Restraint is widely practised within inpatient mental health services and is considered a higher-risk procedure for patients and staff. There is a sparsity of evidence in respect of the efficacy of personal protective equipment (PPE) used during restraint for reducing risk of infection.A series of choreographed restraint episodes were used to simulate contact contamination in research participants playing the roles of staff members and a patient. For comparison, one episode of simulated recording of physical observations was taken. Ultraviolet (UV) fluorescent material was used to track the simulated contact contamination, with analysis undertaken using established image registration techniques of UV photographs. This was repeated for three separate sets of PPE.All three PPE sets showed similar performance in protecting against contamination transfer. For teams not utilising coveralls, this was dependent upon effective cleansing as part of doffing. There were similar patterns of contamination for restraint team members assigned to specific roles, with hands and upper torso appearing to be higher-risk areas. The restraint-related contamination was 23 times higher than that observed for physical observations.A second layer of clothing that can be removed showed efficacy in reducing contact contamination. PPE fit to individual is important. Post-restraint cleansing procedures are currently inadequate, with new procedures for face and neck cleansing required. These findings leave scope for staff to potentially improve their appearance when donning PPE and engaging with distressed patients. The COVID-19 pandemic creates challenges for infection control in mental health inpatient units. One particular concern is the risk of infection when engaged in physically restraining a patient, often referred to in mental health practice as ‘physical intervention’ (PI). PI is a difficult area of practice which can be very distressing for patients and staff, with significant risks of injury. There are national programmes for reducing the need for PI.1At times PI is necessary to contain serious risks arising from acutely disturbed behaviour. Within the UK, there were around 60,000 episodes of restraint reported in mental health services between 2016 and 2017.25Preliminary review of the literature indicates that there is a sparsity of evidence in respect of personal protective equipment (PPE) used during PI in mental health settings. The majority of higher-quality evidence for PPE used in health care is focused upon the effective use of PPE in general medical hospitals (GMH).This paper describes a systematic evaluation of PPE undertaken to improve the understanding of the performance of PPE during episodes of PI. The project design was developed by the Gloucestershire Health and Care NHS Foundation Trust in collaboration with the National Association of Psychiatric Intensive Care Units (NAPICU), 3D Imaging Lab, Sheffield Teaching Hospital NHS Trust and Avon Wiltshire Partnership NHS Trust.6 However, no specific additional recommendations are made for the mental health procedure of PI.Public Health England (PHE) has issued guidance for PPE use in mental health settings.7 These recommendations were based on basic tests of PPE undertaken by PI training teams. The tests primarily considered which PPE was most likely to remain in place during PI.NAPICU published guidance on managing acute disturbance in mental health which included recommendations for PPE in PI.At the time of writing, high-quality evidence or evaluation of PPE effectiveness in PI procedures within the mental health setting is absent. This contrasts with general medical settings where specific types of PPE have been evaluated for use in specific procedures, e.g. aerosol-generating procedures.The PPE currently specified by PHE for use in mental health inpatient settings is focused upon carrying out procedures that would also be carried out in a GMH setting. Examples of this include procedures which require staff to be within 2 m or in contact with a patient, such as assistance with activities of daily living and recording physical observations.There are procedures carried out in mental health settings that are comparable to general medical settings which require airway support, for example, resuscitation and electro-convulsive therapy, with the latter requiring an anaesthetic. For these procedures, current evidence supporting use of specific PPE is considered largely applicable to mental health settings.8Providers of mental health inpatient services, particularly ‘locked door services’, have an authorised approach to managing disturbed/aggressive behaviour which can extend to PI. For the UK, a broad description of PI is contained within Chapter 26 of the Mental Health Act Code of Practice (2015).A team of staff (often three) with specific roles, i.e. number one responsible for supporting the head and numbers two and three assigned to securing of the patient’s arms;A defined series of techniques that can be employed by members of staff to physically intervene to restrict the movement and contain serious risk represented by the behaviour of a patient;PI for relocating a person presenting various levels of resistance;PI methods for containment of risk within a de-escalation process resulting in the discontinuing of the need for PI;PI within which it is possible to administer medication parenterally.There are a variety of different systems for PI used by mental health service providers in the UK, although all share similar characteristics. These are:PI carries its own risks of injury to patients and staff. These include uncertainty as to the level of control that can be achieved in any given situation. Furthermore, PI episodes can often be difficult to predict in terms of the levels of resistance, amount and nature of close contact, or the length of time an episode will take to conclude.10PI is considered amongst the highest-risk procedures used in mental health settings and is governed by law. It often causes distress and should only be implemented when there are no less restrictive alternatives. Reducing risk of injury for all those involved is a central international consideration for the application of PI.12It has long been recognised that at times additional risks to the staff can include increased possibility of infection from close proximity spitting, biting and scratching.13Since first being identified in December 2019, COVID-19 has proven to be highly infectious, leading to unprecedented measures of ‘lockdown’ and mandatory social distancing. Specific details of how COVID-19 spreads remain subject to study. It has been established that contact contamination represents a significant infection method. The extent to which COVID-19 can spread by aerosol remains subject to debate.Episodes of PI require very close physical contact between staff and patient. Within the process of close contact, there is also potential for physically challenging struggle during which opportunities for contact transmission of COVID-19 are increased.Bodies are in physical contact with each other, particularly hands, providing direct opportunity for contact contamination.The extent to which any PPE will remain in place and able to withstand high levels of demanding physical activity.The potential for any PPE to be purposely damaged or attempts at removal made by the recipient of PI.Potential for very close proximity between the head/oral and nasal region between those involved in PI. This can range from 50 mm to 500 mm.Potential for very close-proximity directed projection of larger droplets of oral fluid, e.g. by spitting.14Potential for very close-proximity shouting, coughing and raised voice projecting smaller particles of respiratory and oral secretions.Increased respiratory rate and depth resulting from physical exertion, increasing the possibility of secretion and/or inhalation of virus-containing material.The following are common characteristics of PI which may be considered to increase the risk of COVID-19 and other infections:How does PPE perform in mitigating contact contamination during PI?What is the pattern of contact contamination arising from PI?What are the specific PI issues for the robustness and comfort of PPE?This paper aims to address the following questions:This paper aims also to identify suggestions for improvement.We searched nine databases including BNI, PsycInfo, Cinahl, Embase, Medline, Emcare, Google Scholar, World Health Organization, Global research on Coronavirus disease (COVID-19) database and medRxiv/bioRxiv COVID-19 SARS-CoV-2 preprints. We searched for all published evidence within these databases before 6 May 2020. We used search terms of Coronavirus, COVID, infection, infection control and physical restraint, physical intervention, restraint, mental health, psychiatry. In total, 276 published papers were retrieved; however, none contained evidence specifically covering PPE and infection control during restraint in mental health.Twelve participants, divided into three groups of four, were used to undertake three episodes of simulated PI. Each of the episodes involved one participant representing the infection source (patient) and the remaining three representing members of a PI team (staff). In each episode the simulated PI followed a predetermined choreography, and in each episode the group playing the role of staff donned different PPE.The participant representing a patient infected with COVID-19 had ultraviolet (UV) material placed in the areas of the body most likely to contain infectious material, i.e. lower face, upper chest, arms and hands. Spitting of oral fluid was simulated using UV fluorescent material consistent with training aids for infection control. All three episodes were completed using different staff in physically separate areas to remove the possibility of cross-contamination between the three episodes.Following the PI episode, the amount and location of contact transfer of UV fluid between simulated patient and staff was recorded by UV photography.The extent to which the PPE remained in place.Potential or actual hazards arising from the PPE.The ease with which the different PPE sets could be donned and doffed.Comfort of the PPE during use.The following was also subject to evaluation:7 Pre-evaluation tests determined that the following criteria for the selection of the PPE should be formally evaluated:PPE generally considered mainstream with commercial mass production.PPE items were likely to remain in place.PPE items less likely to increase risk in the context of PI (e.g. slip hazard).PPE items known to have some infection control value.PPE worn as standard by staff during shifts.In the PI scenario, the extent to which PPE will remain in place may be equally as important as the infection control specification.F. Efficacy in preventing transfer of simulated infectious material.G. Time taken to don and doff.H. Potential for cross-contamination while doffing.The items were likely to have differences in the PI context worthy of comparison. These included:Under the supervision of an Infection Control Specialist Nurse (ICSN), the same amount of UV fluorescent substance was placed on three different participants assigned the role of patient on the areas likely to contain infectious fluid on a COVID-19-positive patient. These areas were hands, nasal and face region, and the anterior aspect of patient torso. The face and arms were contaminated using two different colours .Point One: spray bottle 150 mm away from the target.Point Two: spray bottle 300 mm away from the target.At two points during the PI episodes spitting was simulated. This was achieved by means of a spray bottle containing UV material of a third colour (red). This was directed towards a participant’s face to simulate transfer of oral fluid by spitting.After standard training following an Infection Control Action Card, nine participants who were staff from the Physical Interventions training team assigned to the role of TSM (staff members) donned and doffed the three different PPE sets in Donning and doffing for all TSMs took place in an area of 4 square metres with two chairs present and a standard size peddle bin. Each team of three TSMs had a separate area. Time taken to don was recorded. Donning and doffing was observed by the ICSN, who recorded any issues against a standard checklist. Any correction advice needed or offered by the ICSN was also recorded.In order to simulate the cleaning facilities available at the site of PI episodes in mental health inpatient facilities, universal wipes (branded Clinell) were used for personal cleansing.Immediate containment of assault;Relocation;De-escalation.The following PI procedures were choreographed and enacted in three episodes as a linear progressive sequence of similar time duration (4.5 min).Pre-donning PPE;Post-donning PPE;Post-PI simulation with PPE;Post-doffing without PPE.A darkened photograph booth was constructed which was illuminated with visible and/or UV light. Within controlled parameters, full-body photographs were taken of the test subjects at the following points:PPE Set C did not require donning of PPE as they were already wearing scrubs and a mask, representing the standard uniform of a member of staff on an inpatient mental health ward. As such, there were only three sets of photos required for this group.Comparison of the simulated contamination was made between the different PI episodes and contamination types using a standardised analysis of the UV photographs. Established deformable image registration techniques were implemented  to align all visible light photographs for the definition of nine standard regions . These were then applied to the UV photographs to measure the regional distribution of the contamination.k-means clustering with UV images converted to the CIE L*a*b* colour space).15 Here the dominant colour of each dye (and other background features) was found from photographs of the ISTS prior to the PI. These colours were then used to partition the TSM photographs to identify areas of contamination. Combining this segmentation with the regional body contours permits a zonal analysis. Some manual editing was required to exclude regions of clothing which appeared blue in the UV image.Detection of the coloured dyes was performed by segmenting the UV photographs using a clustering methodology (Each TSM was observed during and interviewed following the three simulations. The three episodes were video recorded (including donning and doffing). The videos were reviewed by a panel of Infection Control and PI specialists to identify any issues arising.There was evidence of contact transfer during the simulated episodes of PI .2 saturation) were taken. The same method for tracking contact contamination was applied. When compared with taking physical observations, 23 times more contact contamination occurred following an episode of PI .For general reference, a participant not involved in the PI episodes was ‘contaminated’ in the same manner as the simulated PI patient with UV substance. Following this their general physical observations , there is some level of UV fluorescence similar to contamination detected prior to donning of the PPE. This was considered likely to have originated from the Clinell wipes used to clean prior to donning.Using the standardised regions, the regional distribution of contamination is shown for each team in the schematic representation of The spit contamination (red) is concentrated around the head for all groups, although for team A and B some was also deposited on the upper torso. Team A showed some contamination remaining post-doffing.The contamination derived from the hands and arms (green) of the patient was spread much more widely, but none was detected on the face and neck for any team. Team A, post-doffing had virtually no contamination, with only a very small amount remaining on the hands of subject A2. Teams B and C, however, show contamination remaining post-doffing, and this is concentrated on the hands and to a lesser extent on the arms and lower torso (Team B).The contamination from face and torso (blue) showed a wider distribution including the head and neck. Similarly, the post-doff distribution was also wide, with the highest level found in the hands, but for team B contamination was still detected on the face. In all teams it was found that the PPE and skin-cleaning product was clearly visible in the UV images, which in some cases was difficult to differentiate from the contaminant dye.By aggregating the post-PI results for team members one, two and three over all the three groups, it is possible to see where the contamination is predominately derived from . The redAll three PPE sets showed similar performance in protecting against contamination transfer. For teams not utilising coveralls, this was dependent upon effective cleansing as part of doffing. There were similar patterns of contamination for restraint team members assigned to specific roles, with hands and upper torso appearing to be higher-risk areas. The restraint-related contamination was, on average, 23 times higher than that observed for the recording of physical observations.Goggles: The goggles remained in place, but on one occasion there was evidence of UV material breaching the goggles. The goggles quickly misted up and severely restricted vision. TSM reported some discomfort after 20 min.Masks: There were no issues with FFP3 and fluid-resistant masks in terms of comfort or hazards. They remained in place.Coveralls: The zip marginally opening during the PI procedures caused high levels of heat and perspiration. TSM reported that the coveralls were very uncomfortable from overheating.Disposable scrubs: There was one incident of the disposable scrubs ripping due to inappropriate sizing. However, they remained in place and were felt to be comfortable.Scrubs: Remained in place, were comfortable, but had to replaced by alternative clothing post-doffing.All three of the PPE sets showed similar performance in the amount of transfer of contaminant. Pre-doffing, the location of containment was surprisingly similar between the teams.Only PPE set A  provided protection of the arms. This set showed less UV contaminant in contact with skin in the arms area post-PI compared with Set B and Set C.Of particular note was contaminant located in axilla region (coloured green) of numbers two and three in the PI teams . This arose from contact with the infection source hands and arms when they were secured using PI techniques. Numbers two and three (left and right side of the patient) in the teams received less contamination (coloured blue) compared with team member one originating from the face and chest area of the infection source.Number one in the PI team (responsible for the controlling the head) had more UV material than the other team members on their hands and wrist which transferred from the face and torso area (coloured blue) of the infection source. Number one in the teams received less contamination from the hands and arms of the infection source (coloured green) compared with the other two team members.Following doffing, all three PPE sets showed similar performance in protecting against contamination transfer. For teams not utilising coveralls, this was dependent upon effective cleansing as part of doffing. One of the test subjects clearly missed cleansing an area of contact contamination in the arms. If the PPE had provided arm coverage, it is likely that this area would not have remained contaminated post-doffing.The spitting simulation (coloured red) presented particular challenges for the PPE. Both the fluid-resistant and the FFP3 mask were successful in preventing contact contamination. However, spit contaminant did breach the goggles and was found on the lower eye area of one of the test subjects . Contaminant was also found on the neck area, and automatic analysis identified small regions over the torso in a number of subjects.Careful review of the video recording could not clearly establish how the contaminant breached the goggles. Theories include the front panel of the goggles becoming dislodged from the frame or the face seal of the goggles becoming displaced during the PI episode.Spitting demonstrated the clear need for face and neck cleansing procedures that currently do not feature in standard cleansing advice for health care.This evaluation has demonstrated that PPE needs to be properly fitted to the wearer. Examples of this include the coverall zips travelling downwards during simulation as a result of being oversized for the individual. There was also an episode of disposable scrubs ripping on donning as a result of them being too small for the test subject’s legs.Two areas of major concern include the overheating experienced by the test subjects from the coveralls, and the goggles misting up to the point of severely restricting the vision of the PI team members.It has been long established that donning and doffing PPE is crucial to its effective performance. This also requires PPE to be specifically fitted to the individual rather than limited to a small number of sizing options. This evaluation showed diminished performance for some poorly fitting PPE.For mental health inpatient practice, face and neck cleansing procedures are required and are possibly more important than previously thought. These are required to deal with the risk of spitting or experiencing pressure of speech while unable to observe social distancing.Debate continues as to the extent to which COVID-19 is airborne, although this was not the focus of this evaluation. Given the specific characteristics of PI , FFP3 standard masks would likely be preferable to fluid-resistant masks.The specific context of PI involving high levels of physical exertion is a central PPE consideration. This requires PPE to be robust, comfortable and well secured. Of specific note is the need for eye protection to be robust and include mitigation against misting. This could be achieved by selecting robust goggle designs and preparing them with an anti-mist spray.While offering higher levels of protection, overheating caused by coveralls as well as more difficult doffing observed in this evaluation suggested that alternatives could return satisfactory performance while mitigating these problems.The evaluation suggests that a second layer of clothing which can be removed can be effective in minimising contact contamination following PI. This is, however, dependent upon there being effective cleansing equipment and procedures available following PI. Exactly what the second layer is may be less relevant than its presence.This could be an important consideration in selecting PPE, providing more options including those with improved appearance for patients who may already be anxious and frightened in the acute mental health context. The role of each PI team member within the procedure was shown to be relevant to the higher-risk areas for contact contamination. For the staff member in charge of controlling the head, the hand and wrist areas were at higher risk of contamination. For those assigned to each side of the patient, then the respective side of the axilla region as well as hands and wrist were higher-risk areas.This evaluation demonstrates the need for further high-quality evidence which is derived from the unique characteristics of mental health inpatient practice. The simulated PI used in this study is specific to inpatient mental health services. However, our findings may also have relevance to other public service sectors such as the police, prison service, care home and residential settings where PI may be implemented.Members of the local ethics committee were consulted. The evaluation design did not involve any patients or patient-related data and therefore is considered part of standard procedure and equipment evaluation not requiring ethical approval. Valid consent to take part in the evaluation was provided by all involved.The evaluation was restricted to contact transfer of potential virus-containing material from the patient to the staff acting in the PI. The evaluation did not consider the potential for infection to be transferred from the staff to the patient in similar circumstances. Nevertheless, the conclusions drawn may guide support for patients in the areas of cleansing and changing clothes.The evaluation did not consider potential for aerosol transmission of smaller material.The transfer of UV material cannot be considered as an accurate representation of the volume of potentially virus-containing material transferred, although it does give an indication of the contact transmission areas that could contain virus material.Some items of PPE (notably gloves and goggles) and the universal wipe used to clean the skin were clearly visualised in the UV light photographs with a colour close to the fluorescent dye used to represent contamination. This made automatic segmentation of the dye distribution difficult.Regions of contamination appeared overexposed in the UV photographs, with a bright white appearance. This was difficult to distinguish from lower-level contamination of the blue fluorescent dye.The high-resolution close-up photographs showed that some contamination was not detected in the 32 whole-body images, albeit only very small amounts.


Although species with larger body size and slow pace of life have a higher risk of extinction at a global scale, it is unclear whether this global trend will be consistent across biogeographic realms. Here we measure the functional diversity of terrestrial and freshwater vertebrates in the six terrestrial biogeographic realms and predict their future changes through scenarios mimicking a gradient of extinction risk of threatened species. We show vastly different effects of extinctions on functional diversity between taxonomic groups and realms, ranging from almost no decline to deep functional losses. The Indo-Malay and Palearctic realms are particularly inclined to experience a drastic loss of functional diversity reaching 29 and 31%, respectively. Birds, mammals, and reptiles regionally display a consistent functional diversity loss, while the projected losses of amphibians and freshwater fishes differ across realms. More efficient global conservation policies should consider marked regional losses of functional diversity across the world. Anthropogenic extinctions are driving functional shifts in biological communities, but these changes might differ considerably among taxa and biogeographic regions. Here the authors show that projected losses of functional diversity among land and freshwater vertebrates are unevenly distributed across the world. For instance, 198 extinctions of vertebrates have been recorded since 1900, which is at least ten to hundred times higher than the natural baseline rate3. The most recent IPBES report estimates that about 1 million of the world species  might be at risk of extinction in the next decades4. However, counts of threatened species do not fully reflect the ecological impacts of extinctions, because species’ contributions to ecosystem functioning depend on their functional traits6 (i.e. key characteristics involved in organisms’ response to the environment and their effects on the ecosystem properties7).The loss of global biodiversity is accelerating throughout the world11. For several groups of organisms, many species have evolved toward similar ecological strategies resulting in a limited set of functions permitted within the possible range of trait values. For vertebrates, functional traits can be summarized in a functional space with a few dimensions in which species are clumped around some prevalent strategies11. However, those global studies considered all the species together, whereas regions of the globe  host distinct types of ecosystems and faunas13, which possibly cover different portions of the functional space. For instance, Carmona et al.11 showed that among the global mammal functional spectrum, higher primate species (suborder Simiiformes) represent a functional hotspot . However, the uneven and localized distribution of the higher primates might critically differentiate the regional functional space of mammals.Previous mappings of the global diversity of functional traits have attempted to describe the dimensionality of the functional space of different taxa by defining a set of independent dimensions that provide non-redundant information11. Considering that species and their threats are not equally distributed across the world14, the functional traits of threatened species in a specific region might differ from the global pattern. As the extinction of threatened species will neither occur simultaneously across the world or at the same pace, identifying which regions are more inclined to suffer larger losses of taxonomic and/or functional diversity might help to effectively target conservation goals. Moreover, the most threatened species  are likely to disappear earlier than species with a lower risk of extinction , highlighting the need to take account the IUCN category when measuring the functional diversity at the regional scale . For instance, if CR species exhibit a unique set of functional traits, their extinction would cause a drastic loss of functional diversity17. Thus, prioritizing the conservation efforts to maintain those species, might efficiently contribute to tackling the loss of biodiversity. Instead, if the most threatened species do not host a unique set of functional traits, their extinction will not drastically alter the functional diversity of species. In this case, the conservation policies aimed at conserving functional diversity should be oriented toward long-term goals, on a broader range of threatened species.In the context of global changes, potential extinctions could cause dramatic erosion and rearrangement of ecological strategies globallyHere, we examined the distribution of the functional diversity of five groups of vertebrates across the main six terrestrial biogeographic realms by testing how regional taxonomic discrepancies translate into functional differences among realms. We then quantified how the potential extinction of species will affect the functional diversity patterns by simulating a progressive loss of threatened species from the most threatened species to the less endangered species. We characterized the functional diversity of each taxonomic group using a set of functional traits associated with different key aspects of their ecology and their life-history characteristics. The analyses reveal that the realm functional diversity differs within and between taxonomic groups. Moreover, we show that all realms are not similarly affected by the loss of threatened species but Indo-Malay and Palearctic realms are particularly inclined to experience a drastic loss of functional diversity. Understanding the regional patterns of functional diversity loss as well as the functional diversity supported by endangered species is key to improve our predictions on identifying the most vulnerable regions of the globe and hence better target the conservation policies of threatened vertebrates.19. We collected information on traits related to ecological functions  for the five vertebrate groups using the most recent functional trait databases22. In addition, we retrieved the most comprehensive available species lists compiled at the realm scale25. Because the functional trait databases for none of the groups were complete, a specific imputation procedure was used to complement this missing information  was higher in the Afrotropical, Neotropical and Indo-Malay realms and lower in the Australian, Nearctic and Palearctic realms Fig. . Functio27, we performed null models to estimate whether the amount of functional space occupied by an assemblage within a realm is smaller  or larger  than expected given the number of species in each biogeographic realm. These results showed a generalized pattern of higher-than-expected functional redundancy (or no difference from expected), with the only exception being birds in the Nearctic . For birds and mammals, three and four realms, respectively, did not differ from expected, showing that similar amounts of functional space are filled in each realm, regardless of the differences in species richness  while reptiles were the least impacted  and allows them to extend their range across large distances. In addition, birds have the highest mobility and are least affected by geographical dispersal barriers compared to the other groups, which might explain the highest functional and taxonomical similarity between realms. For mammals, however, strategies related to large species with a slow pace of life are missing in the Palearctic and Nearctic and to a lesser extent in the Neotropical, which can be related to the decline of the megafauna during the Pleistocene11, highly threatened in Indo-Malay, Afrotropical and Neotropical realms32 but absent from the other realms. Endangered species include our closest biological relatives in Africa, such as chimpanzees (Pan troglodytes), bonobos (Pan paniscus) and gorillas (Gorilla spp.), as well as orang-utans (Pongo spp.) in the Indo-Malay realm, and some spider (Ateles spp.) and capuchin monkeys (Cebus spp.) in the South-American tropics32. All these species are threatened by multiple anthropogenic pressures including hunting and deforestation, which might increase with ongoing and future global changes30. The loss of functional diversity would also be higher in the Indo-Malay realm for birds  or the Indian vulture (Gyps indicus) are edging very close to extinction, mainly due to habitat loss and degradation15. Thus, Indo-Malay does not only support a higher than expected proportion of threatened species14, but also a higher than expected proportion of threatened species with unique functional traits. This stresses the ultimate importance of biodiversity conservation in the Indo-Malay realm. Habitat degradation coupled to a particular geographical environment (e.g. insularity of South-East Asia) is likely to increase the threats of the large-bodied birds and mammals. As demonstrated for threatened species14, our results also suggest that the functional diversity of some taxonomic groups in some realms  is inherently more vulnerable to the effects of anthropogenic pressures. Despite the similar intensity of loss in functional diversity , one of the largest reptile species, while the loss of the vipers (Vipera spp.) could erode the functional space of the Palearctic. Given the importance of those large species, which are often critical consumers and keystone species33, for ecological processes, their extinction might affect ecosystem functioning in the near future35.Despite the high functional similarity among realms for mammals, birds and reptiles, the loss of threatened species will lead to uneven functional changes among realms. Indeed, the loss of threatened species would result in a combined effect of the diversity of functions supported by the species inhabiting each realm and the number of threatened species in the same realm. For instance, the loss of threatened mammal species would have contrasting effects, with some realms  being highly impacted, with others (e.g. Nearctic) being less vulnerable. Even under a low extinction risk scenario, the potential loss of functional diversity in the Indo-Malay realm was significantly higher than the potential loss in the other biogeographic realms Figs. . The cauity Fig. . For ins36 and lower long-distance dispersal abilities than endothermal organisms like birds or mammals. For instance, the recolonization of Central and Western Europe by fish species from Ponto-Caspian Europe after the Last Glacial Maximum37 could explain the low functional diversity of the Palearctic realm for freshwater fish fauna. In contrast, fewer climatic restrictions and the higher rates of speciation reported in tropical realms across evolutionary times could have contributed to the exploration of unique parts of the functional space that are not realized in northern realms. For instance, Neotropical Siluriformes have unique ecological strategies, ranging from algae browsing Loricaridae  to parasitic Trichmycteridae catfishes9.The current occupation of the functional space for amphibians and freshwater fishes is more uneven among biogeographic realms—each realm hosts a low proportion of the world’s functional diversity. This is particularly striking for amphibians and freshwater fishes where the realms’ functional spectra largely differ from the global patterns. This might reflect a functional adaptation to local environmental conditions. Although our data do not allow disentangling the processes behind such distinctiveness, we can hypothesize that the strong link of amphibians and freshwater fishes to the aquatic environment has limited their dispersal. Accordingly, amphibians and freshwater fishes have smaller distribution ranges11. For instance, many large-bodied freshwater fishes, such as sturgeons (Acipenser spp.), are threatened in the Palearctic, whereas many small-bodied species, such as suckermouth armoured catfishes (Chaetostoma spp.), are threatened in the Neotropics25. Moreover, the high current functional distinctiveness between biogeographic realms also explained the differences in the loss in functional diversity among realms. Indeed, some threatened species host a unique set of functional traits in some realms, while being functionally redundant with non-threatened species in other realms. For example, the functional traits, such as viviparity and low fecundity of some threatened amphibian species such as salamanders  are unique in the Palearctic, the realm that could experience the strongest loss in functional diversity, whereas similar traits are filled by another amphibian species in tropics, such as the caecilians in Afrotropical, Neotropical and Indo-Malay38. Although these results were hardly affected by a potential bias due to different sampling efforts among realms and/or among functional traits, some results should be considered with caution. For example, only 21% of the freshwater fishes in Neotropical have been evaluated by IUCN versus 44% worldwide. More studies are thus required to better evaluate the influence of the IUCN Red List completion on the predicted losses of functional diversity39 for specific taxonomic groups in some incompletely evaluated realm faunas.For reptiles, amphibians and freshwater fishes, the changes of functional diversity among realms were stronger in both intensity and direction than those reported at the global scale11, discrepancies arise at the realm scale, with threatened species supporting distinct parts of the functional space in different realms. Indeed, the loss of some small-sized threatened species such as geckos (Sphaerodactylus spp.) in the Neotropical realm or moss frogs (Arthroleptella spp.) in the Afrotropical realm would contribute to the erosion of the functional richness of those two realms , Nearctic, and Indo-Malay realms;42 their loss might reduce the services they provide to humanity. Although non-native species introductions might partly compensate for those functional losses, the detrimental and unpredictable effect of biological invasions43 makes this a high-risk strategy. The Indo-Malay realm currently experiences multiple threats including increasing habitat degradation through deforestation and damming45, as well as a strong overharvest of both terrestrial and aquatic vertebrate faunas48. Such a steep increase of environmental disturbances combined with the functional vulnerability of the Indo-Malay realm for all groups of vertebrates should elicit coordinated and efficient conservation strategies in this region. This is further exacerbated in the Indo-Malay realm due to the strong dependency on wild animal resources for food supply49.Even if it is not possible to predict the ecological consequences of functional loss at the realm scale, the loss of functional richness might trigger a loss of stability in the ecosystems and weaken their capacity of resistance and resilience to ongoing global changes. For instance, large-sized fish species are recognized as key species controlling food webs through predationToward a global scale conservation perspective, the global loss of species should be tackled differently according to the organisms. Mammals and birds occupy similar parts of their functional space in all biogeographic realms and display similar responses to the projected species extinction. This allows a global functional approach to alleviate the predicted biodiversity loss for these two taxonomic groups. In contrast, reptiles, amphibians and freshwater fishes are distributed in different parts of their functional space in each realm, likely a result of evolution within smaller units, higher dependence on environmental conditions and/or low dispersal capacities. For these types of organisms, the projected loss of functional diversity differs from global assessment and requires more regional or local approaches to better manage the functional consequences of species extinctions.15. By doing this, we assigned a realm for 5489 mammal species, 10,787 bird species, 5489 reptile species and 5833 amphibian species. Since IUCN spatial database does not cover all species, we completed our database with two additional sources of species occurrences: (1) the WWF WildFinder species database23, except for mammals where we used the latest version of the species distribution provided by ref. 24. If (1) was not available, we used (2) the global biodiversity information facility (GBIF). Using WWF WildFinder, we assigned a realm for 1634 bird species, 7378 reptile species and 2006 amphibian species. 437 mammal species were assigned using ref. 24. From GBIF, we downloaded all the records belonging to the four classes of animals . Before using the spatial data, we cleaned the dataset following a cleaning procedure that was similar to but more conservative than other currently available methods . First, records were screened, and only those with (1) coordinates; (2) a taxonomic rank of “species” were kept. From this list, we filtered out the records with clearly false locality coordinates ). Those are the most common errors encountered with GBIF occurrence data55. In addition, we removed the records from living specimens , conserved specimens (i.e. museums), and unknown sources. We also excluded the species with less than 50 records within each realm as a low number of records can be due to misidentifications, which might have strong effects on our analyses. We finally refined the dataset by overlaying the occurrences within the six biogeographic realms (see below) and dropping the species that fall outside of the polygons. This spatial overlay process was conducted using the ‘sp’ library56 in R. The number of species for which realm was assigned using GBIF was 1 (<0.02%) for mammals, 442 (3.4%) for birds, 572 (5.3%) for reptiles and 25 (3.2%) for amphibians.We collected species occurrences from the most accurate and available source of data for each taxonomic group. For mammals, birds, reptiles and amphibians, we used the IUCN spatial database to assign realm identity for each species24 for mammals) was higher than 95% at a global and realm scales for all groups  influenced the functional space of each group in each realm and the world . For that, we calculated the functional overlap between the functional spaces built with all species and the functional space built with all species except the species retrieved from the IUCN spatial data  and currently represents the most detailed spatial information on this taxa.For freshwater fishes, we collected the occurrences database from ref. 58, the Afrotropical, Australian , Nearctic, Neotropical, Indo-Malay and Palearctic . Notice that the Australasian and Oceania realms are considered as one combined realm in our analyses since the number of records was too low in Oceania for some taxonomic groups (e.g. fishes) to consider it as an independent realm. We thus called it the Australian realm, following the terminology of ref. 57. Our final database of spatial occurrences for the six realms encompassed 5927 mammals, 12,863 birds, 13,439 reptiles, 7864 amphibians and 13,008 freshwater fishes occurring in at least one biogeographic realm  and mostly related to life-history characteristics. For mammals, birds and reptiles, we used the AMIOTE database20 including data for 4953 species of mammals, 9802 species of birds, and 6567 species of reptiles. For mammals, we selected a subset of eight traits for which at least 1000 species were informed11. These traits were: litter size (number of offspring per litter), number of litters per year, adult body mass (g), longevity (years), gestation length (days), weaning length (d), time to reach female maturity (days), and distance from the tip of the snout to the tail base (cm). For birds, we selected a total of eight traits: clutch size (number of eggs), number of clutches per year, adult body mass (g), incubation time (days), longevity (years), fledging age (days), egg mass (g) and distance from the tip of the beak to the opening of the cloaca (cm). For reptiles, we selected a total of six traits with sufficient information: clutch size (number of eggs), number of clutches per year, adult body mass (g), incubation time (days), longevity (years) and distance from the tip of the snout to the opening of the cloaca (cm). For amphibians, we used the AmphiBIO database21 to get data for 6776 species of amphibians. Within this dataset, we selected five traits with enough information: age at maturity (years), the maximum number of reproduction events per year, body size (mm), maximum litter size  and offspring size (mm). Finally, for freshwater fishes, we used the last updated version of the most comprehensive database on morphological traits, available for 10,705 species of freshwater fishes22. The link between morphological traits and ecological functions is well documented for freshwater fishes61, making morphological traits a relevant proxy to describe the functional diversity of this group. This database encompasses 11 traits describing the size and shape of body parts involved in food acquisition and locomotion. The fish body shape and weight were described through the size using the standard length (cm) and body mass (g) taken directly from FishBase62, body elongation (ratio between body length and body depth) and body lateral shape (ratio between the head depth and body depth). The other traits describing the position and the size of each part of the fish were eye size and position, mouth size and position, pectoral fin size and position, and caudal peduncle throttling and were measured on fish pictures60.We collected information on traits related to ecological functions for the five groups of vertebrates. All the traits have been selected for their ecological relevance and gathered from published studies . We obtained published phylogenies for each of the taxonomic groups68. Species that were not present in the phylogeny were added to the root of their genus, using the ‘add.species.to.genus’ from the R package phytools69. For birds and mammals, 1000 phylogenetic trees were available representing the phylogenetic uncertainties65. We considered the phylogenetic uncertainties by calculating the eigenvectors as average the eigenvector obtained for each phylogeny.Since none of the trait databases assembled was complete, we completed this information by performing trait-imputation procedures generated using the missForest R packageIt is important to note that, compared to traditional imputation procedures, we aimed to characterize the position of species in the corresponding trait space rather than estimating the values of the original traits . Thus, we tested the accuracy of our trait imputation procedure by comparison of the position of the species with complete trait values in the functional space  with the position of the same species for which we had artificially removed traits in the different dimensions of the functional space. We estimated the performance of the imputation using the normalized root mean square error (NRMSE), which expresses the average distance between real and imputed positions of species as a proportion of the range of values of species in the corresponding dimension. To do it, we artificially removed 10% of trait values from a subset of species with complete information and selected one random species with incomplete information and superimposed its pattern of missing values. Thus, we kept constant the pattern of missing values as the one in the original dataset. We performed the phylogenetically-informed imputation procedure as described above using the entire dataset consisting of all the species with non-complete trait information and the species with complete trait information . This way, the ratio between missing and complete data was higher in the simulations than in the original dataset, therefore ensuring a conservative test of the quality of our imputation procedure. In addition, we repeated this examination 100 times for each taxonomic group and measured the standard error of the NRMSE, which was always <0.5% demonstrating that these repetitions gave similar results (Supplementary Table 15) using the R package ‘rredlist’70. For each taxonomic group, we used the IUCN classes: CR: critically endangered; EN: Endangered; VU: Vulnerable; NT: Near Threatened; LC: Least Concern and DD: Data Deficient. In total, we retrieved the status for 6388 mammals, 11,158 birds, 8295 reptiles, 7167 amphibians and 20,295 fish species (including marine species).We collected the conservation status of species from the IUCN Red List (version 2020-315), were standardized using the R packages ‘taxize’71. All names were resolved against the GBIF Backbone Taxonomy. Among taxonomic groups, the proportion of species described by spatial and functional trait databases varied between 53% for reptiles  and 74% for mammals (4408 species with trait information out of 5926 species). At the realm scale, the proportion of species with trait information within each taxonomic group was congruent with the proportion of species observed using only spatial data  realm coverage and (2) trait distortion. (1) We used chi-squared tests between the occurrence dataset (considered as the most complete database), the functional dataset and the IUCN Red List dataset  on the log-transformed and scaled functional traits of each taxonomic group. Spectra were built using all species for which we had trait information. Two dimensions were needed for all taxonomic groups but freshwater fish, for which four dimensions were needed . We estimated the probabilistic distribution of the species within the functional spaces using all species with spatial and functional information by performing multivariate kernel density estimations with the ‘TPD’26 R package. Although TPD functions are continuous, in order to perform operations with them it is more practical to divide the functional space into a D-dimensional grid composed of many equal-sized cells . Then, the value of the TPD function is estimated for each cell. The value of the TPD function in a given point of the space reflects the density of species in that particular area of the space . The kernel for each species was a multivariate normal distribution centred in the coordinates of the species in the functional space and bandwidth chosen using unconstrained bandwidth selectors from the ‘Hpi’ function in the ‘ks’72 package (see details in ref. 11). To facilitate comparisons, the kernel for each species was kept constant for generating the functional space of each biogeographic realm.The construction of the spectra of each taxonomic group was similar to the procedure followed in ref. 15. The species classified among the most threatened species category ) have a higher risk of extinction than the species with a lower threat (vulnerable (VU), endangered (EN), near threatened (NT)). We started removing the species with a higher risk of extinction (-CR), then we removed successively the species with lower threatened risks . These simulations mimicked a gradient of extinction risk from a scenario where only the most endangered species went extinct to a more dramatic scenario where all threatened species (including the NT species) went extinct. For convenience, we named the scenarios according to the least threatened category considered. We also ran the last scenario considering the extinction of all threatened and near-threatened species plus all the data deficient species . This scenario considers the eventuality that all data deficient species are threatened and therefore represents an extreme scenario compensating for potential incompleteness in species threat evaluation by the IUCN.To test how functional diversity can be affected by species loss at different biogeographic realms, we simulated the loss of threatened species in a progressive framework according to the IUCN status of the species15, and another TPD function after removing the species classified as threatened. Whereas the TPD functions of all species assessed by IUCN reflect the current spectra, the TPD functions after removing threatened species reflect the potential spectra if threatened species go extinct. TPD functions are probability density functions so that they integrate to 1 across the whole functional space, a property that permits the comparison of different TPD functions18. We applied a quantile threshold of 99% to reduce the potential effect of outliers on the estimation of the amount of functional space occupied by the different spectra. After thresholding, the TPD functions were rescaled, and the probabilities were expressed in terms of quantiles to ease the interpretability of the results18.We estimated in each biogeographic realm how functional diversity will change in case of extinction of threatened species. For that, in each functional space, we estimated a TPD function considering all the species assessed by IUCNWe represented the impact of simulated extinctions by subtracting in each point of the functional space, the quantile value of the TPD function after removing threatened species from the quantile value of the TPD function of IUCN-assessed species. Negative values in this index indicate a decrease in the relative abundance of the trait values corresponding to a functional space cell and vice versa. To quantify how much the functional spectra of each group will change after extinctions, we estimated for each cell the absolute value of this quantile difference and averaged these values across cells. With this approach, we could also characterize which functional space cells become empty after extinctions .73. The Jaccard dissimilarity index measures, for two assemblages, the proportion of unique species and ranges from 0  to 1 (completely different species)74. The overlap-based dissimilarity between two assemblages reflects the degree of overlap between the probabilistic distributions of species in the functional space between the two assemblages and ranges from 0 (complete overlap) to 1 (no overlap)26.Taxonomic diversity was calculated as the number of species in each biogeographic realm  and endemicity was calculated for each realm as the proportion of the species occurring only in that realm. Functional diversity was measured as the amount of functional space occupied by the spectra . We also calculated taxonomic (TDiss) and functional dissimilarity (FDiss) between biogeographic realms using the Jaccard dissimilarity index (for taxonomic diversity) and overlap-based dissimilarity  as implemented in the ‘betapart’ and ‘TPD’ R packages, respectivelyP-values to indicate the statistical significance of the rank. P-values higher than 0.975 indicate that the observed FRic is significantly higher than expected, P-values lower than 0.025 indicate that the observed FRic is significantly lower than expected given the number of species.For each biogeographic realm, we compared the distribution of species within the functional space with a null model where the same number of species were randomly selected from the world’s pool of species. For each taxonomic group and realm, we drew 999 simulated assemblages and compared the functional richness of those 999 assemblages to the observed FRic. We then calculated standardized effect sizes (SES) as the difference between the observed value and mean of the simulated ones standardized by the standard deviation of the simulated values. We ranked the observed FRic against the simulated FRic and calculated the To test whether the SES values were correlated to the species richness in each realm, we performed linear regression models between the SES and the number of species in each realm for each taxonomic group. A negative relationship shows that species-rich assemblages tend to be functionally clustered while species-poor assemblages tend to be overdispersed. A positive relationship shows the opposite pattern, species-rich assemblages will host even more functionally diverse species while species-poor assemblages are functionally clustered.P-values to indicate the statistical significance of the rank.To assess if the impacts of potential extinctions in each realm are different from what would be expected if extinction risk is not related to species’ traits, we also compared the observed changes in functional diversity to a null model where the extinct species are randomly selected within the realm’s pool of species. For each scenario of extinction risk, we compared the loss of functional diversity to 999 losses of functional diversity where the species traits of threatened species were randomly selected among the realm pool of species. This strategy allowed us to ascertain whether losing threatened species reduces more or less than expected the functional spectra of the different taxonomic groups. For each scenario, we created 999 TPD functions simulating cases in which the same number of species were lost at random from the total set of IUCN-assessed species from the corresponding realm (i.e. including both non-threatened and threatened species rather than only threatened ones). We performed similar comparisons between each of these simulated spectra and the spectra of the IUCN-assessed species and calculated the For each taxonomic group, we tested whether the SES values of each biogeographic realm were significantly different from each other using multiple pairwise comparison tests. This allowed us to compare the differences between multiple normal distributions. To obtain distributions from SES values, we used a bootstrapping procedure where we calculated 99 SES values from a sample of 99 FRic values among the 999 simulated assemblages. We used this procedure for both the SES of the current FRic patterns and the changes in FRic under the loss of threatened species for each scenario. Pairwise comparisons results are shown by a compact letter display of all pairwise comparisons with a significance level at 5%.Further information on research design is available in the Supplementary InformationDescription of Additional Supplementary FilesSupplementary data 1Supplementary data 2Reporting Summary


Along with emergence of the organoids, their application in biomedical research has been currently one of the most fascinating themes. For the past few years, scientists have made significant contributions to deriving organoids representing the whole brain and specific brain regions. Coupled with somatic cell reprogramming and CRISPR/Cas9 editing, the organoid technologies were applied for disease modeling and drug screening. The methods to develop organoids further improved for rapid and efficient generation of cerebral organoids. Additionally, refining the methods to develop the regionally specified brain organoids enabled the investigation of development and interaction of the specific brain regions. Recent studies started resolving the issue in the lack of non-neuroectodermal cells in brain organoids, including vascular endothelial cells and microglia, which play fundamental roles in neurodevelopment and are involved in the pathophysiology of acute and chronic neural disorders. In this review, we highlight recent advances of neuronal organoid technologies, focusing on the region-specific brain organoids and complementation with endothelial cells and microglia, and discuss their potential applications to neuronal diseases. The central nervous system (CNS) comprises the brain and spinal cord, and mediates most activities in body and mind processes. Although surgically isolated and postmortem brains from the patients are invaluable resources to study the pathology of the neuronal diseases, access to these human brain samples is very limited due to ethical and practical reasons. Thus, mouse models have been widely applied to molecular studies in the brain development and the drug response. However, in genetic and molecular levels, rodents differ from humans, displaying a vastly dissimilar developmental program. Brain organoids  are three-dimensional (3D) brain models in a laboratory dish. They have arisen as innovative model systems to study human brain development and diseases. In this review, we will provide overview how the brain organoids are produced, especially focusing on the region-specific brain organoids. We will also discuss the challenges in brain organoid fields and recent implementation of non-neuronal cells in brain organoids to complete organization of brain composition.When human pluripotent stem cells (hPSCs) are cultured in the 3D aggregates, they differentiate into various brain cell types and spontaneously organize into structures recapitulating the developing human brain. hPSC-derived brain organoids represent the promising resources to investigate molecular mechanisms of brain development and disorders. At gastrulation stage, the primary central nervous system (CNS) appears as a neural plate that is composed of embryonic neuroectoderm cells . The neuSince the serum-free floating culture of embryonic body (SFEB) has provided a blue-print to form 3D structures of brain organoids from Dr. Sasai’s group . The forEBs are forced to develop into the early neuroectoderm (NE) with single or dual SMAD inhibition  that blocks mesenchymal differentiation Fig. . The neuThe striatum integrates neuronal signals from multiple cortical regions and connects downstream neuronal circuits in the basal ganglia to control voluntary movement, emotion, and cognitive functions. The striatum mainly originated from the ventral telencephalon, in particular the lateral ganglionic eminence (LGE). Brain organoids specified to the striatum area were recently derived by prolonged culture of LGE-like cell aggregates that are generated by retinoid X receptor stimulation , Activin A stimulation, and WNT inhibition . The LGEThe hippocampus is a part of the limbic system and plays a main role in learning, memory, and emotion and is vulnerable to metabolic and cytotoxic insults including traumatic injury, ischemia, and aging. Atrophy and hyperexcitability of hippocampus areas are frequently detectable in patients diagnosed with Alzheimer’s disease (AD) or schizophrenia, and are associated with memory loss and cognitive impairment. Whereas the cortex and basal ganglia are derived from dorsal  and ventral TE , respectively, hippocampus arises from dorsomedial TE . The medial patterning of TE aggregates can be induced by transient exposure (3 days) of the GSK3β inhibitor and BMP4 to modulate the dorsal midline formation . Medial An alternative protocol was recently established to direct the organoids more toward the choroid plexus region with higher concentrations of BMP4 . The choThe thalamus and hypothalamus are located under the cerebral cortex and develop from the caudal and rostral diencephalon region, respectively , 16. In +/NKX2.1+ dopaminergic neuron and agouti-related protein–releasing neurons [NE aggregates robustly differentiate into the rostral diencephalon (rDI) under growth factor–free suspension culture. Because of the inhibitory effect of insulin signaling on the rostralization, the inhibitors for AKT signaling , which is an insulin-downstream pathway, promote hypothalamic differentiation , 29. An  neurons .The pituitary gland is another homeostatic center in the brain and anatomically lies beneath the hypothalamus. In particular, the anterior pituitary regulates growth, stress, and sexual reproduction by synthesizing and releasing their related hormones and is governed by regulatory hormones from the hypothalamus via the hypophyseal portal system. The aberrant hypothalamic-pituitary axis gives rise to the disorders of metabolism and fertility, such as obesity, hypothyroidism, and hypogonadism. Despite the functional and spatial relationship, the anterior pituitary emerges from a different origin from the hypothalamus. The pituitary organogenesis is initiated by the formation of Rathke’s pouch that arises from an upward invagination of non-neuronal oral ectoderm adjacent to the hypothalamic origins in the developing embryo. Early BMP4 exposure to the ventral hypothalamic organoids facilitates the superficial formation of the oral ectoderm layer that subsequently invaginates to form Rathke’s pouch . The pitThe retina is the extension of the CNS and originated from the optic vesicle that evaginates from a part of the diencephalon (DI). The optic vesicle undergoes invagination to form cup-like structures with two-cell layers: neural retina (NR) in the inner wall and retinal pigment epithelium (RPE) in the outer wall. The optic cup formation of 3D hESC aggregates was initiated by WNT inhibitors that promote rostralization and counteract caudalizing effect of knockout serum replacement in the medium . Short tThe midbrain constitutes the rostral part of the brainstem controlling the motor ability and is anatomically connected caudally to the cerebellum and rostrally to the basal ganglia and the thalamus/hypothalamus. The mesencephalon (ME) patterning is accomplished by the treatment of WNT, SHH, and FGF8 into NE aggregates , 30. TheThe cerebellum is essential for motor control including equilibrium and posture and arises from the rhombencephalon (RH). Early FGF2 treatment together with insulin into NE aggregates promotes their caudalization and the formation of isthmic organizer–like structures . SubsequCerebellar neurodegeneration manifests with symptoms of motor abnormalities including ataxia, difficulty in speaking, and tremor. The cerebellar organoids recapitulate early developmental stage of cerebellar organization. Thus, it is good to model cerebellar diseases in neonatal phase like congenital malformation and neurodevelopmental disorders, such as Dandy-Walker syndrome and Joubert syndrome. Since neurodegeneration in the cerebellum has been observed in Huntington’s disease, the cerebellar organoids are also promising model system for neurodegenerative diseases.Primary sensory information about the external environment is received from the skin and muscle and transmits signals into the spinal cord and up to the brain. Cortical motor signals that are mainly produced from the motor cortex are returned into the peripheral tissues throughout the spinal cord. Thus, the spinal cord is essential for most bodily functions, including speech, sensation, and muscle movement, so damage to the spinal cord devastates the motor abilities and the quality of life of patients permanently. Two-dimensional (2D) differentiation of spinal motor neurons from hPSCs is initiated with dual SMAD inhibition followed by activation of Wnt/β-catenin signaling via GSK3β inhibition  . The comSMN1 gene. A recent study demonstrated that the ventral spinal cord organoids from SMA patient–derived iPSCs show decline of the motor neuron differentiation [SMN1 expression activates cell cycle–related genes and promotes re-entry into the cell cycle in the motor neurons. Interestingly, treatment of CDK4/CDK6 inhibition  can attenuate the reduction of motor neuron differentiation. Therefore, the spinal cord organoid is a useful tool to investigate the pathological mechanism and development of new medical approaches for neuromuscular disorders.Spinal muscular atrophy (SMA) is a genetic neuromuscular disorder that is characterized as degeneration or developmental defect of spinal motor neurons. In particular, neonatal onset of SMA, called Werdnig-Hoffmann disease, is caused by homozygous mutations or deletions in the ntiation . The depMyasthenia gravis (MG) is an autoimmune disorder that disrupts transmission of nerve impulse in neuromuscular junctions (NMJs). Despite the potential applications to various neuromuscular diseases, the spinal cord organoid cannot generate skeletal muscle cells that are divergent from mesodermal lineage. Derivation of NMJ organoid was recently accomplished from neuromesodermal progenitors (NMPs) that are bipotent axial stem cells and can be derived from hPSCs with GSK3β inhibitor and FGF2 in 2D culture conditions . NMPs arMost of the brain and spinal cord organoid protocols are optimized to direct hPSCs into neuroectodermal lineage. Endothelial cells and pericytes develop from mesodermal cells and constitute cerebral vasculature network that carries oxygen and nutrients through the whole brain. Endothelial cells are essential components of the blood-brain barrier (BBB) that tightly regulates the movement of ions, molecules, and cells between blood and brain parenchyma. Microglia cells are brain-resident macrophages and act as the primary immune defense system of the brain. Since the function of BBB and microglia is tightly regulated at human brain pathogenesis, traditional brain organoids that lack these cells are still insufficient.During embryonic brain development, several cell types, such as interneurons and oligodendrocytes, appear in specific brain areas and migrated into other areas. Dynamics of cell migration is important for establishment of primary excitatory-inhibitory balance and protection of nerve system from brain injury and intercepted in autism spectrum disorder. Unlike surgically isolated brain samples and rodent models, region-specific brain organoids do not contain these migrated cell types. To overcome the issue of depauperate cell types in the organoids, scientists recently have modified the existing protocols to permit the derivation of oligodendrocyte , vasculaIn mouse prenatal brain, oligodendrocyte precursor cells (OPCs) first arise from MGE and anterior entopeduncular area of the ventral TE around embryonic day 12.5 (E12.5) and gradually spread from ventral to dorsal side . SubsequAlthough the fusion with the ventral forebrain organoid has potential to establish oligodendrocyte myelination in the cortex, some recent protocols directly induce oligodendrocyte-like cells into the dorsal cortical organoid using growth factors and small molecules governing oligodendrocyte differentiation , 42. PlaThe Pelizaeus-Merzbacher disease (PMD) is a monogenetic leukodystrophy that is mainly caused by mutations in the PLP1 X-linked gene. The oligodendrocyte-containing organoids from iPSCs of PLP1 point mutation (254T>G) PMD patients exhibit severe reduction of MYRF-positive oligodendrocyte . PLP1 isThe brain organoids can grow up to 4 mm in diameter around 2 months and be maintained around 1 year. Despite their capacity of long-term maintenance, the brain organoids cannot grow bigger than this size due to a limited exchange of oxygen, nutrient and cellular waste in the inner-most regions of the organoid. The absence of a vascular system is fatal to the organoids and leads to the induction of apoptotic cell death with long-term culture. In addition, the stimulation from vascular endothelial cells is essential for the differentiation of neuroprogenitor cells.One of the first studies to vascularize the brain organoids was to engraft human brain organoids into the mouse brain . The traFunctional vascularization of the brain organoids was also modeled in in vitro systems. Under 2D culture, derivation of endothelial cells from hPSCs is initiated by mesodermal formation with WNT activation  . Subsequ1-42 oligo disrupted the tight junctions in the vascularized brain organoids, decreasing the perfusability. Furthermore, the transplantation of the vascularized organoid into the mouse brain displayed the functional connection of the human vasculature with the host blood flow network, indicating that in vitro formation of the vascular system is essential not only to mimic physiological environment of the human brain but also for subsequent applications of brain organoids to disease modeling and drug testing.E26 transformation–specific (ETS) family proteins are involved in the transcriptional regulation of genes related to endothelial and hematopoietic differentiation. Among ETS transcription factors, ectopic induction of ETV2 alone sufficiently converts human dermal fibroblasts into vascular endothelial cells . FurtherHuman umbilical vein endothelial cells (HUVECs) harbor the property to form capillary-like structures and commonly used primary cells to study the vasculogenesis and angiogenesis. An alternative vascularized organoid can be archived by spontaneously inducing brain organoids with mixture of hESCs and HUVECs . Within Organoids cultured in vitro are more vulnerable to the cellular stress and damage than in vivo brain . MicroglNon-guided whole brain organoids are known to produce cell types of mesodermal origin that are represented by expression of myogenin and myosin genes  , 7. ReceSingle-cell transcriptional profiling (scRNA-seq) is often coupled with the organoid studies to address the molecular features and heterogeneity of individual cells , 39, 42.Current brain organoid protocols utilize different combinations of signaling inhibitors and were previously categorized by their cortical patterning level: non-directed , least dIn addition to the induction of vasculogenesis, researchers have made an effort to ameliorate interior hypoxia and nutrient starvation of the organoids by retaining scalability of in vitro system. One of the advanced approaches is slicing of the brain organoid into a disk shape that allows the exposure of the innermost regions to the external medium environment . After tAlong with the emergence and improvement of 3D brain culture systems, it has been concerned that lab-grown brain organoids become conscious like premature babies . The actIn conjunction with the brain organoids, the generation of other peripheral tissues has been archived and refined. Most of the organ functions are governed by the nervous system with inputs from the brain and spinal cord to peripheral neurons. Amyotrophic lateral sclerosis (ALS) patients suffer from the abnormality of heart electrical pacing, detoxification, and breathing by the functional defect of motor neurons in the central nervous system (CNS). Neuropathy exhibits the symptom of chronic liver disease and kidney failure with the damaged peripheral nervous system. Stomach and intestinal functions are susceptible to anxiety and stress in the brain. Unlike in vivo organs, the peripheral tissue organoids do not have neuronal cells and are not available to understand how the tissue functions are controlled by the nervous system and invaded by multiorgan diseases. The fusion culture system of brain organoid allows axonal and neuronal projection into the other types of organoids . The cocTogether, the organoid technologies have rapidly progressed and opened new avenues for biomedical applications, but are still growing. In particular, further expansion and maturation of the brain organoids to late fetal and perinatal stage brain development, at which synaptic pruning, myelination, and cortical layer formation begin, are required. The multidisciplinary approaches to build biomimetric organoid culture system are also exciting opportunities. The “vascularization,” “immunization,” and “neuronization” will improve the maturation of organoid systems and be more amenable to in vitro pathological researches and eventual regenerative medicines.


Seed metabolites play key roles as a source of nutrients for humans and livestock, or as a supply of raw materials for industries. Besides this, the seed benefits from a wide range of molecules that are timely produced and utilized to promote the transition from the quiescent state of the dry seed to active germination, and later on to facilitate post-germinative growth and seedling establishment. Such a complex developmental strategy requires sophisticated control mechanisms carried out by specific metabolites acting as signal molecules. The latter include carbohydrates  and lipids . The seed ability to withstand abiotic and biotic stresses is also related to distinctive metabolic profiles  resulting from the integration of exogenous signals with endogenous signaling pathways, finally triggering protective responses. The recent advances in this field of plant science have contributed to disclose several important features of the seed metabolome, highlighting the specific role of different classes of compounds in seed quality and health. To date, there is still a consistent gap of knowledge on these issues that will be possibly overcome through the in-depth, multilevel investigation of seed physiology and biochemistry. All the efforts devoted to elucidate the genetic and molecular bases of seed quality traits will pave the way to powerful applications in crop breeding and seed technologies. This Research Topic provides the latest new insights on the contribution of seed metabolites and related physiological and molecular processes to seed and seedling resilience.Watt et al. provides an exhaustive up-date on transcription factors that modulate genes responsible for multiple traits, with focus on abiotic stress tolerance and grain size. This is a promising research field that holds a strong potential significance for cereal crop improvement, in view of the current and future climate challenges. The review underlines critical issues, e.g., species-specific components and synteny, and the need to better understand the way microRNAs regulate transcription factors. Seed size issues can be afforded from different perspectives. The work by Curtidor et al. aims at understanding how carbon composition changes during embryo development, contributing to final seed or embryo size. The study was performed using Arabidopsis thaliana accessions with medium and large seed sizes. Based on metabolic profiles, the “torpedo-to-mature embryo” and “mature embryo-to-desiccation stage” transitions disclosed distinct carbon markers. Such changes were pronounced in the accession with the large seed size. Also the study  reports on issues of seed size, looking in this case at the molecular pathways linked to cytokinin action in the model plant A. thaliana. The subject of this investigation is the multifunctional AtENO2 gene encoding the enolase enzyme and AtMBP-1 (c-Myc binding protein 1-like). A mutation in AtENO2 results into decreased seed size and weight, starch and cytokinin levels whereas secondary metabolic pathways were significantly enriched. An interesting model that includes the factors responsible for the impact of AtENO2 on the seed size and weight is also presented.The review by An et al. reports on the current state of the art dealing with embryo-endosperm interaction, the main process controlling grain filling, crucial for yield and quality of the three most important cereals worldwide, rice, wheat, and maize. There are still several open questions regarding the cross-talk and signaling between these two tissues, the role of abscisic acid (ABA) and gibberellin (GA). The review underlines the urgent need for cooperative actions involving geneticists, breeders, physiologists, and agronomists to overcome such constraints.The review by Mahmood et al. describe the beneficial effects of He-Ne laser treatments on sunflower seeds, in terms of enhanced drought stress tolerance. Notably, the treatment promoted the accumulation of nutraceutical phenolic compounds and improved the unsaturated-to-saturated fatty acid ratio of the oil as well as carotenoids and total soluble phenolics.In line with this claim, the Research Topic also includes two original works focused on seed metabolites in the context of seed vigorization, thus linking fundamental and applied research. Forti et al. investigated the impact of seed hydropriming on the pre-germinative metabolism in the eggplant crop wild relative Solanum villosum Miller (hairy nightshade). Hydropriming followed by dry-back resulted in synchronized germination. The treatments triggered increased tocopherols levels and accumulation of the naturally occurring antioxidant phenolic compounds chlorogenic acid and iso-orientin, found in the dry seeds and ex novo accumulation of rutin. The dynamic changes of the pre-germinative metabolism induced by hydropriming are discussed in view of future applications that might boost the use of eggplant CWRs for breeding, upon upgrade mediated by seed technology.Sistani et al. using an integrative metabolomics-proteomics approach applied to Pisum sativum L. in symbiotic association with Arbuscular mycorrhizal fungi (AMF). Seeds of mycorrhizal pea plants were strongly enriched in terms of secondary metabolites with nutritional, medicinal, and pharmaceutical properties, as well as active roles in pathogen response. The reported data show that AMF exert a significant influence on the pea seed metabolome.Finally, the role of seed metabolites in pathogen resistance has been investigated by All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.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.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Background: Tentorial dural arteriovenous fistula is a rare subtype of intracranial dural arteriovenous fistula (DAVF) with a deteriorating natural course, which may be attributed to its pial angioarchitecture. TDAVF often harbors feeders arising from pial arteries (FPAs). Reports have revealed that, if these feeders are not obliterated early, the restricted venous outflow during the embolization process may cause upstream congestion in the fragile pial network, which increases the risk of hemorrhagic complications. Because most reported cases of TDAVF were embolized through feeders from non-pial arteries (FNPAs), little is known of the feasibility of direct embolization through FPAs.Methods: We present three patients with medial TDAVFs that were embolized through the dural branches of the posterior cerebral and superior cerebellar arteries. Findings from brain magnetic resonance imaging, computed tomography, angiography, and clinical outcomes are described. Furthermore, we performed a review of the literature on TDAVFs with FPAs.Results: The fistulas were completely obliterated in two patients; both recovered well with no procedure-related complications. The fistula was nearly obliterated in one patient, who developed left superior cerebellum and midbrain infarct due to the reflux of the embolizer into the left superior cerebellar artery. Including our cases, eight cases of TDAVFs with direct embolization through the FPAs have been reported, and ischemic complications occurred in three (37.5%).Conclusions: Advancing microcatheter tips as close to the fistula point as possible and remaining highly aware of potential embolizer flow back into the pial artery are key factors in achieving successful embolization. Balloon-assisted embolization may be an option for treating TDAVFs with FPAs in the future. Intracranial dural arteriovenous fistulas (DAVFs) account for 10–15% of intracranial vascular shunts . CompareIn most reports, treated TDAVFs were embolized through feeders from non-pial arteries (FNPAs). In this report, we present our experience of direct embolization through the dural branches of pial arteries in three patients with medial TDAVF and review the literature on views on and treatment of TDAVFs with FPAs.This retrospective study was conducted in accordance with the Declaration of Helsinki; it was approved by the Institutional Review Board of Chang Gung Memorial Hospital (IRB: 202100129B0). Three patients with TDAVFs underwent TAE in 2020 after providing informed consent.The procedure was conducted through bifemoral arterial access under general endotracheal anesthesia with heparinization. A 5F guide catheter  or a triaxial guide catheter system consisting of a 6F guide catheter  and 5F intermediate catheters  was positioned at the vertebral artery. An angiographic catheter was positioned at the carotid artery or the contralateral vertebral artery for a control angiogram. Under road-mapping guidance, the FPAs were super selected with a microcatheter  over a 10-inch micro guide wire  as close to the fistula point as possible. After the micro guide wire was removed, a contrast medium was injected through the microcatheter to confirm the proximity of the microcatheter tip to the fistula and that the brain parenchyma was not stained. Embolization was performed by injecting ethylene vinyl alcohol copolymer  through the microcatheter using the “plug and push” technique until the fistula was obliterated or flowback or penetration of Onyx to undesired vessels occurred, which was confirmed with regular angiography performed through the intermediate catheter, guiding catheter, or angiographic catheter.A 57-year-old man with hypertension presented with intermittent weakness of the left limbs , a left facial burning sensation, and left upper limb numbness persisting for 3 weeks. Brain magnetic resonance angiography (MRA) revealed serpentine vessels at the quadrigeminal cistern. Angiography revealed a DAVF at the edge of the tentorial incisura that was draining mainly through the vein of Galen to the straight sinus and partly draining through the cerebellar cortical vein to the transverse sinus. The feeders were the bilateral occipital arteries, right middle meningeal artery (MMA), marginal tentorial artery from the right internal carotid artery (ICA), dural branch of the bilateral posterior cerebral arteries (PCAs), and left artery of the falx cerebelli.The dural branch of the P1 segment of the right PCAs was superselected with a Marathon microcatheter to approach the fistula point at the falx cerebelli. Embolization was performed with 3-ml Onyx to complete obliteration of the fistula. Post-embolization angiography images revealed complete obliteration of the patient's fistula .The subsequent course of the patient was uneventful after the operation; the muscle strength of the left side improved to Grade 5 on the Medical Research Council scale. The patient had a modified Rankin Scale (mRS) score of 1 at 9-month follow-up.A 49-year-old woman with history of diabetes mellitus reported sudden-onset severe headache and dizziness. Brain computed tomography revealed diffuse subarachnoid hemorrhage. Angiography revealed a TDAVF with feeders from the bilateral superior cerebellar arteries (SCAs), left posterior meningeal artery, and bilateral occipital arteries. The TDAVF was draining through a varix in the draining vein at the tentorial edge of the vein of Galen.The right medial tentorial artery from the right SCA was superselected with an Apollo microcatheter close to the fistula at the varix. Embolization was performed with Onyx to obliterate most of the fistula. A Marathon microcatheter was then navigated from the left SCA to the left medial tentorial artery to perform further embolization. However, during the second embolization, a reflux of Onyx to the left SCA orifice was noted .The patient had right hemiplegia after intervention. Emergent brain MRA revealed acute infarction in the left superior cerebellum and the tectum of the midbrain. At 6-month follow-up, the patient still presented with right hemiplegia and had an mRS score of 4.A 67-year-old man with hypertension presented with sudden-onset nausea, vomiting, dizziness, dysarthria, ataxia, and general weakness. Brain computed tomography revealed intracerebral hemorrhage at the cerebellar vermis with rupture into the fourth ventricle. An external ventricular drain was placed. Angiography revealed a DAVF located at the falx cerebelli with the medial tentorial branches of the bilateral SCAs as feeders and that drained through a varix to the vein of Galen and the straight sinus. A venous stenosis was also noted at the venosinus junction. The dural branch of the left SCA was superselected with an Apollo microcatheter to approach the fistula point at the falx cerebelli; the right SCA was superselected with an Echelon-10 microcatheter. Complete obliteration of the fistula was achieved through Onyx injection .The patient presented with no new neurological deficits after TAE. The patient actively participated in a post-acute care rehabilitation program and had an mRS score of 4 at 9-month follow-up.In this report, we demonstrated that obliteration of TDAVF could be achieved with embolization through the dural branches of the pial arteries, the PCA [or the artery of Davidoff and Schechter (ADS)], and the SCA .The goal of DAVF treatment is to occlude the shunting points of the fistula while minimizing the effect on the normal vascular system. A careful examination of the angioarchitecture of the TDAVF  should be performed before endovascular treatment is initiated to tailor treatment strategies to the patient.A TDAVF may receive a blood supply from different locations: anteriorly from the tentorial artery; inferiorly from the PCA or SCA; superiorly from the falcine and MMA; or posteriorly from the occipital, posterior meningeal, or falx arteries . DAVFs wLawton and Tan  divided The PCA and SCA have rarely been selected as the primary access points for TAE of TDAVF because of the risk of causing brainstem and cerebellar infarction; the proximal SCA has perforating branches that supply blood to the rostral cerebellum and brainstem, and anastomotic channels exist between the collicular branch of the PCA and the medial terminal stem of the SCA . TherefoBhatia et al.  proposedWu et al.  suggesteOsada and Krings  categoriPrevious reports have generally selected the MMA, when available, for TAE of TDAVFs. The MMA runs a long course in the dura and can tolerate a long reflux of embolizer; however, the reflux should not approach the foramen spinosum, where damage to the trigeminal and facial nerves can occur if the petrosal branch of the MMA is embolized. The occipital artery is occasionally used for TAE. However, this artery is surrounded by loose connective tissue and may run a tortuous course, especially when accommodating a high flow, which renders distal catheterization difficult . The marTransvenous embolization may be considered if the microcatheter can be navigated to the fistula point. However, we did not attempt transvenous embolization because the presence of stricture or a tortuous course hampered easy access to the fistula points. Moreover, partial occlusion in the draining vein may precipitate venous hypertension and create a risk of hemorrhage in the upstream pial network. Therefore, transvenous embolization may serve as an alternative treatment or a combined treatment with TAE for TDAVFs , 16.Tentorial dural arteriovenous fistulas are deeply located DAVFs with a deteriorating natural course; they are frequently supplied blood by branches of the PCA or SCA, which can be directly catheterized for TAE to achieve complete obliteration of fistulas. Advancing microcatheter tips as close to the fistula point as possible and remaining highly aware of potential embolizer reflux are key factors in achieving successful embolization. Furthermore, balloon-assisted embolization through FPAs may be an option for treating TDAVFs in the future.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by Institutional Review Board at Chang Gung Memorial Hospital. Written informed consent for participation was not required for this study in accordance with the national legislation and the institutional requirements. Written informed consent was obtained from the individual(s) for the publication of any potentially identifiable images or data included in this article.C-LC and Y-CC: conceptualization, investigation, writing—original draft, review, and editing. C-TL, S-CC, C-HY, and Y-MW: writing—review and editing. T-HL: writing—review and editing and supervision. H-FW: conceptualization, resources, writing—review and editing, and supervision.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.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.