Its combination with graphene has gained increasingattention due to its potential applicability in novel optoelectronicdevices. By using a combination of spatially resolved photoemissionspectroscopy and low-energy electron microscopy, we are able to providea direct visualization of the massive charge transfer from grapheneto α-RuCl3, which can modify the electronic propertiesof both materials, leading to novel electronic phenomena at theirinterface. A measurement of the spatially resolved work function allowsfor a direct estimate of the interface dipole between graphene andα-RuCl3. Their strong coupling could lead to newways of manipulating electronic properties of a two-dimensional heterojunction.Understanding the electronic properties of this structure is pivotalfor designing next generation low-power optoelectronics devices.We investigate the electronic properties of a grapheneand α-rutheniumtrichloride (α-RuCl In turn, this offers a route to explore the physicsof charge-doped Mott insulators.12In recent years, there has beena surge in interest in heterostructures composed of different two-dimensional(2D) materials.3 is a layered transition metal compound withahoneycomb lattice structure similar to graphene. However, unlike graphene,it is a Mott material with insulating behavior arising from strongelectronic correlations.13 At low temperatures,the complex competition of magnetic interactions ultimately stabilizesa zigzag antiferromagnet in RuCl3.14 However, the influence of doping, for instance by photoinduced chargedcarriers, is predicted to stabilize ferromagnetic order.15 Additionally, RuCl3 is classifiedas a Kitaev material due to its strong spin–orbit coupling,crystal field, and electronic correlations, which lead to anisotropicexchange interactions that favor the formation of a quantum spin liquidexpected to host Majorana fermions.20 These quasiparticles have non-Abelian statisticsand are essential for topological quantum computation.21 Nonetheless, the evidence for such behaviorin RuCl3 is still under debate22 and seems to be strongly affected by the presence of crystal defects,which promote impurity scattering and non-Kitaev interactions.23RuCl3, a chargetransfer occurs between the two materials due to their different workfunctions and electronic structures.8 Thischarge transfer can modify and hybridize the electronic propertiesof both materials10 as well as influencethe magnetism in RuCl3.24 The couplingbetween graphene and RuCl3 can modify the electronic bandstructure of RuCl3 and enhance its spin–orbit coupling,potentially impacting the Kitaev physics in the material.24 Anomalous quantum oscillations have been reported in the Gr/RuCl3 heterostructure and explained within the Kitaev-Kondo latticemodel.27 The strong charge transfer has also been used tocreate modulation-doped graphene where a lateral thickness variationof a tunnel barrier changes the magnitude of the charge transfer betweengraphene and RuCl3,11 enablingultrasharp (less than 5 nm wide) p–n junctions,12 which were also observed in nanobubbles of grapheneon RuCl3.28 The interactionbetween graphene and RuCl3 can also lead to plasmon polaritonsat the interface.29 The coupling betweenplasmon polaritons and the Mott physics in RuCl3 couldunlock new ways of manipulating light and electronic properties, withpotential applications in sensing, imaging, and communication. Moreover,by leveraging the unique passive doping control (no gating needed)of RuCl3 over graphene, we envision the creation of low-powerdevices that exhibit enhanced light-harvesting capabilities and precisecontrol over optical signals.30When graphene is brought in contact withRuCl3 and graphene. Nanometer-scale spatially resolved photoemissionspectroscopy (nanoXPS) and low-energy electron microscopy (LEEM) areused to explore the electronic properties of the heterostructure,allowing for a direct visualization of its charge transfer.Here,we employ a combination of experimental techniques to betterinvestigate the electronic properties of the interface between RuCl31 LEEM allowsimaging of the morphology and electronic properties of heterostructureswith high spatial resolution. By using low-energy electrons to probethe surface of the material, we can investigate local variations inelectronic properties to study their evolution over time. By comparingour experimental results with the calculations present in the literature,we validate our findings and provide a more complete understandingof the electronic properties of the heterostructure.It is possible to effectively map the core levels of 2D systemsand the dispersive electronic band structure of the heterostructurewith submicron spatial resolution via nanoXPS and angle-resolved photoemissionspectroscopy (nanoARPES).3, clearly visible in the nanoARPES data and reflectedin the core levels measured via nanoXPS. LEEM analysis provides avalue of the shift in work function that is much lower than the bandshift measured via nanoARPES, consequent to the charge transfer betweenthe layers. This discrepancy can be attributed to the presence ofa dipole at the interface that greatly affects the work function value.32 Moreover, the appearance of a band below theFermi level, not observed in other ARPES experiments, can be attributedto the effect of charge transfer on RuCl3. The most straightforwardinterpretation suggests that electrons from graphene have partiallyoccupied the typically unoccupied upper Hubbard band, causing it toshift below the Fermi level. Alternatively, in a study of adatom-dopedRuCl3, Zhou et al. identified the appearance of such aband between the lower Hubbard band and Fermi level as an unconventionalMott transition driven by the charge transfer.33 Either way, this new spectral weight shows that chargetransfer from graphene to RuCl3 induces significant changesin the electronic structure of the system. The presence of this bandprovides further evidence of the complex electronic interactions occurringat the interface and highlights the role of charge transfer in drivingunconventional electronic phenomena in this system.The experimentaldata show a massive charge transfer from grapheneto RuCl3 (Supporting Information. The device has three distinct regions: one with graphene on thickh-BN as a reference (Gr/h-BN), one with graphene directly on RuCl3 (Gr/RuCl3), and one with a thin h-BN flake sandwichedbetween graphene and RuCl3 (Gr/h-BN/RuCl3).The thin h-BN acts as a buffer to decrease the Gr-RuCl3 interactions.28 The thick h-BN substrate providesa stable and flat surface for other materials and minimizes the effectof the underlying substrate on the electronic properties of the heterostructure.A sketch of the three regions is reported in 3 at a binding energy of −1.3 eV. The contrast allows for identifyingthe three regions described above. The color scheme for the threecolored squares on the map is consistent with the sketch in panel(b) and confirmed by the core level analysis via XPS. We focus onthe peaks originating from Cl, Ru, and C core levels, reported in 3 region is theCl 2p core level. Its signal decreases when the h-BN buffer is presentand disappears entirely outside the RuCl3 flake. The Ru3d3/2 core level partially overlaps with that of C 1s.It is possible to fit and track the evolution of the C 1s peak forthe three different regions  substrate, with three other materials exfoliated ontop: graphene, thin h-BN (2 nm), and RuCl3 and Gr/RuCl3 regions, respectively. Notably, the graphene bands in theseregions are shifted upward by approximately 500 meV where the h-BNlayers separate the graphene and RuCl3 crystals and by750 meV where RuCl3 is in direct contact with graphene,indicating a progressive p-doping when reducing the distance betweengraphene and RuCl3. The charge transfer from graphene toRuCl3 is responsible for the shift of the Dirac cone andis similarly manifested in the position of the C 1s core level, wherethe observed chemical shift, as illustrated in 35 The influenceof charge transfer and the resulting interface dipole is evident inthe upward shift of the h-BN bands (indicated by white arrows) byapproximately 1 eV, relative to the region without RuCl3 as shown in To directly visualize the electronic propertiesof the system,we conducted a nanoARPES study. This study enabled us to observe theelectronic band structure in three specific regions highlighted in 3 bands are visible only in the region where grapheneis in direct contact, with no intervening h-BN buffer. The RuCl3 bands are identified by studying the energy distributioncurves (EDCs) taken along the dashed line in panels (a–c) of 3 electronicstructure, highlighted by three red arrows, displays two dispersionlessbands centered at binding energies of ∼0.5 and ∼1.3eV and a third more dispersive band at a deeper binding energy (∼3.8eV).Because of the short mean free path of the photoelectrons,theRuCl9 However, it is worth noting that the observedenergy position of the lower Hubbard band may appear to be lower thanthat predicted by computational models. This discrepancy could beattributed to the challenges in accurately estimating the energy gapusing, e.g., density functional theory (DFT) calculations. Factorssuch as electron–electron interactions and correlation effects,which are not fully captured in DFT calculations, can influence theenergy position of the lower Hubbard band.The band centered at −1.3 eV is very likely to correspondto the lower Hubbard band, as indicated by Biswas et al.3 structure.33Regarding the bandat −3.8 eV, it is identified as an in-planeorbital and labeled as Cl p bands, originating from the Cl orbitalswithin the RuCl3,36 suggests that its emergence is a result of the interaction betweenRuCl3 and graphene. This band can be understood as theupper Hubbard band, which is typically unoccupied, being partiallyfilled by electrons transferred from graphene and consequently shiftedbelow the Fermi level. Another explanation put forth by Zhou et al.proposes that the introduction of dopants on the surface of RuCl3 leads to the population of new bands near the Fermi level.33 These bands are attributed to an unconventionalMott transition, as described by the authors. It is possible thatRuCl3 undergoes a similar Mott transition when in contactwith graphene, as observed in the case of Rb and K doping in ref  collected along the dashedlines on the Fermi surface are displayed in 40 we can extract the amount of charge tunneling from graphene to RuCl3, about 4.1 × 1013 cm–2,consistent with previous, if indirect, experimental and computationalobservations.29 When the spacing between layers is increased with a few h-BN layers,the tunnel barrier thickens, resulting in a decreased charge transferand therefore a lower p-doping level in graphene (∼1.7 ×1013 cm–2).A more quantitativeestimate of the total amount of charge transferredbetween the layers, with and without the h-BN buffer layer, is givenby considering the Fermi surface for each of the three regions, asreported in 3. It is possible tocompute the magnitude of the dipole by measuring the variation ofthe work function across the different regions of the system. By applyinga positive voltage to the sample, the incident LEEM electrons transitionfrom mirror mode, with the electrons reflecting before touching thesample surface, to LEEM mode, where the electrons are scattered fromsample surface with a landing energy proportional to the applied samplebias. In LEEM mode, the incident electrons can be accepted into unoccupiedbands of the sample surface causing a lower reflected intensity thanin the mirror mode. The inflection point of this drop in intensityfrom mirror mode to LEEM mode can be interpreted as the work functionof the sample surface when accounting for the work function of theLEEM cathode.Finally, we can quantifythe electric dipole generated at the interfaceby the charge transfer to the RuCl3 andGr/h-BN. When the h-BN buffer layer is also present, the shift inthe work function is reduced by 160 meV. This difference with respectto the Gr/h-BN region agrees with the 70 meV difference between theGr/h-BN/RuCl3 and Gr/RuCl3 regions. Previously,Yu and co-workers demonstrated that the work function of graphenecan be substantially affected by the dipole formed by surface adsorbates.32 Analogously, here we estimate the magnitudeof the electric field at the interface knowing the value of the chemicalpotential and the value of the work function, with respect to pristinegraphene. In the presence of a dipole at the interface, the work functionof graphene can be written asWD is the offset of work function due to the dipoleat the interface, Wgr0 is the intrinsic work functionof the undoped graphene, and EF is the position of the Fermi level.32 We can therefore evaluate the magnitude of the electric dipole withrespect to the pristine sample simply considering the measured workfunction difference across the different regions and adding this tothe corresponding difference in the EF position with respect to the Dirac point. This resultsin an electric dipole energy of ∼1 eV and ∼660 meV forGr/RuCl3 and Gr/h-BN/RuCl3 regions, respectively.In 3 heterostructure. The resultsshowed direct evidence of significant charge transfer from grapheneto RuCl3, leading to a doping-induced Mott transition andpotential enhancement of the Kitaev physics. LEEM measurement alsoallowed us to provide an estimate of the dipole moment formed at theinterface between RuCl3 and graphene, instrumental forcomprehensive device modeling. This work lays out valuable insightsinto the electronic properties of Gr/RuCl3 heterostructuresand its potential for future applications, where the passive controlof the doping level in graphene is at the foundation of low-powerelectronics and light-harvesting devices.In conclusion, we used a combination of experimentaltechniques,including nanoXPS and nanoARPES and LEEM, to investigate the electronicproperties of the Gr/RuCl


Current data on infectious complications of chimeric antigen receptor (CAR) T-cell therapy are limited to single center retrospective cohort studies. Presented here is a subset of patients from a multicenter retrospective cohort study including recipients of CAR T-cell therapy at Duke University, University of North Carolina at Chapel Hill, and Stanford University.In a retrospective cohort of 66 patients who received CD19 CAR T-cell therapy Duke University between January 1, 2018 and August 31, 2021 rates and characteristics of bacterial, viral, and fungal infections within the first year after CAR T-cell infusion are described. Demographic, baseline clinical, and outcome variables are compared between patients who developed infection and those who did not.Forty-nine total infections occurred in 29 (43.9%) patients within 1 year of CAR T-cell infusion. Patients who developed infection were more likely to have an underlying malignancy other than diffuse large B-cell lymphoma , and to have developed immune effector cell-associated neurotoxicity syndrome . Viral  and bacterial  pathogens predominated and infections most often involved the bloodstream  and lung .There were no statistically significant differences between rates of bacterial and viral infection based on time since CAR T-cell infusion (≤30 days vs 30 – 90 days vs >90 days), though fungal infections only occurred after 90 days in patients with relapse of their underlying disease.Infectious complications after CAR T-cell therapy are common within the first year of infusion. Bacterial and viral infections predominate while fungal infections are rare and occurred only in patients with relapsed disease.Melody Smith, MD, MS, BMS: Advisor/Consultant Krista Rowe-Nichols, RN, MSN, AOCNS, Bristol Myers Squibb: Advisor/Consultant|Kite Pharmaceuticals: Advisor/Consultant|Kite Pharmaceuticals: Speaker Bureau Christopher Kelsey, MD, Colgate-Palmolive: Expert Testimony|Johnson and Johnson: Expert Testimony Taewoong Choi, MD, Janssen biotech: Honoraria Matthew McKinney, MD, ADC Therapeutics: Advisor/Consultant|ADC Therapeutics: Honoraria|Beigene: Grant/Research Support|Beigene: Honoraria|Epizyme: Advisor/Consultant|Genentech: Advisor/Consultant|Genentech: Grant/Research Support|Genentech: Honoraria|Gilead/Kite: Advisor/Consultant|Gilead/Kite: Honoraria|Incyte: Grant/Research Support|Novartis: Advisor/Consultant|Seagen, Inc.: Advisor/Consultant|Takeda: Advisor/Consultant|TG therapeutics: Advisor/Consultant


Benefit-sharing mechanisms have been instrumental in securing the support of local communities living on the edge of protected areas to implement protected area goals and enhance biodiversity conservation outcomes. Understanding the acceptability of the types of benefit provided among diverse communities is crucial for co-designing benefit-sharing approaches that accommodate local perspectives. Here, we used quasi-structured questionnaires and focus group discussions (FGD) to assess the acceptance of the types of benefit received by the communities in the Greater Serengeti Ecosystem (GSE) in Tanzania and the effectiveness of the benefits in securing community support for conservation reserves. We found that the categories of social service provision, livelihood support, and employment described all the benefits provided across conservation institutions operating in the GSE. However, the types of benefit within these categories varied significantly among conservation institutions, in terms of level and frequency of benefits received by communities. Overall, student scholarships were highly rated by respondents as the most satisfying benefit received. Respondents who were dissatisfied with the benefits received thought that the benefits did not compensate for the high costs arising from wildlife incursions onto their land. Communities’ acceptance of the benefits received varied greatly among villages, but only a small proportion of pooled respondents (22%) were willing to support the existence of a protected area without benefit. This study suggests that local people are willing to support conservation outcomes but require conservation institutions to give greater consideration to the costs incurred by communities, their livelihood needs, and access to natural resources or other benefits. We recommend that benefit-sharing be tailored to the local circumstances and cultures of people living close to protected areas, particularly communities expressing more negative views, to ensure adequate and appropriate compensation is provided.The online version contains supplementary material available at 10.1007/s10531-023-02583-1. With highly diverse wildlife and spectacular landscapes, the GSE is a leading global tourism destination and a major source of revenue for the country.With over 30% of its land included within protected areas, Tanzania contributes more than most countries in the world to Aichi Target 11 in terms of the proportion of its land area protected for conservation purposes WPA . One of However, this success has come at a cost to local communities in and around the GSE  Regulation , and by one professional association, the Frankfurt Zoological Society (FZS).The local communities in and around the GSE Fig.  vary in The smallest community participating in the research were the hunter-gatherer Hadzabe communities , believed to have occupied the eastern part of the GSE over 10,000 years ago Marlowe . In the The second community grouping is the Maasai and Datoga pastoralist communities, which migrated from South Sudan to Kenya in the 15th century and later settled in northern Tanzania Nilsson . During The third and largest community grouping is the agro-pastoralist communities who live along the western border of the GSE and identify as Kurya, Ikoma, Sukuma, Isenye, Ngoreme, Zanaki, Jita and Luo , the Datoga pastoralists were from Olpiro Village and the agro-pastoralists communities lived in the villages of Bisarara, Park Nyigoti and Robanda. Before data collection, we obtained a research permit from Tanzania’s Commission for Science and Technology (COSTECH) No. 2021-476-NA-2021-170 and an introductory letter from the respective district councils, Serengeti and Karatu. Within each village, households were selected based on their accessibility and availability of household heads for interviews. The research was conducted under Charles Darwin University Human Research Ethics Committee Permit number H21058.We surveyed 296 households, i.e. 13.2% of the total households  in the study areas. This included 60 hunter/gatherers (~ 60%), 86 in a pastoralist village (25%) and 50 from each of three agro-pastoralist communities . With each household randomly selected, we undertook a quasi-structured questionnaire, defining a household as a group of people living together, mostly with a single person who self-identified as its head. If the head of the household was not available, we chose the nearest household instead. The interviews began by seeking verbal approval of respondents after they were informed of their right to withdraw from the interview at any stage, and that whatever had been recorded would be redacted from the research records, if they did decide to withdraw. At each household we asked respondents the following questions: (i) What types of benefit did you receive as a result of living in or near a protected area? and (ii) Did you find the benefits received acceptable?  Strongly reject; (2) Reject; (3) Neither reject nor accept; (4) Accept; (5) Strongly accept). We also asked respondents to name the benefit with which they were most satisfied. We then asked respondents a mix of closed and open-ended questions about their perspectives on the effectiveness of the benefits received in securing community support for the conservation of protected areas.Interviews were largely conducted in the Kiswahili language, a national language in Tanzania in which the lead author is fluent, with a translator employed to conduct discussions in the local language when required. The interviews were recorded, transcribed, and translated into English for analysis by the lead author.To cross-reference survey results, we conducted a Focus Group Discussion (FGD) with a small group of participants (~ 10) in each of the villages visited for household questionnaire surveys. Two FGDs were held in each village for people aged over 20 years, one for men and one for women, making a total of 12 in all. The discussion was guided by a checklist of questions drawn from the research objectives to stimulate participants’ contributions. Specifically, we focused on the types of benefit received from conservation institutions, how often the communities received benefits, and who within the village benefited. We tabled the benefits mentioned during the questionnaire survey for confirmation in each village’s FGD. The first author facilitated and ensured that participation and the discussion proceeded until participants ran out of questions/queries on each topic Newing , 104. Associal services provision, livelihood support benefits, and employment of the local people.Categories of benefits were based on key research questions Patton  and grouSocial services provision included all social benefits received such as an investment in schools , health centers, ambulance services, village office buildings and funds received for village development projects. Livelihood support included benefits which fulfill locals’ necessities of life such as food, water for domestic use or livestock, vaccination for livestock, loans, health insurance, training , or access to non-timber forest products (NTFP). The Employment category included any employment received from the protected area, regardless of whether this included pensionable terms (permanent) or non-pensionable terms (temporary).Mean household acceptability for the different types of benefit was derived from the Likert scores in each village. We then employed binomial Generalized Linear Models (GLM) using R 3.6.2 . This did not improve the model fit and was not followed further (data not shown). AICc was used for model selection. Model validation was performed using the DHARMa R package Hartig . Model rAbout 40% of respondents from the households were female, and the rest were male, within the age groups of 18–35 years (30%), 36–50 years (45%), and over 50 years (25%). Most household respondents (65%) had some formal education  but 35% had not attended school. A large majority (85%) of respondents were born in the villages surveyed.The reported benefits received by each surveyed village varied in type, magnitude and frequency Table . For exaAcross all communities, the average acceptance level (Likert score) across all benefits was 2.2 (out of a maximum of 5). Social service benefits were ranked the highest (mean score of 2.8), followed by livelihood benefits (mean score of 2.4) and the least was employment (mean score of 1.6) for the pooled data Fig. .The benefits most appreciated by respondents varied greatly between communities Fig. . Three vMost respondents (72%) felt encouraged to support protected area conservation goals by the benefits they received Appendix 4) but support varied significantly among villages and between genders (P < 0.001). Age and education levels were not associated with any of the agreements (P > 0.05). The most supportive were pastoralist respondents (98%), and the least supportive were the Agro-pastoralist 1 respondents (only 5%)  whereas, only a very small proportion of respondents from Agro-pastoralist 1 village agreed , which has a set of criteria and priorities . However, the NCAA is obliged to support only 25 villages, all inside the protected area URT . In contMost respondents viewed the benefits as effective in encouraging them to support the nearby protected area, and agreed that the benefits help reduce illegal activities in the protected area. Further, most respondents were not willing to support the existence of a protected area without benefits. Communities receiving more benefits from conservation also tend to be more aware of conservation activities, and have a more positive perception of the protected area, than communities receiving little benefit  (Chamba Crocuta crocuta), lion (Panthera leo), and leopard (Panthera pardus)  Chamba , livestoThe conflict between the protected areas’ management or government and the community also influences communities’ perspectives toward the benefits received from protected areas in the GSE. For example, some communities have a strong fear of eviction from Ngorongoro Conservation Area. The same concern was reported by Melubo and Lovelock  for the Another point of conflict was the location of the boundary of a protected area. Respondents from two communities in our study claimed that the boundary of Ikorongo Game Reserve was wrongly placed by the game reserve authority under the use of force. A similar claim has been recently reported by Matata et al. . The comThe penalties given to wildlife offenders by the court under the Wildlife Conservation Act No. 5 of 2009, perceived to be substantial, can be another source of conflict between protected areas and nearby communities. Penalties include confiscation of livestock found illegally in the protected areas, a minimum sentence of 20 years in prison for possession of bushmeat, even though bushmeat hunting is an important livelihood strategy for people in the western Serengeti  costs that the local communities experience from living with wildlife. Accordingly, institutional policies need to be investigated to identify where they serve as a barrier to improving the benefit-sharing mechanisms in the GSE. For example, business opportunities for local people need to be expanded, and barriers to local business development understood and minimized. Involvement of both genders in conservation decision making is also vital in gaining overall community support for conservation. We recommend that future research should focus on conservation-related local community demands and aspirations, and on developing co-designed benefit-sharing mechanisms to deliver effective conservation outcomes as well as enhance community livelihoods. In addition, the impact of wildlife on the local people’s livelihood strategies in the GSE should be thoroughly investigated to find solutions for delivering effective conservation programs.Supplementary material 1 (DOCX 14.6 kb)Supplementary material 2 (DOCX 14.6 kb)Supplementary material 3 (DOCX 14.7 kb)Supplementary material 4 (DOCX 38.8 kb)Below is the link to the electronic supplementary material.


Background: Idiopathic pulmonary fibrosis (IPF) is a fatal and irreversible interstitial lung disease. The specific mechanisms involved in the pathogenesis of IPF are not fully understood, while metabolic dysregulation has recently been demonstrated to contribute to IPF. This study aims to identify key metabolism-related genes involved in the progression of IPF, providing new insights into the pathogenesis of IPF.Methods: We downloaded four datasets  from the Gene Expression Omnibus (GEO) database and identified differentially expressed metabolism-related genes (DEMRGs) in lung tissues of IPF by comprehensive analysis. Then, we performed GO, KEGG, and Reactome enrichment analyses of the DEMRGs. Subsequently, key DEMRGs were identified by machine-learning algorithms. Next, miRNAs regulating these key DEMRGs were predicted by integrating the GSE32538 (IPF miRNA dataset) and the miRWalk database. The Cytoscape software was used to visualize miRNA-mRNA regulatory networks. In addition, the relative levels of immune cells were assessed by the CIBERSORT algorithm, and the correlation of key DEMRGs with immune cells was calculated. Finally, the mRNA expression of the key DEMRGs was validated in two external independent datasets and an in vivo experiment.Results: A total of 101 DEMRGs (51 upregulated and 50 downregulated) were identified. Six key DEMRGs  were further identified using two machine-learning algorithms (LASSO and SVM-RFE). In the lung tissue of IPF patients, the expression levels of ENPP3, ENTPD1, and PDE7B were upregulated, and the expression levels of GPX3, PNMT, and POLR3H were downregulated. In addition, the miRNA-mRNA regulatory network of key DEMRGs was constructed. Then, the expression levels of key DEMRGs were validated in two independent external datasets (GSE53845 and GSE213001). Finally, we verified the key DEMRGs in the lung tissue of bleomycin-induced pulmonary fibrosis mice by qRT-PCR.Conclusion: Our study identified key metabolism-related genes that are differentially expressed in the lung tissue of IPF patients. Our study emphasizes the critical role of metabolic dysregulation in IPF, offers potential therapeutic targets, and provides new insights for future studies. Idiopathic pulmonary fibrosis (IPF) is a progressive, life-threatening, chronic interstitial lung disease of unknown etiology . It is cAn increasing number of studies have recently demonstrated the role of metabolic dysregulation in IPF. For instance, Kang et al. reported altered glycolysis and glutamine metabolism in human lungs with severe IPF . FurtherOur study first analyzed GSE32537, GSE110147, GSE150910, and GSE92592 from the Gene Expression Omnibus (GEO) database and identified differentially expressed metabolism-related genes (DEMRGs) in the lung tissue of IPF patients. Subsequently, we conducted a functional enrichment analysis of DEMRGs. Then, we used two machine-learning methods, least absolute shrinkage and selection operator (LASSO) regression and support vector machine recursive feature elimination (SVM-RFE), to identify six IPF signature genes as key DEMRGs: ENPP3, ENTPD1, PDE7B, GPX3, PNMT, and POLR3H. The expression of ENPP3, ENTPD1, and PDE7B was significantly upregulated in IPF patients’ lung tissue, and the expression of GPX3, PNMT, and POLR3H was significantly downregulated. Afterward, we predicted miRNAs regulating key DEMRGs using the miRWalk database, combining it with the GSE32538 dataset (miRNA microarray expression profiles of IPF) to construct a miRNA-mRNA regulatory network. Next, the relative levels of immune cells were assessed by the CIBERSORT algorithm, and the correlation of key DEMRGs with immune cells was calculated. Finally, we validated the expression patterns of six key DEMRGs by analyzing the external independent dataset GSE53845 and performing qRT-PCR.https://www.ncbi.nlm.nih.gov/geo/) database  (The metabolism-related genes (MRGs) were obtained from the Molecular Signatures database  . Specifivia the “oligo” R package for background correction and normalization of the raw data in the two datasets. Subsequently, differentially expressed genes were identified using the “limma” R package. GSE150910 and GSE92592 were RNA-seq datasets that were generated using the Illumina platform. We first downloaded their raw gene count matrix files. Then, we performed differential expression analysis on the gene expression matrix normalized by the vst function of the “Deseq2” R package. An adjusted p-value <0.05 was set as the threshold for identifying differentially expressed genes.GSE32537 and GSE110147 were based on the GPL6244 platform of Affymetrix Human Gene 1.0 ST Array. We used the RMA algorithm After acquiring the DEMRGs from each of the four GEO datasets, we used the Venn diagram to search for the common upregulated DEMRGs and the common downregulated DEMRGs. The “ggvenn” R package was applied to plot the Venn diagrams of common DEMRGs of the four datasets.https://david.ncifcrf.gov/)  . We perfrf.gov/) .To identify the most critical DEMRGs, we used two machine-learning algorithms: least absolute shrinkage and selection operator (LASSO) regression and support vector machine recursive feature elimination (SVM-RFE). The LASSO algorithm is a regression analysis method that minimizes regression coefficients through successive shrinkage operations to reduce the possibility of overfitting, thereby reducing redundancy and eliminating uncorrelated genes from these analyses . The SVMp-value <0.05. Since the IDs of the miRNAs in this dataset were derived from an older version of miRBase, we updated the miRNA IDs using the miEAA 2.0 database   to obtain differentially expressed miRNAs (DEmiRNAs). The significance threshold was set at an adjusted database . Subsequerg.de/) . If a DEerg.de/) .http://cibersort.stanford.edu/). First, we evaluated the relative expression of immune cells in all samples and plotted a histogram of immune cell content for each sample. Subsequently, we compared the content of each immune cell between IPF patients and healthy controls and plotted a boxplot for visualization. The Shapiro-Wilk test was performed to examine the normality of data, and the t-test or Mann-Whitney Wilcoxon test was used to conduct comparisons between groups based on the results of normality test . We compared the mRNA expression levels of the key DEMRGs between IPF patients and control groups. We performed the Shapiro-Wilk test to check the normality of the data before making comparisons between groups. Based on the normality results , we usedsoftware .The animal study was approved by the Laboratory Animal Welfare Ethics Committee of Central South University. Mice of the C57BL/6 strain  were purchased from Hunan SJA Laboratory Animal Co., Ltd. . Mice were housed in pathogen-free conditions with a 12 h dark/light cycle and were given access to food and water without restriction.Single tracheal instillation of bleomycin (BLM) was applied to construct the pulmonary fibrosis model . Mice we−ΔΔCT method. Triplicates of all experiments were performed. Total RNA was extracted from lung tissue using TRIzol reagent , and qRT-PCR was performed using the ABI ViiA 7 real-time PCR system. GAPDH mRNA was used as an internal control for the key DEMRGs, and the relative fold differences were calculated using the 2p-values <0.05 were considered statistically significant.Relative expression levels of the key DEMRGs were plotted in a barplot using the GraphPad Prism 8 software. Based on the normality results calculated by Shapiro-Wilk , the difWe obtained 949 unique MRGs through MSigDB. Subsequently, we performed differential expression analysis on lung tissue samples from IPF patients and healthy control individuals from four GEO datasets , and thus obtained the differentially expressed MRGs (DEMRGs) between IPF patients and healthy controls in each dataset. As a result, in GSE32537, GSE110147, GSE150910, and GSE92592, we detected 203, 279, 263, and 267 upregulated DEMRGs, respectively. In addition, we identified 336, 402, 231, and 211 DEMRGs that were downregulated in GSE32537, GSE110147, GSE150910, and GSE92592. The heat map shows the distribution of DEMRGs in the four datasets . The redWe performed a functional enrichment analysis of these 101 common DEMRGs through the DAVID database. In order to identify key DEMRGs, the LASSO regression analysis was used to screen the gene signatures for the 101 common DEMRGs , yieldinAfter updating the miRNA IDs by the miEAA 2.0 database, we identified 59 upregulated miRNAs and 103 downregulated miRNAs in IPF from the GSE32538 dataset . Using tp = 0.0023). Therefore, ENPP3 might be potentially associated with increased levels of M2 macrophages in the IPF process.We compared the expression levels of six key DEMRGs in IPF patients and controls in two independent external datasets (GSE53845 and GSE213001). According to the results, ENPP3, ENTPD1, and PDE7B were significantly upregulated in the lung tissues of IPF patients , while GAccording to the results of qRT-PCR, the expression levels of ENPP3, PDE7B, and ENTPD1 were elevated, while the expression levels of PNMT, GPX3, and POLR3H were decreased in the lung tissues of bleomycin-induced pulmonary fibrosis mice compared with the sham group . The resOur study aims to identify key metabolism-related genes of IPF. First, we performed the differential analysis of the four GEO public datasets  and integrated metabolism-related genes from the MSigDB dataset, resulting in 51 DEMRGs that were commonly upregulated and 50 DEMRGs that were commonly downregulated in the four IPF datasets. Subsequently, we performed functional enrichment analysis on these 101 DEMRGs, and the results indicated that these genes were involved in various metabolism-related terms. Then, two machine-learning algorithms were utilized to screen the key DEMRGs, resulting in six genes  as key DEMRGs. We further combined the miRNA expression profile dataset of IPF and the predicting results of the miRWalk database to construct the miRNA-mRNA network regulating the key DEMRGs. Next, we performed an immune infiltration analysis and identified an elevated M2 macrophage level in IPF patients, which reflects the enhanced M2 polarization-mediated fibrosis phenotype. In addition, the mRNA expression of the key DEMRGs was validated in two external independent datasets (GSE53845 and GSE213001). Finally, the gene expression pattern was validated by qRT-PCR, demonstrating that the key DEMRGs might have potentially significant roles in IPF.The immune cell infiltration results showed increased levels of M2 macrophages in the lung tissue of IPF patients. As the most abundant immune cells in the lung (approximately 70%), macrophages play a critical role in pulmonary fibrosis-related airway remodeling . ActivatGPX3 encodes glutathione peroxidase 3, which is expressed mainly in the lung and kidney . Recent PDE7B encodes a phosphodiesterase that hydrolyzes cAMP and downregulates its signaling effects . In addiThe advantage of this study is that we have identified key metabolism-related genes that are commonly differentially expressed in IPF lung tissue using multiple bioinformatics approaches and validation in animal models. These genes may be a potential focus for future research on IPF metabolic disorders. However, several shortcomings of our study need to be acknowledged. First, the general profile of the IPF population cohort and the healthy control population cohort in the original dataset was not identical; for example, the mean age of the case group in the original study of GSE32537 was 62.6 years, whereas the mean age of the control group was 47.5 years. Therefore, it is unclear whether these differential gene expressions could be influenced by age. Nevertheless, our findings were obtained based on the analysis and validation of multiple datasets, thus minimizing the effect of potential confounding factors. The second limitation of this study is that although the identified key DEMRGs are commonly differentially expressed in IPF lung tissues, the specific degree of their impact on IPF needs to be clarified. Therefore, it will be important to interpret the findings with caution until they are validated by functional experimental research, despite the fact that they were based on reliable bioinformatics data.Overall, through a comprehensive analysis of public datasets and experimental validation, we identified key metabolism-related genes that are differentially expressed in the lung tissue of IPF patients. Our study emphasizes the critical role of metabolic dysregulation in IPF, offers potential therapeutic targets, and provides new insights for future studies.


ChatGPT, a large language model, has shown good performance on physician certification examinations and medical consultations. However, its performance has not been examined in languages other than English or on nursing examinations.We aimed to evaluate the performance of ChatGPT on the Japanese National Nurse Examinations.We evaluated the percentages of correct answers provided by ChatGPT (GPT-3.5) for all questions on the Japanese National Nurse Examinations from 2019 to 2023, excluding inappropriate questions and those containing images. Inappropriate questions were pointed out by a third-party organization and announced by the government to be excluded from scoring. Specifically, these include “questions with inappropriate question difficulty” and “questions with errors in the questions or choices.” These examinations consist of 240 questions each year, divided into basic knowledge questions that test the basic issues of particular importance to nurses and general questions that test a wide range of specialized knowledge. Furthermore, the questions had 2 types of formats: simple-choice and situation-setup questions. Simple-choice questions are primarily knowledge-based and multiple-choice, whereas situation-setup questions entail the candidate reading a patient’s and family situation’s description, and selecting the nurse's action or patient's response. Hence, the questions were standardized using 2 types of prompts before requesting answers from ChatGPT. Chi-square tests were conducted to compare the percentage of correct answers for each year's examination format and specialty area related to the question. In addition, a Cochran-Armitage trend test was performed with the percentage of correct answers from 2019 to 2023.The 5-year average percentage of correct answers for ChatGPT was 75.1% (SD 3%) for basic knowledge questions and 64.5% (SD 5%) for general questions. The highest percentage of correct answers on the 2019 examination was 80% for basic knowledge questions and 71.2% for general questions. ChatGPT met the passing criteria for the 2019 Japanese National Nurse Examination and was close to passing the 2020-2023 examinations, with only a few more correct answers required to pass. ChatGPT had a lower percentage of correct answers in some areas, such as pharmacology, social welfare, related law and regulations, endocrinology/metabolism, and dermatology, and a higher percentage of correct answers in the areas of nutrition, pathology, hematology, ophthalmology, otolaryngology, dentistry and dental surgery, and nursing integration and practice.ChatGPT only passed the 2019 Japanese National Nursing Examination during the most recent 5 years. Although it did not pass the examinations from other years, it performed very close to the passing level, even in those containing questions related to psychology, communication, and nursing. ChatGPT is a large language model developed by OpenAI . Based oArtificial intelligence (AI) applications have been used in the medical field, including medical chatbots, and applications that analyze and summarize electronic medical record systems, perform image diagnosis, analyze and organize the medical literature, and perform patient monitoring ,4. ReleaAlthough physicians and nurses both play critical roles in the health care system, their specialties and responsibilities differ. Physicians focus on diagnosing and treating illnesses, whereas nurses focus on providing direct patient care and support. Nurses monitor patient health, administer medications, assist with activities of daily living, and provide emotional support to patients and their families. Nurses also communicate with other health care professionals to ensure that patients receive the appropriate care. Therefore, their training and responsibilities generally focus more on patient care and communication than on diagnosis and treatment.While passing the national examination does not guarantee the ability to practice in a clinical setting, a different scenario arises when considering the performance on registered nurse licensing examinations. These examinations feature questions that emphasize on patient emotions and communication, contrasting with those found in physician licensing examinations. Notably, if excellent performance can be demonstrated in these nursing examinations, it is likely to pave the way for a significant expansion of AI applications in the medical field. However, the performance of ChatGPT on nursing licensing examinations has not yet been evaluated.We aimed to evaluate the performance of ChatGPT on national examinations for registered nurses in Japan.The data sets included questions and answers from the National Nurse Examinations in Japan from 2019 to 2023 . These eEach year, the Ministry of Health, Labor and Welfare (MHLW) of Japan, which certifies the qualification of registered nurses nationwide, reviews questions among conducted examinations, which cannot be answered with just 1 answer, or questions for which no correct answer exists, based on the MHLW’s own checks and comments from a third-party organization—the Japan Nursing School Association. Then, the MHLW deems these as “inappropriate questions” and removes them from the examinations. The inappropriate questions were excluded from this study. In addition, all questions were screened, and questions containing visual assets, such as clinical images, medical photography, and graphs, were removed because ChatGPT (GPT-3.5) is an interactive language AI that does not support image recognition.Because prompt engineering significantly affects generative output, we standardized the input formats of the questions . QuestioPrompt 1: Simple-choice questionsPlease answer the following questions briefly and by number.Question: <Questionnaire contents>1. <Option 1>2. <Option 2>3. <Option 3>4. <Option 4>Prompt 2: Situation-setup questionsBased on the following situation setup, please answer the questions briefly and by number.Situation-setup: <Situation-setup contents>Question: <Questionnaire contents>1. <Option 1>2. <Option 2>3. <Option 3>4. <Option 4>Based on the scoring criteria of the official nursing examination, the percentage of correct answers provided by ChatGPT (GPT-3.5) was calculated separately for basic knowledge and general questions. We calculated the percentage of correct answers for each of the simple-choice questions  and the situation-setup questions  and conducted a chi-square test to compare the percentage of correct answers between the 2 prompts. Finally, the percentage of correct answers was calculated for each of the 32 subject areas, and areas with higher and lower percentages of correct answers compared with the overall mean and 1 SD were extracted. All statistical analyses were performed using R .This study did not require ethics approval because we only analyzed data from a published database.Five years of the National Nurse Examination data showed that the largest number of inappropriate questions occurred in 2019, with 10 questions having been excluded from the scoring and 2 or 3 inappropriate questions in the other years. The number of questions with figures and tables ranged from 6 to 16. Thus, the number of questions analyzed in this study was 214 of 240 in the lowest year and 232 of 240 in the highest year .The 5-year average percentage of correct answers provided by ChatGPT was 75.1% (SD 3%) for basic knowledge questions and 64.5% (SD 5%) for general questions . ThroughComparing simple-choice questions (prompt 1) and situation-setup questions (prompt 2), the average percentage of correct answers for prompt 1 was 66.3% (SD 3%) and 65.9% (SD 7%) for prompt 2. Differences in the proportions of correct answers between prompts 1 and 2 were not observed throughout the study period . HoweverP=.36; On comparing the percentages of correct answers for each subject area among all questions included in the analysis, the average percentage of correct answers for all areas was 65.9%  lack of up-to-date data (ChatGPT only studied records until 2021) and (2) increased question complexity. Although GPT-3.5 learned data only up to 2021, it is crucial to highlight that ChatGPT is able to answer first-time questions; in other words, it is not simply filling in holes using existing internet sources, as there was no sharp decrease in scores in the 2022 and 2023 examinations. The fact that the situation-setup questions were also answered correctly without significant difference indicates that ChatGPT seemed to do well on the questions dealing with the human mind, such as those involving conversations with patients. Meanwhile, the possibility of losing track of relevant issues in complex situational settings and of having limitations such as difficulty recognizing certain expressions, including the frequent use of hyphens and other expressions, were also shown. If the current version of ChatGPT were used in nursing practice, it could be difficult to assess patients whose situations are complex, such as those requiring treatment for multiple diseases or those with socioeconomic problems. However, this is likely to depend on the amount of information that ChatGPT can store in its short-term memory, which would be resolved in the future models.This study used all questions from the 2019-2023 National Nursing Licensing Examinations in Japan, and the results were highly reliable for the performance assessment of the ChatGPT’s answers with low variability. However, this study has some limitations. First, questions with figures and tables were excluded. Although GPT-3.5, which was used to measure the performance in this study, was unable to judge figures and tables, Wang et al  reportedIn principle, judgments were made based on the content of choices, and computational questions were judged as being correct if the formula and the results of the calculation were correct.In general, AI using a large language model is known to perform better in English than in other languages , althougIn the future, if additional data in the areas of poor performance are acquired and tuned so that questions and options can be understood appropriately without prompt engineering or supplementary human explanation, it is highly likely that the passing criteria will be exceeded in a stable manner. More advanced tools, such as GPT-4 or Bard (developed by Google), superseding the capabilities of ChatGPT, continue to be released and are expected to be used in many clinical situations such as diagnosis, explanation of treatments and drugs, and communication with patients. However, further research will be needed on ethical issues such as the division of roles between human nurses and AI, decision-making responsibilities, and the risks for patients when applied in clinical practice.ChatGPT passed or performed very close to the passing level on the Japanese National Nurse Examinations. With additional learning, prompt engineering, and tuning of ChatGPT, it will likely exceed the passing criteria. ChatGPT has the potential to assist nurses with decisions based on data regarding the patient’s physical condition, and to provide support for psychological issues.


A novel function for the L1 cell adhesion molecule, which binds the actin adaptor protein Ankyrin was identified in constraining dendritic spine density on pyramidal neurons in the mouse neocortex. In an L1-null mouse mutant increased spine density was observed on apical but not basal dendrites of pyramidal neurons in diverse cortical areas  in the L1 cytoplasmic domain was critical for spine regulation, as demonstrated by increased spine density and altered spine morphology in the prefrontal cortex of a mouse knock-in mutant (L1YH) harboring a tyrosine (Y) to histidine (H) mutation in the FIGQY motif, which disrupted L1-Ankyrin association. This mutation is a known variant in the human L1 syndrome of intellectual disability. L1 was localized by immunofluorescence staining to spine heads and dendrites of cortical pyramidal neurons. L1 coimmunoprecipitated with Ankyrin B (220 kDa isoform) from lysates of wild type but not L1YH forebrain. This study provides insight into the molecular mechanism of spine regulation and underscores the potential for this adhesion molecule to regulate cognitive and other L1-related functions that are abnormal in the L1 syndrome. Dendritic spines on cortical pyramidal neurons receive 80%–90% of excitatory glutamatergic synapses in the neocortex , which is highly conserved among L1 family members . An L1 knock-in mouse harboring the FIGQH mutation displays axon targeting errors , Neuron-glial related CAM (NrCAM), and Neurofascin are transmembrane recognition molecules that perform diverse functions in neural development including axon growth and guidance, neuronal migration, cell survival, and synaptic plasticity  are null mutants. L1-/y and wild type (WT) male littermates were analyzed in this study. L1Y1229H (L1YH) mice, mutated in the Ankyrin binding motif FIGQY  at P10–P13 as described , as normal distributions were not assumed. Spine morphologies are classified as mushroom, stubby, and thin spines based on the relative size of the spine head and neck . The proportion of spine morphological types was similarly analyzed in EGFP-labeled cortical neuron cultures.Adult mice (50–150) were anesthetized with isoflurane, brains were isolated and processed for Golgi impregnation using the FD Rapid Golgi Stain Kit (FD NeuroTechnologies) as described . The Sholl center was defined as the midpoint of the cell body at a soma detector sensitivity of 1.5 μm, and the automatic tracing mode was used to seed and trace dendritic arbors. Images in DAT format were subjected to Sholl analysis using Neurolucida Explorer with a starting radius of 10 μm and radius increments of 10 μm. Two factor ANOVA was used to assess significant differences in the mean number of crossings at each distance from the soma with significance set at RRID:AB_300798, host chicken), L1  and MAP2  for 2 h at 4°C. AlexaFluor-conjugated secondary antibodies  were incubated with sections for 2 h at room temperature. Sections were mounted on Superfrost Plus slides with Prolong Glass mountant, cured for 48–72 h, imaged confocally, and deconvolved using AutoQuant 3 software (Media Cybernetics). Microscopy was performed in the UNC Microscopy Services Laboratory .Nex1Cre-ERT2: RCE mice were induced with tamoxifen at P10-P13 and brains were harvested at P20. After transcardial perfusion with 4% PFA, fixed brains were isolated, sectioned, permeabilized, and blocked in 10% normal donkey serum containing 0.3% Triton X-100 in PBS. Sections were stained with antibodies directed against GFP  were precleared for 30 min at 4°C using Protein A/G Sepharose beads. Precleared lysates  were incubated with 2.5 μg nonimmune IgG (NIgG) or L1 monoclonal antibody 2C2 (Abcam #ab24345) together with 1.25 μg L1 monoclonal antibody 5G3 (BD/Pharmingen #554273) for 2 h on ice. Protein A/G Sepharose beads were added for 30 min before washing with RIPA buffer. Samples were subjected to SDS-PAGE (6%) and transferred to nitrocellulose. Membranes were blocked in TBST containing 5% nonfat dried milk and incubated overnight with monoclonal antibodies  against Ankyrin B , washed, and incubated with HRP-secondary antibodies  for 1 h. Blots were developed using Western Bright ECL Substrate (Advansta) and exposed to film for times yielding a linear response of the signal.For preparation of mouse cortical lysates, forebrains (P30) were homogenized in RIPA buffer and centrifuged at 16,000× post-hoc testing (p < 0.05).Cortical neuron cultures were prepared from the forebrains of WT and L1YH embryos at E15.5 as described  in means between independent controls and experimental subjects as described  to discriminate significant differences  due to its importance in cognitive functions Yizhar, . In addiPyramidal neurons in layer 2/3 of the PFC were further analyzed to determine if loss of L1 affected spine morphology or dendritic arborization. Dendritic protrusions acquire different morphologies classified as mushroom, stubby, and thin spines based on the relative size of the spine, head, and neck  of L1-/y mice compared to WT.To determine if the Ankyrin binding site on L1 was required for dendritic spine regulation we focused on layer 2/3 pyramidal neurons in the PFC of L1YH knock-in mice, in which a histidine substitution for tyrosine at position 1229 causes deficiency in binding the actin-spectrin scaffold protein Ankyrin  or L1-/y PFC . Tamoxifen exposure of these mice at P10–P13 induces the expression of EGFP in postmitotic, postmigratory pyramidal neurons, enabling visualization of spines, as well as dendrites, axons, and soma as described  as described . In L1YH neuronal cultures Sema3B-Fc and Sema3F-Fc also induced a significant degree of spine retraction compared to Fc-treated L1YH neurons . Spine densities of L1YH neurons treated with Sema3B-Fc (*p > 0.99) or Sema3F-Fc (*p = 0.91) were not significantly different from similarly treated WT neurons. Spine morphology of WT and L1YH cortical neurons was quantified in Fc-treated cultures to determine if mutation of the L1 Ankyrin binding motif altered the proportion of spine types in vitro. There were no significant differences in the fraction of mushroom, stubby, or thin spines relative to total spines in cultured WT neurons . The relative proportion of spine types in vitro differed somewhat from that in vivo, where mature mushroom spines were more predominant. Cultures contain a diversity of neuronal types that may be altered in their state of differentiation and lack factors present in vivo, which likely influence spine morphology. In summary, these results supported the interpretation that Sema3B- and Sema3F-induced spine retraction is not substantially mediated by L1 interactions at FIGQY in vitro. It should be noted that L1-null (L1-/y) neuronal cultures were not assayed, because breeding requires mating WT males with heterozygous L1 females, yielding litters with a low percentage of L1-/y male embryos.To investigate whether L1-FIGQY interactions were required for Sema3B- or Sema3F-mediated spine retraction, cortical neuron cultures were prepared from forebrains of WT and L1YH embryos (E15.5) and cultured for 14 days Here we show in L1-null mice that the neural cell adhesion molecule L1 constrains dendritic spine density in pyramidal neurons in diverse areas  of the cerebral cortex. This novel function for L1 was restricted to apical dendrites of cortical pyramidal neurons. The Ankyrin binding motif in the L1 cytoplasmic domain (FIGQY) was required for constraining spine density as demonstrated by increased spine density in layer 2/3 pyramidal neurons of the PFC in the L1 mouse mutant harboring a tyrosine to histidine substitution (FIGQH) in the motif. This mutation impaired L1-Ankyrin binding and is a known variant associated with the human L1 syndrome of intellectual disability  in its Ig1 domain necessary for Sema3F-induced spine retraction (Mohan et al., L1 is known to be expressed at the highest levels during postnatal stages in the mouse cortex and to decline with maturation (Demyanenko et al., In conclusion, the present study extends the role of L1 family members in dendritic spine regulation to the prototype of the family, L1. The increased spine density on apical dendrites due to L1 deficiency or perturbation of its Ankyrin binding site may alter excitatory/inhibitory balance in cortical circuits and affect overall behavior. The phenotypes observed in the L1 mouse genetic models studied here may also shed light on the molecular basis of cognitive and other L1-related functions that are abnormal in the L1 syndrome.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The animal study was reviewed and approved and all mice were handled according to the University of North Carolina Institutional Animal Care and Use Committee policies in accordance with NIH guidelines.KM, SW, JS, VM, BD, EZ, and YP propagated L1YH mouse strains, performed experiments, and analyzed data. DL and MS supervised breeding of L1-/y mice, performed Golgi staining on brain sections of L1-/y mice, and provided advice on experimental design. PM supervised research, analyzed data, and wrote the article. All authors contributed to the article and approved the submitted version.


Heat stress poses a significant challenge to egg production in layer hens. High temperatures can disrupt the physiological functions of these birds, leading to reduced egg production and lower egg quality. This study evaluated the microclimate of laying hen houses using different management systems to determine the impact of heat stress on productivity and hen health. The results showed that the ALPS system, which manages the hen feeding environment, effectively improved productivity and decreased the daily death rate. In the traditional layer house, the daily death rate decreased by 0.045%, ranging from 0.086% to 0.041%, while the daily production rate increased by 3.51%, ranging from 69.73% to 73.24%. On the other hand, in a water-pad layer house, the daily death rate decreased by 0.033%, ranging from 0.082% to 0.049%, while the daily production rate increased by 21.3%, ranging from 70.8% to 92.1%. The simplified hen model helped design the indoor microclimate of commercial layer houses. The average difference in the model was about 4.4%. The study also demonstrated that fan models lowered the house’s average temperature and reduced the impact of heat stress on hen health and egg production. Findings indicate the need to control the humidity of inlet air to regulate temperature and humidity, and suggest that Model 3 is an energy-saving and intelligent solution for small-scale agriculture. The humidity of the inlet air affects the temperature experienced by the hens. The THI drops to the alert zone (70–75) when humidity is below 70%. In subtropical regions, we consider it necessary to control the humidity of the inlet air. Heat stress is a significant issue that affects egg production in layer hens. When layer hens are exposed to high temperatures, their physiological functions are disrupted, which can decrease egg production and quality. This, in turn, results in significant economic losses to poultry farmers. Several factors can affect the quality of eggs, and heat stress is just one of them. Previous studies have shown that the health conditions of layer hens, the thermal environment they are in, the microbial biomass of the breeding environment, and the type of cages they are housed in can all impact egg quality. The thermal environment is a crucial factor that affects the overall health and productivity of poultry. Poultry animals are highly sensitive to changes in temperature and humidity levels, and the thermal environment can impact various aspects of their physiology and behavior. Previous studies have shown that the quality of eggs is affected by multiple factors, such as the health conditions of hens, the thermal environment, the microbial biomass of the breeding environment, and the type of cage. Among them, the thermal environment is the key main factor ,2,3,4. IDue to economic considerations, the traditional battery cage is the primary type of cage used in Taiwan. However, the high temperature can cause heat stress, which can negatively affect the hens and result in poor egg production . Animal Previous studies have highlighted the importance of an appropriate air temperature, humidity, air velocity, and air quality for optimal hen performance. When ambient temperatures are too high or low, a mechanical ventilation system with cooling and heating capabilities is necessary. Moreover, the ventilation system should be adjusted according to the season. The water-pad ventilation system, also known as the tunnel ventilation system, utilizes mechanical ventilation to regulate the indoor temperature of the layer house in response to seasonal changes ,13,14. TTo develop a water-pad system suitable for small-scale agriculture in Taiwan, this study utilized computational fluid dynamics (CFD) to investigate the impact of fan mode on indoor climate control in the water-pad system. A newly constructed layer house at a university experimental farm was chosen as the site for examining the influence of fan mode on indoor temperature distribution. Additionally, we selected two commercial laying hen houses and integrated our self-designed intelligent system into their management system to examine the effect of the intelligent system on the management mode of small-scale agriculture.We developed an Artificial Intelligence Layer Production System (ALPS) to manage farms and installed it in commercial laying houses in Taiwan for intervention. ALPS helps us collect information on the indoor climate in layer houses to validate our numerical computations.Our investigation focused on the effect of humidity and three different fan-operation modes on temperature distribution in a layer house at a university in Taichung, Taiwan. We conducted CFD validation, analyzed the flow of three fan arrangement models, and calculated the Temperature–Humidity Index (THI).First, we built a CFD model based on information collected on-site. We then used the CFD model to calculate the results for the three fan modes. Finally, we compared the temperature distributions of the three models and calculated the THI, taking into account the humidity, to estimate the actual temperature experienced by the hens. In this study, we developed a preliminary version of an Artificial Intelligence Layer Production System (ALPS), as depicted in The diagram in 2, temperature, and humidity sensors  was installed in the house to monitor the environment.TM, DFW Airport, TX, USA) was implanted into the subcutaneous tissue of the nape of 36 hens, and a scanner read the temperature measured by the chip. The temperature recording period was from July 2021 to December 2021. In total, 46 pieces of temperature data were obtained for each hen. The measurement results are shown in The microchip Bio-ThermTM  was used to analyze the flow in space. Equation (1) is the equation of mass conservation.Generally speaking, the airflow in the spaces is incompressible flow. Therefore, the density of the air in the spaces is constant. Therefore, we modify Equation (1) to (2).Equation (3) is the steady-state momentum equation called the Navier–Stokes equation.Equation (4) is the steady-state equation of energy conservation.Twp = 23 °C. The temperature Tlayer was set to 40 °C (the body-surface temperature around the layer) according to the measurement of 36 layers. The velocity of the fans, v, was set to 2, 4, and 6 m/s. Other boundaries, such as the walls, roofs, etc., in the house were set as wall boundaries, and the wall temperature Twall = 25 °C.Boundary conditions were set to solve the Equations (2)–(4) in the study. The boundary conditions were set as follows. The water-pad system at the front of the layer house was set to Twp) to 23 °C.Water pad inlet: Set the front-end water curtain of the hen house as a pressure outlet boundary condition during the simulation, and set the outlet temperature (Tlayer is set to 40 °C (layer surface temperature).Layers: Set the hen flock as a heat source, so that the heat dissipation of the layers mainly comes from the surface of the layer and the heat generated by respiration. The specific heat dissipation value is affected by the environmental temperature and the physical condition of the layer. The temperature Tf set to 30 °C.Fan: The fan outlet is a velocity outlet with temperature Twall to 25 °C.Other boundaries: Set the walls and roof inside the house as wall boundaries, and ignore the evaporation of water vapor on the floor and water tank inside the house. Set the wall temperature This study used commercial software Ansys Fluent R19  and SolidWorks Flow Simulation  as the CFD tools. For this study, the flow model was set to a three-dimensional, fully turbulent, and incompressible flow model under the assumptions of RANS (Reynolds-averaged Navier–Stokes equations). The CFD tools use a semi-implicit method to handle the pressure-linked equations in the velocity-pressure coupling algorithm, and employ a second-order upwind discretization. In the turbulence model, the study used the standard k-ε turbulence model to model the turbulence characteristics of the fluid. The standard k-ε model is the most commonly used model for simulating turbulence in computational fluid dynamics , and is x = 5 m, 50 m, and 95 m, respectively. A total of 15 monitoring points were used to obtain wind speed data as independent references for each grid size. The grid independence results showed that for grid sizes above 13,835,801, the values of the 15 monitoring points reached a convergence criterion of 10−3. The grid independence was shown in The mesh of the model was shown in The temperature–humidity index (THI) was used to assess the impact of environmental conditions on the thermoregulatory mechanisms of animals ,20,21. model of the tunnel structure and conducted field measurements. We took this a step further in order to improve heat build-up and adjusted the fan modes. The results showed that using only the lower exhaust fan (Model 3) was an effective fan mode in this study. However, our findings indicated that water-pad systems might not effectively reduce the thermal stress on animals in areas with high humidity. When the outdoor humidity exceeds 70%, the indoor temperature–humidity index (THI) falls into a dangerous zone. The higher the humidity, the less heat that can be dissipated through evaporation, resulting in a higher perceived temperature for the animals. This is particularly relevant in Taiwan, a country that has a subtropical climate with high temperatures and high humidity; this significantly impacts the heat stress on animals subjected to indoor breeding methods using water-pad structures.The novelty of this study is the use of computational fluid dynamics (CFD) to investigate the impact of the fan mode on indoor climate control in the water-pad system. This represents a new approach to studying the indoor climate of the water-pad system and provides insights into how to optimize the system for improved temperature distribution.We came to the following conclusions for indoor microclimate control in layer houses:The CFD of the simplified model can quickly obtain the assessment of indoor climate control. We verified the calculation results of the CFD model through on-site measurements, and the average error was controlled at 4.4%. We verified that the results show that the hen shape can be simplified to obtain results close to the actual measurement.The temperature in the rear was higher than in the front, an inherent problem in designing the tunnel-type water-pad system layer house. Because the temperature of the hens in the front was transferred to the rear due to the airflow, the hens in the back were exposed to a higher temperature than those in the front. In addition, the air in the rear could not be discharged quickly for the first time due to the fan unit’s resistance, so the rear temperature was in a high-temperature state. According to the results of this study, the length of the layer house was controlled within 30 m, and the Model 3 design was used to control the temperature difference between the front and rear to 0.4 °C.The apparent temperature of the hens was increased due to increased inlet humidity. The THI was located in a dangerous zone when the inlet air humidity was over 80%. When the humidity is below 70%, THI drops to the alert zone. The humidity of the inlet air affected the temperature distribution throughout the house. In subtropical regions, we considered it necessary to control the humidity of the inlet air. This way, the house’s temperature and humidity could be controlled entirely.This study analyzed a layer house with a water-pad system with three fan modes. Based on the conditions of Taiwan’s average relative humidity, which is about 60–80%, and the needs of small-scale agriculture, the research results suggest that Model 3 should be used as the operation mode of layer houses with a water-pad system in small-scale agriculture. It provides small-scale agriculture in Taiwan, or areas with a similar climate to Taiwan’s, with a convenient and usable layer house with a water-pad system that considers the need for energy-saving and intelligent agriculture. It provides solutions for small-scale farming in terms of industrial upgrade and reduced energy consumption in the era of global warming and climate change.A limitation of this study is that the humidity control design has yet to be implemented in commercial layer houses. The main reason for this is that farm operators are concerned about affecting current egg production. Furthermore, we know that increasing wind speed can help reduce perceived temperature. However, the effect of wind speed on the animal’s perceived temperature cannot be directly obtained from the animal’s response, which is research that needs to be continued in the future.


In the present work, the thermal conductivity and thermal expansion coefficients of a new morphology of Ni/graphene composites are studied by molecular dynamics. The matrix of the considered composite is crumpled graphene, which is composed of crumpled graphene flakes of 2–4 nm size connected by van der Waals force. Pores of the crumpled graphene matrix were filled with small Ni nanoparticles. Three composite structures with different sizes of Ni nanoparticles  were considered. The thermal conductivity of Ni/graphene composite was associated with the formation of a crumpled graphene structure (with a high density of wrinkles) during the composite fabrication and with the formation of a contact boundary between the Ni and graphene network. It was found that, the greater the Ni content in the composite, the higher the thermal conductivity. For example, at 300 K,  Nowadays, the demand for new materials with unique mechanical and physical properties is constantly growing. The search for graphene/metal composites is a new and actively developing branch of composite technologies ,2,3,4. TTo date, the mechanical properties of metal matrix composites reinforced by graphene have been widely studied ,13,14,15 mA cm−2 . These nIn addition to high strength, graphene shows unique thermal properties—these include high thermal conductivity and a negative thermal expansion coefficient . It has been experimentally shown that the thermal conductivity of single-layer graphene at room temperature is in the range from 4840 W/(K m) to 5300 W/(K m) ,25,26. TA negative TEC for graphene has been observed in the temperature range from 200 to 400 K ,35,36. TThus, together with the improvement of mechanical properties, graphene reinforcement results in the improvement of thermal properties. The study of such properties of graphene/metal composites is of high importance for the fabrication of new electronic devices such as chemical sensors, energy storage, catalysis, hydrogen storage, batteries, and supercapacitors ,4,40. ThOne of the interesting graphene/metal composite morphologies is a graphene network filled with metal nanoparticles. Such graphene networks, also called graphene aerogel/fiber/crumpled graphene or 3D graphene, exhibit very different morphologies with random porous structures and a pore size from tens to hundreds of nm . For somSuch metals as Cu, Ni, Ag, and Au are of great interest from this point of view, and, in the present work, Ni was chosen as the filler for crumpled graphene. It was shown that Ni nanoparticles grow densely and uniformly inside graphene aerogel . For exaA promising method for estimating the phonon thermal conductivity of such composites is nonequilibrium molecular dynamics (MD). There are several algorithms for calculating the thermal conductivity using the LAMMPS  ,55 packaIn the presented work, the thermal conductivity and TEC of graphene/Ni composites were studied in a wide temperature range by MD simulation. The methodology for the calculation of thermal properties of graphene/metal composites was developed and tested on the graphene/Ni composite. The proposed methodology will further allow for calculations of the thermal properties of such complex structures as graphene networks filled with other atomic clusters. Both metallic fillers and other elements  can be added to the graphene network. This methodology can be applied to calculate the thermal conductivity of crumpled graphene itself, since this structure is proposed for new supercapacitors ,4,40, whIn x-, y-, and z-axis to obtain the three-dimensional (3D) composite precursor, which was then exposed to 300 K temperatures for 20 ps to obtain a more realistic structure with crumpled graphene flakes. After that, hydrostatic compression at temperatures of 300 K was applied to remove empty space from the structure. It should be noted that, during this step, new covalent bonds are not formed between neighboring GFs, since higher temperatures are required , [010], and [001] crystallographic directions, respectively. The lattice parameter was 3.518 Å. The number of Ni atoms was 13,500 and 54,000 atoms, respectively. The equilibration and thermalization of Ni samples were carried out under the same conditions as for the composite.Note that, in this work, the results obtained for a composite were compared with that for pure, single-crystal Ni. Thus, pure Ni fcc crystals with the sizes of 51 × 51 × 51 Å and 205 × 51 × 51 Å were considered. The x-, y-, and z-axis. For each state, three to five measurements of the TCC and TEC values were carried out. The standard deviation of these values ranged from about 5–7% at each temperature. To visualize the structure, the molecular graphics program in VMD  was used, which allows for the display and analysis of structural changes at certain simulation stages 26], the The thermal properties of Ni/graphene composites, where crumpled graphene is a matrix and metal nanoparticles are fillers, were studied by molecular dynamics. It has been shown that the coefficients of thermal expansion and the thermal conductivity of composites strongly depend on the Ni content.The thermal conductivities of the Ni/graphene composites were much lower than for pure Ni, but much higher than for crumpled graphene. The formation of a strongly deformed structure of crumpled graphene during its fabrication resulted in the formation of wrinkles and ripples, which significantly decreased the thermal conductivity in comparison with graphene. An increase in the amount of Ni atoms in the composite resulted in an increase in thermal conductivity due to the addition of an element with higher thermal conductivity. The formation of new contact boundaries between the nickel and carbon phases also had a strong effect on the thermal conductivity of the composite. The thermal conductivity of the Ni/graphene composite can be controlled by the changing Ni content.T > 300 K, it was considerably increased. The higher the Ni content in the composite, the higher the TEC value. At low temperatures, the thermal expansion was defined by the contribution of nickel nanoparticles, while, at high temperatures, the graphene network affected thermal expansion more.The addition of nickel to the crumpled graphene led to an increase in thermal expansion. The TEC value of the CG-8 at.% Ni composite  at a low temperature was closer to the TEC of crumpled graphene, while, at The obtained results, in combination with previously published data on the improvement of the mechanical properties of Ni/graphene composites ,108,109,This work is just the start of the complex study of the physical and mechanical properties of graphene-based composites, which can be used for new electronics and protective coatings. In the future, we plan on considering the thermal properties of Me/graphene composites with metals with a higher thermal conductivity and TEC value than Ni nanoparticles, such as Cu and Al. This will expand the understanding of the thermal properties evolution of Me/graphene composites with metal fillers. This morphology is very new, and additional research is required for a better understanding of their future potential.


Therefore, the primary aim of this study was to assess the changes of lumbar angles, evaluated in a supine position using magnetic resonance imaging (MRI), on a mattress Twenty healthy subjects , aged 32.3 ± 6.5 (mean ± standard deviation), with body mass index 22.4 ± 2.9, completed three evaluations: (i) spine MRI in supine position on a mattress (MAT); (ii) spine MRI in supine position on rigid surface (CON); and (iii) biplanar radiographic imaging in standing position. The following indexes were calculated for both MAT and CON: lumbar lordosis angles L1–L5, L1–S1, L5–S1, and the sacral slope (SS). Further, pelvic incidence (PI) was calculated from the biplanar radiographic images.Main findings were (i) L1–L5 and SS were greater in MAT than CON ; (ii) L5–S1 was lower in MAT than CON (−1.6°); (iii) L1–S1 was greater in MAT than CON only for male subjects (+2.0°); (iv) significant and positive correlations between PI and L1–L5, L1–S1 and SS were observed in both CON and MAT.The use of a mattress determined small but statistically significant changes in lumbar angles.The use of a mattress determines small but statistically significant changes in radiological angles describing the sagittal alignment of the lumbar spine when lying in the supine position.• Lordosis angle L1–L5 was greater in MAT than in CON condition (+2.9°).• Sacral slope was greater in MAT than in CON condition (+2.0°).• Lordosis angle L5–S1 was lower in MAT than in CON condition (−1.6°). Humans  quality , 3.The National Sleep Foundation, USA, highlighted that 93% of people recognize a comfortable mattress as an important instrument being able to get quality sleep . Thus, tThe mechanical characteristics of the mattress are crucial for sleep quality and body comfort. If the mattress is too soft, the mechanical support to musculotendinous structures may be lower leading to higher tension to posterior soft tissue elements, while the intervertebral discs will be under tension at the anterior side. Conversely, if the mattress is too firm, the lumbar section of the spine will not smoothen immediately when lying down . Since sDifferent methods have been described in literature to evaluate bed comfort, such as spine shape reconstruction , 11, eleTo the best of our knowledge, no previous studies examined the changes in spine angles in healthy adults while lying down on surfaces with different rigidities in a supine position. Therefore, the primary aim of this study was to assess the changes of the L1–L5, L1–S1, L5–S1, and sacral slope (SS) angles, evaluated by MRI, between two conditions: (1) in a supine position on a mattress; (2) in a supine position on hard surface. The second aim was to analyze the correlation between the spine angles evaluated in a standing position, using the EOS system, and L1–L5, L1–S1, L5–S1, and SS angles in both supine conditions (mattress and control). We hypothesized to detect significant changes in spine angles between mattress and control conditions.This observational, cross-sectional, pilot study was approved by the Ethics Committee of San Raffaele Hospital (Ref. 158/INT/2020). All procedures were performed in compliance with current national and international laws and regulations governing the use of human subjects (Declaration of Helsinki). The study protocol was registered at clinicaltrials.gov (Ref. No. NCT04638374). All subjects received clear explanation of purpose, methods, potential risks, and benefits of the study, and before the beginning of the experimental procedures, written informed consent was obtained from all participants. The study was conducted at the IRCCS Istituto Ortopedico Galeazzi , in accordance with the STROBE guidelines  for crose.g., scoliosis); contraindications to MRI . Therefore, healthy subjects aged between 18 and 45 years old who met the inclusion criteria were included in the study. All subjects completed the following clinical evaluations to assess the spine angles: (i) spine MRI in supine position on a mattress (MAT); (ii) spine MRI in supine position on hard surface, as control condition (CON); (iii) EOS imaging .Subjects were invited to participate in the study at the Radiology Service of IRCCS Istituto Ortopedico Galeazzi, Milan, Italy. Exclusion criteria were recent fracture; surgery within the past 12 months; history of low back pain in the previous 12 months; spinal disorder including degenerative disease and deformities . Height was rounded to the nearest 1 cm and body mass to the nearest 0.5 kg. Body mass index (BMI) was calculated using the standard formula (weight in kilograms divided by height in meters squared).The mattress used in this study was a medium firm mattress composed of a single layer of polyurethane . The mattress size was adapted for the 1.5-T MRI scanner ; in detail: 50 cm width and 190 cm length. Total mattress thickness was 22 cm. The rigid surface utilized in this study was the standard MRI scanning bed. Figure Standard T2-weighted sagittal images of the lumbar spine were performed with turbo spin-echo sequences for the assessment of the radiological angles . The following indexes were calculated for each subject on the midsagittal slice in both MAT and CON: (i) lumbar lordosis angle (L1–L5) ; (ii) L1–S1 angle; (iii) SS, i.e., the angle between the horizontal line and the superior endplate of the sacrum; and (iv) pelvic incidence (PI), i.e., the angle between the line perpendicular to the sacral plate and the line connecting the midpoint of the sacral plate to the midpoint of the femoral heads.Digitized images of the thoracolumbar spine and pelvis were performed with the EOS Imaging System , which simultaneously acquires images in coronal and sagittal planes, with subjects in standing position. No further calibration procedures were required. The following indexes were calculated for each subject, again using the IDS7 system measurement tools: (i) L1-L5 angle; (ii) L1Statistical analysis was performed using GraphPad Prism version 6.00 .t test.The normality of the distribution of the anthropometric and demographic variables , both for the entire study sample and for male and female subjects separately, was checked using graphical methods and the Shapiro–Wilk test. Since all variables were normally distributed, the baseline differences between genders were checked with unpaired Student’s Four different investigators  manually performed all measurements on MRI and EOS images for all subjects and the analysis was repeated two times, a month away, by only one investigator (S.B.). Intra- and inter-rater reliability was tested for each studied outcome in MAT and CON. For the inter-rater reliability, intraclass correlation coefficient (ICC) estimates and their 95% confidence intervals (CIs) were calculated based on a single rating, consistency, 2-way mixed-effects model. Further, for the intra-rater reliability, ICC estimates, and their 95% CIs were calculated based on a single rating, absolute agreement, 2-way mixed-effects model. As previously described , values –L5 and SS, evaluated by the EOS system, for females. Differences between MAT and CON were tested through paired Student’s t test; further, delta values (MAT minus CON) were calculated for males and females and were compared using unpaired Student’s t tests or with the Mann–Whitney rank test for non-normally distributed variables. Significance was set at p < 0.05. Effect sizes (ES) were used to determine the magnitude of the effect for all significant outcomes of pairwise comparison using Cohen’s d and considered to be either trivial (< 0.20), small (0.21–0.60), moderate (0.61–1.20), large (1.21–2.00), or very large (2.00) [The normality of the distribution of each MRI and EOS measurements, both for the entire study sample and for male and female subjects separately, was checked using graphical methods and the Shapiro–Wilk test. All variables were normally distributed with the exception for L1 small 0.–0.60, mo–L5, L1–S1, L5–S1, and SS in MAT or CON  was tested by the means of the Pearson correlation coefficient. Correlations were considered significant when r > 0.25 and p < 0.05.The existence of a correlation between PI, as evaluated by the EOS system, and L1–L4 level as diagnosed by an expert radiologist (L.M.S.). Data on age, height, body mass, BMI, and spine angles evaluated in a standing position by the EOS are presented in Table Twenty-one subjects met the inclusion criteria and were included in the study . One male subject was dropped a posteriori because of the presence of spondylolisthesis at L3Table –L5 was greater in MAT than CON (+2.9°) with the only exception for female subjects whereas L1-S1 angle was +2.0° greater in MAT than CON only for males . SS was always significantly greater in MAT than CON (+2.0°) and, conversely, L5–S1 was lower in MAT than CON only for the entire sample . Further, no significant differences were observed in the comparisons of delta values between male and female subjects .Table –L5, L1–S1, L5–S1, and SS in CON, MAT, and associated delta values. Significant and positive correlations between PI and L1–L5, L1–S1, and SS were observed both in CON  and MAT  whereas no significant correlations between PI and L5–S1, both in MAT and CON, and between PI and delta values were detected.Multipanel Fig. versus a rigid surface. Further, we also evaluated possible correlations of lumbar angles between a standing and a supine condition (both in MAT and CON).The lack of sleep negatively impact an individual’s cognitive and physical performances, mood, quality of life, social interaction and can lead to a decreased work productivity and increased injury risk too , 22, 23.–L5 and SS were greater in MAT than CON; (ii) L5–S1 was lower in MAT than CON; (iii) L1–S1 was greater in MAT than CON only for male subjects; iv) significant and positive correlations between PI and L1–L5, L1–S1, and SS were observed. Our initial hypotheses were only partially confirmed. The mattress used in the study was composed of a single layer of polyurethane and is typically considered a medium firm mattress. In the comparison between CON and MAT, significant differences were observed for three variables  but not for the L1–S1 angle. Namely, L1–L5 and SS increased when participants were on the mattress, indicating that the mattress significantly influenced some of the lumbar angles, ultimately contributing to an increased sense of comfort. The underlying mechanisms explaining these differences are likely related to effect of mattress firmness on muscular stiffness and pressure distribution when lying supine [i.e., L1–L5) increased whereas lumbosacral lordosis  decreased when subjects were laying down on the mattress compared to the control condition. Further, L1–L5 angle, but not L5–S1, positively correlated with PI in standing position for both MAT and CON, with no significant differences between the two conditions. This result is in line with previous studies showing that the proximal lumbar lordosis has a stronger correlation with PI than distal lumbar lordosis, both in standing and supine positions [The main findings of this study were (i) L1g supine . Noteworositions , 30.–L5, L1–S1, and SS angles in MAT whereas female subjects registered a significant increase only in SS. These results are only partially in line with previous studies showing that range of motion of spine segments during different motion tasks is significantly greater for females than for males [–L3, L3–L4, and L4–L5 compared to males. Legaye et al. [–L5 values than women (61.4° ± 10.2° versus 58.1° ± 10.8°) while Bailey et al. [versus 53.0° ± 1.4°). Korovessis et al. [versus 43° ± 12°). Conversely, the lordosis angle was not significantly different in a supine position . On the contrary, our data suggest no significant differences between sex in L1–L5 angle , L1–S1 angle , SS , and PI .For what concerns gender differences, male participants showed a significant increase in L1or males . Furtheror males  assessede et al.  reportedy et al.  evaluates et al.  found th–S1 in CON, SS in CON, L1–S1 in EOS). Second, a validated assessment with MRI was used in the study so the high quality of the data makes the results reliable and repeatable. In addition, x-ray-based images were acquired with the EOS imaging system, which allows for low-dose biplanar images obtained in standing position. The full trunk, femoral heads, and the pelvis were included in the images with the advantage of having a non-conical projection that is typical of standard x-ray studies. PI, L1–L5, L1–S1 and SS indexes were therefore measured in a highly reproducible way [–L5, L1–S1, SS, but not with L5–S1, measured in both CON and MAT.One strength of this study is that both intra- and inter-rater reliability were tested for each outcome in MAT and CON. In detail, the intra-rater ICC was classified as excellent (> 0.9) for each variable, while the inter-rater ICC was classified as good (0.75–0.9) in seven variables and excellent in three variables ; however, the participants were recruited on the basis of specific inclusion and exclusion criteria, therefore the study sample was very homogenous, and the gender distribution was equal . Additionally, we evaluated how the spinal alignment changed in the very short term, but we don't know how the column behaves may have changed after a full night of sleep. It is likely that the muscles relax with subsequent greater change in spinal angles. Further studies are warranted to evaluate this aspect.Some limitations need to be acknowledged. First, only one type of mattress was used in the study and the patients were evaluated only in the supine position; the results could change by using a mattress with different characteristics  or with the patient laying in a prone position. Second, the sample size is small in the study , the changes of lumbar angles comparing rigid surface and mattress. We observed that the use of a mattress determines small but statistically significant changes in radiological angles describing the sagittal alignment of the lumbar spine when lying in the supine position. Our results do not have a direct clinical impact but they could represent the basis of future studies for the improvement of comfort and sleep quality. Authors also highlight the importance to design short- and long-term randomized controlled trials aiming to study spine alignment, sleep quality, and pain using different sleeping surfaces with various levels of firmness.In conclusion, this is the first study describing, with an accurate protocol and high-quality evaluations (