Hematene in the pristine form emerges out to be a magnetic semiconductor with a bandgap of 1.0/2.0 eV for the majority/minority spin channel. The dependence of magnetic anisotropy energy (MAE), TC, and the bandgap on compressive and tensile strains has been scanned exclusively. It is examined that TC depends firmly on the compressive strain and increases up to 21.1% at a compressive strain of 6% whereas it decreases significantly for tensile strain. The MAE is negatively correlated with the tensile and compressive strain. The value of MAE for all compressive strain cases is more than that of the pristine hematene. These results summarize that the studied 2D hematene has broad application prospects in spintronics, memory-based devices, and valleytronics.As a newly exfoliated magnetic 2D material from hematite, hematene is the most far-reaching ultrathin magnetic indirect bandgap semiconductor. We have carried out a detailed structural analysis of hematene  TC.Hematene: 3 atoms thick 2D material with high  Its unique superlative physical and chemical properties make it a promising material for various electronic and photonic applications.3–5 Since 2004, many experimental and theoretical studies have been performed over graphene-based materials and they have been proved masters in almost every field especially in catalysis, electronics, photovoltaics, electrochemistry, and optoelectronics.6,7 As a result of these tremendous applications, material scientists have started to explore the worth of 2D materials for magnetism as well. According to the Mermin–Wagner theorem,8 long-range ferromagnetic order present in a bulk material can easily be destroyed by the thermal fluctuations for dimensions, d ≤ 2. The animation of an aligned arrangement of magnetic moments over macroscopic length scales is the trademark of magnetism with an intuitive disturbing of time-reversal uniformity. This is frequently determined by the interplay of the adjacent spins (exchange interactions) that leads to accommodate only some specific correlative orientations among all. At zero temperature, this local order can extend over macroscopic length scales. With a rise in temperature, thermal fluctuations tend to obstruct the alignment of magnetic moments in the neighboring regions. In this way, long-range order is destroyed above the Curie temperature (TC). This is the main parameter that decides whether the magnet can be used in numerous theoretical, experimental, and technological advances such as the study of topology, the fluctuation driven in new phases, electronic manipulation, and detection of spin.9–12 Therefore, TC must be at least of the order of room temperature to serve as a new platform for various technological applications. These facts disheartened material scientists; however, they have not completely given up over this area.Research interests in two-dimensional (2D) materials have rapidly been increasing over the past two decades.3 and CrGeT3 which have also been fabricated successfully.13,14 After that, researchers regained their passion for 2D magnetism and hematene is the latest member in this category which is 2D analog of hematite ore. Hematene has several properties in common with graphene. As graphene is a thin sheet of carbon atoms, hematene is also a thin sheet of iron and oxygen atoms. Researchers at Rice University synthesized 2D hematene with the liquid-phase exfoliation technique.15 Balan et al.15 confirmed the 2D morphology of hematene with the help of transmission electron microscopy. They reported the existence of long-range ferromagnetic order in hematene while its parent hematite is antiferromagnetic in nature. They also showed that hematene on loading with arrays of titanium nanotubes may serve as an enhanced excellent photocatalyst. The atoms in hematene are found to be held together by strong chemical bonds rather than weak van der Waals interactions. Therefore, hematene has the potential to serve as a replacement for well-known 2D materials such as graphene, black phosphorous, and MoS2 in various applications where the structures of these eminent materials can be easily disrupted. This fact revolutionized the scenario of emerging 2D magnetic materials.The other members of the family of next-generation 2D materials belong to the monolayers of CrIet al.16 investigated the nature of magnetic ordering in this 2D metal oxide hematene. They explained that it has a stripped ferrimagnetic ground state with a feeble overall magnetic moment. They also attempted to modulate its magnetism with alloying and by substituting chromene (Cr2O3) instead of Fe2O3. Gonzalez et al.17 studied the structural, optical and electronic properties of hematene. Further, they also explored the functionalization of hematene with Au (111) and stanene . The interaction with the former is weak, involving a long bonding distance, just like a typical van der Waals system whereas the interaction with the latter leads to the hybridization of the p orbital of stanene with the unoccupied dz2 orbitals of hematene, turning the resultant heterostructure into a ferrimagnet. Chen et al.18 discussed tuning the magnetism of two-dimensional hematene by ferroelectric polarization and found that the control of magnetism depends on the interface terminations. This occurs mainly due to change in the Fe–O bond length, which is determined by relative displacement of Fe and O atoms and electron transfer between five nonequivalent 3d orbitals.Bandyopadhyay TC in 2D materials presents a major challenge. In these critical situations, hematene may be the only 2D material having a TC of the order of room temperature with a remarkable breakthrough for advanced magnetic applications. In the present study, we focus on the novelty of predicting the TC of pristine hematene first by ab initio simulations. Further, the 2D materials crystals can sustain large strains compared to their bulk counterparts. For example, single-layer FeSe can sustain strains as large as 6%, whereas single-layer MoS2 can be strained as high as 11%.19–21 Therefore, strain engineering can also be a practical approach to tune the properties of 2D materials. The second aim of our study is to check the effect of compressive and tensile strains up to 6% on the magnetic response and TC. To our firm belief, this is the first report on the prediction of TC of hematene and its strain-induced magnetism.The search for high 2ab initio calculations were carried out by means of the projector augmented wave (PAW) method based on density functional theory (DFT) as implemented in the Vienna ab initio Simulation Package (VASP).22,23 The coulomb corrected generalized gradient approximation (GGA + U) within Perdew–Burke–Ernzerhof (PBE) parameterization was employed in order to take into account exchange-correlation potentials.24 The Hubbard parameter, U = 4 eV was used for correcting onsite Fe-d electron correlation in hematene.25 Further, the value of the U-parameter was also optimized and U = 4 eV was selected because it reproduces the experimental bandgap of pristine hematene quite well  was calculated by using Blöchl correction in the tetrahedron method with the same smearing width. The thermal stability of hematene at a temperature of 300 K was checked in VASP by employing the NVT ensemble.26 The phonon spectrum was obtained by utilizing the Density Functional Perturbation Theory (DFPT) approach and the post data were processed with the phonopy code.27 Bader charge analysis was executed to study the charge transfer and charge redistribution.28 The value of TC was estimated within mean-field theory by computing various exchange constants that are related to the magnetic interactions between the Fe atoms of the same and different sublattices with the help of Heisenberg Hamiltonian. In order to predict MAE, non-collinear calculations were carried out with the inclusion of spin–orbit coupling (SOC) non-self consistently by changing the direction of the quantization axis.The 33.1I) is enclosed in an octahedron of six O-atoms whereas the other one (FeII) is surrounded by three O-atoms as depicted in 15 however, hematene (001) possesses exciting lattice symmetries and is found to be more stable. Therefore, we considered mainly this phase. It has a triangular lattice with a lattice constant, a = 5.09 Å.17The novel 2D material hematene is not a single atom thick sheet like monolayer graphene; however, it has a thickness of three atoms only . But, this can still be categorized as a monolayer. The first Fe atom  and thus, to consider ferromagnetic and antiferromagnetic states, five configurations A1, A2, A3, A4 and A5 as depicted in Efor) were evaluated by using the following expression:EHema, EFe and EO are the ground state energies of hematene in a particular magnetic configuration, Fe and O atoms, respectively. The negative value of formation energies for all cases, as listed in 5  is found to be most stable. To ensure the dynamical stability of this configuration, phonon dispersion spectrum was analyzed  with a time step of 1 fs by using ab initio Molecular Dynamics (AIMD) simulations.26 After running 5000 steps, the movements of Fe and O atoms were found to be just minor with negligible changes in the bond length. Also, there was no trace of breaking of bonds, and the variation in total energy is also very small  of FeI as 3.896 μB and FeII as 4.156 μB, aligning in antiparallel arrangement with the total spin magnetic moment (μstot) as 0.52 μB. This confirms its ferrimagnetic nature which is in accordance with the previous results by Balan et al.,15 Bandyopadhyay et al.16 and Gonzalez et al.17 The magnetic interaction mechanism is quite compatible with the kinetic exchange system in this case. Although the FeII–FeII distance is large, yet Coulomb repulsions decrease rapidly like hoping strength. Also, FeI–FeI and FeII–FeII interactions are intervened by the O-atom leading to magnetic superexchange as the bond angle of FeI–O–FeI is ∼90°. But the O-atom here is threefold coordinated and forms sp2 bonding, halting its strong FM nature. This result is in accordance with the experimental hysteresis data17 which shows that when the external field is removed, hematene has a total spin moment of 4 μB which is about 1/4 of that of A1 state. So, this experimental remanence and feeble net magnetic moment in A5 are due to the difference in the charge distribution of Fe atoms from both sublattices which is explained later on in electronic properties. On the contrary, bulk hematite is antiferromagnetic in nature and its Morin transition occurs at 260 K with a Neel temperature of 955 K. Being the most stable configuration, we have considered A5 configuration only for detailed analysis of electronic properties and the magnetic response.The magnetic response of hematene as governed by DFT simulations under GGA + U indicates the spin magnetic moment  and bandstructure of pristine hematene  indicateEF). The main contribution in the total DOS comes only from Fe-d and O-p states , and magnetocrystalline anisotropy (MCA). As hematene has inversion symmetry, so, no DMI is expected in its pristine form. The magnetism in 2D materials has been examined extensively for decades and serves as a testbed for their study in different aspects such as critical behavior and dimensional crossover of magnetic ordering. The interplay of MCA and dimensionality is considered to be a promising route for the development of magnetism in 2D materials as it is only MCA that can stop thermal fluctuations to destroy long-range magnetic order. In this regard, first principles DFT calculations that entail SOC authorize a dominant theoretical tool to compute MCA. Howbeit, the tiny magnitude of the associated MAE enforces stringent convergence to the calculations at the rate of high computational cost. This purpose was realized by first characterizing the Hamiltonian of the system including the scalar relativistic term self-consistently and then including the SOC non-self-consistently within the force theorem3.2i.e. compressive and tensile strain (in the range: −6% to +6%) were considered. The relaxations of strain-induced structures do not induce any distortion in the shape of hematene. The stability of these strained structures was confirmed with the help of formation energy and the phonon spectrum. The negative values of formation energies and real phonon frequencies for all cases illustrate their stability  is given by:m runs over each Fe atom, θm is the angle between the mth Fe atom and normal to the symmetry plane. λ is the quadratic contribution to the energy. We have also examined in which manner, MAE changes with the biaxial strain  that are related to the magnetic interactions between Fe atoms of the same and different sublattices are computed using Heisenberg Hamiltonian within the mean field theory33 as follows:The various exchange constants . This opens up new avenues for hematene to serve in spintronic and memory-based magnetic devices.The calculated e strain . The ult4TC which forbids 2D materials from showing their potential towards magnetism related applications has been overcome in hematene because for its pristine form and all strain-induced structures, high TC is observed. On applying compressive and tensile strains, the structural and electronic properties do not change much but the magnitude of MAE and μstot increase for all compressive strain cases as compared to the pristine case. We hope that our results will further stimulate experimental studies on hematene and pave the way for its application in magnetic tunneling junctions and magnetic random access memory devices.In summary, we have examined 2D-hematene by strain engineering with an aim to upgrade the magnetic response using first-principles calculations based on DFT. Pristine and all strain-induced hematene structures in the ground state are found to be a magnetic and indirect semiconductor with different bandgaps for the majority and minority spin channels. The main drawback of low There are no conflicts to declare.NA-002-D0NA00556H-s001


The specimen, biological structure, and spatial data that define the HRA are disparate in nature and benefit from a visually explicit method of data integration. Virtual reality (VR) offers unique means to enable users to explore complex data structures in a three-dimensional (3D) immersive environment. On a 2D desktop application, the 3D spatiality and real-world size of the 3D reference organs of the atlas is hard to understand. If viewed in VR, the spatiality of the organs and tissue blocks mapped to the HRA can be explored in their true size and in a way that goes beyond traditional 2D user interfaces. Added 2D and 3D visualizations can then provide data-rich context. In this paper, we present the HRA Organ Gallery, a VR application to explore the atlas in an integrated VR environment. Presently, the HRA Organ Gallery features 55 3D reference organs,1,203 mapped tissue blocks from 292 demographically diverse donors and 15 providers that link to 5,000+ datasets; it also features prototype visualizations of cell type distributions and 3D protein structures. We outline our plans to support two biological use cases: on-ramping novice and expert users to HuBMAP data available via the Data Portal , and quality assurance/quality control (QA/QC) for HRA data providers. Code and onboarding materials are available at https://github.com/cns-iu/ccf-organ-vr-gallery#readme.The Human Reference Atlas (HRA,  We present the HRA Organ Gallery, a VR application implemented to explore the 3D organ models of the HRA in their true scale, location, and spatial relation to each other. We focus on two use cases: on-ramping novice and expert users to the HuBMAP data available via the HuBMAP Data Portal and quality assurance/quality control (QA/QC) for HRA data providers. The HRA effort  led by HThe disparate data defining the HRA benefits from a visually explicit method of data integration. VR offers unique means to explore spatial and abstract data in a unified, immersive, and presence-enhancing environment beyond traditional WIMP interfaces, i.e., windows, icons, menu, pointer . While eRecent advancements in VR development have led to an interest in applying VR to biomedical research and practice. VR allows dynamic exploration and enables viewers to enter visualizations at various viewpoints , and to In recent years, researchers have also explored VR as a method for users to engage with virtual models of human anatomy, and thus a tool for medical education . VR has Not only does VR present advantages for 3D modeling, it also offers novel means of exploring and presenting data. For instance, information-rich virtual environments (IRVEs) are effeVR offers a unique method of visualizing and interacting with the diverse data types that constitute the HRA in a unified 3D environment. The design of the HRA Organ Gallery uniquely focuses on enriching the analysis experience with presence and immersion while simultaneously preserving the spatiality of biological data. Not only is the HRA Organ Gallery the first open-source immersive application to allow users to view, explore, and analyze 55 3D reference organs in an integrated VR environment, it is the first such application to also integrate varying types of data and data visualizations while affording users with a broad range of actions .HRA Organ Gallery research and development are driven by two concrete bioinformatic use cases:On-ramping of novice and expert users: The HuBMAP Data Portal  quickly identify what spatial metadata is missing for how many organ-specific tissue blocks and (b) which tissue blocks are registered incorrectly, e.g., the size, location, or rotation of a tissue block is different from that a surgeon remembers. The HRA Organ Gallery makes it possible to quickly identify and correct wrongly registered tissue blocks.https://unity.com) for the open-source OpenXR platform (https://www.khronos.org/openxr). To facilitate interactions with the tissue blocks and organs, we use the XR Interaction Toolkit 2.0.4 . We targeted and optimized for the Meta Quest 2 with Oculus Touch controllers (https://www.meta.com/quest/products/quest-2). The majority of the development was performed on a Dell Precision 7560 running Windows 11 Pro, with 64 GB RAM, an 11th Gen Intel i9, 2.60 GHz processor, and a Nvidia RTX A2000 GPU.The HRA Organ Gallery v0.6 was developed using Unity 2021.3.11 (https://ccf-api.hubmapconsortium.org) to access the biological structure, spatial, and specimen data needed to construct the 3D scene from the CCF.OWL 2.1.0 , they can use teleportation using a red ray interactor. To minimize the time needed to arrive at the desired organ, white teleportation anchors on the virtual floor transport the user ~50 cm in front of the organ and adjust their orientation towards the organ when clicked.Showing all 55 organs at the same time creates performance issues for the targeted VR hardware. The default view in v0.6 thus only shows eight organs . However, a filter menu on the left lets the user turn organs on and off through checkboxes.https://ccf-api.hubmapconsortium.org/#/operations/scene). The JSON response is then deserialized into a custom C# class to capture relevant data fields, such as the tooltip name for organs and the list of anatomical structures with which tissue blocks collide. While 3D organ models are stored in local memory once the application has been run for the first time, it requires an internet connection to retrieve the most recent set of tissue blocks.When loading, the app performs a call to the CCF API . The SceneBuilder runs a suite of methods before or during the first frame or asynchronously: Methods in orange and dark green load and place organs and tissue blocks. Methods in light green add an OrganData component to each organ, which lets components across the app access its metadata. Methods in pink do the same for tissue blocks. Once completed, the user can start interacting with the organs. When running on the Meta Quest 2, it takes about 20 seconds for the entire scene to load.lder see , the datlder see  defines The goal of the UIs of the HRA is (1) to enable tissue providers to generate, verify, and (if needed) correct existing tissue block registrations, and (2) to allow HuBMAP Data Portal users to semantically and spatially explore datasets curated at high quality through unified pipelines. The HRA Organ Gallery aims to support this essential on-ramping use case with a series of VR superpowers.As a <computational biologist or clinical researcher>, I want to <be able to pick tissue blocks with my hands in VR> so I can <examine the many anatomical structures that comprise them, explore what tissue blocks were registered where, and save relevant organs/blocks out for later examination>.We are prototyping a wrist pocket see , where tAs a <computational biologist or clinical researcher>, I want to <be able to examine multiple overlapping tissue blocks> so I can <understand the spatial distribution of tissue blocks in tight anatomical structures better>.We developed a prototype of a probing sphere see , tied toAs a <computational biologist or clinical researcher>, I want to <move, rotate, and scale organs and the tissue blocks inside them> so I can <assess whether the reference organ fits the requirements for registering my tissue blocks>.We are planning an organ pullout feature, where the user can grab, move, rotate, and rescale all 55 3D reference organs using the most natural input device: their hands (via VR controllers). This enables the user to switch between true, increased, and decreased scaling, default and custom rotation, and any position for the organ the user can reach with their hands. This enables the user to check if the reference organs will be a good fit for registering their tissue blocks. Additionally, it allows the user to inspect the organ and its tissue blocks from any angle–a non-trivial feature on the 2D interfaces of the EUI, where restrictions on camera movement prevent the user from overly experimental 3D camera movement. Note that all three functionalities described so far are neither available nor feasible in the EUI.As a <computational biologist or clinical researcher>, I want to <see cell type populations for a selected tissue block> so I can <decide if a tissue block is relevant for my work>.We are prototyping a cell type population explorer see  that useAs a <computational biologist or clinical researcher>, I want to <view 3D models of proteins characterizing the cells of a selected tissue block> so I can <explore HRA data across scales>.The planned 3D protein viewer will allow experts to view selected proteins in VR see . In the As a <newcomer to a HuBMAP team or a data provider>, I want to <be able to walk through the HRA in a co-op mode with another person> so I can <learn from a more experienced individual>.The ability to collaborate in a co-op mode see  will allAs of February 9, 2023, 19 users from 15 data providers have registered 1,203 tissue blocks from 292 donors using MC-IU’s forementioned RUI; out of these, 407 tissue blocks from 153 donors with 508 tissue sections and 1,421 datasets by 10 data providers have been published; all others are somewhere in the QA/QC process. While users register the spatial location of tissue blocks with great care, mistakes happen. Resulting errors in spatial or specimen data are difficult to identify when reviewing the data in 2D. However, they become immediately apparent in VR, where 3D organs and 3D tissue blocks can be examined in 3D up close from all angles and at different scales of magnification. Thus, the HRA Organ Gallery makes it possible for different end users  to perform QA/QC using an intuitive and efficient VR user interface.As a <data provider>, I want to <see cell type populations for a selected tissue block> so I can <check if the tissue block was registered correctly>.The 3D display of reference organs in VR can be enhanced by adding auxiliary 2D data visualizations of biological structure data (such as the number of cells per cell type) or specimen data  for a tissue block of interest see , based oAs a <data provider>, I want to <correct a wrongly registered tissue block> so I can <save time as opposed to correcting the registration in a separate 2D application>.We are envisioning a fix-on-the-spot feature see , where tWe will expand on the training and onboarding materials already created by producing a series of video tutorials and other documentation (written and in-app). These materials will guide and orient users, especially those unfamiliar with VR headsets, by explaining the controls and features in the application. They will also document how the HRA Organ Gallery ties into the evolving HRA and the data used to build it. We continue disseminating these training materials via YouTube and other social media platforms and will ship them as part of every release. We will explore deploying the HRA Organ Gallery to classrooms for anatomy and science students and the general public. This way, the HRA Organ Gallery becomes a valuable tool for HuBMAP outreach but also for ‘in-reach’ to train existing HuBMAP and other researchers working on the HRA. For example, we demonstrated the app at a Common Fund Data Ecosystem (CFDE) kick-off meeting in the Washington, DC area in November 2022, where we were able to show how tissue blocks from four different funded efforts  can be harmonized, presented, explored, and queried within one coherent, immersive 3D space.We will solicit further feedback from subject matter domain experts at the NIH, and its funded efforts. This will allow us to introduce and involve a wide variety of experts in the HRA effort, including experts in bioinformatics, 3D modeling, medical illustration, anatomy, data curation, and biology. We also plan a user study to gather quantitative data about performance for different QA/QC error detection methods and completion time and to test the usability, engagement, and presence afforded by the app.https://www.oculus.com/experiences/quest). We will develop extensive training materials  to enable a diverse array of users to utilize the HRA Organ Gallery for their own research and data curation.The HRA Organ Gallery provides an immersive view of HRA construction and usage, and with the organs and tissue blocks in their true size, enabling effective QA/QC using modern VR headsets and hand-held extended reality devices that are well documented and affordable (~$600 per set). It will soon be made available for free to more than 15 million VR devices across th


The rest of genes (107) are potential candidates for BAT, being also closer to known BAT genes and more expressed in BAT biopsies than expected by chance . The resulting new list of predicted human BAT genes should be useful for the discovery of novel BAT genes and metabolic pathways.Brown adipose tissue (BAT) is a promising therapeutic target against obesity. Therefore, research on the genetic architecture of BAT could be key for the development of successful therapies against this complex phenotype. Hypothesis-driven candidate gene association studies are useful for studying genetic determinants of complex traits, but they are dependent upon the previous knowledge to select candidate genes. Here, we predicted 107 novel-BAT candidate genes in silico using the uncoupling protein one (UCP1) as the hallmark of BAT activity. We first identified the top 1% of human genes predicted by the human gene connectome to be biologically closest to the UCP1, estimating 167 additional pathway genes (BAT connectome). We validated this prediction by showing that 60 genes already associated with BAT were included in the connectome and they were biologically closer to each other than expected by chance (p < 2.2 × 10 Therefore, research on the genetic architecture of BAT could be key for the development of successful therapies against obesity. Hypothesis-driven candidate gene association studies are a useful approach to study the genetic determinants of complex traits4. However, this approach depends on the previous knowledge to formulate and test association hypothesis. Here, we used the human gene connectome (HGC) to predict novel-candidate genes for BAT. The HGC is a set of biological distances and routes of interaction predicted in silico by a shortest distance algorithm applied to the full human genome network, which is conceptually similar to GPS navigation5. Therefore, these biological distances are based on information about protein interactions. In particular, the HGC uses protein–protein interaction data from the String database (https://string-db.org/), considering only binding interactions that have been previously described. It has been shown in previous studies that the HGC is an effective tool for predicting novel disease-causing genes, according to their biological distance to known disease-causing genes9. Here we extended the HGC approach to increase the list of known BAT pathway genes into predicted BAT genes.Brown adipose tissue (BAT) is a possible therapeutic target against obesity and related disorders due to its ability to oxidise glucose and lipids and to dissipate energy as heat10 could be used to detect new BAT candidate genes, by ranking all human genes on the basis of their biological proximity to already known BAT genes , assuming the most highly ranked genes to be the most likely related to BAT. A list of novel potential BAT candidate genes, rigorously identified on the basis of biological relevance to BAT phenotype, would be useful and easy for most BAT investigators to use.The HGC and its associated server1. Therefore, its relevance and exclusivity on brown adipocytes is beyond doubt. Genes biologically close to UCP1 should therefore be strong candidates for being BAT-associated. Consequently, we used the HGC to extract the top 1% of human genes biologically closest to UCP1. We found that 60 genes already associated with BAT were included in that list, being the rest of them (107) the set of novel candidates.We generated a list of in silico-predicted novel human BAT candidate genes. We considered uncoupling protein 1 (UCP1) gene as the unique core gene. UCP1 is considered a hallmark of brown adipocyte, and is involved in the uncoupling of the electron-transport chain in the mitochondria inner membrane5. From this connectome, we extracted only the genes in the top 1% of all human genes by p-value and obtained a list of 168 genes (167 excluding UCP1 itself). We considered these genes as the connectome of BAT .We used, as an input, the connectome of UCP1, being the set of all human genes ranked according to their biological proximity to UCP1. These biological distances are predicted using protein–protein interaction patternsWe searched in PubMed and Web of Science for studies analysing the association of any of the 167 identified genes and BAT (known BAT genes hereafter). We considered as novel candidates those genes for which we found no evidence of relationship with BAT (candidate BAT genes hereafter). We are aware that some genes with a relationship described with BAT could not have been included in our review of BAT genes. However, an increase in the number of known BAT genes into the connectome would further support its predictive power and the relevance of the remaining candidates , but their aggrupation based on the functional biological distance between them.The biological-interrelatedness between known and candidate BAT genes was estimated with the functional genomic alignment phylogenyWe assessed the predictive power of BAT connectome by testing whether the known BAT genes included in the connectome were significantly closer to each other than to other genes. First, we calculated the median distance between known BAT genes (3540 distances considered). Then, we sampled 1 million random sets of 3540 biological distances between known BAT genes and other genes. A p-value was estimated by determining the proportion of simulated random gene sets with a median distance to the known BAT genes equal or smaller to the distance between known BAT genes.12.We further evaluated the predictive power of the connectome by testing whether known BAT genes were biologically closer to BAT candidates compared to other human genes. The median distance between candidate and known BAT genes inside the connectome (based on 6420 distances) was compared with the median distance between known BAT and random genes (1 million sets of 6420 random distances). Then, we estimated a p-value by determining the proportion of random sets with a median biological distance equal or smaller to the distance between candidate and known BAT genes. These and previous analyses were performed with R13 through the “ArrayExpress” R package14 and included raw data in order to be processed (see below). Two of these studies analyzed biopsies from human brown adipose tissue in the deep neck (around the carotid sheath) and the thyroid gland (close to the isthmus region), respectively16. The other two studies generated brown adipocytes from human mesenchymal stem cells, induced pluripotent stem cells or fibroblasts18. In all studies, we considered only those groups of arrays  that showed BAT features like the expression of BAT markers ), an increase of mitochondrial content, etc18. We only considered studies for which raw microarray data was available in order to perform the same processing procedure for all datasets. In particular, we applied the Robust Multichip Average processing methodology as implemented by the “oligo” R package19. After the processing, we analyzed the quality of the arrays using the “arrayQualityMetrics” R package20. We removed those samples identified as outliers and/or close to controls, i.e., non-BAT samples, in order to increase the probability of retaining only BAT-like samples. In any case, we compared the results with and without these samples and we found no qualitative differences (data not shown). Then, we matched expression data from the microarrays probes to specific genes using the “AnnotationDbi” R package21. We removed any probe associated with several genes, as it could not be ambiguously annotated. For the remaining probes, we calculated the median expression of all probes associated with each gene in order to get an average value of expression per gene. In this way, we obtained a value of expression for around 20,000 genes in each study. We ranked genes based on their expression in each study and selected those with an expression higher than the 95th percentile. Finally, we count the number of BAT connectome genes that were in the top of expression in any of the studies considered. We repeated this process with 1 million random sets of genes in order to test the significance of the results. A p-value was estimated by determining the proportion of random sets having a number of genes in the top of expression equal or greater than the BAT connectome.We further validated our results using previously published microarray studies (gene expression related to BAT). We considered 4 microarray datasets that we were able to retrieve from ArrayExpress11. This analysis showed that the BAT candidate genes identified in this study were evenly distributed over the whole range of known BAT genes . Known BAT genes were also biologically closer to BAT candidates compared to random set of genes that were outside of the connectome . These results are consistent with the hypothesis of tight functional interrelatedness between BAT genes. It was expected that genes belonging to the same pathway would display small biological distances between each other. Confirming this hypothesis makes it possible to infer novel genes related to BAT based on HGC-predicted biological distances to known BAT genes.We found that the median biological distance between known BAT genes inside the connectome was 4.72. None of the simulated sets of random genes had a median distance to known BAT genes equal or smaller than 4.72 , suggesting that the genes included in the BAT connectome tend to be more expressed in BAT than expected under the null expectation. When focusing on BAT-candidates, we found that 18 candidates were in the top of expression of the studies considered, being this number only marginally significant . This lack of significance is possibly caused by a lack of power. This is supported by the fact that only 19 known-BAT genes were present in the top of expression . In other words, there were still 42 known-BAT genes not included in the BAT top of expression. Note that all these genes are known to be implicated in BAT functioning. Therefore, although these analyses detected a signal of association with BAT, they did not have enough power to detect the full connection between these genes and BAT expression, thus being conservative. We hypothesized that the experimental procedures used to differentiate cells to brown adipocytes could introduce noise, so we repeated the analyses but considering only the two studies analyzing biopsies of brown adipose tissue. Considering these studies, we found even stronger support for the enrichment of BAT-expressed genes within the BAT connectome . This is congruent with an increase of power after focusing specifically on actual BAT biopsies. Interestingly, the signal also becomes stronger in the subset of BAT candidates, existing a significant enrichment of genes expressed in BAT among these candidates . In other words, the BAT candidates also tend to be more expressed in BAT than expected by chance when focusing on the gene expression of BAT biopsies  of any of the 4 studies considered. This number was higher than expected by chance ). Interestingly, Ucp1 was not more expressed in the inguinal white adipose tissue (WAT) from Gab2-KO mice, suggesting that Gab2 is BAT-specific. They also showed that Gab2 has an impact in brown-adipocyte differentiation. The deletion of Gab2 increased the transcription of Ucp1 in brown preadipocytes, leading to a higher Ucp1 content at the end of differentiation along with higher expression of other thermogenic genes. Therefore, recent evidence suggests a role of GAB2 in the development and functioning of BAT, as predicted by the BAT connectome. The histamine H4 receptor (HRH4), one of the proteins that mediates the effect of histamine, is another BAT candidate. The HGC also predicted this protein to be biologically close to UCP1, but again, no evidence supported a direct connection until recently. Zhao et al.23 has reported an increase of HrH4 expression in the subcutaneous WAT of mice exposed to cold conditions. They also reported an impairment of cold-induced browning in WAT of HrH4-KO mice. KO mice showed an impaired up-regulation of thermogenic genes, including Ucp1 and Pgc-1α, along with a worse regulation of temperature and lower heat production in response to cold. Interestingly, the treatment with 4-methylhistamine, an specific agonist of HrH4, increased the browning of subcutaneous WAT in mice. This suggests a role of HRH4 as a novel regulator of browning and cold-induced thermogenesis in white adipose tissue. Therefore, this protein might also play a role in UCP1-related thermogenesis in BAT, as predicted by the HGC. In summary, recent evidence is starting to support the predictions of the BAT connectome.Evidence about their implication in adipose tissue thermogenesis has recently emerged for some of these BAT candidate genes. This is the case of the Grb2-associated binding protein 2 (GAB2), a protein implicated in amplification and integration of signalling pathways in the cytoplasm. The HGC predicted that GAB2 is biologically close to UCP1, but no evidence supported a direct link between GAB2 and UCP1 or BAT at the moment our analyses were done. Wang et al.IGFBP7) would be an example of this. According to the HGC, IGFBP7 interacts with C-Jun (JUN) that in turn interacts with UCP1, thus being IGFBP7 biologically close to UCP1. However, there is currently no direct link between IGFBP7 and UCP1. In this regard, Hu et al.24 analyzed the impact of IGFBP7 on cattle subcutaneous preadipocytes. They found that knockout preadipocytes for IGFBP7 had lower lipid accumulation and triglycerides formation during maturation compared to controls. In addition, these knockout adipocytes showed lower expression of adipogenic-related genes. Conversely, cattle preadipocytes overexpressing IGFBP7 showed the opposite pattern, i.e., higher lipid accumulation, triglyceride formation and expression of adipogenic-related genes. One of these adipogenic genes was the Peroxisome proliferator-activated receptor gamma gene (PPARG), which is also included in the BAT connectome as a known-BAT gene. Therefore, IGFBP7 may be also implicated in the differentiation of brown adipocytes. The integrin alpha-V gene (ITGAV) would be another example of a potential BAT gene that has not been directly associated with BAT yet. ITGAV interacts with the Insulin receptor substrate 1 (IRS1) according to the HGC, which in turn interacts with UCP1, but no direct link to brown adipose tissue has been reported. Morandi et al.25 studied the role of ITGAV in the differentiation of adipose derived stem cells obtained from human subcutaneous adipose tissue. They found that the silencing of ITGAV enhanced adipogenic differentiation. In contrast, ITGAV overexpression decreased the mRNA levels of adipocyte marker genes, namely Adiponectin (ADIPOQ) and PPARG. They also tested the impact of an ITGAV inhibitor (Cilengitide). As expected, it influenced adipocyte differentiation in a similar way than the silencing of ITGAV, i.e., enhancement of adipogenesis and strongly induction of PPARG, which is a known-BAT gene in the BAT connectome. Therefore, ITGAV seems to be implicated in white adipocyte differentiation and may be also relevant in the differentiation of brown adipocytes. These results, along with the fact that both IGFBP7 and ITGAV are above the 95th percentile of expression in human BAT biopsies, support the relevance of these genes as strong candidates for BAT functioning and development.There are BAT candidates with no direct link to BAT but for which current evidence suggests that the association is plausible. Insulin-like growth factor-binding protein 7 gene  were minimized—an aim that is expected to be achieved by selecting only one core gene strongly implicated with the functioning of BAT, i.e., UCP1. This approach has as counterpart a higher probability of false negatives , but we preferred to prioritise the exclusion of false positives given the type of study for which this connectome is mainly aimed: candidate gene association studies. These type of studies use a limited list of genes whose relationship with interest phenotype has to be suggested by previous knowledgeSupplementary Information.


Stroke has the highest disability-adjusted life-years lost in any disease, and approximately one-third of the patients get aphasia. Computers and tablets are innovative and aid in intensive treatments in speech rehabilitation for patients with aphasia. However, mechanical training limits the help to patients.This study aims to provide a framework for an integrated virtual reality (VR) app to provide speech rehabilitation for patients with aphasia.The content was generated through an in-depth literature review and discussion with experienced rehabilitation physicians and occupational therapists. We then conducted a 2-round Delphi study with 15 experts from hospitals and universities to rate the content using a 5-point Likert scale. The app was developed by an interdisciplinary team involving VR, medical science of rehabilitation, and therapeutic rehabilitation. Pilot usability testing of this novel app was conducted among 5 patients with aphasia, 5 healthy volunteers, 5 medical staff, and 2 VR experts.We designed 4 modules of speech rehabilitation: oral expression, auditory comprehension, cognition, and comprehensive application. Our VR-based interactive and intelligent app was developed to provide an alternative option for patients with aphasia. Pilot usability testing revealed user satisfaction with the app.This study designed and tested a novel VR-based app for speech rehabilitation specifically adapted to patients with aphasia. This will guide other studies to develop a similar program or intelligent system in a clinical setting. Aphasia is an acquired language impairment following acquired brain injury (ABI) that affects some or all language modalities, including the expression and understanding of speech, reading, writing, and gestures . ABI is Approximately one-third of stroke patients experience aphasia . PatientMechanical training limits the help to patients. Communication activity in real-world settings is more effective for patients in improving their communicative ability . NeitherThis study aims to provide a framework for an integrated VR app and perform a preliminary test of its usability and safety. Few apps are explicitly designed to meet the requirements of engagement, functionality, aesthetics, and information quality . TherefoFor the development of speech rehabilitation content based on VR for patients with aphasia after stroke, a 2-round Delphi study was conducted . The DelThe Ethics Review Committee of Nursing and Behavioral Medicine Research, School of Nursing, Central South University, approved the study .Before round 1 of the Delphi study, we conducted a literature review using the search terms “aphasia,” “speech,” “speech treatment,” “language therapy,” “communication,” and “rehabilitation” via the Wan Fang Database, Chinese National Knowledge Infrastructure (CNKI), PubMed, and Web of Science in September and October 2020 to identify available content and modes related to language rehabilitation. We developed the content by learning from a model of cognitive communication competence . It inclIn November 2020, we held group discussions with experienced rehabilitation physicians and occupational therapists at the Brain Hospital of Hunan Province and the Hunan Cancer Hospital to determine the relationship between brain regions and language production, the rehabilitation process of speech, and their opinions about current speech treatment. In December 2020, we held an online meeting with 2 experts in the Department of Computing at the Hong Kong Polytechnic University to generate ideas about potential content based on VR. Based on the information collected, we developed an initial draft of 20 submodules in 6 modules. We designed a questionnaire for experts to review and use a 5-point Likert scale to evaluate which part should be included. Each piece was described with a name, specific content, and a short description of the training modes.The panel consisted of experts with both theory-based and practice-based backgrounds to obtain a variety of insights from researchers. Experts with a theory-based background all published papers in the speech treatment or rehabilitation fields. Experts with a practice-based background were selected based on their practical experience and publications. Additionally, we approached our network and asked responding experts to provide the names of essential experts in this field, such as a snowball sampling technique. The experts who participated in the first Delphi round were also approached in the second round. In total, 21 experts were invited by email, WeChat, or an in-person meeting to participate in the 2 rounds of the Delphi study. It contained detailed information about the goal, study procedure, first-round questionnaire, experts’ demographic information, and their judgment and familiarity with the corresponding field. Of the 21 experts, 15 (71.4% response rate) responded to the invitation.The first-round questionnaire was composed based on the contents identified by the literature review and group discussions. This resulted in a questionnaire consisting of 6 structured modules and 20 submodules. Experts were invited to rate these contents on a 5-point Likert scale ranging from 1  to 5 (extremely important). They were asked to modify, add, or delete content, as appropriate, and to provide suggestions they supposed were reasonable. The experts’ demographic information, as well as their judgment and familiarity with the corresponding field, was required. We reminded those who did not complete the survey following the initial invitation at 2-3-week intervals by WeChat or email.P value of <.05 [Microsoft Excel 2003 and IBM SPSS Statistics (version 26.0) were used for data analysis and processing. The responses of the experts were independently analyzed by 2 researchers, and the data were double-entered to minimize typing errors. We listed and combined similar recommendations. The mean, SD, and coefficient of variation (CV) were calculated. The CV is defined as the SD divided by the mean used to describe the relative dispersion degree of the items' importance evaluation by experts . A lower of <.05 . Cs repr of <.05 . The def of <.05 . We thenIn February 2021, we held an online meeting with 2 experts in the VR field to discuss the content based on VR before the second round. Experts who completed the first-round questionnaire were invited to participate in round 2. Again, we reminded those who did not complete the survey following the initial invitation at 2-3-week intervals by WeChat or email. The experts rated each revised section again. They were asked to rate how much they agreed that each element could be used and to comment on each part. Additional advice about the content that they assumed reasonable was required.P value of the Kendall coefficient W test of no more than .05 among experts that the contents should be included. The mean, SD, and CV values were calculated for each part. The Delphi survey was completed when all the items in the questionnaire met the aforementioned criteria.Analysis following round 2 aimed to identify any consensus on the contents and determine whether an additional round was needed. Microsoft Excel 2003 and IBM SPSS Statistics (version 26.0) were used for data analysis and processing. We predefined a mean score of no less than 4, a CV no more than 0.2, and a 2-tailed In November 2020 and February 2021, we held online meetings with 2 experts in the VR field to discuss this project. A variety of devices and components can deliver a VR experience, with the main categories including smartphone VR headsets, tethered personal computer (PC)-based VR headsets , or stand-alone VR headsets . HTC Vive and Oculus Quest create a more immersive experience and provide the user with a stronger sense of presence than smartphone VR owing to resolution, frame rate, and sufficient input mechanisms . To provThe VR scene was deployed on mobile phones using the Google Cardboard VR plug-in. Combining Google Cardboard equipment and mobile phones makes it a portable and cheap VR equipment that most patients can afford. The software can be used to receive language rehabilitation whenever and wherever possible. The entire VR scene was developed and constructed using the Unity game engine, and Adobe Photoshop was used for 2D assets. Unity is a bridge connecting artificial intelligence (AI) platforms, VR scenes, and back-end data storage. It uploads the designed text content to the Baidu AI platform and obtains a synthesized voice. The synthesized voice is used in the VR scene as a voice instruction to guide the user. Cardboard VR delivers pictures, words, videos, and synthesized voices to patients that substitute therapists in traditional treatment methods. The patients will see the designed questions and relevant materials in the VR scene and will record their voice answers using a mobile phone microphone or register their head rotation as an input for selecting answers in multiselection questions. Unity would then record their responses according to their head rotation or send the recorded voice to the Baidu AI platform to get the recognized text back. The results will be stored in an Excel sheet and sent to the therapist's email address with the patient’s name. We conducted feasibility exercises of the app on 5 patients with aphasia, 5 healthy volunteers, 5 medical staff, and 2 VR experts between 25 and 65 years of age . All 5 patients with aphasia were hospitalized for speech rehabilitation for 2 weeks, and all of them had ischemic stroke. The aphasia quotient (AQ) of the Chinese version of the Western Aphasia Battery ranged from 58 to 75 after 2 weeks of face-to-face training. This feasibility exercise allowed us to better understand and potentially reduce the likelihood of adverse effects in patients with aphasia who may have been more sensitive to sensory effects . Interviews were the measurements used in the experiments. The outline of interview questions after the participants’ experience with the mobile-based VR speech rehabilitation app is as follows: (1) What groups do you think this mobile-based VR speech rehabilitation app is suitable for? (2) How does the mobile-based VR speech rehabilitation app differ from face-to-face language rehabilitation? (3) What would encourage or hinder app adoption after using the mobile-based VR speech rehabilitation app? (4) What is your overall rating of the mobile-based VR speech rehabilitation app? All interviews were recorded using a digital voice recorder and transcribed verbatim within 24 hours. The researcher explored participants’ feelings about the app by asking questions in a private environment, and they were encouraged to put forward new questions and content.P<.001). Of the 6 modules and 20 submodules rated by the panel members in the first round  was 0.204 . Finally, a consensus was reached on 4 modules and 17 submodules. One expert suggested that patients should complete answering within the allotted time. Some respondents suggested additional executive functioning, reading, and writing training. However, this app is meant to improve patients' oral expression abilities, so reading and writing are beyond our consideration. The revised content contained 4 modules and 17 submodules.The results of the second round are presented in All 17 (100%) participants could open the app. A user-centered design philosophy was implemented to create additional interactive and customizable features that indicated a high degree of usability for patients with aphasia. Participants' comments were collected and descriptively analyzed based on the interview outline. The 4 responses were classified based on their feedback. In response to question 1, “What groups do you think this mobile-based VR speech rehabilitation app is suitable for?”, 12 (70.6%) of 17 participants suggested that the user group could include patients who were discharged or currently hospitalized. Participants stated that with this app, they could correct and refine their language skills, and 5 (29.4%) of 17 participants recommended that the user group should include people, especially those who are under significant financial pressure and do not have easy access to medical treatment but would prefer an opportunity to learn.In response to question 2, “How does the mobile-based VR speech rehabilitation app differ from face-to-face language training?”, all 17 (100%) participants mentioned the advantages of in-home apps. Participants stated that this app reduces the difficulty of going to the hospital and is more convenient as they do not have to worry about weather and traffic. In addition, 15 (88.2%) of 17 participants reported a high level of immersion and engagement and thought that patients could thoroughly enjoy themselves when using this app without judgment from others. Additionally, there is no need to worry about self-abasement because of the inability to speak fluently. Moreover, 4 (23.5%) of 17 participants critiqued the effectiveness of this app because patients could not receive immediate feedback, and the efficiency of using this app could not be guaranteed.In response to question 3, “After using the mobile-based VR speech rehabilitation app, what would encourage or hinder your adoption of the app?”, 15 (88.2%) of 17 participants mentioned that the design and arrangement met patients’ daily communication needs; they felt more secure because the training content was designed by a professional rehabilitation team. In addition, 15 (88.2%) participants mentioned that the app was easy to use, and 3 (17.6%) reported low levels of motion sickness.In response to question 4, “What is your overall rating of the mobile-based VR speech rehabilitation app?”, all 17 (100%) participants indicated that the app was novel, and 5 (29.4%) of 17 participants gave some recommendations, such as considering different training difficulties and increased functionality. All patients expressed interest in language rehabilitation using the new technology.This study developed the first accessible and cost-effective mobile-based VR speech rehabilitation app in China. Through a 2-round Delphi study, panel members reached a consensus on 4 modules and 17 submodules that benefit patients in gaining essential daily communication language skills and various skills in everyday life. Patients get opportunities to interact with the virtual environment and can practice at any time. A review systematically identified and evaluated a series of mobile apps for speech-language therapy and found a lack of interactive and engaging elements in the apps, which failed to make patients self-manage . The conWe developed the content by learning from a model of cognitive communication competence . LanguagLanguage rehabilitation combined with cognitive rehabilitation can improve patients' communication skills . The cogTo emphasize the communication demands of an individual's life, the contextual domain is placed at the top of the cognitive communication competence model . A CochrThis study had some limitations. First, a range of brain regions are related to attention and executive functioning, and these nonlanguage domains play a role in the abilities of patients with chronic aphasia . Owing tWe conducted a Delphi study and developed a mobile-based VR app. This study constitutes a step toward the development of a combination of health and VR. We believe there is significant potential to make our app an in-home app in the future, contributing to the automation of rehabilitation administration. However, further studies are needed to evaluate the feasibility and efficacy of our app. We hope that our research provides guidelines and references for others in the medical field.