Due to the high cost and short term of passive immunization against the respiratory syncytial virus, the main virus causing acute viral bronchiolitis, predicting epidemic regions and epidemic months is extremely important. The objective of this study is to identify both the month when the seasonal peak begins and Brazilian regions and states with the highest incidence of monthly hospitalizations due acute viral bronchiolitis.Based on data obtained from DATASUS, monthly hospitalization rates due acute viral bronchiolitis were calculated for every 10,000 live births to children under 12 months of age in all Brazilian states and the Federal District between 2000 and 2019. Seasonal autoregressive integrated moving average models were estimated to forecast monthly hospitalization rates in 2020.A higher incidence of hospitalizations was found for male children, especially under six months of age. As for Brazilian regions, between 2000 and 2019, the South region registered the highest incidence of hospitalizations, followed by the Southeast, Midwest, North and Northeast regions, in this order. Considering the seasonal peak, the period between March and July 2020 comprised the highest expected hospitalization rates.Palivizumab is suggested to be started between February/March and June/July for most Brazilian states, with the exception of Rio Grande do Sul, which, in addition to presenting the highest rates of hospitalizations for acute viral bronchiolitis per 10,000 live births, has the longest seasonal peak between May and September. AVB can also be caused by Mycoplasma pneumoniae, rhinovirus, Chlamydia pneumoniae, human metapneumovirus and coronavirus viruses. In Brazil, RSV is considered the main cause of mortality of children under 12 months of age, being responsible for 60 to 75% of all cases of AVB.Acute viral bronchiolitis (AVB) is the result of inflammation and obstruction of the bronchioles, caused by respiratory viruses. Among viruses that cause AVB, the most common are: respiratory syncytial virus (RSV), adenovirus (ADV), influenza virus types A and B and parainfluenza virus (PIV) types 1, 2 and 3. considering that these groups present with prolonged illness and greater risk of death. Other risk factors include early weaning, exposure to cigarette smoke, age below six months, school-aged sibling living in the same household, socioeconomic status, being black and male.AVB has a low mortality rate (<1%), although it can be higher (30%) in high-risk groups of children such as premature infants younger than 32 weeks and/or with bronchopulmonary dysplasia, children under two years with congenital heart disease and/or immunocompromised, malnourished, In this sense, periods with low temperatures contribute to the higher incidence of AVB cases related to RSV infections. Studies report the occurrence of a seasonal peak of RSV infection in the city of Rio de Janeiro from March to May, in the city of São Paulo from April/May to July/August and, in Salvador, between May and July. The seasonal peak of RSV infections in Porto Alegre occurs from June to September. Despite knowing the seasonal behavior of BVA in some Brazilian capitals, we lack a mapping covering the entire national territory, with the purpose of indicating AVB seasonal peak periods in each state.Incidence of viral infections may vary depending on local climatic conditions. AVB, in particular, is a seasonal disease that coincides with epidemics of secondary infections and viral respiratory pathogens. The incubation period can last five days, and viral infection varies according to disease intensity and the child’s immune status. In addition to preventive measures to avoid contagion, there is a drug for the prevention of AVB, Palivizumab. It is not a vaccine, but an immunoglobulin—an antibody—that induces specific passive immunization against RSV. Immunization with Palivizumab is made in monthly intramuscular doses of 15 mg/kg/dose during epidemic months. Usually, this medication is administered in five doses during the five months of highest incidence of the virus, as it lasts for 30 days. The effectiveness of passive immunization is 1/200, reducing hospitalization rates for AVB by 55%.RSV transmission takes place through eye or nasal contact with contaminated secretions, and the virus can be recovered after more than one hour on infected surfaces. Therefore, it is essential to carry out studies that identify the period of the year in which the highest incidence of AVB cases occurs in each Brazilian state and/or region, with the purpose of indicating the best month to start medication according to local characteristics, enabling an efficient funding in the fight against AVB.Due to the high costs, Palivizumab is restricted to greater risk cases.That being said, this study aims to identify the AVB seasonal period by analyzing a time series of hospitalization rates between 2000 and 2019 in each Brazilian state and the Federal District, in order to suggest the best month to start immunization with Palivizumab. Classic time series models were adjusted and used to predict monthly rates of hospitalizations for AVB in 2020.This time series consists of children younger than 12 months, born between January 2000 and December 2019 and diagnosed with AVB, distributed across 26 Brazilian states and the Federal District. The number of hospitalizations for AVB and the number of live births between 2000 and 2019 were obtained from the website of the Department of Informatics of the Unified Health System in Brazil (DATASUS). Then, the monthly rates of hospitalizations for AVB per 10,000 children were calculated based on the ratio between monthly hospitalizations for AVB and annual live births. Rates were multiplied by 10,000 to facilitate interpretation of results. Data on AVB hospitalization were accessed with the International Classification of Diseases, 10th revision — ICD J21.0, J21.8 and J21.9, which comprise AVB caused by RSV, other specified microorganisms, and unspecified cases. was conducted to compare incidences of hospitalizations for AVB in different Brazilian regions.To assess the significance of the monotonous linear trend of hospitalizations for AVB from 2000 to 2019 in different Brazilian regions, the Cox-Stuart trend test was applied. In addition, the incidence (in %) of hospitalizations for AVB between 2000 and 2019 was calculated for each Brazilian region based on the ratio between total hospitalizations for AVB and total live births in the region. The result was multiplied by 100 to obtain the percentage incidence. Thus, considering the incidences (in %), two-proportion comparison tests were used to verify the possible influence of children’s biological sex on the rates of hospitalizations for AVB in each region. Furthermore, the test of comparison of multiple proportions by the method of Benjamini and YekutieliThen, the time series modeling process based on the Autoregressive and Moving Average (ARMA) class models and their extensions was carried out on the series of hospitalization rates by AVB in the 26 Brazilian states and the Federal District, to predict the behavior of the series for the months of 2020. ARMA models are a cautious alternative to describe time series involving a minimum number of parameters.The ARMA class models, proposed by Box and Jenkins (1970), are capable of modeling stationary time series, that is, series that do not show behavioral patterns over time and develop randomly around a constant average. The difference of ARIMA models is the “integration” of a step in which successive differences occur in the series of interest, transforming it in a stationary series. In addition to a graphic analysis of the time series, it allows to identify the behavior of an increasing or decreasing linear trend through hypothesis tests such as the Cox-Stuart test .In general, real time series present in non-stationarity form, such as a trend. In order to model a trended time series, an extension of the ARMA model class is used, the Autoregressive Integrated Moving Average (ARIMA) models. In addition to simple successive differences intended to remove a trend, these also create seasonal differences in order to remove seasonality and obtain stationarity. The process of modeling time series with models of the ARMA class and its extensions occurs firstly by identifying a SARIMA model for each location, following the usual procedure by Box and Jenkins (1970).Another form of recurrent non-stationarity in real series is seasonality. To model time series with seasonality, another extension of the ARMA model class is used: the Seasonal Autoregressive Integrated Moving Average (SARIMA) models. The next step is to estimate the parameters by the maximum likelihood method, since the εtεt are normally distributed. The model selection was aided by information such as Akaike Information Criterion (AIC) and Bayesian Information Criterion (BIC), likelihood ratio test, lower error variance and parsimony. To verify the adequacy of the model, the Ljung-Box tests were used to detect autocorrelation and the Jarque-Bera tests to verify the normality of residuals. Finally, a prediction was made. This study took place as recommended by Resolution 466/2012 of the National Health Council. The project was approved by the Research Ethics Committee of Universidade Estadual de Maringá (Opinion 739.422/2014), and the Free and Informed Consent Term was not used, since data were obtained from secondary sources. The analysis was performed using R language. In preparing data, the packages read.dbc, dplyr, data.table, Hmisc and tidyr were used; For the adjustment and forecasting of the series, the forecast package was used; maps were designed through XML packages, RCurl, maptools, RColorBrewer, maptools and Maps.The number of simple differentiations d and seasonal differentiations D (s=12) is selected to obtain a stationary series. Once the series become stationary, the sample autocorrelation (SAC) and partial sample autocorrelation (PSAC) functions are used to specify the order p of the polynomial of autoregressive terms and the order q of the polynomial of terms of moving-averages, necessary for the adequate representation of the time series being modeled.The study cohort comprised 615,657 children under 12 months of age diagnosed with AVB. Of these, 367,580 (60%) were males and 248,077 (40%) were females. In addition, 449,395 (73%) children were younger than six months and, 98,442 (16%) being boys and 67,820 (11%) being girls. On the other hand, 166,262 (27%) children were older than six months, of which 269,138 (44%) were boys and 180,257 (29%) were girls. The children diagnosed with AVB were distributed according to the five regions of Brazil. Of the total, 51,524 (8%) lived in the North Region, 107,237 (18%) in the Northeast Region, 288,715 (47%) in the Southeast Region, 125,331 (20%) in the South Region and 41,954 (7%) in the Midwest Region.After categorizing the interval between 2000 and 2019 into four periods , we could see the increase in hospitalizations for AVB over the periods, both in terms of percentage and of estimated annual trends, which were statistically significant (p<0.0001) in all Brazilian regions, as shown in  method (p<0.0001), noting that the South region has the highest incidence, followed by the Southeast, Midwest, North and Northeast regions.Using the two-proportion comparison test to examine the incidence of AVB hospitalizations according to sex, a statistically significant difference was found for all regions (p<0.0001), with a higher incidence among males. According to In identifying the model to be adjusted, given the general presence of seasonal effect, seasonal differentiation was adopted for the 27 series of monthly rates of hospitalizations by AVB, transforming them into stationary series and providing access to their autocorrelation in the SAC and PSAC. After identifying a model for each series, the parameters were estimated, the suitability of the model was verified and the behavior of the time series was predicted for the months of 2020.The adjustment and forecast of SARIMA models in the series of the 26 Brazilian States and the Federal District are shown in The number that follows the horizontal dotted line, in blue and also located on the left side of each graph, refers to the average monthly rates of hospitalizations for AVB between January 2000 and December 2019.The monthly rates of hospitalizations for AVB per 10,000 live births in Brazil for 2020 are shown in The expected AVB hospitalization rates for 2020 were categorized into seven hierarchical intervals: up to 5, >5 and ≤10, >10 and ≤20, >20 and ≤30, >30 and ≤40, >40 and ≤ 50 and finally >50. The intervals are represented in The darkest scales are concentrated between March and July 2020, like in In the South Region, the State of Rio Grande do Sul stood out with the highest rates of hospitalizations for AVB predicted for 2020. Its seasonal peak had the longest duration among all States, with high rates from May to October 2020, during fall, winter and part of the spring. Its rates range from 35.46 to 68.82 hospitalizations for AVB per 10,000 live births. In the Northeast Region, the State of Alagoas had the lowest rates predicted for 2020, reaching a maximum of 3.74 hospitalizations per 10,000 live births. Furthermore, the states of Amazonas and Pernambuco showed predicted rates of hospitalizations for AVB lower than 22 hospitalizations per 10,000 live births.This study verified the increase in incidence of hospitalizations for AVB over 2000 through 2019 in all Brazilian regions. The South Region had the highest rates of hospitalizations for AVB predicted for 2020. The seasonal peak of hospitalizations expected for 2020 in the State of Rio Grande do Sul comprised the fall, winter and part of the spring, with rates ranging from 35.46 to 68.82 hospitalizations per 10,000 live births.In all Brazilian regions, male children aged less than six months had a higher incidence of hospitalizations for AVB. Studies show that boys develop respiratory tract infections more often than girls, with the exception of sinusitis, otitis externa, and probably tonsillitis. Anatomy and behavioral/socioeconomic characteristics may explain this difference between genders. There is evidence that the peripheral airways are disproportionately narrower during the first few years of life in boys, which may predispose them to lower respiratory tract infections. There is also a lack of coverage of all hospitalizations in the country, as the data stored in the Hospital Information System refer to hospitalizations funded by the Unified Health System in public or private hospitals. Another limitation stems from the possible underreporting of hospital admissions, which depend on each federative unit.It is worth mentioning that this research is naturally subject to some limitations, since it works with secondary data. One of them is the non-immediate updating and possible incompleteness of the data available on the DATASUS platform.In France, hospital records from 2009 were analyzed in search of cases of hospitalizations for AVB of children under one year of age and factors associated with death. Cases were described based on the variables: age, sex, underlying conditions , length of hospital stay, recurrent hospitalizations, intensive care unit (ICU) admission, and use of assisted ventilation. The hospitalization rate was 35.8 per 1,000 children under one year of age in 2009. Approximately 10% of hospitalized children required treatment in ICU.In Brazil, an analysis of the seasonality of hospitalization rates for bronchiolitis and pneumonia resulting from RSV between 2005 and 2012 suggests the beginning of Palivizumab prophylaxis in January for the North, Midwest and Southeast regions, in February for the Northeast region, and in March for the South Region. In the South Region, the peak of RSV infections occurs later, between June and July, concomitant to the influenza season. In this study, coincidentally, the forecast for the peak of hospitalization rates for AVB in Brazil in 2020 was between March and July, starting with fall, in March, and covering part of the winter in the months of June and July.Studies addressing the prevalence and circulation of RSV in children with acute respiratory diseases in different states point to a greater circulation of the virus from April to May in the Southeast, North, Northeast and Midwest regions.It is important to consider that the public data available on DATASUS is delayed by up to two years. Thus, the use of 2020 data is not suitable because they are partial and still being entered into the system. Even if they were available, it would be necessary to develop an interrupted series analysis or intervention analysis, since an external event (pandemic) influenced the occurrences and records of the outcome. After two years of the pandemic, with daycare centers and schools open again and the relaxation of social isolation, the previous pattern of RSV tends to establish itself again over time. Anticipating occurrences is of great importance, especially for public management. The present study is not limited to a forecast for 2020, but indicates the historical pattern of regions and periods/months of the year at greatest risk. If a period/month was identified, it means the pattern has become evident over the years. The most critical months are different in each region—another reason for attention and more accurate decision-making, enabling optimization and cost reduction with the administration of the monoclonal antibody and with hospitalizations.An epidemiological surveillance system to monitor RSV activity is of utmost importance for the implementation and maintenance of passive immunization programs with monoclonal antibodies in risk groups. Since there is a well-defined pattern of viral circulation, knowing local epidemiological data, considering regional climatic characteristics and paying attention to eventual changes over the years and the emergence of new pathogens, leads to better management of RSV prevention programs.


Hypertrophic scar (HS) is an abnormal fibrous hyperplasia of the skin caused by excessive tissue repair in response to skin burns and trauma, which restricts physical function and impairs patients’ quality of life. Numerous studies have shown that pressure garment therapy (PGT) is an effective treatment for preventing hypertrophic scars. Herein, we found that mechanical stress stimulates the neuropilin 1 (NRP1) expression through screening GSE165027, GSE137210, and GSE120194 from Gene Expression Omnibus (GEO) database and bioinformatics analysis. We verified this stimulation in the human hypertrophic scar, pressure culture cell model, and rat tail-scar model. Mechanical compression increased LATS1 and pYAP enrichment, thus repressing the expression of YAP. Functionally, the knockdown of NRP1 promoted the expression of LATS1, thus decreasing the expression of YAP and inhibiting endothelial cell proliferation. Furthermore, co-immunoprecipitation analysis confirmed that NRP1 binds to YAP, and mechanical compression disrupted this binding, which resulted in the promotion of YAP relocation to nuclear. In conclusion, our results indicated that NRP1 transduces mechanical force inhibition by inhibiting YAP expression. Mechanical pressure can release YAP bound to NRP1, which explains the phenomenon that mechanical stress increases YAP in the nucleus. Strategies targeting NRP1 may promote compression therapy with optimal and comfortable pressures. Hypertrophic scar (HS) is a skin abnormal fibrous hyperplasia response to skin burns and trauma, restricting physical function and impairing patients’ quality of life . DespiteRestricting scar blood flow is considered an essential mechanism of PGT . It reveSeveral studies have focused on the use of mechanical sensors that transmit mechanobiological influences, such as biological sense systems, responding signals, and control of their mechanical surroundings . MechanoNeuropilin 1 (NRP1) is a cell surface transmembrane protein that is expressed in many cell types and acts as a co-receptor for mechanical stimulation , 11. A rYes-associated protein (YAP) senses physical properties such as mechanical forces . YAP regIn this study, we identified a mechanically sensitive NRP1 positively associated with mechanical force stimulation in vivo and in vitro. We investigated the regulatory role of NRP1 in YAP expression through control of the YAP upstream regulator LATS1. We uncovered that mechanical stress disrupts YAP binding to NRP1, resulting in YAP transportation into the nucleus under mechanical force stimulation. This study demonstrated a novel NRP1 regulatory model of YAP that transduces mechanobiological functions, which might enhance the effects of compression therapy with optimal and comfortable pressure. Our findings provide new insights into the treatment of hypertrophic scars.Several human cell types are sensitive to mechanical stress and can translate mechanical signals into biochemical signals due to specific gene mutations and modifications . Therefo2 = 0.95) as the soft thresholding parameter to ensure that the network was scale-free. Based on the topological overlap, 15 modules were identified by hierarchical clustering.  and dark green modules   and constructed a co-expression module using weighted gene co-expression network analysis (WGCNA). We selected power of β = 18  Fig. . NRP1 waNext, we verified this result in other GEO datasets. Differentially expressed genes (DEGs) were identified for the upregulated genes to determine the changes in cells under mechanical stress in GSE137210 (glioblastoma cells) and GSE120194 (hepatocellular cells) datasets Fig. . The intPGT inhibits HS formation by decreasing blood flow  however,NRP1 is a sensory co-receptor of intercellular forces  and sheaTo determine whether NRP1 transduces mechanical forces in HDMECs, we used NRP1-shRNA to knockdown NRP1 in HDMECs and evaluated proliferation and tube formation in the control and NRP1-deficient cells. NRP1 knockdown was confirmed using western blotting Fig. . ImmunosA novel and reliable rat tail model with biological features similar to those of human HSs has been developed and used for morphological and molecular studies , 19. To Our immunostaining results showed that compression therapy significantly reduced YAP expression and promoted LATS1 expression in both human hypertrophic scars and rat tail scars tissues Fig. . These fSince we observed that mechanical forces significantly inhibited YAP expression, we had to investigate the relationship between NRP1, YAP and LATS1. Immunoblotting analysis revealed a concurrent increase of LATS1, MST1, Salvador family WW domain-containing protein 1 (SAV1), and Mps one binder 1 (MOB1) expression, while a reduction in YAP expression under 30 mmHg compression Fig. .LATS1-dependent signaling is widely recognized as a regulator of YAP inhibition and cytoplasmic localization . HoweverAdditionally, we evaluated the effects of mechanical compression on the upstream YAP regulators at various mechanical strengths (0 mmHg and 30 mmHg) matching the pressure of clinical PGT  for 24 hPressure clothing therapy (PGT) is the current standard for preventing and treating post-burn scars . It workNRP1 is a mechanoreceptor on the endothelial cell membrane that can regulate atherosclerotic plaque formation by sensing shear stress resulting from blood flow , 11. It The Flexercell FX-5000 compression system is a recognized cell pressure culture system for biomechanical effects studies . In thisTo explore the downstream mechanism that NRP1 mediates mechanobiological effect, we analyzed the effects of the inhibition of NRP1 on YAP and Hippo signaling pathways. It is widely believed that LATS1 is the upstream regulator of YAP and enhances YAP phosphorylation to inhibit its transportation to the nucleus in response to mechanical signals . MeanwhiThe current view implicates mechanical force as a direct activator of the YAP nuclear localization . Since YIn conclusion, we identified a gene, NRP1, that is related to response to mechanical signals, using online databases, clinical hypertrophic scar specimens, pressure-cultured HDMECs models, and pressure-inhibited rat tail-scar models. Mechanosensitive transmembrane NRP1 mediates the biological effects of mechanical stress through the LATS1/YAP signaling axis. NRP1 mediates the interference of mechanical stress on YAP, but mechanical stress is another essential inhibitor of YAP expression. Therefore, targeting NRP1 may promote compression therapy with optimal and comfortable pressures. Our findings can help improve the curative effect of PGT and the quality of life for patients with HS.This study was approved by the institutional review board of the First Affiliated Hospital of Sun Yat-sen University, China. This study observed seven patients with pathological scars who required surgery. Four of the patients were male and had been scalded by hot water. Their ages ranged from 5 to 11 years old, and the time since their injuries ranged from one year to nine years. The hyperplastic scars were located on various parts of their bodies, including the right forearm, left upper limb, right upper limb, and left thigh. All four male patients had been treated with PGT for at least nine months. Before surgery, the Vancouver Scar Scale (VSS) scores were 12, 5, 6, and 10. In addition to these male patients, there was also a seven-year-old girl with contracture scar tissue on her right foot from a hot water burn six years prior (VSS: 10). The other two cases of hyperplastic scar tissue came from a 12-year-old boy who had suffered from hot water burns on his face and neck (VSS: 11) for over ten years. A 4-year-old girl with hot water burns on her neck (VSS: 8) for three years.Representative FFPE sections were prepared for epitopes available for antibody binding, including dehydration, deparaffinization, and antigen retrieval, after which endogenous peroxidases were quenched before antibody staining. The sections were fixed in 4% paraformaldehyde for 30 min, washed thrice with PBS, and incubated with 0.01% (v/v) Triton X-100 in 5% bovine serum albumin (BSA) at 37 °C for 30 min. Slides were incubated with anti-rabbit NRP1  at 4 °C overnight. The SignalStain® Boost Detection Reagent  was equilibrated to 37 °C for 60 min. The antibody solution was removed and the sections were washed thrice with TBST for 5 min each. Subsequent experimental steps were performed in the dark. One-hundred microliter Opal 570 Reagent  was added per slide and incubated for 10 min at 37 °C in a humidified chamber. The slides were then washed thrice with TBST (5 min per wash).Subsequently, the antigen unmasking step was repeated for stripping. Using a microwave, the slides were heated in 10 mM sodium citrate buffer (PH 6.0) at sub-boiling temperature for 10 min and then cooled to 37 °C for 30 min. The slides were then incubated with anti-LATS1 , anti-rabbit YAP (D8H1X) , and anti-rabbit CD31 antibodies  overnight at 4 °C. The SignalStain® Boost Detection Reagent  was equilibrated, sections were washed, 100 µl Opal 520 Reagent  was applied, and then washed thrice with TBST. All slides were counterstained with DAPI for 5 min and images were captured using an epifluorescence microscope .6 HDMECs suspension after neutralization. After 6–8 h of incubation, each gel was transferred to a well of a BioPress Compression Plate. The pressure force was set at 0.12 and 0.20 at 1 Hz frequency (static wave). For the control, the gels were transferred but unloaded. HDMECs were isolated from these hydrogels using type I collagenase  at a 1 mg/mL dilution for subsequent experiments.Human dermal microvascular endothelial cells (HDMECs), purchased from ScienCell Research Laboratories Inc. , were cultured in Dulbecco’s modified Eagle’s medium nutrient mixture F-12  supplemented with 10% FBS. For the 3D cell culture hydrogels, various ingredients were mixed in one well of a 24-well cell culture plate. A mixture of 18 µl NaOH (0.1 mg) and 42 µl 10 × PBS was added to 300 µl type I collagen from rat tail tendon  and then mixed with 1 mL DMEM/F-12 with 10% FBS and 2 × 102 for 4 h. Tube formation was captured using an inverted epifluorescence microscope  and analyzed by measuring the total tube length and nodes by ImageJ .In the tube formation assays, cells were seeded in μ-Slide Angiogenesis dish . Briefly, 10 µl Matrigel  was added to each well and incubated for 1 h at 37 °C. Compression-treated or other treated HDMECs were resuspended in 50 µl F12-DMEM with 1% FBS and seeded in the wells at a density of 5000 cells. The slides were incubated at 37 °C in 5% COHuman NRP1 short hairpin RNA (shRNA) lentiviral vectors were purchased from VectorBuilder. The IDs used were VB900072-0600gbe (pLV-shRNA#1-Puro), VB900072-0593myd (pLV-shRNA#2-Puro), and VB900072-0589fxv (pLV-shRNA#3Puro). The sequences of the sense oligonucleotides were CAGCCTTGAATGCACTTATAT (shRNA#1), TAAATGTGGCGATACTATAAA (shRNA#2), and TGTGGATGACATTAGTATTAA (shRNA#3). HDMECs were grown to 30–40% density and co-transfected with polybrene (5 µg/mL) and pNRP1 vectors. Western blotting was used to validate the transfection efficiency of shRNA#3 for subsequent experiments.We followed the stretch-induced rat tail scarring module designed by the Shanghai Ninth People’s Hospital . The AniRat tail pressure garments were designed and made by the Rehabilitation Department of the First Affiliated Hospital of Sun Yat-sen University. We used a densely woven nylon elastane material to fabricate the garments. We applied a FlexiForce Sensor system  to ensure that the pressure applied on the wounds was consistent.P-value for each gene to define the correlation between gene expression and compression time gradients. We selected the top two most significant modules related to mechanical compression according to P < 0.01 and the higher GS value. NRP1 was included in this module.To explore the genetic change in response to mechanical compression, we selected GSE165027, which includes the responses of chondrocytes to mechanical compression. We then constructed a co-expression network using the WGCNA algorithm with the R package , 29. AftP < 0.05. The Venn plot displays the common genes after mechanical compression stimulation in three different human sources.We selected GSE120194 and GSE137210 datasets, which include the prominent features in response to mechanical compression, from the Gene Expression Omnibus (GEO) database. GSE120194 included differentially expressed mechanically responsive mRNAs in human hepatocellular carcinoma cell lines. GSE137210 compared gene expression changes in glioblastoma cells (LN229) treated with 23 Pa compressive solid stress (CSS) or no CSS. The data were normalized via log2 transformation for analysis. Data filtering, original annotation, quality control, normalization, and DEG identification were performed in RStudio (version 1.2.5042) using the GEOquery, limma, stringr, and ggplot2 packages. The thresholds were |logFC | > 1 and GeneMANIA Cytoscape plugin  was usedTotal protein was extracted using radioimmunoprecipitation assay (RIPA) buffer  containing a protease inhibitor cocktail and phosphatase inhibitor cocktail . Briefly, 20 µl of each sample was loaded onto sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The membranes were blocked with 5% BSA for 1 h at 37 °C. Membranes were incubated with the following primary antibodies overnight at 4 °C: NRP1  and the Hippo Signaling Antibody Sampler Kit . Horseradish peroxidase (HRP)-linked secondary antibody was incubated with the membranes. Immobilon Western HRP Substrate  was used to detect protein bands. Its intensity was quantified by densitometry using the ImageJ software .The distribution and localization patterns of NRP1, LATS1, YAP, and Ki67 in HDMECs cultured in 3D cell culture hydrogels were detected using immunofluorescence. The gels were fixed in 4% paraformaldehyde for 30 min, washed thrice with PBS, and incubated with 0.2% (v/v) Triton X-100 in 5% BSA at 37 °C for 1 h. The hydrogels were incubated with anti-rabbit NRP1 , anti-rabbit LATS1 , anti-rabbit YAP (D8H1X) , anti-rabbit Ki67  at 4 °C overnight, washed thrice with PBS, and incubated with Alexa Fluor 555- and 488-conjugated goat anti-rabbit secondary antibodies  at 37 °C for 1 h in the dark. All sections were counterstained with 4′,6-diamidino-2-phenylindole (DAPI)  for 5 min. Images were captured using a Zeiss LSM 880 confocal microscope and processed using LSM image software.P < 0.05 was considered to indicate a significant difference. At least three independent replicates were used for each experiment. The results are expressed as the means ± standard error of mean (SEM).SPSS 22  was used for statistical analyses. Graph the statistical results using GraphPad Prism 8 . A two-tailed Student’s t-test was used for comparisons between two groups. One-way ANOVA was used for comparing multiple groups. Original Data File


Strategies to overcome toxicity and drug resistance caused by chemotherapeutic drugs for targeted therapy against hepatocellular carcinoma (HCC) are urgently needed. Previous studies revealed that high oxidored-nitro domain-containing protein 1(NOR1) expression in HCC was associated with cisplatin (DDP) resistance. Herein, a novel dual-targeting nanocarrier system AR-NADR was generated for the treatment of DDP resistance in HCC. The core of the nanocarrier system is the metal–organic frameworks (MOF) modified with nuclear location sequence (NLS), which loading with DDP and NOR1 shRNA (R). The shell is an A54 peptide inserted into the erythrocyte membrane (AR). Our results show that AR-NADR efficiently internalized by tumor cells due to its specific binding to the A54 receptors that are abundantly expressed on the surface of HCC cells and NLS peptide-mediated nuclear entry. Additionally, DDP is more likely to be released due to the degradation of Ag-MOF in the acidic tumor microenvironment. Moreover, by acting as a vector for gene delivery, AR-NADR effectively inhibits tumor drug resistance by suppressing the expression of NOR1, which induces intracellular DDP accumulation and makes cells sensitive to DDP. Finally, the anti-HCC efficacy and mechanisms of AR-NADR were systematically elucidated by a HepG2/DDP cell model as well as a tumor model. Therefore, AR-NADR constitutes a key strategy to achieve excellent gene silencing and antitumor efficacy, which provides effective gene therapy and precise treatment strategies for cisplatin resistance in HCC. Hepatocellular carcinoma (HCC) is a common malignant tumor characterized by aggressiveness and rapid progression, with high morbidity and mortality . PatientNOR1) gene , first isolated from nasopharyngeal carcinoma (NPC) [P33.4, is specifically expressed in normal tissues such as the testis, placenta, trachea and brain [NOR1 displays moderate to strong expression in HCC [NOR1 is related to advanced clinical stage and predicts poor prognosis of HCC [NOR1 also could activate hepatic stellate cells (HSCs), contributing to liver fibrosis in vitro through the Wnt/β‑catenin signaling pathway [NOR1 gene is associated with resistance to imatinib [NOR1. Knocking down NOR1 expression effectively sensitized DDP-resistant HCC cells to DDP by reducing drug pumping and increasing the accumulation of DDP. Therefore, the NOR1 gene seems to have emerged as a crucial target for sensitizing HCC cells to chemotherapeutics.The oxidored-nitro domain-containing protein 1 (ma (NPC) , and locnd brain , 10. Pren in HCC  and promn in HCC , 13. Eles of HCC . NOR1 al pathway . Moreove pathway . It has imatinib . However+) and L-histidine were used to synthesize a porous Ag-MOF that can effectively load chemotherapeutic drugs (DDP) and NOR1 shRNA (R). To enter the nucleus from outside a cell, nanocarriers require special modifications [The RNA interference (RNAi) technique is considered a promising therapeutic method for cancer treatment because of its ability to effectively silence genes connected with overexpressed cancer markers and to subsequently resensitize cancer cells . Howeverications . The surications . NLS-modications , 38, 39.As an exogenous substance, nanomaterials are easily identified and cleared by the mononuclear phagocytic system, resulting in lower blood circulation times . To solvConsequently, we propose to structure a tumor-targeting, pH-stimulated drug/shRNA codelivery system (AR-NADR) Fig.  that comNOR1 gene was designed by GeneChem (China). Polycarbonate porous membrane syringe filters (200 nm) (BS-PES-22) were obtained from Biosharp (China). Penicillin and streptomycin cocktail (PB180120), fetal bovine serum (FBS) (164210-50) and medium (Dulbecco's Modified Eagle Medium (DMEM)) (PM150210) were obtained from Procell (China). TRIzol reagent (15596018) was purchased from Thermo Fisher Scientific (USA). The Cell Counting Kit-8 (CCK-8) (C6005) was manufactured by NCM Biotech (China). Calcein/PI Cell Viability Assay Kit (C2015S), Annexin V-FITC/PI Apoptosis Assay Kit (C1062M), ATP Detection Kit (S0026) and Lipo8000™ Transfection Reagent (C0533) were purchased from Beyotime Biotechnology (China). The Reactive Oxygen Species (ROS) Assay Kit (AKCE002) and Mitochondrial Membrane Potential (MMP) Assay Kit (AKOP013) were purchased from Boxbio (China). The antibody against NOR1 (HPA028436) was manufactured by Sigma-Aldrich (USA). Goat anti-rabbit IgG H&L (HRP) (511203) was purchased from ZENBIO (China).Silver nitrate (S116266) was purchased from Aladdin (China). Cisplatin (IC0440) and dialysis membrane (2 kDa) (YA1036) were obtained from Solarbio (China). L-histidine (A604351), succinic anhydride (A607835), the MuLV First Strand cDNA Synthesis Kit (B532435) and SYBR Green PCR Mix (B110031) were obtained from Sangon Biotech (China). NLS peptide and A54 peptide were designed by QYAOBIO (China). The shRNA plasmid used to interfere with the 2. We chose six-week-old female nude mice and bought the mice from Hunan SJA Laboratory Animal Co., Ltd. (China).HepG2 cells and HepG2/DDP cells were obtained from the Institute for Advanced Study, Central South University (China). The cells were cultured in DMEM containing 10% FBS and 1% penicillin–streptomycin at 37 °C and 5% CONOR1 in DDP-resistant HCC, the total RNA in HepG2 cells and HepG2/DDP cells was extracted using TRIzol reagent. A NanoDrop 2000  was applied to measure the concentration and purity of the extracted RNA. Sequencing libraries were generated with a Hieff NGS Ultima Dual-mode mRNA Library Prep Kit for Illumina  following the manufacturer’s recommendations. The library quality was assessed on the Agilent Bioanalyzer 2100 system. Subsequently, the samples were sequenced by an Illumina NovaSeq6000. Finally, the raw reads were further processed with a bioinformatics pipeline, namely, the BMKCloud (www.biocloud.net) online platform. Differential expression analysis of the two conditions/groups was performed using DESeq2. Genes with differences in expression levels that exhibited adjusted P values of < 0.05 along with |log2FC|≥ 1.5 were identified as significantly differentially expressed genes. The CCK-8 assay was used to calculate the half-inhibitory concentration (IC50) of HepG2 cells and HepG2/DDP cells before/after transfection with NOR1 shRNA.To measure the expression of 3, 0.1 g) were added to 5 mL of double distilled water (ddH2O). Next, silver nitrate solution was added dropwise to the L-histidine solution. Before the solution changed to milky-white to form Ag-MOF (AM), the solution was placed in a reaction pot at 37 °C and magnetically stirred. Then, the solution was centrifuged at 12,000 rpm for 5 min and washed 3 times with ddH2O. The AM powder was dried in a vacuum freeze dryer for storage.L-histidine (0.4 g) and silver nitrate  was added. The carboxylated Ag-MOF was obtained by centrifugation  after magnetic stirring overnight. Carboxylated Ag-MOF (1 mg) was dissolved in 5 mL of ddH2O, and then 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride , N-hydroxysuccinimide  and NLS peptide  were added and the solution was stirred overnight at 37 °C. Unincorporated EDC, NHS and NLS were filtered by dialysis to obtain NA.Ag-MOF (1 mg) was dissolved in 5 mL of ddH2O, PEI (0.1 g) was added to generate positively charge NA, and 50 µg of shRNA plasmid, which was used to interfere with NOR1 gene expression, was added to react for 2 h with NA/shRNA (NAR). Then, 1 mg of DDP was added, the solution was magnetically stirred at 37 °C in the dark overnight, and NADR was obtained by centrifugation. The absorbances of different concentration gradients of DDP at 301 nm were determined, and the unbound DDP in the supernatant was calculated using standard curves. The calculation formulas for EE and LE were as follows: EE (%) = amounts of DDP loaded on nanocomposite/amounts of DDP added initially × 100. LE (%) = quantity of DDP loaded on the nanocomposite/total quantity of this nanocomposite × 100.NA (1 mg) was dissolved in 1 ml of ddH2 (5 mg), EDC (2.8 mg), and NHS (1.7 mg) were dissolved in PBS (5 ml) and the solution was magnetically stirred at 37 °C for 24 h. The unbound EDC, NHS and A54 peptide were removed using a dialysis bag. Later, DSPE-PEG2000-NH2-A54 (0.1 ml) and the RBC membrane (0.9 ml) were mixed and stirred for 2 h at 37 °C. Eventually, the A54 peptide inserted into the RBC membrane (AR) was synthesized.As mentioned above, the whole blood samples obtained from mice were centrifuged at 3000 rpm for 5 min, and then the supernatant was removed. Finally, the membrane precipitate was washed repeatedly with PBS (3 times) . After wAR was added to an equal volume (0.5 ml) of NADR by ultrasound . The mixture was then extracted repeatedly 20 times using a porous membrane syringe filter (200 nm). The excess AR in the supernatant was removed through centrifugation , and the precipitate obtained was the AR-NADR.Transmission electron microscopy  was used to confirm the size and morphology of Ag-MOF, NLS-Ag-MOF, NADR, AR, and AR-NADR. The zeta potentials of various nanocomposites were detected using a Zetasizer Nano ZS . The X-ray diffraction patterns of Ag-MOF sample are obtained through X-ray diffraction instrument . Fourier-transform infrared spectroscopy (FTIR) was used to study the molecular functional groups of Ag-MOF . UV−vis spectrometry  was used to confirm the construction of AR-NADR. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS‒PAGE) was performed to identify erythrocyte membrane proteins. The gel was stained with Coomassie Blue staining solution (P1300) . Finally, the gel imaging system was used for analysis after decolorization.To determine whether DDP could be more easily released from AR-NADR once it reached the acidic tumor microenvironment (TME), drug release tests were performed. In short, 1 ml of AR-NADR (the DDP concentration was 1 mg/mL) was placed into a dialysis bag (molecular weight cutoff of 2 kDa) and which was immersed in PBS (20 ml) at pH 7.4 and pH 6.5 separately with magnetic stirring at 37 °C. A 1 mL sample of the dialysate was collected from the incubation medium at specific time points , and equal volumes of fresh PBS were placed into the incubation medium. The absorbance of the dialysate was detected at 301 nm (DDP characteristic peak) to measure the concentration of DDP released from the AR-NADR using standard curves.The immune evasion of AR-NADR was assessed in a RAW264.7 cell uptake assay. RAW264.7 cells were incubated with Cy5.5-labeled NADR, R-NADR and AR-NADR (20 µL) for 6 h and later stained with DAPI. Finally, an inverted fluorescence microscope  was used for imaging.3 cells/well) for 24 h. Various concentrations of NADR, R-NADR and AR-NADR (0–100 µg/mL) were added and incubated for 24 h. Then, 10 µL of CCK-8 solution was added to each well and incubated for 2 h, and the absorbance was measured at 450 nm.The cytotoxicity of AR-NADR was determined using a CCK-8 in Human Umbilical Vein Endothelial Cells(HUVECs). In short, the cells were seeded in 96-well plates  = /(absorbance of positive control−absorbance of negative control) × 100.Hemocompatibility was detected with a hemolysis assay. Various concentrations of AR-NADR (0–100 µg/mL) were incubated with 5% red blood cells at 37 °C for 2 h. The supernatant was collected after centrifugation , and a microwell plate detector  was applied to detect the absorbance at 545 nm. The positive and negative controls were ddHThe targeting effect of the A54 peptide on different tumor cells was detected. First, HepG2 cells (A54 + cells) were treated with 20 μL of RBCm@NLS-Ag-MOFs (RBC-NA) or A54-RBCm@NLS-Ag-MOFs (AR-NA) for 12 h. In addition, the A54 peptide competition test was used for further verification. In brief, the A54 peptide (20 μg) was pretreated for 2 h to block the HepG2 cell surface receptors, and then the cells were treated with 20 μL of AR-NA for 12 h [NOR1 gene silencing efficiency was sequentially evaluated. HepG2 cells and HepG2/DDP cells were cultured in 6-well plates (5 × 105 cells/well) for 24 h. Later, HepG2/DDP cells were treated for 48 h with PBS, free shRNA, Lipo8000-NC (Lipo8000-negative control plasmid), Lipo8000-shRNA and AR-NAR (AR-NA/shRNA), in which the polyplexes contained the NOR1 shRNA plasmid at a shRNA concentration of 2.5 µg well−1. The GFP of the plasmid could emit green fluorescence, which was used to evaluate transfection efficiency by an inverted fluorescence microscope . In addition, total RNA was extracted from cultured cells with TRIzol reagent and reverse transcribed into cDNA with a MuLV First Strand cDNA Synthesis Kit. Then, real-time quantitative PCR (RT‒qPCR) was implemented with a SYBR Green PCR Kit, and the fluorescence was measured using a LightCycler 96 . All samples were analyzed in triplicate and normalized to GAPDH levels. The relative fold variation in mRNA expression  was tested on the basis of the 2−ΔΔCt method. The sequences are provided as follows. For HS-GAPDH, the forward primer was CAGGAGGCATTGCTGATGAT; the reverse primer was: GAAGGCTGGGGCTCATTT; For NOR1, the forward primer was: CACTCCTCATCTTCTTCCAA; the reverse primer was: CCTCTTCTTCTTCTTCACCTT. In addition, the mRNA obtained from cells was evaluated by agarose gel electrophoresis . Furthermore, the protein obtained from cells was extracted with RIPA buffer and ultrasound, and the protein concentration was measured with a BCA protein assay kit. Following the Western blotting steps, we detected the expression of NOR1 in the cells with the corresponding antibody. ImageJ software was used for semiquantitative analysis of the agarose gel electrophoresis bands and Western blot bands. To demonstrate that reducing NOR1 expression could sensitize HepG2/DDP cells to DDP, the IC50 was calculated by the CCK-8 assay carried out using samples subjected to the above treatments. Furthermore, the cell efflux of DDP at 4, 6, 12, and 24 h was detected. HepG2 cells, HepG2/DDP cells and HepG2/DDP cells (treated with Lipo8000-shRNA) were seeded in 12-well plates (5 × 104 cells/well) for 24 h. Then, DDP (15 μg/mL) was added. After culturing for 4, 6, 12, and 24 h, the medium supernatant was collected at the indicated time points, and the absorbance was measured. The rate of cell efflux of DDP was calculated with a standard curve.Agarose gel electrophoresis was carried out to estimate NOR1 shRNA loading in NA. Different NA/shRNA weight ratios (w/w) (the weight of shRNA was 5 µg) were applied to an agarose gel  in TAE buffer containing YeaRed Nucleic Acid Gel Stain . A UV transilluminator and a digital imaging system  were applied to obtain images. The 3 cells/well) for 24 h. Then, the cells were exposed to AR-NA, AR-NAR, DDP, AR-NAD, or AR-NADR (the concentration of DDP was 0–40 μg/mL) for 24 h; subsequently, CCK-8 solution (10 µL) was added to each well for incubation at 37 °C for 2 h. The relative cell viability was calculated according to the absorbance at 450 nm. Additionally, the IC50 was calculated in cells treated with DDP, AR-NAD and AR-NADR. Moreover, live/dead cell staining was performed to estimate the antitumor effects of AR-NADR in vitro. HepG2 cells and HepG2/DDP cells were cultured in 6-well plates (5 × 105 cells/well). Next, the cells were exposed to PBS, AR-NA, AR-NAR, DDP, AR-NAD, or AR-NADR (the concentration of DDP was 15 μg/mL) for 6 h. After the medium was removed, calcein-AM and PI were used for live/dead staining, and the cells were observed with an inverted fluorescence microscope .First, the toxicity of AR-NADR was determined by the CCK-8 test. HepG2 cells and HepG2/DDP cells were cultured in 96-well plates (5 × 105 cells/well) for 24 h and later exposed to PBS, AR-NA, AR-NAR, DDP, AR-NAD and AR-NADR (the concentration of DDP was 15 μg/mL) for 12 h. Subsequently, the cells were collected. The JC-1 Assay Kit was used to measure the mitochondrial membrane potential (MMP) of cells detected by flow cytometry . The intracellular adenosine triphosphate (ATP) and reactive oxygen species (ROS) level were detected with ATP quantification kit and ROS assay kit respectively. An Annexin V-FITC apoptosis detection kit was used to evaluate cell apoptosis by flow cytometry to comprehensively reveal the antitumor effect of AR-NADR in vitro.To investigate the mechanism by which AR-NADR overcomes drug resistance, HepG2/DDP cells were inoculated in 6-well plates (5 × 106 cells/100 μL) were subcutaneously used to inoculate nude mice to prepare a DDP-resistant hepatocellular carcinoma model. The tumor volume was measured using the following formula: volume = length × width2/2. The mice were deemed ready for further therapeutic study after the tumor volume reached 100 mm3. To evaluate the target of AR-NADR in vivo, 100 μL each of Cy5.5-NADR, Cy5.5-R-NADR and Cy5.5-AR-NADR  was injected into HepG2/DDP tumor-bearing mice via the tail vein. The fluorescence signals of Cy5.5 from the mice, extracted organs and tumors were further detected with an In Vivo Imaging System  at 6, 24, and 48 h after administration.Six-week-old female nude mice were obtained from Hunan SJA Laboratory Animal Co., Ltd. (China). All the protocols for the proposed in vivo experiments were approved by the Animal Use and Care Committee of Central South University. HepG2/DDP cells (5 × 10HepG2/DDP tumor-bearing mice were randomly divided into six groups (n = 5 per group) and intravenously injected with PBS, AR-NA, AR-NAR, DDP, AR-NAD, or AR-NADR in the tail vein 5 times . The tumor size and body weight of the mice were recorded every second day. At 14 days, all the animals were euthanized, and the major organs , tumors and blood samples were collected for histological analysis (hematoxylin–eosin (H&E) staining) and blood tests. Western blotting and immunohistochemical analyses (IHC) were also performed to measure NOR1 expression in tumor tissues. Apoptosis in tumor tissues was detected via terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) assays based on the standard protocol.p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001).Data are expressed as the mean ± standard deviation (SD) after at least three independent experiments and were assessed by GraphPad Prism software. Intergroup differences were assessed with one-way analysis of variance (ANOVA) and Tukey’s post hoc test.  and HepG2/DDP cells (n = 3). As shown in Fig. NOR1 (OSCP1) gene was significantly increased in the HepG2/DDP groups. After transfection with NOR1 shRNA, the IC50 in HepG2/DDP cells was significantly lower than that in HepG2 cells, indicating that the overexpression of the NOR1 gene was probably related to DDP resistance in HepG2/DDP cells  and NOR1 shRNA (R) to constitute a NADR nanocore, and II) the shell, in which the A54 peptide is inserted into a red blood cell membrane vesicle (AR) and is wrapped around the NADR to construct the whole core–shell structure of AR-NADR.As indicated in Fig. −1 to 1611 cm−1, and C–N stretching vibration was shifted from 1148 cm−1 to 1183 cm−1 and 1144 cm−1. This indicated that Ag+ in AgNO3 reacted with C–N in imidazole ring of histidine to form Ag–N chemical bond, which changed the infrared spectrum absorption peak of histidine. In the UV‒vis spectrum , AR-NADR specifically combines with the A54 peptide receptor expressed in HCC cells. The cellular uptake behavior was analyzed to determine its specific binding capability in various tumor cells. The RBCm@NLS-Ag-MOFs (RBCm-NA) and A54-RBCm@NLS-Ag-MOFs (AR-NA) were labeled with FITC expressing green fluorescence. As shown in Fig. To detect the effect of NLS nuclear targeting, the cellular uptake behavior of AR@Ag-MOFs (AR-AM) and AR@NLS-Ag-MOFs (AR-NA) was investigated in HepG2 cells. Cy5.5 was used to label AR-AM and AR-NA so that they displayed red fluorescence. As revealed in Fig. To investigate the binding capability of NA for NOR1 shRNA, an agarose gel electrophoresis assay was adopted. Different NA/shRNA weight ratios (w/w) were used to prepare NAR, and the movement of shRNA was observed with agarose gel electrophoresis. As shown in Fig. 50 value (25.5 ± 0.5 µg/mL) than in HepG2 cells (IC50 = 16.04 ± 1.18 µg/mL). NOR1 expression in cells was associated with sensitivity to DDP, and inhibition of NOR1 expression reversed drug resistance in HepG2/DDP cells. The IC50 values in the Lipo8000-shRNA and AR-NAR treated groups were 13 ± 0.98 µg/mL and 12.8 ± 1.2 µg/mL, respectively, which were dramatically lower than those in HepG2/DDP cells in the nontreated group. Furthermore, as shown in Fig. 50 of DDP in HepG2/DDP cells and decreased the efflux of DDP. Thus, AR-NADR presumably reverses chemoresistance by suppressing NOR1expression.Furthermore, the in vitro gene transfection efficiency of AR-NADR was evaluated. GFP in the plasmid emitted green fluorescence, which was used to evaluate the transfection efficiency. As shown in Fig. 50 value of DDP in HepG2 cells was observably lower than that in HepG2/DDP cells . Moreover, AR-NAD had a stronger HepG2 cell killing effect than DDP alone, with an IC50 value of 13.17 ± 0.46 µg/mL. Because of the EPR effect as well as the target function of the nanocarrier, the internalization of DDP drugs by cells was higher [50 = 12.8 ± 1.2 µg/mL), and was almost twofold higher than that of DDP alone. This result further revealed that the effective reduction in the IC50 value in the AR-NADR-treated group in HepG2/DDP cells was mainly due to the downregulation of NOR1. Similar to other results (Fig. CCK-8 assays and live/dead cell staining were performed to investigate the cytotoxicity of AR-NADR against HCC cells. Compared with the PBS group, the DDP, AR-NAD and AR-NADR groups had significantly inhibited proliferation of HepG2 cells (Fig. s higher . MoreoveTo thoroughly investigate the mechanism of the anticancer efficacy of AR-NADR against DDP-resistant cells, we sequentially investigated the MMP, ATP, ROS and apoptosis of HepG2/DDP cells. Mitochondrial injury characterized by decreased MMP is one of the hallmark events that occurs during the early stage of apoptosis. The change in MMP after apoptosis induction in cells allows the membrane permeability to be altered . As showThe efficiency of drug accumulation in tumors is a significant element that needs to be considered in drug delivery systems. To investigate the tumor targeting capability of AR-NADR in vivo, its biodistribution in a tumor-bearing mouse model was evaluated. As indicated in Fig. The therapeutic efficacy of AR-NADR in vivo was examined in subcutaneous HepG2/DDP cell tumor-bearing nude mice Fig. A. The tuThe biocompatibility of nanomaterials is attracting increasing attention for addressing medical and biological problems . In thisIn this research, we revealed the potential relationship between NOR1 and DDP-resistant HCC. We successfully constructed a novel nanosystem, namely, AR-NADR, which contained A54-modified RBC membrane vesicles as the shell and an NLS modified Ag-MOF loaded with DDP and NOR1 shRNA as the core for the treatment of cisplatin resistance in HCC. This drug delivery system possesses many advantages, such as double targeting, satisfactory biocompatibility, and release properties in response to weak acidic environmental stimuli, effective gene transfection and synergistic antitumor effects. Consequently, AR-NADR is regarded as a reversal agent providing extraordinary potential for overcoming cisplatin resistance in tumors via multiple mechanisms. AR-NADR not only offers an effective strategy for the clinical application of gene therapy but also unveils a novel approach with applications in the biomedical field.


Rehabilitation post-knee arthroplasty is integral to regaining knee function and ensuring patients’ overall well-being. The debate over the relative effectiveness and safety of outpatient versus home-based rehabilitation persists.A thorough literature review was conducted adhering to Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines across four databases. Two researchers independently identified eligible studies centering on knee arthroplasty patients undergoing either outpatient or home-based rehabilitation. Study quality was assessed using the Cochrane Collaboration’s risk of bias tool, while continuous outcomes were subject to meta-analyses using Stata 17 software.Our analysis identified no significant differences in primary outcomes, including Range of Motion, Western Ontario and McMaster Universities Arthritis Index, Knee Injury and Osteoarthritis Outcome Score, Oxford Knee Score, and the Knee Society Score, between home-based and outpatient rehabilitation across different follow-up points. Adverse reactions, readmission rates, the need for manipulation under anesthesia, reoperation rate, and post-surgery complications were also similar between both groups. Home-based rehabilitation demonstrated cost-effectiveness, resulting in substantial annual savings. Furthermore, quality of life and patient satisfaction were found to be comparable in both rehabilitation methods.Home-based rehabilitation post-knee arthroplasty appears as an effective, safe, and cost-efficient alternative to outpatient rehabilitation. Despite these findings, further multicenter, long-term randomized controlled trials are required to validate these findings and provide robust evidence to inform early rehabilitation choices post-knee arthroplasty. Knee arthroplasty is a renowned surgical procedure for end-stage osteoarthritis (OA) that has proven its worth over time . With glPostoperative rehabilitation plays a pivotal role in fostering adherence to the therapeutic regimen, expediting the recovery of knee function, and heightening patient satisfaction . HoweverThis knowledge gap necessitates a comprehensive investigation into the relative effectiveness and safety of these two prevalent rehabilitation paradigms. Accordingly, our study conducted a systematic review and meta-analysis to delve into this issue. Our goal is to equip healthcare providers, patients, and policymakers with evidence-based insights to facilitate informed decision-making in selecting the optimal early postoperative rehabilitation approach after knee arthroplasty. In the broader context, our findings will contribute to the ongoing discourse on enhancing patient-centric care. By aligning rehabilitation strategies with individual patient preferences, circumstances, and needs, we can ensure more tailored and effective healthcare solutions. These endeavors ultimately aimed to optimize the overall outcomes of knee arthroplasty, significantly improving the quality of life for those affected by end-stage OA.During the systematic review process and subsequent reporting of our results, we maintained adherence to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines . Since tFour electronic databases PubMed, Embase, Web of Science, and Cochrane Library were searched on May 6, 2023 and no time limitation was applied. Vocabulary and syntax were specifically adapted according to the database. The specific search terms of PubMed were:  AND (“Rehabilitation” [Title/Abstract] OR “Rehabilitation” [MeSH Terms]) AND ((“Outpatient” [Title/Abstract] OR “Ambulatory Care” [MeSH Terms]) OR (“Home-Based” [Title/Abstract] OR “Home Care Services” [MeSH Terms])). No language limitation was applied. Reference lists of relevant articles were also screened manually for any additional possible records.Studies included in the systematic review needed to meet the following criteria: (1) The subjects of the study must be patients who have undergone knee arthroplasty; (2) The intervention measures should be: the experimental group undergoes outpatient rehabilitation, while the control group undergoes home-based rehabilitation; (3) Outcome indicators should include range of motion (ROM), Western Ontario and McMaster Universities Arthritis Index (WOMAC), Knee Injury and Osteoarthritis Outcome Score (KOOS), Oxford Knee Score (OKS), and Knee Society Score (KSS); (4) If a study involving the same population is published more than once, select the study with the largest sample size or the newly published study. The subjects of the study must be patients who have undergone total knee arthroplasty. The study did not include patients who had undergone unicompartmental, bilateral, or revision arthroplasty procedures.The exclusion criteria were as follows: (1) Studies that are duplicates or redundant publications; (2) Studies that do not include the required outcome indicators; (3) Studies with data that is unable to be extracted or with incomplete literature; (4) Case reports, commentaries, expert opinion, and narrative reviews.Data extraction was carried out independently by two systematically trained researchers based on the inclusion and exclusion criteria. The information extracted from the literature included basic information , intervention measures, outcome indicators, and measurement time points for each study. When there was no data of interest in the published report, we contacted the investigators of the original study by email to request the unpublished data.The quality of the included studies was assessed by the Cochrane Collaboration’s risk of bias tool . Two revP > 0.05, I2 < 50%), a fixed-effects model will be used for the analysis. If statistical heterogeneity was present , a random-effects model will be applied to combine the effect sizes. If the heterogeneity is too obvious and its source is indeterminable, a descriptive analysis will be carried out instead. Differences will be considered statistically significant at P < 0.05.Meta-analyses were conducted using Stata version 17 . For this study, all the outcome indicators to be combined are continuous variables. As the outcome indicators are measured using the same method and identical units, results will be represented by the mean difference (MD) and 95% confidence intervals (CI). Firstly, heterogeneity tests were performed on the study results. If there is no significant statistical heterogeneity between the studies  such as WOMAC and KOOS, where participants, aware of their group assignments, were their own assessors. This potential for performance bias might have influenced the outcomes of these studies, and hence, the interpretation of the results needs to take this factor into account. For objective measures like ROM, blinding of the outcome assessment is essential and more feasible. For such measures, the risk of performance bias is significantly reduced when the assessor is unaware of the participant group assignments. Moreover, in 21% of the included randomized controlled trials, a high risk of selective reporting bias was observed. This indicated that the possibility of incomplete or selective outcome reporting might have affected the overall results of these studies , and a fixed effect model was used for meta-analysis. There were no statistically significant differences in the changes in knee flexion between the home rehabilitation group and the outpatient rehabilitation group at 1–1.5, 3, 6, 12 months post-surgery ; 3 months: MD = 0.12, 95% CI ; 6 months: MD = 0.16, 95% CI ; 12 months: MD = 0.06, 95% CI  , so a fixed effect model was used for meta-analysis. There were no statistically significant differences in the changes in knee extension between the home rehabilitation group and the outpatient rehabilitation group at 1–1.5, 3, 6, 12 months post-surgery ; 3 months: MD = 0.03, 95% CI ; 6 months: MD = 0.01, 95% CI ; 12 months: MD = 0.02, 95% CI  , so a random effect model was used for meta-analysis. There were no statistically significant differences in the changes in WOMAC between the home rehabilitation group and the outpatient rehabilitation group at 1–1.5, 3, 6, 12 months post-surgery ; 3 months: MD = − 2.68, 95% CI ; 6 months: MD = − 0.26, 95% CI ; 12 months: MD = 1.56, 95% CI .Variation in osteoarthritis index score. Six studies –19, 23 rP = 0.65, I2 = 0%), so a fixed effect model was used for meta-analysis. There were no statistically significant differences in the changes in KOOS between the home rehabilitation group and the outpatient rehabilitation group at 1–1.5, 3, 6, 12 months post-surgery ]; 3 months: MD = 5.61, 95% CI ; 6 months: MD = − 0.16, 95% CI ; 12 months: MD = 0.31, 95% CI .Variation in knee injury and osteoarthritis outcome score. Four studies , 12, 13 P = 0.16, I2 = 36%), so a fixed effect model was used for meta-analysis. There were no statistically significant differences in the changes in OKS between the home rehabilitation group and the outpatient rehabilitation group at 3, 6, 12 months post-surgery ]; 6 months: MD = 0.26, 95% CI ; 12 months: MD = 0.52, 95% CI .Variation in Oxford knee score. Four studies , 20, 21 P = 0.69, I2 = 0%), and a fixed effect model was adopted for the meta-analysis. No statistically significant differences were found in the changes in KSS between the home rehabilitation group and the outpatient rehabilitation group at 1–1.5, 3-, 6-, and 12-month post-surgery.Knee Joint Function Score Variation. Three studies , 19, 23 Five studies , 15, 23 Three studies , 11, 23 Seven studies , 17, 20 The funnel plots constructed with the observed study showed symmetry, and no significant publication bias was detected in funnel plots Fig. .Fig. 5FuOur study, through a meticulous systematic review and meta-analysis, reveals that home-based rehabilitation is on par with outpatient rehabilitation in restoring knee function following knee arthroplasty, thereby corroborating several findings reported in the existing literature , 17, 24.Outpatient rehabilitation provides an environment conducive to specialized recovery for patients, particularly those discharged early with conditions like stroke, cardiac diseases, respiratory system diseases, hip fractures, and geriatric ailments. Studies have shown that outpatient rehabilitation can significantly reduce the risks of mortality and adverse prognoses for patients . AlthougOsteoarthritis (OA) is a primary cause of disability. Total knee arthroplasty (TKA) effectively alleviates pain, corrects deformities, and enhances functionality. Atrophy and voluntary activation (VA) are the primary reasons for quadriceps strength loss, where the impact of VA is nearly double that of muscle atrophy. VA is one of the critical factors for strength loss post-TKA; if a patient’s motor unit cannot achieve its maximal firing rate, a reduction in VA would significantly affect muscle output. However, research on the link between the decline in VA post-TKA and myocardial infarction is still scarce . SeveralAt present, both outpatient and home-based rehabilitation play pivotal roles in the field of rehabilitation. Outpatient rehabilitation offers a professional environment, catering to patients with more intricate needs, while home-based rehabilitation provides patients with more convenient and personalized support. For those who require specialized modalities or equipment, such as thermotherapy or the use of short-crank bicycles, home-based rehabilitation might not be entirely adequate. Within an outpatient setting, the provision of professional equipment and techniques ensures that patients receive appropriate treatments. Indeed, numerous studies have indicated that specific equipment and techniques are indispensable in expediting the recovery process . For insSafety concerns surrounding home-based rehabilitation stem from a lack of professional supervision. However, telemonitoring systems , 13, 15 The rehabilitation journey post-knee arthroplasty is a long-term commitment. Patients’ needs vary based on their educational level, socioeconomic status, and social support system. Outpatient rehabilitation offers specialized care, but its feasibility can be limited for older patients, those with mobility issues, or those with limited access to healthcare resources. Our analysis indicates that home-based rehabilitation is cost-effective, without a compromise on patient quality of life and satisfaction , making However, outcomes can differ based on patient adherence, and the absence of direct supervision might pose risks . ImplemeIn this study, “home-based rehabilitation” encompasses diverse strategies within a home setting, from professional services to unsupervised methods. This broad categorization might introduce clinical heterogeneity affecting our findings. Some reviewed studies reported participants transitioning between or simultaneously initiating home-based and outpatient therapy, which might have impacted treatment distinctions. In future research, we will aim for a more detailed categorization of rehabilitation strategies.Our study has limitations. We only considered English and Chinese literature, risking potential publication bias. A limited number of studies were included without a bias analysis, increasing clinical heterogeneity. Particularly, ROM measurements in the meta-analysis presented high heterogeneity due to unclear active or passive measurements in primary studies. Additionally, the small sample size for safety indicators suggests the need for more extensive studies for validation.In conclusion, our systematic review and meta-analysis provide compelling evidence that home-based rehabilitation following knee arthroplasty provides comparable outcomes to outpatient rehabilitation, with satisfactory safety and patient satisfaction, while also offering potential cost savings. This approach does not compromise patient satisfaction, potentially making it more accessible for a broader patient population. However, it is crucial to recognize that the success of home-based rehabilitation relies heavily on the adherence of patients to the rehabilitation plan, highlighting the importance of rigorous protocols and regular telemonitoring to ensure patient safety. Further multicenter, long-term randomized controlled trials are warranted to confirm these findings and provide robust evidence to guide the selection of early rehabilitation methods post-knee arthroplasty. Given the evolving landscape of healthcare delivery and the increasing emphasis on patient-centered care, our findings have significant implications for the management of patients after knee arthroplasty.Home-based rehabilitation after knee arthroplasty is an effective, safe, and cost-effective alternative to outpatient rehabilitation. These findings, however, need to be confirmed with further multicenter, long-term randomized controlled trials to provide robust evidence to guide the selection of early rehabilitation methods post-knee arthroplasty.


Bell’s palsy is an acute, ipsilateral facial paralysis secondary to inflammation of cranial nerve VII. This condition is classically caused by herpes simplex virus (HSV); however, many providers will make a diagnosis in the setting of other underlying conditions that are known to cause similar symptoms. The annual incidence of Bell’s palsy is 11.5-53.3 per 100,000 persons, with a small subset of individuals being contact sport athletes. A unique challenge to treating Bell’s palsy in collegiate athletes is finding a way for these players to return to their sport in a timely fashion, while also avoiding traumatic ocular injuries. Athletic goggles may provide a potential alternative option for athletes to return to the play of their respective sport prior to the physical symptoms subsiding. Due to the prolonged duration of most Bell's palsy symptoms, athletic goggles have the ability to save up to a full season of eligibility for a player. Aside from ocular injuries, a further challenge which encompasses all cases of Bell’s palsy is the negative psychosocial effects which accompany the physical symptoms of this condition. Both the patient's physical and psychosocial health considerations must be taken into consideration. In this case report, we review the utility of ocular protection in helping collegiate athletes with unilateral facial paralysis return to play prior to the resolution of symptoms. Bell’s palsy, described in the 19th century by Sir Charles Bell, is associated with acute, ipsilateral facial paralysis. The diagnosis of Bell’s palsy is one of exclusion through a detailed patient history, physical examination, and laboratory or imaging studies. Although these symptoms are thought to commonly be due to an underlying herpes simplex virus (HSV) infection, facial paralysis has also been associated with neoplasms, trauma, infections, and iatrogenic causes [The annual incidence of Bell’s palsy ranges from 11.5 to 53.3 per 100,000 persons across multiple populations . SymptomA specific subset of individuals who experience Bell’s palsy are contact sport athletes. In this patient population, Bell’s palsy presents a unique hurdle for sports medicine physicians due to the difficulty associated with safely returning the athlete to their respective sport. The National Collegiate Athletic Association (NCAA) allows only four seasons of eligibility for student athletes who do not complete a red shirt year, meaning that the four-to-six week average duration of symptoms is enough to sideline a player for a significant portion of their athletic career . CurrentAn 18-year-old collegiate men’s soccer player presented to the student health clinic with acute onset left-sided facial paralysis, which began upon awakening that morning. He denied any other neurological symptoms and had no pertinent past medical history.His vital signs were in normal range , and he appeared in no acute distress. His gait and station were normal, however, cranial nerve testing revealed facial droop and an inability to smile or raise his forehead on the left side of his face. Upper and lower facial paralysis on the left side was evident. There was no excessive lacrimation or drooling, and there was no change in hearing or taste. Gross sensation and monofilament test were intact. Deep tendon reflexes were 2+ bilaterally throughout and the finger-to-nose test was intact. Romberg’s sign was negative, and he showed no tremors. Muscle tone and strength were both 5/5 bilaterally.The patient was diagnosed with Bell’s palsy and started on valacyclovir 1 g by mouth once a day for seven days, along with a prednisone taper for management of symptoms. The patient was instructed to return for care if his condition worsened.The following day, the patient was evaluated at the university athletic training facility by an athletic trainer and the team physician. The examination by the team physician found that the patient was able to close his left eye, but only with significant effort. This along with the other facial symptoms supported the diagnosis of Bell’s palsy. Due to the risk of eye injury, the team physician deemed it unsafe for the patient to participate in men’s soccer until he regained the ability to completely close his left eye with ease. Alternatively, however, while the athlete waited for his facial paralysis to resolve, it was suggested that wearing protective eyewear may allow him to return to play without missing time. An eye patch was discussed as a possible solution; however, due to its negative impact on depth perception, a pair of goggles was ultimately decided upon and ordered for the patient. The goggles came with an adjustable elastic strap as well as scratch resistant lenses. While this specific model was not air-tight, it did include foam lining around the circumference of the plastic frame to maximize comfort for the wearer. When the patient tried on the goggles, he felt self-conscious with his appearance and made the decision not to wear them. Consequently, the patient was held from men’s soccer while his symptoms subsided, rather than returning to play immediately while wearing the goggles.Bell’s palsy is described as an acute unilateral paralysis of the face, with 70% of patients experiencing complete paralysis -3. The eWith a lifetime risk of 1 in 60, the median onset of Bell’s palsy is roughly 40 years of age, however, it can be seen in all age groups. Risk factors for Bell’s palsy include pregnancy, preeclampsia, obesity, and hypertension . Bell’s The diagnosis of Bell’s palsy is one of exclusion and although there is no specific time requirement for onset, symptoms begin quickly and typically peak within 72 h . NasolabThe American Academy of Neurology (AAN) recommends treating all Bell’s palsy patients with oral corticosteroids to maximize the probability of complete recovery of the facial nerve. Oral antiviral therapy, although not proven to be beneficial, is also commonly prescribed in combination with corticosteroids due to the high suspicion of HSV infection as an etiology . This paWhile not utilized in this case, an osteopathic manipulative treatment (OMT) is another modality that can be considered in the management of athletes with Bell’s palsy. Considerations to cranial, lymphatic, cervical, and sacral somatic dysfunctions with subsequent osteopathic manipulative corrections have been the focus of several case reports -15. SpecBell’s palsy symptoms typically stabilize after approximately three weeks then gradually improve by two to three months. Although many patients experience a full recovery, some patients see lasting adverse effects. Roughly 30% of those diagnosed are left with some degree of permanent facial asymmetry . This loAn essential precaution in patients with Bell’s palsy, especially athletes, is ocular protection. Due to Bell’s palsy causing an inability to close the affected eye, the risk of traumatic eye injury increases substantially . With neStudies have shown that up to 90% of sports-related ocular injuries could have been prevented with appropriate eye wear, including sport goggles . The NCABell’s palsy is a unique condition that often resolves spontaneously over weeks to months. Oral corticosteroids are well accepted as treatment to decrease the duration of Bell’s palsy symptoms. Antiviral agents, while commonly used, have inconsistent results with large variations in reducing overall recovery time between individual patients. For athletes, specifically at the collegiate level, additional treatment may come with use of athletic goggles for ocular protection. The prolonged recovery period of Bell’s palsy is less than desirable due to sports eligibility being limited to only four years. To decrease the risk of ocular injuries in these athletes while also allowing them to return to their respective sports in a timely fashion, adequate eye protection can be considered. This added protection, although potentially effective, can further contribute to the already negative effects Bell’s palsy has on mental health and quality of life. Our objective with this case report is to bring further attention to the management of Bell’s palsy with respect to ocular injury risk and mental health. We believe that athletic goggles, although somewhat stigmatized by the athletes themselves, provide an alternative option to expedite the athlete's return to play. It is our opinion that protective eyewear should be considered in all cases of Bell's palsy which occur in contact sport athletes. Further studies should be considered to investigate alternative solutions which not only offer adequate eye protection for athletes, but also limits the negative social effects already present with facial asymmetry.


AR leads to a rapid hyperpolarization and a higher excitation threshold due to an increase in membrane Cl− permeability. The synaptic GABAAR is mostly composed of two α(1–3), two β, and one γ subunit with the most abundant configuration α1β2γ2. Recently, antibodies (Abs) against α1, β3, and γ2 subunits of GABAAR were detected in a severe form of autoimmune encephalitis with refractory seizures, status epilepticus, and multifocal brain lesions, affecting gray and white matter. Experimental studies confirmed multiple mechanisms and direct functional effects of GABAAR Abs on neurons with decreased GABAergic synaptic transmission and increased neuronal excitability. The expression of GABAAR on astrocytes is well established. However, extensive studies about the effects of autoimmune GABAAR Abs on astrocytic GABAAR are missing. We hypothesize that GABAAR Abs may lead additionally to blocking astrocytic GABAARs with impaired Ca2+ homeostasis/spreading, astrocytic Cl− imbalance, dysfunction of astrocyte‐mediated gliotransmission  and accumulation of excitatory neurotransmission, all this contributing to seizures, variable clinical/MRI presentations, and severity. The most abundant expressed GABAAR subunits in rodent astrocytes are α1, α2, β1, β3, and γ1 localized in both white and gray matter. Data about GABAAR subunits in human astrocytes are even more limited, comprising α2, β1, and γ1. Overlapping binding of GABAAR Abs to neuronal and astroglial receptors is still possible. In vitro and in vivo animal models can be helpful to test the effects of GABAAR Abs on glia. This is from an epileptological point of view relevant because of the increasing evidence, confirming the glial involvement in the pathogenesis of epilepsy. Taken together, autoimmune disorders are complex and multiple mechanisms including glia could contribute to the pathogenesis of GABAAR encephalitis with seizures.The γ‐aminobutyric acid (GABA) is the main inhibitory transmitter in the central nervous system and GABA receptors mediate the inhibitory synaptic transmission. GABA binding to neuronal GABA AR and GABACR, both ligand‐gated chloride (Cl−) channels, and the G‐protein‐coupled GABABR, inhibiting neuronal activities via reduction of exocytosis/transmitter release, membrane hyperpolarization, and depolarization shunt. The neuronal GABABR is coupled to K+ or Ca2+ channels by second messenger systems including G‐proteins, resulting in the inhibition of presynaptical transmitter release or in hyperpolarization of postsynaptical neurons.AR leads to a rapid hyperpolarization and a higher excitation threshold, for example, an inhibitory postsynaptic potential, due to an increase in membrane Cl− permeability.AR is composed of five subunits which form a heteropentamer from 19 isoforms .AR is mostly composed of two α(1–3), two β, and one γ subunit with the most abundant configuration α1β2γ2. Otherwise, the peri‐ or extrasynaptic receptors consist of α4/α6 with β and δ.ARs.ARs mediating tonic inhibition differ from those mediating phasic inhibition with regard to receptor localization and subunit composition. GABAARs mediating phasic inhibition are synaptically localized and have a low affinity for GABA, resulting in transient, rapidly desensitizing GABAergic conductance. In contrast, tonic inhibition is mediated by extrasynaptic GABAARs, which have a high affinity for GABA, leading to persistent GABAergic conductance.ARs, especially the tonic currents, play a crucial role in vital physiological functions, for example, regulation of neuronal excitability, network oscillations, synaptic plasticity, neurogenesis, neuronal development, information processing, higher motor function, vigilance, and cognition.ARs are the target of different drugs, for example, barbiturates  and benzodiazepines , which have different subunit specificities and bind to distinct sites on the receptor complex, differentially regulating the opening of the Cl− channel.AR pathology are possible. Epilepsy and neuropsychiatric disorders including anxiety disorders, schizophrenia, depression, and substance abuse have been reported to be caused by dysfunctional GABAARs, for example, due to mutations of α1 or β3 subunits.AR have been detected in a severe form of autoimmune encephalitis with refractory seizures and status epilepticus.AR. This raises the question of whether other patients may have Abs to other isoforms of the GABAAR and why only a subset of subunits is immunogenic.AR encephalitis, a rare form of autoimmune encephalitis, occurs at any age and presents generally with characteristic clinical‐radiologic patterns including encephalopathy and refractory seizures (to anticonvulsive drugs) with multifocal brain lesions, affecting both gray and white matter.AR encephalitis is treatable.AR Abs themselves contribute to the brain lesions or other immune mechanisms or cells  are involved. According to the distinctive neuroimaging phenotype of GABAAR encephalitis, a positive correlation between the distribution of brain MRI lesions and gene expression level of β3 subunit–containing GABAAR was identified.AR encephalitis could be distinguished based on the topology/distribution of lesions and comprise the confluent type with bilateral confluent lesions in limbic, frontal and temporal lobes, and spotted type with multiple scattered small‐to‐medium patchy lesions.AR in neuronal cultures.AR Abs on the hippocampal CA1 and CA3 pyramidal neurons with decreased GABAergic synaptic transmission and increased neuronal excitability, contributing to seizure generation in GABAAR autoimmune encephalitis.AR Abs led to a reduced number of spontaneous inhibitory postsynaptic currents (sIPSCs) generated in CA1 pyramidal neurons but did not affect their amplitude.AR Abs decreased the number and amplitude of sIPSCs.AR function on a short time scale, diminishing GABA currents with an increase in network excitability.AR localization away from synapses was found to be triggered by the Abs. Otherwise, Abs against α1γ2 subunits exerted no direct effects on GABAAR function.AR encephalitis, indicating that Abs can mediate their effect through many mechanisms within the same disease.AR encephalitis directly competed with the neurotransmitter, changing the receptor in a resting‐like state, and the second Ab antagonized the benzodiazepine potentiation.AR encephalitis in a human patient.The γ‐aminobutyric acid (GABA) is the main inhibitory transmitter in the central nervous system (CNS) and GABA receptors mediate the inhibitory synaptic transmission.2+ level.2+ elevation in astrocytes can remain within the cell or spread to neighboring astrocytes via gap junctions, contributing to astroglial networking.2+ increase can trigger the release of gliotransmitters such as glutamate, adenosine triphosphate (ATP), taurine, glycine, and D‐serine, which can activate pre‐ and postsynaptic receptors in neurons and regulate development and remodeling of synapses as well as the further synaptic neurotransmission.ARs and GABABRs on astrocytes is well established, for example, in the soma, the synapse‐surrounding processes, and the brain vessel‐contacting endfeets.A subunits in astrocytes are α1, α2, β1, β3, and γ1, which were partly demonstrated in both white and gray matter.AR subunits including α1–5, β1–3, γ1–3, and δ have been identified in cultured primary astrocytes from rodent.ARs has been confirmed electrophysiologically, immunohistochemically, and biochemically in cultured astrocytes, brain slices, acutely isolated hippocampal slices, and membrane fractions of mature rodent brain.ARs were demonstrated to share similar channel properties and pharmacology with the neuronal receptors.ARs were expressed by astrocytes in intact tissue from rat hippocampus and showed similar pharmacological properties as previously described for cultured rat astrocytes, supporting that glial GABAARs are not an artifact of cell culture.ARs, resulting in enhanced GABA responses.AR subunit composition, for example, due to the presence of γ1 subunit in astrocytic receptors instead of the γ2 subunit of most neuronal GABAARs.ARs in protoplasmic and fibrous astrocyte subtypes from rat spinal cord cultures.ARs, whereas protoplasmic astrocytes show positive modulation by DMCM, which was attributed to the specific subunit composition of astrocytic GABAARs as mentioned above.ARs were reduced with in vitro aging and cerebral ischemia in activated astrocytes.ARs were detected in membrane patches from the soma and processes of glial fibrillary acidic protein (GFAP)‐positive astrocytes with radial processes  in the adult dentate gyrus of mice, performing whole‐cell recordings and using bath application of the specific GABAAR agonist muscimol with subsequent reversible blocking of the responses by the GABAAR antagonist bicuculline.ARs were mainly composed of the subunits α2/α4, β1, and γ1/γ3.Astrocytes are the main glia cell population in the CNS, forming complex connections with neurons, blood vessels, and other glial cells, and contributing to the maintenance of normal brain functions. Astrocytes regulate synaptic neurotransmission by being part of the tripartite synapse, which is a functional unit composed of pre‐ and postsynaptic neurons as well as perisynaptic astrocytic processes.AR via binding of extracellular GABA resulted in the opening of Cl− channels in astrocytes from primary cell cultures and rodent hippocampal slices.−‐induced depolarization caused Ca2+ influx from the extracellular space through voltage‐gated Ca2+ channels.AR encephalitis cortical and subcortical changes in brain MRI are described in comparison with other forms of autoimmune encephalitis.AR Abs on astrocytic GABAAR are missing.AR encephalitis showed a neuronal specificity of GABAAR after colocalization of neuronal and glial markers.AR Abs. An astrocyte‐microglia co‐culture model of inflammation was developed by Faustmann et al. (2003), representing physiological and inflammatory conditions in the brain, and allowed to study the effects of different substances.AR Abs on glia in vitro. Of course, the phenotype of glia can be affected by these culture conditions and the relevance of such experiments to better understand human pathology is limited. For this reason, neuropathologic analysis of autoimmune encephalitis cases associated with GABAAR Abs can be used to research underlying mechanisms similar to cases with multiple sclerosis, another autoimmune CNS disease. However, neuropathological studies in the case of GABAAR Abs are so far not available, because this rare disease can be identified early and patients can be treated effectively, biopsies are no longer required, and most patients survive.AR Abs may lead additionally to dysfunction and reduction of astrocytic GABAAR complexes  receptors in the hippocampal dentate gyrus and on presynaptic metabotropic glutamate receptors (mGluRs) in the hippocampal CA3‐CA1 neurons, potentiating excitatory transmission.2+ and glutamate can spread via astrocytic gap junctions, composed of connexin (Cx) hexamers that form functional channels between neighboring cells, contributing to large intercellular networks. Cx43 is one of the predominant gap junction proteins in astrocytes.2+‐dependent glutamate gliotransmission from hyperexcitable astrocytes led to upregulation of excitatory neurotransmission in epileptic hippocampus.AR via GABA with subsequent intracellular Ca2+ increase also regulates the release of ATP and adenosine from astrocytes, modulating neuronal function.− for regulation of the excitation‐inhibition balance was demonstrated, because astrocytic Cl− led to a modulation of neuronal signaling in vivo.ARs expressed in several brain regions including the cortex are often localized around inhibitory synapses with the consequence that GABA‐activating astrocytic GABAARs close to the synaptic cleft may provide Cl− to maintain inhibitory transmission.− (the major inhibitory anion in the CNS) that can be recruited by activation of astrocytic GABAAR, contributing to modulation of neuronal signaling by replenishing Cl− during periods of prolonged neuronal activity. In summary, these findings support that long‐lasting inhibition is depending on astrocytic GABAARs and intracellular astrocytic [Cl−]i because a decrease in either case can contribute to a lower threshold for seizure induction.AR by Abs can lead to dysregulation of Ca2+ homeostasis and spreading via gap junctions, astrocytic Cl− imbalance, dysfunction of astrocyte‐mediated gliotransmission  with the accumulation of excitatory neurotransmission. Abnormal hyperexcitability and hypersynchronous activity in neuronal circuits can trigger neuroinflammation and reactivate more astrocytes and microglia, resulting in gliosis. The reactive astrocytes undergo physicochemical and morphological changes such as hypertrophy, upregulation of many proteins, for example, metabotropic glutamate receptor (mGluR) subtypes 3, 5, and 8, the pro‐inflammatory proteins COX2 and CXCR4, nerve growth factor and its receptors, cytokines including transforming growth factor β (TGFβ), tumor necrosis factor α (TNFα), interleukins (IL) 1, 4, 6, and 10, as well as enzymes, for example, inducible nitric oxide synthase (iNOS), cathepsins D and G.+ with impaired balance and osmolarity could lead to hyperexcitability and seizures. Several findings suggest that both the water channel aquaporin 4 (AQP4) and the inwardly rectifying K+ channel (Kir4.1) are involved in the regulation of K+ and water levels in the brain.+ buffering. As a consequence of impaired astrocytic K+ buffering, the K+ clearance is expected to be attenuated, resulting in a lower seizure threshold and seizure generation.AR on microglia promoted a neuroprotective microglial phenotype.AR can cause microglial activation and additional neurotoxicity. Taken together, these findings highlight the complexity of autoimmune disorders and support the hypothesis that multiple mechanisms including glia could contribute to the pathogenesis of autoimmune encephalitis associated with GABAAR Abs and seizure generation.Interestingly, there is accumulating evidence of functional heterogeneity within the astrocyte population in the CNS.These considerations are from an epileptological point of view interesting because there is increasing evidence, confirming the involvement of glia in the pathogenesis of epilepsy.None of the authors has any conflict of interest to disclose.We confirm that we have read the Journal's position on issues involved in ethical publication and affirm that this report is consistent with those guidelines.