CorA structures in closed (Mg2+-bound), and open (Mg2+-free) states, together with functional data showed that Mg2+-influx inhibits further Mg2+-uptake completing a regulatory feedback loop. While the closed state structure is a symmetric pentamer, the open state displayed unexpected asymmetric architectures. Using high-speed atomic force microscopy (HS-AFM), we explored the Mg2+-dependent gating transition of single CorA channels: HS-AFM movies during Mg2+-depletion experiments revealed the channel’s transition from a stable Mg2+-bound state over a highly mobile and dynamic state with fluctuating subunits to asymmetric structures with varying degree of protrusion heights from the membrane. Our data shows that at Mg2+-concentration below Kd, CorA adopts a dynamic (putatively open) state of multiple conformations that imply structural rearrangements through hinge-bending in TM1. We discuss how these structural dynamics define the functional behavior of this ligand-dependent channel.CorA, a divalent-selective channel in the metal ion transport superfamily, is the major Mg Yet, the first CorA crystal structure in the absence of divalent cations showed little or no changes compared to the fully Mg2+-bound closed channel structure, with only a slight kink between TMDs and intracellular domains , the transition from the closed (Mg2+-bound) state to the conductive unliganded conformation is still unclear. At 7.1 Å, the putative open CorA asymmetric cryo-EM structures were solved only from a relatively low number of particles   , and due2+-depletion experiments. HS-AFM is unique in its ability to concomitantly characterize molecular structures and dynamics under native-like conditions. In agreement with electrophysiological, spectroscopic and biophysical data  and the asymmetric (Mg2+-depleted) states interspersed by a wide plateau of conformational fluctuations of a highly flexible molecule, which likely represents the conductive state.In order to elucidate the sequence of events underlying gating-related CorA conformational changes, we used high-speed atomic force microscopy (HS-AFM)  to captucal data , we foun2+-concentration changes in real-time. For this, wild-type (wt) CorA was reconstituted at low lipid-to-protein ratios (LPRs between 0.2 and 0.4) in the presence of Mg2+. The resulting proteo-liposomes were adsorbed onto freshly cleaved mica under saturating Mg2+ (10 mM) condition and imaged at an acquisition rate of 1–2 frames s−1 with a resolution of 0.5 nm pixel−1. CorA-containing vesicles were generated from protein reconstitutions in POPC/POPG . When imaged by HS-AFM, vesicles spread on the mica support resulting in large membrane patches with densely packed CorA , in which all channels are expected to be in the closed conformation. Subsequently, membranes were imaged at reduced Mg2+-concentration near the reported apparent Kd for Mg2+ (~2 mM), and finally at 0 mM Mg2+ to focus on the dynamically open Apo form.We then monitored the structural changes of the channel upon Mg2+-depletion was achieved by live injection of EDTA, while continuously monitoring CorA. Using the closed state as reference, no structural changes of the periplasmic face of CorA were observed at 10 mM Mg2+, at 2 mM Mg2+ (after ~10 min) and in absence of Mg2+ (after ~12 min) (−1) any putative changes in the conformation of the periplasmic face of the channel are beyond the current resolution limit of HS-AFM . This is consistent with the ~7 Å cryo-EM structures, which show little or no Mg2+-dependent changes on the periplasmic face (Mg~12 min) . At the mic face .2+  . Analysis of the distribution of symmetric and dynamically asymmetric CorA particles pointed to a Kd of ~2 mM Mg2+, which is in good agreement with the reported affinity , and 0 mM Mg2+ (putative).In stark contrast, previous EPR  and cryon state) . First, affinity  in our eaffinity , with whaffinity . Next, w2+-liganded cryo-EM structure  CorA structures, where individual subunits move towards the former 5-fold axis and thus stand taller  gradually increased (i.e. switching back and forth between the high (open) state(s) and the state of lower height) cumulated earlier during the titration experiment  the dynamics of the channels reach a maximum (2+-currents in Xenopus leavis oocytes decay within 15–20 min (2+ (2+ the average time spent in the closed (low height) state is almost twice (~60 s) the averaged dwell-time in one of the putatively open (elevated height) conformations. After ~10 min in Mg2+-free condition, the situation is reversed, and more molecules show long dwell-times in the elevated state  conformation(s) are favored when Mg2+ is depleted from all binding sites. In between, the channel is in a highly dynamic regime, probably reflecting Mg2+-unbinding and -rebinding events . We also acquired movies at 250 ms frame acquisition and found that individual sequential frames displayed different apparently unrelated conformations, an indication that the conformational fluctuations are even faster  class, representative of a highly mobile and flexible molecule that switches fast between numerous sub-structures that could not be classified to any of the conformations described by cryo-EM. After full depletion, re-addition of Mg2+ could partially recover ~35% the symmetric closed-state structure , the channel adopts a stable 5-fold symmetric state reminiscent of the high-resolution structures (phase 1). After several minutes of exposure to a Mg2+-free solution, CorA transitions into a set of asymmetric architectures, which are characterized by elevated cytoplasmic domains (phase 3). These structures are adopted through major conformational changes implicating hinge-bending of TM1 and reorientation of the cytoplasmic domains. This picture is wholly consistent with data derived from functional (electrophysiology), biochemical (crosslinking), biophysical  and structural (cryo-EM) approaches. However, these results contrast with recent crystallographic data of CorA Mg2+ binding site mutants that reported limited or no structural changes compared to the closed wt channel, thereby challenging the hypothesis that Mg2+-depletion leads to large conformational changes and channel opening . Active subunits change and the entire molecule displays high structural variability, likely adopting many more conformations than the three states so far identified by cryo-EM. Indeed, while we can assign the fully 5-fold symmetric state with certainty, our assignment of molecules to state open-I and open-II must be considered as putative. Perhaps more importantly, at intermediate Mg2+-concentrations ~ 50% of the molecules could not be assigned and were pooled in a class of undefined quaternary structures (open-+). In this context, the details of the cryo-EM study become relevant: open-I and open-II only pooled 26,271 and 27,416 particles, respectively, of the total 173,653 particles, and thus a significant portion of the particles remained outside of these classes  is located right at the periplasmic end of the channel, whereas amino acids towards the cytoplasmic face of TM1 are less conserved  first transmembrane helix (TM1), ranging all the way from the periplasmic face to the surface of the cytoplasmic Mgc domain . Thus, ic domain , allowedonserved .2+, CorA molecules adopt a rather stabilized asymmetric state resembling the putatively open state cryo-EM structures. However, a living cell under physiological conditions is unlikely to ever reach Mg2+-concentrations below ~1 mM. Thus we hypothesize that in cellula the physiologically relevant open state is a collection of fluctuating conformational states. Thus, the conformational energy landscape of CorA gating would consist of two deep energy minima representing the stable closed (symmetric) and putatively open (asymmetric) conformations connected by a wide plateau in which a variety of open conformations are adopted through permanent molecular fluctuations. The compelling utility of HS-AFM for the study of macromolecular dynamics at high spatio-temporal resolution and in native-like conditions is demonstrated, elucidating a process so far inaccessible to other structural or biophysical techniques. The present data set and our proposed mechanistic interpretation of the conformational dynamics of Mg2+-dependent CorA gating sets the stage for an unprecedented understanding of CorA as a ‘reverse’ polarity ligand gated ion channel with an unique gating mechanism.The idea that there are multiple conductive conformations of CorA, or, as a paradigm shift, the conductive conformation of CorA is a fluctuating molecule, is favored by both, the subunit movement analysis and state transition analysis. In the long-term absence of MgT. maritima was expressed and purified as previously described . The concentrated protein sample  was homogenized by gel filtration  and equilibrated in 50 mM HEPES, pH 7.3, 200 mM NaCl, 20 mM MgCl2, and 1 mM DDM).CorA from escribed . BrieflyFor CorA reconstitution into liposomes, the protein was gently mixed with freshly prepared solubilized POPC-POPG (3:1) or DOPC-DOPE-DOPS (4:5:1) lipids (Avanti Polar Lipids) at low lipid to protein ratios (LPR) between 0.2–0.4 (w:w) at a total protein concentration of 1 mg/mL. After 4 hr equilibration, detergent was removed by addition of biobeads overnight.2+.A 1.5 mm diameter muscovite mica sheet was glued on a HS-AFM glass rod sample support and mounted on a HS-AFM scanner. Reconstituted CorA membranes were adsorbed on freshly cleaved mica for ~5 min. Subsequently, the sample was rinsed with imaging buffer  containing 10 mM Mg−1 and at a resolution of 0.5 nm pixel−1. The energy input by the AFM tip  was mini2+-saturated to low/no Mg2+-conditions was achieved by depleting Mg2+ by either adding EDTA into the measuring solution or alternatively by buffer solution exchange to Mg2+-free buffer using an integrated constant-pressure and constant-flow pump system  state or the putatively open, activated (asymmetric and elevated) state. Such, the height/time traces did also not discriminate which of the open states was assumed. Notably however, our experiments showed that structures exposing increased protrusion heights are likely representatives of Mg2+-depleted molecules and thus detection of elevated height is a valuable fingerprint for the state transition. For conformational transition analysis membrane patches of ~20 CorA channels were imaged at Mg2+ concentrations of 10 mM, 3 mM, 0 mM, and after re-addition of 25 mM. Extracted CorA molecules from high-resolution movie sections  were manually assigned to different structural states of (C) symmetric flower-shaped, (O-1) asymmetric elevated, (O-2) dome-shaped, elevated and (O-+) all others. The significance of the occurrence of each conformation under the four tested Mg2+ concentrations was tested using a two-tailed students test.HS-AFM images were first-order flattened and contrast adjusted using laboratory-made routines in Igor Pro software (WaveMetrics). Videos were then drift corrected with respect to the membrane patch, or aligned on individual CorA molecules using an in-house developed analysis software plug-in for ImageJ . Dimensi In the interests of transparency, eLife publishes the most substantive revision requests and the accompanying author responses.Acceptance summary:2+2+ ion channel found in all eukaryotes. CorA function is negatively regulated by Mg2+. These channels are open when the binding site is unoccupied but they close in the presence of high Mg2+. Previous structural studies primarily using EPR and cryo-EM suggest that the channel undergoes a closed to open conformational switch in absence of Mg2+, which results in loss of five symmetry. Cryo-EM studies show that the open channel exists in at least two conformations which could either represent snapshots of a fluctuating channel or intermediates in the gating pathway. Recent crystallographic studies, however, suggest that the unbound channels may not undergo as much of a conformational change as suggested by the cryo-EM and EPR studies.Mg2+ is removed from the channel. HS-AFM technique allows them to track conformational changes in a single CorA channel over time and correlate these changes with putative gating transitions. Functional experiments show that the gating process of the channel is relatively slow – currents decay over 15-20 minutes in electrophysiology experiments. Thus, the conformational changes are expected to be within the bandwidth of these measurements (500 ms per frame). Mg2+ depletion experiments show that the stable starting structure with five-fold symmetry becomes dynamic with time and ultimately settles to one or more asymmetric structures. One of the most interesting aspects of this study is that unlike most ion channels, the open channel conformation is both asymmetric and dynamic. Overall this is a technically work which advances the field both in terms of providing new information about the gating mechanism of CorA and demonstrating the power of HS-AFM to monitor single molecule structural transitions.In this study, the authors have used high-speed AFM to monitor the time-dependent conformational changes as MgDecision letter after peer review:eLife. Your article has been reviewed by three peer reviewers, including Baron Chanda as the Reviewing Editor and Reviewer #1, and the evaluation has been overseen by a Reviewing Editor and Richard Aldrich as the Senior Editor.Thank you for submitting your article "Real time dynamics of gating-related conformational changes in CorA" for consideration by The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.Essential revisions:2+ bound state is the baseline, lowest-height state, why doesn't the "down" state have a Δ-Height of zero? The choice of 2 states affects the dwell time analysis , and without a better rationale, I don't see why this choice is valid. There are other possible reasons for poor fit. For instance, some of the conformational transitions in CorA is much faster than the sampling rate and this could result in aliasing. This is difficult to rule out but should be discussed.1) All of the reviewers are concerned about the two-state model assumption for data analysis. The authors themselves state that there are two open states and one closed state. The model fit shown in Figure 3A is clearly inadequate. The open conformation is quite heterogenous, correct? The kymograph shown really looks like 3 states to me . And if the MgAlso the height histogram on the right side shows a very disperse distribution, only two not very distinct local maxima that were not taken as level thresholds (so why 2-state?). As this data depiction is crucial for all the analysis done in the rest of Figure 3 it needs to be clearly and convincingly communicated.STaSI is a model-independent idealization algorithm but it tends to overfit data. Have the authors tried any of the other HHM based algorithms? Given that they are assuming a two-state model, it would make more sense to use HMM.2+ and find it very interesting that this conformation is conducting. However, we all agree that the assignment of these highly dynamic structures to discrete states is restrictive and perhaps also subjective. It is not clear how the conformational states  taken from EM work were assigned to the conformations recorded with high speed AFM. Was it done manually? What were the objective criteria? How many structures were taken? Figure 5B only gives percentages. Again, this is crucial for all the data analysis done in Figure 5. As the authors have stated, the classification of discrete state in this highly flexible molecule is a challenge. In Figure 5A, it is not clear whether the images shown below are idealized (expected) images or averaged images for each of the states. It should be stated clearly in the legend. The actual averaged image should be shown as an inset. In addition, in some instances, the state assignments are based on changes in the shape rather than height. If both heights and changes in shape are taken into consideration, it appears that the number of possible states will be much more and trying to bin these highly dynamic structures into a few discrete states becomes less meaningful.2) The reviewers are convinced that the CorA molecule becomes highly dynamic in the absence of Mg2+ concentration were achieved for each experiment, and why the experiments were designed the way that they were. It seems like two different methods were used to change Mg2+Mg2+ concentrations: either pipetting in EDTA, or slow perfusion with an EDTA-containing solution over 10's of minutes . .3) My other main critique is that it is very difficult to understand how changes in Mg2+, so that any slow kinetic component is due to channel kinetics. Whereas with perfusion, changes in Mg2+ concentration and the channel's conformational journey from "phase 1" to "phase 3" both occur on over a minutes-long time scale. Because of the slow kinetic component to channel dynamics, it's really important for the logic of the paper to be clear about which method of Mg2+ depletion was used in the data in Figures 2 through 5.I assume that adding EDTA nearly instantaneously removes bulk Mg2+ to low Mg2+ over about 14 minutes with another 12 minutes in Mg2+ free conditions ? In Figure 3, the sharp delineation of the green, blue, and red boxes seem to indicate Mg2+ concentration steps, and the legend refers to images collected at saturating, 2, and 0 mM Mg2+. This is confusing. The text goes back and forth between describing imaging at 2 mM . According to my reading, Figure 5 then uses 3 discrete Mg2+ concentrations, but the use of the color-fade arrows seems to indicate a ramp.Do I understand correctly that many  of the experiments in Figure 3 involved a ramp from high Mg2+ concentrations provided. Given the amount of functional data already available for this channel, including a good idea of the Kd for Mg2+, I might have gone straight to imaging at discrete Mg2+ concentrations. Could the rationale be explained?I'm not clear what advantage ramping the Mg4) Abstract: "finally equilibrates to an asymmetric structure." Is the final state a single asymmetric structure or it is at least two conformations. Based on the state identification matrix in Figure 5, it would seem that at 0 Mg, there are more than one asymmetric structures, unlike the Mg bound state.5) Abstract: – "putative open state adopts multiple conformations through hinge-bending motions". This sentence contradicts the previous sentence and it is not clear whether the data provided in this study show hinge-bending motion. The conformational changes are compatible with hinge-bending but there is no direct evidence here. Essential revisions:2+ bound state is the baseline, lowest-height state, why doesn't the "down" state have a Δ-Height of zero? The choice of 2 states affects the dwell time analysis , and without a better rationale, I don't see why this choice is valid.1) All of the reviewers are concerned about the two-state model assumption for data analysis. The authors themselves state that there are two open states and one closed state. The model fit shown in Figure 3A is clearly inadequate. The open conformation is quite heterogenous, correct? The kymograph shown really looks like 3 states to me . And if the MgCorA actually adopts many more than 2 structural states . As such, the analysis as presented in 3A is not based on explicit structural states nor determines dwell times directly associable with specific structural states. Let us first go over the analysis carried out in Figure 3 in some detail: Each molecule is analyzed as described in 3A and then the behaviors of all molecules are pooled in 3B and 3C.Originally, we made a significant effort to track the movement of the individual subunits and make state assignments by transforming the CorA ring profiles into height cross-sections. Theses profiles were associated to the states described by cryo-EM. However, given the large variability within the molecules , combined to the temporal resolution limits of the experiment (and the bias from the polar transformation), it turned out to be impossible to determine states and/or individual subunit movements in any significant way.2+-depletion experiments. In light of the nature of the structural changes between the closed (Mg2+-bound) and many open (Mg2+-free) conformations, we are confident that height is a reliable measure to track the main closed-open transition. In other words, unable to define several states that could be integrated into a gating model as the reviewer suggests, we use a 2-state model with no other ambition than assigning OFF vs ON, where all of the poorly defined “open” state conformations are “pooled” as the ON state. This is clearly not ideal, but we argue that given the fact that the open state is unlikely to be defined in the classical, “single conformation” sense (as is one of the main conclusions of this work), this simple model is just intended to assess the kinetics of entry and exit of the closed state i.e. the symmetric state vs all other states.Thus, as a subterfuge to define and assign a given structural state to each molecule at any instant, we simply tracked the height increase and fluctuations of CorA during MgHow was this assessed: To determine CorA’s height changes we only used the center area of each molecule (5x5nm) and calculated the relative ΔHeight . The reason that absolute height was not reported is mostly due to the larger variability in absolute height values under a wide number of experimental conditions. The region of interest of this measurement was defined in the center area of each molecule. In cases of limited resolution, the pore might simply not be observed in an otherwise low CorA molecule (ΔHeight <1nm). Hence, when considering straightforward changes in height, CorA formally switches between only 2 states, yet due to resolution limitations, the ΔHeight of the lower state is somewhat widened.2+-depletion. We thank the reviewers for pointing to these uncertainties and have changed the text accordingly as highlighted in the manuscript.The major conformational difference between the closed and the multiple open states, leads to an increased height of the center or the rim by ~2nm ΔHeight in the open state(s) (e.g. one subunit ‘stands up’ in one of the bean-shaped asymmetric conformations). When all subunits are low but there is a central pore  the observed height difference between pore and rim is ~1nm, and poorly resolved closed state molecules have a ΔHeight <1nm. In brief, failing to make significant structural state assignments, we used the best AFM criterion (i.e. height) to assign the closed state versus all other (putatively activated) states as a function of the progression of MgThere are other possible reasons for poor fit. For instance, some of the conformational transitions in CorA is much faster than the sampling rate and this could result in aliasing. This is difficult to rule out but should be discussed.Also the height histogram on the right side shows a very disperse distribution, only two not very distinct local maxima that were not taken as level thresholds (so why 2-state?). As this data depiction is crucial for all the analysis done in the rest of Figure 3 it needs to be clearly and convincingly communicated.-1. In 2+-bound state and Mg2+-free states) at the current speed of image acquisition. In consequence, the only reasonable measure is whether the molecule is in its Mg2+-bound symmetric state (that can be well resolved) or an overlap of Mg2+-depleted dynamic molecules. We discuss the aspect of scan speed, subunit dynamics and the ‘merging of all activated states into one state as defined by height’ in our revised manuscript version. As detailed above and in the response to question 1a), the HS-AFM analysis essentially reduces the data to a 2-state model (inactivated or activated).The reviewer is correct. The fluctuations of the CorA subunits are indeed faster than our sampling rate of about 2 frames sSTaSI is a model-independent idealization algorithm but it tends to overfit data. Have the authors tried any of the other HHM based algorithms? Given that they are assuming a two-state model, it would make more sense to use HMM.In this point we disagree with the reviewer. We performed a thorough analysis how well STaSI performs, down to low signal to noise ratios (SNR=0.5), and found that STaSI makes the correct conservative 2-state assignment with ~95% accuracy. We compared STaSI with vbFRET (another Bayesian based approach for state assignment and state transition fitting) and StaSI performed better in our hands. We like that STaSI uses a minimum description length (MDL) and proposes the number of states in a fully unbiased way, resulting in idealized traces. How many structural states are hidden behind the assigned height-states is of course another question that would need time analysis of these height-states with large sample statistics. This is well beyond the scope of the present work.2+ and find it very interesting that this conformation is conducting. However, we all agree that the assignment of these highly dynamic structures to discrete states is restrictive and perhaps also subjective. It is not clear how the conformational states  taken from EM work were assigned to the conformations recorded with high speed AFM. Was it done manually? What were the objective criteria?2) The reviewers are convinced that the CorA molecule becomes highly dynamic in the absence of MgThis is one of the key points we are trying to make. Here, we elaborate in more detail:2+-free) condition, CorA channels adopt a large variation of conformations and any attempt to automatically assign particular structures, was unsuccessful by lack of resolution, bandwidth and/or the fact that neighboring molecules touched the molecules under scrutiny .1) We agree with the reviewer that there is difficulty to assign snapshots of the highly dynamic molecule to the states observed by cryo-EM. In its dynamic (Mg2) We also argue that the cryo-EM structures (Open-I and Open-II) must represent conformations that sit in some sort of energy well  otherwise, cryo-EM would not have been able to average any kind of structure (even at low resolution). However, the fact that cryo-EM was able to discriminate these two conformations by no means implies that there might not be many more. In fact, we are convinced this is the case and one important reason why it is so hard to make structural assignments under a regime of relatively low bandwidth.3) Given 2), it is not unreasonable to argue that our data are somewhat equivalent to the cryo-EM open state low resolution structures.4) How have we assigned molecules to individual classes? Manually. We used the following criteria (underlined) that correspond to features observable in the cryo-EM structures: “The channel adopts several structures that we classified into 4 categories: First, the fully-liganded channel corresponding to the 5-fold symmetric closed conformation. Second, a structure of reduced diameter with three asymmetrically distributed protrusions of increased height, assigned to the cryo-EM open-I state. Third, a round- or star-shaped conformation with an elevated center. This HS-AFM topography can be easily identified as it only displays small structural variations and resembles the cryo-EM open-II state.”th class.5) However, we came to the same conclusion as the reviewers that an explicit assignment to the cryo-EM open states was too restrictive. Hence, we introduced another class of “not assigned” molecules: “Finally, given that our tentative assignment to open-I and open-II states only covers a fraction of all molecular observations, a fourth category must necessarily include the remaining range of structural variations, which we named Open-+ states…”. Indeed, we see that the majority of the molecules are in this 42+-bound to a more-or-less stabilized Mg2+-free state over a large range of dynamic conformations. Thus, we do not think that we were restrictive trying to ‘push’ our findings towards assignment with the cryo-EM structures.6) Finally, a key conclusion – see Figure 5C – is that the channel goes from a stabilized MgHow many structures were taken? Figure 5B only gives percentages. Again, this is crucial for all the data analysis done in Figure 5. As the authors have stated, the classification of discrete state in this highly flexible molecule is a challenge.2+-condition between ~1,000-2,000 molecular representations were considered for classification into the 4 subgroups and then normalized to the total number of frames. In particular 943 HS-AFM images of mostly stable closed CorAs were analyzed in saturating 10mM Mg2+-condition, 1,122 frames at 3mM Mg2+, 2,394 CorA snapshots for 0mM Mg2+, and 1,104 representations were classified for the recovery experiment after re-addition of 25mM Mg2+. These numbers are now mentioned in the Materials and methods section and the corresponding figure legend.For each MgIn Figure 5A, it is not clear whether the images shown below are idealized (expected) images or averaged images for each of the states. It should be stated clearly in the legend. The actual averaged image should be shown as an inset.The CorA molecules shown in Figure 5A bottom and Figure 5B left and top are sketches depicting the main structural classes of CorA. To avoid confusion, we now clarify this it in the figure legend.In addition, in some instances, the state assignments are based on changes in the shape rather than height. If both heights and changes in shape are taken into consideration, it appears that the number of possible states will be much more and trying to bin these highly dynamic structures into a few discrete states becomes less meaningful.We agree with the reviewers and believe that there are many more structures in the dynamic open state of CorA, considering the observed structural variations in the presented HS-AFM videos (see our responses to comments above).2+-concentrations ~50% of the molecules could not be assigned and were pooled in a class of undefined quaternary structures (open +).”, and, “We expect that given a larger number of particles to process (in cryo-EM) or improvements in the spatial and/or temporal resolution of HS-AFM, additional states are likely to emerge.”This is why we state: “Active subunits change and the entire molecule displays high structural variability, likely adopting many more conformations than the 3 states so far identified by cryo-EM. Indeed, while we can assign the fully 5-fold symmetric state with certainty, our assignment of molecules to state open-I and open-II must be considered as putative. Perhaps more importantly, at intermediate Mg2+- and time- depended sequence of conformational events for CorA: First, a transition into highly mobile, elongated structures occurs, followed by a transition into features resembling to those of the Mg2+-free cryo-EM structures, these molecules have a clearly increased height. As detailed above, the height changes  are used to automatically detect activation of the molecules only, while the specific shapes are assigned later (see response to 2a) to describe the transition in more detail. We have added additional text in the Discussion section to point out these limitations.Due to the current limitations in high-resolution single particle HS-AFM data in this study  more work is required to further optimize sample preparation, which will allow more observations, and HS-AFM technology to distinguish between possible different sub-structures in the open state. However, our experiments observed a clear Mg2+ concentration were achieved for each experiment, and why the experiments were designed the way that they were. It seems like two different methods were used to change Mg2+ concentrations: either pipetting in EDTA, or slow perfusion with a EDTA-containing solution over 10's of minutes . .3) My other main critique is that it is very difficult to understand how changes in Mg2+, so that any slow kinetic component is due to channel kinetics. Whereas with perfusion, changes in Mg2+ concentration and the channel's conformational journey from "phase 1" to "phase 3" both occur on over a minutes-long time scale. Because of the slow kinetic component to channel dynamics, it's really important for the logic of the paper to be clear about which method of Mg2+ depletion was used in the data in Figures 2 through 5.I assume that adding EDTA nearly instantaneously removes bulk MgYes, the reviewer understood the two concentration change protocols and we have fully and explicitly described them now in the main text.2+ to low Mg2+ over about 14 minutes with another 12 minutes in Mg2+ free conditions ? In Figure 3, the sharp delineation of the green, blue, and red boxes seem to indicate Mg2+ concentration steps, and the legend refers to images collected at saturating, 2, and 0 mM Mg2+. This is confusing. The text goes back and forth between describing imaging at 2 mM . According to my reading, Figure 5 then uses 3 discrete Mg++ concentrations, but the use of the color-fade arrows seems to indicate a ramp.Do I understand correctly that many  of the experiments in Figure 3 involved a ramp from high Mg2+-concentrations and the depletion methods used. We only used the fast and manual EDTA titration procedure to deplete Mg2+ in discrete steps. In all but 1 experiment  shown in Figure 3, Mg2+ was fully depleted in a single titration step (i.e. addition of ~10mM EDTA). For further analysis  only molecules monitored at 0mM Mg2+ were analyzed. For the in-depth analysis of the individual structural classes the distinct Mg2+ concentrations of 10mM, 3mM, 0mM and re-addition of Mg2+ to 25mM were achieved pipetting. We clarify the distinct Mg2+ concentrations in the text and all figure legends.We thank the reviewer for pointing out the confusion of analyzed Mg2+ concentrations provided. Given the amount of functional data already available for this channel, including a good idea of the Kd for Mg2+, I might have gone straight to imaging at discrete Mg2+ concentrations. Could the rationale be explained?I'm not clear what advantage ramping the Mg2+ in our particular experimental set-up (reconstituted and densely packed CorA channels adsorbed on a sample surface), independent of previously determined apparent affinities. Concerning the Kd, the ramp experiments merely corroborate the findings from functional data. Anyway, ramps help us make sure that the addition of EDTA does not physically disturb and influence the measurements. We agree with the reviewer that the transition analysis itself should be performed at distinct Mg2+ concentrations, particularly when considering the long transition times. Accordingly, all CorA transition experiments used for in-depth analysis and presented in the main manuscript  were recorded and analyzed at distinct Mg2+ concentrations. We clarify this by rephrasing the main text in the revised manuscript and as discussed above (point 3).We performed the ramping experiments using a high-precision buffer exchange system first, to test for the affinity of Mg4) Abstract: "finally equilibrates to an asymmetric structure." Is the final state a single asymmetric structure or it is at least two conformations. Based on the state identification matrix in Figure 5, it would seem that at 0 Mg, there are more than one asymmetric structures, unlike the Mg bound state.2+-sensor domain after spending ~10 minutes in Mg2+-free conditions. Due to the flexibility and the fast dynamics of the large and elevated substructures of CorA we are resolution limited, and thus can only make tentative assignments to the two found cryo-EM structures. Our high resolution CorA analysis at the onset of complete Mg2+-depletion  is highly suggestive of more than two open states of elevated heights. To this end, we changed the Abstract to “… that equilibrates to asymmetric structures of increased membrane protrusion height level.”We realize that the expression “equilibrates to an asymmetric structure” might be misleading. Our HS-AFM experiments revealed multiple, dynamically fluctuating asymmetric structures, that show an elevated Mg5) Abstract: "putative open state adopts multiple conformations through hinge-bending motions". This sentence contradicts the previous sentence and it is not clear whether the data provided in this study show hinge-bending motion. The conformational changes are compatible with hinge-bending but there is no direct evidence here.2+ sensor are likely translated to the channel pore via hinge bending in TM1.Good catch. As mentioned in point 4, we corrected the misleading phrasing of a single asymmetric state (point 4 above) and have also clarified in the revised version of the manuscript that the observed CorA subunit movements in the putative open state are consistent with the hinge-bending movement detected by cryo-EM. As discussed in the text and Figure 5—figure supplement 1, TM1 is the only connection between the TM region and the cytoplasmic domain. Logically, the large rearrangements in the Mg


Many studies, including the discovery of the transcription factors autoimmune regulator (AIRE) and fasciculation and elongation protein zeta family zinc finger (FEZF2), have shown that a defect in thymus central self‐tolerance is the earliest event promoting autoimmunity. AIRE and FEZF2 control the level of transcription of many neuroendocrine self‐peptides and TRAs in the thymic epithelium. Furthermore, AIRE and FEZF2 mutations are associated with the development of autoimmunity in peripheral organs. The discovery of the intrathymic presentation of self‐peptides has revolutionized our knowledge of immunology and is opening novel avenues for prevention/treatment of autoimmunity.Confirming Burnet's early hypothesis, elimination of self‐reactive T cells in the thymus was demonstrated in the late 1980s, and an important question immediately arose about the nature of the self‐peptides expressed in the thymus. Many genes encoding neuroendocrine‐related and tissue‐restricted antigens (TRAs) are transcribed in thymic epithelial cells (TECs). They are then processed for presentation by proteins of the major histocompatibility complex (MHC) expressed by TECs and thymic dendritic cells. MHC presentation of self‐peptides in the thymus programs self‐tolerance by two complementary mechanisms: (1) negative selection of self‐reactive “forbidden” T cell clones starting already in fetal life, and (2) generation of self‐specific thymic regulatory T lymphocytes (tT reg cells. There is no doubt that thymus‐based novel strategies could alleviate in the future the weight of so many known and still unknown autoimmune diseases that remain the heavy tribute, mainly paid by mankind, for the performance and extreme diversity of its highly complex adaptive immunity.Presentation of self‐peptides in the thymus is the basic mechanism that underlies T cell differentiation, negative selection of self‐reactive T cell clones, and generation of tT Thymus cunila (savory or “sarriette” in French). Galen thought that the thymus had no other function than providing protection between the sternum and superior vena cava. Galen also observed that the size of the thymus was larger in young animals and decreased with aging.The word “thymus” first appeared in the manuscripts of Claudius Galen (129−around 216 A.D.) and was described as an excrescence having some morphological analogy with the leaf of the plant Over many centuries, the anatomy of the thymus was the essential focus of the studies on this organ until the Scottish embryologist John Beard 1858−1924), a pioneer of today's theory of cancer stem cells, wrote that he considered the thymus to be the source of some white blood cells in the body., a pioneIn 1890, the German anatomist Wilhelm Waldeyer 1836−1921) noticed that in elderly people islets of normal thymic tissue could be observed even after adipose involution of this organ. Jan‐August Hammar  also showed that, although thymic development is maximal at puberty, normal thymic tissue persists until advanced age. He observed that animal castration before puberty prevents thymic involution with age and that thymic hypoplasia is associated with pregnancy, undernourishment, and some infectious diseases. He showed that, conversely, thymic hyperplasia is associated with autoimmune Graves’ thyroid disease, Addison's adrenal insufficiency, myasthenia, and acromegaly.−1921 notThe first immunological function of the thymus was discovered when French‐born Australian immunologist Jacques F.A.P. Miller showed that thymectomy performed in mice immediately after birth rendered them highly susceptible to infections and provoked their premature death. He also observed a marked lymphopenia in blood, spleen, and lymph nodes of these mice. These animals were also unable to reject a foreign skin graft, an essential hallmark of the immune response at that time. Miller concluded that the thymus was the organ responsible for the development of immunocompetent cells that constitute a specific cell population, thymus‐dependent (T) lymphocytes.In the perspective of hematopoietic growth factors identified at that time, Miller advanced the hypothesis of one or several soluble thymic factors that would be responsible for driving T cell differentiation.tolerance to characterize one of the cardinal properties of the adaptive immune system with diversity and memory. In marked contrast with Medawar's statement, during a conference at the University of London in 1962, Burnet prophesized: “If, as I think, the thymus is the site where occurs proliferation of lymphocytes in clones with precise immunological functions, we have also to consider another function: elimination or inhibition of clones with reactivity to self.”Since the foundation of immunology at the end of the 19th century, prediction of autotoxicity/autoimmunity has been intimately associated with the discovery of antitoxins/antibodies. As soon as in 1900, Paul Ehrlich  proposed the aphorism “horror autotoxicus” to claim the impossibility that one organism could be attacked under normal conditions by its own cells in charge for its defenses. Ehrlich thought then that either structures or mechanisms should exist to avoid autotoxicity, and this should be of the highest importance for individual health and species survival.30 cumulated TCR and BCR combinations, the majority of which are able to recognize self‐antigens. In normal conditions, however, the adaptive immune system does not aggress self and, for a long time, immunology was defined as the science of self−nonself discrimination. Burnet was actually speaking about immunology as the science of self and nonself recognition. Indeed, without “training,” lymphocytes are not able to discriminate between self‐ and nonself‐peptides. In 1987 and 1988, the research groups of Douarin,before the acquisition of T cell immunocompetence. Therefore, the thymus is not only responsible for the generation of diversity of the TCR repertoire, but is also primarily a cemetery for early T cells expressing a TCR specific for self‐antigens that are presented to differentiating T cells by proteins of the major histocompatibility complex (MHC) expressed by TECs and thymic dendritic cells (DCs).Afterward, the molecular mechanisms responsible for the stochastic recombination of gene segments encoding the variable domains of the immunoglobulin B cell receptor for the antigen (BCR)reg cells that are able to inhibit in the peripheral self‐reactive T cells having escaped negative selection in the thymus. The Foxp3 transcription factor, through its expression in Treg cells, is essential for the prevention of autoimmunity as shown in the human immune dysregulation, polyendocrinopathy, enteropathy, X‐linked (IPEX) syndrome, as well as in scurfy and −/−Foxp3 mice.reg cells also depends on the presentation of self‐antigens by thymic MHC proteins. How the same mechanism of MHC‐mediated self‐peptide presentation promotes CD4+ fates that are so distinct during thymic T cell differentiation (negative selection of self‐reactive T cells and generation of self‐specific tTreg cells) is still a matter of active exploration.In 1972, Richard Gershon (1932−1983) from Yale identified immunosuppressive cells regulating competent lymphocytes.(OXT) transcription coincides with its expression in the hypothalamus.Following these seminal studies about the powerful tolerogenic mechanisms occurring inside the thymus, the real nature of self‐peptides that are presented by thymic MHC proteins was then scrutinized. Previously, it was largely assumed that proteins circulating in the blood were captured somewhat “passively” in the thymus  and then presented to differentiating T lymphocytes during their transitory residence in this organ. In 1978, immunoreactive neurotensin (NT) and somatostatin were identified in the chicken thymus epithelium,Most importantly, in the thymus, the processing of OT precursor does not lead to classic neurosecretion as established a long time ago for the hypothalamo−neurohypophysial axis. Thymic OT is not located in classic secretory granules, but is diffuse within TEC/TNC cytosol, in large clear vacuoles, and around perimembranous space.The hypothesis that a thymic neuropeptide actually behaves as a self‐peptide was further investigated with NT, a 13‐amino acid linear neuropeptide. Intrathymic MHC‐I presentation of NT was demonstrated according to the biochemical rules of MHC‐I presentation as established by Hans‐Georg Rammensee's laboratory in Tübingen.In the early 2000s, Bruno Kyewski, Jens Derbinski, and Ludger Klein in Heidelberg further proposed that promiscuous gene expression of tissue‐restricted antigens (TRAs) in the thymus was a novel key to understand immune self‐tolerance and autoimmunity.IGF2 transcription in TECs,The epigenetic regulation of promiscuous gene expression in the thymus is currently under intense investigation.While the unique physiological function of the thymus in programming central self‐tolerance is now well established, more and more experimental observations point to a disturbance of thymus‐dependent tolerance as a key event in the early development of organ‐specific autoimmune diseases Fig. . In 1973reg cells. If this hypothesis was verified, it would be clear that a defect in thymic tolerogenic function might determine early the development of organ‐specific autoimmune diseases. Our laboratory investigated this question more particularly for the pathogenesis of autoimmune insulin‐dependent T1D. Concerning the members of the insulin gene family, IGF2 is highly expressed in TECs in the cortex and medulla of the thymus from different species. IGF1 transcripts are detected in all TECs as well as in thymic macrophages, while INS is transitorily transcribed in rare subsets of mTECs.Igf2 transcription is detected in the thymus of BB‐DR rats but is absent in the thymus of ±85% of BB‐DP rats, in close coincidence with the incidence of autoimmune diabetes in BB‐DP rats.reg cells), as well as defective programming of central tolerance to insulin‐secreting islet β cells.Several groups then addressed the fundamental question whether inhibition or a significant decrease in intrathymic self‐presentation could result in a continuous release of “forbidden” self‐reactive T cells accumulating in the peripheral repertoire, as well as to a decrease in the generation of self‐reactive tTIns1 and Ins2. Ins2 transcription predominates in murine mTECs, while Ins1 is mostly expressed in islet β cells. These contrasted profiles explain why insulitis and autoimmune diabetes are accelerated in Ins2−/− NOD mice,Ins1−/− NOD mice.Ins2 transcription in the thymus also modulate insulin‐specific T cell tolerance.Ins2 transcription is independent of glycemia and is markedly enhanced by an anti‐lymphotoxin‐β monoclonal antibody.Mafa also promotes thymic Ins2 transcription and Mafa inactivation was shown to reduce thymic Ins2 expression and to stimulate in parallel the generation of autoantibodies against anti‐islet β cells.INS transcripts are quantified at a lower level in the thymus of human fetuses with the genetic marker insulin‐dependent diabetes mellitus 2 (IDDM2) of susceptibility to T1D.A specific topography and hierarchy also exist in the expression of the two mouse insulin genes, AIRE), a member of the zinc‐finger gene family, played a major role in further evidencing the central role played by a thymus dysfunction in the pathogenesis of organ‐specific autoimmunity.AIRE mutations determine a rare recessive congenital syndrome called autoimmune polyglandular syndrome type 1 or autoimmune polyendocrinopathy‐candidiasis‐ectodermal dystrophy syndrome.Aire transcription is maximal in mTECs, and −/−Aire mice develop several autoimmune processes in parallel with a marked decrease in the intrathymic expression of numerous neuroendocrine self‐peptides  and many TRAs.IDDM2 and AIRE transcription determine the level of INS transcription in the human thymus.Aire‐expressing mTECs is regulated by RANK signals from thymic CD4+CD3− lymphoid tissue inducer cells.Aire‐expressing cells were recently identified as distinct bone marrow−derived tolerogenic cells that could anergize in secondary lymphoid organs effector self‐reactive CD4+ T cells having escaped thymic negative selection.The identification of the autoimmune regulator gene .More recently, it has been shown that the gene TSHR transcripts than carriers of the protective allele.With regard to the most frequent autoimmune endocrine disease, all major thyroid‐specific autoantigens, such as thyroperoxydase, thyroglobulin, and thyrotropin receptor (TSHR), are also transcribed in human TECs in normal conditions.H1 effector response toward peripheral nerves.A defect in α‐myosin expression in TECs exerts a major role in the physiopathology of autoimmune myocarditis,Rag1 and Rag2 in jawed fishes was responsible for the appearance of a novel and highly complex system of immune defenses, the adaptive immunity. The subsequent development of the combinatorial immune system has been sometimes regarded as the immunological “Big Bang.” Because of its high risk of autotoxicity inherent to its diversity, the emergence of adaptive immunity exerted an evolutionary pressure so strong that, according to Ehrlich's prediction of horror autotoxicus, novel structures and mechanisms appeared with the specific role of orchestrating immune self‐tolerance. The first unique thymus also appeared in sharks and rays but it was preceded by thymoid lymphoepithelial structures located in the gill baskets of lamprey larvae.Foxn4), the paralog of Foxn1, which is responsible for the differentiation of the thymic epithelium in most vertebrates. Thus, Foxn4/Foxn1 determined the emergence of the thymic epithelium, which is an absolute requirement for T cell differentiation and programming of central immune self‐tolerance.The innate immune system evolves in parallel with the neuroendocrine system in all living species without any sign of autotoxicity/autoimmunity Fig. . Toll‐liThe hierarchy observed in the organization of the thymic repertoire of neuroendocrine self‐peptides has also evolutive implications. Neuroendocrine functions having been installed before adaptive immunity, they had to be protected against autoimmunity. OT is implicated at different steps of the reproductive process and is therefore fundamental for the preservation of animal and human species. Because of its predominance in the thymus, OT is much more tolerated than VP, its neurohypophysial homolog, which mainly regulates water homeostasis and vascular pressure. This lower immunological tolerance of VP may explain why rare cases of autoimmune central diabetes insipidus have been repeatedly observed.−/−Igf2 mice.In the insulin family, insulin is the primary T1D autoantigen and no autoimmunity has been reported against IGF‐2, which is fundamental for fetal growth and development. Nevertheless, because of their homology, thymic neuroendocrine self‐peptides program immune cross‐tolerance to their whole family, and tolerance of insulin is indeed decreased in Ins2 is expressed at very low levels in rare mTEC subsets, proinsulin per se does not exert any tolerogenic properties that could be used to reprogram immunological tolerance toward islet β cells. With the exception of only two studies (that were not confirmed until now),INS transcription in mTEC subsets. The risk of hypersensitivity or anaphylaxis following administration of an autoantigen was also reported.Until now, the development of anti‐infective vaccines was essentially based on immunogenic and memory properties of the adaptive immune system. Future vaccines against autoimmune diseases could be developed on the basis of the initial property of self‐tolerance programmed for this system and recent knowledge about the potent tolerogenic properties of the thymus, which have not been exploited until now. As reported above, although Igf2 is a dominant member of the insulin family expressed in the thymus.Igf2 transcription is defective in the thymus of BB‐DP rats.11−25 and insulin B9−23 (InsB9−23) compete for binding to DQ2 and DQ8 , the MHC‐II alleles conferring the highest genetic susceptibility to T1D.IGF‐2 B9−23, IGF‐2 B11−25 presentation by peripheral blood mononuclear cells isolated from DQ8+ diabetic patients induces a tolerogenic cytokine profile with marked IL‐10 induction.Contrary to InsBIGF‐2 mediates significant cross‐tolerance to insulin.Igf2 transcription and IGF‐2 production.Infection of a murine TEC line with the diabetogenic coxsackievirus B4‐E2 inhibits reg and Breg cells.IGF‐2 induces the activation of TIGF‐2 could provide a more efficient basis than insulin for developing a specific “reverse/tolerogenic self‐vaccination”This promising concept of reverse tolerogenic self‐vaccination is under current development with the active support of Wallonia DGO6 Win2Wal THYDIA Project.reg cells. There is no doubt that thymus‐based novel strategies could alleviate in the future the weight of so many known and still unknown autoimmune diseases that remain the heavy tribute, mainly paid by mankind, for the performance and extreme diversity of its highly complex adaptive immunity.Far from being a useless “vestigial” organ, numerous studies performed worldwide have definitively demonstrated that the thymus is crucial for ensuring the global homeostasis of the adaptive immune system and is unique in programming central immune self‐tolerance. Presentation of self‐peptides in the thymus is the basic mechanism that underlies T cell differentiation, negative selection of self‐reactive T cell clones, and generation of tTThe authors declare no competing interests.


The calculated results agree well with the corresponding experimental data, and the reliability of the resulting positional selectivity was verified by the corresponding experimental data.Electrophilic aromatic bromination is the most common synthetic method used to prepare aryl bromides, which are very useful intermediates in organic synthesis. To understand the experimental results in electrophilic aromatic brominations,  Th. Th17 intert-butyldimethylsilyloxyl are quite comparable strong activating substituents, the electrophilic aromatic bromination of compound 21 at its lowest effective temperature was found to be highly para-selective with respect to a strong activating substituent to afford exclusively isomer 22a in 92% yield (23) [As methoxyl and 2% yield  62], wh, whtert-33a, 33b), (−)-jorumycin (33c) and (−)-jorunnamycin A (33d) +: 359.0259 & 361.0238. Found: 359.0263 and 361.0246.To a solution of compound 27b  in acetonitrile  at −10 °C was added N-bromosuccinimide  in one portion. The resulting mixture was stirred at 0 °C for 8 h, quenched with water (10 mL), and extracted with dichloromethane . The combined organic layers were washed with brine, dried over anhydrous sodium sulfate (Na2SO4), filtered, and concentrated. The residue was purified by column chromatography on silica gel (100–200 mesh) to afford compound 28b (336.7 mg) in 97% yield. Yellow oil; 1H-NMR  δ 7.21 , 3.79 , 3.78 , 2.26 , 1.55 ; 13C-NMR  δ 153.9, 151.4, 150.3, 140.7, 127.6, 124.0, 110.9; FTIR (film): 2980, 2937, 1760, 1471, 1417, 1370, 1272, 1255, 1223, 1147, 1099, 1067, 1004, 969, 882, 753 cm−1. HRMS (TOF MS ES+) m/z: Calcd. for C14H19BrO5Na [M + Na]+: 369.0314. Found: 369.0318.To a solution of compound ab initio calculations provide clear indication for the positional selectivity tendencies in an electrophilic aromatic bromination. Without considering any other factors, a π-donor substituent would direct an electrophilic aromatic bromination to the position in the preferential order of para > ortho > meta, whereas a π-acceptor substituent would direct this substitution to the position in the preferential order of meta > ortho > para. Although direct comparison is not always valid due to the different reaction conditions, the tendency is nevertheless quite clear. The regioselective electrophilic aromatic bromination has facilitated the synthesis of various bioactive natural products, which might allow organic chemists to think more clearly about their related research. Moreover, the theoretical analysis could also be used for understanding the regioselectivity of electrophilic aromatic brominations in the related publications.In summary, the results based on


Rugby league is a full-contact collision sport with an inherent risk of concussion. Wearable instrumented technology was used to observe and characterize the level of exposure to head impacts during game play.To verify the impacts recorded by the x-patch™ with video analysis.Observational case series.The x-patch™ was used on eight men’s semi-professional rugby league players during the 2016 Newcastle Rugby League competition (five forwards and three backs). Game day footage was recorded by a trained videographer using a single camera located at the highest midfield location to verify the impact recorded by the x-patch™. Videographic and accelerometer data were time synchronized.M = 76.1 g, SD = 17.0) than impacts that did not result in a concussion .The x-patch™ sensors recorded a total of 779 impacts ≥ 20 g during the games, of which 732 (94.0%) were verified on video. In addition, 817 impacts were identified on video that did not record an impact on the sensors. The number of video-verified impacts ≥ 20 g, per playing hour, was 7.8 for forwards and 4.8 for backs (range = 3.9–19.0). Impacts resulting in a diagnosed concussion had much greater peak linear acceleration (The vast majority (94%) of impacts ≥ 20 g captured by the x-patch™ sensor were video verified in semi-professional rugby league games. The use of a secondary source of information to verify impact events recorded by wearable sensors is beneficial in clarifying game events and exposure levels. Wearable instrumented technology has the potential to quantify the kinematic responses of the head when exposed to head impact forces during contact and collision sports.The vast majority of high acceleration impacts (≥ 20 g) recorded on sensor were verified on video review of the games, and impacts resulting in a medically diagnosed concussion had greater peak linear acceleration than impacts that did not result in a concussion.There was a substantial number of possible false-positive high-acceleration impacts recorded on the sensors before, during, and after the games. It is recommended that until sensor technology improves, head impact sensor data are used in conjunction with video.Rugby league is a high-intensity collision sport  with a rUsing technology to assist in identifying head impacts and concussion, diagnosis has been increasingly common. For instance, sideline video review and analysis has been introduced in a number of professional leagues worldwide to improve the recognition of a possible concussion . SeveralA growing body of research has combined the use of impact sensors with game video to verify the accuracy of impacts recorded by this wearable technology. However, a more complete assessment of the validity of all impacts using additional sources of information is required , 50. ForData were prospectively collected from a men’s semi-professional rugby league team during the 2016 Newcastle Rugby League season. A total of 8 players  from a single club consented to participate. The participant’s playing position consisted of five forwards and three backs. A typical rugby league team formation involves 13 players  on the field at one time. During the course of the season, there were six medically diagnosed concussions in four players. Concussion was diagnosed by a medical practitioner. The operational diagnosis was consistent with the Concussion in Sport Group   definitiAll consenting participants wore x-patch™ sensors (X2 Biosystems) during all games that they participated in during the 2016 Newcastle Rugby League season. The x-patch™ sensors were attached to the skin covering the right mastoid process of each player because previous literature has suggested that sensor positioning over the mastoid process is crucial to ensure that it is not activated by soft tissue effects during impacts . Each sep = .14) [p = .0027), which is statistically different from the expected 1.0 relationship. Therefore, we decided to not report in detail the angular head kinematic data . The x-patch™ does not measure impact force and cannot differentiate between a direct head impact and an impact to another part of the body that results in acceleration of the head, i.e., impulsive or inertial loading. The x-patch™ records data when linear acceleration exceeds 10 g; at which point, the x-patch™ saves 10 ms prior to the impact and 90 ms after, with a maximum of 1000 data points per channel. During the study, each x-patch™ was removed immediately after each game in the change room, and data were downloaded to the Injury Management Software . The IMS produces a time-stamped line output for each “impact” which included PLA, PRA, PAV, HIC, and other variables. The x-patch™ does not measure impact force, and therefore, any perturbation of the wearer that causes a linear head acceleration greater than 10 g is recorded as a “head impact,” even when direct head impact did not occur. The sensors were cleared of data before being charged and stored for the next game.The x-patch™ contains a triaxial accelerometer and gyroscope that measure linear and angular kinematics. These are applied to estimate the head’s kinematic responses, e.g., resultant linear acceleration and peak linear acceleration (PLA), peak rotational velocity and acceleration (PRV), and location of impact. The slope of the relationship between the actual PLA in a laboratory setting and the PLA measured by the x-patch™ has been reported as 0.972, which is similar to the expected relationship of 1.0 (p = .14) . Howeverattacking—running with the ball, defending—tackling, and off-the-ball incidents—no player was in possession of the ball when contact between players was made). They were also characterized into number of tacklers , wrestling impacts that happen after the initial contact (yes/no), side of contact , area of contact , and whether an impact appeared to be direct contact with the sensor (yes/no). The type of game-play scenario was also considered. As previously described in the video review of rugby league game play [Each game was digitally recorded by a trained videographer. One single-view high-definition camera was positioned at the highest possible vantage point at the field’s midline . Close-up shots panned left and right following the ball to maximize the visibility of game play, players, and potential impacts. Game footage was analyzed in conjunction with data obtained from the x-patch™. Video time was synchronized with time stamps from the sensors using the first three impacts from each player. Each half of the game was viewed from start to finish using QuickTime X (Apple Inc.) by one author (LC). The reviewer has experience in watching professional rugby league. Video was played back at an appropriate speed to verify whether or not an impact occurred. This consisted of pausing, replaying, and using slow motion as needed. Every impact on video was matched to the sensor data for validation. An impact was defined as “any contact, to the head or trunk/torso, made to the player wearing an x-patch™ by another player or the playing surface.” Sensor-recorded impacts not verified with video were identified. Similarly, impacts on video that did not correlate with x-patch™-recorded impacts were identified. Impacts were categorized as either direct (defined as an impact that made direct contact with the head) or indirect . Impacts were also characterized by play characteristics  of peak linear acceleration (PLA) and peak rotational velocity (PRV) for all verified head impacts ≥ 20 g were calculated by player position and game-play situation. Similar to criteria applied in previous studies , 43, thet test was used to compare the rate that forwards and backs sustained verified impacts. Exploratory analyses compared the acceleration  between verified/non-verified impacts, direct/indirect impacts, and concussion/non-concussion impacts using non-parametric tests because PLA was not normally distributed. The Mann-Whitney (MW) test was used when there was homogeneity of variances, and the Kolmogorov-Smirnov (KS) test was used when variances were unequal. All analyses were performed using SPSS 23 (IBM Corp.).Players engaged in game play for different amounts of time over the course of the season. We calculated verified impacts per minute. Cross tabulations were conducted between the impact sensors’ estimated head impact location and the characterization conducted using video analysis. Data were also reviewed for playing position  and play characteristics . An exploratory M = 19 min, SD = 16.46, range = 15–50; forwards M = 12 min, SD = 19.38, range = 2–51). In addition, there was a 135-min game time lost due to faulty sensors .During the 2016 Newcastle Rugby League competition, eight participants were instrumented with the wearable sensors and game video was captured during all games. These athletes played a total of 91 games across the season (89.1 player game hours/5346 player game minutes). Stratified by playing position, data from forwards accounted for 39.6% (2117 min) and data from backs accounted for 60.4% (3229 min) of playing time. The x-patch™ became detached from each player at least once during the season, for a total of 183 min of lost data due to detached sensors for the season .There were 2997 rs Table . Of thosrs Table . The vidsensor-recorded impacts to the 8 players . The number of impacts recorded in the first half (49.3%) compared to the second half of games (50.7%) was similar. Players sustained an average of 0.10 direct impacts per minute played , which equates to an average of one verified impact per 10.0 min. Forwards sustained more verified impacts per minute compared to backs . An exploratory independent sample t test suggests that forwards and backs did not statistically differ in the rate they sustained direct impacts (t(7) = 1.04, p = .33). However, effect size analysis shows a large, meaningful difference between the rates they sustained impacts based on playing position (Cohen’s d = 0.83). The individual player data by playing position, game time impact, and video verification are provided in Table Of the 732 video-verified impacts, 536 were identified as direct head impacts. Of the 536 video-verified direct head impacts, the ball carrier (attacker) recorded 261 (48.7%) and the tackler (defender) recorded 253 (47.2%), while 22 were recorded during an off-the-ball incident  by the location of impact from the x-patch™, the most impacts occurred to the front  and side , with fewer to the back  and top  of the head. When examining the location of the verified impacts on video review, the most impacts were determined to occur to the side  and front , with fewer impacts to the back . The x-patch™ accurately recorded the location of video-verified direct head impacts in 55.6% of video-verified impacts to the front of the head, 38.0% of video-verified impacts to the side of the head, and 54.5% of video-verified impacts to the back of the head. The sensor recorded 39 impacts to the top of the head, whereas no impact was verified by video review as impacting the top of the head , as well as greater PRV compared to indirect impacts . Secondary impacts during a tackle  accounted for 261 (35.7%) of total impacts (Table n = 301) of all x-patch™ recorded events. Of the hit-up plays, forward positions accounted for 44% (n = 132) of those impacts, while backs accounted for approximately 56% (n = 169) of impacts. A summary of the total impacts per play and the total impacts per play for forward and back positions is provided in Table Direct impacts (as determined by video review) had a greater PLA compared to indirect impacts  than video-verified direct impacts that did not result in a concussion that were > 20 g . Figure There were six diagnosed concussions during the season. All six concussions 100%) occurred as a result of a direct head impact. The PLA of the impacts that resulted in a diagnosed concussion was much greater  than impacts that did not result in a concussion . However, as seen in Fig. The purpose of this study was to verify the information recorded by wearable impact sensors to determine the reliability of the data, in addition to describing the playing characteristics and game-play situation of the video-verified impacts in semi-professional men’s rugby league. Eight semi-professional male rugby league players wore wearable impact sensors during a single season. The vast majority of high acceleration impacts during games were verified on video. Specifically, there were 779 in-game sensor-recorded impacts ≥20 g, of which 732 were verified on video . It is difficult to determine the true level of false-negative data because video review does not enable determination of the observed impacts to the head that occur above or below the 10 g threshold . In addition, given that for 101 (17%) tackles, there was more than 1 impact  registered by the x-patch™, the true level of false-negative data could also be much greater than the 817 because on video review, it was only the initial impact that was considered as being missed, and not any subsequent impact that may have been recorded during the same tackle.raises concern that impact sensor studies that do not verify impacts may be over-reporting both the level of exposure of athletes  and the severity of the head impacts .Our reported rate of video-verified true sensor-detected impacts may be an overestimate, and it might be confounded by player position. Rugby league is a demanding physical contact game with frequent body contact. Defensive players and “hit-up” players  may have a greater body contact rate than backs. If the x-patch™ records spurious head impact events, as appeared to be the case before and after match play, by chance, the video review is likely to identify an impact event  when the x-patch™ records a spurious head impact. Rugby league may not be the ideal setting for validating all characteristics of head impact sensors. The rate of false-positive sensor-detected impacts cannot be determined. There were 295 impacts recorded on the sensors after the game had ended. Of those, 88 were ≥ 20 g impacts. It seems unusual that there would be so many large impacts recorded after the game, and we cannot determine the extent to which the sensors yield false-positive findings before, during, or after the game. In general, the rate of false-positive impacts, in addition to the possibility that the device could register greater linear acceleration than was actually the case, t test did not show group differences, but this non-significant finding is likely an artifact of the small sample size in our pilot study; effect size analysis showed a large, meaningful difference in the number of impacts stratified by playing position (Cohen’s d = 1.15). We have previously reported that the concussion interchange rule  was used more commonly in forwards than in backs at the professional level  [On average, players sustained 1 verified impact every 10.0 min. The rate that these athletes sustained impacts appeared to differ based on their playing position, with forwards sustaining impacts more frequently than backs. Our exploratory (and underpowered) independent sample cks 43%)  and the cks 43%) . In the n = 301) of all verified x-patch™-recorded impacts, with the tackler accounting for 46% (n = 279) and 5% (n = 32) coming from off-the-ball incidents, a similar result as the professional level. In addition, the hit-up play data revealed that forward positions accounted for 44% (n = 132) and backs approximately 56% (n = 169) of impacts recorded.On-field play characteristics may also be associated with head impacts and potential concussions. A prior study showed that the concussion interchange rule was used to remove the tackler 55% and 61% of the time compared to 43% and 38% for the ball carrier at the professional and national youth level, respectively . In the The current study offers some insights into the level of impacts sustained in the sport of rugby league at the semi-professional level. Although the results are preliminary given the small sample size, it is clear that there appears to be variations in impact exposure by position (forwards versus backs), type of play , game situation , and number of tacklers involved in a tackle. These findings also offer further insights into the previous rugby league video analysis studies that have been conducted at the professional level –23 examiIn the current study six concussions were diagnosed in players wearing the x-patch™. There was a large, statistically significant difference between the PLA of concussive impacts versus non-concussive impacts. Future studies should aim to collect a larger number of concussion events in order to calculate the possible thresholds of PLA (as well as other parameters) and sensitivity and specificity of those impact thresholds to determine the potential clinical application of the x-patch™ or similar devices for assisting in the clinical diagnosis of concussion.There are a few limitations to the current study design. First, using only eight players is the small sample size and limits the generalizability of the findings. Second, the false-negative incidences only considered a single  impact as missing, as such, the possibility that a second or subsequent impact registration was also not coded by the impact sensor was not calculated as a “missed impact” resulting in an underestimation of the potential false negatives. Third, each video was coded by a single researcher. It is possible that some impacts were missed by the researcher when reviewing video. Fourth, the current findings may not be generalizable to all levels of rugby league or other sports. Fifth, the current findings are limited to the x-patch™ impact sensor only and may not reflect the capabilities of other impact sensors. Finally, there is no accepted standard as to what head impact-related PLA represents a “subconcussive” hit  apart from a strict clinical outcome, nor is it understood how or whether these impacts affect brain microstructure or function. Future studies with larger sample sizes may consider reviewing the role cumulative impacts may have on the vulnerability of an athlete to future concussive events. In addition, in view of the literature that suggests that cumulative head impact exposure is a predisposing factor for the onset of concussion , 55, revThese data are consistent with the findings from Cortes and colleagues  who foun


Researchers are increasingly investigating the drinking behavior and associated consequences among Asian-Americans and Pacific Islanders (APIs) in the United States. Among adolescents, APIs generally have lower rates of alcohol consumption and appear to be at lower risk for alcohol abuse compared with other ethnic groups. Similarly, the rates of drinking and heavy drinking have been found to be lower among API college students than among other ethnic groups. Among adult Asian-Americans, Japanese-Americans have the highest and Chinese-Americans have the lowest lifetime prevalence of drinking and heavy drinking. Southeast Asians  living in the United States appear to be at high risk for heavy drinking. Numerous factors help determine the drinking patterns of APIs, including their economic status, educational attainment, and degree of acculturation as well as genetic and environmental factors, such as drinking norms and alcohol availability. In the past few decades, people of Asian and Pacific Islander (API) ethnicity have been the fastest growing minority in the United States, with their numbers almost doubling between 1980 and 1990 , the poverty rate among API families also was higher than among Caucasian families  . These oThe socioeconomic disparity among APIs reflects differences among, and even within, ethnic groups in both educational preparation  and length of stay in the United States. Thus, within various ethnic groups , poverty rates are higher among families with a shorter length of stay in this country than among families with a longer length of stay . For somSeveral surveys have assessed alcohol consumption and its associated problems among various age and ethnic groups of APIs. Most of these surveys were conducted in the 1980s and involved participants recruited primarily in California, Hawaii, and New York, where most APIs reside.The drinking patterns of adolescents, including those of API heritage, were investigated in several surveys. For example, in a study conducted in New York City, 27,000 junior and senior high school students were asked about their alcohol and other drug (AOD) use  do exist in this population. For more accurate information, school-based alcohol use surveys should be supplemented by information on treatment referral and treatment use of API adolescents as well as through surveys of key informants .Drinking patterns among APIs also have been investigated using college student samples. Across surveys, both the percentage of drinkers and the percentage of heavy drinkers invariably were lower among API students than among Caucasian students . These fOne study also assessed the influence of the degree of acculturation on the students’ drinking behavior. The level of acculturation was determined from the number of generations the student’s family had been in the United States and from the student’s ability to speak the ethnic language. In that study, the more strongly acculturated API students reported higher levels of alcohol consumption than did the less strongly acculturated students  . SimilarAs previously discussed, Southeast Asians, particularly Vietnamese and Cambodians, emigrated to the United States in two waves, each of which comprised people of vastly different educational and cultural backgrounds. The first wave, which arrived shortly after the Vietnam war in 1975, included mostly well-educated people who had lived in urban areas. Conversely, immigrants who arrived with the second wave, during the 1980s, frequently had been peasants living at a subsistence level. Most of these immigrants were illiterate in their native language as well as in English and were ill equipped for life in the United States . In addi2Small-scale studies have indicated that Southeast Asians living in the United States are at high risk for heavy alcohol consumption. For example, a study of Vietnamese living in California demonstrated that although the percentage of drinkers was similar among Vietnamese males and among males in the general American population, binge drinking was twice as common among Vietnamese males as among non-Southeast Asian males see . SeveralMany Southeast Asian cultures do not consider alcohol a harmful drug but, rather, a substance with healing properties.As mentioned previously, many Southeast Asian immigrants suffer from depression, PTSD, and other psychological problems. They may use alcohol as self-medication to provide temporary relief from their symptoms and from memories of traumatic experiences.Alcohol is more readily available in the United States than in the Southeast Asian countries.Many Southeast Asians living in the United States are children of American soldiers and Southeast Asian mothers who came to this country to locate their fathers. Many of these people did not find or were rejected by their American families; consequently, they often feel marginalized in American society.The role of these factors in contributing to alcohol problems is supported by findings from studies that evaluated Southeast Asians who were undergoing alcoholism treatment. For example, in an investigation at the addiction treatment center of the University of Minnesota Hospital and Clinic, 38 of 50 Southeast Asians who sought treatment reported that they used AODs specifically to relax and enjoy life . SimilarTo date, the alcohol consumption patterns of Pacific Islanders have not been examined in detail. The available studies generally have focused on native Hawaiians, comparing them with other ethnic groups living in Hawaii. For example, one study has suggested that Hawaiians have more serious AOD problems than do members of other major ethnic groups see . In thatNot much is known about the determinants of drinking among APIs, especially because these determinants may differ substantially among, and even within, ethnic groups. Moreover, in many studies the sample sizes are too small to control for major demographic variables . Various studies, however, have focused on the influence of acculturation on drinking behavior. For example, When evaluating and interpreting the findings of these studies, however, it is important to keep in mind that factors other than acculturation may contribute to the observed differences in drinking behavior, including the followingBoth in the United States and in some Asian countries, drinking behaviors have changed substantially over the past few decades. These changes may confound the effect of acculturation.The socioeconomic status of the descendants of API immigrants has changed over the years . These factors may influence drinking behavior independently of the degree of acculturation.APIs who immigrated to the United States were not representative of the general population of their homelands. Accordingly, the drinking practices of the immigrants may have already differed from those of the general population in those countries.Demographic variables  of the API immigrants and their descendants may differ from those of other ethnic groups  in the United States. These differences also may account for differences in drinking behavior and confound the influence of acculturation.1 variant produces a functional enzyme, whereas the ALDH22 variant produces an inactive enzyme. Each person inherits two copies of the ALDH2 gene, one from the father and one from the mother. People who inherit two copies of the ALDH22 gene or one copy of the ALDH21 gene and one copy of the ALDH22 gene produce an impaired ALDH enzyme and are susceptible to the flushing reaction  alcohol dehydrogenase, which converts alcohol to acetaldehyde, and (2) aldehyde dehydrogenase (ALDH), which converts acetaldehyde to acetate. ALDH inactivity results in the accumulation of acetaldehyde in the body, which leads to the so-called flushing reaction. This reaction is characterized by numerous symptoms, such as facial flushing, nausea, headache, dizziness, and rapid heartbeat. Several genes code for the ALDH enzyme, including the ALDH2 gene. Two variants of the ALDH2 gene exist. The ALDH2reaction . Because2 gene variant is not equally common among all ethnic groups. Few Caucasians but up to 50 percent of Asians carry one or two copies of the ALDH22 gene and are therefore susceptible to flushing , Japanese women had the second highest mortality rates, followed by white men and white women. Because the average alcohol consumption among the Japanese women was very low, the differences in cirrhosis mortality rates reflect a higher prevalence of viral hepatitis infections in Japanese than in whites in the United States  and suggAPIs, particularly Asian-Americans, are the fastest growing ethnic minority in the United States. Moreover, the demographic profile of the Asian-American population is changing rapidly. Filipino-Americans now outnumber Chinese-Americans, who once were the largest Asian-American group. Likewise, the Vietnamese-American population is expected to exceed the Chinese-American population by the year 2000. Other Southeast Asian populations also will likely continue to grow. However, the drinking patterns and consequences of alcohol use in these populations have not been studied adequately in recent years. Future studies must address the determinants and characteristics of alcohol use among Asian-Americans in order to develop and provide adequate treatment services to these populations. Such studies also must consider the heterogeneity of the Asian-American population as well as the cultural differences within ethnic groups. Additional studies also should focus on Pacific Islanders, because little information is currently available regarding their drinking practices.


S. pneumoniae in diseases such as meningitis. While some capsular serotypes are more often found in invasive disease, high case fatality rates are associated with those serotypes more commonly found in asymptomatic colonization. We tested whether growth patterns and capsule size in human cerebrospinal fluid depends on serotype using a clinical isolate of S. pneumoniae and its capsule switch mutants.The polysaccharide capsule is a major virulence factor of We found that the growth pattern differed markedly from that in culture medium by lacking the exponential and lysis phases. Growth in human cerebrospinal fluid was reduced when strains lost their capsules. When a capsule was present, growth was serotype-specific: high carriage serotypes  grew better than low carriage serotypes . Growth correlated with the case-fatality rates of serotypes reported in the literature. Capsule size in human cerebrospinal fluid also depended on serotype.We propose that serotype-specific differences in disease severity observed in meningitis patients may, at least in part, be explained by differences in growth and capsule size in human cerebrospinal fluid. This information could be useful to guide future vaccine design. Streptococcus pneumoniae is a leading cause of disease ranging from mild to severe manifestations. Invasive pneumococcal disease (IPD) includes the life-threatening conditions of bacteraemia and meningitis, both of which have high mortality rates [S. pneumoniae is also a leading cause of pneumonia. A major virulence factor of S. pneumoniae is the polysaccharide capsule and, based on the biochemical properties of the capsule, S. pneumoniae is categorized into different serotypes. Currently, approximately 100 serotypes are known [ty rates , 2 and Sre known . Severalre known –7. Therere known , 9.The pneumococcal capsule has been a target of research for several decades and current vaccines are composed of capsule polysaccharides. The most commonly used, pneumococcal conjugate vaccine 13 (PCV13) and pneumococcal polysaccharide vaccine 23 (PPSV23), contain serotype-specific polysaccharides for 13 and 23 serotypes respectively. The large-scale use of PCVs has led to changes in disease and carriage prevalence of individual serotypes over time and geographically –13. ThesspxB gene resulting in apoptosis [Serotypes differ in their ability to cause severe disease –18 and tpoptosis .Supporting the experimental findings, studies show that, although the outcome of pneumococcal disease can be affected by factors such as age, immunodeficiency and other host characteristics, even when controlling for these factors, certain serotypes are associated with more severe disease –18. SimiS. pneumoniae in hCSF in vitro is scarce. We tested whether differences in ability to grow in hCSF in vitro and differences in capsule size correlate with serotype-specific disease severity.It is not fully understood how the different serotypes are responsible for differences in disease severity. Therefore, here we studied the growth behaviour and capsule size of different serotypes in human cerebrospinal fluid (hCSF) rather than culture media to reflect more closely the environment the bacteria would encounter in a meningitis patient. Although other studies have looked at hCSF parameters in the setting of pneumococcal meningitis –27, dataS. pneumoniae is able to grow and how it behaves in hCSF in vitro, we performed growth analysis over 40 h of the strain 106.66 (serotype 6B) in BHI + FCS and hCSF. The growth pattern of strain 106.66 in hCSF lacked a discernible exponential phase and the autolysis phase typically seen when grown in BHI + FCS medium  as well as the wild type strain 51,114 L with its nonencapsulated spontaneous mutant, 51,114 S. Both the encapsulated strains 106.66 (serotype 6B) and 51,114 (serotype 19F) grew in hCSF to a higher OD than their nonencapsulated mutants . When pooling data for the four high carriage and four low carriage serotypes , the lopes Fig. b. No sigmax in hCSF  , particWe also found significant differences in capsule size between 19F, 7F and 6B serotypes in hCSF. Of note, the 19F serotype, which is associated with severe disease , 9, and A limitation of our study is that, although growth was monitored in human CSF, all analyses were performed in vitro. We collected and froze the hCSF samples as soon as possible following lumbar puncture and did not refreeze samples but we cannot exclude the possibility that the composition of the hCSF changed during the time after removal from the patients. In vivo there would be a turnover of hCSF which would likely provide more nutrients to the bacteria. Therefore, with our in vitro experiments we may be underestimating the amount of pneumococcal growth in hCSF. This could be overcome by development of an in vitro system where the hCSF is replenished, if sufficient hCSF was available. Another major difference between our experiments and the situation in vivo is the absence of immune cells in our system. As meningitis progresses the number of immune cells in the hCSF might be expected to increase and any effect of this on bacterial growth and capsule thickness is not accounted for in our experiments. We deliberately chose to use hCSF from patients without inflammation to model early disease when pneumococci first reach the hCSF. However, it would also be interesting to study pneumococcal growth and capsule thickness in hCSF from patients with meningitis as we speculate that these may be affected by a nutritionally richer environment. We have made the assumption that the hCSF we have used is a model of healthy hCSF but the samples were taken from patients rather than healthy volunteers, although routine parameters were within normal ranges. Due to availability of samples, our experiments were limited to hCSF from 3 patients. Further testing in hCSF from more patients would be beneficial, although we note that the results we found were consistent between the hCSF of the three different patients.A further limitation is the number of pneumococcal strains tested. It would be interesting to expand the study to further high and low carriage serotypes including those with large mucoid capsules such as serotypes 3 and 8 if the challenges of making capsule switch mutants of such serotypes could be overcome. However, we note that the pattern of growth, particularly the lack of autolysis was consistence for all strains tested.We found that in hCSF there was no autolysis and that serotype influenced growth and capsule size. Growth correlated positively with previously published serotype CFR ranges. Our findings contribute to the understanding of the role of serotype in pneumococcal disease, particularly meningitis. We further suggest that growth and capsule size in hCSF in vitro may be useful attributes to predict severity of disease caused by different serotypes to guide future vaccine design.n = 1), 7F (n = 2), 14 (n = 1), 9 V (n = 2), 15B/C (n = 1), 18C (n = 2), 23F (n = 1) and 19F (n = 3)). Production of the 106.66 capsule switch mutants and the 106.66 capsule deletion mutant (106.66 Janus) are described in a previous publication [Swiss pneumococcal strain 106.66 (serotype 6B) is a clinical isolate from the nasopharynx of a child from a nationwide surveillance program collecting nasopharyngeal and invasive isolates , 38. 106lication .2) showed two colony phenotypes: large  and small colonies  with appearance of encapsulated and nonencapsulated pneumococci, respectively. The two phenotypes were separated and purified by three consecutive passaging steps where each time one single colony was picked and streaked on a CSBA plate. Separation as well as presence or absence of capsule was confirmed by serotyping and FITC-dextran exclusion assay .Additionally, a clinical isolate of serotype 19F  recovered from the hCSF of an adult meningitis patient in 2017 from the South African GERMS national laboratory based surveillance program was provided by the National Institute for Communicable Diseases (NICD) in Johannesburg. This serotype was chosen due to its repeated association with meningitis and high CFR , 9. PlatFor all strains, Quellung reaction was used to confirm serotype. The serotypes were categorized as low carriage / high invasive potential  or high carriage / low invasive potential  according to a previous paper .Residual hCSF from three adult patients undergoing routine lumbar puncture in 2018 and 2019 due to idiopathic intracranial hypertension (non-inflammatory) was used for growth and capsule analysis. Lumbar puncture was performed by trained personnel. hCSF samples and related patient data were anonymized by the treating physician prior to any research use in accordance with the Swiss Human Research Law . Routine hCSF parameters were within normal ranges for all samples . Samples were stored initially at 4 °C until collection and were aliquoted and frozen at − 80 °C within 48 h after lumbar puncture. All samples were used for analysis within 3 months of freezing. They were thawed 15 min before use. There was no pooling of CSF samples from different patients.2 atmosphere. Three to 10 colonies were picked and used to inoculate tubes containing 5 ml brain heart infusion  supplemented with 5% fetal calf serum  (BHI + FCS). The culture was then incubated until OD600nm reached 0.8–0.9. From this culture, a 250 μl sample was added to 750 μl fresh BHI + FCS. The culture was grown until OD600nm reached 0.4 to ensure bacteria were in the exponential growth phase.Bacteria were stored at − 80 °C in Protect bacterial preservers . Bacteria were plated on CSBA and grown overnight in a 37 °C, 5% CO600nm of 0.4, the culture was centrifuged for 5 min at 3000 g and the supernatant discarded. The culture was then re-suspended in 5 ml phosphate buffered saline pH 7.4 (PBS). Growth was assessed as described previously [450nm measured using a VERSAmax microplate reader (Molecular Devices). SoftMax Pro Software, version 5.3 was used to record measurements. OD was measured at 30 min intervals over 40 h with 5 s of automatic shaking preceding each reading. Condensation was prevented by pre-treating the plate lid with 3 ml 0.05% Triton X-100 in 20% ethanol [450 (ODmax) over 40 h.When bacterial culture reached ODeviously . 8 μl ofeviously  (with 4 2 atmosphere and colonies counted the following day.To determine colony forming units (CFU), dilutions of bacterial cultures were plated onto CSBA plates and cultured overnight at 37 °C, 5% COmax that each serotype reached in hCSF or BHI + FCS over 40 h against the corresponding serotype case-fatality rate (CFR). The CFR was taken from a previous publication by Sjöstrom K, et al. (2006) [To analyze whether the ability to grow in hCSF in vitro correlated with disease severity we plotted the OD. (2006)  in which600nm of 0.4 the culture was centrifuged at 3000 g for 5 min. The supernatant was discarded and the pellet re-suspended in 5 ml PBS. A sample of 50 μl bacterial suspension was used to inoculate 600 μl of BHI + FCS or hCSF. The tubes were incubated in a water-bath at 37 °C. FITC-dextran exclusion assay was performed at time 0 and 9 h after incubation. To perform the FITC-dextran assay, 10 μl of the bacteria culture was removed and mixed with 2 μl FITC-dextran . The mixture was pipetted onto a microscope slide and a coverslip was applied. A Zeiss Axio Imager M1 fluorescence microscope with a 100 x objective was used to view the slides. The Zeiss AxioCam HRc camera was used for photographing the slides. Capsule size was measured from at least 5 randomly selected bacterial cell bodies over five images per group of each of the three independent images, adding up to a total of 15 images per group.The FITC-dextran method of Gates et al. , in whicmax value in hCSF.We performed FITC-dextran exclusion assays of wild type strain 106.66 serotype 6B, its 7F capsule switch mutant (106.66cps208.41) and a capsule switch mutant of 19F serotype (106.66cps111.46) due to the association of serotype 19F with meningitis and a high CFR [p-value, two-tailed, of ≤0.05 was considered significant. The comparisons of ODmax were performed using a non-parametric Mann Whitney, two-tailed comparison test. A value of p ≤ 0.05 was considered significant.Statistical analysis and graphs were performed using the GraphPad Prism software . The same software was used for linear regression analysis for which a Additional file 1: Table S1. List of 106.66 capsule switch mutants used in experiments [Figure S1. Capsule thicknesses of strains 106.66 and 51,114 with and without capsule in BHI + FCS. Figure S2. Growth pattern of S. pneumoniae wild type strain 106.66 and 106.66 capsule switch mutants representing high carriage serotypes  and low carriage serotypes  in BHI + FCS1  and hCSF2  over 40 h. Figure S3. Growth of S. pneumoniae wild type strain 106.66 in BHI + FCS, CDM3 [Figure S4. Colony forming units (CFU) after 6 h of growth in human CSF (hCSF) for South African strain 51,114 L (serotype 19F) and its spontaneous capsule loss mutant 51,114 S. Figure S5. Maximum OD values of wild type 106.66 and capsule switch mutants in BHI + FCS. Figure S6. Colony forming units (CFU) after 6 h of growth in human CSF (hCSF).