Introduction: A multi-degree-of-freedom ankle rehabilitation robot with an adjustable workspace has been designed to facilitate ankle joint rehabilitation training. It features a rotation center adapted to the human body, making it suitable for patients with ankle dysfunction following a stroke.Method: In this study, a multi-degree-of-freedom reconfigurable ankle rehabilitation robot (RARR) with adaptable features, based on the principles of ergonomics, has been proposed to cater to the varying needs of patients. This robot offers an adjustable workspace, allowing for different types of ankle joint rehabilitation exercises to be performed. By adjusting the assembly of the RARR, personalized and targeted training can be provided to patients, circumventing issues of redundancy in degrees of freedom during its use. A kinematic model of the robot has been established, and finite element simulation has been employed to analyze the strength of critical components, ensuring the safety of the robot. An experimental platform has been set up to assess the smoothness of the rehabilitation process with RARR, with angle measurements conducted using an Inertial Measurement Unit (IMU).Results and discussion: In conclusion, both simulation and experimental results demonstrate that the robot offers an adjustable workspace and exhibits relatively smooth motion, thereby confirming the safety and effectiveness of the robot. These outcomes align with the intended design goals, facilitating ankle joint rehabilitation and advancing the field of reconfigurable robotics. The RARR boasts a compact structure and portability, making it suitable for various usage scenarios. It is easily deployable for at-home use by patients and holds practical application value for wider adoption in rehabilitation settings. Stroke is an acute cerebrovascular disorder characterized by a high incidence rate, elevated mortality, and a range of complications . In receAnkle rehabilitation robots have extensive applications in the field of rehabilitation medicine, including but not limited to ankle injury rehabilitation, athlete rehabilitation, and elderly population rehabilitation, among other application contexts. Traditionally, ankle rehabilitation robots can be categorized into two main types: wearable and platform-based systems . PlatforWhen developing ankle rehabilitation devices, it is essential to prioritize user needs, such as device adjustability and adaptability, the capability for personalized rehabilitation plans, a comfortable user experience, and low usage costs, among other aspects. However, this presents several challenges: 1. Precision and Safety: Precision and safety are fundamental prerequisites for designing such devices. The robot must ensure the effectiveness and safety of the equipment. 2. Cost and Accessibility: The manufacturing cost of the robot and the accessibility of the equipment pose a challenge. Rehabilitation robots need to be produced within a reasonable price range to ensure widespread availability to medical institutions and patients. 3. Software Development and Algorithm Design: Complex control algorithms need to be developed to enable the robot to provide personalized rehabilitation plans. The causes of ankle injuries in patients are diverse, and each patient may require different rehabilitation approaches. Therefore, the development of a personalized and reconfigurable ankle rehabilitation robot tailored to individual patient needs holds significant importance.This study presents a multi-degree-of-freedom ankle rehabilitation robot with an adjustable workspace for post-stroke lower limb ankle rehabilitation. The key feature of this robot is its ability to be personalized and assembled according to the specific rehabilitation needs of different users, involving the selection of varying numbers of actuators and assembly modes to achieve different workspaces for the robot. This customization aims to provide personalized treatment, avoid redundancy of degrees of freedom, and alleviate the financial burden on users. The organization of the remainder of this study is as follows: This section provides a detailed account of the design process for the RARR system, comprising two main aspects: the mechanical structure of RARR and the control system. The mechanical structure can be further subdivided into three key components: reconfigurable design, adjustable design, and rotation center matching design. The RARR’s mechanical structure encompasses three degrees of freedom, and its adaptability is achieved by selecting different numbers of actuators and diverse assembly configurations to accommodate adjustable robot workspaces. These configurations can be set to enable three single-axis motion modes: dorsiflexion/plantarflexion, inversion/eversion, and internal/external rotation; three dual-axis motion modes: dorsiflexion/plantarflexion with inversion/eversion, dorsiflexion/plantarflexion with internal/external rotation, and inversion/eversion with internal/external rotation; and one three-axis motion mode. The adjustable design allows for user-specific adaptability, facilitating both left and right foot interchangeability and catering to users of varying heights. The rotation center matching design is implemented to prevent patients from incurring secondary injuries while using the system. The control architecture of RARR is composed of four main modules: the central control module, sensing module, selection module, and actuation module.The RARR model is illustrated in In the process of rehabilitation training, multifunctional or reconfigurable robots are more appealing to patients as they can reduce healthcare costs and improve the effectiveness of rehabilitation training . To cateTo accommodate users with varying lower leg lengths, the robot can be adjusted. By changing the position of the sliders, the functional sections of the robot can be adjusted forward and backwards along the guide rail to suit users of different heights. In RARR, adjustable limit structures are employed at the internal/external rotation and inversion/eversion rotational joints, allowing patients to switch between left and right foot rehabilitation training, as depicted in An important consideration in the design of rehabilitation robots is ensuring that the robot’s rotation center aligns with the ankle joint’s rotation center. Mismatched rotation centers can lead to patient discomfort or even secondary injuries, it is significant to figure out the issue of axis alignment of human-robot coupling . EnsurinThe RARR control system consists of four main modules: the central control module, sensing module, selection module, and actuation module, as depicted in In the course of a patient’s rehabilitation exercise, the controller governs the motion based on received signals and control algorithms. Subsequently, the actual joint angles of the robot are fed back to the central control module through motor encoders and posture sensors, forming a closed-loop control system. This system enables real-time and continuous monitoring and adjustment of the patient’s rehabilitation status.RPY angles. Initially, a stationary coordinate system {A} is set at the robot’s virtual rest center as a reference frame. Its Z-axis is parallel to the footplate plane, pointing toward the far end of the footplate, the X-axis is perpendicular to the footplate plane, pointing upward, and the Y-axis is parallel to the footplate plane, pointing to the right of the footplate. Simultaneously, a moving coordinate system {B} is established at the same origin, moving with the platform, with its axes oriented similarly to coordinate system {A}, representing the post-motion footplate plane, as shown in B relative to coordinate system A can describe the state of an object.The kinematic analysis of rehabilitation robots is of significant importance for workspace analysis, motion trajectory planning, and the determination of the robot’s feasibility. Therefore, it is necessary to establish a kinematic model for the analysis of relevant kinematics . In the γ, β and α represent the rotation angles of coordinate system B around RPY angles is as follows:Here, {B} rotates around When performing dorsiflexion/plantarflexion motion, coordinate system Similarly, we can derive the kinematic models for the other modes as follows:Inversion/Eversion Motion Mode:Internal/External Rotation Motion Mode:Dorsiflexion/Plantarflexion and Inversion/Eversion Motion Mode:Dorsiflexion/Plantarflexion and Internal/External Rotation Motion Mode:Inversion/Eversion and Internal/External Rotation Motion Mode:Three Degrees of Freedom Motion Mode:XYZ fixed-angle representation in RPY. Let:Given the rotation matrix, we can deduce the Through derivation, we can obtain:By dividing Based on the normal range of motion of the human ankle joint and the mechanical limit design of the robot, we can determine that Where The workspace of the robot is a crucial metric for evaluating its feasibility, as it reflects the robot’s performance and directly impacts its practical application value . To visuOne of the primary objectives of this study is to reduce the volume and overall weight of the ankle rehabilitation robot by simplifying the mechanical structure and optimizing materials. This approach aims to minimize psychological stress on patients during the training process and reduce energy consumption. Considering factors such as material yield strength and mass density, 6,061 aluminum alloy was chosen as the primary material for key components of the RARR. The main material properties of 6,061 aluminum alloy are outlined in To validate the reliability and rationality of the mechanical design and material selection in this study, finite element analysis was conducted on selected key components of the designed mechanism, as illustrated in 2 and 3.8147e+007N/m2, respectively. These values are significantly below the material’s yield strength of 5.51485e+007N/m2. The maximum strains are 7.2267e-005 m/m and 5.7207e-004 m/m for Part 1 and Part 2, respectively. Additionally, the maximum displacement values are 1.762e-006 and 3.9592e-006 m for Part 1 and Part 2, respectively, indicating minimal deformation. These results suggest that the chosen materials for Key Components 1 and 2 are appropriate, and the dimensional design of the components meets the design requirements.From the information presented in the figures, it is evident that the maximum stresses locally experienced by Part 1 and Part 2 are 4.975e+006N/mIn accordance with the design plan, the prototype was successfully manufactured, and seven different assembly modes were realized through various assembly methods, as illustrated in One healthy subject  was recruited to undergo passive rehabilitation training. This study was conducted with the approval of the Ethics Review Committee of Shanghai University of Medicine and Health Sciences , and all procedures adhered to the standards outlined in the Helsinki Declaration.Passive rehabilitation training is suitable for early rehabilitation of ankle joint dysfunction patients who may exhibit symptoms of muscle weakness, low muscle strength, or high muscle tone. Hence, the experimental setup involved lower joint operating speeds to ensure system safety. After donning the experimental prototype, the subject underwent passive control rehabilitation training to verify the effectiveness and stability of joint motion during the testing, as shown in During the training process, first, zeroing the IMU device was performed to address errors caused by the installation of the IMU by the experimenter . SubsequThis study has successfully developed and evaluated a multi-degree-of-freedom, reconfigurable ankle rehabilitation robot with a variable working space to assist in the rehabilitation training of patients with ankle dysfunction following a stroke. The robot’s variable working space and reconfigurable characteristics allow it to adapt to seven different modes, catering to the diverse rehabilitation needs of users. The mechanism incorporates three rotational degrees of freedom around the human ankle’s rotation center, reducing discomfort for users during operation. Theoretical analysis was conducted to determine the relevant parameters of the robot and validate its performance. Subsequent experiments demonstrated the robot’s reconfigurability and the smoothness of training trajectories, aligning with the design objectives. Designed with principles of human factors engineering, the robot features a compact structure, portability, and cost-effectiveness, making it suitable for home use by patients. The modular design helps reduce equipment usage and maintenance costs, depending on the robot’s assembly mode, its manufacturing cost ranges from $800 to $1200. As a result, the developed Reconfigurable Ankle Rehabilitation Robot (RARR) holds practical application value in ankle rehabilitation and is primed for widespread use. Furthermore, we believe that the design methods/principles of this device can provide valuable insights for the design of devices for other body parts. For example, in the design of upper limb exoskeleton robots, the interchangeable and adjustable design can facilitate patients in performing rehabilitation exercises for different limbs. Additionally, the adjustability of robot mechanism lengths can accommodate various patients. The application of a simple, compact, and portable design concept not only reduces the production cost of the robot but also allows patients to use it in different settings. In the future, we will continue to enhance the device’s practicality and aesthetics, integrating various human-machine interaction modes such as voice control and a plantar electrostimulation system to increase its utility. Clinical trials will also be conducted to further enhance the rehabilitation effectiveness in clinical practice.


This nanocompartment-confined strategy breaks the shackles of conventional polymerization, providing a universal platform for in vivo synthesis of polymers with diverse structures and functions.Polymerization in living systems has become an effective strategy to regulate cell functions and behavior. However, the requirement of high concentrations of monomers, the existence of complicated intracorporal interferences, and the demand for extra external stimulations hinder their further biological applications. Herein, a nanocompartment-confined strategy that provides a confined and secluded environment for monomer enrichment and isolation is developed to achieve high polymerization efficiency, reduce the interference from external environment, and realize broad-spectrum polymerizations in living systems. For exogenous photopolymerization, the light-mediated free-radical polymerization of sodium 4-styrenesulfonate induces a 2.7-fold increase in the reaction rate with the protection of a confined environment. For endogenous hydrogen peroxide-responsive polymerization,  Polymerizations in living systems can effectively regulate cell functions and behaviors, but their uses have been hindered by the existence of complicated intracorporal interferences, the needs of high concentration of monomers and extra stimulates. Here, the authors address these issues by developing a nanocompartment-confined strategy to achieve broad-spectrum polymerizations in living systems. Until now, various exogenous chemical reactions have been achieved in living systems for the synthesis of fluorescent probes or active drugs including click reaction, transfer hydrogenation, and removal of protective groups8. Polymerization reaction, a well-known method to produce multiple synthetic macromolecules with diversity and tunability properties, provides great possibilities for chemists to understand, analyze and regulate cell performance9. To date, some proof-of-concept covalent and supramolecular polymerizations have been achieved in living cells to create new functional capabilities to modulate biological responses11. For illustration, Bradley et al. described the direct free radical photopolymerization inside living cells to manipulate cellular behavior12; Deisseroth and Bao et al. genetically engineered specific living neurons to synthesize electrical functional polymers to regulate the electrophysiological behaviors13; Weil et al. designed a platinum(II)-containing tripeptide that assembled into intracellular fibrillar nanostructures to disrupt energy homeostasis and cellular metabolism after response to endogenous hydrogen peroxide (H2O2)14. Despite these encouraging findings, artificial biological polymerization still encounters certain problems: (1) transportation of high concentrations of monomers is always required; (2) the ubiquitous bioactive and metabolic substances in the living system might quench the polymerization process; (3) most polymerizations demand extra stimulates (ultraviolet (UV) irradiation, metal ions, etc.) to initiate the reaction; and (4) the synthetic polymers suffer from functional limitations to modulate additional biological responses5. The existence of these challenges hampers the pace of polymerization in living systems, thereby motivating researchers to actively seek solutions.Living organisms are replete with a wide variety of important chemical reactions in both intracellular and extracellular microenvironments, e.g., the biological synthesis of polysaccharides, proteins, and nucleic acids, to constitute the elementary components, confer essential functionalities, and modulate the biological process Fig. 1–5. Unti2O2-responsive polymerization) have been investigated  sulfosuccinate), FDA-approved anionic oil-soluble surfactant, was exploited to investigate the vesicle-directed confined polymerization , fluorescence enhancement for the NaSS@AOT group (5.5-fold) was 2.9 times higher than that of NaSS group (1.9-fold), validating the nanocompartment-confined effects  represent appealing stimuli to trigger biological polymerization16. Horseradish peroxidase (HRP), an enzyme known to catalyze H2O2-dependent one-electron oxidations, could promote the polymerization of natural or unnatural aromatic monomers to obtain corresponding polyaromatic products15. Previous research has demonstrated the effectiveness of AOT anionic vesicles in facilitating HRP-catalyzed reactions, particularly when utilizing aniline analogues as the monomers17. Here, aromatic amine monomers , p-phenylenediamine (PPD), m-phenylenediamine (MPD), p‑aminodiphenylamine (ADPA), 4,4’-iminodianiline (NDA), and ADPA in the form of amine hydrochloride salt (ADPAH)) was separately entrapped in the cavity of the AOT vesicle, and H2O2 was utilized to trigger the oxidative polymerization as it could transport through the bilayers due to the low polarity  . In the NIR-II window, the combination of low absorption by main endogenous absorbers and the reduced scattering from various tissues facilitates numerous advantages, including deep tissue penetration capabilities (at least 20 mm), high maximum permission energy, and excellent sensitivity24. From this perspective, after NIR-II laser (1064 nm) irradiation, the temperature of ADPAH@AOT with the addition of H2O2 (100 μM) quickly increased 42.3 °C in 10 min, 3.5 times higher than that of free ADPAH monomer at the same condition  and followed by treatment of ADPAH@AOT. An upregulated CRT expression was shown in the CLSM images of ADPAH@AOT/NIR-II group, while negligible red signals were observed in the PBS group , e.g., calreticulin (CRT), high mobility group box 1 (HMGB-1), and adenosine triphosphate (ATP)oup Fig. . The sigoup Fig. . Moreoveoup Fig. .2O2 (100 μM) and followed by a NIR-II laser irradiation, then the immature bone marrow-derived dendritic cells (BMDCs) were seeded in the lower chambers  maturation. 4T1 cells in the upper chambers were pre-treated with ADPAH@AOT in the presence of Hers Fig. . In conters Fig. . Besidesers Fig. . These r2O2-triggered polymerization, indicating successful oxidative polymerization in living system. This endogenous oxidative polymerization also contributed to activatable PTT as revealed by the increased temperature (11.6 ± 0.1 °C) in ADPAH@AOT treated tumors after NIR-II irradiation compared with a slight temperature increase (3.9 ± 0.1 °C) in PBS group after 5 min of irradiation  provides a collagen network and serves as a natural barrier for immune evasion and attenuation of antitumor therapy30. Bromelain, a protease, not only possesses anti-inflammatory, anticancer, and immunomodulatory activities, but also exhibits a preferred catalytic temperature of ~45 °C32. This temperature preference makes it suitable for a photothermal-mediated enzyme switch in efficient collagen digestion33. In this regard, bromelain was incorporated in ADPAH@AOT to promote ECM degradation, assist antitumor activity, facilitate immunomodulatory, and enhance T cells infiltration into the tumor tissues. The collage degradation efficacy of ADPAH@AOT-Bro/NIR-II group, expressed as gelatin digestion units (GDU), was calculated to be 3253.6 ± 169.8 GDU g−1, higher than that without NIR-II laser irradiation , compared to that of ADPAH (32 ± 2.5%) and PBS (25 ± 1.6%) group, respectively  and ADPAH@AOT group (82.6 ± 10.3 pg mL−1), respectively. These data implied that the H2O2-responsive polymerization activated effective NIR-II PTT at tumor sites, which could also activate immunogenic cell death, promote DC maturation, and assist with ECM degradation bromelain to facilitate the T cell infiltration  staining of both the primary and abscopal tumors. The fluorescence intensity of CY3-labeled anticaspase-3 antibody, representing the degree of cell apoptosis, was higher in ADPAH@AOT-Bro/NIR-II group than that in other groups on both flank sides Fig. . In deta2O2-responsive polymerization were selected to study nanocompartment-directed biological polymerization. The illumination of NaSS in AOT vesicle induced a 2.7-fold increase in the rate of polymerization and a 2.9-fold enhancement of fluorescence in living cells than those of NaSS group, respectively.In summary, a nanocompartment-confined strategy was established to achieve various types of polymerizations in living systems. This compartmentalization provides a confined environment for monomers enrichment and isolation, offering the possibility to increase the polymerization rate and decrease the potential toxicity. The commonly used exogenous light-mediated free-radical polymerization and endogenous H2O2 overexpressed tumor sites and activated an effective NIR-II PA imaging and PTI; (2) enabled a 6.4-fold higher reaction rate for ADPAH@AOT group than that of free ADPAH group; (3) induced a 3.5-fold temperature increase after ten-minute NIR-II irradiation, offering the potential for dosage mitigation and toxicity reduction. After the assistance of bromelain to digest collage in ECM, the infiltration of T lymphocytes in tumor tissue for ADPAH@AOT-Bro/NIR-II group was enhanced by 2.0-fold for the primary tumor and 2.1-fold for the distant tumor than those of ADPAH@AOT/NIR-II group, respectively. In particular, ADPAH@AOT/NIR-II group also showed the highest antitumor efficiency for both the primary and distant tumors among all groups. We envision that this confined polymerization approach would greatly promote the feasibility of conducting multiple polymerization reactions in living systems to confer new functionalities, modulate biological processes, and even fabricate cellular compartments.The nanocompartment-directed oxidative polymerization: (1) occurred at HMouse mammary carcinoma cell 4T1 and NIH-3T3 fibroblasts were obtained from the American Type Culture Collection (ATCC). Balb/c female mice (5-6 weeks) were purchased from InVivos Pte. Ltd. (Singapore). All animal experiments were reviewed and approved with the Guidelines for Care and Use of Laboratory Animals of the Institutional Animal Care and Use Committee of Nanyang Technological University (NTU-IACUC) with a protocol number of A19016.NaSS  and Irgacure 2959  were added into deionized water (2.0 mL). AOT  was added to the above solution under vigorous stirring to obtain the NaSS@AOT. Then, NaSS and NaSS@AOT were illuminated using a UV lamp (365 nm) and their emission spectrum (excitation at 480 nm) was recorded every minute.2 incubator. 4T1 cells were then grown in an eight-well plate (1 × 104 cells per well) overnight and then added with NaSS or NaSS@AOT (containing 25 mM of NaSS and 1 mM of Irgacure 2959). After incubation for 4 h, cells were washed three times with PBS solution and then illuminated at 365 nm for 5 min. Finally, the nuclei were stained with Hoechst 33342 and the samples were subjected to CLSM observation.Murine mammary carcinoma cells (4T1) were grown in RPMI 1640 culture medium containing 10% fetal bovine serum and 1% antibiotics (penicillin and streptomycin) at 37 °C in a 5% COHCl (6 mol) was slowly added to a solution of monomer  in tetrahydrofuran (THF) solution (10 mL). After stirring for 0.5 h, the sediment was filtrated to get ADPAH as a brown solid. Other substrates  in the form of amine hydrochloride salt were obtained using the same method.ADPAH  and HRP  were added into deionized water (2.0 mL). Then, AOT  was added to the above solution under ultrasound conditions to obtain the ADPAH@AOT. Other monomers@AOT  were obtained using the same method.2O2-responsive polymerization, the UV–vis/NIR absorption spectra of OPD@AOT, PPD@AOT, MPD@AOT, ADPA@AOT and NDA@AOT before and after 8 h-incubation with 100 μM H2O2 were measured. For the kinetics study, all of the above reactions were performed by measuring the absorbance of OPD@AOT (430 nm), PPD@AOT (555 nm), MPD@AOT (490 nm), ADPA@AOT (655 nm), NDA@AOT and ADPAH@AOT (1003 nm) at different reaction time points, and the reaction rate of each polymerization was measured from the slope of the absorbance. For the interference experiment, 10% of fresh cell supernatant was added to the above solution. Then their absorbance at different reaction time points was measured and the reaction rate was calculated.To study the H2O2-responsive polymerization of ADPAH@AOT, various concentrations of H2O2  were incubated with ADPAH@AOT for 8 h with gentle stirring, followed by UV–vis/NIR absorption spectra measurement. To study the specificity response to H2O2, ADPAH@AOT (50 μg mL−1) was incubated with various reactive oxygen species  (100 μM) for 10 min with gentle stirring, followed by UV–vis/NIR absorption spectra measurement.The further investigation of H29. The 6-31 + G(d) basis sets were chosen to describe all atoms. For open-shell singlet, the keyword ‘guess=mix’ was used. Frequency calculations were carried out at the same level of theory to identify all of the stationary points as minima (zero imaginary frequency) and to provide the thermal correction to free energies at 298.15 K and 1 atm. For comparing the free energies of different conformations and calculating redox potentials, the higher accuracy single-point energy calculations were performed at the M06-2X level with the 6-311 + G for all atoms, using the Solvation Model based on Density (SMD) with water as the solvent. The redox potentials were corrected to E0(SHE) = 4.28 V. The energies of molecular orbital and excitation energy (using TD-DFT) were calculated at the CAM-B3LYP-D3(BJ)/6-311 + G level with SMD. All calculations were performed with the Gaussian 16 (Revision A.03) software package.The initial structures of all molecules were generated by performing conformational searches using Grimme’s programs xTB 6.3 and CREST 2.10.2, and further optimized using the (U)B3LYP functional with Grimme’s D3(BJ) dispersion correction2O2-dependent photothermal property, ADPAH@AOT aqueous solution (50 μg mL−1) with different concentrations of H2O2  was irradiated using a 1064 nm laser (1 W cm-2) for 10 min. The temperature of ADPAH@AOT solution was recorded by a thermal infrared imaging camera . For the concentration-dependent photothermal property, ADPAH@AOT aqueous solution with different concentrations of ADPAH  was irradiated by 1064 nm laser for 10 min, and the temperature change was recorded by using a thermal camera. Subsequently, ADPAH@AOT solution  with different power densities  was irradiated by 1064 nm laser for 10 min, and the temperature was recorded every 30 s. To investigate the photostability of polymerized ADPAH@AOT, 1.0 mL of ADPAH@AOT (50 μg mL−1 with the addition of 100 μM) in water was exposed to 1064 nm laser (1 W cm−2) irradiation for 10 min, and then cooled down for 15 min. This procedure was performed for four consecutive cycles.For the HThe biocompatibility of ADPAH@AOT was evaluated using MTT -2,5-diphenyl-2H-tetrazolium bromide) assay. NIH-3T3 fibroblasts were grown onto a 96-well plate (7000 cells/well) and followed by incubation overnight. Then, the medium was replaced with a fresh medium containing ADPAH@AOT of known concentrations. After 24 h, the cells were incubated in a cell medium (100 μL) containing an MTT solution (10 μL). After another 4 h incubation, DMSO (150 μL) was injected to replace the medium. The absorbance was measured by a microplate reader at the 490 nm wavelength. The relative cell viability (%) was determined by comparing the absorbance values of sample wells with that of control wells.2O2 100 μM to mimic the tumor microenvironment). After 24 h, the cells were incubated in a 100 μL cell medium containing MTT solution (10 μL). After another 4 h incubation, DMSO (150 μL) was injected to replace the medium. The absorbance was measured by a microplate reader at the 490 nm wavelength. The relative cell viability (%) was determined by comparing the absorbance values of sample wells with that of control wells. As for the ADPAH@AOT/NIR-II group, 4T1 cells were treated with ADPAH@AOT (20 μg mL−1) for 4 h and then exposed to 1064 nm (1 W cm−2) light irradiation. After another 20 h incubation, cell viability was detected using an MTT assay kit.The cytotoxicity of ADPAH and ADPAH@AOT was also evaluated using an MTT assay. 4T1 cells were grown onto a 96-well plate (7000 cells/well) and followed by incubation overnight. Then the medium was replaced with a fresh medium containing ADPAH@AOT of known concentrations (containing H−1) for 4 h and then exposed to 1064 nm (1 W cm−2) laser irradiation. After another 20 h incubation, the supernatants were collected for ATP quantification and the cells were washed, fixed , permeabilized (0.1% Triton X-100 for 10 min), and blocked (3% BSA for 60 min). Then anti-CRT antibody (or HMGB-1 antibody) was incubated with the cells at 4 °C overnight. After washing with PBS three times, the cells were incubated with the corresponding secondary antibody at room temperature for 2 h. Subsequently, the nuclei were stained with Hoechst 33342 and the samples were observed by CLSM.For immunofluorescence staining of CRT (or HMGB-1) expression, 4T1 cells were grown onto an eight-well plate  and followed by incubation overnight. Thereafter, 4T1 cells were treated by ADPAH@AOT  in the presence of H2O2 (100 μM) and followed by a NIR-II laser irradiation, then the BMDCs were seeded in the lower chambers. After coincubation for 6 h, the DCs were harvested and co-stained with APC- conjugated anti-mouse CD11c, FITC-conjugated anti-mouse CD80, and PE-conjugated anti-mouse CD86 antibodies, followed by flow cytometry. At the same time, the media supernatants were also collected for cytokines detection.Here, a 24-well transwell system was utilized−1) with different concentrations of H2O2  was added into photoacoustic glass tubes to collect the PA intensity under 1064 nm laser. For in vivo PA imaging, ADPAH@AOT solution was intratumorally administrated into 4T1 tumor-bearing mice at the dosage of 3.0 mg kg–1 for PA imaging scans under 1064 nm laser irradiation.ADPAH@AOT solution  were added into deionized water (2.0 mL). Then, AOT  was added to the above solution under vigorous stirring to obtain the ADPAH@AOT-Bro.ADPAH , HRP , and bromelain  was placed into 5 mL tubes. Then, 1.5 mL of ADPAH@AOT or ADPAH@AOT-Bro solution (2 mg mL−1) was added into the tubes and the solution was mixed homogeneously. The tubes were exposed to a 1064 nm laser (10 min) and hydrogen peroxide solution  was added to stop the reaction. Subsequently, the pH of the solution was adjusted to 6.0 with NaOH and then formaldehyde  was added to the solution. Titration was performed with 0.05 N NaOH until the pH reached 7.8 and the titration volume of the test solution was recorded. For the blank, gelatin solution  was placed into 5 mL tubes containing H2O (1.5 mL) and then treated with hydrogen peroxide solution . Titration was performed as above, and the titration volume of the blank solution was also recorded. Enzymatic activity was calculated as follows: GDU = (test volume−blank volume) × 700/mg enzyme.The enzymatic activity of ADPAH@AOT-Bro towards gelatin was defined as Gelatin Digestion Unit (GDU): one unit digests 1.0 mg of amino nitrogen from gelatin in 40 min6 cells) on the right side. After five days, the left flanks were injected with 4T1 cells (1 × 106 cells) and designed as distant tumors. Once the primary tumor volume reached ~50 mm3, ADPAH@AOT-Bro was intratumorally injected into the primary tumors at the dosage of 3.0 mg kg−1 (n = 3). At 4 h post-injection, primary tumors received 1064 nm NIR light  irradiation. On day 7, mice were sacrificed and blood, tumors, and spleens of mice were harvested. Blood was placed at room temperature for 2 h and then centrifuged at 300 × g for 5 min to obtain serum for IFN-γ detection. The tumors on the right side were collected for CRT and HMGB-1 staining, and the tumors from both sides were collected for CD4 and CD8 immunofluorescence staining. At the same time, the tumors were collected and cut into small pieces and incubated with the solution  at 37 °C for 2 h, and then the mixture was filtered through a strainer to get single cell suspension. Thereafter, the cell suspensions were co-stained with Alexa Fluor®700 anti-mouse CD45, FITC-conjugated anti-mouse CD3, PE-conjugated anti-mouse CD4, and APC-conjugated anti-mouse CD8 for 20 min, and followed by flow cytometry analysis. Spleen and tumor-draining lymph nodes were pressed gently and filtered through a strainer to get a single-cell suspension. Then, the cell suspensions were stained with APC anti-mouse CD11c, FITC-conjugated anti-mouse CD80, and PE-conjugated anti-mouse CD86, followed by flow cytometry analysis.Mice were subcutaneously injected with 4T1 cells (1 × 106 cells) on the right side. After five days, the left flanks were injected with 4T1 cells (1 × 106 cells) and designed as distant tumors. Once the primary tumor volume reached ~50 mm3, mice were divided into five groups (n = 5 in each group): (1) PBS, (2) ADPAH@AOT, (3) ADPAH@AOT-Bro, (4) ADPAH@AOT/NIR-II, and (5) ADPAH@AOT-Bro/NIR-II. The primary tumors were intratumorally injected with ADPAH@AOT and ADPAH@AOT-Bro at the dosage of 3.0 mg kg–1. At 4 h post-injection, mice in groups (4) and (5) were exposed to a 1064 nm (1 W cm−2) light for 5-min irradiation and the temperatures were recorded every minute via a thermal camera. The tumor volume was measured with a caliper every other day, which was calculated as followings: tumor volume (V) = a × b2 / 2, where a is the major axis and b is the minor axis of the tumor. The tumors for both sides were harvested for caspase-3 and H&E staining on day 14 after the mice were sacrificed.Mice were subcutaneously injected with 4T1 cells (1 × 10p < 0.05, **p < 0.01, and ***p < 0.001 were considered to be statistically significant. All statistical calculations were conducted using GraphPad Prism V 9.5.0.Results were expressed as mean ± SD. Shapiro-wilk test was used to assess the normality of the data. Statistical comparisons were conducted by one-way ANOVA with a Tukey post-hoc test. For all results, *Further information on research design is available in the Supplementary InformationReporting SummaryPeer Review FileSource Data


Here, we assume X and Y both are independent random variables of progressively first failure censored (PFFC) data following inverse Pareto distribution (IPD) as stress and strength, respectively. This article deals with the estimation of SSR from both classical and Bayesian paradigms. In the case of a classical point of view, the SSR is computed using two estimation methods: maximum product spacing (MPS) and maximum likelihood (ML) estimators. Also, derived interval estimates of SSR based on ML estimate. The Bayes estimate of SSR is computed using the Markov chain Monte Carlo (MCMC) approximation procedure with a squared error loss function (SELF) based on gamma informative priors for the Bayesian paradigm. To demonstrate the relevance of the different estimates and the censoring schemes, an extensive simulation study and two pairs of real-data applications are discussed.The stress-strength reliability (SSR) model  X and Y are random variables measuring the strength and stress of a system, respectively. Then the system’s SSR is described as ϕ = P(Y < X), and the system will fail if X ≤ Y. This concept was first suggested by ), where t is chosen such thatx] is the integral part of x.Here, using the generated MCMC samples, we obtained the HPD credible interval of SSR  help of  algorithϕ is obtained under SELF by incorporating gamma prior distributions. The following steps are carried out for the simulations as follows:The efficiency of the various estimators covered in this work is examined using a comprehensive Monte Carlo simulation study. The average values (AV) and mean squared errors (MSE) are used to compare these estimators (MSE). In addition, the interval estimates are compared with average lengths (AL). The Bayes estimate of SSR n = n1 = n2 with same group sizes k = k1 = k2. Also, we assumed same prefixed number of failures m = m1 = m2 with same prefixed censoring schemes Considered number of groups The different sample sizes with different prefixed censoring plans for computation purposes are tabulated in α1, α2) =  and  =  are chosen. Consequently, the corresponding true values of SSR ϕ become, ϕ = 0.80 and ϕ = 0.60, respectively.Two sets of true parameters )k from IPD.Generate m, using  algorithϕ. Also, compute the ACI of SSR based on ML estimators.Compute MPS and ML estimates of SSR r1, s1) = ,  = } and { = ,  = } with the assumption that the prior means equal to true parameter values, respectively. M = 10, 000 posterior samples are generated using M-H algorithm, and M0 = 2, 000 samples are discarded as burn-in-period. Then, compute Bayes estimate and HPD credible interval of SSR ϕ.For Bayesian computations, the hyper-parameters are chosen as { and 20 mm (say Y1) by dividing each observation by 10. The transformed data sets are given by discusseX1 (15 mm): 59.440, 20.275, 16.837, 57.486, 22.565, 7.638, 15.667, 12.781, 81.387, 56.239, 46.847, 13.509, 7.224, 49.794, 35.556, 56.907, 64.048, 20.042, 55.042, 74.875, 48.966, 67.806, 45.771, 10.673, 71.630, 4.266, 8.040, 33.922, 7.009, 19.342.Y1 (20 mm): 7.146, 41.902, 28.464, 58.557, 45.660, 11.385, 18.785, 68.816, 66.266, 4.558, 57.862, 75.670, 59.429, 16.649, 9.972, 70.736, 76.514, 18.713, 14.596, 35.070, 54.744, 11.699, 37.581, 58.160, 11.986, 4.801, 20.016, 3.675, 24.453, 8.355.p-values based on ML estimates are computed. The ML estimates of unknown parameters α1 and α2 are computed as 19.2748 and 16.4732, respectively. The KS statistics  are computed as 0.2097 (0.1232) and 1926 (0.1894), respectively. Based on the p-values, we can say that the considered data sets are good fits for the IPD model. Further, after randomly grouping the considered data sets into n = 15 groups with k = 2 items within each group, and then consider the first failure censored samples. The bold observation shows the first-failure observation in the respective groups as shown in To begin, we examine the goodness of fit to see if the IPD can be used to analyze these data sets separately. The Kolmogorov-Smirnov (KS) statistics along with associated Finally, the first failure-censored samples of the considered data sets, respectively, are given byX1 (15 mm): 4.266, 7.009, 7.224, 8.040, 10.673, 13.509, 15.667, 16.837, 19.342, 20.042, 20.275, 45.771, 46.847, 49.794, 56.239.Y1 (20 mm): 3.675, 4.558, 4.801, 8.355, 9.972, 11.699, 14.596, 18.713, 18.785, 20.016, 24.453, 28.464, 37.581, 54.744, 59.429.m = 10. The four different common prefixed censoring schemes and their associated PFFC samples are as follows:Now, applying four different common prefixed censoring schemes on the above ordered first failure censored samples with effective sample size Scheme 1: Scheme 2: Scheme 3: Scheme 4: ri = si = 0.0001; i = 1, 2 as we don’t have any prior information. We generate 10,000 posterior samples from the marginal posteriors Now, we obtain the ML, MPS, and Bayes estimates of SSR under consideration of four different censoring schemes. Also, computed asymptotic confidence and HPD credible intervals of SSR, see, In this illustration, two different types of electrical insulation failure times (measured in seconds) under continuous-increasing voltage stress are considered. Two electrical insulation’s are tested and recorded each of size 30. These data are studied by . Here, wX2 (in seconds): 0.97, 0.14, 0.3, 1.34, 2.4, 0.84, 1.46, 0.24, 0.45, 0.04, 0.99, 2.77, 4.72, 0.94, 0.23, 1.46, 0.3, 0.31, 1.04, 1.05, 0.36, 0.65, 0.22, 0.98, 1.78, 0.59, 0.14, 0.07, 0.07, 2.86.Y2 (in seconds): 1.99, 2.52, 1.03, 4.55, 1.35, 3.48, 3.21, 1.66, 0.4, 0.27, 5.19, 2.7, 0.08, 0.3, 0.84, 2.36, 3.15, 1.77, 2.68, 1.8, 7.96, 2.45, 7.03, 0.45, 0.17, 8.21, 9.42, 3.14, 2.81, 6.52.p-value based on ML estimates are computed. The ML estimates of m = 8, which are tabulated in First, we check whether IPD fits these data sets. We find KS statistics along with associated The concept of estimating SSR for IPD using PFFC samples from both the classical and Bayesian prospective was tackled in this study. In the case of the classical estimation procedure, two estimation methods, the ML and MPS methods, are used to estimate SSR. The MPS method for PFFC data has not yet been discussed in the literature. Also, 95% of asymptotic confidence and HPD credible intervals of SSR were constructed. Extensive simulations were examined to see the performance of different estimation procedures. The outcomes of the simulation results suggest that the Bayes estimator is more precise than the ML and MPS estimators. In addition, the performance of the MPS estimator is quite better than the ML estimator. Thus, for all practical purposes, the Bayes estimator can be a good choice when the prior information is available; otherwise, the MPS or the ML method is commended. Finally, to demonstrate the methodologies considered in this study, we analyzed two different pairs of real data sets as illustrative examples. The approach and estimation results presented in this paper will be valuable to reliability practitioners in real-world situations.


Augmented reality and virtual reality technologies are witnessing an evolutionary change in the 5G and Beyond (5GB) network due to their promising ability to enable an immersive and interactive environment by coupling the virtual world with the real one. However, the requirement of low-latency connectivity, which is defined as the end-to-end delay between the action and the reaction, is very crucial to leverage these technologies for a high-quality immersive experience. This paper provides a comprehensive survey and detailed insight into various advantageous approaches from the hardware and software perspectives, as well as the integration of 5G technology, towards 5GB, in enabling a low-latency environment for AR and VR applications. The contribution of 5GB systems as an outcome of several cutting-edge technologies, such as massive multiple-input, multiple-output (mMIMO) and millimeter wave (mmWave), along with the utilization of artificial intelligence (AI) and machine learning (ML) techniques towards an ultra-low-latency communication system, is also discussed in this paper. The potential of using a visible-light communications (VLC)-guided beam through a learning algorithm for a futuristic, evolved immersive experience of augmented and virtual reality with the ultra-low-latency transmission of multi-sensory tracking information with an optimal scheduling policy is discussed in this paper. With the enormous growth in commercialized fifth-generation 5G) cellular systems and 6G technology on the horizon by 2030 , there iG cellulain AR/VR . The expThe end-to-end latency requirements for an AR/VR system include an interactive remote environment with a latency often ranging between  content . Though  content . Therefo content  in enabl content . Such a  content  coupled  content .There are several factors responsible for the association of delays with the simulation environment and the AR/VR devices while presenting an enhanced view of the surroundings to the users in the virtual world. The low end-to-end delay in AR/VR devices arises due to the steps involved in sensor sampling, data processing and fusion, image rendering and encoding, transmission and decoding, and displaying of each frame. These are some of the main causes for the occurrence of the motion-to-photon (MTP) latency, as shown in  latency  acts as al world . It has al world  that a ltrollers . Figure trollers  or post-trollers , have beWith the unprecedented growth of VR HMDs following the announcement of the Meta Oculus , there sTo meet the latency requirements of various AR and VR applications, a comprehensive discussion and quantitative analysis of the specifications are required. Novel communication designs with ultra-high speed, low power, and ultra-low latency specifications should be envisioned and developed. This paper discusses existing technologies, as well as several breakthrough solutions and advancements in ultra-low-latency communications in the 5G and Beyond era. Technologies such as WiFi 7, mobile edge computing (MEC), and the Tactile Internet will unlock new possibilities in remote control, mobility, automation, and industrial control, motivating communications service providers to further upgrade users’ experiences in real-time use-cases such as cloud gaming and AR/VR applications. Using such wireless technologies for network design, low-latency connectivity can also be achieved by focusing on finding an optimal end-to-end packet transmission path from transmitter’s Packet Data Convergence Protocol (PDCP) to the receiver’s PDCP from the device all the way to the application, wherever it is located , and back. This leads to a necessity to meet all the requirements of an optimal message transmission , technolThe remainder of this paper is organized as follows. In In this section, we review the main categories of applications that are using AR and VR technology as summarized in With the need for a new approach to the modern economy, the concept of Industry 4.0 has been introduced for innovation and technological development in the next industrial revolution by utilizing machines/robots to mimic the work of humans . IndustrAR/VR plays an important role in industrial innovations and contributes to their increased growth by bridging the existing gaps between the physical and virtual spaces by allowing people to exist in multiple different realities, mediums, and forms. Recently, investors have accelerated their interest in the AR/VR industry, and it is predicted that the industry will grow by more than USD 125 billion by 2024 . In AR, AR and VR provide maximum benefit in the entertainment industry with their applications based on improving the gaming experience and enhancing cinema, events, shows, and museums, hence attracting more customers. The gaming industry is investing a massive amount to deploy virtual environments in entertainment as the gaming market size is expected to grow at a compound annual growth rate of Research . AR/VR pResearch . ArtistsResearch .The authors in  providedAR/VR is gaining popularity in the healthcare sector, and the market for AR/VR in healthcare isexpected to reach nearly USD  5 years . The usa 5 years . The int 5 years ,38 that  5 years  to medic 5 years . While p 5 years . These A 5 years .AR/VR technology has a huge impact on the entire end-to-end process involved in the design, production, sale, and even marketing of automobiles. Moreover, the usage of the 5G network makes the adoption of these futuristic AR/VR technologies for high-speed data transfers through smartphones and Internet penetration with very low latency easier. The classical robotics methods used for risk assessment in road safety have been dominated by the usage of AR/VR technologies in autonomous vehicles to enhance road safety and improve the general driving experience . Three tAs the outbreak of COVID-19 has resulted in a huge loss in revenue for the tourism industry, leaders are resorting to AR/VR devices as a powerful tool for boosting the post-pandemic tourism industry . With thAR and VR have gained popularity by providing students an enhanced authentic classroom experience equipped with instructions from the educator. Utilizing AR/VR technologies in classrooms has been proven to be successful in engaging students with truly unique and quality content with 360-degree views. VR tools are used to supplement the materials of the educators by helping them enhance classroom instruction by overcoming discipline restrictions and offering the students an immersive learning experience. CAVE, an immersive virtual reality environment, is considered a suitable choice for classrooms as it can be easily used inside a room whose walls, ceilings, and floors may have projection screens . CAVE haIn this section, we review the major requirements for enabling a low-latency immersive experience from the hardware and software perspective of AR/VR technologies, as shown in The AR/VR industry has witnessed an evolution with the release of several consumer-grade VR headsets  in recent years . These dit (GPU) .Motion-to-photon (MTP) latency is the difference between the time of the motion captured by the headset sensor and the time of the appearance of the image on the headset’s display. Long MTP latency can cause various side-effects for users, such as nausea or motion sickness. Inertial measurement unit (IMU) sensors play an active role in sampling the motion data consisting of the user’s pose information, which will be later transmitted to the rendering engine to generate a framebuffer . A time on (PDM)  techniquications , provide-on-Chip  has beenA new strategy to determine total system latency was introduced in  to measumilar to  was perfThe selection of an optimal simulation platform plays a pivotal role the analysis and study of different types of latency. For the modeling of a simulation platform, adequate training is required by interacting with various complicated or costly scenarios of real outdoor environments. The simulator utilizes VR to simulate a complete AR system, which reduces the latency of the system. The simulator creates an arrangement responsible for precisely controlling the registration of virtual objects and allowing testing for the presence of registration error . An apprAR/VR utilizes 3D lifelike virtual avatars known as codec avatars  to mimicA small-scale system-on-a-chip (SOC) prototype was presented in  for low-pipeline . There apipeline  to updatpipeline  was modepipeline , by a flpipeline , was intpipeline  using a Both 5G and the WiFi 6 enhancements are acting as potential technologies to satisfy the market demands with their increased throughput and quality of service (QoS) requirements. The limitations of capacity and coverage occurring in 5G technology due to the lack of resources can be overcome by utilizing the free unlicensed spectrum in WiFi technology. Orthogonal frequency-division multiplexing (OFDM) modulation facilitates the coexistence of both technologies to enable higher speeds, high capacity, and low latency for enhanced QoS performances . WiFi isironment . WiFi 7 ironment . An imprA task group to enable AR/VR applications known as IEEE 802.11bf  has beenWith the proliferation of the IoT for many sensing devices in AR/VR applications, these devices suffer from limitations regarding the data, resources for computing, and communications . It has The TI is an innovative tool used for AR/VR applications comprising sensors, actuators, robotics, computation components, and dedicated hardware  to empower tactile and haptic  feedback in Internet-based applications . The TI nication .In this section, we describe several factors that are responsible for maintaining the performance parameter trade-offs in order to enable a low-latency and reliable wireless network for AR and VR applications. In The design of a smart 5G wireless network, as shown in ol plane  for provol plane . The latol plane , where tA UAV-aided wireless network design  can be udetector  in AR/VRdetector  also posdetector  to accelace link . A latenace link  to localace link  is consiThe reliability of the traffic behavior of information or video frames is being questioned in the wireless scenario of AR/VR network design due to the temporary outages occurring from the impairments in the signal-to-interference-plus-noise ratio (SINR). However, increasing the reliability always results in additional delay as re-transmission to send the rectified frames at the physical layer by using the parity increases the latency. The most-important reliability aspect in 5G to ensure a smooth and immersive AR/VR experience is obtaining an ultra-high success rate of tracking message signaling with a maximum packet error rate (PER) of shown in  to satisshown in  was consSeveral studies have been conducted to analyze the quality of experience (QoE) , which ius Rift) , providiers’ QoE .With the advancement of 5G technology, AR/VR requires significant resources for real-time processing, i.e., rendering, vision, and physics engines, at end user devices to maintain its ubiquitous nature across heterogeneous networks, including 4G, 5G, and WiFi. Usually, in mission-critical networks, the delivery of small information packets (32 to 200 bytes) within a latency of 1 ms  results T of 1ms . The reqnication . With thnication ,118 to iWe divide the environment into simple scenarios  and hard scenarios  based on the movement of the user’s HMD.For simple scenarios, we can consider reinforcement learning , where tFor hard scenarios, we can apply the combination of reinforcement learning and supervised learning, known as imitation learning (IL) , to tackTo implement delay-aware semantic communication involving a proactive transmission strategy of data packets for high delivery rates of video frames (VFs), interference-aware directional beamforming technology is an essential tool to generate highly optimized directional beams. Moreover, this approach requires some preprocessing steps to overcome the bandwidth limitations when handling packet overloading , which cThe algorithms as shown in This article provided insights into the various contributions of technological advancements in 5G and B5G networks to enable ultra-low latency for a high-quality immersive experience in AR/VR technologies. The main categories of AR/VR applications along with their benefits in each category were discussed. The major requirements of the powerful technologies in different emerging AR/VR use cases were highlighted from the hardware and software perspectives to enable a low-latency AR/VR system. Several factors responsible for maintaining the trade-offs of the performance parameters in order to enable a low-latency and reliable wireless network were presented. An innovative wireless network design integrated with ground-breaking technologies such as WiFi 7, the Tactile Internet, and edge computing were shown as the key enablers of 5G and the IoT in AR/VR applications. The usage of these technologies was highlighted as the most-powerful tools to perform intensive tasks while operating through an ultra-reliable and sub-millisecond-latency communication to enable a high-connectivity ecosystem. Future directions with a focus on high update rates for package delivery for low latency transmission were explored in this article. An optimal transmission queuing strategy was described with the use of several advanced technologies, such as the deployment of MIMO-based mmWave beamformers and intelligent reflecting surfaces for the timely delivery of the user’s tracking information. Moreover, the combination of VLC with holomorphic MIMO was presented as a potential solution to enable high reliability and data throughput in an AR/VR wireless architecture. An accurate route for transmission can be obtained by using this optimal strategy by exploring the semantic level of features by selecting or predicting the shortest path for high delivery of video frames. Visionary solutions, including the introduction of learning algorithms using RL and IL based on different scenarios, were described to achieve an optimal selection of the best scheduling policy for an evolved and extremely immersive AR/VR experience.


Adductor spasmodic dysphonia (AdSD) disrupts laryngeal muscle control during speech and, therefore, affects the onset and offset of phonation. In this study, the goal is to use laryngeal high-speed videoendoscopy (HSV) to measure the glottal attack time (GAT) and glottal offset time (GOT) during connected speech for normophonic  and AdSD voices. A monochrome HSV system was used to record readings of six CAPE-V sentences and part of the “Rainbow Passage” from the participants. Three raters visually analyzed the HSV data using a playback software to measure the GAT and GOT. The results show that the GAT was greater in the AdSD group than in the normophonic group; however, the clinical significance of the amount of this difference needs to be studied further. More variability was observed in both GATs and GOTs of the disorder group. Additionally, the GAT and GOT time series were found to be nonstationary for the AdSD group while they were stationary for the normophonic voices. This study shows that the GAT and GOT measures can be potentially used as objective markers to characterize AdSD. The findings will potentially help in the development of standardized measures for voice evaluation and the accurate diagnosis of AdSD. This is due to the low frame rate of videostroboscopy, which can only produce an illusory slow-motion of a vibratory cycle . The synAdductor spasmodic dysphonia (AdSD) is a type of focal dystonia that affects the laryngeal muscles and interferes with vocal fold vibrations and voice production. Perceptually, AdSD results in a strained, broken, breathy, and strangled voice  and is cCurrent literature studying AdSD using HSV has focused on steady-state phonation ,18. PateThe purpose of the current study is to expand on the above-mentioned studies by obtaining connected speech samples in patients with AdSD and those with no voice disorder  voices and quantitatively analyze the beginning and end of each phonation. Toward this goal, the glottal attack time (GAT) and glottal offset time (GOT) were measured for AdSD and normophonic voices in the connected speech samples. The GAT was defined as the time between the first oscillation and the first contact of the vocal folds at the phonation onset . AdSD is2.2.1.The data were obtained from five normophonic voices  and five with AdSD . HSV data were collected during the reading of the six CAPE-V sentences and the first six sentences of the Rainbow Passage. The HSV system consisted of a Photron FASTCAM mini AX200, M4 (32GB), monochrome high-speed camera , coupled with a flexible nasolaryngoscope. The recordings were conducted at a frame rate of 4000 frames per second (fps) with a spatial resolution of 256 × 224 pixels. HSV recordings of each participant included 200,000–400,000 frames in total and were stored as mraw files (Photron camera format) on an MSI GL63 8SE Laptop (the camera control system) for further analysis.2.2.The AdSD participants were recruited among the treatment-seeking population at Mayo Clinic, Scottsdale, AZ, diagnosed with AdSD  with a playback speed of 30 fps. They were allowed to adjust the playback speed as low as 1 fps to select the accurate frame numbers for the contact. The raters did not go below 15 fps for detecting the oscillation frame. The raters applied a Gaussian filter (with a kernel size of 3 × 3) and adjusted the gamma, gain, brightness, and contrast of the frames to facilitate the visual measurements. This filter was used to remove the pattern noise due to the camera lens and endoscope being slightly out of focus. The three raters completed their measurements for four participants and compared their measurements for every vocalization after completing the analysis. To do so, they met over video call and went over the vocalizations for which their measurements were more than 10 frames apart for the contacts and 20 frames apart for the oscillations. Accordingly, the measurements of GAT and GOT by each pair of raters were subtracted from each other , and the Wald 99% confidence interval was calculated for inter-rater reliability assessment. The raters watched the videos together and came to a consensus about their measurements. Based on this consensus meeting, Rater 3′s measurements were found to be the most reliable measures. Rater 3 improved the accuracy of her measurement based on the consensus meeting and completed the data analysis for the remaining five participants. In this study, Rater 3′s measurements for all participants were used for the data analysis. Additionally, two independent evaluators compared the measurements completed by the three raters to assess the similarity/dissimilarity of measurements among raters and determined the sources of discrepancies. The three Raters reviewed more than 2.6 million HSV images (frames) for the data analysis. Participant N1 was excluded from the analysis due to extremely dark HSV data.In addition to the descriptive statistics, parametric (ANOVA) and non-parametric tests  were used to compare the GAT/GOT between the normophonic and AdSD groups. Moreover, a generalized linear model with repeated observations was used based on the individual measures from the participants. For a regular general linear model (GLM or ANOVA), the estimations are carried out with the covariance matrix forced to sphericity (homoskedasticity); however, this assumption is violated when considering the individual data in this study. Therefore, in this study, rather than using the GLM, the models were built using the PROC MIXED (SAS 9.4) procedure with repeated observations. Instead of the least squares solution, the generalized linear model used a maximum likelihood solution without the sphericity assumption . In the repeated measures model, additional variables were introduced including gender, age, order of measurements, and interaction terms. At last, the dynamics of the GAT and GOT were evaluated using a time-series analysis.3.The three raters’ measurements for the first and last oscillation, also the first and last contact were within the limits of +/− 18 frames based on the Wald 99% confidence interval. The following is based on what the raters and evaluators concluded as the reasons for their agreement/disagreement with the measurements. More agreement was observed in the measurement of the contact frame number than in the oscillation frame number. More discrepancies in the measurement of the oscillation frame number were found at the offsets of vocalization than at the onsets. The raters were found to be more in agreement when they went further in the analysis of one participant’s data. Generally, more agreement was observed for the measurements of normophonic voices. One of the challenges in the measurement of the disordered voices was determining whether an onset belonged to a new phonation or that there was a delayed onset due to a phonatory break during the same phonation. In the measurements by Rater 1, in several instances, a continuation of phonation after an obstruction of the vocal folds was considered as a new phonation by mistake, which was more apparent for the disordered participants. The partial obstructions of the view of the vocal folds during phonation were found to be the main source of missing measurements by some raters. Both Rater 1 and 2 found Rater 3′s measurements more accurate for the four participants. Rater 3 adjusted her measurements further during the consensus meeting with the other raters. Rater 3 completed the analysis of the remaining participants considering what she had learned during the consensus meeting.The average GAT in the normophonic group varied from 13.5 to 15.4 ms, and from 11.2 to 22.3 ms for the AdSD group  and 2. TWe also investigated the relationship between GAT and GOT measures separately for the two groups. Based on the aggregate data, the correlation between the GAT and GOT was very high (Pearson r = 0.635 for the normophonic and r = 0.79 for the AdSD group). The bar graph of the GATs for the normophonic and AdSD group is shown in p = 0.037). The GOT averages for the AdSD group were slightly higher than the normophonic group, but the difference was not statistically significant. The results were confirmed with both parametric tests (ANOVA) and non-parametric tests .The number of onsets/offsets for which the GAT/GOT were measured were compared between the AdSD and normophonic groups. Moreover, the comparison of the number of GAT/GOT with values equal to zero was conducted. None of the differences were statistically significant, but the direction of the effect sizes was as follows: The AdSD group had a lower average number of onsets (42.6 vs. 54) and offsets (50.4 vs. 62.3), a higher number of onsets with zero GATs (4.6 vs. 1.75), and a higher number of offsets with zero GOTs (8.6 vs. 4.8). The statistical tests were repeated after removing the GATs and GOTs with zero values associated with hard glottal attacks/offsets. Accordingly, the AdSD group showed statistically significantly higher average values for GAT than the normophonic group , and the difference was statistically significant. The repeated measures model for the GOT did not achieve a clear picture of the effect of the disorder, as it did not show statistically significant differences between the two groups. No effect of gender or age was observed. The GAT and GOT of the normophonic male participants were higher than those of the normophonic females.No statistically significant differences were observed for the GATs/GOTs between the true and false vocal folds onsets/offsets for S3M. However, the average GATs/GOTs of the false vocal folds were longer with higher variation than those of the true vocal folds. The average GAT was 19.4 ms (std of 5.2 ms) for the true vocal folds and 25.9 ms (std of 18.9 ms) for the false vocal folds in S3M. The average GOT was 25.4 ms (std of 14.1 ms) for the true vocal folds and 30.8 ms (std of 17.1 ms) for the false vocal folds. The CV value of 73% for the GATs of the false vocal folds and 27% for the true vocal folds was also indicative of the much greater variability of the GAT of the false vocal folds. The distributions of the GAT and GOT for the true and false vocal folds are shown in The dynamics of the individual GAT and GOT time series were analyzed visually in the order they appeared in each participant see . Figure Overall, there was a big difference in the dynamics of the GAT time series, representing consecutive onsets, between patients. The contrast between the normophonic and AdSD groups for GOT was slightly different than that of the GAT. The normophonic participants’ GOT dynamics were still stationary but there was a certain large variability (heteroskedasticity). However, the GOT dynamics for the AdSD patients were extremely nonstationary and extremely volatile (constant rapid changes from one offset to another) to a much larger degree than what was observed for the normophonic group.4.The results show the GATs are longer in the AdSD group than in the normophonic group, which was confirmed by the generalized linear model analysis. The average GAT was 16.8 and 14.7 ms in the AdSD and normophonic groups, respectively. This was supported by previous research that showed a longer latency between the muscular activation and the phonation onset in AdSD compared to the normophonic voices . MoreoveA greater variability was observed in both the GAT and GOT in the AdSD group, which was expected as the disorder led to more uncertain behaviors and irregularity in the vibrations of the vocal folds. Considering that the AdSD patients have less neurological control over their laryngeal muscles during voice production –32, the The comparison of GAT/GOT measured for the true and false vocal folds in S3M showed some overlap. As the false vocal folds’ vibration is a compensatory strategy, more variability and extreme values were observed for GATs/GOTs of the false vocal folds. The GAT and GOT values for the false vocal folds in S3M were the highest among the AdSD group. Although S3M was a male, and this observation might be explained by the lower fundamental frequency of the voice, a similar observation was not made for the GAT/GOT values of the true vocal folds in this participant. Therefore, the longer GAT/GOT of the false vocal folds could be related to the vibratory characteristics of these folds, which will be further investigated in the future.The inter-rater reliability assessment showed that the measurements by the three raters were reliable as the upper limit of the 99% confidence interval was less than 18 frames. This frame number (criterion) was considered acceptable in the current study since the fundamental frequency of the voices ranged between 100 and 250 Hz. Some discrepancies between the measurements among the raters were expected due to the nature of the data and the manual measurement. Since the glottal closure can last for several frames, it was possible for the raters to select a slightly different contact frame number. Indicating the first/last oscillation was also challenging since the raters needed to select the frame while the video was being played, and, depending on when the video was stopped, the selected frame could have been different. Additionally, due to the small amplitudes of oscillations, more discrepancies between the oscillation frame numbers by the three raters in comparison with the contact frame numbers were expected. These discrepancies were more noticeable at the offset of phonation due to the longer GOTs than the GATs and more (difficult to detect) miniature oscillations at the offset. The software settings, the computers, and the room brightness were different among the raters, which could have been another source for some of the discrepancies. As the raters became more comfortable with the anatomy/physiology of vocal folds, their measurements became more reliable.Although the observations of the GAT and GOT in this study are in line with our understanding of the AdSD, the dynamic nature of the GAT and GOT measurements in the HSV data limits the accuracy of manual measurement. The detection of the oscillation/contact frames depended on the quality of the data, the image adjustments by the raters, and the visual acuity of the raters. Additionally, the playback speed of the video and the small amplitudes of the vibrations impacted the accurate measurement of the oscillation frames. These challenges became more noticeable when analyzing the AdSD data due to the higher irregularity, unexpected behavior in the vocal fold vibration, and occurrence of phonatory breaks. In future, an automated method will be developed to overcome the challenges of manual measurements. A more detailed comparison of the GAT/GOT values for equivalent vocalizations needs to be performed to provide more accurate assessments of GAT/GOT differences between the normophonic and AdSD voices, which is among our future goals. Additionally, the relationship between GAT/GOT measures and the severity of AdSD, measured by perceptual voice assessment, will be studied. These works are important for indicating the clinical significance of these measures, as the statistical significance alone is not sufficient to interpret the findings of the study . The sma


Location-based Augmented Reality applications are increasingly used in many research and commercial fields. Some of the fields that these applications are used are recreational digital games, tourism, education, and marketing. This study aims to present a location-based augmented reality (AR) application for cultural heritage communication and education. The application was created to inform the public, especially K12 students, about a district of their city with cultural heritage value. Furthermore, Google Earth was utilized to create an interactive virtual tour for consolidating the knowledge acquired by the location-based AR application. A scheme for evaluating the AR application was also constructed using factors suitable for location-based applications: challenge, educational usefulness (knowledge), collaboration, and intention to reuse. A sample of 309 students evaluated the application. Descriptive statistical analysis showed that the application scored well in all factors, especially in challenge and knowledge . Furthermore, structural equation modeling (SEM) analysis led to a model construction that represents how the factors are causally related. Based on the findings, the perceived challenge significantly influenced the perceived educational usefulness (knowledge)  and interaction levels . Interaction amongst users also had a significant positive impact on users’ perceived educational usefulness , which in turn influenced users’ intention to reuse the application . Location-based augmented (LBAR) applications have existed for quite some time, but their use for various communication purposes has become widespread after the advent of two popular augmented-reality location-based games, namely Pokemon Go and Ingress, created by Niantic in 2014 and 2016, respectively. Location-based augmented reality apps  are currently used in a number of fields, such as entertainment, education, and marketing. An application can also be used to fulfill more than one purpose. For example, an LBAR mobile application can be used for tourism marketing, but at the same time, it can also provide information that educates visitors about a tourist destination.Location-based augmented reality applications typically run on mobile devices (smartphones or tablets) and make use of the GPS sensor of the mobile device to guide users to specific locations. When these locations are reached, digital content is activated. Digital content can be in many forms, such as text, video, 2D and 3D graphics, and animations. Location-based augmented reality can also be combined with image-based augmented reality to superimpose the real world with digital content when specific locations are reached, and the camera of the mobile device is pointed at objects from the real environment  .Cultural heritage communication is increasingly taking advantage of innovations in digital technologies, and LBAR applications can be utilized for creating informative cultural tours. Using LBAR applications, users are prompted to visit cultural heritage monuments and places where significant historical events have occurred. When the users get close to these locations, digital content appears on the screens of their mobile devices, informing them about various aspects of these locations, such as historical facts, architectural and artistic trends related to human constructions, etc. Applications designed for augmenting the environments with cultural heritage content are numerous. Overlaying the physical world with historical information is present in many initiatives e.g., ,3,4).,4.3,4]).This study aims to present a location-based augmented reality application for cultural heritage communication and education and the considerations that have taken place in the pre-production phase. The study also presents a Google Earth interactive application intended for use after the augmented reality experience to refresh and strengthen the knowledge acquired during the cultural tour. Another aim of this study is to construct a suitable evaluation scheme for assessing the efficiency of the location-based AR application and to evaluate multivariate causal relationships among various factors using structural equation modeling. The factors which were used in the evaluation were outdoor activity, challenge, ease of use, interaction/collaboration, knowledge, and intention to reuse.To create and present a location-based AR experience for cultural heritage education that is engaging to learners and could act as an example for educators wishing to employ AR in their cultural education tours.To present the steps for developing such applications, giving emphasis to the decisions taken in the pre-production phase.To create and present a VR Google Earth project that would be used after the AR experience to consolidate the acquired knowledge.To construct a suitable evaluation scheme based on past research for assessing the factors related to a location-based AR experience.To evaluate multivariate causal relationships among various factors using structural equation modeling (SEM).To summarize, the research objectives of this study were the following:This article is organized in the following way: A brief literature review is provided in Then a Google Earth interactive application that was created to refresh the knowledge obtained from the AR tour is presented in Augmented Reality (AR) has a long history. The following subsection presents some important milestones in the AR evolution.The first AR technology, an AR head-mounted display, was developed in 1968 at Harvard  by a sciIn the following decades, the growth of AR was even more remarkable. A significant milestone in AR evolution was in 2000 when an open-source library called AR toolkit was released, providing developers with tools for rapid augmented reality application development. In 2009, Esquire magazine introduced AR in print media, and in 2013, Volkswagen created the MARTA app, which gave technicians step-by-step instructions by augmenting the vehicle parts with text and animations. In 2014, Google introduced its augmented reality glasses, and in 2016, Microsoft started shipping its AR head-mount device HoloLens  = 0.949) since the GFI, CFI, and TLI values exceeded the 0.90 criterion proposed by Byrne in 2010 [First, we tested the measurement model and calculated the goodness-of-fit measures as well as the construct, composite, and discriminant validity of the latent constructs used. The measurement model consisted of four latent variables  and ten observed variables. The goodness-of-fit measures suggested a good fit for the model  and ranged from 0.577 to 0.857, exceeding the 0.50 cut-off value). The average variance extracted (AVE) for all constructs ranged from 0.532 to 0.65, exceeding Fornell and Larcker’s (1981) [The four scales were evaluated for their convergent and discriminant validity. The standardized factor loadings of the items of the four scales were all significant   cut-off s (1981) . Thus, tp = 0.000, χ2/df = 2.51, CFI = 0.967, GFI = 0.955, IFI = 0.967, TLI = 0.949). Moreover, the RMSEA value was 0.07. Thus, the study’s model showed an acceptable fit to the data. The hypothesized relationships were then tested by examining the standardized estimates of the path coefficients  and positive predictor of the player’s perceived usefulness (knowledge)  and interaction levels . On the contrary, challenge did not significantly influence (p > 0.05) players’ re-usage intentions in a direct manner . Thus, hypotheses H1 and H2 are accepted, while H3 is rejected. Interaction and collaboration with other players had a significant positive impact on players’ perceived usefulness/knowledge . The impact of interaction/collaboration on players’ re-usage intention was non-significant . Hence, H4 is accepted, while H5 is rejected. In addition, perceived usefulness/knowledge had a positive impact on users’ intention to reuse the application  in the future, supporting H6.Based on the results , the conThe equations for the structural model are given in Furthermore, we tested the indirect effects of the constructs using the bootstrap method on AMOS 21.0. Results are presented in The descriptive statistical analysis and the structural equation modeling analysis results apply to the specific application and the specific location (Doltso district), as well as the specific educational settings , and cannot be generalized. In order to obtain a broader perspective on how AR location-based applications designed for cultural heritage education using Taleblazer affect factors such as challenge, knowledge, collaboration, and intention to reuse, more similar evaluations will be needed in order to compare the results and provide more generalized insights. The research team of this paper has already designed similar applications for other districts of the city of Kastoria that have cultural heritage value, archeological sites, as well as tours of environmental interest and pure touristic interest. These applications are currently being evaluated by a large number of students that take place in the educational programs of the Center for Education for the Environment and Sustainability of Kastoria as well as visitors of the area.Another limitation of the study concerns the VR Google Earth tour that is presented in this study. Google Earth tour was utilized to create an interactive project for consolidating the knowledge acquired in the augmented reality tour, but in this study, only the augmented reality application was evaluated since the VR tour was optional for the teachers and students to use in classroom time or at home. In the future, we plan to evaluate the whole experience (AR application and Google Earth VR tour) in terms of knowledge gains and engagement.From the analysis, it became evident that the collaboration and interaction amongst students scored lower than other factors, such as challenge and educational usefulness/knowledge. Taking this in mind, the research team plans to explore new ways to encourage collaboration and interaction and design a new application version that enhances this dimension. One possible way of doing this is by assigning groups and instructing students that belong to the same group to take on different roles. Each student will collect points by answering questions specific to the role assigned to them, and these points can be added up in the end to make the group total. The students will be free to collaborate within their group. The teacher, in the end, will acknowledge the winning group. This is an example of a procedure that could enhance student collaboration and interaction.Furthermore, the MIT Taleblazer research team is exploring the prospect of adding a multiplayer option . This opIt presents an AR location-based application for cultural heritage communication and education and the steps followed for developing the application. In this way, the study also aims to inspire educators wishing to create their own AR tours and provide them with ideas and guidance on how to proceed with such tasks.It presents a method, using a VR Google Earth project, for repeating the cultural tour remotely, consolidating in this way the knowledge acquired during the AR experience.It proposes an evaluation scheme for location-based AR educational tours and presents descriptive statistical analysis results from this evaluation.It presents the results of a structural equation modeling analysis, determining the causal relationships between the evaluation factors.This study presents a location-based augmented reality application with gamification elements for cultural heritage communication and education. We believe this study contributes to the existing literature in the following ways:More specifically, the study presents the stages for implementing the application, emphasizing certain decisions made in the requirements analysis and application design phase. The application’s target audience is mainly primary and secondary education students following a specific educational program.The application guides the users through a city district with historical and cultural interest where many old constructions, mainly from the Byzantine and post-Byzantine eras, are preserved. During the AR tour, the users are prompted to take on different roles , observe their surroundings, and answer questions related to these roles. A Google Earth interactive web application was also developed in order to be utilized after the AR tour for revising and consolidating the acquired knowledge.In order to evaluate the application, an evaluation scheme was developed mainly by using constructs derived from the related literature. A sample of 309 students evaluated the AR application. The findings show that the students felt intense feelings of challenge and perceived the application as highly useful in terms of knowledge gains. Furthermore, structural equation modeling analysis revealed the causal relations among the study’s constructs. The results from the structural equation analysis showed that the constructs of challenge and users’ interaction/collaboration had a significant and positive impact on the users’ perceived knowledge. It can also be argued that although challenge and interaction did not have direct effects on re-usage intentions, they had in-direct effects through the construct of educational usefulness/knowledge, which can be regarded as an important mediator of the above relationships.


Dermatophagoides farinae (Der f)-IgE and eosinophil in mothers with AR at delivery and the eosinophil levels and AR incidence in children.The identification of allergic rhinitis (AR) in early life is important for the target of intervention. AR is caused by various environmental factors, including house dust mites. We investigated the relationship between the The study participants were 983 mother–child pairs from the COhort for Childhood Origin of Asthma and Allergic Diseases. AR was diagnosed by a doctor at delivery in mother and at 3 years of age in offspring. The association between eosinophil level and AR was assessed using logistic regression analysis.The Der f-IgE level in mother having AR at delivery was associated with the mother’s eosinophil level, and the mother’s eosinophil level was associated with the child’s eosinophil level both at age 1 and 3. The risk of AR at age 3 in children was increased according to increased eosinophil levels in mothers at delivery and in children both aged 1 and 3 years . The risk of childhood AR at the age of 3 is increased when both mothers and children have high eosiniophils .Der f-IgE in mothers at delivery was related to eosinophil levels in mothers with AR and higher level of eosinophils in both mother and children was associated with the increased risk of AR incidence at the first 3 years of life of children.The online version contains supplementary material available at 10.1186/s12887-023-04156-1. Allergic rhinitis (AR) is a chronic respiratory disease characterized by symptoms such as rhinorrhea, nasal itching, and sneezing . The preDermatophagoides farinae (Der f). Maternal AR along with environmental exposure is a familial risk factor for infant AR. The risk of AR is well known to have a tendency to transmit between mother and their offspring. However, there are a few studies suggesting that maternal Der f-IgE and eosinophil is associated with their offspring’s early life AR in humans.House dust mites (HDMs), a representative environmental factor, are the most common triggers of AR , 7. The Therefore, the present study aimed to find the relationship between Der f-IgE levels and eosinophil counts in mother with AR and the risk of AR in their offspring’s early life.This study was conducted as part of the Cohort for Childhood Origin of Asthma and Allergic Disease (COCOA). COCOA is a general prospective population study as a hospital-based birth cohort designed to reveal the impact of environmental exposure in South Korea. COCOA includes demographic variables such as maternal height and weight, disease history, smoking history, tracks children’s height, environmental questionnaire responses, clinical symptoms, and disease history every year. A detailed description of the COCOA study design has been published previously . A totalIgE  levels and leukocyte levels in the mother were measured at the time of the infant’s birth. IgE  levels and leukocyte levels in the children were measured at 1 and 3 years of age. Total serum IgE levels and serum levels of specific IgE to egg and milk were measured using a fluorescent enzyme immunoassay . Leukocytes were calculated using an automatic blood-cell counter  .The questionnaire based on the International Study of Asthma and Allergies in Childhood (ISAAC) was translated into Korean. The parents of infants filled out a modified version of the questionnaire. The questionnaire consists of (1) demographic factors such as gender, date of birth, height, and weight; (2) symptom history related to AR; (3) environmental factors related to allergic diseases such as residential patterns and indoor air quality .SPT was performed in the mother, 6 months old infant, and in the child on the third birthday. SPT was conducted for 18 allergens  in children and 14 allergens  in mothers using normal saline and histamine as a negative and positive control, respectively. A mean wheal size greater than 3 mm was considered a positive reaction.AR diagnositic information was used when the child was 3 years old. AR was suspected when the participant complained symptoms of AR, such as runny nose and stuffy nose. AR was diagnosed by a physician considering SPT results (Supplementary Tables http://www.r-project.org/foundation/main.html). Summary statistics are expressed as mean ± standard deviation (SD) or number (percentage). Two groups were compared by t-test. ANOVA was performed to determine whether maternal blood parameters differed by the maternal Der f-IgE levels at delivery. The children were divided into three groups according to the tertiles of mothers’ Der f-IgE and eosinophil levels. ANOVA was also performed to compare blood parameters in children aged 1 and 3 years according to the Der f-IgE and eosinophil levels of the mothers classified into three groups. The values divided into groups are as follows: (1) the maternal Der f-IgE: low(≤ 0.01), middle(> 0.01 to ≤ 0.92 ), and high(> 0.92); (2)maternal eosinophil: low(≤ 0.6), middle(> 0.6 to ≤ 1.2 ), and high(> 1.2); (3) infant’s 1y eosinophil: low(≤ 2.0), middle(> 2.0 to ≤ 3.5 ), and high(> 3.5); (4)infant’s 3y eosinophil: low(≤ 2.1), middle(> 2.1 to ≤ 3.7 ), and high(> 3.7). Also, logistic regression analysis was performed to determine whether maternal and childhood eosinophil counts were associated with 3-year-old AR in children. Three types of logistic regression models were constructed. The models are as follows: (i) Model 1, maternal age, BMI, total IgE at birth and secondhand smoke in pregnancy; (ii) Model 2, sex, and total IgE at 1 year old; (iii) Model 3, sex, and total IgE at 3 years of age. A logistic regression was performed to examine the association between higher eosinophilic levels in both mothers and children and the risk of AR incidence at age 3, using sex, maternal age at birth, and secondhand smoke in pregnancy as confounder variables. Significance was set at p-value < 0.05.All statistical analyses were performed using the R program  when exposed to HDM for several weeks have been observed in in vivo studies , 18. It Multiple studies have identified relationships between the immune system in mothers and children –22. EosiWhen an allergen, such as an HDM, enters the body, T helper 2 (Th2) cells are activated, and eosinophil levels increase . This inIn addition, studies on the prevalence of AR in infants and children of various races have shown that the prevalence ranges from 6 to 32.1% –35. In KThis study has several limitations that should be considered during interpretation. We obtained retrospectively the information on environmental exposures. Also, we calculated the prevalence of AR symptoms based on responses to a questionnaire. Therefore, recall bias could be occurred. However, we have many strengths. Our modified questionnaire form of ISAAC primarily concerned for responses about AR symptoms ever but less so for responses about AR symptoms in the 12 months before survey completion. Our group has validated the questionnaire in many studies and conducted the analysis of risk factors in previous studies. Also, the sample size of the present study was relatively large and the response rate was high. Moreover, we longitudinally examined every year at the same hospitals by the same researchers , 16, 37.In conclusion, our study demonstrates that maternal AR at delivery is associated with the increase of Der f-IgE and eosinophil levels and maternal and their childhood eosinophil counts increased the incidence of AR in early life of offspring. Therefore, our findings for higher eosinophils in mothers and their offsprings can be helpful in the early prediction, prevention, and management of AR in childhood.Below is the link to the electronic supplementary material.Supplementary Material 1