. Super-resolution imaging showed that CK666 treatment resulted in a loss of actin foci, which were instead replaced by abundant actin fibers . In contSimilar to primary mouse B cells, treatment of Ramos cells with CK666 reduced foci numbers , Video 5ARPC2, an essential component of the complex. All three gRNAs greatly reduced the average levels of the ARPC2 protein in the targeted cell population  specific for pulation . All thrpulation . Thus, ADIAPH1 was successfully targeted in Ramos cells with one gRNA and FMNL1 with two gRNAs alasin D . Similaralasin D . Thus, talasin D . The uptalasin D . In addialasin D , signifialasin D , consistalasin D .Thus, thTo understand how inhibition of Arp2/3 or formins compromises antigen extraction, we analyzed the mechanical activity of the B cells in the immune synapses. Pulling on the antigen during extraction can be visualized by imaging the deformation of the antigen-coated PMSs using the lipid dye DiI . Ramos cTo understand the role of Arp2/3 and formins in pulling on the antigen-presenting membrane, we analyzed PMS deformations induced by the Ramos cells in the presence of CK666 or SMIFH2. We found that the overall number of membrane pulling events was not affected by either of the inhibitors compared to the DMSO control . HoweverOur data show that after initial cell spreading, the B cell synapse contains a stochastic pattern of foci and fibers. These structures are generated locally in the synapse by transient activities of the Arp2/3 complex and formins, respectively and are important for antigen extraction. Despite their different mechanism of production, the foci and fibers are closely related as they are often generated from each other during their constant turnover. This is also illustrated by colocalization of both Arp2/3 and formins with the actin foci, leading to production of both branched actin and linear filaments from the same sites. This actin behavior produces network-like connectivity that is distinct from the actin structure of CD4  and CD8 We speculate that when B cells extract antigens from live antigen-presenting cells, actin foci and fibers form nodes and edges in a loosely connected network, whose function is required for efficient antigen extraction. In this model, stochastic Arp2/3 activation produces actin foci by local bursts of branching of preexisting short filaments. Branched actin polymerization may either push on the membrane to generate protrusions, similarly as it happens in podosomes , or syneThis model is supported by our observation and manipulation of the two major regulators of actin polymerization, the Arp2/3 complex and formins. The foci are selectively marked by stoichiometric incorporation of ARPC2 and they disperse after inhibition of Arp2/3 function, consistent with a branched-actin structure. The importance of the Arp2/3 complex in foci formation is consistent with previous data linking activators of the Arp2/3 complex, WAS WIPF1 and ITSN2, with foci formation in T cells  and in BOur data also show that Arp2/3-generated actin foci are intimately associated with antigen extraction, as seen in instances of their presence at the sites of antigen endocytosis and their close, albeit stochastic, relationship to BCR tyrosine-phosphorylated proteins and CCPs. Our model thus suggests that forces that lead to antigen extraction are dependent on new actin polymerization in actin foci. This hypothesis is supported by previous observation that F-actin is continuously accumulating on individual membrane invaginations during B cell antigen uptake . If the Nevertheless, our data also indicate that the branched actin polymerization in the foci is not sufficient to extract antigen as inhibitions of formin and myosin IIa activities also inhibit antigen uptake. Since formin inhibition altered foci dynamics, we hypothesize that formins together with myosin IIa provide stability to actin polymerization in the foci that helps the branched actin to expand in between the future base and tip of the invagination, generating an inward force, as opposed to a lateral force and a comet-like movement along the plasma membrane. A similar stabilizing role for myosin contractility was suggested for function of CCPs in other cells types . AlternaThe actin organization and mechanism of antigen extraction studied here pertains primarily to the synapses formed by naive and memory B cells, where antigen extraction is directly followed by endocytosis . AnotherUnexpectedly, our data show that the formation of the actin foci in naive B cells does not require antigen stimulation. The actin foci may thus be generated continuously as a general property of the B cell cortical cytoskeleton. In agreement with this observation, the majority of actin foci in the synapse do not associate with antigen clusters, or with CCPs. However, a small fraction of actin foci did associate with antigen clusters and to a lower degree with CCPs. Since BCR signaling regulates actin dynamics , includiOur data establish a new view on the architecture of B cell synapses and on the molecular basis driving its formation. This information can be used to better understand the role of F-actin not just in B cell antigen endocytosis, but also in receptor signaling at the plasma membrane. The organization of the BCR, and other signaling receptors on B cell surfaces critically depend on actin structure  and defeC57BL/6 mice and heterozygous Lifeact-EGFP mice on a C57BL/6 background were used as a sources of primary mouse B cells. All mice were between 1–6 months old and were male and female. Mice were bred and treated in accordance with guidelines set by the UK Home Office (project license number 7008844) and the Francis Crick Institute Ethical Review Panel.Primary mouse B cells were isolated from spleens by mashing tissue through a 70 μm cell strainer, followed by red blood cell lysis using ACK Lysing Buffer (Gibco), and negative selection using anti-CD43 microbeads (Miltenyi Biotec).HEK293T and Ramos cell lines were provided, authenticated and mycoplasma-tested by the Francis Crick Institute Cell Services.2(Southern Biotech) for mouse splenic B cells and goat F(ab’)2 anti-humanFc5μ (Jackson Immunoresearch) for Ramos cells. The antibodies were biotinylated with 20-fold molar excess of NHS-LC-LC-biotin (Pierce) and labeled with either Cy3 or Cy5 Monoreactive dyes , or with AlexaFluor 405 or AlexaFluor 647 NHS esters (ThermoFisher) in Sodium Carbonate buffer. Excess dye was removed using Zeba 7K MWCO desalting columns .Anti-BCR antibodies used to stimulate B cells on PMSs and in solution were goat anti-mouse Igκ F(ab’)All antibodies used in this study were:Inhibitors were dissolved in dimethyl sulfoxide . For all inhibitor experiments, B cells and inhibitors were incubated in HBSS with 0.01% BSA. Appropriate concentrations of DMSO were used for control experiments. The inhibitors and their concentrations were:Plasma membrane sheets were generated as described . In brieIsolated B cells (1.5 × 106) or cultured Ramos cells (2 × 105) were washed and resuspended in 50 μl of warm HBSS-BSA, added to each well and incubated at 37°C for 20 min to allow immune synapses to form. For live cell imaging, samples were kept at 37°C by a heated chamber and an objective heater. For inhibitor experiments in live cells, inhibitors were added directly to the center of the imaging well at this time. Alternatively, cells were fixed in 2% paraformaldehyde  for 20 min at room temperature (RT) and washed with HBSS-BSA.Epifluorescence and TIRF imaging were carried out either on a Nikon Eclipse Ti microscope with an ORCA-Flash 4.0 V3 digital complementary metal-oxide semiconductor (CMOS) camera (Hamamatsu Photonics) and 100x TIRF objective (Nikon), or on an Olympus IX81 microscope with an Andor iXon electron multiplying charged-coupled device (EMCCD) camera and 100x objective (Olympus). Both microscopes were controlled through Metamorph software (Molecular Devices) and illumination was supplied by 405, 488, 552 and 637 nm lasers (Cairn) through an iLas2 Targeted Laser Illuminator (Gataca Systems) which produces a 360° spinning beam with adjustable TIRF illumination angle. Acquired datasets were analyzed using ImageJ and Matlab (Mathworks). All images were aligned and, where necessary, corrected for background, flatfield illumination and photobleaching.To label F-actin for super-resolution imaging, samples prepared as above blocked for 1 hr using 10% BSA and 5% vol/vol normal mouse serum in HBSS and then incubated with phalloidin-AF647 (1:10 dilution) in HBSS-BSA overnight at 4°C. Just before imaging, cells were washed with PBS and incubated with STORM imaging buffer composed of 6.8 mM Tris (pH 8.0), 1.36 mM NaCl, 1.36% Glucose (wt/vol), 13.4 mM cysteamine (Sigma), 34 mM HCl, 4.76 μM Type II Glucose Oxidase (sigma), 185 nM Catalase from bovine liver (sigma), 2 mM COT (Sigma). STORM imaging was carried out on a Nikon N-STORM microscope (Eclipse Ti-E Inverted Microscope) with an Andor iXon EMCCD camera and 100x/1.49 numerical aperture (NA) oil-immersion TIRF objective. Cells were imaged under TIRF illumination, with a 640 nm laser and 405 nm laser. The 405 nm laser was used to maintain photo-blinking by gradually increasing its power. Fluorescence was collected at wavelengths between 640 and 790 nm. 10,000 to 20,000 frames were recorded at 18 ms exposure time per frame. For 3D STORM, astigmatic lens in the Nikon N-STORM microscope was slotted into the imaging path and cells were imaged under near-TIRF illumination. 3D calibration was carried out using the Nikon Elements software using fluorescent microspheres adsorbed on a glass coverslip. Molecular coordinates were localized using ThunderSTORM software , drift-cFoci in F-actin images were quantified by a combination of image filtering and tracking in individual cells using Matlab . All celTo compare automated segmentation of actin foci to their manual identification, two primary B cell timelapses were segmented by the automated analysis and by four researchers knowledgeable of the actin structures, but blinded to the results of the automated segmentation . We compFoci and fibers were first segmented in automatically detected cells in all images as described above. The enrichment of fluorescence signal of interest in actin foci and fibers was calculated from the fluorescent intensity in these structures, normalized to mean intensity in the synapse. Coincidence of actin foci with ARPC2 spots, DIAPH1 spots or CCPs and vice versa was calculated as the fraction the foci that overlapped with the center of the other structures. As a control, centers of the same number and size of structures were placed without overlap at random location in the same cell. A mean of ten of the randomization runs was taken as the random value for each cell.Mechanical activity in the synapse was visualized using DiI-labeled PMSs as described . BrieflyFor quantification of antigen uptake from PMSs, cells were incubated with inhibitors for 15 min at 37°C before incubation with PMSs in the continued presence of the inhibitors for 30 min. Large-scale image datasets and their processing was performed as described . In brieIsolated B cells or Ramos cells taken from culture were incubated with a LIVE/DEAD marker in PBS for 20 min. They were then washed and incubated with fluorescently labeled, biotinylated surrogate antigens at 1 μg/ml in PBS with 1% BSA and 2 m EDTA for 30 min on ice. Alternatively, the cells were incubated with fluorescently-labeled, biotinylated transferrin (Rockland). Cells were washed and incubated with inhibitors for 20 min on ice. Samples were then split into three and incubated either on ice or at 37°C for 10 or 30 min before fixation with 2% PFA on ice for 20 min. After fixation, cells were washed and stained for 20 min on ice with fluorescently labeled streptavidin, to label the ligands remaining on the cell surface. Samples were analyzed on an LSR Fortessa (BD Biosciences). Internalization was quantified as the percentage of antigens remaining at the cell surfaces using FlowJo (TreeStar).Human ARPC2-mRuby3 was constructed from pLVX-ARPC2-GFP and pOPINF2-WIPI2B-human-mRuby3 and cloned into pLVX for lentivirus-mediated transfection. To construct mRuby-DIAPH1, we cloned human DIAPH1 cDNA from Caco-2 cells using RNAeasy micro and QuantiTect Reverse Transcription kits (Quiagen) and inserted it into pVLX-mRuby3. For Lifeact-GFP transfection we used pLVX-LifeactGFP plasmid with the resistance gene switched to neomycin. Clathrin light chain A-mCherry was constructed from mouse Clta-GFP  and inseGuide sequences were:Recombinant replication-incompetent lentiviruses were produced by co-transfecting HEK293T cells using transIT-LT1 (Mirus)-mediated pMD2.G and psPAX2 helper plasmids together with the appropriate lentiviral plasmid. Lentivirus-containing media were harvested 48 and 72 hr following transfection and used to spin-infect Ramos cell lines for 90 min at 1350 x g in the presence of 5 μg/ml polybrene (Sigma). Sixty hours after the spinfection, cells were selected using 2.5 μg/ml of puromycin or 500 μg/ml of geneticin for one to two weeks. Cells expressing the fluorescent markers were further purified using Avalon cell sorter (Propel Labs).Ramos cells were lysed with RIPA buffer (Sigma), containing 1% protease inhibitor cocktail (Sigma), for 10 min on ice and then centrifuged at 13,000 rpm for 10 min at 4°C. Samples were heated in 1X NuPAGE LDS Sample Buffer (Invitrogen) and 1X NuPAGE Sample Reducing Buffer (Invitrogen) at 95C for 5 min and then separated using ExpressPlus PAGE Gels 4–20% (GenScript). Proteins were transferred to a polyvinylidene fluoride (PVDF) membrane and then blocked for 1 hr in 50% Odyssey Blocking Buffer (LI-COR) and 50% PBS. The membrane was incubated with the appropriate concentration of primary antibodies in PBST overnight on a rocker. The membrane was washed with PBST and incubated with the fluorescently conjugated secondary antibody in 50% PBST, 50% Odyssey Blocking Buffer and 0.05% sodium dodecyl sulfate (SDS) for 1 hr. After washing with PBST, the membrane was incubated in PBS and imaged using an Odyssey CLx scanner (LI-COR). In the interests of transparency, eLife publishes the most substantive revision requests and the accompanying author responses.Acceptance summary:Your work greatly advances the working model for how the actin cytoskeleton is organised to enable antigen extraction by B cells, which is critical to their function in antigen presentation. You have shown how the Arp2/3 branched actin and formin linear actin nucleation factors and myosin cooperate to generate a network of transient foci that overlie the extraction sites and are interconnected by the linear filaments that also incorporate myosin II based contractile units. While important questions remain, this work sets a high standard for quantitative analysis of these dynamic structures generating a strong foundation for future exploration.Decision letter after peer review:eLife. Your article has been reviewed by three peer reviewers, and the evaluation has been overseen by a Reviewing Editor and Anna Akhmanova as the Senior Editor. The reviewers have opted to remain anonymous.Thank you for submitting your article "B cells extract antigens using a dynamic network of Arp2/3-generated actin foci interconnected with linear filaments" for consideration by The reviewers have consulted with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.Summary:Your manuscript "B cells extract antigens using a dynamic network of Arp2/3- generated actin foci interconnected with linear filaments" presents an analysis of actin structures associated with BCR signaling and early phases of antigen extraction by B cells on model antigen presenting surfaces. You use cutting edge imaging methods to show suggestive results that BCR signaling is associated with distinct actin structures . Subsequently you assemble circumstantial evidence using imaging and CRISPR generated knockdowns that these structures may be implicated in antigen extraction. However, a number of key experiments and critical controls are required to establish your novel claims and the reviewers have consulted to suggest experiments that can be accomplished in 2 months.Essential revisions:1) Using foci and linear actin tracking in live cells, you should show that the Arp2/3-dependent foci and the Formin- dependent filaments are part of a crosslinked network. It will be crucial to include a membrane marker in this experiment to discern filopodia from formin-based linear filament population.2) Using your previously published tension sensors, and Arp2/3 inhibition/knockdown, the authors should measure forces exerted by the foci during antigen endocytosis. Knowing the magnitude of the forces at the antigen site would bridge the missing connection between the localization of foci and antigen endocytosis.3) By examining the endocytosis of a generic clathrin pathway marker such a transferrin, you need to substantiate the specific impact of Arp2/3 inhibition on antigen internalization.4) You should provide a supplementary figure detailing the workflow utilized to extract foci, and how the Gaussian filter size for enhancing foci was determined/ chosen. The efficiency of this foci extraction method also needs to be shown.eLife. Your revised article has been evaluated by Anna Akhmanova as the Senior Editor, a Reviewing Editor and three peer reviewers.Thank you for resubmitting your work entitled "B cells extract antigens using Arp2/3-generated actin foci interspersed with linear filaments" for further consideration by The manuscript has been improved but there are some remaining issues that need to be addressed before acceptance, as outlined below:Please see the comments below. After consultation, we think that an excellent title would be "B cells extract antigens at Arp2/3-generated actin foci interspersed with linear filaments" but you can suggest other titles that accommodate reviewer 2 concerns.Reviewer #1:eLife.The manuscript has substantially improved after the revision and provides new insights into antigen extraction at B cell synapse. I have no further experimental suggestions for the authors, and strongly recommend the manuscript for publication at Reviewer #2:The authors have addressed many of the major comments from the previous review. Their improved description of the determination of foci and fibers, inclusion of controls, and analysis of PMS deformation to infer the location and directionality of forces make the manuscript more complete. The characterization of this connected network in B cells is novel and suggestive of a role for this network in B cell signaling.However, the significance of the observed phenomena and its role in B cell function is still not clear to this reviewer. The experiments in the original and the additional experiments in revision seem to imply that these actin foci and filaments are a general feature of B cell cytoskeleton and only mildly dependent on BCR signaling.So the statement: "We propose that when B cells extract antigens from live antigen-presenting cells, actin foci and fibers form nodes and edges in a loosely connected network, whose function is required for efficient antigen extraction. " is speculative as best.As stated by the authors, these structures do not assemble in an obligatory manner either at antigen clusters or at CCPs, which are the sites of endocytosis. Therefore, their relevance for Ag gathering is not apparent. The data in Figure 5 suggests that the loss of Arp23 or mDia function leads to generic deficits in Ag gathering, which are consistent with general defects in endocytosis as shown in control Tf data. From all of these it is difficult to conclude that these foci play any significant role in exerting forces to test Ag quality or lead to efficient Ag extraction.Given these concerns, the authors have not convincingly demonstrated that "B cells extract antigens" using these actin structures, and therefore the authors should revise the title to reflect their findings more accurately.However, the identification of these structures and quantification of their origins and dynamics will be of sufficient interest to the community.Reviewer #3:To our opinion, the authors successfully answered most of the requests of the editor. However, we fill that the two following points should be addressed prior to final acceptance.1) The authors use the DIL membrane marker to observe the PMS invaginations. In Figure 6A they show a figure where DIL patches are visible even outside of the B cell area. How do the authors explain this? Are DIL spots within the synapse more important in number and intensity than the ones outside? This should be clarified.2) An article describing actin patches at the B cell immune synapse has been published during the time the present article was revised. Although the two articles are complementary, the authors should imperatively cite it and eventually discuss it . Essential revisions:1) Using foci and linear actin tracking in live cells, you should show that the Arp2/3-dependent foci and the Formin- dependent filaments are part of a crosslinked network. It will be crucial to include a membrane marker in this experiment to discern filopodia from formin-based linear filament population.Our data mainly focused on the functional synergy between then Arp2/3 and formin mediated actin polymerization. To substantiate the physical connections between the actin foci and actin fibers within the synapse, we extended our analysis of Ramos cells co-expressing Lifeact-GFP and ARPC2-mRuby. We now show in a new part of Figure 3 that ARPC2-containing actin foci emanate fibers that often transiently connect to other actin structures including other foci . We think that this finding illustrates the closely interconnected nature of the actin network in the synapse. This result is also consistent with the detectable presence of DIAPH1 in actin foci , and with the reduction of interspersed fibers by the formin inhibitor SMIFH2 . Nevertheless, given the transient nature of the network-like connections, we agree that it is more appropriate to think of the foci-fiber relationship as a mixed pattern with a cooperative function, rather than a “crosslinked network”. We therefore refer to this architecture as a “functional network” throughout the manuscript and toned down the statements on the importance of the physical interconnection of the foci by linear filaments.To address the possibility that the fibers in the synapse represent filopodia, rather than filaments within the cell, we labeled the plasma membrane of the Lifeact-GFP/ARPC2-mRuby co-expressing Ramos cells with DiD and imaged their immune synapses. The results are summarized in a new Figure 3—figure supplement 1. The DiD showed dynamic undulations of the plasma membrane in the synapse in pattern that partly corresponded to the Lifeact signal. This is consistent with the actin-dependent mechanical activity of plasma membrane of the cells. Filopodia at the cell edges were also visible in these experiments as thin, Lifeact-positive, DiD-positive protrusions. However, despite of the general correspondence of the Lifeact and DiD signals in the synapse, we could find many Lifeact-positive, ARPC2-negative filaments in the synapse that did not accumulate any extra DiD signal. An example is shown in the figure. We conclude that most of the filament dynamics relevant for this paper and for antigen extraction occurs within the cortical cytoskeleton rather than within filopodia.2) Using your previously published tension sensors, and Arp2/3 inhibition/knockdown, the authors should measure forces exerted by the foci during antigen endocytosis. Knowing the magnitude of the forces at the antigen site would bridge the missing connection between the localization of foci and antigen endocytosis.We agree that the relationship of the actin architecture to the location of the pulling forces is important. We decided not to use the DNA sensors, however, as their force signal on PMSs is weak because the tension is buffered by movement of the PMS during pulling. Instead, we used our previous technique of directly analyzing the force-mediated PMS deformation using labeling with the lipid dye DiI . As cells pull on the PMS, DiI fluoresce locally increases as the deformed PMS accumulates at the sites of pulling. The DiI fluorescence appears either as spots, which we previously showed are vertically oriented membrane invaginations, or as ridges or tubules, depending on the direction of the pulling and range of the movement. Although the strength of the forces cannot be determined by this analysis, it allows us to robustly detect the sites of the pulling forces, their direction and also the overall vigor of the mechanical activity. We used Lifeact-expressing Ramos cells for these experiments to improve the spatial resolution due to the bigger size of the cells, but pilot experiments suggest that the results translate to primary cells as well. The data are presented in the new Figure 6, in Videos 13-16, and in the corresponding sections of the Results and Materials and methods.We found that the sites of pulling associated with both actin foci and actin fibers . Because fibers cover greater area than foci in the synapse, most of the pulling activity associated with fibers . However, correcting for the difference in area, foci were the more efficient structure at pulling . Although both actin structures pulled on the PMSs, there was a difference in the shape of the DiI structures indicating difference in the direction of pulling. While foci were typically associated with DiI spots, which grew inwards, fibers often produced elongated structures, tubules and ridges , that moved laterally. This explains why foci were found to be associated with antigen movement into the cell, but fibers were not.These results were corroborated by inhibitor treatment. We found that the overall pulling activity was not significantly reduced by either CK666 or SMIFH2 . Since these drugs did inhibit antigen extraction, these results indicate that mechanical activity per se is not sufficient for antigen internalization. In addition, treatment of the Ramos cells with CK666 led to enhanced generation of tubules/ridges at the expense of spots, with a significant fraction of cells producing excessive tubulation within the synapse . This further confirms that fibers, which are enhanced in CK666-treated cells, are not sufficient for antigen internalization. Possibly, because of their orientation along the plasma membrane, the fibers generate lateral, instead of inward-oriented movement. Because of the length of the tubules generated by fibers, we speculate that the fibers are producing longer range of membrane movement than foci. Although the DiI imaging did not reveal this clearly, we believe that extension of the inward antigen movement by fibers associated with foci contributes to antigen extraction and is part of the reason why formin inhibition with SMIFH2 also blocks antigen uptake. Overall, we believe that these data add important information to the manuscript, namely that while either Arp2/3 or formin activity apply forces on the antigen, their concerted action is needed for pulling to proceed to antigen internalization.3) By examining the endocytosis of a generic clathrin pathway marker such a transferrin, you need to substantiate the specific impact of Arp2/3 inhibition on antigen internalization.To address the effects of the inhibitors of on general clathrin-mediated endocytosis, we measured internalization of soluble transferrin. We show in a new Figure 5—figure supplement 1 that the inhibitors had a very modest effect, similar to the modest effect on internalization of the soluble surrogate antigen. We conclude that most of the effects that these drugs exert on the uptake of membrane antigen is specific to the immune synapse due to increased demands on the actin cytoskeleton.4) You should provide a supplementary figure detailing the workflow utilized to extract foci, and how the Gaussian filter size for enhancing foci was determined/ chosen. The efficiency of this foci extraction method also needs to be shown.To illustrate the image segmentation more clearly, we created a new figure , which shows the workflow of the algorithm, the shape and size of the filtering elements, and the intermediate results that are used to compute the final segmentation masks identifying foci and fibers. In addition to explanation in the figure legend, we also extended the description of the technique in the Materials and methods and we include the Matlab source code used for the segmentation and for foci counting.We agree that understanding of the performance of the segmentation is important to interpret the results. We therefore compared the automated segmentation with manual segmentation by four human researchers who were knowledgeable of the actin structure (they were co-authors of the manuscript), but had no prior knowledge of the results of the computerized segmentation. We focused on foci segmentation in two Lifeact-GFP timelapses of primary B cells as fibers were found to be too laborious to segment manually even for such a limited number of images. We compared the results of the segmentation by the computer to that of the humans and also the humans to each other. The results are shown in Figure 1—figure supplement 1B. We found that computerized segmentation detected more foci than manual segmentation, indicating that it was more sensitive. To evaluate the positions of the detected foci, we calculated “true positive rates” of the computerized segmentation as the percent of manually segmented foci that were also detected by the computer, and “false positive rates” as the percent of foci detected by the computer, but not detected manually. We calculated these rates for each computer-human pair and also for all human-human pairs. The results showed that the computer-human pairs could not be distinguished in their true and false positive rates from the human-human pairs. Thus, although not perfect, the automated segmentation performs just as well as any experienced researcher, while being consistent and higher throughput. It is also more sensitive, which is likely because human researchers are quickly fatigued by the process after a few image frames.The manuscript has been improved but there are some remaining issues that need to be addressed before acceptance, as outlined below:Please see the comments below. After consultation, we think that an excellent title would be "B cells extract antigens at Arp2/3-generated actin foci interspersed with linear filaments" but you can suggest other titles that accommodate reviewer 2 concerns.Reviewer #2:[…] However, the significance of the observed phenomena and its role in B cell function is still not clear to this reviewer. The experiments in the original and the additional experiments in revision seem to imply that these actin foci and filaments are a general feature of B cell cytoskeleton and only mildly dependent on BCR signaling.So the statement: "We propose that when B cells extract antigens from live antigen-presenting cells, actin foci and fibers form nodes and edges in a loosely connected network, whose function is required for efficient antigen extraction. " is speculative as best.As stated by the authors, these structures do not assemble in an obligatory manner either at antigen clusters or at CCPs, which are the sites of endocytosis. Therefore, their relevance for Ag gathering is not apparent. The data in Figure 5 suggests that the loss of Arp23 or mDia function leads to generic deficits in Ag gathering, which are consistent with general defects in endocytosis as shown in control Tf data. From all of these it is difficult to conclude that these foci play any significant role in exerting forces to test Ag quality or lead to efficient Ag extraction.Given these concerns, the authors have not convincingly demonstrated that "B cells extract antigens" using these actin structures, and therefore the authors should revise the title to reflect their findings more accurately.However, the identification of these structures and quantification of their origins and dynamics will be of sufficient interest to the community.We agree that the relationship of the actin structures to antigen extraction is complex, however, we think that we show sufficient evidence that actin foci are sites of antigen endocytosis, despite their stochastic appearance. We have therefore changed the title to the one suggested by the editor to better reflect our conclusions: “B cells extract antigens at Arp2/3-generated actin foci interspersed with linear filaments”.To acknowledge the speculative nature of the statement on line 356, we edited the sentence to start with “We speculate that …”Reviewer #3:To our opinion, the authors successfully answered most of the requests of the editor. However, we fill that the two following points should be addressed prior to final acceptance.1) The authors use the DIL membrane marker to observe the PMS invaginations. In Figure 6A they show a figure where DIL patches are visible even outside of the B cell area. How do the authors explain this? Are DIL spots within the synapse more important in number and intensity than the ones outside? This should be clarified.Indeed, DiI spots were observed on PMSs outside of B cell synapses. These are spots present on the PMSs before interaction with B cells. We believe that these spots are pre-existing ruffles, bulges or tethered vesicles of the PMSs. For this reason, to analyze the mechanical activity within B cell synapses we used live cell imaging and tracked only DiI spots that newly appeared or moved during the recording time. Thus, the results in Figure 6 reflect only the pulling activity of the B cells and are not affected by the preexisting DiI spots on the PMSs unless the B cells pull on them as well. This is explained in “Materials and methods” under “Quantification of B cell mechanical activity”. Please note that some of the cells lost LifeAct-GFP expression and thus they are not visible, yet still pull on certain areas of the PMSs outside of the analyzed synapses.2) An article describing actin patches at the B cell immune synapse has been published during the time the present article was revised. Although the two articles are complementary, the authors should imperatively cite it and eventually discuss it .We have added a citation of the paper by Kumari et al. along with a brief discussion at the end of the fourth paragraph of “Discussion”.


Plasmopara halstedii) in sunflower (Helianthus annuus L.) is conferred by major resistance genes, denoted Pl. Twenty-two Pl genes have been identified and genetically mapped so far. However, over the past 50 years, wide-scale presence of only a few of them in sunflower crops led to the appearance of new, more virulent pathotypes (races) so it is important for sunflower varieties to carry as wide a range of resistance genes as possible. We analyzed phenotypically 12 novel resistant sources discovered in breeding pools derived from two wild Helianthus species and in eight wild H. annuus ecotypes. All were effective against at least 16 downy mildew pathotypes. We mapped their resistance genes on the sunflower reference genome of 3,600 Mb, in intervals that varied from 75 Kb to 32 Mb using an AXIOM® genotyping array of 49,449 SNP. Ten probably new genes were identified according to resistance spectrum, map position, hypersensitive response to the transient expression of a P. halstedii RXLR effector, or the ecotype/species from which they originated. The resistance source HAS6 was found to carry the first downy mildew resistance gene mapped on chromosome 11, whereas the other resistances were positioned on chromosomes 1, 2, 4, and 13 carrying already published Pl genes that we also mapped physically on the same reference genome. The new genes were designated 23–Pl32Pl according to the current nomenclature. However, since sunflower downy mildew resistance genes have not yet been sequenced, rules for designation are discussed. This is the first large scale physical mapping of both 10 new and 10 already reported downy mildew resistance genes in sunflower.Resistance to downy mildew ( Plasmopara halstedii can occur in most countries where sunflowers (Helianthus annuus L.) are grown. It is an obligate parasitic oomycete belonging to the Peronosporales showing physiological races or pathotypes (Pl) have been identified in sunflower  Progress and Novinka which were resistant to downy mildew in Russia. When research on sunflower started in Canada, accidental crosses with wild H. annuus in 1962–1963 showed that resistance was available in this weedy species  was not the same as that in the United States (300)  . Betweenes (300)  may wellwer seed . Progreshus spp.  providedhus spp. .Studies have been made of quantitative, perhaps pathotype-non-specific, resistance  but thisH. annuus and other Helianthus species, and research for new downy mildew resistance genes has been made in crosses of this material with cultivated sunflower. The genes introduced into cultivated sunflower up to 2008 were summarized by 14Pl , SSR (simple sequence repeats)  and more recently SNP (single nucleotide polymorphisms) . Pl22 was the first resistance gene to be physically mapped to a region of 1.7 Mb of chromosome 13  in resistant sunflower lines and not in near-isogenic susceptible lines  or whether they are inherited independently, the new lines were crossed firstly with lines representing the resistance genes clusters on chromosomes 1, 8, and 13 together with COQ (HA458) whose gene had not yet been mapped  under the control of the 35S promoter was infiltrated in sunflower leaves of entire plants in Agrobacterium-mediated transient expression experiments, as previously described (A construct consisting of the escribed . The conescribed . The triescribed .® (Affymetrix) according to the manufacturer’s instructions. Genotyping was performed using a 50K SNP AXIOM® array (1), following the Best Practices Genotyping Workflow. Samples with a dish quality control (DQC) value ≥ 0.9 and QC call rate ≥ 0.90 thresholds were considered to have passed the quality control assessment. SNP quality control parameters and thresholds were defined as follows: polyploid species-type; cr-cutoff ≥ 90; fld-cutoff ≥ 3.6; het-so-cutoff ≥ -0.3; het-so-otv-cutoff ≥ -0.3; hom-ro-1-cutoff ≥ 0.6; hom-ro-2-cutoff ≥ 0.3; hom-ro-3-cutoff ≥ -0.9. For mapping, only PolyHighResolution SNPs considered to be reliable and informative were retained. A final SNP filter focused on genotype data of parental lines was applied: for either or both parents, markers that failed SNP calling, or were genotyped as heterozygous, or showed divergences of allele calling between technical replicates were discarded from the analysis.From the phenotype segregation patterns, the numbers of F3 progenies chosen for genotyping each resistance source varied from 27 to 72, according to apparent similarity with another line or novelty, and to number of seeds available . The genetic distances between markers were calculated using the lkh 1 -1 function and the genetic maps were obtained with the bestprintd function. Published linked markers of Pl genes were positioned on the sequenced sunflower genome , linkage was not considered. Ninety four of the 122 progeny segregations agreed with those expected (non-significant X2). In some cases, SII or RII plants could have caused some misinterpretation. Pathotype 710 was used for the majority of tests and gave the highest percentage of rejection of the null hypothesis (significant X2): 33% compared with 27% for pathotype 304 and 12% for pathotype 334. Among the lines used in the crosses, COQ (HA458) and HAS40 gave the largest numbers of significant X2, but in the case of HAS40, there were more segregations with unexpectedly low numbers of susceptible plants than those with excess susceptible plants. All X2 agreed with the hypotheses proposed for the line HIS32. From these results, the resistances of HIS33, INTER35 and HAS62 showed linkage to ARGPl on chromosome 1, those of IDAHO, HIS32, HIS36 and HAS85 linkage to 17Pl on chromosome 4 and those of HAS42 and HAS54 linkage to 5Pl on chromosome 13, whereas resistances of HAS103 and HAS6 appeared independent of all the known Pl genes tested. It was only for HAS40 that no conclusion could be drawn; it showed few susceptible plants in crosses with ARGPl but also in crosses involving 17Pl.Supplementary Table 6Pl conferring resistance to pathotype 710. Three lines, HAS62, HIS32, and HAS54 were found to show segregations indicating the presence of two genes for resistance to pathotype 334 although 6Pl is not effective against this pathotype. The three lines are from quite different origins, but they appear to contain a 2Pl-type gene in addition to that giving resistance to a large number of pathotypes. When tested with pathotype 714, the progenies showed segregations agreeing with the hypothesis for a single dominant gene. These segregations were used to define the resistance genotype of each F2 plant used for mapping.Table P. halstedii showed that only three of the 12 resistant lines responded by an HR: HAS103, HAS6 and HAS85, as well as the positive control line PMI3 . The resistance of HAS40 was quite precisely mapped on the genome sequence in a region of 75 Kb, but surprisingly corresponding to a large genetic region of 16.8 cM. This genomic region does not appear to include any RGC in XRQ, where there may have been a deletion.The resistances in HIS33, INTER35, HAS62, and HAS40 were mapped on chromosome 1 Figure . The firPlARG  in an arPlARG . In contand Pl16  but diffand Pl16 . Comparis marker  Pl or by their resistance phenotypes   .Pl genes on the physical map of chromosome 13 according to their linked published markers. 5Pl 2 cM and 8Pl 4.5 cM from marker NTIR7H3 using two different populations. However, recombination in this area appears to vary between crosses, 8Pl in one cross and 1.2 cM in another. Taking into consideration mapping of 5Pl and 21Pl below ORS316 but above ORS630 by 22Pl is above ORS316, with 5Pl and 21Pl between the two SSR markers and then 8Pl, and the resistance genes in HAS42 and HAS54 in the same zone, covering 8 Mb  that could not be localized by phenotypic studies was mapped successfully with the array on chromosome 1.Using the AXIOM® array, of 12 sources of resistance effective against at least 16 downy mildew pathotypes. The results suggest that these sources may be considered as providing new resistances carried by 10 novel resistance genes.The results presented here describe mapping, on the sunflower reference genome , using aPl genes are in one or different clusters. Seventy seven percent of tests reported here did not differ from the Mendelian segregations 3R: 1S, 7R:1S or no segregation. Of those with significant X2, some were probably due to the wide use of the highly aggressive pathotype 710, which, in breeding programs, has occasionally been found to give symptoms in plants containing resistance genes but with background genotypes that are unfavorable for expression of resistance. Concerning parental lines, COQ (HA458) and HAS40 showed the highest proportion of “abnormal” segregations (6/13 and 8/17 respectively). For COQ, there was an excess of susceptible plants in five tests out of the six, and it may be suggested that this is due to background susceptibility to pathotype 710. In contrast, for HAS40, five of the eight “abnormal” segregations showed an excess of resistant plants. In three cases they were in crosses with lines  that were confirmed by the SNP genotyping to have resistance genes on the same region of chromosome 1. These segregations thus probably corresponded to loose linkage between the genes, which could not be estimated precisely with the numbers of plants phenotyped for their downy mildew resistance. It was concluded that phenotyping by resistance tests provided first indications of the genetics of the downy mildew resistances studied but that molecular studies were necessary to confirm distinction and precise location.Before the availability of molecular analyses, determination of linkage or independence of downy mildew resistance genes depended on resistance tests on seedlings with adequate pathotypes. The present results show that a large number of crosses and progenies must be tested for analyses of segregations to permit decisions as to whether ® array used for genotyping has theoretically 49,449 SNP. Over the 12 crosses, we found an average of 5953 high-quality polymorphic SNPs, between a minimum of 4138 for HAS40 and a maximum of 8548 for HIS33 . Although some physical information was lost according to the cross, there were always sufficient markers to map correctly the resistances in regions of 10s of Mb, and sometimes in 10s of kb (HAS40). This was quite surprising since HAS40 had a small number of polymorphic markers on the chromosome concerned (221). However, the F3 population used for mapping was large (70 plants) making it possible to take account of a sufficient number of recombination events. In addition, the segregation ratios observed for resistance were close to theoretical values, suggesting that mapping population size and phenotyping quality are important criteria for precise fine mapping. On the contrary, the cross involving HAS103 had 50% more polymorphic markers on the chromosome concerned, but the mapping population was smaller (45) and segregation for resistance not optimal. Together with a lack of recombination events on chromosome 2 in the HAS103 x XRQ population, the result is poor physical mapping.The AXIOMH. annuus ecotypes or from different Helianthus species. In the first report mapping ARGPl, chromosome 1 appeared much shorter in a cross involving H. argophyllus and cultivated sunflower  devoid of SNPs, whereas the SNPs selected for the study provide relatively regular chromosome coverage. Thus, in the populations studied, the recombination frequency may be high in these regions compared with the reference genome. Alternatively, there may have been deletions in the reference genome compared with the genotypes used for mapping. Second, we observed inversions between genetic and physical maps, for example on chromosome 4, again suggesting differences between the parental genotypes used to map the resistances and the reference genome. It would be interesting to have the sequence of more than one genome to allow better comparisons with genotypes of various origins and to understand sunflower genome evolution.Pl genes that were previously genetically mapped in the same regions. The genetic regions defined for formerly mapped Pl genes appear to be very large on the physical map, encompassing up to 10 Mb for ARGPl and up to 20 Mb for 14Pl, both on chromosome 1. ARGPl on chromosome 1. However, it should be kept in mind that gene order in some genotypes could be slightly different from that in the reference genotype, making fine mapping difficult.Mapping and positioning physically on the reference genome made it possible to compare localizations of new downy mildew resistance genes from different genetic backgrounds and also P. halstedii.Even with the availability of a sunflower genome sequence, all the questions regarding the genetic and molecular architecture of resistance to downy mildew will not be solved in the near future, so it appears of interest to fix a rule to designate genes in sunflower involved in the interaction with Pl5, but they showed slight differences in phenotyping reactions to pathotypes: PM17 is susceptible to a Spanish isolate of pathotype 330 to which XRQ is resistant, so it was considered to have 5-Pl whereas 803.1 is resistant to the US isolate of 330 to which XRQ is susceptible so its gene was defined as 5+Pl  located in the same region of chromosome 4, since only HAS85 responded by an HR. It would be of interest to test the reaction of a line carrying 19Pl to this effector to determine whether the resistance of HAS85 is distinguished from this gene. On chromosome 13, effector reaction also distinguishes 22Pl in line PMI3  for the gene in HAS40 to 32 Mb (640 genes) for the other resistances on chromosome 1, in HIS33, INTER35 and HAS62. At this scale, we cannot conclude between close genes or one gene so we have tried to fix some rules for naming the genes identified in the present study.17Pl. For the lines selected from interspecific breeding pools, detailed history is not available and downy mildew reactions were not studied during fixation. With no proof of difference, and the resistances of HIS33 and INTER35 (from H. resinosus) both being mapped in the same area on chromosome 1, it appears most logical to consider that only one resistance gene was introgressed. Therefore, we propose to call the gene from 23H. resinosus Pl. For the other lines, several resistances map in the same zone as known genes, and others appear to co-localize, but, in all cases they come from different species or from ecotypes collected in different geographical origins. We therefore suggest that they should be given different names, at least for the present (24–Pl32Pl) and we include in the name the source of the resistance. These are listed in Table Firstly, those that map in the same area and come from the same origin are given the same name: IDAHO, whose gene mapped to chromosome 4, was bred from the same origin as HA458 so it was considered that it carries With more than 30 downy mildew resistance genes identified and positioned on the sunflower genome, simple numbering according to order of identification is not helpful to avoid errors. It might be useful to include at least chromosome number in gene names. Of course, international agreement would be necessary, as for the triplet denomination of downy mildew pathotypes .Pl regions  that can range from a few nucleotides to 100s of kb, are more difficult to identify. By aligning genetic maps on the reference genome sequence, we identified such polymorphisms which suggest that some resistances, originating from wild sunflowers, could be due to structural variations. In order to facilitate the sequencing of the downy mildew resistance genes or other loci of interest, it would be very useful to produce new tools or new genomic resources in sunflower. For example, from a pan-genome, it would be interesting to develop a genotyping tool based on SNPs identified on this pan-genome in order to identify large structural variations. At a finer scale, making the optical maps of the genomes from 10s of accessions would be a useful resource too. Finally, on an even finer scale, assembling high quality genome sequences of sunflowers and wild Helianthus would considerably speed up research in sunflower, which could become a model plant species in many scientific fields .We performed the colinearity studies of the genetic maps using the genome sequence of the cultivated sunflower line XRQ . HoweverHelianthus species, and more particularly wild H. annuus are quite rich in broad spectrum downy mildew resistance genes. Use of combinations of those described here should help to provide some reasonably durable resistance, protecting the sunflower crop against changes in downy mildew pathotypes.The present results suggest that Sunflower plant material used in this study was grown from seeds made available by INRA as part of INRA sunflower genetic resources. The lines Idaho, COQ and U8Q were obtained by selfing from USDA sources.® array. YP and LG acquired genotyping data. YP mapped the resistance genes. FV, LG, SM, and YP wrote the manuscript. All authors were involved in revising the manuscript critically and approved it.YP, FV, and LG conceived the project. FV produced the plant material and carried out phenotyping. CP-S and YP produced the plant material for genotyping. SM designed the 50K-SNP AXIOMThe authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Nontuberculous mycobacterial (NTM) pulmonary disease has conventionally been classified on the basis of radiology into fibrocavitary and nodular-bronchiectatic disease. Whilst being of great clinical utility, this may not capture the full spectrum of radiological appearances present. The aim of this study was to use latent class analysis (LCA) as an unbiased method of grouping subjects with NTM-pulmonary disease based on their CT features and to compare the clinical characteristics of these groups.Individuals with NTM-pulmonary disease were recruited and a contemporaneous CT scan obtained. This was scored using an NTM-specific scoring system. LCA was used to identify groups with common radiological characteristics. The analysis was then repeated in an independent cohort.Three classes were identified in the initial cohort of 85 subjects. Group 1 was characterised by severe bronchiectasis, cavitation and aspergillomas, Group 2 by relatively minor radiological changes, and Group 3 by predominantly bronchiectasis only. These findings were reproduced in an independent cohort of 62 subjects. Subjects in Group 1 had a lower BMI and serum albumin, higher serum CRP, and a higher mortality.These findings suggest that NTM-pulmonary may be divided into three radiological subgroups, and that important clinical and survival differences exist between these groups.The online version of this article (10.1186/s12890-018-0675-8) contains supplementary material, which is available to authorized users. Pulmonary non-tuberculous mycobacterial disease is a challenging infection which is associated with a high mortality –3. TreatThe aim of this study was to characterise the radiological features of NTM-pulmonary disease using an NTM-specific scoring system, to use latent class analysis (LCA) as an unbiased method to identify subgroups sharing common patterns of radiological features, and to examine the clinical characteristics associated with any such patterns.Individuals with NTM-pulmonary disease were recruited prospectively from the outpatient department of the Royal Brompton Hospital and Chelsea and Westminster Hospital between September 2012 and November 2013. All met American Thoracic Society 2007 disease criteria . SubjectCT scoring was performed as previously described  by a speStatistical analysis was performed in the R environment version 3.4.0 . Latent To examine the reproducibility of the groups identified by LCA and their radiological characteristics, the analysis was repeated on an independent cohort from another tertiary centre. Subjects with NTM-pulmonary disease at Papworth Hospital who underwent CT scanning at as part of their clinical care were identified retrospectively, their anonymised imaging and clinical data were retrieved and data analysed using the exact methodology used for the original cohort.A total of 85 subjects were recruited, their clinical and radiological characteristics are shown in Additional file Three latent classes were identified using the AIC  and cavitating nodules which were present in 22.5%. The third class, referred to as “Bronchiectatic” disease had the most extensive bronchiectasis (high in 71.2%) which was of high severity in the majority (56%), and the highest frequency of tree-in-bud changes (medium or high levels in 28%), although cavitation and nodules were uncommon.P < 0.001.The composite CT score Fig.  was highP = 0.003 vs Nodular, P = 0.079 vs Bronchiectatic groups). They had a higher prevalence of semi-invasive aspergillosis, were more likely to have received treatment for NTM and over one-third were receiving systemic corticosteroids, although these differences were not significant. The mean C-reactive protein level was higher than both other classes , body mass index was lower  and serum albumin was lower than in the Nodular (P = 0.007) but not the Bronchiectatic (P = 0.246) group. The mean corrected carbon monoxide transfer factor (TLCOc) was the lowest of the three groups, but this did not reach significance. Dyspnoea and quality of life scores were not significantly worse than the other two classes.The clinical characteristics of the latent classes are shown in Table P = 0.307 vs Cavitary, P = 0.008 vs Bronchiectatic groups).Compared to the other groups, the Nodular group had a more even mix of underlying respiratory pathology and 21% had no underlying disease. Lung function indices were the highest of the three groups. This group had the shortest mean duration of NTM disease prior to enrolment (Significantly more subjects (75.8%) in the Bronchiectatic group had previously diagnosed underlying bronchiectasis and 21.2% were chronically infected with Pseudomonas, which was uncommon in the other groups. The Bronchiectatic group had the highest rates of antibiotic prophylaxis and the lowest rate of current NTM treatment.P = 0.011, Fig. Survival data were available for 78 subjects with a median follow-up time of 126 weeks. There were significant differences in survival seen between groups, with the Cavitary group showing the highest mortality of 42.9% compared with the bronchietatic (9.1%) and Nodular (16.1%) groups  although this was less than the universal presence seen in the Brompton cohort. Another point of difference was the high level of severe tree-in-bud change, seen in 87% but not seen in the Brompton cohort. Class 2 corresponded to the Nodular group, with generally mild changes except cavitating nodules which were present in 14.9% and severe cavitation in 20%. In contrast to the Brompton cohort, aspergillomas were present in 10% and a medium level of tree-in-bud changes in 40.2%. Class 3 corresponded to the Bronchiectatic group with all subjects having high severity bronchiectasis and 87.3% having highly extensive bronchiectasis. In contrast to the Brompton cohort the majority (90.5%) had a medium or high degree of tree-in-bud changes and no subjects had medium or high nodule scores compared with 12% in the Brompton cohort. Similarly, the vast majority had no cavitating nodules or severe cavitation, and aspergillomas were not seen. As with the Brompton cohort, class 1 showed the highest composite CT scores (mean 3.95), class 2 the lowest (mean 0.97) and class 3 intermediate values (mean 1.99). All differences were significant with 001 Fig. .The mean age was youngest in class 2 and highest in the class 1, although this difference was not significant. There was significantly more underlying bronchiectasis in class 3. There was significantly more MAC (87.5%) in class 1 compared to other classes, whereas in the corresponding Cavitary group from the Brompton cohort this species accounted for only 50% of subjects.N = 43), two classes were identified by LCA representing 58 and 42% of the group  three classes were identified representing 62, 21 and 16% of the group. The features of these groups  and those with low composite CT scores and low scores for most CT features (corresponding to the Nodular group). A higher proportion of subjects fell into the Bronchiectatic group, likely due to the higher overall prevalence of underlying bronchiectasis in this cohort.The only major difference to the Brompton cohort was the higher prevalence of tree-in-bud changes, which were moderate or severe in 76% compared with 17%. The prevalence was higher in all three disease subgroups and does not appear to be due to the increased prevalence of underlying bronchiectasis. As the scoring of the validation cohort was performed separately to the Brompton cohort observer bias may be responsible for this difference, although this was performed by the same individual, alternatively it may be that differences in treatment, coinfection, or other clinical factors between centres is responsible. There was also more severe cavitation and aspergilloma in the nodular Papworth group compared with the Brompton, however only four subjects accounted for this difference.In contrast to other studies mentioned previously, the presence of a separate consolidative pattern of NTM-pulmonary disease was not seen in this study. The vast majority of subjects in both cohorts had only mild consolidation, although this feature was slightly more prominent in the Cavitary group. Only one subject had consolidation and no other feature of NTM disease. Our findings may in part be explained by the absence of immunocompromised or critically ill subjects from our cohorts. When the analysis was restricted to subjects with known underlying bronchiectasis only two subgroups were identified, with the Bronchiectatic group being split in half dividing the cohort into groups more consistent with the conventional ‘fibrocavitary’ and ‘nodular-bronchiectatic’ patterns. However, in the validation cohort the three subgroups were still clearly identified.The principle limitation of the study is the heterogeneous nature of the cohort, comprising subjects with differing underlying respiratory diseases, NTM species, antibiotic treatments and infecting co-pathogens. Nevertheless the findings were replicable in an independent cohort suggesting they are not merely related to the specific case mix at a single centre. Furthermore, such a mix of subjects is representative of real-life clinical practice, although as both cohorts come from tertiary centres the proportion of subjects with severe bronchiectasis and aspergillomas may be higher than the wider population of individuals with NTM-pulmonary disease. A number of subjects had previously undergone HRCT and declined further imaging, therefore in some subjects the collected clinical data may not reflect the point in time when the HRCT was performed and in 10% of cases more than a year had elapsed from imaging to study enrolment. A single radiologist was responsible for performing CT scoring and despite the use of an objective scoring system this remains a potential source of bias, although the scoring system was based on the Bhalla score  which haThis study provides validation of the existence of cavitary disease as a distinct phenotype of NTM-pulmonary disease associated with a poor prognosis. In addition they suggest that ‘nodular-bronchiectatic’ disease is formed of two separate groups with important radiological and clinical differences, which may possibly reflect differences in underlying lung disease or duration of infection. These data underline that fact that NTM-pulmonary disease is a heterogeneous condition and more precise phenotyping will be valuable in clinical decision making and stratification in clinical trials.Additional file 1:Supplementary materials. (DOCX 1280 kb)


The aim of this study was to describe the technical structure of the 9-1-1 call-taking system and to describe its vulnerabilities that could lead to compromised patient care.9-1-1 calls answered from mobile phones and landlines use a variety of technologies to provide information about caller location and other information. These interconnected technologies create potential cyber vulnerabilities. A variety of attacks could be carried out on 9-1-1 infrastructure to various ends. Attackers could target individuals, groups, or entire municipalities. These attacks could result in anything from a nuisance to increased loss of life in a physical attack to worse overall outcomes owing to delays in care for time-sensitive conditions. Evolving 9-1-1 systems are increasingly connected and dependent on network technology. As implications of cybersecurity vulnerabilities loom large, future research should examine methods of hardening the 9-1-1 system against attack. The catalyst for creating the 9-1-1 system in the United States came in 1957, when the National Association of Fire Chiefs recommended using a single number to report fires. In 1967, the Federal Communications Commission worked with the American Telephone and Telegraph Company to establish a single emergency number [Today, emergencies can be reported through 9-1-1 from 96% of the geography of the United States, placing an estimated 240 million calls annually. There are 5783 public safety answering points  in 3135 counties throughout the United States [Although the original implementation of basic 9-1-1 services relied mostly on telephone technology, enhanced 9-1-1 (E9-1-1), deployed in the 1980s, introduced significant new elements aimed at identifying the caller’s telephone number and location. Around the same time, new tools were developed to assist dispatchers in handling emergency calls: advanced hardware and software to receive and display the caller’s number and location data, computer-aided dispatch (CAD) systems to assign and track field responders, and mapping software to display both caller and responder locations. These tools greatly enhanced emergency response capabilities but required additional computer, network, and server infrastructure beyond the rudimentary elements of the telephone network. As capabilities further expanded, systems that were once safely within the walled garden of the telephone company were increasingly connected to less well-regulated networks such as the public internet. Though undoubtedly valuable from a patient outcome perspective, this also introduced new risks.According to the Department of Homeland Security, the emergency services sector will likely become more of a target for cyberattacks as systems become more interconnected and dependent on information technology for daily operations . DisruptAn emergency call placed from a landline is the simplest scenario for emergency call taking see . When thThe call is answered at the PSAP by a dispatcher, often with the help of a CAD system. One of the dispatcher’s first priorities is to determine the location of the call. The CAD system uses the ANI to search a database, which returns Automatic Location Information (ALI). The ALI record contains the address, phone number, and which services are assigned to the caller’s position.A wireless emergency call’s routing varies depending on the E9-1-1 implementation. In a phase 1 implementation, the call is sent from a cell tower to a mobile switching center see . The mobIn a phase 2 implementation, additional equipment determines the caller’s location more precisely see . The exaVoice-over-IP (VoIP), or voice communications delivered by internet-connected networks from providers such as Skype  and Vonage , presents a special problem for 9-1-1 location services see . In thisInformation security vulnerabilities fall under 3 main categories: confidentiality, integrity, and availability—often referred to as the CIA triad.An attack on confidentiality results in the disclosure of previously private information or communications to one or more parties who are not authorized to receive or view it. Surveillance of the 9-1-1 system could provide attackers with information valuable to planning an incident. Metrics such as call volumes and response times could be used to maximize damage during a physical attack or increase the likelihood of success of criminal acts.An attack breaching integrity violates the correct functioning of a system, producing aberrant results desired by the attacker. An example of this is an attack called swatting, where an individual misdirects law enforcement resources to target another individual, often for personal reasons such as revenge. A December 2017 swatting incident in Wichita, Kansas, led to the shooting of an unarmed man, resulting in his death . SwattinTeletypewriter (TTY) services are intended as communication relays between deaf and hearing persons. A deaf person connects to the relay service using a special terminal with a keyboard or an online service, then requests to an operator who they would like to speak with. The operator connects the call, then verbatim reads messages from the terminal to the caller, and types messages from the caller to the terminal. If an attacker connects to a TTY service and describes an emergency to the operator, the operator will call 9-1-1 and report exactly what the TTY user is typing. Using a TTY service removes some of the attacker’s potentially identifying information (such as their voice). In addition, TTY relay services are required to ensure user confidentiality and are prohibited from keeping records of the contents of any conversation . This meOther integrity attacks include spoofing apps or services that generate false location or caller identification (ID) information by modifying the ANI or ALI record. This method will not work with a landline because the ANI/ALI is not dynamic. Many spoofing services treat emergency calls differently and will replace the correct ANI data before connecting the call. In addition, when an emergency call is placed from a spoofing service, the service provider ignores any ANI information received from the caller and inserts the correct data. In some rare circumstances, VoIP providers have been found to forward user-provided ANI data without verification or alteration, which could allow spoofing. In addition, this information can seemingly be updated without verification.If an attacker successfully spoofed ANI information, they could circumvent automatic PSAP routing to target a specific PSAP. The routers that forward emergency calls have a direct dial phone number that is used to transfer calls between PSAPs when a caller is incorrectly routed or if fire and police services are dispatched separately. Although the direct dial numbers for PSAPs’ 9-1-1 network connections and 10-digit emergency lines are kept secret , they can sometimes be discovered during a 9-1-1 call that involves a transfer if the PSAP equipment is not configured to mute the dialing tones used to connect the call. If an attacker utilized a spoofed ANI to call the router at the newly discovered direct dial number, they could place a seemingly authentic call to a specific PSAP. Targeting a specific PSAP could improve an attacker’s chances of getting the desired response to a call, such as a swatting attack.All location determination mechanisms rely on the PSAP querying the ALI database. If an attacker altered this database, they could change the ALI information for any phone number to their target’s address or change the address associated with their target’s phone number. If an attacker denied access to the ALI database, dispatchers would rely entirely on the information provided by the caller. It is notable that the ALI database represents a single point of failure in every location-determining scheme.The VSP database that maintains the mapping between the subscriber and location could also be targeted. Modification of the database or a denial of service attack would have the same effect as an attack on the ALI database, but may be secured differently.Mobile handsets without an active service plan can still be used to place 9-1-1 calls. These nonservice initiated (NSI) devices do not provide a call back number because they do not have a service plan. Instead, they provide a phone number with area code 9-1-1 with the last 7 digits of the device’s electronic serial number or international mobile equipment identity number in place of a phone number. Such a device could be purchased with cash and used to place a call, removing the standard information links between the 9-1-1 call and caller.Wireless phase 2 calls utilize the location data from the mobile handset itself. It is possible to inject arbitrary coordinates into the handset, either by global positioning system spoofing or modifying the device firmware such that it reports prespecified coordinates. The call will be routed to the appropriate PSAP based on the cell tower location but the ALI record will contain the arbitrary location which is then displayed at the PSAP. This is not foolproof, however, as most cellular providers also use cell tower triangulation as part of the location-determination process.Attacking the location-determination systems (as described above) or denying service to the entire 9-1-1 infrastructure could delay response to an incident to increase collateral damage. Modifying the VSP or ALI records for an individual could lead to emergency response being directed to the wrong location, delaying emergency services to a targeted individual.Another integrity attack could target first responders or enhance damage from a physical attack. By misdirecting resources throughout a municipality, an attacker could delay emergency response to a planned physical attack, increasing collateral damage. In addition, an attacker could create a major incident that concentrates emergency responders in a specific location for the purpose of directly attacking responders. These 2 types of attack could also be used in combination. Altering or denying service to the VSP or ALI database could increase the credibility of calls used to initiate such incidents.An attack on availability would make a PSAP unavailable to the public. This could be accomplished by a telephone denial of service attack, as occurred during March 2016 in Phoenix. An attacker released a malicious script via a link on Twitter that caused devices to repeatedly call the PSAP without allowing the user to hang up. This produced thousands of false 9-1-1 calls, filling up the phone lines and preventing the public from reporting emergencies . In a DeAll 9-1-1–focused attacks to date have been linked to criminal hackers rather than state-sponsored actors . HoweverCurrently, there is a national effort to upgrade traditional telephone-based 9-1-1 architecture to Next Generation 9-1-1 (NG 9-1-1). This is a collection of technologies that allow PSAPs to receive short message service text messages, images, and even live video streams in addition to existing landline, mobile, and VoIP calls. NG 9-1-1 promises increased capabilities for precisely locating callers and responding to mass casualty incidents, infrastructure disruptions, and natural disasters. However, converting to a system dependent on internet-connected networks raises the possibility of inheriting the same cybersecurity vulnerabilities that plague existing connected infrastructure while continuing to be susceptible to the threat models affecting telephone-based systems that we have already discussed.Inundating a PSAP with phone calls can make the phone system unavailable. The same attack can be applied to computer systems with the internet or network traffic in a distributed denial of service (DDos) attack. A March 2015 DDos attack on the City of Madison, Wisconsin, targeted municipal websites with the unintended consequence of disrupting 9-1-1 infrastructure. In March 2018, a ransomware attack  made the City of Baltimore’s CAD system unavailable to dispatchers for almost 24 hours while systems were restored. Although the PSAP was still reachable, call takers had to gather information by hand that would have normally been displayed automatically, slowing the dispatching process [The National Public Safety Telecommunications Council has outlined the future of EMS communications . SoftwarFortunately, centers upgrading to NG 9-1-1 have the potential to mitigate cybersecurity vulnerabilities and related disruptions. Important best practices include frequently patching vulnerable software applications, segmenting networks to protect infrastructure from a direct connection to the internet, and developing incident response plans and teams to react to cybersecurity events that occur .A variety of attacks could be carried out on the 9-1-1 infrastructure to various ends. Attackers could target individuals, groups, or entire municipalities. These attacks could result in anything from a nuisance to increased loss of life in a physical attack to worse overall outcomes owing to delays in care for time-sensitive conditions. Evolving 9-1-1 systems are increasingly connected and dependent on network technology. As implications of cybersecurity vulnerabilities loom large, future research should examine methods of hardening the 9-1-1 system against attack—whether through the technical work of designing and developing increasingly secure software platforms utilizing next generation encryption and authentication methodology to further increase trust of network communications, through implementing regulatory frameworks with incentives for procurement and operational guidance, or through, perhaps most feasible in the short term, raising awareness among individual and regional centers of existing security challenges and corresponding cybersecurity best practices.


Previous studies showed that the number of berries is a major component of the compactness level of the grapevine clusters. Variation in number of fruits is regulated by events occurring in the fruitset, but also before during the flower formation and pollination, through factors like the initial number of flowers or the gametic viability. Therefore, the identification of the genetic bases of this variation would provide an invaluable knowledge of the grapevine reproductive development and useful tools for managing yield and cluster compactness. We performed the phenotyping of four clones (two compact and two loose clones) of the Tempranillo cultivar with reproducible different levels of cluster compactness over seasons. Measures of reproductive performance included flower number per inflorescence, berry number per cluster, fruitset, coulure, and millerandage indices. Besides, their levels of several hormones during the inflorescence and flower development were determined, and their transcriptomes were evaluated at critical time points (just before the start and at the end of flowering). For some key reproductive traits, like number of berries per cluster and number of seeds per berry, clones bearing loose clusters showed differences with the compact clones and also differed from each other, indicating that each one follows different paths to produce loose clusters. Variation between clones was observed for abscisic acid and gibberellins levels at particular development stages, and differences in GAs could be related to phenotypic differences. Likewise, various changes between clones were found at the transcriptomic level, mostly just before the start of flowering. Several of the differentially expressed genes between one of the loose clones and the compact clones are known to be over-expressed in pollen, and many of them were related to cell wall modification processes or to the phenylpropanoids metabolism. We also found polymorphisms between clones in candidate genes that could be directly involved in the variation of the compactness level. Vitis vinifera L.) selection programs. Berries inside the cluster are organized as a thyrse, which is unique to grapevine among economically important crops. The thyrse, etymologically derived from Dionysus’s staff, is a type of mixed compound inflorescence. It is a raceme inflorescence type where flowers are replaced by a cyme , where all the vines were treated in the same way. The four clones used in the analysis originated from a otstocks . Two of otstocks . Samplinotstocks ]. FlowerEach clone was phenotyped during 2–5 years using morphological variables of the cluster and the berry related to compactness trait as described in 51, GA4, GA1, G29, and GA8. Hormones were analyzed at E-L 13–14, E-L 16–17, E-L 18–19, and E-L 26 stages.Hormones were analyzed at the Servicio de Cuantificación de Hormonas Vegetales in the IBMCP in Valencia, Spain with a UHPLC-mass spectrometer  from at least 100 mg of flower and inflorescence material. Hormones included IAA, ABA, JA, SA, and the GAs GA1) as recommended by manufacturer. DNase I digestion was carried out with the RNase-free DNase Set (QIAGEN). RNA integrity and quantity were assessed with a Nanodrop 2000 spectrophotometer (Thermo Scientific) and an Agilent’s Bioanalyzer 2100. RNA samples were processed to build strand-specific cDNA libraries  using Illumina TruSeq RNA Library Preparation Kit . Sequencing of all 24 libraries (3 replicates ∗ 4 clones ∗ 2 stages) was conducted on two sequencing lane using Illumina HiSeq 2500 v4 platform  to produce 19–24 million strand-specific 125 bp paired-end reads per library. Sequencing was performed at the Center for Genomic Regulation .Total RNA was extracted from samples using the Spectrum plant total RNA kit ] between clones was performed using the RNA-seq alignments bam files. Variant calling vcf files were obtained with the variant caller utility implemented in the SAMtools package v1.2  . The vcfvcftools . 605 polvcftools  were convcftools .p < 0.05)   adapted Phenotypic analyses showed a large amount of variability within Tempranillo cultivar for many traits related to the reproductive development Table . In seveFinally, several variables related to the reproductive performance of the vine seem relate to compactness. Based on 5-year data, the number of normal (seeded) berries in the cluster was similar in the two compact clones, and significantly lower in the loose clones, where there is still a difference between VP25 and VP11, the clone with the lowest number. This is a reflection of the fruitset rates, with similar values for VP2 and RJ51, followed by VP25 and, with the lowest value, VP11. An inverse trend is observed for the number of flowers and coulure, where VP11 showed the highest value, although the differences between the other clones are not significant. Other variables showed no significant differences between clones (like Millerandage), or no relation with compactness trait (like the length of pedicel).In order to evaluate the relation between each measured parameter and cluster compactness, we performed a principal component analysis (PCA) based on the phenotypic data Figure . The PCAp < 0.05) most abundant in E-L 18–19 (just before flowering) against the three other stages and significantly less abundant at E-L 13–14 against the three other stages  higher in E-L 18–19 versus the three other stages, which showed no differences between them  global levels were significantly higher at E-L 26, with no differences between the other stages  were found in comparisons compact vs. loose clones, supporting the hypothesis that the mechanisms for loosening the clusters may be different in VP11 and VP25, and must be studied separately. The stages E-L 18–19 and E-L 26 were chosen for gene expression analysis because these two stages showed the most relevant differences between clones. Most remarkably, the higher abundance of GA1 in the loose clone VP11 at EL 18–19 discriminated it from both compact clones. VP11 also showed the lowest levels of the two active GAs at EL26, and the highest of GA8. VP25 only showed differential hormone levels with respect to the compact clones (and VP11) for ABA at E-L 26.In summary, considering all the clones together, significant differences between stages were found for all the hormones studied excepted SA, indicating their possible role during inflorescence growth and flowering. No differences  presenting compact clusters, as expected for the variety, and two presenting loose clusters (VP25 and VP11). Our hypothesis is that each of these two clones present loose clusters due to a genetic mutation originally produced in a single Tempranillo plant, which was vegetatively propagated. This mutation makes it differ from the normal plants with compact clusters but in a, basically, identical genetic background. The contrast in compactness was reproducible over the years and the clones were grown in the same conditions and parcel, at few meters from each other. Therefore, we expected that the differences between clones in phenotypic traits, hormones, and gene expression levels had a genetic origin that could be isolated by monitoring the gene expression and their polymorphism with little “noise” in the gene expression. Besides, in this work VP25 and VP11 have shown different phenotypic, hormonal, and gene expression characteristics, indicating that their loose phenotypes result from distinct mechanisms and should be studied independently.Our previous findings showed that the two main components affecting the cluster compactness in a multi-cultivar frame were the cluster architecture and the number of berries . Within For targeting the most appropriated phenological stages for the transcriptome evaluation, we first analyzed the hormones evolution during the inflorescence and flower development to pinpoint key stages with dramatic changes.1, GA4 and GA8, which we only observed for VP11 for GA1. We could not relate this observation to the loose phenotype of VP11 because the authors performed the experiment on Pinot Gris clone R6, a somewhat compact clone. Interestingly, GAac applied on 1 cm inflorescence (around early E-L 13) induced an increase of flower number and branching  Using the RNA sequences from the sequencing data, we could identify transcripts with SNP leading to modification of the protein integrity, such as the inclusion of a stop codon. (ii) Some genes showed a complete impairment of expression in at least one clone (no more than three detected reads), with possibly severe direct damage in their ability to be transcript due to variation in their regulator sequence. (iii) Other differentially expressed genes that showed identical expression between clones in both stages; their expression might not be influenced by environmental or physiological factors and the difference might be constitutive. We identified several genes fulfilling these criteria. However, functional analysis revealed that many of these three types of genes have an unclear or unknown function. It might mean that mechanisms important for compactness and related traits are yet to be studied and deciphered but we gathered some evidences for some genes of potential functional roles that can be discussed.Among the 52 genes bearing variation between clones, four showed polymorphisms predicted to cause a high impact in the protein sequence. Vitvi14g00503 has clearly two polymorphic SNPs in VP2, one leading to a non-functional allele with a stop gain. This gene codes for a phosphoribosylaminoimidazole carboxylase but no evidence of a specific involvement in plant phenotype has been described in the literature.Vitvi17g00268 has two polymorphic SNPs in VP25 . It corresponds to a protein phosphatase 2C that presented high expression in almost all tissues in the grapevine atlas in cv. Corvina , specifiVitvi08g02244 is homologous to a high mobility group protein B1. One of the alleles in RJ51 has a splice-site donor variant leading to different 5′-UTR. Homologous genes have a described impact in the phenotype in other species. In Arabidopsis, mutant lacking HMGB1 had a slightly delayed and reduced germination rate, reduced root length, and enhanced sensitivity to methyl methane sulfonate (MMS) . HoweverWe used two strategies to highlight genes potentially presenting polymorphisms in their regulatory sequence. The first one was the identification of the genes only expressed or no expressed at all (less than three reads) in one loose clone in any stage. We hypothesized that promoters regions in those genes would be altered to reverse their expression in the affected clone.Few of these genes presented a clear function Table , none amMost of the genes with differential expression between clones but not presenting variation between the two stages had an undefined or unclear function Table . There wThe genes described in the previous section exhibited dramatic differences of expression or sequence polymorphisms. Many other genes that were differentially expressed at a lower magnitude give invaluable information on the behavior of networks of genes involved in molecular mechanisms differentially affected in the studied clones. More specifically, we identified major changes in some hormones biosynthesis and signaling, mechanisms related to differences in the cell wall structure and the biosynthesis of flavonoids.Two of the analyzed hormones, JA and SA, will not be further discussed, as their quantities were stable between clones and we did not found in the expression analysis any element that would involve them.1 in VP11, the clone with a lower number of seeds, and ac treatment in a grapevine cultivar induced seed abortion. As mentioned before, GAs are known to play a role in flowering initiation but there was no difference of expression in all the known genes involved in the flowering pathway. We also never observed any difference between clones in flowering timing, which allowed us to discard the flowering regulatory network as a responsible event for the differences observed in cluster compactness or fruitset between clones.Gibberellins promote flowering through the activation of genes encoding the floral integrators in long-day plants such as Arabidopsis . In grapac application are different to those promoted by other stimuli, such as shading (C-starvation)  of the enzyme catalyzing the last step of the active GA biosynthesis pathway, the GA 3-beta dioxygenase, were more abundant in the compact clone RJ51 than in the two loose clones at E-L 18–19, levels were always higher in VP2 too but difference is only significant with VP11 for Vitvi09g00452. ac.Although the molecular mechanisms specific to grape responses to GAs are not fully known, some advances are in progress, and it has been recently shown that grape flower abscission mechanisms triggered by GArvation) . In addi1 is more abundant in VP11 clone, while GA4 could only be detected in VP2. This might be explained by a higher turnover and rate of degradation. Three isoforms  of the enzyme degrading active GAs into inactive GAs, the GA 2-beta dioxygenase, were differentially expressed. ac treatment, but they were other isoforms, on chromosomes 7 and 10. Vitvi06g00659 expression fitted the profiles of GA1 in clones and stages. At E-L 18–19, it was more abundant in loose clones (only significant between VP11 and RJ51) while a drop of expression in VP11 only was observed at E-L 26. At E-L 18–19, Vitvi05g00163 was also more abundant in loose clone VP11 but less abundant in VP2 than RJ51. Vitvi19g02230 is specifically down regulated in VP11 in E-L 18–19. These two genes did not show differences between clones at E-L 26.Hormones analysis at E-L 18–19 showed that, of the two active GAs, GA1. Such a process has been hypothesized before in maize and Arabidopsis GA2ox4, was able to catalyze in vitro all 13-hydroxylated and non-13-hydroxylated GA substrates . Vitvi19g02230 (GA2ox3) was able to catalyze active GA1 and GA4 (but not GA9 and GA20), as all the GA2ox studied.The substrate specificity of some of the enzymes has been studied in grapevine by ac significantly increased wall extensibility in the wheat non-mutant controls but had no effect on the near-isogenic GA-insensitive genotypes  indicates a lower activity in the cell wall formation in VP11. It is possibly related to disruptions in cellular multiplication in VP11, which is also confirmed by the higher expression of transcripts related to cytoskeleton regulation in compact clones. However, unlike at E-L 26 in Garnacha , the genArabidopsis revealed that both cell wall metabolism and cytoskeleton were strikingly over-represented in pollen in preparation for the progamic phase, the pollen tube growth through the pistil . This regulation also plays an important role in the pollen tube growth but little is known of its variation during earlier events in flower. A transcriptome analysis in e pistil . As for a patens . Two isoac treatment in grapevine flowers led to significantly higher polyphenol and anthocyanin content in wine , might be favored as a side effect by an overproduction of phenylpropanoids and/or anthocyanin, likely since flowers stages. Studies done on the polyphenols contents in musts produced from these clones did not show the same tendency, and VP11 showed the lowest values of anthocyanins and total polyphenols [(At least one isoform of every gene in the phenylpropanoids metabolism from the phenylalanine to the anthocyanin was found differentially expressed and more abundant at E-L 18–19 in VP11 than in RJ51, and many of them also vs. VP2 and even vs. VP25 Figure . Interes in wine . The mucd organs . GAs wer hybrida  while in hybrida . Cheng ephenols [ and pers1 and reduced expression of genes involved in cell wall metabolism. As in VP25, a number of genes differentially expressed could also play a role in the phenotypical differences and are worthy to be further investigated.We described here for the first time in grapevine intra-cultivar differences at three levels: phenotypical, hormonal, and transcriptional, among four clones, two producing compact clusters and two producing loose clusters. Evolution of hormonal levels during inflorescence development have been shown in Tempranillo, with clear differences between hormones and few differences between the clones. Considering all the analyses, loose clone VP25 presented few differences with the compact clones, giving no clues about general mechanisms or gene networks involved in its loose phenotype. Although several differentially expressed or polymorphic genes versus compact clones might be involved in looseness, even though their role is currently unknown. On the contrary, clone VP11 showed large differences with the compact clones in several aspects, mainly phenotypical and in gene expression. Stage E-L 18–19, corresponding to the start of flowering, was the most informative stage, and gave some indications based on the coincidence of higher levels of active GAJI and JG designed the study and drafted the manuscript. JG, SI, JT, EB, and JI performed sampling and phenotyping. JI performed phenotypic analysis. JG performed the hormonal and gene expression analysis and interpretation. All authors read and approved the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.