However, AR‐induced neonatal heart regenerative phenomenon is controversial due to the variation of operative details in different laboratories. Here, we provide an optimized AR operation procedure with stable regeneration and high survival rate by achieving heart exposure, normalizing myocardium cut‐offs, and reducing operation duration. We also established a whole‐heart‐slice approach to estimate the myocardial regeneration after the AR operation, which ensures no false‐negative/positive results. The combination of the optimized AR operation and the whole‐heart‐slice analysis provides a stable system to study neonatal heart regeneration and cardiomyocyte proliferation  To avoid false judgment of heart regeneration, we employ a whole‐heart‐slice approach to evaluate the myocardium growth after AR operation. With the optimized AR operation and whole‐heart‐slice approach, researchers can obtain a stable regenerative phenomenon after neonatal cardiac injury.22.1C57BL/6J wildtype (WT) mice were obtained from Vital River Laboratory Animal Technology Co. Ltd. The IL‐6 knockout (KO) (B6.129S2‐Il6tm1Kopf/J) mice were acquired from the Jackson Laboratory. All experiments involving animals were conducted in accordance with the Guide for the Use and Care of Laboratory Animals. All animal protocols were approved by the Institutional Animal Care and Use Committee (IACUC), Fuwai Hospital, Chinese Academy of Medical Sciences.Neonatal 1‐day‐old mice of both sexes were used in our study. The 2.2Mice were killed at 21 days post‐resection (dpr), and the hearts were gently harvested without any other extraction. Dissected hearts were rinsed in sterile PBS until they stopped beating and then fixed in 4% paraformaldehyde at 4°C for 48 hours. Hearts were dehydrated automatically in ethanol and dimethyl benzene after washing by fluent water. Dehydrated heart tissue was embedded in paraffin and sectioned. Whole heart slice was applied afterwards, and each section was mounted on a separate glass slide for further pathological staining.t test was used to show the differences.Masson's trichrome staining was performed on heart section slides following manufacturer's instructions (Sigma‐Aldrich). Images were captured using the Automatic glass scanning system (Zeiss) and were identified myocardium (red) and fibrotic tissue (blue) by at least two researchers. GraphPad Prism software (6.0) was used for statistical analysis and Student's 2.3t test was used for assessing statistical differences between the two groups, whereas comparisons among more than two groups were performed using the analysis of variance. The results with P <.05 were considered statistically significant.All data were expressed as the mean ± standard error of the mean. Student's unpaired 33.1The size of the amputated ventricular apex greatly influences the heart regeneration after AR operation.Approximately, a 1‐cm incision thoracotomy was sufficient for the consequent operation Figure , includiSeveral researchers do not use stereoscope nor exteriorize the heart out of the chest,3.2It has been generally accepted that a cut‐off of 10%‐15% of the heart apex by measuring the weight of the amputated part is proper in the AR model.Due to the inadequate standardizations of the cutting size, some researchers cannot guarantee a stable regenerative phenomenonBesides the size of the resected apex tissue, the cutting angle is also an important factor affecting the capacity of regeneration in the AR model. The optimal angle of amputating the apex was 60‐80° to the long axis (LA) of the heart Figure A. The cuDuring the operation, the performer should remove the heart apex by one cut only, which requires a lot of practice to achieve. Superfluous cuts3.3IL‐6 KO) is harmful for neonatal heart regeneration after AR injury.Measurement of myocardial regeneration after AR operation is essential to evaluate whether the AR model is successfully established. Previous studies appraise neonatal mouse heart regeneration at 21 days after the AR operation in 1‐day‐old mice by pathological staining with few tissue sections.IL‐6 KO mouse hearts at 21 dpr even fibrosis regions were thinner than 100 μm (Figure IL‐6 KO mouse hearts could recover the pumping function at 21 dpr, which was consistent with the whole‐heart‐slice results (Figure To avoid a false judgement of heart regeneration after AR injury, here we established a whole‐heart‐slice approach Figure . We slicm Figure . Our echs Figure .4We provide a stable and feasible operation for neonatal mouse cardiac AR injury model, which includes exteriorization of the heart, standardization the size of the resected cardiac tissue, and specification of cutting angles. A whole‐heart‐slice approach was established to estimate myocardial regeneration after AR injury, which could avoid false‐negative/positive results. The combined application of the optimized AR operation and the whole‐heart‐slice approach would assist researchers to obtain more stable myocardial regenerative response to AR injury in neonatal mice, which might be helpful to reduce the controversy of AR‐induced neonatal heart regeneration.5C57BL/6 mice, which is the most common strain to establish transgenic mice. It is necessary to reappraise the myocardial regenerative ratio when researchers want to perform AR operation following our procedures on the other mouse strains. However, the most procedures we optimized are generally applicable, such as exteriorization of the heart, specification of cutting angles and the whole‐heart‐slice approach.Here, we optimized AR operation on The authors declare that they have no conflicts of interest with the contents of this article.LYD and NY designed the study. LYD collected and analysed the data. FJ and LY performed the histological staining. LYD and FJ drafted and wrote the manuscript. NY and HSS revised the manuscript. All authors read and approved the final manuscript.Fig S1Click here for additional data file.Fig S2Click here for additional data file.Fig S3Click here for additional data file.Supplementary MaterialClick here for additional data file.


Cladophora cellulose (CLAD). As a benchmark, low surface area microcrystalline cellulose (MCC) was used. The solid-state properties of mixtures were characterized with X-ray diffraction, Fourier-transform infrared spectroscopy, and differential scanning calorimetry. The dissolution behavior of mixtures was studied in three biorelevant media, i.e., fasted state simulated gastric fluid, fasted state simulated intestinal fluid, and fed state simulated intestinal fluid. Additional thermal analysis and dissolution tests were carried out following 4 months of storage at 75% RH and room temperature. Heated mixtures of FFA with CLAD resulted in complete amorphization of the drug, whereas that with MCC produced a mixture of up to four different polymorphs. The amorphous FFA mixture with CLAD exhibited rapid and invariable fasted/fed state dissolution in simulated intestinal fluids, whereas that of MCC mixtures was highly dependent on the biorelevant medium. The storage of the heated FFA-CLAD mixture did not result in recrystallization or changes in dissolution profile, whereas heated FFA-MCC mixture showed polymorphic changes. The straightforward dry powder formulation strategy presented here bears great promise for reformulating a number of problem drugs to enhance their dissolution properties and reduce the fasted/fed state variability.Flufenamic acid (FFA) is a problem drug that has up to eight different polymorphs and shows poor solubility. Variability in bioavailability has been reported in the past resulting in limited use of FFA in the oral solid dosage form. The goal of this article was to investigate the polymorphism and amorphization behavior of FFA in non-heated and heated mixtures with high surface area nanocellulose, i.e.,  Enhancement of solubility of poorly soluble drugs is one of the most intensively investigated topics within the pharmaceutical formulation science today. The issues of solubility-limited bioavailability can be further complicated by polymorphism and solid-state instability, presenting great challenges for the industry. Therefore, extensive studies on relatively challenging molecules are required to develop new formulation strategies that can both enhance the bioavailability of the drug and reduce the inter-individual variability. The present study focuses on the formulation of flufenamic acid (FFA) as a model poorly soluble drug with high degree of polymorphism. In particular, as many as nine polymorphs have been described for FFA .Flufenamic acid, together with mefenamic acid (MFA) and tolfenamic acid (TFA), belongs to the class of fenamates, which were developed in the 1960s . Being aTraditional uses of FFA and other fenamates include analgesic treatment of rheumatoid arthritis, migraine, and, especially, primary dysmenorrhea. The distinctive utility in dysmenorrhea treatment has been linked to a dual mode of action involving not only inhibition of prostaglandin synthases, but also antagonism to prostaglandin itself . BecauseHigh inter-individual variability of pharmacokinetic parameters in humans has been cited as one of the main limiting factors for FFA’s practical use . AlthougCladophora cellulose (CLAD). The formulation of FFA with low surface area cellulose, i.e., microcrystalline cellulose (MCC), does not achieve a similar effect.We have previously shown that heat-assisted mixing of poorly soluble drugs from varying pharmacological classes with high surface area nanocellulose results in enhanced solubility and dissolution rate in biorelevant media. In particular, the latter effect was shown for NSAIDs from arylpropionic acid derivatives, e.g., ibuprofen, flurbiprofen, ketoprofen, and naproxen , as wellCladophora cellulose was provided by FMC Biopolymers . Microcrystalline cellulose (Avicel PH101) and flufenamic acid (FFA) were purchased from Sigma Aldrich . Biorelevant media of simulated gastric fluid (SGF), fasted state simulated intestinal fluid (FaSIF) and fed state simulated intestinal fluid (FeSIF) were prepared using the powder purchased from Biorelevant  according to the manufacturer’s instructions.The structure and physicochemical properties of FFA are presented in FFA powder was used as supplied without sieving to separate a specific particle size fraction. The mixture was prepared by dry blending of FFA with cellulose. The weight ratio between FFA and cellulose was 1:9. Typically, 5 mg of FFA was mixed with 45 mg of cellulose in a glass vial using a Turbula mixer  for 15 min.Following preparation of physical mixtures, the sealed vials were heated to 134 °C for 1 h.−2 mbar and 35 mA, and the sputtering time was 30 s.SEM images of cellulose powder samples were acquired with a scanning electron microscope . The membranes were sputtered with Au/Pd prior to analysis to minimize charging effects of the samples. A Polaron sputter coater  was used. The sputtering settings were 4 × 10An accelerated stability study was conducted on FFA-cellulose mixtures to follow solid-state transitions. Glass vials containing the heated sample were stored at constant relative humidity (75%) over a saturated NaCl solution at room temperature for 4 months in a desiccator.An X-ray diffractometer  with Bragg−Brentano geometry  was used. Operating current settings were 40 kV and 40 mA. The 2θ angle was varied between 10° and 45° at 0.02° scan steps. The data were collected on flat powders placed in reduced background specimen holders supplied by the manufacturer .The DSC measurements were performed with a Mettler Toledo DSC 3 instrument . The samples were first cooled from room temperature to −30 °C, and then heated to 160 °C at the 10 K/min heating rate. Constant nitrogen gas at a flow rate of 60 mL/min was purged throughout the measurements. Typically, 1 mg of 1:9 drug-cellulose mixture was used per measurement. For pure substances, 1 mg of FFA and 1 mg of cellulose were used per measurement. The aluminum pan containing the sample was punctured to avoid overpressure. The collected data were treated with the eStar analysis software provided by Mettler Toledo.mixtureH is the enthalpy of FFA melting in the mixture, ΔdrugH is the melting enthalpy of a pure drug, and a is the correction factor corresponding to drug content, i.e., a = 1 for a pure drug, and a = 0.1 for a 10% w/w mixture.Crystallinity index was calculated as follows:−1 corresponding to the stretch of C=O bonds. The measurements were performed with a Bruker Tensor 27 FTIR  according to the pellets technique with potassium bromide (KBr). The amount of FFA in the drug-cellulose mixture (1:9 w/w) was about 2 mg. The amount of KBr used was around 200 mg. The collected data were treated with the OPUS Data Collection Program (V 1.1).FTIR absorbance spectrum was acquired to follow the interactions between FFA and cellulose. The most informative region relevant for this study included the area from 1800 to 1400 cmThe dissolution test was performed in a SOTAX  apparatus using 500 mL of the biorelevant medium per dissolution vessel. The temperature for each dissolution vessel was maintained at 37 °C. The same amount of mixed samples was used in all timings and all different media (100 mg FFA + 900 mg cellulose). Each group of non-heated and heated samples was run in triplicate. The paddle speed for each dissolution vessel was 50 rpm. The sampling times for all media were 5, 15, 30, 45, 60, 90, and 180 min, respectively. At each time point, 5 mL of the medium was withdrawn and filtered through a 0.45-μm PTFE filter after discarding the first 2–3 mL. A total of 1.5 mL of the remaining sample was transferred in glass vials for further analysis by HPLC.® STAR RP-18e (2 µm) Hibar® HR 50–2.1 mm column . The column temperature was 50 °C, and injector temperature was 20 °C. The mobile phase A was 0.1% formic acid in water and mobile phase B was 0.1% formic acid in acetonitrile. The flow rate was 0.8 mL/min. The injector wash was 50% acetonitrile. The retention time was 4.45 min, and run time was 8.0 min. UV detection at the wavelength of 280 nm was used. Calibration samples were run prior to the analysis of the study samples by preparing the FFA stock solution in the biorelevant medium at a concentration of 250 μg/mL. The stock solution was used as the calibration standard by a subsequent gradual dilution of 1:250.A Hitachi Chromaster HPLC-UV system was optimized for the detection of FFA prior to the analysis. The liquid chromatography system used was a Hitachi Chromaster pump 5110 with a Hitachi Chromaster 5260 autosampler and Purospher2 gas sorption analysis.The cellulose batches used in this study were identical to those used previously ,13. Nota−1 is seen as compared to the FFA-MCC-N sample. On the other hand, in the FFA-CLAD-H mixture, a clearly visible shift of the stretch of C=O bonds is observed from 1651 cm−1 to 1670 cm−1 as compared to the non-heated mixture. The position of the C=O bond at 1651 cm−1 in the normal mixture normally coincides with that of the pure FFA.Based on the XRD, DSC, and FTIR results, it can be concluded that heated mixtures of FFA with CLAD show significant degree of FFA molecular rearrangement and render the latter amorphous. In the previous studies, amorphization of poorly soluble drugs in mixtures with CLAD resulted in enhanced solubility and dissolution rate.We reported previously that the amorphization of poorly soluble aromatic pharmaceutical compounds in high surface area CLAD cellulose is sufficiently stable over time to enable practical applications. The solid-state stability for high challenging polymorphic substances therefore needs to be elucidated. The produced mixtures were stored at 75% RH and room temperature for over 4 months. Following the storage, the mixtures were studied with DSC to monitor phase transitions. Furthermore, the FFA release was re-evaluated after storage in all 3 biorelevant media.The results of the present study confirm the previous conclusions on the usefulness of high surface area nanocellulose to induce and maintain amorphization of poorly soluble aromatic pharmaceutical compounds, which was previously observed for profens, e.g. ibuprofen, and nifedipine. That the observed effect can be maintained for a highly challenging polymorphic substance is novel. The underlying mechanism for this effect is most likely the interplay between H-bonding and hydrophobic (π-π and π-OH) interactions between cellulose and drug molecules, which has been confirmed by molecular dynamics studies . The strThe results of the present study show that the heated mixtures of FFA-CLAD produce stable amorphous mixtures which exhibit enhanced dissolution rate and reduced fasted-fed state variability in biorelevant intestinal media. Heated FFA-MCC mixtures, i.e., with low surface area, do not produce a similar amorphization effect, especially with respect to the fasted-fed state variability. Instead, FFA recrystallizes into 4 different polymorphs rather than stable amorphous state as is the case with the FFA-CLAD-H sample. The different polymorphic entities and variable dissolution profile result in unpredictable dissolution behavior of FFA in MCC mixtures. The solubility of FFA in the SGF is very low, and ≤ 1% of the loaded drug, i.e., ≤ 2 μg/mL, is dissolved. The amount of FFA dissolved from MCC sample in the SGF is slightly higher than that from CLAD mixtures, which could be ascribed to stronger molecular interactions with the high surface area nanocellulose.


Filaments of intense vapor transport called atmospheric rivers (ARs) are responsible for the majority of poleward vapor transport in the midlatitudes. Despite their importance to the hydrologic cycle, there remain many unanswered questions about changes to ARs in a warming climate. In this study we perform a series of escalating uniform SST increases  in the Community Atmosphere Model version 5 in an aquaplanet configuration to evaluate the thermodynamic and dynamical response of AR vapor content, transport, and precipitation to warming SSTs. We find that AR column integrated water vapor (IWV) is especially sensitive to SST and increases by 6.3–9.7% per degree warming despite decreasing relative humidity through much of the column. Further analysis provides a more nuanced view of AR IWV changes: Since SST warming is modest compared to that in the midtroposphere, computing fractional changes in IWV with respect to SST results in finding spuriously large increases. Meanwhile, results here show that AR IWV transport increases relatively uniformly with temperature and at consistently lower rates than IWV, as modulated by systematically decreasing low‐level wind speeds. Similarly, changes in AR precipitation are related to a compensatory relationship between enhanced near‐surface moisture and damped vertical motions. Aquaplanet atmospheric rivers grow in size and intensity as sea surface temperatures increaseIncreases in column water vapor beyond Clausius‐Clapeyron rates mask drying at upper levelsWeakening dynamics damp changes in atmospheric river vapor transport and precipitation TherefoIn all, this study seeks to contextualize AR climate change statistics—whether those established from observations or from studies in comprehensive climate models—by isolating the impacts of warming SSTs. We will not only test physical linkages between SST increases and AR statistics but will break down more complex AR processes  into simple constituents  to further our understanding.A secondary goal of this manuscript is the introduction of a simple and novel AR detection tool (ARDT) developed as part of the TempestExtremes framework  Version 2 with Community Atmosphere Model version 5 (CAM5) physics , aerosol cloud interactions are turned off, and the only aerosol emissions come from sea salt, which the model diagnoses from surface wind. The zonal and hemispheric homogeneity of the boundary conditions, along with the lack of seasonality, allows for many more ARs under the same boundary conditions and forcing, so the sample size reduces confidence bounds. Medeiros et al.  found th2.2p0= 1,000 hPa, pT=300 hPa, g is gravitational acceleration, q is specific humidity, and u and v are the zonal and meridional wind velocities, respectively.The American Meteorological Society (AMS) definition for AR contains little quantitative guidance AMS, , allowinThe grid point is poleward of 20°N/S.The Laplacian of IVT at the grid point is h et al.  as well h et al. .The grid point is part of The grid point does not belong to a tropical cyclone  software suite  within a 2.0° great‐circle distance.Their SLP increases by at least 375 Pa over a 3.6° radius.Their 300 minus 500 hPa geopotential height thickness decreases by six meters over 7.5°. This criterion acts so as to only detect warm core storms and not eliminate extratropical cyclones.One pitfall associated with the TE AR detection criteria used here is the identification as ARs of bands of high IVT associated with TCs. While ARs can occur in association with TCs and extratropical transition . This is analogous to a series of seasonal‐length ensembles.Due to zonal uniformity and symmetry of SSTs across the equator, AQPs lend themselves nicely to meridional distributions of zonal mean statistics. Hence many statistics are presented in this form and due to the aforementioned hemispheric symmetry, are shown only for the northern hemisphere . Furthermore, since we do not track ARs in time, all analysis on AR variables  is performed on a grid point basis, rather than over the entire AR object throughout all or part of its lifecycle. Therefore, all meridional distributions of AR variables discussed here may be better characterized as distributions of variables under “AR conditions”  in which all non‐AR grid points are excluded from analysis. We evaluate significance throughout this manuscript at the 95% confidence level with respect to 3‐monthly ensemble members with the northern and southern hemispheres treated as independent samples , upper subtropics , lower midlatitudes , and upper midlatitudes . The relatively large gap between LST and UST exists because of a complex precipitation response in the area between those bands, as seen in Figure  3We begin our discussion in section 3.1The midlatitude circulation provides the large‐scale dynamical background for ARs. We characterize the midlatitude circulation in terms of three metrics: the Hadley cell (HC) edge, the subtropical jet (STJ), and the eddy‐driven jet (EDJ). The purpose of this section is to provide a broad overview of the major circulation responses to SST increases in order to provide context for the AR responses described in later sections.The HC edge defines the poleward extent of the region of subsidence associated with the HC's descending branch, where strong static stability suppresses precipitation. To locate it, we follow the work of Davis and Birner  who defiWhereas the HC edge broadly defines regions of convergence and divergence through the tropics and subtropics, the tropospheric jets act as dynamical guides against which cyclones, and the closely related ARs, form and propagate. Statistics related to AR occurrence, landfall location, and moisture transport are impacted by the prevailing jet regime for a given ocean basin   Duchon, . We then Duchon, . As the  Duchon, . Hence, Last, we find the STJ as in Davis and Birner , who def3.2Figure  section . In termWe investigate changes in zonal mean IVT first by breaking it up into its zonal (uIVT) and meridional (vIVT) components Figure . PerhapsIn non‐AR regions, the poleward shift in IVT is exclusively due to an increase and poleward shift in westerly transport (uIVT), as meridional transport remains small , a change likely facilitated by the poleward shift in the EDJ Figure . Given t3.3Figure Similar “super‐CC” moistening rates in ARs have been observed before , which we decompose into histograms of zonal and meridional winds taken over all AR points within each subregion Figure . These dr is the residual value. We plug in the area‐weighted fractional changes for IVT, IWV, and As stated earlier, we roughly consider IVT to be the product of IWV and low‐level wind speed. With this in mind, the decreased magnitude of zonal mean IVT increases compared to IWV increases suggests that slowing winds attenuate the IVT response. We compactly show the compensatory relationship between IWV and low‐level winds on IVT by returning to the weighted area‐mean fractional changes of IVT and IWV Figures  and 6 fon IVT asΔIVTIVT=Δr values in AR grid points indicates that absent changes in wind speed, AR IVT increases are approximated by those in AR IWV, a result which agrees well with several previous analyses  and dynamics  in modulating AR and large‐scale vapor transport. In particular, it allows us to see the opposition between the thermodynamic and dynamic components under warmer SST regimes. The generally small magnitudes of the 3.5We move on now to perhaps the most widely associated feature of ARs. We begin by examining zonal means within AR and non‐AR grid points, as in previous sections. From here, we examine the drivers of precipitation and its changes first by applying a simplified physical model of precipitation analogous to that found in Table 3.5.1Figure P) of AR grid points over the ocean is roughly equal to the vertically integrated condensation rate; we can thus characterize it with the following proportionality:qsfc∗; since ARs are approximately saturated near the surface  are the most informative metric, especially when the most extreme AR events can lead to catastrophic flooding  with uniformly increased SSTs of +2K, +4K, and +6 K over the control “QOBS” profile  and a systematic damping in midtroposphere vertical velocities (Table Despite systematic increases in AR IWV and IVT, AR precipitation has a much more varied response across latitudes (es Table . Furtheres Table . While Aes Table , the enhes Table  shows thes Table . Nonethees Table  and 12c.We approached this study with the goal of leveraging an idealized model to investigate the sensitivity of AR statistics to isolated SST warming. In fact, the idealizations present in this experiment design provide a means to more clearly elucidate the underlying physical processes and explain the relevant theory. The analyses of AR IWV and precipitation described here provide physical context for results obtained in comprehensive climate models, where compensatory or compound effects can confound interpretation and leave open questions about the mechanistic relationships between forcings and impacts.Text S1Click here for additional data file.


The ankle joint complex (AJC) is of fundamental importance for balance, support, and propulsion. However, it is particularly susceptible to musculoskeletal and neurological injuries, especially neurological injuries such as drop foot following stroke. An important factor in ankle dysfunction is damage to the central nervous system (CNS). Correspondingly, the fundamental goal of rehabilitation training is to stimulate the reorganization and compensation of the CNS, and to promote the recovery of the motor system’s motor perception function. Therefore, an increasing number of ankle rehabilitation robots have been developed to provide long-term accurate and uniform rehabilitation training of the AJC, among which the parallel ankle rehabilitation robot (PARR) is the most studied. The aim of this study is to provide a systematic review of the state of the art in PARR technology, with consideration of the mechanism configurations, actuator types with different trajectory tracking control techniques, and rehabilitation training methods, thus facilitating the development of new and improved PARRs as a next step towards obtaining clinical proof of their rehabilitation benefits.A literature search was conducted on PubMed, Scopus, IEEE Xplore, and Web of Science for articles related to the design and improvement of PARRs for ankle rehabilitation from each site’s respective inception from January 1999 to September 2020 using the keywords “ parallel”, “ ankle”, and “ robot”. Appropriate syntax using Boolean operators and wildcard symbols was utilized for each database to include a wider range of articles that may have used alternate spellings or synonyms, and the references listed in relevant publications were further screened according to the inclusion criteria and exclusion criteria.Ultimately, 65 articles representing 16 unique PARRs were selected for review, all of which have developed the prototypes with experiments designed to verify their usability and feasibility. From the comparison among these PARRs, we found that there are three main considerations for the mechanical design and mechanism optimization of PARRs, the choice of two actuator types including pneumatic and electrically driven control, the covering of the AJC’s motion space, and the optimization of the kinematic design, actuation design and structural design. The trajectory tracking accuracy and interactive control performance also need to be guaranteed to improve the effect of rehabilitation training and stimulate a patient’s active participation. In addition, the parameters of the reviewed 16 PARRs are summarized in detail with their differences compared by using figures and tables in the order they appeared, showing their differences in the two main actuator types, four exercise modes, fifteen control strategies, etc., which revealed the future research trends related to the improvement of the PARRs.The selected studies showed the rapid development of PARRs in terms of their mechanical designs, control strategies, and rehabilitation training methods over the last two decades. However, the existing PARRs all have their own pros and cons, and few of the developed devices have been subjected to clinical trials. Designing a PARR with three degrees of freedom (DOFs) and whereby the mechanism’s rotation center coincides with the AJC rotation center is of vital importance in the mechanism design and optimization of PARRs. In addition, the design of actuators combining the advantages of the pneumatic-driven and electrically driven ones, as well as some new other actuators, will be a research hotspot for the development of PARRs. For the control strategy, compliance control with variable parameters should be further studied, with sEMG signal included to improve the real-time performance. Multimode rehabilitation training methods with multimodal motion intention recognition, real-time online detection and evaluation system should also be further developed to meet the needs of different ankle disability and rehabilitation stages. In addition, the clinical trials are in urgent need to help the PARRs be implementable as an intervention in clinical practice. The ankle joint complex (AJC) mainly consists of the tibia, fibula, talus and calcaneus, as shown in Fig. Therefore, it is of vital importance to replace traditional ankle rehabilitation training by developing ankle rehabilitation robots, which have the benefits of providing long-term accurate and uniform rehabilitation training as well as the ability to adaptively modify the difficulty of rehabilitation training according to real-time feedback from training . In addiVarious reviews of robotic devices for ankle rehabilitation have been performed , 14–17. The purpose of this paper is to provide a systematic review of the state of the art in PARR technology, with consideration of the mechanism configurations, actuator types, and rehabilitation training methods, thus serving as a tutorial for engineers who will design or control PARRs, making them aware of the advantages and limitations of different mechanical and control choices. From a research point of view, this paper also proposes a taxonomy expansion and a review of future research directions. The following sections review the PARRs with regard to the mechanism configurations, actuator types with different trajectory tracking control techniques, and rehabilitation training methods; we also compare, analyze and summarize them separately. Finally, the research hotspots and trends are discussed, and we present the takeaways regarding PARRs as the findings of this review.The systematic review was conducted by performing a literature search with PubMed, Scopus, IEEE Xplore, and Web of Science, and the search was limited to English-language articles  published from January 1999 to September 2020. The reason that we chose January 1999 as the starting point for searches is that the first PARR was developed in 1999, named “ Rutgers Ankle” . The eleThe device described is a parallel mechanism with at least 2-DOFs;The study focuses on ankle rehabilitation;The PARRs involved are developed with prototypes, not simply theoretical concept designs;The involved paper is a scientific article written in the English language and accessible to the authors.Studies were eligible for inclusion if the following criteria were met:Devices such as wearable ankle rehabilitation robots, ankle-foot prostheses and 1-DOF robotic ankle rehabilitation systems were excluded;Studies not related to ankle recovery were excluded as well;Studies with insufficient information on the PARR design were excluded;Non-English articles were excluded.The final search queries for the review were completed on September 30, 2020. A total of 65 articles representing 16 unique PARRs met the inclusion criteria, and the associated full articles were obtained by downloading them from electronic databases. Inside the selected articles, 57 out of the 65  shown in Fig. f-locked ; the 3-Dn-DOF motion of the PARR. By contrast, the electric motor can be easily controlled to rotate in the forward and reverse directions accurately, which means that there is no need for the electrically driven PARRs to be driven redundantly; in addition, they can have a smaller size while achieving the same function as that of the pneumatic-driven PARRs. However, the compliance of an electrically driven PARR needs to be realized by adopting compliance control algorithms [RUS/RRR PARR [For all these PARRs, the most commonly used actuator of pneumatic-driven PARRs is the pneumatic muscle actuator (PMA) due to its superiority in terms of low weight and high power/volume ratios, which can provide an intrinsic softness to make joint compliance possible , 70. Howgorithms , 60–62. gorithms  and the RRR PARR , 38. HowAs we summarized above, there are two types of PARRs based on actuator type, namely, pneumatic-driven PARRs and electrically driven PARRs. The PARRs of Fig. ectively . The resectively . In addiectively .Through the abovementioned summary, we can see that most pneumatic-driven PARRs were developed with trajectory tracking control to improve rehabilitation accuracy , 52, 54 The main advantage of automated rehabilitation systems is the ability to perform a large number of repetitions, which has been proven to be extremely beneficial in the treatment of neuromuscular injuries . TherefoPS/RRR PARR, the passive rehabilitation training trajectories were preset with the control system built [For the electrically driven PARRs, Yoon et al. proposed a wide set of exercise modes to improve rehabilitation training, ranging from passive exercise to proprioceptive training such as balance exercise based on position and impedance control theories . The ARBem built , 66, andem built .For the pneumatic-driven PARR, the Rutgers Ankle adopted a position controller for passive training to drive the patient’s AJC along certain trajectories and a force controller for active resistive exercise , 58, forThrough the abovementioned summary, we can see that safety is always the most important for robot-assisted neurological rehabilitation, not only compliance control in the task space but also compliance of the actuators and the robot itself are of crucial importance to ensure safety, because the patients can provide moderate torque to initiate the active motion when they have certain muscle strength, and excessive interaction forces may occur in this situation if the PARR is not flexible. In this respect, pneumatic-driven PARRs have more advantages because the pneumatic muscle has a lightweight and compliant nature, which makes it more appropriate for natural interaction with patients . On thisThe aim of PARRs is to improve ankle rehabilitation performance, therein assisting the AJC in regaining its ROM and muscle strength. Therefore, a designed PARR must have a sufficient workspace and excellent kinematic performance, such as velocity transfer performance and force transfer performance. Because the AJC has 3-DOFs, many PARRs are designed with 3-DOFs, such as the PARRs in , 43–45, In conclusion, there are three main considerations for the mechanical design and mechanism optimization of PARRs. The first is to choose a better actuator type, whether pneumatic-driven or electrically or whether the actuator is below or above the PARRs. The second is to cover the all 3-DOFs of the AJC, thus ensuring that one PARR can effectively carry out rehabilitation training in the DO/PL, IN/EV and AD/AB DOFs. The third is to optimize the kinematic design, actuation design and structural design of the PARRs to make them more rational to use in rehabilitation training.From the results of trajectory tracking control techniques and the rehabilitation training methods, we can see that all of these are related to the ankle rehabilitation training modes. In this regard, the control framework of Saglia et al., as shown in Table In addition to these, joint force control  and hierIn brief, the trajectory tracking and interactive control are both used for the different rehabilitation training methods. On the one hand, the trajectory tracking accuracy must be high enough to drive the AJC along the required trajectory precisely, which may be somewhat difficult for the pneumatic-driven PARRs due to the nonlinearity of the PMAs and is the reason why many trajectory tracking control algorithms, such as the FBDO, NIFT, and ABS-SMC , 52, 54,Based on the abovementioned systematic review and summary, we can see that many core aspects of PARRs have converged on common solutions, especially for PARRs using the same actuator types, even though their mechanical designs are often quite different. The parameters of the 16 abovementioned PARRs are summarized in detail with their differences in Table Ankle rehabilitation training is a small branch of rehabilitation robotics. Very significant results have been achieved in upper-limb rehabilitation robots  and loweExisting PARRs all have their own disadvantages, such as insufficient or redundant DOFs, the rotation center of the AJC not coinciding with the mechanism’s rotation center while ankle rehabilitation requires varying positions of the AJC and synergistic movement of the lower limb from the patient, a complex mechanism with branches that easily lead to interference, poor driving modes and inconvenient use. Therefore, designing a PARR with only three DOFs and whereby the mechanism’s rotation center coincides with the AJC rotation center, which also has a simple structure, enough workspace, ease of use and small size, is a key problem in the mechanism design and optimization of PARRs. In addition, the actuator types are very important. As we discussed, the pneumatic-driven PARRs have the advantage of intrinsic compliance, but they are difficult to control precisely owing to the nonlinear and time-varying characteristics of PMAs, while the electrically-driven PARRs can be controlled precisely but lack the intrinsic compliance and require complex compliance control algorithms. Therefore, the design of actuators combining the advantages of the two actuator types will be a research hotspot, and some new actuators may be used for the development of PARRs.Many PARRs have been developed with compliance control by using impedance control, admittance control and their derivative compliance control algorithms. However, it is not convenient to use compliance control strategies with fixed parameters because the mechanical impedance of the AJC changes both with the stage of rehabilitation training and significantly between patients. Although some researchers have studied adaptive admittance control , 71 and The current rehabilitation training methods of PARRs are almost all within the control framework shown in Table From the above review, we can see that none of the PARRs use sEMG signal information for active rehabilitation. A commonly used sensor in PARRs at the present stage is the force/torque sensor , 67, 71.PARRs are used to help patients regain their limited ROM, restrength their weak muscles, recover their dynamic balance, and restore their motion functions. However, most PARRs have been tested using only healthy subjects, while few have been tested on patients with AJC dysfunctions –71. TherThis study aims to provide a systematic review of the state of the art in PARR technology. Therefore, ankle rehabilitation robots without parallel mechanisms, such as the ankle-foot orthosis (AFO) , 87, 88,This review mainly summarized the state-of-the-art PARRs in terms of pneumatic-driven and electrically driven PARRs, with consideration of the mechanism configurations, actuator types with different trajectory tracking control techniques and rehabilitation training methods. By comparing, analyzing and summarizing the differences and similarities among the reviewed PARRs, we can see that more work needs to be done to perfect the mechanical design and interactive control strategies to maximize patient safety and improve rehabilitation outcomes, although significant progress has been made in the development and improvement of PARRs over the last two decades, with the mechanical designs and control strategies being greatly improved for both pneumatic-driven and electrically driven PARRs. Designing a PARR with only three DOFs and whereby the mechanism’s rotation center coincides with the AJC rotation center is of vital importance in the mechanism design and optimization of PARRs. For the control strategy, compliance control with variable parameters should be further studied, with sEMG signal included to improve the real-time performance. And multimode rehabilitation training methods with multimodal motion intention recognition, real-time online detection and evaluation system should be further developed to meet the needs of different ankle disability and rehabilitation stages. In the foreseeable future, there will be PARRs that combine the best of both pneumatic-driven and electrically driven PARRs. The research hotspots and trends in this field will mainly center on mechanism optimization, compliance control with variable parameters, multimode rehabilitation training methods specific to different ankle disabilities and rehabilitation stages, multimodal motion intention recognition, and the evaluation and selection of the optimal exercise therapy.The other thing to note is that few of the developed PARRs have been subjected to clinical trials, all the control strategies and rehabilitation training modes are in the stage of testing, with none being used in clinical applications. Some simple ankle rehabilitation devices have already been in commercial use, such as the AnkleMotus from Shanghai Fourier Intelligent Technology Co., Ltd, China, and the Minitalus from EasyTech, Italy. However, they were designed for training with a specific function or for a single AJC’s DOF. The PARRs with more comprehensive rehabilitation training function are developing rapidly, and several are already in clinical trials. It is expected that within a year or two, PARRs with full rehabilitation training capabilities will be implementable as an intervention in clinical practice.Generally speaking, the study provides a systematic review of the PARRs’ researches and technologies. Our goal in this review is to guide future researchers in the development of better devices for patients with ankle dysfunctions. We hope that this review will serve as a useful resource for future developers and facilitate the evolution of the field.


Lepidotrigona terminata  is a stingless bee that distributed in Eastern Asia. The complete mitogenome of L. terminata (GenBank accession number MN737481) is 15,431 bp in size, including 13 protein-coding genes, 22 transfer RNAs, two ribosomal RNAs genes, and a noncoding D-loop region. The D-loop region is located between ND4L and tRNAMet, different from the other two stingless bee mitogenomes previously reported. The base composition of the whole L. terminata mitogenome is 38.18% for A, 11.67% for G, 38.32% for T, and 11.83% for C, with a high AT bias of 76.50%. The present data could contribute to a detailed phylogeographic analysis of this valuable economic insect for further study in differentiating closely related species. Lepidotrigona terminata  (Hymenoptera: Meliponinae) is a species of stingless bee distributed in China  and deposited in the insect specimen room of Research Institute of Resource Insects with an accession number RIRI-w-20191012. Sequencing work of the complete mitogenome of L. terminata was performed using Illumina Nextseq500 in Beijing Microread Genetics Co., Ltd., with a total data volume 4 G (150 bp Reads). High-quality reads were assembled from scratch using IDBA-UD and SPAdes , and several genes have been rearranged. The mitogenome is 15,431 bp in size (GenBank accession number MN737481), including 13 typical invertebrate PCGs, 22 transfer RNA genes, two ribosomal RNA genes and a noncoding control region (D-loop). The A + T content of the whole L. terminata mitogenome is 76.50%, showing an obvious AT mutation bias tRNA could be folded into the typical cloverleaf secondary structures. The unusual Ser (UCU)tRNA lacks the dihydrouridine (DHU) arm.All 13 PCGs use standard ATN as a start codon. As for the stop codon, nine PCGs had the common mitochondrial stop codon TAA, whereas L. terminata with 17 other Hymenoptera insects (Lepidotrigona terminata was clustered with the other two stingless bees (Melipona scutellaris and Melipona bicolor), and the phylogeny tree indicates that stingless bee has a closer relationship with bumblebees (Bombus) than with honeybees (Apis). This mitogenome data might be also useful for further phylogeography analyses in Meliponinae.Based on the concatenated 13 mitochondrial PCGs sequences of 18 species from Hymenoptera, the neighbour-joining method was used to construct the phylogenetic relationship of  insects . The phy insects . Lepidot


This special report was developed to communicate policy and procedures for free-standing acute inpatient rehabilitation hospitals (AIRHs) to protect patients and healthcare personnel and to prevent further spread of severe acute respiratory syndrome coronavirus 2. The recommended policies were developed in conjunction with the New Mexico Department of Health and hospital leadership. As we attain additional knowledge and experience during this pandemic, suggestions of best practice will continue to evolve for AIRHs. The authors encourage readers to work with local regulatory officials to ensure regulatory compliance as well as respect of the availability of local resources. This special report communicates potential policy and procedure modifications for free-standing acute inpatient rehabilitation hospitals in response to the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)/coronavirus disease (COVID-19) pandemic. The goal of the communication is to help protect patients and healthcare personnel and to prevent further spread of SARS-CoV-2. The recommendations were developed in conjunction with the New Mexico Department of Health and hospital leadership. A comprehensive response policy to SARS-CoV-2/COVID-19 should include facility changes, enhanced personal protective equipment policy, enhanced admission policy and enhanced surveillance testing policy. In the setting of rapidly changing state and federal guidelines, hospital staff should work with state officials to ensure that potential policy and procedure modifications are in line with state and federal recommendations. The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which is the infectious source for coronavirus disease COVID-19) was first reported on 31 December 2019 9 was fir. In BernThe rapid spread and sheer number of cases have exacted a global and national toll on the healthcare system as healthcare professionals balance the need for continued patient care with the need to control the spread of the virus . This haSupplementary material to this article and can be found online [Our free-standing acute inpatient rehabilitation facility is in a unique position because it is one of only four such facilities in the state of New Mexico capable of providing complex acute inpatient rehabilitation services. Our rehabilitation hospital is the only hospital in New Mexico accredited by the Commission on Accreditation of Rehabilitation Facilities for 3 years in six programs: Comprehensive Integrated Inpatient Rehabilitation Program, Inpatient Brain Injury Program, Inpatient and Outpatient Spinal Cord System of Care, Inpatient Stroke Specialty Program, Outpatient Medical Rehabilitation Program, and Outpatient Medical Rehabilitation Program (Children and Adolescents). The hospital has 265 employees with 43 contractors. There were 62 licensed acute inpatient rehabilitation beds and 1470 inpatient admissions in 2019. The hospital provides a full continuum of inpatient and outpatient rehabilitation care, including physical therapy, occupational therapy, speech and language pathology, rehabilitation nursing and case management services. The inpatient program is designed for patients with a variety of rehabilitation needs, offering general inpatient medical rehabilitation and three specialized programs: brain injury rehabilitation, stroke rehabilitation and spinal cord injury rehabilitation. Details of the program are supplied as d online .On 6 July 2020, senior hospital leadership was informed of a patient who discharged on 3 July 2020 then tested positive for COVID-19 two days after being discharged. In response to this notification, all patients and staff were tested that same week as part of a swift and organized response. Testing resulted in identification of three patients and four staff members with positive COVID-19 tests. Given the acute inpatient rehabilitation population at our facility  is at high risk of being affected by respiratory pathogens such as COVID-19, a comprehensive assessment was made with resulting action plans .Our hospital has an established infection prevention and control program. It became clear, however, that new measures were needed in the setting of this unique pandemic. The medical director, in conjunction with state officials from the Department of Health and senior hospital leadership, developed new procedures and policies as detailed below.The goal of the SARS-CoV-2/COVID-19 specific policies and procedures is to protect patients and healthcare personnel and to prevent further spread of the virus. These policies and procedures may be of benefit to other free-standing acute inpatient rehabilitation facilities.Identify SARS-CoV-2 exposuresContain SARS-CoV-2 exposuresSafely maintain average daily censusThe specific aims of the program are as follows:The efficient and timely identification of SARS-CoV-2 exposures mitigates risk of an outbreak across our hospital system and protects the vulnerable patient population. Containment limits exposures to a single unit, allowing for continued patient flow in other units, ultimately allowing for uninterrupted admission and the ability to maintain the average census. This enables ongoing, high-level rehabilitation services while also allowing admission from overwhelmed acute care hospitals.To help prevent or reduce airborne transfer of infectious organisms and viruses through airborne transmission, all hospital filters were changed to minimum efficiency reporting value-13 (MERV-13). The MERV-13 filters are reported to filter 90% of particles in the 3–10 μm range, 85% of particles in the 1–3 μm range and 50% of particles in the range 0.3–1 μm range .Enhance personal protective equipment (PPE)  were delivered and deployed to hospital staff. This enhanced PPE protocol was made mandatory; the use of cloth, homemade masks was initially discouraged and later banned.All potential patients must be free of symptoms typically associated with SARS-CoV-2 for 7 days before admission, unless attributed to other etiologies All potential patients not previously identified as COVID-19 positive are required to have at least one negative test within 72 h before admissionPatients previously identified as COVID-19 positive require two negative tests >24 h apart before admissionAll newly admitted patients are tested upon admission regardless of the last COVID-19 testAll admitted patients tested weeklyWeekly rotating surveillance testing of 25% hospital staffCohorts established with designated units, rehabilitation gym space, nursing, therapy and ancillary staff Each cohort consisting of 31 patient rooms (double occupancy) with dedicated nursing, therapy (assigned to unit) and ancillary staff (assigned to cohort)Nursing, therapy and ancillary staff cannot visit/aid units not in designated cohortSocial distancing procedures  are enfoPatients identified as positive through the admission testing or weekly surveillance testing process will be immediately transferred to acute care hospital for medical triage and treatmentAffected unit would be quarantined for at least 2 weeks: — Comingling between adjacent units in cohort will cease immediately and continue to be prohibited until such time as the quarantine is lifted — No new patients admitted to affected unit and no patients will be transferred out of affected unit other than to discharge Designated staff responsible for cohort in which patient was assigned will undergo immediate testing and subsequent weekly testing until no new positive results are detectedThe goal of the developed policy and procedures is to protect patients and healthcare personnel and to prevent further spread of SARS-CoV-2. The policies were developed in conjunction with an infectious disease epidemiologist at the New Mexico Department of Health. The authors feel that these processes and procedures may be of benefit to other free-standing acute inpatient rehabilitation facilities.These suggestions for updated facility policies in response to the SARS-CoV-2/COVID-19 pandemic are in line with the experience of other facilities . As we aThe development of our hospital's policies and procedures follows current recommendations and prior experience with respiratory pandemics. In the setting of rapidly changing state and federal guidelines we also worked with New Mexico state officials to ensure our recommendations were in line with state and federal recommendations. The authors encourage readers to work with local regulatory officials to ensure any polices or procedures developed are within regulatory compliance, respects the availability of local resources and addresses specific local conditions.There will be a need to develop detailed federal and national guidelines for acute inpatient rehabilitation hospitals in response to SARS-CoV-2/COVID-19 pandemic and future viral pandemics. These guidelines will ensure rehabilitation hospital policies and procedures are within regulatory compliance, respect the availability of national and local resources and appropriately address the evolving pandemic. In the absence of such guidelines, rehabilitation hospitals will have to work with state officials to develop guidelines in response to the SARS-CoV-2/COVID-19 pandemic.Policy and procedures should be developed for acute inpatient rehabilitation hospitals to protect patients and healthcare personnel and to prevent further spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)/coronavirus disease (COVID-19).In the setting of rapidly changing state and federal guidelines hospital staff should work with state officials to ensure recommendations are in line with state and federal recommendations.A comprehensive response policy to SARS-CoV-2/COVID-19 should include facility changes, enhanced personal protective equipment policy, enhanced admission policy and enhanced surveillance testing policy.


Staphylococcus species and Th2 cytokines in allergic rhinitis (AR) models.The host-microbial commensalism can shape the innate immune responses in respiratory mucosa and nasal microbiome also modulates front-line immune mechanism in the nasal mucosa. Inhaled allergens encounter the host immune system first in the nasal mucosa, and microbial characteristics of nasal mucus directly impact the mechanisms of initial allergic responses in nasal epithelium. However, the roles of the nasal microbiome in allergic nasal mucosa remain uncertain. We sought to determine the distribution of nasal microbiomes in allergic nasal mucosa and elucidate the interplay between nasal microbiome Staphylococcus aureus (AR-SA) and S. epidermidis (AR-SE) were isolated from the nasal mucosa of patients with AR. The influence of nasal microbiome Staphylococcus species on allergic nasal mucosa was also tested with in vitro and in vivo AR models. Pyrosequencing data showed that colonization by S. epidermidis and S. aureus was more dominant in nasal mucus of AR subjects. The mRNA and protein levels of IL-33 and TSLP were significantly higher in AR nasal epithelial (ARNE) cells which were cultured from nasal mucosa of AR subjects, and exposure of ARNE cells to AR-SA reduced IL-33 mRNA and secreted protein levels. Particularly, ovalbumin-driven AR mice inoculated with AR-SA by intranasal delivery exhibited significantly reduced IL-33 in their nasal mucosa. In the context of these results, allergic symptoms and Th2 cytokine levels were significantly downregulated after intranasal inoculation of AR-SA in vivo AR mice.Staphylococcus species was more dominant in allergic nasal mucosa, and nasal commensal S. aureus from subjects with AR mediates anti-allergic effects by modulating IL-33-dependent Th2 inflammation. The results demonstrate the role of host-bacterial commensalism in shaping human allergic inflammation.Colonization by  Allergic rhinitis (AR), an IgE- and T helper (Th)2-mediated inflammatory nasal disease, is caused by sensitized immune responses to inhaled allergens. This allergen-specific immune response is thought to arise from an imbalance in Th1-Th2 immune regulation that results in increased levels of Th2 cytokines . AllergiHuman mucosal surfaces are in direct contact with the external environment and are susceptible to invasion and colonization by various allergens and pathogens –9. RespiStaphylococcus species, including S. epidermidis and S. aureus, as the most abundant constituents in allergic nasal mucus. Particularly, S. aureus isolated from the nasal mucus of AR subjects accelerated the reduction of IL-33 in AR nasal epithelium. Furthermore, allergic nasal commensal S. aureus prevented allergic inflammation in vivo in mice by reducing IL-33-related Th2 immune responses in the nasal mucosa. Overall, our study presents evidence that Staphylococcus species colonization is significantly increased in the nasal mucus of AR subjects. In addition, administration of S. aureus isolated from human allergic nasal mucus suppressed allergic inflammation in nasal mucosa by reducing IL-33 secretion.Based on our previous evidence of microbial distribution in healthy nasal mucus, we assessed the microbial composition in allergic nasal mucus and subsequently investigated whether nasal commensal bacteria in allergic nasal mucus contributes to Th2 inflammatory responses in nasal epithelium. The present study identified Additional methodological details are available in the online supplement.Information on 17 subjects with AR enrolled in this study and the exclusion criteria are described in the online data supplement. Participation was voluntary, and written informed consent was obtained from all subjects. The Institutional Review Board of Seoul National University College of Medicine (No. 1709–049-883) and Gyeongsang National University Hospital (No. 2019–05-004) approved the protocol for this study.Mucus and/or nasal mucosa from the middle turbinate of human subjects was collected and assessed for quality as described in the online data supplement .Staphylococcus epidermidis and S. aureus strains from five individual subjects with AR were used in the study.To isolate bacterial colonies, the mucus was placed in lysogeny broth (LB) plates. After 1 day of incubation, bacterial colonies were obtained from LB plates. The species of each colony was identified using GS-FLX 454 pyrosequencing with 16S rRNA gene amplification, as described previously . StaphylNormal human nasal epithelial (NHNE) and allergic rhinitis nasal epithelial (ARNE) cells were each cultured from five subjects using an air-liquid interface method. Cells were used 14 days after creation of the air-liquid interface (ALI). Isolated AR-SA and AR-SE were inoculated as described previously . DetailsExperiments with four-week-old female wild type (WT) BALB/c mice  were carried out according to guidelines approved by the Institutional Animal Care and Use Committees of Seoul National University Hospital (No. 2016–1470). Microbiome depletion, AR-SA and AR-SE inoculation, nasal lavage (NAL) sample collection, and nasal tissue harvesting are described in the online supplement.S. aureus inoculation. The negative control group (WT) was sensitized and challenged with phosphate-buffered saline (PBS), and the positive control group (AR-OVA) was sensitized and challenged with ovalbumin (OVA). Both WT mice and AR-OVA mice were inoculated with human nasal microbiome S. aureus by intranasal delivery. The schematic figure for allergen sensitization, intranasal challenge, and S. aureus inoculation is shown in Fig. Mice were divided into four groups depending on AR mouse serum samples were stored at − 70 °C before use for measurements of total IgE and OVA specific IgE, as described previously . DetailsMice heads were fixed in 10% formalin, decalcified, and embedded in paraffin wax. Nasal tissues were sectioned and stained with hematoxylin and eosin (H&E) for inflammatory cell counting, Sirius red stain for eosinophil counting, and periodic acid-Schiff (PAS) stain to indicate secretory cells of the nasal epithelium.Bacterial samples were serially diluted ten-fold with phosphate-buffered saline (PBS). Next, 10 μl of each diluted sample was plated on an LB agar plate. The plates were incubated for 24 h at 37 °C. The numbers of AR-SA and AR-SE colonies growing on the agar surface were counted manually. Bacterial growth was reported based on the colony-forming units (CFUs) for each sample.femAs specific for S. aureus (femA-SA) and S. epidermidis (femA-SE) were determined using real-time PCR (RT-PCR), as described in the online supplement.Levels of transcripts encoding human IL-33 and TSLP, mouse IL-4, IL-5, IL-13, and IL-33, or IL-33 levels were assessed by western blotting using human IL-33 antibody . Details are available in the online supplement.Secreted mouse IL-33 (DY3626) was quantified using the DuoSet® ELISA kit from R&D Systems according to the manufacturer’s instructions. To quantify IL-33, nasal lavage (NAL) fluid of a murine model was collected. The working range of the assay was 15.6–1000 pg/mL.P-value of < 0.05 was considered statistically significant. All statistical analyses were performed with GraphPad Prism software .At least three independent experiments were performed with cultured cells from each donor, and data are expressed as mean ± standard deviation (SD) of three independent experiments. Groups were compared by the Mann-Whitney U test, and differences among treatment groups were evaluated by analysis of variance (ANOVA) with a post hoc test. A N = 17) were evaluated by analyzing cultured bacterial colonies and 16S rRNA gene sequencing, as previously described [Staphylococcus epidermidis, S. aureus, Enterobacter aerogenes, Corynebacterium accolens, Micrococcus luteus, Corynebacterium pseudodiphtheriticum, and Klebsiella pneumoniae were among the most commonly detected species. S. epidermidis demonstrated the highest abundance, accounting for 58.04% of mapped sequences, and S. aureus exhibited the largest increase in presence of microbial distribution in allergic nasal mucus  and S. aureus (AR-SA) in allergic nasal mucus. Both AR-SE and AR-SA strains were isolated from four AR subjects to assess the mechanistic link between dominant allergic nasal commensals and pathogenesis of AR in nasal mucosa.The microbial compositions of middle turbinate mucus in human subjects with AR N = 7 were evfemA-SA and femA-SE genes were then measured using RT-PCR. The results showed that femA-SA and femA-SE mRNA levels increased significantly from 8 h after inoculation, with the highest expression observed at 48 hpi  and were the highest at 48 hpi (1.28 × 109 ± 3.20 × 108 CFU/mL)  and were the highest at 48 hpi (1.85 × 109 ± 3.53 × 108 CFU/mL) . The mRNA levels of S. aureus targets IL-33 gene and protein expression in ARNE cells and compared expression levels in ARNE cells with those of AR-SA-inoculated NHNE cells. The IL-33 mRNA levels of ARNE cells were significantly higher than those of NHNE cells regardless of AR-SA inoculation . Reduced IL-33 gene expression was only observed in AR-SA inoculated ARNE cells until 48 h after inoculation . BALB/C mice (N = 5) were inoculated with human nasal microbiome S. aureus at 2 days  following nasal microbiota depletion  using 30 μl of an antibiotic cocktail   Fig. a. Nasal S. aureus mRNA levels were measured in in vivo nasal mucosa to confirm effective colonization of AR-SA in WT mice and AR-OVA mice after inoculation. RT-PCR results revealed that femA mRNA levels and colony counts of S. aureus were significantly higher in WT and AR-OVA mice with S. aureus inoculation than in WT and AR-OVA groups without inoculation  and 17.01 ± 1.43 (AR-OVA), respectively. These scores were significantly reduced in AR-OVA mice with human nasal microbiome AR-SA inoculation  , IL-33 activates target cells to produce Th2 cytokines that regulate local and systemic allergic inflammation , 33. BotS. aureus colonization enhances resistance to allergic inflammation in human nasal epithelium by reducing IL-33 expression in nasal mucosa. Thus, intranasal delivery of human nasal commensal S. aureus represents a potential therapeutic approach for preventing and treating allergic inflammation via reduction of epithelial cell-derived Th2 cytokines.Our study increases understanding of whether the nasal microbiome of subjects with AR reduces Th2 inflammation to protect AR nasal epithelium from IL-33-mediated inflammation. We showed that abundant Additional file 1. A video file of the nasal swab for sample collection.Additional file 2. Supplementary material and method.Additional file 3. Supplementary figure.