This study aims to investigate and explain the magnetite-accelerated stress corrosion cracking phenomenon of Alloy 600 under caustic conditions, based on the electrochemical behavior. After the SCC test that lasted for 300 h, no cracks were observed in any of the magnetite-free specimens, whereas cracks with a depth of 150 to 280 μm were generated in all the magnetite-deposited specimens. Furthermore, the electrochemical behavior of magnetite and Alloy 600 demonstrated that Alloy 600 behaved as an anode in the coupling system with magnetite. In this coupling system, the electrochemical potential of Alloy 600 can be shifted into the range potentially susceptible to stress corrosion cracking. Heat-transfer tubing made of nickel-based Alloy 600 has undergone a variety of corrosion-related damages in the secondary environments of steam generators (SG) in pressurized water reactors (PWRs): stress corrosion cracking (SCC); intergranular corrosion; wastage; pitting ,2. BecauSCC is well known as the major mechanism of Alloy 600 SG tubing failure occurring in the secondary environment of PWRs. This degradation mode mainly occurs in heated crevices covered with porous magnetite deposits, because a severe corrosion environment can be formed in these crevices as a result of high impurity concentrations caused by local boiling ,5,6,7. EThe reason for the occurrence of rapid SCC failure in Alloy 600 remains a problem that must be solved in this field. To explain the root causes for this, the fact that the real corrosion-related degradation is generated on the porous magnetite-deposited tube surface needs to be considered carefully. Specifically, only the tube surface connected through the pores inside porous magnetite deposits is locally exposed to the crevice environment. At the same time, the tube surface is in electrical contact with the magnetite deposit. Magnetite is well known as a conductive material, and its electrical conductivity indicates almost metallic behavior ,14. Cons°C for 150 s to dissolve the carbon in the matrix and control the microstructure (average grain size: about 27 μm), and then quenched in water at room temperature. These specimens were abraded using SiC paper up to #1000 grit, and then fabricated into a U shape  and was formed into a plate by hot-rolling in the temperature range of 1050 to 1150 °C. The plate thickness was finally reduced to about 1.5 mm through a cold-rolling process. The cold-worked plate was machined into specimens with dimensions shown on the left side of 2(SO4)3, and 0.1 M triethanolamine by using a three-electrode system under the following conditions: ED potential = −1.05 VSCE; ED time = 120 min; ED temperature = 80 °C. The details of the ED process and characteristics of the magnetite film are given in our previous reports [To investigate SCC behavior caused by the coupling effect of Alloy 600 and magnetite, we needed to prepare SCC specimens electrically connected with magnetite. To achieve this, a thick magnetite film was grown on the entire surface of the prepared SCC specimen using the electrodeposition (ED) method, except for an area with a width of approximately 800 μm in the apex region  .The SCC tests for the magnetite-free and magnetite-deposited specimens were carried out separately in deaerated 10 wt% NaOH solutions for 300 h at 315 The Alloy 600 specimen for the potentiodynamic polarization test was machined into a rectangular shape (6 mm × 6 mm × 1.5 mm) and was spot-welded to an Alloy 600 wire that had been covered with a heat-shrinkable polytetrafluoroethylene (PTFE) tube. This specimen was used as an Alloy 600 electrode. A magnetite electrode was prepared by ED of a magnetite film on the entire surface of the prepared Alloy 600 electrode under the same conditions as had been used for the preparation of the magnetite-deposited SCC specimen.°C by using a static Ni-autoclave with a capacity of 1 L shown in °C, polarization scans were performed from the OCP toward the positive or negative direction at a scan rate of 30 mV/min. The potentiodynamic polarization test was performed in the same solution used for the SCC test at 280 SHE represents the electrode potential versus SHE and Eobs is the measured electrode potential. T is the experimental temperature, and t is the room temperature (25 °C). In addition, the reproducibility of the polarization curve was confirmed by repeatedly performing the tests at least three times using newly prepared electrodes and test solutions.The measured potentials were converted to the standard hydrogen electrode (SHE) scale according to the following relationship ,21:(1)ES2 was also presented in 2. The corrosion potential of the coupled Alloy 600 will be more polarized to the active–passive transition region with increasing the magnetite surface area, resulting in the greatly increased anodic current. Therefore, it is considered that these changes in the electrochemical behavior also occur on the exposed Alloy 600 surface in the magnetite-deposited specimen shown in Particularly, the alloy which exhibits an active–passive behavior in the anodic polarization curve is known to be more susceptible to SCC in its active–passive transition region . In thisMany investigations related to the SCC phenomena of SG tubing occurring in crevices covered with magnetite deposit have mainly focused on water chemistry factors, such as pH, temperature, and impurity concentration. However, it should be noted that SG tubes are always corroded under the situation which is in contact with the porous magnetite deposit in the secondary side of PWRs. Especially, in the crevice region between the SG tubes and their support plate, flow-induced vibration can lead to tube damage by fretting-wear due to impact and sliding movement between the tubes and their support plates ,27,28. TThis work confirms that the caustic SCC of Alloy 600 is significantly accelerated when the alloy is in electrical contact with magnetite. From the perspective of electrochemical behavior, it is demonstrated for the first time that the galvanic coupling with magnetite can shift the electrochemical potential of Alloy 600 into the more susceptible potential region to SCC. Therefore, the magnetite-accelerated stress corrosion cracking (MASCC) mechanism should be considered as a new acceleration factor of SCC in Alloy 600 SG tubes in secondary side conditions, in addition to the impurity concentration within the porous magnetite deposits.The SCC behavior of Alloy 600, coupled with magnetite, has been investigated under a high-temperature caustic condition. Magnetite in contact with Alloy 600 induced rapid SCC of Alloy 600. Regarding electrochemical behavior, Alloy 600 and magnetite acted as an anodic and cathodic members, respectively, in the coupling system. In this couple, the electrochemical potential of Alloy 600 was shifted into the active–passive transition region of the anodic polarization curve, which is the range susceptible to SCC. Based on the experimental results, a galvanic corrosion mechanism between magnetite and Alloy 600 is proposed as a new acceleration contributor to a rapid cracking of Alloy 600 in the PWR secondary environment.


Neurons in the orbitofrontal cortex (OFC) fire in anticipation of and during rewards. Such firing has been suggested to encode reward predictions and to account in some way for the role of this area in adaptive behavior and learning. However, it has also been reported that neural activity in OFC reflects reward prediction errors, which might drive learning directly. Here we tested this question by analyzing the firing of OFC neurons recorded in an odor discrimination task in which rats were trained to sample odor cues and respond left or right on each trial for reward. Neurons were recorded across blocks of trials in which we switched either the number or the flavor of the reward delivered in each well. Previously we have described how neurons in this dataset fired to the predictive cues ; here we In the So, do OFC neurons signal reward predictions errors or do they signal reward predictions? Here we tested this question directly by analyzing the firing of OFC neurons recorded in an odor discrimination task in which rats were trained to sample odor cues and respond left or right on each trial for reward. Neurons were recorded across blocks of trials in which we switched either the number or the flavor of the reward delivered in each well. Previously we have described how neurons in this dataset fired to the predictive cues ; here we2.2.1Male Long-Evans rats were obtained at 175–200 g  from Charles River Labs, Wilmington, MA. Rats were tested at the University of Maryland School of Medicine in accordance with School of Medicine and NIH guidelines.2.2Surgical procedures followed guidelines for aseptic technique. Electrodes, consisting of drivable bundles of eight 25-um diameter FeNiCr wires  electroplated with platinum to an impedance of ~ 300 kΩ, were manufactured and implanted as in prior recording experiments. Driveable electrodes were implanted in the left orbitofrontal cortex of six rats  in each rat. At the end of the study, the final electrode position was marked, the rats were euthanized with an overdose of isoflurane and perfused, and the brains were removed from the skulls and processed using standard techniques.2.3.Recording was conducted in aluminum chambers, on one wall of which was a panel with an odor port and two fluid wells arranged below it see . The odoRats were trained extensively before they were implanted with electrodes. After implantation, we retrained rats to work while attached to the recording cable. Each training session included as many trials as a rat would perform before quitting, ~150–250. This initial shaping phase gradually introduced all elements of the task (described below), and thus rats could learn the associative structure of the task over this period. Recording was begun when rats could complete five blocks of trials  with the cable. Total number of pre-recording training sessions averaged 32.5 (ranging from 24 to 43).Each recording session consisted of a series of self-paced trials organized into five blocks. Rats could initiate a trial by poking into the odor port while the house light was illuminated. Beginning 500 ms after the odor poke, an odor would be delivered for 500 ms. If the rat withdrew from the odor port before completion of the 1000 ms preodor + odor period, the trial would be aborted and the houselight turned off. The end of the odor served as a go-response indicating that rats could respond by moving from the odor port to the left fluid well or the right fluid well, after which they had to wait for 500 ms before fluid delivery began. The identity of the odor specified whether they could receive reward at the left well (forced-choice left), the right well (forced-choice right), or either well (free-choice). The identity and meaning of these odors remained the same across the entire experiment. Odors were presented in a pseudorandom sequence such that the free-choice odor was presented on 7/20 trials and the left/right odors were presented in equal numbers . In addition, the same odor could be presented on no more than 3 consecutive trials.Rewards were either one drop or three drops of chocolate or vanilla milk, with drop size ~0.05 ml and 500 ms between drops. Response-reward contingencies were consistent within blocks of trials, such that the same reward would be delivered for every correct right response, either free- or forced-choice, and a different reward would be delivered for every correct left response, free- or forced-choice. Upon each block transition, either the number of drops would change on both sides (1 drop to 3 drops and vice versa) and flavor remained constant, or the flavor would change on both sides (chocolate to vanilla and vice versa) and the number of drops remained constant. These block transitions were not explicitly signaled and could not be predicted based on the exact number of trials. The first block, consisting of on average 43 ± 16 (SD) trials, was used to set the rats’ expectations before the first transition. The length of the last four blocks varied non-systematically around 65 ± 11 (SD). The reward schedule was arranged so that in each block, reward features available on one side were always paired with the opposite reward features on the other side – thus when one drop of chocolate milk was available on the left, three drops of vanilla was available on the right, etc., resulting in a total of four different reward combinations. The reward combination in the first block was randomly chosen, after which the block order followed a set scheme consisting of a drop-number transition, a flavor transition, another drop-number transition, and another flavor transition , we compared consumption of the chocolate vs. vanilla milk solution in two-bottle tests. All rats were tested for ten total minutes, with the location of the bottles swapped every 30 s. Two rats were given five 2-min tests while the other four rats were given one 10-min test each.2.5Procedures were the same as described previously . Wires w2.6Units were sorted using Offline Sorter software from Plexon Inc. , using a template matching algorithm. Sorted files were then processed in Neuroexplorer to extract unit timestamps and relevant event markers. These data were subsequently analyzed in Matlab .t-test across all trials, p < .05). We designed the subsequent analyses using two different epochs that surrounded the timepoint of each drop delivery (the timestamp at which the solenoid was activated to begin reward delivery) or omission . The reward response epoch was from 100 ms to 300 ms after the specified reward timepoint. The complementary reward anticipation epoch began 200 ms before the reward timepoint and ended 100 ms after it. For each analysis, we calculated a prediction error or prediction score, defined as the difference between the average firing rate in the early trials of the block in question and that in the late trials of the same block or the previous block, as appropriate. We then performed t-tests on the distribution of scores across all included neurons (p < .05). For the analyses of flavor shifts presented in t-test, p < .05); (c) the error rate on forced-choice trials was significantly different on the last ten vs first ten trials on either side ; (d) the response latency was significantly different in the last ten vs. first ten forced-choice trials on either side . For these flavor change analyses, scores were calculated for each neuron on each shift. For all figures, population averaged activity was baseline subtracted on each trial before averaging.We first screened neurons by whether they showed a significant increase in firing rate in from 100 to 500 ms after delivery of the first drop of reward compared to baseline, defined as the last two seconds of the preceding inter-trial interval . This ANOVA had factors side (i.e. direction), number of drops, and flavor. We then analyzed neurons with a main effect of flavor by comparing the magnitude of changes across flavor shifts  with those across the block after flavor shifts .2.7We compared these data with recordings from dopamine neurons recorded in rats in the same task, as reported earlier . Figures3.We recorded single-unit activity from OFC as rats performed a task with changing amounts and flavors of a milk solution reward , they were no different immediately after flavor shifts vs. the end of those same blocks .A similar pattern in which OFC neurons signaled reward predictions rather than prediction errors was evident when reward flavor changed unexpectedly. At the start of blocks 3 and 5, 3 drops of chocolate milk solution replaced 3 drops of vanilla milk solution (or vice versa) on one side, and 1 drop of chocolate replaced 1 drop of vanilla milk solution (or vice versa) on the other side. Although the two flavors were equally preferred, DA neurons still showed a robust phasic increase after this shift, which declined with learning in the block like a prediction error . This inIn contrast to the lack of prediction error signaling after unexpected flavor changes, we still observed reward predictive signaling immediately after these shifts. This was evident in some blocks after the flavor shifts on the 1-drop side. Here, the flavor that had previously been delivered in three drops (e.g. chocolate or vanilla) was delivered as only a single drop. If rats were attending to flavor, that particular flavor might be seen as predicting two additional drops right after the shift. Indeed, we have previously reported that rats’ behavior can subtly reflect this prediction . This is4.As noted at the outset, it has become widely accepted that the OFC is important for signaling information about expected rewards . Such anHere we addressed this question directly by analyzing single unit activity recorded in OFC in rats performing a task that we have previously used to identify reward prediction error correlates in midbrain DA neurons . We founThe complementary relationship identified here between activity in OFC and the DA neurons replicates prior findings in a variant of this task in which we manipulated reward value by changing either number or timing . In that


Pemphigus foliaceus (PF) is one of the two main forms of pemphigus and is characterized by circulating IgG to the desmosomal cadherin desmoglein 1 (DSG1) and by subcorneal blistering of the skin. The pathomechanism of blister formation in PF is unknown. Previously we have shown that PF IgG induces aggregation of DSG1, plakoglobin (PG), and IgG outside of desmosomes, what in immunofluorescence of PF patient skin visualizes as a granular IgG deposition pattern with a limited number of coarse IgG aggregates between cells. Here we have investigated the fate of these aggregates in skin and found that these are cleared by endocytosis. We performed double immunofluorescence staining on snap-frozen skin biopsies of six PF patients for the following molecules: IgG, the desmosomal proteins DSG1 and DSG3, desmocollins 1 and 3, PG, desmoplakin and plakophilin 3, and for the endosomal marker early endosomal antigen 1 and the lysosomal markers cathepsin D and lysosomal-associated membrane protein 1. Endosomes were present in all cells but did not make contact with the aggregates in the basal and suprabasal layers. In the higher layers they moored to the aggregates, often symmetrically from two adjacent cells, and IgG, DSG1, and PG were taken up. Finally these endosomes became localized perinuclear. Endocytosis was only observed in perilesional or lesional skin but not in non-lesional skin. Older immunoelectron microscopic studies have suggested that in PF skin endocytosis of detached desmosomes takes place but we found no other desmosomal proteins to be present in these endosomes. Double staining with cathepsin D and LAMP-1 revealed no overlap with IgG, DSG1, or PG suggesting that lysosomes have no role in the clearing process. Collectively, our results show that endocytosis is part of the pathogenic process in PF but that no detached desmosomes are taken up but instead the deposited IgG is taken up together with DSG1 and PG. DSG1 isin model –9. Injecin model . Waschketivation . Recent tivation . Steric tivation . We havetivation . In healormation , 9. Receormation . The cluormation . In skinormation . The reds effect .www.nanotomy.org). In perilesional skin they are present in the lower epidermal layers and they spread upwards to higher layers in lesional skin , positive binding of serum IgG (1/40 dilution) to monkey esophagus in an epithelial cell surface (ECS) pattern, and serum anti-DSG1 antibodies by MESACUP-2 ELISA test for anti-DSG1 . The skin specimens had been immediately frozen in liquid nitrogen and stored at −80°C. Skin obtained from breast reduction was used as healthy control skin. According to national regulations this type of retrospective non-interventional study on leftover tissue from diagnostics does not need approval from the local medical ethical committee.The procedure for immunofluorescence staining and image collection has been described in detail before . For visIn order to examine the fate of DSG1, PG, and IgG clusters, we double stained the perilesional and lesional biopsies for EEA1 and IgG. In 4 of the 6 biopsies EEA1 colocalized with IgG demonstrating that IgG is taken up in endosomes. This is most prominent in the higher epidermal layers, next to and beneath the blister . In moreApart from IgG and PG also DSG1 is present in the same endosomes. In order to examine if other desmosomal components are also endocytosed we examined the localization of other desmosmal proteins in relation to the EEA1/DSG1/PG clusters. No overlap was found. In the basal layer endosomes do not contact the IgG clusters . UpwardsDouble staining of DSG1 and CNX43 showed that both were present in intracellular vesicles but these did not colocalize, implying they originate from different mechanisms . SurprisIn this study we demonstrate that the clustered deposits of IgG in PF patient skin are cleared by endocytosis together with the co-clustered components DSG1 and PG . AlthougTada and Hashimoto suggested that binding of PF autoantibodies induce internalization of intact desmosomes and gap junctions in form of curvicircular structures that contained IgG, DSG1, PG, and CNX43 . Our douIn contrast with PF, PV was extensively studied in cultured cells and it is shown that PV IgG upon incubation is taken up by endocytosis together with DSG3 and PG –23. AfteTaken together we propose the following sequence of the events in PF patient skin. PF IgG binding to non-desmosomal DSG1 induces clustering and reorganization of non-desmosomal DSG1 together with PG which is bound to the cytoplasmic tail of DSG1. As other molecules as DP are absent desmosomal DSG1 is likely not included in these clusters. This causes lack of DSG1 in desmosomal turnover resulting in depletion of DSG1 from desmosomes and reduction of their size. Reorganized DSG1-PG-IgG are trapped into clusters which are likely the double membrane structures seen by electron microscopy . ProteinAll datasets generated for this study are included in the article/DO, HP, and MJ contributed to the design of the study. DO, DK, and ES performed the experiments. DO and ES wrote the manuscript. HP and MJ revised the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Plasmodium falciparum invasion of erythrocytes. However, EBA-175 is shed post invasion and a role for this shed protein has not been defined. We show that EBA-175 shed from parasites promotes clustering of RBCs, and EBA-175-dependent clusters occur in parasite culture. Region II of EBA-175 is sufficient for clustering RBCs in a GpA-dependent manner. These clusters are capable of forming under physiological flow conditions and across a range of concentrations. EBA-175-dependent RBC clustering provides daughter merozoites ready access to uninfected RBCs enhancing parasite growth. Clustering provides a general method to protect the invasion machinery from immune recognition and disruption as exemplified by protection from neutralizing antibodies that target AMA-1 and RH5. These findings provide a mechanistic framework for the role of shed proteins in RBC clustering, immune evasion, and malaria.Erythrocyte Binding Antigen of 175 kDa (EBA-175) has a well-defined role in binding to glycophorin A (GpA) during  Plasmodium falciparum is a causative agent of malaria that alternates between an insect vector and a human host. Within the human host, the parasite undergoes an asymptomatic exo-erythrocytic cycle followed by a symptomatic erythrocytic cycle. During the erythrocytic cycle, merozoite stage parasites invade red blood cells (RBCs) to initiate asexual replication. This is followed by parasite egress from cells and subsequent reinvasion of uninfected RBCs by daughter merozoites. Invasion of RBCs by parasites is a multistage process characterized by 1) initial weak engagement of the RBC by the parasite, 2) apical reorientation of the parasite and strong anchoring to the RBC, 3) tight junction formation, 4) active invasion utilizing an actin-myosin motor, and 5) shedding of the surface proteins of the parasite  and this interaction has a well-defined role in anchoring the parasite during invasion of erythrocytes (Erythrocyte Binding Antigen of 175 kDa (EBA-175) is a hrocytes . EBA-175hrocytes . During hrocytes .Plasmodium proteins that includes DBP, EBL-1, EBA-140, and EBA-181  family of  EBA-181 . EBL fam EBA-181 , and the EBA-181 . The min EBA-181 , which t EBA-181 . Binding EBA-181 . This bin of GpA . Removal culture . These sphorin B .50 of 0.1 mg/mL for parasites and can block binding of EBA-175 to GpA  family of proteins contains multiple members; each recognizing distinct receptors on RBCs during invasion. RH5, a member of the RH family, binds to the RBC receptor basigin, which is essential for invasion (Merozoites use a diverse array of proteins to ensure attachment to and invasion of RBCs. Many of these proteins are involved in establishing a tight junction during invasion and antibodies to these proteins are known to prevent invasion and reduce parasite growth. The interaction with GpA can be blocked by antibodies (Abs) that target the dimer interface and glycan binding pockets of EBA-175 . Ab-217,5 to GpA . Ab-218,5 to GpA . These sinvasion . Post-atinvasion . Both RHinvasion .Plasmodium falciparum parasites. EBA-175-mediated clustering may facilitate the high parasite burdens observed during severe malaria, protect parasites from antibody recognition, and prevent infected erythrocyte clearance.In this study, we establish a role for shed EBA-175 in RBC clustering and examine the biological consequences of this phenomenon. We embarked on this line of investigation after observing potent clustering of red blood cells upon addition of the binding domain of EBA-175 . Here, wEBA-175 is shed by the parasite during invasion. To identify if EBA-175 has an additional role in infection after being shed post invasion, native EBA-175 was purified from a culture of Dd2 schizonts that had undergone rupture and reinvasion overnight. EBA-175 was immunoprecipitated (IP) using Ab-218 that is specific for EBA-175. Purified native EBA-175 was observed on a Coomassie stained protein gel as a band at ~175 kDa in the anti-EBA-175 lane . The conThe elutions from the control or the α-EBA-175 pulldown were added to RBCs in PBS for one hour at room temperature and the overall particle size of the sample analyzed by flow cytometry. The native EBA-175 induced clustering of RBCs while the control elution showed no size change . The freTo assess whether endogenously shed EBA-175 could induce RBC clustering in a culture environment, NF54-GFP-Luc cultures at high parasitemia were analyzed for RBC clustering by flow cytometry analysis of particle size by FSC-A and parasite viability by GFP signal . High paShed EBA-175 is comprised of the entire extracellular domain of EBA-175 from regions I through VI . Of thesThe individual DBL domains that comprise RII, F1 and F2, were also tested for their ability to cause clustering of RBCs by flow cytometry . IncubatRBC clustering was dependent on GpA on RBCs as neuraminidase treatment, an α-GpA monoclonal Ab, or Ab-217, all prevented red cell clustering . Neurami2 shear stress which mimics the flow rate found in the microvasculature. Clusters were observed at all concentrations tested and the mean cluster area , and mean number of clusters , increased in response to protein concentration , where almost all RBCs were contained within clusters, with very few remaining free  and could be seen up to 10 nM  were added to the parasite cultures. Ab-217 blocked the RII-mediated growth enhancement  while thinvasion  and did Microscopy analysis of NF54-GFP-luc cultures in the presence of nanomolar RII revealed large clusters of uninfected RBCs associated with infected RBCs . More thWe asked whether RBC clustering confers parasites a survival advantage, other than growth enhancement, in the presence of known neutralizing Abs during subsequent rounds of invasion following cluster formation. We assessed the effect of RII-mediated red cell clustering on parasite viability by α-AMA1 neutralizing Abs (N3-2D9 and N4-1F6), and the α-RH5 neutralizing Ab 9AD4 (36). The two α-AMA1 antibodies blocked the growth of 3D7 parasites by 20% and 50%, respectively in the absence of RII. Strikingly, addition of 30 nM RII strongly reduced the ability of these Abs to neutralize parasites . The FVOWe next assessed a recombinant α-RH5 mouse Ab that includes the variable region of Ab 9AD4 which potently blocks RH5 engagement of the receptor basigin . In our Plasmodium infections. However, neither the molecular basis for this process nor how it might relate to disease pathogenesis have been fully defined. The results support a model , N3 and N4 (anti-AMA1), and 8C2 were purified from hybridoma supernatants using Protein A agarose resin . Antibodies were buffer exchanged into PBS after purification and sterile filtered for use in parasite cultures. The heavy and light chain variable segments of 9AD4 as deposited in PDB 4U0R  were graRBCs at 2% hematocrit in PBS were utilized in the flow cytometry assays. Elutions from the two immunoprecipitation reactions were added to RBCs for 1 hr at room temperature. The samples were analyzed by a BD FACSCanto  machine with one million events recorded for each technical replicate. Each biological replicate was read five times for a total of five technical replicates. Three biological replicates each consisting of five technical replicates were analyzed by FlowJo and gated according to the no protein control.NF54-GFP-luc parasites were grown without media change for 5 days. Antibodies Ab-217 and Ab-218 were added to cultures 12 hr prior to analyzing with a BD FACSCanto , prior to the next round of egress. One million events were recorded for each technical replicate. Five biological replicates each consisting of three technical replicates were collected. All data was analyzed by FlowJo and gated according to the no protein control.Red blood cells at 2% hematocrit in PBS were utilized in the flow cytometry assays. RII, F1, or F2 were added at varying concentrations to the RBCs for 30 min at room temperature and then washed three times with PBS. The samples were then run using a BD FACSCanto  and analyzed with FlowJo software. One hundred thousand events were recorded for each biological replicate with three biological replicates total. Antibodies at varying concentrations were preincubated with RII at room temperature for 30 min before incubation with RBCs before analysis. For experiments with neuraminidase, 50:50 (v/v) RBCs in RPMI were treated with 10 mU/mL neuraminidase  for 1 hr at 37°C. All data was analyzed by FlowJo and gated according to the no protein control. For those experiments with O, A, and B blood types the same procedure was followed as described above with one million counts read per sample.2, which mimics the wall shear stress in the microvasculature (https://ibidi.com/). The μ-slide VI chambers  were blocked overnight at 4°C with PBS/1% BSA. The following day, 5 ml 1% Ht 0 + RBC in RPMI (pH7.2–7.4) was prepared. The suspension was added to the reservoir syringe and allowed to flow over the μ-slide VI for 5 min at 1 dyn/cm2, before stopping the flow and immediately capturing both bright-field images from 10 consecutive fields with a 20 × objective, using a Leica DM1 inverted light microscope. In each experiment, the RBC without protein sample was tested first, followed by the samples containing EBA-175 RII protein starting from the lowest concentration (1 nM) followed by 10 nM then 100 nM.The cluster formation assays under flow were carried out as described previously  with minculature . To genehttp://rsb.info.nih.gov/ij/), each of the 10 images corresponding to 10 fields per sample were analysed, and the size of clusters was measured. In preparation for measurement, the threshold settings were adjusted to highlight the clusters, in order to allow the margins of the cell clusters to be identified more clearly. Briefly, the captured images were processed using brightness and contrast, followed by threshold to enhance the visualization of clusters against the background. Clusters were selected using the Analyze/Measurement settings, with the ‘wand’ tool, which highlights the circumference of the selected rosette and calculates the area (µm2). All of the measurements from each image were added and the results expressed as mean cluster area (µm2) and mean number of clusters from 10 fields, from three independent experiments.Using ImageJ software . Mean cluster values from at least three independent experiments for each concentration were compared by one-way ANOVA, with p<0.05 being taken as statistically significant.P. falciparum strains 3D7, Dd2, FVO/FCR1, and HB3 were cultured in fresh human O+red blood cells  at 2% hematocrit in rich medium . For assessing blood type influence on RII-mediated growth, parasites were cultured in fresh human A+, B+, or O+red blood cells at 2% hematocrit in rich medium. Synchrony of parasites was maintained with successive rounds of 5% sorbitol treatment as described previously  was cultured in rich medium with 10 nM WR99210 . For assessment of natively shed EBA-175, NF54HT-GFP parasites were cultured without media change for 5 days to allow accumulation of shed EBA-175 in the growth media.The eviously . A trans3H]-hypoxanthine (0.5 μCi/well) was then added and parasites were incubated for a further 18–24 hr in assay medium. The assay duration (96 hr) corresponds to approximately two intraerythrocytic developmental cycles. The assay is stopped by harvesting the well contents onto a filter paper (Skatron FilterMAT 11731) using a semiautomatic cell harvester . After air dried, the mats were analyzed using Beckman LS6000 IC liquid scintillation counter. Each [3H]-hypoxanthine incorporation measurement was performed in duplicate  for three independent times.Semi-synchronous 3D7, Dd2, FVO/FCR1, and HB3 in trophozoite/schizont stages at 2% hematocrit were plated in 96-well plates with a starting parasitemia of 2.5–3.0% and the cultures were kept shaken throughout the experiment to mimic in vivo conditions and limit the number of multiple invasion events into a single RBC. Purified RII and/or antibodies were immediately added to cultures after plating. These cultures were allowed to progress through 1.5 cycle (72 hr) in assay medium (90% hypoxanthine-free rich medium mixed with 10% rich medium). eLife. Your article has been reviewed by three peer reviewers, one of whom is a member of our Board of Reviewing Editors, and the evaluation has been overseen by a Senior Editor. The following individuals involved in review of your submission have agreed to reveal their identity: Richard Culleton (Reviewer #2).Thank you for submitting your work entitled "Shed EBA-175 mediates red blood cell clustering that enhances malaria parasite growth and enables immune evasion" for consideration by eLife.Our decision has been reached after consultation between the reviewers. Based on these discussions and the individual reviews below, we regret to inform you that your work will not be considered further for publication in Plasmodium falciparum EBA-175 mediates red cell clustering during parasite invasion and hence promotes parasite growth and also enables immune evasion. Although EBA-175 has been widely investigated and many aspects of this glycophorin binding protein are known, the function of the shed EBA-175 has not been defined. By performing a series of well-designed experiments, the authors expanded our understanding of this protein crucial for the survival of the parasite and a possible target for malaria vaccine.In this nicely written manuscript, the authors show that shed Plasmodium falciparum infection is indeed attractive. Therefore, the in vitro phenomenon should be replicated in vivo under physiological conditions and that may necessitate a substantial body of extra work and revision to validate the main hypothesis. In addition, validation of the purification of shed EBA-175, description and validation of flow cytometry data, and the number biological replicates needs to be included.The reviewers have also expressed major concerns and those are included in each individual review attached below. The main issue surrounds the lack of results in support of the authors claim concerning EBA-175-mediated clustering as a possible mechanism that may be occurring in vivo. The hypothesis that EBA-175 aggregated red blood cells may in fact contribute to pathogenesis in natural Should the authors perform the suggested experiments and provide data in support of their claims, they are encouraged to submit their manuscript as a new submission.Reviewer #1:Plasmodium infection of red blood cells. This shed EB-175  not only potentiates the growth of the parasites by inducing agglutination or clustering of RBC, but it also causes or leads to immune evasion by preventing antibodies to access the parasite.The authors describe their observations concerning an interesting phenomenon, namely the shedding of EB-175 during From my quick read of this manuscript I was quite excited about the EBA-175 story from Niraj Tolia's lab and thought the paper worthwhile for an external review. Upon closer reviewing and particularly examining the results and the approach, my enthusiasm has somewhat diminished. They support their claim of the shed EBA-175-induced clustering by showing that antibodies against EBA-175 do prevent this clustering, whereby in the absence of these antibodies, the parasites do expand/grow. This is shown in Figure 3 with Ab-217 that prevent parasite growth. However, I am not clear about this particular representation of their data mainly because nowhere in the manuscript do the authors provide adequate explanations about this particular manner of data presentation, stats, normalization, etc.In essence, I have some concern about the veracity of some of the results given the number of biological replicates as in the case of results shown in Figure 1C, which represent a single biological replicate, of 5 technical replicates. Similarly, the results in Figure 2B that demonstrate the clustering effect brought about by RII, at titrated concentration, and not by F1 or F2, appear to be from a single biological replicate; the figure legend is not particularly informative. Similar concern is directed to results in Figure 2D where the authors show that treating Plasmodium RBC cultures with NA and with antibodies to GpA also inhibits clustering. It is not clear if the differences between untreated and treated are significant? Subsection “EBA-173f RII promotes RBC clusters in a GpA dependent manner” – the results are poorly described. In general, although the in vitro results are interesting, some results are difficult to understand.P. falciparum infection and that this shedding may lead to malaria-induced pathogenesis. Although an attractive hypothesis, the authors claim that it would be impossible to test it in humans because the shed EBA-175 could not be measured in sera of malaria patients as the protein would be bound to RBC. Could clustered RBC be detected in P. falciparum infected persons?The authors propose that this mechanism of EBA-175 shedding may actually occur in persons suffering from I would think that the authors could have tested this hypothesis in vivo studies in mice, e.g., mice with deleted GpA in which case the shed EBA-175 would not bind to RBC? Alternatively, would it be possible to have used CRISPR/Cas9 to edit the EBA-175 gene? Hence, although the results are potentially interesting and the suggestion that such mechanism may in fact operate to promote the spread of malaria infection, in my opinion, the results are not fully convincing.Reviewer #2:P. falciparum, and that it benefits the parasite by increasing replication rates through reducing the amount of time required for daughter merozoites to encounter new RBCs to invade, and through shielding of the iRBC from the action of growth and invasion inhibitory antibodies.This manuscript by Paing et al. is a nicely written account of a series of well designed and simple experiments that show that clustering of red blood cells in in vitro culture is mediated by region 2 (RII) of EBA-175 shed by the parasite during invasion. The authors hypothesise that this clustering of non-infected red blood cells (RBCs) around infected RBCs (iRBCs) also occurs in natural infections of The experiments that show that clustering occurs in in vitro cultures, and that this can be mediated through shed RII of EBA-175, are on the surface well thought out and conducted, but could be made greatly more robust by validation of the purification of shed EBA-175, and description and validation of the flow cytometry data.Moreover, the author's interpretation of the utility of this phenomenon to the parasite is based on the assumption that clustering also occurs in vivo. I think that the authors need to present evidence that 'clusters' are viable under the large stresses and shear forces encountered in the host blood stream, and that they remain viable under these conditions for the 48 hours required for the daughter merozoites to egress from the original iRBC. Apparatus and protocols for measuring the effects of flow on malaria parasite iRBCs are well established , and would benefit this work immensely. I feel that without evidence that clustering could occur in vivo, the authors are overreaching in their interpretation of the significance of these results. I recommend that the authors test the robustness of these clusters under relevant physiological conditions in flow chamber experiments. There may also be scope to investigate clustering in rodent malaria parasites.Introduction:Clumping of erythrocytes by bivalent antibodies is well described and, indeed, has been the basis of erythrocyte typing. A dimer of shed EBA-175 might be hypothesized to confer a similar activity, based upon a bivalent structure and affinity for GpA. Although it has been presented many times in the literature, I think that a schematic of the domain structure of EBA-175, would introduce the general reader to the arrangement of RII, F1, F2, low complexity region, RVI, and the TM domain. Perhaps a model comparing the predicted structure and dimensions of bivalent shed EBA-175 with a bivalent antibody, to support the hypothesis of a clumping capacity.Plasmodium literature has a long history describing erythrocyte rosetting, this should also be introduced, and the parasite proteins proposed to be involved, and distinguished from a new hypothesis involving EBA-175. It would be useful for the reader to provide a thorough definition of 'clustering', as this term may be unfamiliar to many readers. Clustering should be compared to 'rosetting' and 'clumping' and their differences and similarities discussed.The Plasmodium falciparum is also known for the propensity of infection of erythrocytes by multiple ring forms, suggesting that the rupture of a schizont results in multiple events of an adjacent uninfected erythrocyte.Plasmodium to duplicate and amplify gene families. That is, for the parasite to take full advantage of evolutionary selective pressure for rosetting, it might duplicate the EBA-175 gene, use one copy specifically for invasion, and the second copy may evolve toward rosetting. Indeed, the DBL domains of specific PfEMP1 proteins are proposed to be involved in rosetting.I am always wary when two distinct functions are attributed to a single parasite protein. Particularly given the propensity of Results:Figure 1A. I am stunned that the authors can immunoprecipitate sufficient shed EBA-175 to see on a Coomassie stained gel. How much culture was used in this experiment? My recollection is that the visualization methods in the literature are Western blotting or radioisotope labelling, is there precedence otherwise? The identity of the high MW band should be confirmed by Western blotting with an independent antibody, and since the material is so abundant by mass spec or peptide sequencing. As it stands, I am highly skeptical that it is possible to purify such abundant material.Figures 1 and 2: The flow cytometry figure or legend should indicate the number of events sampled, it appears to vary. Many of the plots are blown out, is the frequency of the events so rare? Is the flow cytometry data consistent with the rosetting seen by light microscopy? In Figure 1B I don't understand which indicates clustering, forward or side scatter, and if the authors are arguing that both populations are indicative? For example, the pattern in Figure 1B is different than Figure 2B; and again in Figure 1D. Perhaps a flow cytometry machine which captures images of the particles could be used.Figure 1: It appears that Ab-218 significantly reduces clustering, yet this antibody is known to target a non-functional epitope of EBA-175. What is the explanation for this?Figure 2: Perhaps another region of EBA-175 should be used as a negative control here.Figure 3: Can the authors propose a reason for the fact that the growth of Dd2 is enhanced to greater degree than the other strains? Does Dd2 grow more slowly than the other strains under normal culture conditions? Could it be that the merozoites of Dd2 have shorter viable life spans than merozoites of the other strains?Figure 4: Again, another region of EBA-175 might make an appropriate negative control here.Subsection “Shed EBA-175 purified from parasite culture induces RBC clustering”: "To identify a role for the shed protein…" This is overly presumptive, you should temper it by saying, "To identify if EBA-175 has an additional role after shedding…" Also in the Abstract, second sentence, qualify that a role is speculative.Subsection “EBA-175 mediated RBC clustering is observed in parasite cultures”: "…nanomolar or greater levels." Please support, by reference or provide calculations.Subsection “Clustering of RBCs mediated by RII enhances parasite growth”: It is unnecessary to emphasize "gold standard."Subsection “Clustering of RBCs mediated by RII enhances parasite growth”: "Dd2 has previously been shown…" I don't understand the reasoning here, that the effect of exogenous EBA-175 is somehow correlated with Dd2's dependence on endogenous EBA-175. The clustering effect should be independent, no?Subsection “Clustering of RBCs mediated by RII enhances parasite growth”: Is it necessary to hypothesize that RII might have some direct relationship with hypoxanthine incorporation, that must be controlled for with a second growth assay?Subsection “Clustering of RBCs mediated by RII enhances parasite growth “: "…likely due to…" is speculation.Subsection “Clustering of RBCs mediated by RII enhances parasite growth”, last paragraph: Do you mean to say Figure 3B?Subsection “EBA-175-RII-mediated RBC clustering confers parasites protection from diverse neutralizing Abs”, first paragraph: I don't think that basigin needs to be capitalized.Discussion:In general, additional validation of the EBA-175 purification and flow cytometry experiments is required, and the Discussion is too far reaching in terms of the potential biological significance of this work. Without firm evidence that clustering occurs in vivo under typical physiological conditions, the speculation pertaining to the advantages effects of cluster formation is not justified based on the experimental evidence presented here. The authors have shown convincingly that EBA-175 mediated clustering occurs in in vitro cultures, and that this can lead to increased parasite growth rates, but their experiments do not show that this occurs in natural infections.Discussion, first paragraph: 'uninfected' should be used instead of 'naïve'.P. yoelii is more complicated than the authors describe. The redirected localisation of EBL from the micronemes to the dense granules that occurs as a result of a mutation in R6 of EBL in 17XL, leads to a change in red blood cell tropism; from a predilection for the invasion of reticulocytes to the ability to invade RBCs of all ages. This change in RBC cell invasion preference cannot be explained through enhanced clustering. Furthermore, it has also been shown that another mutation in PyEBL, this time in RII, also increases virulence (again through an increase in the RBC invasion repertoire), but does not lead to localisation away from the micronemes .Discussion, second paragraph: The situation regarding EBL and virulence in References:Abkallo HM, Martinello A, Inoue M, Xangsayarath P, Gitaka J, Yahata K, … Culleton R. Rapid identification of genes controlling virulence and immunity in malaria parasites. PLoS Pathog. 2017 Jul 12;13(7):e1006447. doi: 10.1371/journal.ppat.1006447Dasanna AK, Lansche C, Lanzer M, Scwarz US. Rolling Adhesion of Schizont Stage Malaria-Infected Red Blood Cells in Shear Flow. Biophys J. 2017 May 9;112(9):1908-1919. doi: 10.1016/j.bpj.2017.04.001.Reviewer #3:Plasmodium falciparum invasion of erythrocytes. However, EBA-175 is shed post invasion and a role for this shed protein has not been defined. The authors showed that EBA-175 shed from parasites promotes clustering of RBCs. Region II of EBA-175 is sufficient for clustering RBCs in a GpA-dependent manner. EBA-175-dependent RBC clustering enhances parasite growth and provides a general method to protect the invasion machinery from immune recognition. Finally, they proposed a mechanistic framework for the role of shed EBA-175 in RBC clustering, immune evasion, and severe malaria. I think the experiments were carefully designed and conducted; the results were appropriately discussed, and clearly written. I also agree them that these data will serve as a baseline information understanding novel roles of erythrocyte binding proteins.EBA-175 plays an important role in binding to glycophorin A (GpA) during 1) IntroductionPlease insert one paragraph which describes the reason(s) why they focused on the shed-EBA-175 and RBC clustering? Rosetting should also be mentioned in the Introduction.2) Results Majority in this paragraph contains introductory description. Please bring this to Introduction section.3) Figure 3D clearly indicated that a cluster contains 1 or 2 infected RBC. In this experimental condition, it is very important to know how much% of the RBC clusters contains iRBC? Are there any iRBC negative clusters? Also, it is important to know how much% of the iRBC associated with normal RBC clusters?[Editors’ note: what now follows is the decision letter after the authors submitted for further consideration.]eLife. Your revised article has been favorably reviewed by three peer reviewers, one of whom is a member of our Board or Reviewing Editors, and the evaluation has been overseen by Tadatsugu Taniguchi as the Senior Editor.Thank you for resubmitting your work entitled "Shed EBA-175 mediates red blood cell clustering that enhances malaria parasite growth and enables immune evasion" for further consideration at The manuscript has been improved but there are some remaining issues that need to be addressed before acceptance. For the most part the suggested revisions concern clarifications of the experimental approaches, e.g., rounds of parasite replications, inclusions of more detailed legend explanations, or corrections of typographical errors. The comments from the reviewers are included below.Reviewer #1:The new submission of the revised manuscript by Niraj Tolia is now sufficiently improved. The majority of queries were addressed, particularly by providing results from additional experiments and clarifying entries in the legends and the text concerning biological replicates.There are no substantive concerns regarding this current version of the revised manuscript.Reviewer #2:This manuscript is a revised re-submission of the previous version by Prof. Niraj H Tolia's group. Based on the previous reviewers' comments, the biggest concern was that the clustering phenomenon clearly showed in vitro should be proved under physiologically relevant flow conditions to establish the in vivo relevance of the findings. The authors conducted additional experiments to prove and answer to this critical question described in the Materials and methods section, "Cluster Formation Assays under Flow" and "Quantification of Cluster Under Flow", Figure 2E and Figure 2—figure supplement 2B.I think all the experiments including additional flow experiments were carefully designed and conducted; the results were appropriately discussed, and clearly written. I also agree them that these data will serve as a baseline information understanding novel roles of erythrocyte binding proteins. However, I still have the following comments to improve this re-submitted manuscript.1) Please clearly describe which experiments were conducted in the flow-condition in the respective figure legends."EBA-175 RII induced clusters are viable under physiological flow conditions" Please add data when no RII has added in Figure 2E.2) "for two rounds of replication.…… erythrocytes in circulation."Why did they use two rounds of replication (not one cycle assay)? Please describe the reason.3) "Concentrations of RII greater than 100 nM … both DBL domains ." Should be in the Discussion section. But I felt this part sounds over discussion. Please consider to remove.4) Subsection “EBA-175-RII-mediated RBC clustering confers parasites protection from diverse neutralizing Abs”, last paragraph: To interpret the results described here, the authors should pay more careful attention to the timings when RH5, AMA1 and EBA-175 works during merozoite invasion into the erythrocyte.Reviewer #3:Plasmodium parasites during invasion promotes red blood cell clustering in culture. They show that the clustering is mediated by the glycophorin A binding region of EBA-175, called RII. Intriguingly, they observe that addition of recombinant RII protein to generate RBC clusters both enhances parasite growth and serves as an immune evasion strategy by hindering access of invasion-inhibitory antibodies.In this eloquent manuscript, the authors investigate the phenomenon whereby EBA-175 shed by The authors clearly demonstrate the EBA-175-mediated clustering of RBCs in vitro, and have revised an earlier version of the manuscript to include evidence that clustering occurs under conditions approximating physiological flow conditions. However, there is no in vivo evidence for EBA-175 mediated clustering, such as the observation of clusters from patients, or an estimate of the blood concentration of EBA-175 (which latter the authors posit is not possible). As a result, the authors' hypothesis that EBA-175-mediated clustering plays a role in enhancing parasite growth and immune evasion in severe malaria is speculative. Further, there is controversy surrounding the function of rosetting in vivo – as the authors point out in the Discussion, there is limited experimental evidence that rosetting leads to increased parasitemia or immune evasion. Nevertheless, I agree with the authors' comment that field studies are beyond the scope of this paper. In light of this, I would suggest that the authors make a point in the Discussion that their model still needs to be validated in vivo.Overall I think this work is interesting and novel, and the experiments well-designed, well-executed, and well-presented. [Editors’ note: the author responses to the first round of peer review follow.]We appreciate the in-depth review and have revised the manuscript to address the reviewers’ concerns. The new version includes additional results to show that EBA-175 induced clusters are stable under physiologically relevant flow condition. We also present mass spectrometry data that unambiguously establishes the purified protein is shed native EBA-175. We have also significantly revised the manuscript to improve the description of flow cytometry data and experimental replicates.[…] The reviewers have also expressed major concerns and those are included in each individual review attached below. The main issue surrounds the lack of results in support of the authors claim concerning EBA-175-mediated clustering as a possible mechanism that may be occurring in vivo. The hypothesis that EBA-175 aggregated red blood cells may in fact contribute to pathogenesis in natural Plasmodium falciparum infection is indeed attractive. Therefore, the in vitro phenomenon should be replicated in vivo under physiological conditions and that may necessitate a substantial body of extra work and revision to validate the main hypothesis. In addition, validation of the purification of shed EBA-175, description and validation of flow cytometry data, and the number biological replicates needs to be included.We thank the reviewers for the in-depth review. We have provided additional experiments requested by the reviewers to study the clustering phenomenon under physiologically relevant flow conditions to establish the in vivo relevance of the findings. These are now presented in Figure 2 and Figure 2—figure supplement 2 as well as an independent section in the Results.In addition, as requested by the reviewers, we now provide mass spectrometry data to unambiguously validate the purified protein is shed EBA-175. We have also extensively revised the manuscript to improve the presentation and description of replicates, and to improve the presentation of the flow cytometry data.Should the authors perform the suggested experiments and provide data in support of their claims, they are encouraged to submit their manuscript as a new submission.Reviewer #1:[…] From my quick read of this manuscript I was quite excited about the EBA-175 story from Niraj Tolia's lab and thought the paper worthwhile for an external review. Upon closer reviewing and particularly examining the results and the approach, my enthusiasm has somewhat diminished. They support their claim of the shed FBA-175- induced clustering by showing that antibodies against EBA-175 do prevent this clustering, whereby in the absence of these antibodies, the parasites do expand/grow. This is shown in Figure 3 with Ab-217 that prevent parasite growth. However, I am not clear about this particular representation of their data mainly because nowhere in the manuscript do the authors provide adequate explanations about this particular manner of data presentation, stats, normalization, etc.In essence, I have some concern about the veracity of some of the results given the number of biological replicates as in the case of results shown in Figure 1C, which represent a single biological replicate, of 5 technical replicates. Similarly, the results in Figure 2B that demonstrate the clustering effect brought about by RII, at titrated concentration, and not by F1 or F2, appear to be from a single biological replicate; the figure legend is not particularly informative. Similar concern is directed to results in Figure 2D where the authors show that treating Plasmodium RBC cultures with NA and with antibodies to GpA also inhibits clustering. It is not clear if the differences between untreated and treated are significant? Subsection “EBA-173f RII promotes RBC clusters in a GpA dependent manner” – the results are poorly described. In general, although the in vitro results are interesting, some results are difficult to understand.We apologize that the number of replicates for each experiment was not adequately presented. A thorough explanation of the number of technical and biological replicates in each experiment was added to the manuscript (in figure legends and in text). A minimum of three biological replicates were conducted for each experiment. For those experiments that were analyzed for statistics, at least five biological replicates were conducted.The authors propose that this mechanism of EBA-175 shedding may actually occur in persons suffering from P. falciparum infection and that this shedding may lead to malaria-induced pathogenesis. Although an attractive hypothesis, the authors claim that it would be impossible to test it in humans because the shed EBA-175 could not be measured in sera of malaria patients as the protein would be bound to RBC. Could clustered RBC be detected in P. falciparum infected persons?This would require setting up a field study which regrettably is beyond the scope of this manuscript.I would think that the authors could have tested this hypothesis in vivo studies in mice, e.g., mice with deleted GpA in which case the shed EBA-175 would not bind to RBC? Alternatively, would it be possible to have used CRISPR/Cas9 to edit the EBA-175 gene? Hence, although the results are potentially interesting and the suggestion that such mechanism may in fact operate to promote the spread of malaria infection, in my opinion, the results are not fully convincing. However, I am going to defer my final opinion once we hear from the experts in the field.P. falciparum, and are unlikely to provide insight.Regrettably, these experiments are not possible. This is primarily because the EBA-175/GpA interaction is not replicated in mouse models of malaria. EBA-175 cannot bind murine glycophorin A. This lack of binding is due to a modification on the sialic acid of mouse Oglycan that is not found in humans. In addition, there is no direct ortholog of EBA-175 that can be studied in mice. A CRISPR/Cas9 knock out of EBA-175 would also not add to the manuscript as traditional knock outs of EBA-175 show little to no phenotype in culture. Knockout studies are also confounded by the redundancy in the EBL family of proteins in Reviewer #2:[…] The experiments that show that clustering occurs in in vitro cultures, and that this can be mediated through shed RII of EBA-175, are on the surface well thought out and conducted, but could be made greatly more robust by validation of the purification of shed EBA-175, and description and validation of the flow cytometry data.We agree that a more robust validation of the purification of shed EBA-175 would improve the manuscript. In the revised manuscript we provide mass spectrometry data to establish that the purified protein is shed EBA-175. We also improve the description of the flow cytometry data to improve clarity.Moreover, the author's interpretation of the utility of this phenomenon to the parasite is based on the assumption that clustering also occurs in vivo. I think that the authors need to present evidence that 'clusters' are viable under the large stresses and shear forces encountered in the host blood stream, and that they remain viable under these conditions for the 48 hours required for the daughter merozoites to egress from the original iRBC. Apparatus and protocols for measuring the effects of flow on malaria parasite iRBCs are well established , and would benefit this work immensely. I feel that without evidence that clustering could occur in vivo, the authors are overreaching in their interpretation of the significance of these results. I recommend that the authors test the robustness of these clusters under relevant physiological conditions in flow chamber experiments. There may also be scope to investigate clustering in rodent malaria parasites.We agree that demonstrating the clusters are stable under physiologically relevant shear forces would establish the in vivo relevance of the findings. In the revised manuscript we now show that EBA-175 mediated clusters are stable to the large stresses and shear forces induced by relevant physiological conditions in flow chamber experiments. The new data are included in Figure 2E and Figure 2—figure supplement 2B.Regrettably, rodent models of malaria cannot be used to examine clustering by EBA-175 as mice modify sialic acid residues on glycophorin A, and this modification prevents binding by EBA175. Furthermore, a clear ortholog for EBA-175 has not been identified in rodent malaria parasites. This comment is related to our last response to reviewer 1.Introduction:Clumping of erythrocytes by bivalent antibodies is well described and, indeed, has been the basis of erythrocyte typing. A dimer of shed EBA-175 might be hypothesized to confer a similar activity, based upon a bivalent structure and affinity for GpA. Although it has been presented many times in the literature, I think that a schematic of the domain structure of EBA-175, would introduce the general reader to the arrangement of RII, F1, F2, low complexity region, RVI, and the TM domain. Perhaps a model comparing the predicted structure and dimensions of bivalent shed EBA-175 with a bivalent antibody, to support the hypothesis of a clumping capacity.A domain structure of EBA-175 II, including Ab-217 and Ab-218 binding sites highlighted, is now included in Figure 1—figure supplement 1A. We do not wish to speculate on a biophysical mechanism for clustering and as such we are reticent to add the requested comparison to bivalent antibody binding. While this might be one mechanism for how clustering could be achieved, there are multiple other methods for inducing clusters and we do not wish to bias readers without further biophysical evidence beyond the scope of this manuscript.The Plasmodium literature has a long history describing erythrocyte rosetting, this should also be introduced, and the parasite proteins proposed to be involved, and distinguished from a new hypothesis involving EBA-175. It would be useful for the reader to provide a thorough definition of 'clustering', as this term may be unfamiliar to many readers. Clustering should be compared to 'rosetting' and 'clumping' and their differences and similarities discussed.We use the term clustering/clumping of RBCs to distinguish it from rosetting where a single infected RBC with PfEMP1, STEVORs, or RIFINs on its surface is surrounded by uninfected RBCs. Clustering of RBCs described here is independent of these membrane-tethered proteins.P. falciparum rosetting attributed to the parasite membrane proteins PfEMP1 , RIFINs  and STEVORs . …”A discussion of the differences between rosetting and clustering is included in the revised manuscript: “The clustering phenomenon reported here exhibits similarities to Plasmodium falciparum is also known for the propensity of infection of erythrocytes by multiple ring forms, suggesting that the rupture of a schizont results in multiple events of an adjacent uninfected erythrocyte.The cultures were grown with shaking to minimize this occurrence as per published protocols. Furthermore, the microscopy images clearly show that daughter merozoites invade independent red cells in clusters in Figure 3D.I am always wary when two distinct functions are attributed to a single parasite protein. Particularly given the propensity of Plasmodium to duplicate and amplify gene families. That is, for the parasite to take full advantage of evolutionary selective pressure for rosetting, it might duplicate the EBA-175 gene, use one copy specifically for invasion, and the second copy may evolve toward rosetting. Indeed, the DBL domains of specific PfEMP1 proteins are proposed to be involved in rosetting.We agree that the reviewer that gene duplications result in redundancy and specialization within parasite families. We believe this might occur within the EBL family as well. This will always be a concern for any members of the EBL family due to redundancy.Results:Figure 1A. I am stunned that the authors can immunoprecipitate sufficient shed EBA-175 to see on a Coomassie stained gel. How much culture was used in this experiment? My recollection is that the visualization methods in the literature are Western blotting or radioisotope labelling, is there precedence otherwise? The identity of the high MW band should be confirmed by Western blotting with an independent antibody, and since the material is so abundant by mass spec or peptide sequencing. As it stands, I am highly skeptical that it is possible to purify such abundant material.Four liters of parasite culture was split into equal portions for pulldown with either the control antibody (Ab-8C2) or the EBA-175 specific antibody (Ab-218) following the procedure adapted from O’Donnell et al. (2006). Purified native protein was identified as EBA-175 by immunoblotting  and by mass spectrometry . These results clearly demonstrate that we have successfully purified high quality EBA-175 from parasite culture.Figures 1 and 2: The flow cytometry figure or legend should indicate the number of events sampled, it appears to vary. Many of the plots are blown out, is the frequency of the events so rare? Is the flow cytometry data consistent with the rosetting seen by light microscopy? In Figure 1B I don't understand which indicates clustering, forward or side scatter, and if the authors are arguing that both populations are indicative? For example, the pattern in Figure 1B is different than Figure 2B; and again in Figure 1D. Perhaps a flow cytometry machine which captures images of the particles could be used.We apologize for not being clear about the number of replicates conducted for each experiment. We have revised Figure 1 legend so that it now reads “(B)…Frequency of events (clusters) out of one million counts are located in bottom right corner of dot plot for one representative technical replicate… (D) … Frequency of events (clusters) out of one million counts are located in bottom right corner of dot plot for one representative technical replicate. …”We have also revised Figure 2 legend so that it now reads “(B)…Frequency of events (clusters) out of 100,000 counts are located in bottom right corner of dot plot for each sample when gated according to the no protein control (top left). One representative biological replicate out of three is presented… (D)… Frequency of events (clusters) out of 100,000 counts are located in bottom right corner of dot plot for each sample when gated according to the no protein control (top left). One representative biological replicate out of three is presented…”We also agree that a flow cytometry instrument with the ability to capture images would be a benefit although such an experiment would not add greatly to the manuscript. In lieu of additional flow cytometry data, we conducted experiments under physiological flow conditions and captured images of the clusters .Figure 1: It appears that Ab-218 significantly reduces clustering, yet this antibody is known to target a non-functional epitope of EBA-175. What is the explanation for this?While significant compared to untreated, the inhibition by Ab-218 is slight and likely due to the amount of antibody used. Ab-218 has been shown to have slight inhibition of EBA-175 at high concentrations.Figure 2: Perhaps another region of EBA-175 should be used as a negative control here.We used both F1 and F2 as negative controls. The individual DBL domains serve as controls since either domain alone does not cluster RBCs. These are the most accurate negative control as they are expressed, refolded and purified in the same way as RII. These negative controls also demonstrate that an intact RII is required for the effect. It is unclear what additional negative controls will provide.Figure 3: Can the authors propose a reason for the fact that the growth of Dd2 is enhanced to greater degree than the other strains? Does Dd2 grow more slowly than the other strains under normal culture conditions? Could it be that the merozoites of Dd2 have shorter viable life spans than merozoites of the other strains?All strains used in experiments have between 45-48 hr life cycle, therefore the cycle time and life span is unlikely to contribute to the higher growth rate observed for Dd2. It is evident that the specific increase in growth by Dd2 is due to multiple factors and we do not wish to overly speculate in one direction or another. The exact mechanism for enhanced growth by Dd2 does not alter the main conclusions and focus of the manuscript that EBA-175 clustering enhances parasite growth and protects from neutralizing antibodies.Figure 4: Again, another region of EBA-175 might make an appropriate negative control here.F1 and F2 individual domains of RII have already shown not to induce enhanced parasite growth in Figure 3B. As described in our above response to reviewer 2, these serve as the most accurate negative controls.Subsection “Shed EBA-175 purified from parasite culture induces RBC clustering”: "To identify a role for the shed protein…" This is overly presumptive, you should temper it by saying, "To identify if EBA-175 has an additional role after shedding…" Also in the Abstract, second sentence, qualify that a role is speculative.We agree with the reviewer and have changed the sentence as requested.Subsection “EBA-175 mediated RBC clustering is observed in parasite cultures”: "…nanomolar or greater levels." Please support, by reference or provide calculations.We agree with this reviewer that this is speculative and have removed the sentence.Subsection “Clustering of RBCs mediated by RII enhances parasite growth”: It is unnecessary to emphasize "gold standard."We agree with the reviewer and have revised the sentence accordingly.Subsection “Clustering of RBCs mediated by RII enhances parasite growth”: "Dd2 has previously been shown…" I don't understand the reasoning here, that the effect of exogenous EBA-175 is somehow correlated with Dd2's dependence on endogenous EBA-175. The clustering effect should be independent, no?We agree with the reviewer that this sentence is not clear. We have removed it from the revised manuscript.Subsection “Clustering of RBCs mediated by RII enhances parasite growth”: Is it necessary to hypothesize that RII might have some direct relationship with hypoxanthine incorporation, that must be controlled for with a second growth assay?3H-labelled exogenous hypoxanthine could have on parasite growth.Using multiple assays ensures the results are robust and not artifacts of one particular assay. We are trying to be thorough to rule out the global effects Subsection “Clustering of RBCs mediated by RII enhances parasite growth”: "…likely due to…" is speculation.We have revised the text to: “Concentrations of RII greater than 100 nM gradually caused parasite death. Plausible reasons for the parasite death seen are a block of invasion and/or due to formation of larger and more tightly-packed RBC clusters blocking…”Subsection “Clustering of RBCs mediated by RII enhances parasite growth”, last paragraph: Do you mean to say Figure 3B?We thank the reviewer for identifying this error. We have revised the reference to the correct figure.Subsection “EBA-175-RII-mediated RBC clustering confers parasites protection from diverse neutralizing Abs”, first paragraph: I don't think that basigin needs to be capitalized.We thank the reviewer for identifying this error. We have revised the sentence as requested.Discussion:In general, additional validation of the EBA-175 purification and flow cytometry experiments is required, and the Discussion is too far reaching in terms of the potential biological significance of this work. Without firm evidence that clustering occurs in vivo under typical physiological conditions, the speculation pertaining to the advantages effects of cluster formation is not justified based on the experimental evidence presented here. The authors have shown convincingly that EBA-175 mediated clustering occurs in in vitro cultures, and that this can lead to increased parasite growth rates, but their experiments do not show that this occurs in natural infections.We agree with the reviewer that examining clustering under physiologically relevant flow conditions establishes the in vivo role for this phenomenon. In the revised manuscript we present new clustering data under physiological flow conditions that demonstrate the EBA-175 clusters are stable under physiological stresses.Discussion, first paragraph: 'uninfected' should be used instead of 'naïve'.We agree with this change and have revised the manuscript where appropriate.Discussion, second paragraph: The situation regarding EBL and virulence in P. yoelii is more complicated than the authors describe. The redirected localisation of EBL from the micronemes to the dense granules that occurs as a result of a mutation in R6 of EBL in 17XL, leads to a change in red blood cell tropism; from a predilection for the invasion of reticulocytes to the ability to invade RBCs of all ages. This change in RBC cell invasion preference cannot be explained through enhanced clustering. Furthermore, it has also been shown that another mutation in PyEBL, this time in RII, also increases virulence (again through an increase in the RBC invasion repertoire), but does not lead to localisation away from the micronemes .P. yoelii is complex. We have removed this section as requested.We agree with the reviewer that the relationship of clustering to virulence in Reviewer #3:[…]1) IntroductionPlease insert one paragraph which describes the reason(s) why they focused on the shed-EBA-175 and RBC clustering? Rosetting should also be mentioned in the Introduction.We have added the following to the Introduction to explain the rationale for focusing on this phenomenon:“In this study, we establish a role for shed EBA-175 in RBC clustering and examine the biological consequences of this phenomenon. We embarked on this line of investigation after observing potent clustering of red blood cells upon addition of the binding domain of EBA-175.”We retained the description of rosetting in the Discussion. We feel this is easier for the reader to understand the relevance of rosetting and relationship to clustering once the results have been presented. Moving the description of rosetting to the Introduction does not fit with the focus of the manuscript (i.e. EBA-175) and is confusing as EBA-175 has no role in rosetting.2) Results Majority in this paragraph contains introductory description. Please bring this to Introduction section.We agree and this material was moved to the Introduction.3) Figure 3D clearly indicated that a cluster contains 1 or 2 infected RBC. In this experimental condition, it is very important to know how much% of the RBC clusters contains iRBC? Are there any iRBC negative clusters? Also it is important to know how much% of the iRBC associated with normal RBC clusters?In the culture, both clusters with and without iRBC can be seen. Clusters on average contain 1 to 2 iRBCs although as many as 7 could be seen in a cluster. Not all iRBC can be seen at once as the cluster is a three dimensional structure and would require the use of z-stacks to image every iRBC and uRBC in the cluster. This was not done for this experiment although multiple images were taken of the larger clusters like the one in Figure 3D to observe other iRBCs.While we agree with the reviewer that establishing the number and composition of clusters is an important future aspect for this research, we feel these results are beyond the scope of the current manuscript. In addition, while these results would provide interesting insight they do not add to the current focus of the manuscript which describes the novel phenomenon of clustering.[Editors' note: the author responses to the re-review follow.]Reviewer #2:[…] 1) Please clearly describe which experiments were conducted in the flow-condition in the respective figure legends.2.”We apologize for any confusion in this figure legend. The only experiment in Figure 2 that was conducted under physiological flow conditions was (E). We have revised the legend for Figure 2E to read “Analysis of cluster size for varying concentrations of EBA-175 RII under flow conditions of 1dyn/cm"EBA-175 RII induced clusters are viable under physiological flow conditions" Please add data when no RII has added in Figure 2E.2 values for all observed clusters formed during flow assay experiments, and calculated from the captured images. In the absence of RII, no clusters were observed in flow, as such there were no data to record.The data in Figure 2E comprises the µm2) "for two rounds of replication.… erythrocytes in circulation."Why did they use two rounds of replication (not one cycle assay)? Please describe the reason.[…] The proliferation and/or growth inhibition of the parasites was assessed using the approach of analyzing the incorporation of [3H]-hypoxanthine into parasite nucleic acids after two rounds of replication to get a better signal-to-noise ratio…”We apologize to the reviewer that this was not clear. The growth enhancement effect by RII was detectable after one round, but we used two rounds of replication to achieve minimal noise in the assay. We changed the lines in question to now read “…We assessed whether clustering of RBCs had any effects on parasite growth in culture using diverse laboratory strains to identify strain-transcending effects. 3) "Concentrations of RII greater than 100 nM … both DBL domains ." Should be in the Discussion section. But I felt this part sounds over discussion. Please consider to remove.We agree with the reviewer and have removed this section.4) Subsection “EBA-175-RII-mediated RBC clustering confers parasites protection from diverse neutralizing Abs”, last paragraph: To interpret the results described here, the authors should pay more careful attention to the timings when RH5, AMA1 and EBA-175 works during merozoite invasion into the erythrocyte.We apologize to the reviewer if this section was not clear. We are assessing the advantage of clustering on subsequent rounds of invasion which would follow EBA-175 cleavage and binding back to RBCs forming clusters. To make this more clear we have changed this section to read as follows “…We asked whether RBC clustering confers parasites a survival advantage, other than growth enhancement, in the presence of known neutralizing Abs during subsequent rounds of invasion following cluster formation. We assessed the effect of RII-mediated red cell clustering on parasite viability…”Reviewer #3:[…] The authors clearly demonstrate the EBA-175-mediated clustering of RBCs in vitro, and have revised an earlier version of the manuscript to include evidence that clustering occurs under conditions approximating physiological flow conditions. However, there is no in vivo evidence for EBA-175 mediated clustering, such as the observation of clusters from patients, or an estimate of the blood concentration of EBA-175 (which latter the authors posit is not possible). As a result, the authors' hypothesis that EBA-175-mediated clustering plays a role in enhancing parasite growth and immune evasion in severe malaria is speculative. Further, there is controversy surrounding the function of rosetting in vivo – as the authors point out in the Discussion, there is limited experimental evidence that rosetting leads to increased parasitemia or immune evasion. Nevertheless, I agree with the authors' comment that field studies are beyond the scope of this paper. In light of this, I would suggest that the authors make a point in the Discussion that their model still needs to be validated in vivo.We thank the reviewer for their response and have included the following sentence in the Discussion: “The model and data presented here forms a strong foundation for the future study of clustering and its role in immune evasion in patient populations.”


We found that under elevated CO2 the difference in water loss between the light (650 μmol m-2s-1) and shade (130 μmol m-2s-1) treatment was strongly reduced. Stomatal conductance (gs) decreased under elevated CO2, resulting in an approximate 40–45% reduction in water loss over a 24 h day/night period under high light and high CO2 conditions. Under lower light conditions water loss was reduced by approximately 20% for the CAM bromeliad under elevated CO2 and increased by approximately 126% for the C3 fern. Diurnal changes in leaf turgor and water loss rates correlated strong positively under ambient CO2 (400 ppm) and high light conditions. Future predicted increases in atmospheric CO2 are likely to alter plant water-relations in epiphytes, thus reducing the canopy cooling potential of epiphytes to future increases in temperature.Vascular epiphytes are a major biomass component of forests across the globe and they contribute to 9% of global vascular plant diversity. To improve our understanding of the whole-plant response of epiphytes to future climate change, we investigated for the first time both individual and combined effects of elevated CO The ability of plants to modify their anatomical and physiological traits in response to their aerial and root environment, is a major adaptation to specific habitats. Extreme environments in particular have allowed plants to develop the most peculiar life-history strategies. One such group of plants are epiphytes, these plants grow on other plants (mostly trees) for physical support without extracting any nutrients directly from the host . Most epiphytes are completely detached from the terrestrial environment and spend their entire life-cycle in the canopy. This group of mechanically dependent plants  are also2 across the vertical forest profile  and climate. The upper strata of forests typically experience higher levels of solar radiation and temperatures but lower RH compared to the lower forest strata . Moreove profile . The dyn profile . The non profile . For ins profile . However profile .In an extreme environment like the forest canopy, where plants such as epiphytes are entirely detached from the terrestrial water-source, water conservation becomes even more important to maintain optimal stomatal control. This leads to adaptations such as pseudo-bulbs in orchids, water-holding tanks in bromeliads  or the o2] will continue to increase in the future to levels of 463–623 ppm by the year 2050 (2 uptake is not saturated under current CO2 concentrations (400 ppm), which would suggest that epiphytes in particular, are likely to benefit from this increase by improving their WUE  is approximately 30% less than that of tree foliage. Collecting gas exchange data can therefore be more challenging for epiphytes, which may be the reason for the lack of data available on epiphyte responses to long-term exposure to elevated CO2 at high temporal resolution. Understanding the physiological response of epiphytes to fluctuations in the canopy environment and to future predicted changes in CO2 becomes important when trying to predict the contribution of epiphytes to forest processes.It has been predicted that atmospheric carbon dioxide concentrations , future Rica (2  is likel Rica (2  and redu Rica (2  in respoSB designed and carried out the experiments, analyzed the data, and wrote the manuscript. AH helped with the IRGA data collection and assisted SB with the development of the experiments. RH, AP, and CE-F helped with the project set-up and data collection. WF assisted in the measurement of osmotic pressure and data analysis. All authors provided critical feedback and helped to shape the research, analysis, and manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.