This study compared live instructor-led training with video-based instruction in personal protective equipment (PPE) donning and doffing. It assessed the difference in performance between (1) attending 1 instructor-led training session in donning and doffing PPE at 1 month prior to assessment, and (2) watching training videos for 1 month.This randomized controlled trial pilot study divided 21 medical students and junior doctors into 2 groups. Control group participants attended 1 instructor-led training session. Video group participants watched training videos demonstrating the same procedures, which they could freely watch again at home. After 1 month, a doctor performed a blind evaluation of performance using checklists.Nineteen participants were assessed after 1 month. The mean donning score was 84.8/100 for the instructor-led group and 88/100 for the video group; mean effect size was 3.2 (95% CI: -7.5 to 9.5). The mean doffing score was 79.1/100 for the instructor-led group and 73.9/100 for the video group; mean effect size was 5.2 (95% CI: -7.6 to 18).Our study found no significant difference in donning and doffing scores between instructor-led and video lessons. Video training could be a fast and resource-efficient method of training in PPE donning and doffing in responding to the COVID-19 pandemic. When working with infectious diseases with high consequences, such as Ebola and COVID-19, the simple act of donning (putting on) and doffing (removing) personal protective equipment (PPE) becomes a lifesaving procedure not only for the medical staff, but also for the thousands of people who depend on them.1 Third, the rapid growth in numbers of cases of infection and disruptions in the global supply chain of PPE presents a real risk of shortages of this equipment.2 Therefore, alternative training methods should be considered.In the current COVID-19 pandemic of 2020, with an urgent need to train large numbers of staff in how to don and doff PPE, the use of traditional face-to-face training with an instructor poses a number of challenges. First, it is time-consuming for both trainees and instructors. Second, gathering people together to be trained in person increases the risk of exposing staff to infection.This study compared live instructor-led training with video-based instruction in PPE donning and doffing. It assessed whether there was a performance difference between a control group that was assessed 1 month after receiving instructor-led training in PPE donning and doffing, and a study group that had access to training videos providing similar instruction over the month prior to assessment.The Ethical Committee on Health Research Ethics of the Capital Region of Denmark reviewed the research protocol and waived the requirement for informed consent because the study subjects were healthy volunteers and the study did not involve an intervention.This was a randomized controlled trial pilot study of video versus live instructor-led training.The study was conducted in Denmark. Volunteers responded to poster advertisements and brochures posted at the Faculty of Health and Medical Sciences, University of Copenhagen, at Hvidovre Hospital and Slagelse Hospital. The posters were also included in a magazine for medical students  and posted online in several medical student and medical Facebook groups.Twenty-one participants took part in the pilot study. Inclusion criteria were no previous training in donning high-isolation PPE and 3- to 6-year medical students and junior doctors (1-year doctors before receiving their independent license). Exclusion criterion was previous experience in donning high-isolation PPE (PPE created for diseases with high transmission risk).Randomization was done immediately upon arrival at the training center. Participants who were enrolled into this study were given a sealed envelope containing a unique number. This number was used in data recording throughout the study. The participants were randomly divided into 2 groups by rolling a dice. Those who rolled 1–3 were included in the group with an instructor (control group). Those who rolled 4–6 were included in the group to be trained by watching videos (study group).Training was given by a nurse, and the evaluation was performed by a doctor. Both had worked for 3 weeks at the Port Loko Ebola treatment center in Sierra Leone in 2015 and had attended an intensive 5-day, pre-departure training in the use of PPE prior to their departure, held in England and organized by the Register of Engineers for Disaster Relief. The instructor had participated in adjusting the checklists used in the evaluation. The evaluator had seen the video made by the instructor. In this way, it was ensured that the evaluator was evaluating according to the same criteria that the instructor had used in the training. To ensure that this was a blind study, the evaluator who evaluated the volunteers did not know which students had been trained by the video method (study group) and which had been trained by the instructor (control group).Control group participants each attended one 2- to 3-hour training session during which a demonstration by the instructor of how to don and doff PPE correctly was followed by observation of each of them donning and doffing PPE. The training with the instructor was conducted in groups of 1–4 people and participants donned and doffed PPE several times and received feedback. Before leaving, each participant had to perform sufficient donning and doffing as determined by the instructor, and they needed to confirm that they felt confident with the procedure.The study-group participants watched a pair of videos about how to don and doff the PPE (donning 4:42 minutes and doffing 6:11 minutes). They were required to watch the video immediately after randomization to ensure that they had seen the video at least once. This initial video training session was assisted by an Information Technology specialist who had the role of ensuring that everyone in the video group was able to access the videos from home. The free access allowed them to watch the videos as many times as they wished, and they were asked to record when (and thus the number of times) they watched the videos.https://www.youtube.com/watch?v=laeXzmkX8L0Donning video: https://www.youtube.com/watch?v=AQ-J1HvnquQDoffing video: The videos were made by the same nurse using the same technique and the same equipment that was used in the live training. Both the training and the videos were in Danish. See the following websites:3 The checklists were updated to exactly match the specifics of the PPE used .Coveralls – Lakeland Europe ChemMax 1 special coverall with hood and loops (elastic)Hoods – Microgard hoods with surgical masks WH25/W/00/517/00 Goggles – Progressive safety indirect vent gogglesRespirators – Seton JSP 111 FFP1 disposable dust maskAprons – Seton chemical resistant apron with ties 48” long x 36” wideInner gloves – Abena nitrile medical examination glovesOuter gloves – Gleco medical sterile gynecological gloves, 480 mm long, 0.20 mm thickP-value: 0.54)  group and 12 in the study (video) group. Nineteen returned to be evaluated 1 month later: 9 from the control group and 10 from the study group. In donning, the scores in the instructor group ranged from 67% to 100%, and the scores in the video group ranged from 62% to 100%. The overall mean donning score was 86.5/100; the mean score was 84.8 for the instructor group and 88.0 for the video group. There was no statistically significant difference in the donning score between the instructor and video groups  .P-value: 0.54)  .The average numbers of times the videos were watched were 4.9 for the donning video and 5.2 for the doffing video. The videos were most watched on the first and last days .The average amounts of time spent watching the videos during 1 month were 23 minutes for the donning video and 32 minutes for the doffing video, yielding an average total video training time of 55 minutes. For the instructor (control) group, the training sessions with 1–4 participants took 120–180 minutes.The participants who received video training were on average as competent as those who received instructor-led training in person. The video training took only around one-third of the time taken for a training session with an instructor. Therefore, in responding to the COVID-19 pandemic, video training might be a resource-efficient way of reaching all relevant personnel without requiring face-to-face training.Although video group participants could watch the videos as many times as they wished and whenever they wanted, it was observed that most of them had watched the video on the assessment day, effectively receiving “just-in-time” training. This may have helped their performance and might explain why some participants from the video group performed even better than those who had received training with the instructor 1 month previously.4 However, donning and doffing PPE is much more complicated because it consists of procedures that not only need to be performed correctly, but also in the right sequence, and this is of very high importance. Successful performance by video group participants may have been aided by other factors, such as repetition of the material, not only the possibility to study immediately prior to assessment.Just-in-time training, whereby information is provided at the time it is needed, has been proven to be effective in teaching other procedures. Jones et al. compared differences in just-in-time training between video presentation and small group demonstrations for fit testing N-95 respirator face masks. Fit testing of N-95 is also a part of PPE donning. There were no differences between the groups when assessment was done immediately.5 and this study suggests that the rapid development of video materials to provide training in donning and doffing of PPE is worthy of consideration in the case of the COVID-19 outbreak.Nevertheless, the West Africa Ebola outbreak (2014–2016) showed how training material can be developed quickly during an emergency response and made available for personnel being deployed to the response,To keep the videos down to a reasonable length (donning 4:42 minutes and doffing 6:11 minutes), they needed to be edited. In this process, care was taken not to miss any important aspects. However, feedback from participants revealed that some parts of the videos had been hard to see.https://www.youtube.com/watch?v=o5TCt7QXPQoAdditional video of long glove removal: https://www.youtube.com/watch?v=SX9KxvMuI2kAdditional video of short glove removal: During the group video training on Day 1, participants complained that it was hard to see how to take off the gloves following the “clean on clean, dirty on dirty principle” (see doffing video at 4:56 minutes). Two additional videos were immediately added and all video group participants were given access to them. They gave the same demonstration of how to take off the gloves, but slower and without editing. See the following websites:The video was revised in a rapid time frame to correct a poorly visible action, and, although this invites the criticism that the video group received feedback as an answer to a question in a similar manner to personal feedback during live instructor-led training, we consider that this revision did not impact the results of the study.In the last assessment session, a participant who had been trained by video had watched the doffing video 6 times without noticing that the third ribbon on the neck is untied and therefore received minus 1 point for not untying the ribbon in the assessment. The video was reviewed and it was concluded that the untying of the third ribbon was not properly shown (see doffing video at 2:15 minutes).Although these 2 events did not significantly impact the results of the study, they highlight the importance of testing videos before use in real training, inviting feedback, and making revisions when errors are found or when new information arises.Another limitation was that the sample size was small and therefore the confidence intervals for the effect are large. A larger study might show a statistically significant difference between groups.The study suggests that the PPE donning and doffing competencies of 2 groups were similar where 1 group received live training from an instructor for 1 month before being assessed and the other group watched videos of similar instruction multiple times during the month prior to assessment. The data demonstrate that the video method is time- and resource-effective when training many participants. In responding to the current COVID-19 pandemic in 2020, video training in donning and doffing PPE could provide a means of training large numbers of personnel, while minimizing the amount of time and PPE used in training and ensuring social distancing. This study also highlights the need for training videos to be tested to ensure completeness, accuracy, and clarity of actions.Although the focus of this study was not just-in-time training, the observation that most of the video group participants watched the training videos on assessment day suggests that this may have been a beneficial factor in their performance. The effect of different video training schedules and strategies on performance in donning and doffing PPE would be worthy of further study.


Recent advances in 3D cell culture have provided new opportunities for investigating interactions between cancer cells and their surrounding stromal cells. The 3D culture platform described herein is both time efficient and economical in the study of direct cell–cell interactions. The unique design of our dumbbell model had allowed us to visualize and monitor the entire recruitment process of cancer cells by fibroblasts under an in vitro condition. Suitable for almost every cell type, our model has the potential for a wider application as it can be adapted for use in drug screening and the study of cellular factors involved in cell–cell attraction.Stromal fibroblasts surrounding cancer cells are a major and important constituent of the tumor microenvironment not least because they contain cancer-associated fibroblasts, a unique fibroblastic cell type that promotes tumorigenicity through extracellular matrix remodeling and secretion of soluble factors that stimulate cell differentiation and invasion. Despite much progress made in understanding the molecular mechanisms that underpin fibroblast–tumor cross-talk, relatively little is known about the way the two cell types interact from a physical contact perspective. In this study, we report a novel three-dimensional dumbbell model that would allow the physical interaction between the fibroblasts and cancer cells to be visualized and monitored by microscopy. To achieve the effect, the fibroblasts and cancer cells in 50% Matrigel suspension were seeded as independent droplets in separation from each other. To allow for cell migration and interaction, a narrow passage of Matrigel causeway was constructed in between the droplets, effectively molding the gel into the shape of a dumbbell. Under time-lapse microscopy, we were able to visualize and image the entire process of fibroblast-guided cancer cell migration event, from initial vessel-like structure formation by the fibroblasts to their subsequent invasion across the causeway, attracting and trapping the cancer cells in the process. Upon prolonged culture, the entire population of fibroblasts eventually infiltrated across the passage and condensed into a spheroid-like cell mass, encapsulating the bulk of the cancer cell population within. Suitable for almost every cell type, our model has the potential for a wider application as it can be adapted for use in drug screening and the study of cellular factors involved in cell–cell attraction. A growing tumor contains not only malignant cancer cells but also a large repertoire of stromal cells such as fibroblasts, infiltrated immune cells, blood vessels and a meshwork of extracellular matrix (ECM) macromolecules that lend architectural and biochemical supports to the extracellular milieu . CollectTo study fibroblast–cancer cell interaction, conventional two dimensional (2D) planar culture lacks proper environmental context and structural architecture of an in vivo condition and thus cannot realistically model the complex environment of a living tissue. A mouse model, on the other hand, does not allow interactions between specific cell types to be examined in detail due to the complexity and impenetrability of the tissue environment . The devIn this study, we report the design and development of a physiologically relevant three-dimensional (3D) culture model that can more realistically mimic crucial features of in vivo conditions, especially in relation to fibroblast–cancer cell interactions. Tested on the fibroblastic cell line BHK-21 and four common cancer cell variants, CaKi-1, HeLa, A375 and A549, our model is amenable to modification for all cell types in the study of cell–cell and cell–ECM interactions.®, Shanghai, China. All the cell lines  used in this study were obtained from the Shanghai Cell Bank, Chinese Academy of Science , where authentication was performed by short-tandem repeat (STR) profiling. Unless stated otherwise, all the reagents and chemicals used in this study were purchased from ThermoScientific USA. Matrigel (BD#354234) was a product of BD Biosciences, Franklin Lakes, NJ, USA, whereas lentivirus-carrying mCherry red fluorescent protein (RFP)/luciferase dual reporter proteins was purchased directly from GenomeditechLentivirus-containing RFP reporter genes were transduced into CaKi-1, HeLa, A375 and A549 cancer cells in Dulbecco’s modified Eagle medium (DMEM) supplemented with 8 μg/mL polybrene for a minimum of 8 h prior to blasticidin (5 μg/mL) addition to allow for stable cell selection. To obtain cells with the highest fluorescent intensity, the transductants were subjected to a second round of selection with fluorescence-activated cell sorting cytometry at the nearby Suzhou Nano-Tech and Nano-Bionics Institute (SINANO), Chinese Academy of Science.5 cells/mL for each cell type (1:1 ratio) on ice in 50% Matrigel/DMEM supplemented with 5% fetal bovine serum (FBS). The cell–Matrigel mixture was dispensed as 6 μL micro-droplets onto the center of the wells of a 24-well tissue culture plate and allowed to solidify for at least 30 min in a 37 °C incubator prior to adding 1 mL DMEM/5% FBS. The cells were regularly inspected with a Nikon Eclipse Ti inverted microscope for morphological changes. To ensure the reproducibility of the findings, the experiments were performed in at least 12 replicates for no less than 10 times for every cancer cell type.3D fibroblast–cancer cell co-culture model was set up essentially as illustrated in 5 cells/mL. To create a dumbbell model, the two cell types were dispensed as 6 μL micro-droplets adjacent to each other, leaving a 1 mm gap in between the two. For the ease of explanation, a schematic representation of the model is depicted in Instead of co-culturing in a single droplet, the fibroblasts and cancer cells were suspended independently in separate tubes in 50% ice-cold Matrigel/DMEM at the final concentration of 5 × 104 CaKi-1 cells pre-labelled with mCherry RFP were seeded in triplicate in 8 μm pore size Boyden chambers, while the lower compartment  was filled with either the same number of BHK-21 fibroblasts or no cells (to serve as negative controls) in 1 mL DMEM/1% FBS. Incubation was allowed for 48 h at 37 °C before the membrane was transferred to a new 24-well plate and the number of migrated CaKi-1 cells counted using a Nikon Eclipse Ti inverted microscope under a 588 nm wavelength filter. Statistical significance between the control and study groups was analyzed using the Student’s T test calculator in the socscistatistics.com website.For transwell migration assay, 5 × 105/mL would yield the same spheroid outcomes.In contrast to the 2D monolayer culture condition, BHK-21 fibroblastic cells spontaneously consolidated into a distinct network of vessel-like structures within 5 days of seeding in 50% Matrigel suspension A; day 5.Unlike their fibroblast counterpart, cancer cells such as CaKi-1 , HeLa , A375 (melanoma) and A549 (lung adenocarcinoma) have no ability to develop into vessel-like structures in the same Matrigel suspension. Instead, they proliferated as independent colonies of varied sizes, as shown in To investigate the effects that the fibroblasts have on cancer cell proliferation, CaKi-1 cells pre-labelled with RFP were added to the fibroblast–Matrigel mix before the cells were plated out as droplets. As in previous case, fibroblasts swiftly consolidated into a network of vessels within 5 days of seeding that, upon prolonged culture, evolved into a dense spheroid-like cell mass. The proliferation pattern of the cancer cells changed drastically in the presence of the fibroblasts. Instead of developing into individual colonies of different sizes, almost the entire population of the cancer cells were found to be encapsulated within the spheroid-like cell mass as their fibroblast counterpart underwent morphological changes in Matrigel suspension . Indeed,To find out if the encapsulation effect was anything more complex than simple clogging of the vessel-like meshwork, we sought to monitor every step of the vessel evolvement under a high-magnification microscope. As shown in To visualize and monitor the migration and encapsulation processes in greater clarity, we here introduced a modification to the existing model by plating the fibroblasts and cancer cells as independent droplets away from each other shown in B. To allIncubation for a further two to four days allowed the vast majority of the fibroblasts to reach the end of the causeway and into the cancer cells’ territory . The results confirmed that apart from physical attraction, the fibroblasts were also capable of chemically attracting cancer cells to their vicinity, although the exact chemoattractants in this case are yet to be identified.In conjunction with the dumbbell model above, we have also set up a transwell migration assay to determine whether the fibroblasts were also capable of attracting CaKi-1 cells from a distance by chemoattraction. As shown in The dumbbell model presented herein enables us to robustly capture the morphological changes taking place within the fibroblasts and cancer cells, including the directionality of their interactions, before and during the process of spheroid formation. To achieve the desired effect, it is imperative that the two cell types be seeded in separation to allow the fibroblasts the opportunity to first self-organize into a network of branching vessel-like structures prior to encroaching into the cancer cells’ territory. By coercing the fibroblasts into a cell mass of defined shape along a narrow causeway, we were able to observe the advancing cells as a single collective entity and, thus, gain a clear view of the invasion front, as illustrated in Besides visualizing cell invasion, our approach has provided us with strong experimental evidence to support the notion that cancer cells are innately attracted, physically as well as chemically, to the fibroblasts. Therefore, not only could our model provide an in vivo-like environment for the cells, it also allows the interaction dynamics between the cancer and its surrounding fibroblast cells to be visualized in a clear and unambiguous fashion. Fibroblasts, in particular CAFs, exert a physical force on cancer cells for collective invasion. More specifically, fibroblast–cancer cell co-migration requires direct cell–cell contact and adhesion possibly via the transmembrane molecules N- and E-cadherins ,16,17,185 cells/mL.The model is simple to construct without the need for expensive reagents or specialized equipment. Furthermore, we find the results from the droplet-based platform to be robust and highly reproducible. Of note, the stiffness of the matrices was found not to be critical for both the invading and subsequent cell recruitment events, as concentration of Matrigel anywhere between 25 and 75% would yield the same outcomes. Outside the range, the cell–Matrigel mixture became either too liquidly to gel or viscous for handling. Hence, despite the numerous reports regarding the effects ECM stiffness have on the behavioral pattern of fibroblast cells ,20, the In conclusion, the 3D dumbbell model proposed in this study is a simple and yet efficient way of studying 3D invasion and cell–cell interactions. Not only can it be used for visualization and tracking of the entire process of directed cancer cell migration and, thus, provide invaluable insights into the invasion dynamics that affect metastatic dissemination, the model is readily adaptable for other applications such as drug screening and tissue engineering.


Cognitive impairments can be a significant problem after a traumatic brain injury (TBI), which affects millions worldwide each year. There is a need for establish reproducible cognitive assays in rodents to better understand disease mechanisms and to develop therapeutic interventions towards treating TBI-induced impairments. Our goal was to validate and standardize the radial arm water maze (RAWM) test as an assay to screen for cognitive impairments caused by TBI. RAWM is a visuo-spatial learning test, originally designed for use with rats, and later adapted for mice. The present study investigates whether test procedures, such us the presence of extra-maze cues influences learning and memory performance. C57BL/6 mice were tested in an 8-arm RAWM using a four-day protocol. We demonstrated that two days of training, exposing the mice to extra-maze cues and a visible platform, influenced learning and memory performance. Mice that did not receive training performed poorer compared to mice trained. To further validate our RAWM protocol, we used scopolamine. We, also, demonstrated that a single mild closed head injury (CHI) caused deficits in this task at two weeks post-CHI. Our data supported the use of 7 trials per day and a spaced training protocol as key factor to unmask memory impairment following CHI. Here, we provide a detailed standard operating procedure for RAWM test, which can be applied to a variety of mouse models including neurodegenerative diseases and pathology, as well as when pharmacological approaches are used. Traumatic brain injury (TBI) is a major public health problem worldwide and leads to temporary or permanent physical and cognitive impairments. In particular, people with a history of TBI have an increase risk to develop dementia and neurodegenerative disease , 2.Mechanisms of selective vulnerability to cognitive deficits following a mild TBI are still not well understood, and the use of animal models accelerate a better understanding of the pathological and behavioral outcome associated with a mild TBI. Novel object recognition (NOR) and Morris water maze (MWM) test are the two most popular assays used to evaluate cognitive function after mild TBI . While tIn 1984, Buresova et al. described a “radial maze in the water tank”, making this the first time that water was used as an aversive stimulus in a radial maze  tested tAplysia California [Drosophila Melanogaster [Training schedule affects level of acquisition in humans , rodentslifornia –20, Drosnogaster , bees [2nogaster , and in nogaster . In partnogaster , 24, 25 nogaster .The goal of this paper is to test if mice trained in a spaced RAWM training protocol have the ability to learn the task with a total of 28 trials spaced over 4 days. We hypothesized that reducing the number of trials per day would improve their ability to learn and reduce fatigue. An added benefit is the ability to increase the capability to test more mice in one session. The result of our study, support our hypothesis that few trials per day spaced over 4 days, is sensitive at detecting cognitive deficits following a mild closed head injury (CHI). Our RAWM protocol will likely be widely applicable to detect cognitive deficits in other mouse models of injury or disease.Scopolamine hydrobromide was purchased from Sigma (cat. no.6533-68-2). It was dissolved in double-distilled water, sterile filtered  and administered at a dose of 1 mg/Kg/10 ml. Intraperitoneal (ip) injections were performed 30 minutes before trial 1 every day during the 4-day test. Scopolamine was freshly prepared each day animals were dosed.All animal procedures were approved by the Institutional Animal Care and Use Committee (IACUC) of the University of Kentucky and experiments were conducted in accordance with the standards of proper experimentation in the Guide for the Care and Use of Laboratory Animals and ARRIVE guidelines.The study used 141 adult mice (72/69 ♀/♂) 3–4 months old, C57BL/6J mice . The number of mice used for each experiment is reported in the figure legend. Animals were group-housed (4–5 per cage) in a controlled humidity (43–47%) and temperature (22-23º C) environment and 12/12-h (7 am-7 pm) light/dark cycle with free access to food and water. Behavioral experiments were conducted by the same operator (TM) and performed between 7.30 a.m. and 3.30 p.m.Mice were assigned randomly to groups before the start of each experiment. Each cage had at least one animal from each group/ treatment in a random order. The person performing the scopolamine injections (KNR) and CHI surgery (ADB) blinded the experimental groups from the person performing RAWM test (TM). The person performing the RAWM test (TM), three days before the beginning of the experiment transferred the mice to a clean cage, marked the tail for easy identification, and handled them for the following three days. For the handling, a mouse was allowed to explore the experimenter’s arm and hand for 1–2 min, then returned to its home cage. Mice were never exposed to the maze before the start of the experiment. During the behavioral tests, mice were transferred to the experimental room and relocated in a clean recovery cage without bedding at least 30 minutes before the start of the test. The recovery cage had paper towels inside and was placed half on a heating pad to help the mice recovery from the swimming activity and half off the heat. Also, during the test, wet paper towels were promptly replaced, so mice could have a faster recovery in a dry and warm recovery cage. Male and female mice were tested in separate cohorts.The RAWM test was performed in a circular pool  was used for video recording and scoring behavior.block 1 and block 2 . To encourage mice to learn the location of the platform, an alternation of visible and hidden platform was used during block 1 of training days  and a hidden platform was used during block 2 A step-by-step standard operating procedure has been provided and it is available on The mild closed Head Injury (CHI) model was used in this study to demonstrate the applicability of this new behavioral protocol. CHI was performed as previously described using a digital stereotactically (Stoelting) guided electromagnetic impactor device to produce a highly reproducible TBI with minimal mortality , 28. MicCHI mice received a single controlled midline impact  1.0 mm deep with a controlled velocity of 5.0+0.2 m/s and a dwell time of 100 ms using a stereotaxic electromagnetic impactor  equipped with a 5.0 mm flat steel tip. Sham-injured mice received identical surgical procedures as the CHI group, but no impact was delivered. Following impact, the incision was sutured, body weight was monitored up to 5 days post-surgery. Sutures were removed 1-week post-surgery. Starting at 14 days post-surgery, mice were tested in RAWM test. Exclusion criteria for the CHI model included skull fractures or prominent vestibular disturbances, including tilting of the head, or slight spin when lowered. No mice were excluded from the study following the CHI.JMP Pro software version 14.0  was used for statistical analysis. Graphs were generated using GraphPad Prism version 8.0. Summary values for the learning curves are expressed as mean ± SEM or median ± SEM, as noted in the figure legend. Boxplot were generated using the Tukey method. Scatter plots represent individual mice. Mouse numbers used are indicated in the figure. Differences were considered statistically significant when p < 0.05.The median of errors made by the animal each day was considered for analysis. Only hidden trials were used in the analysis. The AUC was calculated using the trapezoid method, dividing the whole AUC into trapezoidal segments and counting the area of each segment separately, this was done for both errors and distance . The sumWhere:AUC = area under the curvec = errors or distance recorded at a specific time pointn = time of observation of errors or distance at cntn-1 = time of prior observation corresponding of errors or distance at cn-1tA mixed model design using a standard least squares method was used to test for statistical differences between groups. Two models were compared. The first model included: day of testing, experimental group, sex of mice, and all the interactions. The second model did not include sex of mice . Overall, we did not find a statistically significant effect of sex on the outcome measures and did not find that the inclusion of sex improved our statistical model. Therefore, we report the results of the second statistical model without sex of the mice included in the model. We have shown that the sex of the mice affects response to the CHI for some variables ; thus, tThe goal of this experiment was to determine if cues were being used by the mice for the visuo-spatial learning during RAWM test. Mice were tested for a total of 4 days. We evaluated if the use of extra-maze cues and a visible escape platform is essential for the mice to learn the task and has a positive influence on RAWM output, or if the mice were using other extra maze cues or non-visuo-spatial search strategies. A 7-trial protocol was used to address this question, in particular two conditions were considered: 1) mice were exposed to the pool that was equipped with 4 extra-maze cues and a visible platform was used during training days ; 2) in tOur next step was to use a pharmacological approach to validate this 7 trials RAWM protocol. Scopolamine is a “gold standard” non-selective muscarinic compound particularly used for validating learning and memory assay , 32. TheIn this experiment, we evaluated whether our 7-trial protocol was able to detect memory deficits in a mTBI model. In the past, it has been shown that the same animal model of CHI was memory impaired in a 6-arm RAWM when a 15-trial protocol and 2-day test , 36 and Because of the heterogeneity of TBI, RAWM protocols can sometimes not be sensitive enough to detect injury-induced deficits in behavior. The goal of this paper was to provide a revised protocol for an 8-arm 4-day RAWM test. We believe that our protocol will be a useful resource for others attempting to standardize their behavioral assays, and those laboratories interested in detecting subtle differences in cognitive function in mouse models of CNS injury or disease. Our results indicate that using a weaker training protocol, like reducing the number of trials, is more sensitive to identify differences in cognition related to a single mild CHI at 2-week post-injury.A major finding of our study was that the reduction in the number of trials significantly separates the acquisition curves of CHI and SHAM mice and the sex of the mice had no effect on results. Previously, we demonstrated that mice had memory impairment in a 6-arm RAWM at 2 week post-CHI in a 4-day protocol and 15-trial per day  or in 2-Reduction in the number of training per day reduces fatigue and stress in the animals. Literature supports our idea that reducing the number of trials per day and increasing the inter-trial time helps the formation of long-term memory , 25, 26.The current study is not the first to use RAWM in rodent models of TBI. In addition to our studies using RAWM in CHI models of TBI , 30, 36,While we believe that this protocol is more sensitive to detecting changes in mouse models, independent validation of behavioral protocols is important before conducting experiments. Each laboratory should conduct their own validation and include a group of mice to be used as positive control, in order to guarantee that the assay is reliable and consistent. Specially because it has been demonstrated that test standardization does not guarantee the same results across laboratories –49, due In conclusion, we demonstrated that: 1) the use of cues will improve the test acquisition, 2) reduction in the number of trials improves learning, and 3) a single mild CHI in mice could cause cognitive deficits detectable by the reduction of trials. We also provided a standard operating procedure and methods to validate the RAWM behavior for use in phenotyping other mouse models or when a pharmacological treatment would be considered.


Bolivia has the highest prevalence of cervical cancer in South America and the prevalence of viral sexually transmitted infections (STIs) among people in urban cities is increasing. Little is known about the prevalence of viral STIs in rural communities, which generally have limited access to health care. In order to study the prevalence of viral STIs in rural Bolivia, we recruited women from villages and towns in the Department of La Paz in Bolivia.2 test was used to compare variables between the populations and binary logistic regression was used to identify risk factors associated with the positivity of the tests.Three hundred ninety-four female participants were assessed for IgG-antibodies to herpes simplex virus type 2 (HSV-2), human immunodeficiency virus (HIV) and hepatitis B virus , as well as for the presence of HBV surface antigen (HBsAg) in dried blood spots. The prevalence of 12 high-risk types of human papillomavirus (HPV) was assessed by qPCR in dried cervicovaginal cell spots from 376 of these women. χThe seroprevalence of HSV-2 was 53% and of HBV 10.3%. HBAg was detected in 15.8% of women with anti-HBV antibodies indicating chronic infection. The frequency of high-risk HPV infection was 27%, with the most prevalent high-risk HPV types being HPV 56, 39 and 31 followed by HPV 16 and 18. Finally, none of the 394 women were seropositive for HIV, and about 64% of the studied population was positive for at least one of the viral infections.Women in Bolivian rural communities in La Paz show a high prevalence of HBV, HPV and, in particular, HSV-2. In contrast, none of the women were HIV positive, suggesting that the HIV prevalence in this population is low. The pattern of high-risk HPV types differed from many other countries with a predominance of HPV-types not included in the Gardasil vaccine which was officially introduced in Bolivia in April 2017. To investigate and report the prevalence of viral sexually transmitted infections (STIs) other than HIV is important for the introduction or improvement of a wide range of health policies and programs in all regions. It is known that more than 30 microorganisms are sexually transmitted including herpes simplex virus type 2 (HSV-2), hepatitis B virus (HBV), human papillomavirus (HPV) and human immunodeficiency virus (HIV). Estimations according to the World Health Organization (WHO) indicate that more than 1 million STIs are acquired every day.Almost 300 million people are infected with HPV, which if becoming persistent may cause cervical cancer . More thBolivia has a population of around ten million people and 40 to 70% are self-identified as indigenous peoples . An ongoHSV-2 infection is a risk factor for the acquisition of HIV in people who practice unsafe sex, and it is known that the virus is more prevalent in women compared to men. In 2012 the general seroprevalence of HSV-2 of people aged 15–49 years in the Americas is estimated to be 14.4%, while the highest world-wide prevalence is in African populations with an overall estimated prevalence of 31.5% [The Amazonas is an area of high endemicity of HBV, with reported prevalences ranging from 8 to 25% . CountriIn 2017, UNAIDS reported that about 21,000 people are living with HIV in Bolivia with a general prevalence of 0.3%, and that only 36% of those infected are on treatment. A later report indicates an increase of HIV incidence . The genIn rural areas the poor population have limited information about STIs, little/no access to the national health care system, and no resources for treatment. This study therefore seeks to obtain data about the prevalence and possible risk factors of viral STIs in women living in rural areas on the way to the Amazonas of the Department of La Paz in Bolivia, which will help to improve the screening programs and health policies.Our study population was recruited in the rural provinces of Abel Iturralde  and Caranavi (the adjacent lower highlands at 1000 m above sea level) from the Department of La Paz in Bolivia during five field trips; three in the Abel Iturralde province during the dry seasons (July to October) between 2015 and 2017 and two in Caranavi in March 2018 and May 2019. Our research group, a unit working at the Universidad Mayor de San Andrés (UMSA), visited the three villages Tumupasa, San Silvestre and Santa Rosa de Maravilla (mostly indigenous population below 1200 inhabitants) , the smaN = 394) received extended oral and written information regarding STIs and signed an informed consent form. In the case of women under the age of 18 informed consent was signed by their guardians. Demographic data was collected through a questionnaire and a personal interview. Blood samples (N = 389) and cervicovaginal cell samples (N = 376) were collected from female volunteers.Participants were recruited either by direct invitations to small mother groups, unions, local authorities and indigenous leaders or via general invitations by radio and television. The examination and collection of samples was done in the nearest local health center or hospital by laboratory technicians from UMSA. Women who were menstruating or were more than 12 weeks pregnant were excluded from the study. Eligible participants (Because of the lack of sanitary services (including electricity) in some of these areas, sampling was performed using filter papers that preserve the biological material and allowed the transportation and assessment of the samples. Whole blood samples were therefore collected as Dried Blood Spots (DBS) in Whatman 903 filter papers (Protein Saver™ 903 Card) for the evaluation of antibodies against HSV-2, HBV and HIV as well as for HBV antigen. Exfoliated cervicovaginal cells were collected by swabbing the lateral walls using sterile cotton swabs that were then transferred in 1 mL of essential D-MEM medium  and kept at 4 °C. At the laboratory in La Paz city the samples were centrifuged at 1500 rpm for 10 min at 4 °C and suspended cells in 150 μL of medium were transferred and applied on 2 × 2.5 cm Nucleic-card color matrix spots  as dried cervicovaginal cell spots (DCCS). DBS and DCCS were kept at − 20 °C until their further transport to and analysis in Sweden. Data was managed anonymously according to the ethical permission CEI-UMSA 0215 obtained from the Ethical committee at UMSA, La Paz, Bolivia.Blood proteins were extracted from DBS by punching one bloodstained circle of 5 mm diameter from the filter paper as previously described , 15, TheDNA was extracted and purified from DCCS using QIAGEN mini kit as described , One spoElutions of DBS were used to assess antibodies for HSV-2, HBV, and HIV using commercial ELISA kits; HSV-2 , anti-HBV anti-core antibodies , and HIV . In addition, we measured the presence of HBV surface antigens in eluted DBS using ELISA . As the dried blood samples contain lysed red blood cells, which can interfere with ELISA readout and give false-positive results, we increased the cut-off values provided by the manufacturers of the ELISA kits by multiplying with a factor of 1.5 , 18.Equivocal and low positive samples for HSV-2 by FOCUS ELISA were confirmed with the detection of antibodies to the mature portion of glycoprotein G-2 using Western blot as described . BrieflyT) for β-globin below of 37 were included in the analysis.Purified DNA from DCCS were assessed by a Taqman real-time PCR assay which identifies 12 high-risk  and two low-risk (6 and 11) types, by targeting segments of the E6/E7 region . The amp2 test comparing among groups using PRISM 7.0® from GraphPad Software Inc., San Diego, CA. A p value below 0.05 was considered statistically significant.Descriptive data are presented as percentages, median, prevalence estimates and 95% confidence intervals (CIs). Statistical analyses were calculated using χp < 0.1 were included in the adjusted models as in other studies [The associations between the dependent variable  and the independent variables; age, occupation, number of children, current family planning methods, residence and coinfections were tested by binary logistic regression. This analysis was used to estimate crude and adjusted odds ratios and 95% CIs for each category compared to a reference category (Ref). Variables or categories that showed association with the outcome at  studies . Data anWe investigated the prevalence of viral STIs in women from two provinces of the Department of La Paz, which are inhabited mainly by rural and semi-rural indigenous population. Demographic data was taken from the 394 participants through a questionnaire and an interview. The study population Table  was diviThe seroprevalence of HSV-2 was assessed by two methods; all samples were first analyzed using an HSV-2-specific ELISA. Equivocal and low positive samples were confirmed using the “gold standard”, western blot. More than 74% of the samples were positive for HSV-2-specific antibodies using ELISA, but after confirmation using western blot the prevalence was determined to 53% (Table Thirty-eight women out of 389 tested) had HBV core-specific antibodies. Six of these 38 women (i.e. 15.8%) also had detectable HBsAg in their blood indicating a chronic infection. In addition, we found two acute cases of HBV infection, i.e. women that had detectable HBsAg in their blood but no antibodies. Having in total 40 cases, the prevalence for HBV was 10.3%  and 2 low-risk (LR-HPV). Three of the DCCS were excluded as no DNA  was detected. For the remaining 376 samples, 27% were positive for at least one high-risk HPV type Table . Women bThe number of viral STI in the 371 women where all ongoing viral evaluations were performed  was assessed and is summarized in Table . None ofIn Bolivia, the prevalence of viral STIs has increased during the last 10 years, but this has mainly been investigated in larger towns and cities. In the present study, we assessed the presence of viral STIs in 394 women living around villages and towns in the north of the Department of La Paz and found that the burden of HSV-2, HBV and HPV is higher than in larger cities.The HSV-2 seroprevalence varies between South America and Caribbean countries and relate to behavioral and social conditions. It increases with age , 23, andIn female African populations, acquisition of HSV-2 infection is associated with risk factors such as prostitution, limited access to health care, and unnegotiable unsafe sex with their partners due to economic dependence . We haveTwenty-seven percent of the women in this study had an ongoing infection with one or several HR-HPV. This is higher than the 8 to 18% previously reported in rural and urban regions of Bolivia, which indicates that the prevalence of HPV in rural north of La Paz might be higher than in other regions of Bolivia –33. HPV The 12 high risk HPV types we analyzed are the most important ones due to their association with the development of cervical cancer. These genotypes represent the class I carcinogens for cervical cancer according to the (WHO/IARC), and the most common high-risk HPV types we identified were HPV 56, 39 and 31. Previous studies from Bolivian small towns have identified HPV 31 and 58 as the most common HPV high-risk types , were moAlmost 10% of the women in this study had antibodies to HBV, which is similar to the 8% reported in other South American countries such as Colombia and Brazil, and thus confirms the high seroprevalence of HBV in the Amazonas region , 39. NatNo positive cases for HIV was found, which is in agreement with the low estimates by UNAIDS with only a 0.3% of prevalence in adults aged 15 to 49 years, and zero cases from a study of 885 healthy women mostly urban populations in Bolivia . The proAlmost 70% of the rural women included in this study were positive for at least one of the four viral STIs. Approximately 15% of the women had more than one viral STI, and the majority of these co-infections involved HSV-2 and HPV. We did not find a positive association between HSV-2 and HPV infections indicating that these infections do not predispose for each other. However, several studies show that HSV-2 infection is an important risk factor for the development of invasive cervical cancer in HPV-infected women –44. ThusThere are certain limitations to this study. First, STIs are stigmatizing in which might have generated a bias in the recruitment process. Second, many women from villages were afraid to participate because they reported that the Pap smear test was painful, and many of them have never encounter a cytological inspection by medical staff. Third, due to the high rate of illiteracy we could only collect data through oral interviews which might have produced some bias in our data.We conclude that there is an unusually high prevalence of HSV-2 infection in women from villages and towns in the Department of La Paz in Bolivia, and that both HPV and HBV infections are common. In addition, we note that the pattern of high-risk HPV types differ both from what is found in Europe and included in the HPV vaccine introduced in Bolivia in 2017.Additional file 1 Table 1. Types of human papilloma virus detected in cervical samples of 103 participants with HR-HPV and LR-HPV.


This unprecedented result indicates that polyspermy can bypass the triploid block, which is an established postzygotic polyploidization barrier. In fact, we here show that most polyspermy-derived seeds are insensitive to the triploid block suppressor admetos. The robustness of polyspermy-derived plants is evidenced by the first transcript profiling of triparental plants and our observation that these idiosyncratic organisms segregate tetraploid offspring within a single generation. Polyspermy-derived triparental plants are thus comparable to triploids recovered from interploidy crosses. Our results expand current polyploidization concepts and have important implications for plant breeding.Polyploidization, the increase in genome copies, is considered a major driving force for speciation. We have recently provided the first direct in planta evidence for polyspermy induced polyploidization. Capitalizing on a novel  Ever since Darwin published his most famous book on the theory of evolution, scientists have sought to identify the mechanisms that drive the formation of new species. This is especially true for plant biologists who have long been fascinated by the extraordinary diversity of flowering plants.Many species of flowering plant first evolved after a dramatic increase in the DNA content of an individual plant, a process termed polyploidization. Most explanations for polyploidization involve a pollen grain making sperm that mistakenly contain two sets of chromosomes rather than one. Yet, it is difficult to reconcile this explanation with an important aspect of plant reproduction – the so-called “triploid block”.Fertilization in flowering plants is more complicated than in animals. While one sperm fertilizes the egg cell to make the plant embryo, a second sperm from the same pollen grain must fertilize another cell to form the endosperm, the tissue that will nourish the embryo as it develops. This means that sperm with twice the normal number of chromosomes would affect the DNA content of both the embryo and the endosperm. Yet, an endosperm that receives extra paternal DNA typically halts the development of the seed via a process known as the triploid block, meaning it was not clear how often this process would actually result in a polyploid plant.In 2017, researchers reported that plants can, on rare occasions, generate polyploid offspring via a different route: the fertilization of one egg with two sperm rather than one. Now, Mao et al. – who include several researchers involved in the 2017 study – show that this process, termed “polyspermy”, can introduce extra copies of DNA into just the egg cell, meaning it can bypass the triploid block of the endosperm.The experiments involved a model plant called Arabidopsis, and a screen of over 55,000 seeds identified about a dozen with embryos that had three parents, one mother and two fathers. Notably, most of these three-parent embryos developed in seeds that contained endosperm with the regular number of chromosomes and hence escaped the triploid block.These new results show that polyspermy provides plants with a means to essentially sneak extra copies of DNA ‘behind the back’ of the DNA-sensitive endosperm and into the next generation. They also give new insight in how polyploidization may have shaped the evolution of flowering plants and have important implications for agriculture where the breeding of new “hybrid” crops has often been limited by incompatibilities in the endosperm. Arabidopsis plant can generate several polyspermy-induced triploid seedlings      and a bin donors . Pollen e signal . It shousco1 mutant plants expressing either one of the constructs with seeds containing both, the GAL4 activator and the UAS reporter line. This experiment confirmed that only the presence of both constructs complemented the defect resulting in dark green seeds, which exhibited a tdTOMATO signal  and identified 10 normally developed seeds with a change in color  suppress the triploid block in Arabidopsis thaliana , and a two-dimensional multidimensional scaling (MDS) analysis of the fifteen 21,450-dimensional normalized expression profiles. We found that Spearman’s correlation coefficient profiles . Notablyprofiles , and theprofiles  and MDS profiles  plots. TWe first addressed, if there were genes with ploidy-specific expression changes by comparing transcript profiles of BD and BT. This comparison did not yield a single gene with a statistically significant differential expression. This result is in support of previous transcriptome profiling approaches that have uncovered remarkably few changes in plants with different ploidy .We next compared the transcript profiles of BT and TT in order to identify specific expression changes potentially associated with polyspermy. Interestingly, also this approach did not yield genes with statistically significant differential expression. The similarity in the overall transcriptional profiles between TT and BT plants is reflected by strong similarities in various life-history traits, including flower organ size, cell size, and even fertility . Please 2 generation 5 out of 109 plants were found to fall into the near-tetraploids category, while 22 plants segregated a diploid-like profile . By contrast, TT3 plants initiate flowering only after 64.6 ± 5.4 days, which is around 20, 17 and 15 days after the respective parental lines have terminated their flowering phase . The sco1 T-DNA insertion mutant (SALK_025112)  were generated from a cross of 2n female with 2n pollen donor pRPS5a::mGAL4-VP16/-; pUAS::BAR-YFP/-. Stratified seeds  were germinated on soil in a Conviron MTPS growth chamber under long-day conditions (16 hr light/8 hr dark) at 23°C. For HIPODWT, HIPODadm and experiments in Unless otherwise stated, all experiments were carried out using _025112)  was obta1 mutant  was kind1 mutant  was used1 mutant , while badm-1/- and pollen donor plants were generated by using 0.05% colchicine treatment following the one-drop method previously described  (pAt5g40260 with pUAS sequence from pUAS::BAR-YFP (The O::tNOS)  using Pa:BAR-YFP  using AsCENH3 CDS was amplified from leaf cDNA library using primer pair DT231s/DT231as and cloned into p35S::NLS-GFP using AvrII and MfeI to generate p35S::CENH3-GFP.The Agrobacterium tumefaciens  were sown on soil. Germination frequency was scored after 9 days. The experiment was repeated three times.3), 36 (Col-0), 37 (Ler), and 35 (C24) were analyzed. Flowering period was defined as the interval between the formation of the first and the last flower. In independent experiments a total of 4 plants (TT3), 36 (Col-0), 37 (Ler), and 35 (C24) were analyzed. Please note that watering was stopped once the main branch stopped flowering.Flowering time was assessed as days after sowing (DAS), starting from the day of sowing till the opening of the first flower. Within three independent experiments a total of 7 plants , siliques were dissected under Leica S6E or S8apo stereomicroscope  for seed color screening. Seeds with a change in color were analyzed for tdTOMATO signal and embryo and endosperm chromosome number.For the HIPODSample preparation and ploidy determination was performed using the same method as described previously; nuclei were marked with CyStain UV Precise P kit and assessed with CyFlow ploidy analyzer . The triSeeds that contain TT, BT, and BD were selected on ½ MS medium containing 25 μg/ml Glufosinate-ammonium , and the seedlings were transferred to soil 9 DAS. YFP and ploidy analysis was carried out 17 DAS to confirm TT, BT and BD profile. 18 DAS, aerial tissues were frozen and grinded in pre-cooled EP tubes with metal beads and total RNA was isolated following the procedure of GeneMATRIX Universal RNA purification kit.750 ng total RNA per sample was used to prepare sequencing libraries according to the Illumina TruSeq RNA Sample Preparation v2 Guide. Purification of the poly-A containing mRNA was performed using two rounds of poly-T Oligos attached to magnetic beads. During the second elution of the poly-A RNA, the RNA was fragmented and primed for cDNA synthesis. After cDNA synthesis the fragments were end-repaired and A-tailing was performed. Multiple indexing adapters were ligated to the ends of the cDNA fragments and the adapter ligated fragments were enriched by 10 cycles of PCR. After quali- and quantification the resulting sequencing libraries were pooled equimolar and sequenced 75 bp single-read on an Illumina NextSeq500.trimmomatic version 0.38 , multidimensional scaling analysis (MDS), the computation of Spearman’s correlation coefficients, and hierarchical clustering were performed by functions DESeq2, with a subsequent application of the Benjamini-Yekutieli correction procedure and a threshold of the false discovery rate of 5%.The prediction of differentially expressed genes was performed by the Wald test, implemented in SCO1, the embryo was separated from the endosperm in the final fixative step on the slide. The remaining steps were carried out as described in 7 DAP seeds were harvested and their embryo (cotyledon stage) was extracted by applying pressure with syringe needles. The separated embryo and the remaining part of the seed containing the endosperm was individually transferred into 200 μl pre-treatment solution and incubated for 4 hr at RT . For aboImages were taken using Leica DMI6000b epifluorescence inverted microscope, equipped with YFP, DAPI and DsRed filter cube. For YFP expression analysis, dissected sepals were transferred to 10% glycerol and data were collected. For chromosome spread, data from DAPI stained samples were collected with the aid of 100 × oil objective and an immersion oil Immersol 518F  or by using confocal laser scanning microscopy with Airyscan module (Zeiss LSM 880) at excitation of 405 nm and emission of 421 nm. CENH3-GFP localization was observed at excitation of 488 nm and emission of 510 nm. 7 DAP silique images were captured by using Leica S8apo stereomicroscope. In the interests of transparency, eLife publishes the most substantive revision requests and the accompanying author responses.Acceptance summary:As mentioned in our previous decision letter, we and the reviewers considered your work as a conceptual breakthrough, providing highly novel insights and concepts into reproduction system in angiosperms. Your work highlights that polyspermy can circumvent the triploid block in the endosperm and moreover clearly illustrate that polyspermy-derived triploid plants have the potential to form tetraploid progenies in one generation.Decision letter after peer review:eLife. Your article has been reviewed by three peer reviewers, and the evaluation has been overseen by Reviewing Editor Jürgen Kleine-Vehn and Christian Hardtke as the Senior Editor. The following individual involved in review of your submission has agreed to reveal their identity: Takashi Okamoto (Reviewer #2).Thank you for submitting your article "Polyspermy selectively polyploidizes the egg cell thereby bypassing the triploid block" 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.The reviewers considered your work as a conceptual breakthrough, providing highly novel insights and concepts into reproduction system in angiosperms. Your work highlights that polyspermy can circumvent the triploid block in the endosperm and moreover clearly illustrate that polyspermy-derived triploid plants have the potential to form tetraploid progenies in one generation.The reviewers appreciated the high quality of your data and did not raise any major concerns that would require experimental validation. Please consider the reviewer comments (see below) to further strengthen the manuscript. Specifically, please clearly state/discuss the mechanistic advance of this work in relation to your previous publication .Reviewer #1:How polyploidization comes about is a central question in biology. In plants polyploidization is frequently associated with speciation and occurred during the generation of important crop plants, such as wheat. Thus, the authors address a question of high scientific significance and of broad interest.In previous work the authors established that polyploidization in plants can occur via the rare occurrence of polyspermy due to more than one pollen donor contributing to fertilization (as a rule only one pollen tube fertilizes a given ovule). In my opinion, the central advance provided by this paper relates to their finding that during this process two sperm cells, in their set up derived from genetically different pollen donors, fertilize the egg cell. By contrast, the central cell, the other gamete, is fertilized by only one sperm cell. Their data confirm that the resulting embryo is triploid while the endosperm, which develops from the fertilized central cell, is triploid. The authors go on to show that by this mechanism polyspermy circumvents the triploid block, a postzygotic polyploidization barrier, which occurs in the endosperm. It is noteworthy that their genetic method is based on an unbiased approach and does not select for the event, for example through screening for herbicide resistance.Overall, I find this a very exciting paper. It certainly has ramifications as to how polyploidization can come about. Moreover, it has obvious potential implications for evolutionary biology (and application), although I am not sure how relevant that really is given the low observed frequency of the event. I concede, however, that I am not an expert in evolution and speciation.Reviewer #2:Polyploidization has played a major role in the long-term diversification and evolutionary success of angiosperms. Triploid formation among diploid plants, which is generally considered to be achieved by fertilization of an unreduced gamete with a reduced one, has been accepted as a means of polyploid production. However, fusion of an unreduced sperm cell with a reduced central cell forms primary endosperm cell with unbalanced parental genome ratio , and such primary endosperm cell abnormally develops, resulting in arrest of embryogenesis and seed development.Recently, emergence of polyspermy-derived triploid embryo (progeny) has been clearly presented in planta  and in vitro . Moreover, using a novel GAL4/UAS system, termed HIPOD system, it was clearly indicated that triparental progeny is produced through hetero-pollination (polyspermy) in Arabidopsis by Dr. Groß-Hardt's group . The present study is continuous work of the same group, and the authors successfully expanded the HIPOD system to HIPOD-SACO system to analyze developmental profiles of polyspermy-derived embryos and seeds.eLife. Comments from the reviewer are presented below, and the authors can address the comments.In the study, authors showed that hetero-pollination in Arabidopsis resulted in production of seed in which polyspermy-derived triploid embryo (1m:2p) and monospermy-derived endosperm (2m:1p) are formed. This finding is extremely important and interesting, since triploid progeny (embryo) can be developed without effect of triploid block from unbalanced endosperm. In addition, it was also indicated that tetraploid seeds were obtained from self-pollination of the polyspermy-derived triploid plants. This suggests that polyspermy-derived triploid plants has potential to form tetraploid progenies in one generation, being consistent with the triploid-bridge pathway for emergence of tetraploid plants (polyploid plants). Therefore, the contents of the present study provide highly novel insights and concepts into reproduction system in angiosperms, and the reviewer considers that the present study will be a new milestone for basic and applied plant biology. Therefore, the manuscript will be suitable for publication in admetos”).Does polyspermy selectively polyploidize egg cell? , it can be considered that the polyspermy occurs not only in egg cells, but also in central cells with nearly equal efficiency. Two-fold numbers of possible triploid progenies (seeds) were obtained when adm1 donors were employed, compared with wild type donors. This may indicate the possibility that polyspermy occurs both in egg cell and central cell evenly also in case of using wild-type donors, and that, among these variable kinds of fertilized ovaries (seeds), only half of triploid seeds containing 1m:2p embryo and 2m:1p endosperm can survive to mature seeds but remaining half seeds possessing 2m:2p endosperms aborts. In this context, the reviewer feels that the terms 'selective polyploidization' will not be appropriate for presenting the polyspermy-situation in the study. 'Polyspermy-derived selective growth of triparental progeny via triploid block escape' may correctly sound.The reviewer totally agrees that triploid-block can be escaped in the seeds containing polyspermy-derived 1m:2p embryo and monospermy-derived 2m:1p endosperm, and such seeds with triploid progeny develop normally. However, judging from the results of HIPOD experiments using Reviewer #3:This study investigates a potential cause of whole genome duplication and polyploidy in plants as polyspermy (fertilization of an egg by more than one sperm cell). Polyploids in plants are known in many cases to arise from production of unreduced gametes, caused by a change in the progression of meiosis. However, fertilization by unreduced male gametes typically leads to triploid block caused by genome inbalance in the endosperm.The authors develop a specialized assay HIPOD-sco1 to detect polyspermy. This uses a mutation called sco1 that causes seed to be pale green. This is complemented by two different transgenes; (i) RPS5a::GAL4 and (ii) UAS::Tomato-SCO1. In the case of polyspermy both transgenes may be inherited causing complementation of sco1 seed phenotype and expression of the fluorescent TOM-SCO1 protein. I assume pollen is mixed from the two donors and then applied to the sco1 mutant flowers?The authors screened 56K seeds and found 10 seeds that showed evidence of complementation and tomato expression. Using spreads and labeling with CENH3-GFP the authors are able to confirm that the rescued embryos are triploid, and the endosperm is of expected triploid genotype (as in wild type), which implies that polyspermy of the egg cell and not the central cell occurred.admetos”) – is the data seen in adm1 significantly different from wild type. The comparison with data from interploidy crosses is also hard to interpret from the way this is presented.Mutations in the ADM gene suppress the triploid block in Arabidopsis. The authors report – the results of this experiment  by the same authors. Reviewer #2:[…] Does polyspermy selectively polyploidize egg cell? .The reviewer totally agrees that triploid-block can be escaped in the seeds containing polyspermy-derived 1m:2p embryo and monospermy-derived 2m:1p endosperm, and such seeds with triploid progeny develop normally. However, judging from the results of HIPOD experiments using adm1 and wild-type pollen donors (subsection “Polyspermy-induced polyploidization events are often insensitive to triploid block suppression by admetos”), it can be considered that the polyspermy occurs not only in egg cells, but also in central cells with nearly equal efficiency. Two-fold numbers of possible triploid progenies (seeds) were obtained when adm1 donors were employed, compared with wild type donors. This may indicate the possibility that polyspermy occurs both in egg cell and central cell evenly also in case of using wild-type donors, and that, among these variable kinds of fertilized ovaries (seeds), only half of triploid seeds containing 1m:2p embryo and 2m:1p endosperm can survive to mature seeds but remaining half seeds possessing 2m:2p endosperms aborts. In this context, the reviewer feels that the terms 'selective polyploidization' will not be appropriate for presenting the polyspermy-situation in the study. 'Polyspermy-derived selective growth of triparental progeny via triploid block escape' may correctly sound.Thank you for this comment. It is important to discriminate between the seeds generated by selective egg cell polyspermy/polyploidization and the polyploid plants recovered. The latter category commonly traces back to egg cell polyspermy only, because the category of egg and central cell polyspermy, is negatively selected during seed development by the triploid block. However, we understand the concern of this reviewer and agree that this can be misleading. We have accordingly adjusted title and text:Title: "Selective egg cell polyspermy bypasses the triploid block".adm result: "In fact, we here show that most polyspermy-derived seeds are insensitive to the triploid block suppressor admetos." In the remaining text we introduced "can" in the last paragraph of the Introduction and in the subheading “Polyspermy can selectively polyploidizes the egg cell”, and "most" in the last sentence of the subsection “Most Polyspermy-induced polyploidization events are often insensitive to the triploid block suppression suppressor by admetos”.Abstract: We introduced "can" and modified the sentence describing the Reviewer #3:[…] The authors develop a specialized assay HIPOD-sco1 to detect polyspermy. This uses a mutation called sco1 that causes seed to be pale green. This is complemented by two different transgenes; (i) RPS5a::GAL4 and (ii) UAS::Tomato-SCO1. In the case of polyspermy both transgenes may be inherited causing complementation of sco1 seed phenotype and expression of the fluorescent TOM-SCO1 protein. I assume pollen is mixed from the two donors and then applied to the sco1 mutant flowers?sco1/- pRPS5a:mGAL4-VP16/+ and sco1/- pUAS::SCO1_tdTOMATO/+. These two pollen categories were applied onto the stigmatic surface with two brushes."Thank you for this comment. No, we used the same technique applied in Nakel et al., 2017. This was not well specified in the current manuscript and we added the following text to the Materials and methods:"3 days after emasculation, pollination was carried out using pollen grains collected individually from plants of The authors screened 56K seeds and found 10 seeds that showed evidence of complementation and tomato expression. Using spreads and labeling with CENH3-GFP the authors are able to confirm that the rescued embryos are triploid, and the endosperm is of expected triploid genotype (as in wild type), which implies that polyspermy of the egg cell and not the central cell occurred.Mutations in the ADM gene suppress the triploid block in Arabidopsis. The authors report the results of this experiment (subsection “Polyspermy-induced polyploidization events are often insensitive to triploid block suppression by admetos”) – is the data seen in adm1 significantly different from wild type. The comparison with data from interploidy crosses is also hard to interpret from the way this is presented.Yes, we apologize for the confusion. The figure legend has been adjusted to ease understanding. We didn’t do any statistical analysis as we only recovered few data points. However, we now include in a table  depicting the result gained in three independent experiments by three different scientists. From the data, it is evident that there is substantial consistency with respect to fold change.adm results to avoid misunderstandings (see our response to reviewer #2).In addition, we now more carefully phrase the Next the authors perform transcript profiling in triploid plants and consistent with published work observe few if any significant changes.Although the work is well performed – my major reservation is that I don't think a sufficient advance is made over previous work  by the same authors.Here we disagree. Until recently, hardly anybody expected triparental plants to inhabit our planet. In this study, we established a genetic detection assay that allows for the first time to determine the developmental origin of triparental plants. With this tool we were able to show selective polyploidization of the egg cell. This result is particularly unexpected as egg and central cell fertilization are coordinated during monospermy and because the egg cell block is stricter than the central cell block . Moreover, our finding that polyspermy can bypass the triploid block has important evolutionary and agricultural implications: First, it expands current polyploidization concepts, as it provides an explanation of how triploids can arise in the presence of a strict triploid block. Second, three-parent crosses might provide a means to overcome endosperm-induced hybrid incompatibilities by transmitting incompatible sperm along with compatible sperm through the egg cell only. We have rephrased two sentences in the Discussion in order to make this aspect clearer to the reader:"Not only do three parent crosses hold the potential to instantly combine beneficial traits of three rather than two cultivars. […] Scrutinizing the potential of this genetic hitchhiking approach opens up new avenues for future research."We would also like to refer to the comments made by reviewer 1 and 2 on the implication of our work.