Vegetables provide important nutrients but can also induce allergic symptoms. Celery tuber allergy frequently occurs in Central European countries and can cause allergic reactions including fatal anaphylactic shocks. There is little information about allergen content in seeds. Therefore, we analyzed 2 patients with allergic reaction after remoulade sauce consumption who entered the clinic for a diagnostic work-up. The routine diagnostic included serum derived specific IgE testing by ImmunoCAP, ImmunoCAP ISAC, and skin prick tests (SPTs). Furthermore, protein extracts were prepared from both celery tuber and celery seeds and IgE binding capacity of these extracts was assessed by immunoblots, ELISA, and rat basophil leukemia (RBL) assay. We also determined role of cross-reactive carbohydrate determinants (CCDs) by IgE inhibition ELISA. Results revealed distinct protein patterns from celery tuber and seed extracts, suggesting differences in content and quantity of allergenic proteins. IgE antibodies from both sera bound to high molecular weight (HMW) proteins on immunoblots and caused high basophil response, which was also observed upon addition of glycosylated proteins as horseradish peroxidase and Api g 5, respectively. Our results indicate that it is worth considering CCDs from plant foods as a possible allergenic factor and their contribution to the mugwort-celery syndrome. In contrast to the glycosylated form, deglycosylated Api g 5 lacks binding activity of IgE from celery allergic patients’ sera.in vitro diagnostic tests, it seems that the panel of celery allergens remains incomplete. In this study we report on 2 allergic patients, who experienced clinical symptoms after consumption of celery tuber. Testing sera from celery allergic patients enabled us to analyze the relevance of IgE antibodies directed against carbohydrate determinants that can have impact on the allergic reactions. Also the potential impact of CCDs to the mugwort-celery IgE cross reactions was investigated. Furthermore, we analyzed the allergen repertoire present in celery seed and tuber protein extracts by immunoblots.Although a number of celery allergens were identified and are commonly used in Skin prick tests (SPTs) for food allergens were performed using frozen crude extracts (home-made) and SPT with pollens were performed with commercial extract . A positive skin reaction was defined as a mean wheal diameter ≥3 mm. Specific IgE to Ambrosia, Artemisia, nArt v 1, rApi g 1, rPhl p 1 was measured using the ImmunoCAP  with a cut off of 0.35 kUA/L. Additionally, sera were subjected to ImmunoCAP ISAC analysis (Thermo Fisher Scientific). IgE levels >0.3 ISU-E were considered as positive. The study was approved by the ethics committees of Lower Austria (GS4-EK-4/242-2013) and from the French Ministère de la Recherche (DC-2010-1095). Written informed consents were received from all individuals.Celery seed and tuber protein extracts were obtained as previously described.IgE inhibition ELISA was performed as follows: celery allergic patients’ sera (1:20 dilutions) were blocked overnight at 4 °C with 1, 10 and 50 μg/ml of Bet v 1, Api g 1, Api g 5, Art v 1, HRP, celery tuber and seed extract (self-inhibition control), respectively. MaxiSorp plates were coated with 100 μg/ml of celery seed extract. Plates were blocked with 3% BSA and incubated overnight with blocked sera from celery allergic patients. After washing, plates were incubated with AP-conjugated anti-human IgE antibodies, develop with AP substrate and measure optical density (OD) 405. Tests were performed in duplicates and results calculated as percentage of inhibitions.RBL assay was performed as previously described.Two allergic patients with reaction after remoulade sauce consumption entered the clinic for a diagnostic work-up. Remoulade sauce, a typical French sauce, is based on mayonnaise, vinegar, mustard, pickles, and celery. Clinical data of the patients  indicatePatient number 1, a 64 year-old male, was suffering from pollinosis and allergy to parsley and both raw (urticaria) and cooked celery . SPT was positive to raw/cooked celery, parsley, raw carrot, dandelion, fennel, cumin, artemisia, ambrosia, grass, and birch pollen. ImmunoCAP tests were positive for Ambrosia (19.6 kU/l), Artemisia (19.7 kU/l) and Art v 1 (15.2 kU/I), respectively.Patient number 2, a 55 year-old female also experienced urticaria upon consumption of raw celery but she did not report any symptoms to cooked celery. She was also suffering from spring pollinosis and oral syndrome with raw carrot. SPT was positive for raw celery, Artemisia, grass and birch pollen. ImmunoCAP was positive for nArt v 1 (1.52 kU/l), rApi g 1 (1.40 kU/l), and rPhl p 1 (80 kU/l), respectively.Furthermore, patients’ sera were tested by ImmunoCAP ISAC 112 Multiplex Phadia. Serum from patient number 1 predominantly had specific IgE directed against glycosylated allergens. Serum from patient number 2 contained IgE antibodies against Bet v 1 homologues , grass pollen allergens such as Cyn d 1, and major mugwort allergen, Art v 1.Based on the clinical data we decided to investigate the IgE binding patterns of both sera for celery tuber and seed extracts. Celery tuber and seed extracts revealed different patterns of proteins in Coomassie stained 15% SDS-PAGE analysis . Tuber eIgE immunoblots performed with celery seed extract provided a similar pattern when testing both sera. Specific IgE antibodies bound to 2 HMW proteins of 55 kDa and 90 kDa, respectively. Inhibition with glycosylated proteins, horseradish peroxidase (HRP) and nApi g 5, respectively, completely blocked IgE binding. In contrast, preincubation of sera with rApi g 1 did not affect the IgE recognition pattern.Celery tuber extract contained only one HMW protein (65 kDa) recognized by IgE from serum of patient number 1 and incubation with HRP and Api g 5, blocked this binding capacity, whereas inhibition with Api g 1 did not affect this interaction. Serum from patient number 2 contained IgE specific to a protein of molecular mass of approximately 15 kDa, which was inhibited by preincubation of rApi g 1. This result was in line with the data from ImmunoCAP ISAC analysis. Blocking with HRP and nApi g 5 did not affect the IgE against rApi g 1, but depleted a weak signal from HMW proteins.IgE inhibition ELISA experiments with the celery seed extract confirmed the previous results from immunoblot analyses. Specific IgE from serum number 1 recognized seed proteins and was completely inhibited by HRP and nApi g 5, but not by rApi g 1. Preincubation with nArt v 1, displayed more than 25% of inhibition. Blocking with a celery tuber extract showed almost complete inhibition (85% at a concentration of 50 μg/ml) A. Serum ELISA plates were coated with celery seed extract. Sera from patient 1 and 2 (panel a and b) were blocked with the panel of proteins in 3 different concentrations. Bars are presenting percentage of inhibitions. HRP - horseradish peroxidase; CTE – celery tuber extract; SIC – self inhibition control.in vitro. Rat basophil leukemia (RBL) luciferase assay was performed with RS-ATL8 cells as previously described.Due to the comparatively high OD values to glycosylated proteins observed in the IgE ELISA experiments we further investigated whether CCDs could activate basophils For more than 2 decades the role of carbohydrates in an allergic disease has been controversial. Lack of clinical relevance of IgE-binding plant glycans was demonstrated, for example, using human lactoferrin expressed in rice as a model.Different protein patterns suggest differences in the content and quantity of potential allergens. This assumption was confirmed by different IgE recognition patterns in immunoblots for both patients’ sera. Additionally, sera blocked with either tuber or seed extract reacted differently in inhibition ELISA assays. Incubation with celery tuber and seed extracts, gives similar activation level of basophils in case of first serum. The second serum caused inequality in basophils activation, and may also indicate a distinction in allergen contents between tuber and seeds. This phenomenon was already shown, ie, for kiwifruit extracts from pulp and seeds, respectively, differing in allergen quantity and quality.In our study results obtained from immunoblots and inhibition assays provide evidence that both sera contain specific IgE antibodies directed against cross-reactive carbohydrate domains. In the case of patient number 1, complete inhibition on ELISA and immunoblot suggest, that all IgE antibodies were specific for CCDs while serum from patient number 2 had additional antibodies specific for the major celery allergen, Api g 1.Interestingly, the protein responsible for cross reactivity between celery and mugwort is still missing. Limited blocking by antibodies specific for a celery extract by nArt v 1 protein may be a partial explanation of this phenomenon. It is worth noticing that the major mugwort allergen, Art v 1, is characterized by O-glycosylation, which may have an effect on the allergenicity.in vitro diagnosis. Differences between the allergen content in different parts of celery should be also considered as a part of the standardized component resolved diagnosis.To sum up, clinical relevance of sIgE directed against CCDs in celery allergy is controversial. However, individual cases highlight it is worth to include them for CCDs, cross-reactive carbohydrate determinants; CRD, component resolved diagnosis; HMW, high molecular weight; RBL, rat basophil leukemia; HRP, horseradish peroxidase; CTE, celery tuber extract; CSE, celery seed extract; SIC, self inhibition control; OD, optical density; BC, buffer control;, NHS, non allergic human serum.10.13039/501100002428Austrian Science Fund (FWF): DK W 1248-B13 for P.H. and P.D. and FWF: SFB F4603 for K.H.-S.This work is partially funded by the The project outline was drafted by PD, PH and KHS. The experiments were performed by PH, PD, MB, CH, FB, GP, SV, CM-F and KHS. Data analysis and interpretation was done by PH, PD, MB, FB, GP, and KHS. All the authors  provided input to drafting the article and revising it critically for important intellectual content and gave final approval of the version to be published.The study was approved by the ethics committees of Lower Austria (GS4-EK-4/242-2013) and from the French Ministère de la Recherche (DC-2010-1095). Written informed consents were received from all individuals.All authors consented for publication in this study. Authors confirm that the manuscript is original, has not been published before, is not currently being considered for publication elsewhere, and has not been posted to a preprint server.Contact the corresponding author for questions regarding data and materials.The authors declare that they have no competing interests.Authors confirm acceptance of editorial policy.


This study aimed to investigate how the predicted implant fatigue lifetime is affected by the loss of connector screw preload and the finite element analysis method used to simulate preload.A dental implant assembly  was scanned using microcomputed tomography and measured using Mimics software  and an optical microscope. Digital replicas were constructed using SolidWorks software (Dassault Systèmes). The material properties were assigned in Abaqus (Dassault Systèmes). An external load was applied at 30° off-axial loading. Eight levels of connector screw preload  were simulated for DI1. This assembly and an additional model (DI2) having a longer and narrower screw were compared regarding their fatigue limits (using fe-safe software [Dassault Systèmes]) for 2 preloading methods: (1) adding preload torque or (2) adding bolt axial tension.’s recommendation and dropped precipitously between 80% and 70% preload. Adding a preload torque on the screw resulted in a more uniform stress distribution on the screw compared with bolt axial tension, especially for DI2, which had a longer and narrower screw than DI1.The maximum von Mises stresses of DI1 (on the connector screw threads) with and without preload were 439.90 MPa and 587.90 MPa. The predicted fatigue limit was the same for preloads from 100% through 80% of the manufacturerA substantial loss of preload can be accommodated without compromising the fatigue resistance of this dental implant. Computer models should be constructed using torque instead of a bolt axial tension. For structural analysis, it allows detailed visualization of where structures bend or twist on the application of impact or load and indicates the distribution of stresses and displacements. Using FEA, entire structures can be constructed, refined, and optimized before the prototype is manufactured.Finite element analysis (FEA) is widely used for numerically solving differential equations used in mathematical modeling and engineering. It subdivides a larger system into smaller elements by space discretization using a meshing tool. The formulation of a static equilibrium results in a system of algebraic equations for each element. The equations used for modeling these finite elements are then assembled into a larger system of equations that help model the entire problem. Matrix algebra is then used to approximate the solution.6 The loading condition plays a vital role in the performance of dental implants.8 A multicomponent dental implant assembly consists of an implant fixture, an abutment, and a connector screw. Generally, a torque is applied to the connector screw using a torque wrench to fix the abutment to the implant fixture adequately. This tightening develops an axial tension on the surface of the screw that is in contact with the adjacent material. This is referred to as preload, as shown in 9 The magnitude of preload holds major importance in the success of dental implants. Most 2-piece dental implants fail because of screw loosening.19 This may be because of the micromotion between the implant fixture and the abutment that can lead to reduced preload on the connector screw.FEA has been widely used in dentistry to study the stress patterns in various types of prostheses, including dental implants and fixed dental prostheses.22 Hence, applying the manufacturer recommended preload to the screw is essential. Adding the recommended preload may help a screw encounter a more favorable combination of stress amplitude and mean stress, resulting in increased fatigue lifetime.In contrast, excessive preload can cause damage to the screw threads and lead to plastic deformation.This study investigated the effect of preload on the predicted fatigue lifetime of a commercially available dental implant. It was hypothesized that adding a preload will significantly improve the fatigue lifetime of the dental implant assembly. There are 2 ways in which a preload can be applied to a screw in a finite element model. The first method is to apply the preload torque directly to the screw, simulating the tightening of the screw using a torque wrench. The second method is to calculate the bolt axial tension (in N) from the recommended torque using a mathematical formula and then apply the tension on the screw. It was hypothesized that adding the torque would provide a more uniform stress distribution on the screw than the bolt axial tension method.The physical specimens of a dental implant assembly (DI1) including a narrow-diameter dental implant , an abutment , and a connector screw  were scanned using microcomputed tomography (micro-CT) . The scanning parameters used were as follows: an accelerating voltage of 100 kV, current of 100 μA, exposure time of 1,264 ms per frame, aluminum and copper filter, and rotation step at 0.7°. The x-ray beam was projected in a direction parallel to the long axis of the implant fixtures. The image pixel size was 34.6 μm. The x-ray projections were reconstructed to form a 3-dimensional (3D) model, saved as a stack of bitmap-type 3D files using NRecon software (Micro Photonics). Beam hardening correction of 49% and ring artifact correction of 4 were used for the reconstruction. The 3D models were generated in Mimics  through image segmentation from the stacked image data obtained from micro-CT. Mimics organizes all the imported tomograph image slices and displays objects in 3 cross-sectional views . Based on the gray scale values, the objects were modified with the help of segmentation tools. The implant dimensions were measured using Mimics and an optical microscope (Keyence). The length and maximum diameter of the implant fixture were 15.12 mm and 3.40 mm, respectively.23 A cylindrical bone model with 2 layers  was also constructed in SolidWorks based on the dimensions of a simulated bone specimen holder that better mimics the clinical case.24 The bone crest was placed at 3 mm below the implant nominal bone level to simulate the worst-case scenario of crestal bone loss, based on the requirement of ISO 14801.23 An additional model (DI2) with a longer and narrower screw than the connector screw of DI1 was also constructed in SolidWorks. The length and a body diameter of the connector screw of DI1 were 7.34 mm and 2.10 mm, respectively. The length and a body diameter of DI2 were 8.81 mm and 1.67 mm, respectively.The digital replicas of the physical specimens of DI1 were constructed using computer-aided design software (SolidWorks)  software to estimate the fatigue limit using Brown-Miller criteria with Morrow mean stress correction for the lifetime calculation, as detailed in a previous study.3 Constant and cyclic loading  were used for the preload and external load. First, a low load value was used to predict the fatigue limit, which resulted in an infinite lifetime. Then, a high load value was used resulting in a finite lifetime. The load interval was then cut into one-half, and the lifetime was predicted for the intermediate load value. This process was repeated until a threshold value was obtained, and the lifetime of the implant ceased to be infinite. After processing, the results were written to an output .odb file, which was displayed in the postprocessor of Abaqus. This entire process was repeated for the same DI1 model for connector screw preloads of 95%, 90%, 80%, 70%, 50%, 30%, and 0% of the manufacturer’s recommended torque (32 Ncm), and its fatigue limit was estimated in fe-safe.Analysis 1. This analysis was performed to evaluate the effect of preload on the implant fatigue limit. A preload torque of 32 Ncm was applied to the screw of DI1. An external load was applied to the loading cap at an angle of 30° from the implant axis to simulate the bite force (ISO 14801).25:Analysis 2. This analysis was performed to estimate the correct method for preload simulation. For this analysis, both DI1 and DI2 models were taken into consideration. Two methods were followed for preload simulation in each model. The first method was to directly add a preload torque of 32 Ncm on the connector screw and analyze the von Mises stress distribution. A reference point was created at the center of the connector screw head, and a coupling interaction was assigned between the reference point and the screw. The torque was then applied to this reference point that propagated the torque to the entire screw. The second method was to convert the preload torque (in Ncm) to bolt axial tension (in N) and then apply this tension on the connector screw in which the screw head meets the screw shank. The following equation was used to convert torque into bolt axial tensionwhereT = preload torque (Nmm)F = preload (N)p = screw thread pitch (mm)π = the ratio of the circumference of any circle to the diameter of that circlet = coefficient of friction between screw and implantμd2 = pitch diameter of screw thread (mm)β= half angle of screw thread (rad)De = (do + di)/2 (mm), where di is the inner bearing and do is the outer bearing diameter of the nut face (mm)n = coefficient of friction between screw and abutmentμFEA was performed in Abaqus (Dassault Systèmes). 6 cycles, which was substantially longer than the lifetime of the model without preload . The fatigue failure location in both cases was at the implant superficial threads. ’s recommended torque, but it dropped sharply between 80% and 70%.This study establishes the importance of applying adequate preload to the connector screw while predicting fatigue lifetime. From analysis 1, it is evident that adding a preload improved the fatigue lifetime of the dental implant significantly. This is reasonable because applying an appropriate amount of preload ensures the adequate tightening of the screw between the abutment and implant fixture. This will reduce the chances of early micromotion between the components, otherwise leading to a loss of preload resulting in early failures at the connector region from fatigue crack growth under flexure. Although performing simulations or in vitro testing on a commercially available implant, it is important to simulate a preload on the screw. However, the decrease in predicted fatigue limit with decreasing preload was not gradual. The decrease did not begin until the preload was below 80% of the recommended value, and then there was a sudden decrease in the predicted fatigue limit between 80% and 70% of the recommended preload. This provides a useful threshold for future researchers to define preliminary failures when conducting in vitro testing on physical specimens.28 Some studies have simulated preload in the form of bolt axial tension while performing FEA on dental implants.30 In this study, it was observed that simulating preload in the form of bolt axial tension can cause excessive stress concentration around the region of force application, and it may not lead to an effective distribution of stress along the body of the screw. This was more evident in the screw having a greater length and shorter diameter. Hence, it is recommended to simulate preload in the form of torque rather than bolt axial tension while performing fatigue lifetime analysis in FEA to ensure accurate simulation of preload that will improve the fatigue lifetime of the dental implant. Because the application of bolt tension at the location in which the connector screw head meets the screw shank resulted in stress concentration, another plane of tension application was tried . This method did not provide a better result, so the details of the results were not included in this report. Each method has its type of artifact. For the torque application method, In several FEA studies on 2 piece dental implants, it is not clear whether the preload was applied on the connector screw of the implant assemblies, which raises a concern regarding the accuracy of the results reported from those simulations.23 However, FEA studies have their limitations. The materials were assumed to be homogenous, isotropic, and linearly elastic to simplify the FEA study and reduce the calculation time, but that may not reflect the clinical cases. A lack of corrosion was also assumed in the models while performing stress and fatigue lifetime analysis. The cylindrical holder was constructed on the basis of a novel bone specimen holder that attempts to mimic the clinical case,24 but this may not necessarily reflect the anatomic structure of the jaw bone. However, in this study, the focus was to analyze the stresses and fatigue lifetime of the implant assembly; hence, the architecture of the bone did not project a major relevance, and the cylindrical model was used to reduce the complexity of the analysis. FEA studies are based on the approximation of the results and should always be validated by performing mechanical tests. In a previous study, the results of fe-safe software were validated when the load amplitude predicted by FEA to result in failure at 1 million cycle of a narrow-diameter dental implant was within the 95% CI of the load amplitude obtained from the step-stress accelerated lifetime testing on the physical specimens of the dental implant.3 This study was the beginning of a larger project in which we will perform validation once an optimal implant design has been predicted.The simulations in this study were performed according to the ISO 14801 standard; hence, the bone crest was placed at 3 mm below the implant nominal bone level to simulate crestal bone loss.In this study, we investigated the effect of preload on implant fatigue lifetime and compared torque with bolt axial tension to determine the appropriate method to simulate preload. Within the limitations of this study, we concluded that adding a preload improved the fatigue lifetime of a dental implant assembly significantly. However, a substantial loss of preload can be accommodated without compromising the fatigue resistance of the dental implant studied. Adding a torque instead of bolt axial tension to simulate preload can generate a more accurate stress distribution on the connector screw. Hence, both the initial hypotheses were accepted.1


Models of synaptic plasticity have been used to better understand neural development as well as learning and memory. One prominent classic model is the Bienenstock-Cooper-Munro (BCM) model that has been particularly successful in explaining plasticity of the visual cortex. Here, in an effort to include more biophysical detail in the BCM model, we incorporate 1) feedforward inhibition, and 2) the experimental observation that large synapses are relatively harder to potentiate than weak ones, while synaptic depression is proportional to the synaptic strength. These modifications change the outcome of unsupervised plasticity under the BCM model. The amount of feed-forward inhibition adds a parameter to BCM that turns out to determine the strength of competition. In the limit of strong inhibition the learning outcome is identical to standard BCM and the neuron becomes selective to one stimulus only . For smaller values of inhibition, competition is weaker and the receptive fields are less selective. However, both BCM variants can yield realistic receptive fields. One of the hallmarks of the nervous system is it’s adaptive character. Over long time-scales the responses of neurons adapt to the input that it receives. On the neural level the adaptive character is perhaps nowhere more clearly observed than in the primary visual cortex. Manipulation of the visual environment and closure of the eyes have been shown to strongly affect the development of the visual system and the receptive fields that emerge  model was introduced some 40 years ago as a computational theory of synaptic plasticity . In that case u and x as a parameters. The polynomial has highly complicated coefficients, caused by the dependence of This reduction has two applications. First, one can now eliminate Second, the above analysis also yields the critical amount of inhibition above which the FPs merge and the standard BCM solutions become stable. As seen from Fig. nts (Eq. ) but alsnts , the winner-take-all solution carries over from the 2D case. In the steady state the neuron becomes selective to one stimulus only, and remains silent in response to all other stimuli . HoweveWhile weight dependent BCM does not appear to change the number of stable fixed points or the overall learning dynamics, it introduces an additional parameter. This parameter - the strength of feed-forward inhibition - sets the amount of competition. Competition is less selective in the weight dependent BCM model when the inhibition is weak; when inhibition is stronger than a certain threshold value, we retrieve standard BCM-like winner-take-all competition. It would interesting to measure the competition experimentally, ideally using an isolated neuron. We speculate that biology might have exploited this to develop receptive fields with different amounts of selectivity, e.g. create visual receptive fields with different tuning widths Fig. .In regards to the formation of sparse receptive fields, the weight dependent model seems to have little benefit over standard BCM. Both require zero-mean input. While such a pre-processing requirement is common in these type of models, the biology of it is unclear. From our results it seems that fixed feed-forward inhibition does not suffice for this purpose Fig. a. lateral inhibition, while single neurons do not need to be strongly selective (Hertz et al., Competition and selectivity among inputs are generally seen as essential ingredients in networks that perform sensory encoding. So one could wonder if strong competition is preferable from a functional point of view. For individual, isolated neurons there is no need to strongly select specific input patterns at the expense of others inputs as occurs in standard BCM. Competition could arise on the network level only, for instance throughWe assumed that the plasticity occurs exclusively in the excitatory connections and that the feed-forward inhibition is fixed. Knowledge of the inhibitory plasticity and its weight dependence is still scarce, but as experiments progress inhibitory plasticity can be included in the model. Another extension is to consider this modified plasticity rule in recurrent networks.


Study design: Technical note. Objectives: To present a novel C-arm-free technique guided by navigation to insert and place a C1 lateral mass screw. Background and Objectives: Atlantoaxial subluxation (AAS) is a relatively common sequelae in patients with rheumatoid arthritis (RA) and upper cervical trauma. If they present with severe symptoms, surgical intervention such as posterior fusion is indicated. The established treatment for AAS is fixation with a C1 lateral mass screw and C2 pedicle screw (modified Goel technique) to achieve bony fusion. However, this technique requires fluoroscopy for C1 screw insertion. To avoid exposing the operating team to radiation, we present here a novel C-arm-free C1 lateral mass screw insertion technique for AAS. Materials and Methods: A 67-year-old man was referred to our hospital with neck pain, quadriparesis, and clumsiness and numbness of both upper and lower limbs. He had undergone C3–6 posterior fusion previously in another hospital. In physical examination, he had severe muscle weakness of bilateral upper limbs and hypoesthesia of all four limbs. He had hyper-reflexia of bilateral lower limbs and pollakiuria. His Japanese orthopedic score was 8 points out of 17. Preoperative radiograms showed AAS with an atlantodental interval (ADI) of 7 mm. MRI indicated retro-odontoid pseudotumor and severe spinal cord compression at the C1–2 level. The patient underwent posterior atlantoaxial fixation under navigation guidance. To prevent epidural bleeding during the insertion and placement of a C1 lateral mass screw, we have here defined a novel screw insertion technique. Results: The surgical time was clocked as 127 min and blood loss was 100 mL. There were no complications per-operatively or in the postoperative period. The patient showed almost full recovery (JOA 16/17) at two months follow-up and a solid bony fusion was noticed in the radiograms at one year follow-up. Conclusions: This novel surgical procedure and C1 lateral mas screw placement technique is a practical and safe method in recent advances of AAS treatment. Procedurally, the technique helps prevent epidural bleeding from the screw entry point and also allows for proper C1 screw insertion under navigation guidance without exposing surgeons and staff to the risk of fluoroscopic radiation. Atlantoaxial subluxation (AAS) is a relatively common sequelae in rheumatoid arthritis (RA) and in patients who sustained upper cervical trauma. For the patients presenting with worsening symptoms, surgeries such as posterior fusion are indicated. For AAS, the goal of fixation is to achieve a solid bony union . There aThe modified Goel technique, which proposed C1 lateral mass screws combined with C2 pedicle screws, is an established surgical option ,10,11. NThis study was approved by the ethics committee of our institute . The necessary consents were obtained from the patient.A 67-year-old man was referred to our hospital with complaints of neck pain, quadriparesis. He had undergone C3–6 posterior fusion in another hospital 7 years ago due to gait disturbance. He had experienced almost full recovery for 5 years. Gradually, he had clumsiness and numbness of both upper and lower limbs these past two years.In the examination, he presented with severe muscle weakness of bilateral upper limbs and hypoesthesia of both upper and lower limbs. His manual muscle test (MMT) scores were grade 2 for bilateral deltoid muscle and grade 4 for biceps and triceps muscle, and hyper-reflexia was detected in both upper and lower limbs. He was unable to use chopsticks, had pollakiuria, and clinically presented with a Japanese orthopedic association (JOA) score that was 8 points out of 17.Preoperative radiograms showed AAS with an atlantodental interval (ADI) of 7 mm A,B. PreoThe patient underwent posterior atlantoaxial fixation under navigation guidance. To prevent epidural bleeding, to insert a C1 lateral mass screw, we made use of a novel screw insertion technique. We did not choose C1–6 fusion because we wanted to preserve the neck motion of the patient as much as possible. The surgical time was clocked as 127 min and blood loss was 100 mL. The patient experienced no postoperative complications such as CSF leakage, surgical site infection, or neurological compromise.Postoperative radiograms showed good upper cervical alignment A,B PostoThe patient recovered almost fully (JOA 16/17) at two months follow-up. His MMT scores were grade 5 for bilateral deltoid muscle and grade 5 for biceps and triceps muscle. He became able to use chopsticks and had no pollakiuria. His hyper-reflexia and hypoesthesia in both upper and lower limbs were reduced. The retro-odontoid tumor showed a decrease in morphology and spinal cord compression was minimized at four months follow-up A,B.The sR , and integrated.The patient was positioned prone with the neck flexed position on a Jackson frame with a full carbon skull clamp to enable the O-arm scan. The procedure was performed under neuromonitoring. The atlas and axis were exposed with a 5 cm posterior midline incision. The reference frame for the navigation was fixed at the C2 spinous process. The O-arm was then positioned, and three-dimensional (3-D) reconstructed images were obtained, transmitted to the StealthStation navigation system Spine 7After verifying every navigation-mapped spinal instrument, the accuracy of the same should be checked and confirmed. The entry point for the C2 pedicle screw was marked and a high-speed burr was used to make the cortical breach without pushing the C2 or further causing an anterior compression the spinal cord. A navigated pedicle probe was used to ascertain the trajectory and appropriate tapping was conducted, following which the C2 pedicle screw was inserted .The entry point for the C1 lateral mass screw (LMS) was the mid-point  of the C1 posterior arch A,B. A PeFirst, AAS was reduced by changing the neck position from flexion to extension by rotating the Mayfield skull clamp. Second, appropriately sized rods were applied over both sides and the two screws were compressed carefully. When adequate reduction was obtained, the set screws were tightened. An autologous iliac crest bone graft was placed interlminae for osteoinduction. Postoperative radiograms were taken to confirm the correct reduction and placement. A Philadelphia collar was applied to immobilize the neck.AAS is defined as the malalignment with increased mobility between the atlas and the odontoid process, which indicates laxity/rupture of the transverse ligament and loss of its stability. AAS is a common sequela of rheumatoid arthritis (RA) and has an incidence of 10% to 55% in RA patients . It is aDiagnosis is usually made with plain lateral cervical radiograms; however, dynamic views are required to confirm instability. ADI is one of the main diagnostic factors for AAS, which is determined by measuring the distance between the posterior edge of the anterior arch of C1 and the anterior edge of dens. ADI more than 3 mm in children and more than 5 mm in adults are regarded as abnormal . If a suSurgical indications of the AAS include patients presenting with symptoms of progressive cord compression, chronic myelopathy, and neuralgia not relived by conservative management. The Japanese orthopaedic association (JOA) score is used to evaluate the symptoms of cervical myelopathy in patients with AAS, which is highly reliable . In our According to Guo et al. , the meaWith the current advancements in technology, complex cervical spine surgeries are nowadays easily approachable . We haveThough there are numerous benefits of the above technique, it does have a few limitations. The availability of the O-arm navigation system may be sparse by region. Navigated screw placement requires training of the operating personnel. The placement of the reference frame may require care throughout the surgery, displacement of which would require a rescan with the O-arm or cause error in the screw insertion.This novel procedure is a useful and safe technique. It allows for ideal points for the insertion and placement of screws without the risk of VA injury and venous plexus under navigation guidance. This technique is performed without exposing surgeons and staff to the risk of radiation.


Food allergy is a significant public health problem with limited treatment options. As Food Allergy Herbal Formula 2 (FAHF-2) showed potential as a food allergy treatment, we further developed a purified version named EBF-2 and identified active compounds. We investigated the mechanisms of EBF-2 on IgE-mediated peanut (PN) allergy and its active compound, berberine, on IgE production.+ plasma cells, and PN anaphylaxis. Effects of berberine on IgE production, the expression of transcription factors, and mitochondrial glucose metabolism in U266 cells were evaluated.IgE plasma cell line U266 cells were cultured with EBF-2 and FAHF-2, and their effects on IgE production were compared. EBF-2 was evaluated in a murine PN allergy model for its effect on PN-specific IgE production, number of IgEEBF-2 dose-dependently suppressed IgE production and was over 16 times more potent than FAHF-2 in IgE suppression in U266 cells. EBF-2 significantly suppressed PN-specific IgE production  and the number of IgE-producing plasma cells in PN allergic mice, accompanied by 100% inhibition of PN-induced anaphylaxis and plasma histamine release (p<0.001) without affecting IgG1 or IgG2a production. Berberine markedly suppressed IgE production, which was associated with suppression of XBP1, BLIMP1, and STAT6 transcription factors and a reduced rate of mitochondrial oxidation in an IgE-producing plasma cell line.EBF-2 and its active compound berberine are potent IgE suppressors, associated with cellular regulation of immunometabolism on IgE plasma cells, and may be a potential therapy for IgE-mediated food allergy and other allergic disorders. FA anallergy” –26. Persion risk –32. Thervia oxidation (respiration) . IgE is the most heavily glycosylated isotype, with sugar moieties accounting for 12-14% of its molecular weight compared to approximately 3% for IgG  . Alterna for IgG –38. As maffected , 40. Plaaffected  and secraffected , 43. XBPaffected  and is raffected . Loss ofaffected , 47. Thiaffected .Thus, moecretion , but dirFructus mume formula, which has been used to treat parasite infection traditionally, showed a reduction of peanut specific IgE and protected against anaphylaxis in murine models of peanut allergy  on XBP1 expression and mitochondrial metabolism in a human IgE+PC line.Like FA, immunologic responses in parasite infections are associated with excessive production of IgE. FAHF-2 derived from  allergy –53, suggPrunus mume, Zanthoxylum schinifolium, Angelica sinensis, Zingiber officinalis, Cinnamomum cassia, Phellodendron chinense, Coptis chinensis, and Panax ginseng) were extracted using butanol, while the Ganoderma lucidum was extracted using ethyl acetate and the dried extracts were combined to generate the EBF-2 powder substance. Three batches of FAHF-2 and EBF-2 were tested in this study-manufacturing date and shelf life are listed in EBF-2 was generated by purification of FAHF-2 with a safe solvents consisting of butanol and ethyl acetate . 8 herb -5 M β-ME and 0.5% penicillin-streptomycin  at serial concentrations starting at 5 μg/mL were also tested on U266 cells. Supernatants were harvested, IgE levels were determined by ELISA  and cell viability was evaluated by trypan blue exclusion  was grown in complete media containing RPMI 1640 medium supplemented with 10% FBS, 1 mM sodium pyruvate, 1×10ptomycin , 56 at 2xclusion .® (a source of food grade ethanol) to neutralize stomach pH and increase gastrointestinal permeability, three times during week 0  were maintained in pathogen-free facilities at the Mount Sinai vivarium according to standard guidelines . Mice weg week 0 . Thereafg week 0 , PNA micSymptoms were evaluated 30-40 minutes following oral PN challenge as described previously , 59, andvia sub-mandibular bleeding 30 minutes after scoring and measurement of body temperature following oral peanut challenge. Plasma was harvested within 20 minutes after blood collection and stored at -80°C until analyzed. Histamine was measured using a commercial enzyme immunoassay kit  as described by the manufacturer .Mice were sacrificed at week 78 of the protocol and single-cell suspensions of splenocytes were prepared in ice cold staining buffer . First, surface staining with unlabeled anti-IgE (to block membrane IgE), APC anti-CD138, BV711-anti-CD3, and anti-CD16/32 (Fc-block) , CA was performed. Live-dead discriminating dye  was included. Cells were washed and incubated with fixation/permeabilization buffer  for 15 mins, washed with permeabilization buffer , and then incubated with FITC-anti-IgE, in permeabilization buffer. After washing, cells were treated with Cytofix buffer  for 15 mins for post-fixation, washed, and then data were acquired on an LSRII flow cytometer . Flow cytometry analysis was performed using Flow Jo  as follows. Live singlet cells were then analyzed for IgETo evaluate the safety of EBF-2, the sub-chronic toxicity assay was performed on C3H/HeJ mice as in our previous studies . Naïve CHPLC analysis was performed on a Waters 2690 HPLC system coupled with a 2996 PDA detector  for each batch of FAHF-2 and EBF-2 using the method described previously . Each saEBF-2 active compound in tissue samples were analyzed using LC-MS system. Briefly, tissue samples were cut into small pieces and soaked in methanol. The extracts were analyzed on an Agilent 1200 HPLC instrument with an Agilent Eclipse Plus-C18 column , coupled with an Agilent 6130 Single Quad Mass Spectrometry. The samples were eluted with acetonitrile-water (5-95%) containing 0.1% formic acid (v/v) over 100 mins.6 cells/mL) were incubated with or without berberine for 3 days. Cells were harvested and total RNA was isolated using Trizol . The RNA concentrations were quantified by triplicate optical density (OD) readings . Reverse transcription was performed to yield cDNA using ImProm-II™ Reverse Transcriptase  as per the manufacturer’s instructions. The RT-PCR amplification was performed using Maxima™ SYBR Green qPCR Master Mix (2X) kit . Primer sequences of XBP1, BLIMP1, STAT-6, BCL-6 and GAPDH were from previously published literature and listed in U266 cells , carbonyl cyanide-4 (trifluoromethoxy) phenylhydrazone  and rotenone and antimycin  were diluted in the assay medium and loaded into ports A, B, and C of the XF24 assay plate. The machine was calibrated, and the assay was performed using the Mito stress test assay protocol per the manufacturer’s recommendations. The extracellular acidification rate (ECAR) and oxygen consumption rate (OCR) were measured under basal conditions after sequential addition of the above-mentioned drugs.To determine the effect of EBF-2’s active compound berberine on mitochondrial metabolism of an IgE producing plasma cell line, we used the Seahorse mitochondrial stress assay. XF cell Mito stress test kits were obtained from Agilent . Each well of the XF24 cell culture plates was coated with 50 µL of Corning Cell-Tak cell and tissue adhesive at a density of 3.5 µg/cmp-value calculations were two-tailed and a p value < 0.05 was considered as statistically significant.All statistical analyses were performed using GraphPad Prism 9 . One-way ANOVA  was performed followed by Bonferroni correction for all pairwise comparisons. For skewed data, differences between groups were analyzed by one-way ANOVA on ranks followed by Dunn’s method for all pairwise comparisons. Data for symptom score correlation with IgE were analyzed using Spearman correlation. Pearson correlation was used for all other correlation analyses. p<0.05) with complete inhibition of IgE production at 60 μg/mL (p<0.001)  with complete inhibition of IgE production at 500 μg/mL(p<0.001) . Following intragastric challenge, all sham-treated mice developed anaphylactic symptoms, with symptom severity scores ranging from 2-3. In sharp contrast, EBF-2 treated mice were completely protected from anaphylaxis (p<0.05). Hypothermia, a decrease in core body temperature, is a symptom of anaphylaxis in mice. We measured rectal temperatures every 30 minutes after the intragastric challenge, EBF-2 prevented hypothermia, the mean post-challenge body temperature in EBF-2 group was significantly higher than the Sham group and not different from naïve mice (p<0.001 vs. sham). Anaphylaxis is associated with an increase in plasma histamine levels and plasma histamine levels in EBF-2 mice were markedly and significantly lower than in sham mice , whereas body temperatures at challenge were inversely correlated with IgE (p <0.01).We next determined EBF-2’s inhibitory effect on IgE levels and its protective effect against peanut anaphylaxis in a murine model (p<0.001) and maintained consistent levels even after completion of the treatment, up to week 78  (p<0.05), prevented hypothermia (p<0.001), and reduced plasma histamine levels (p<0.001). Furthermore, EBF-2 treated mice showed significantly lower numbers of the IgE+/CD138+ plasma cells from the spleen compared to Sham treated mice (p<0.001). There was a significant correlation between IgE+/CD138+ plasma cells and PN-specific IgE levels (p< 0.0001). These data highlight the persistent protection of EBF-2 from anaphylaxis following peanut exposure.We investigated the long-term protection by EBF-2 and its effect on long-lived IgE producing plasma cells in a peanut anaphylaxis murine model  isolated from FAHF-2 and B-FAHF-2 reduced IgE by plasma cell lines and human PBMCs from food allergic patients , and demin vitro using U266 cells. BBR dose-dependently inhibited IgE production approaching 100% inhibition at 5 µg/mL  which BBR eliminated IgE production, we hypothesized it may act on mechanisms controlling IgE production and secretion. IgE+PCs are under constant stress from antibody production and secretion. XBP1, a transcription factor, promotes and maintains plasma cells antibody production and secretion under ER stress as a compensating response . It is established that a low cellular ATP level allosterically activates the enzyme PFK, one of the rate-limiting enzymes in glycolysis, thus leading to the increase in glycolysis and the subsequent decrease in hexosamine biosynthesis for glycosylation (+PCs by regulating both transcription factors and mitochondrial metabolism.In addition to transcriptional regulation, energy metabolism has emerged as an important regulator of plasma cell survival and function . PreviouIn this study, our goal is to test the potency of a refined botanical medicine in murine model of peanut allergy, we therefore orally sensitized using Th2 adjuvant and orally challenged female C3H/HeJ mice. The advantage is that this murine model showed persistent peanut allergy, which allow us to test the durability of EBF-2 on food allergy and study long-lived IgE producing plasma cells. The reason to use female mice is to consider that females are more susceptible to food allergies  and have+PCs in a murine model. The mechanism of BBR, the active EBF-2 compound in suppressing IgE may be partially associated with its inhibitory effect on XBP1 and mitochondrial metabolism leading to insufficient energy and transcriptional activation for IgE IgH/IgL synthesis and antibody glycosylation. Further understanding of how EBF-2 and BBR regulate established IgE production by IgE+ PCs may lead to new interventions to target key mechanisms of IgE-mediated food allergy.In conclusion, this study demonstrated that a novel botanical medicine, EBF-2, significantly and consistently suppressed IgE production and is markedly more potent than its parent formula FAHF-2. EBF-2 significantly suppressed PN specific IgE production with complete protection against anaphylaxis and long-lasting effects associated with suppression of IgEThe original contributions presented in the study are included in the article/The animal study was reviewed and approved by Icahn School of Medicine at Mount Sinai.NY, ARM, KS, MK, ZW, IM, YS, OF, and YG were significantly involved in conducting experiments, data analysis, and manuscript preparation. JW, DC, DD, and HS were significantly involved in manuscript revision. JZ, MM, and X-ML were significantly involved in study design, data interpretation, and manuscript revision. All authors contributed to the article and approved the submitted version.


Brimonidine tartrate (BRT) is a highly selective α2 adrenergic receptor agonist as treatment for patients with open angle glaucoma and high intraocular pressure. The objective of this study was to formulate an ophthalmic ion-sensitive in situ gel (ISG) of BRT to increase the retention time of the drug and its bioavailability. The optimum formulation of 2 mg/mL BRT-ISG was obtained with 0.45% gellan gum as the gel matrix. In vitro release results showed that the water-soluble drug bromonidine tartrate in ocular in situ gels exhibited a high burst effect and fast release in solution. The results of dialysis membrane permeation showed that there was a significant difference between the commercially available and BRT-ISG groups after 45 min. The results of the pre-corneal retention study indicated that gellan gum can effectively prolong ocular surface retention. Preliminary stability results showed that it should be stored in a cool and dark place, and the formulation under long-term preservation can be basically stable. The pharmacokinetic study of the BRT-ISG in the anterior chamber of the rabbit eye was studied by microdialysis technique, and microdialysis samples were analyzed by LC-MS/MS. The pharmacokinetic study showed that the BRT-ISG reached Cmax (8.16 mg/L) at 93 min after administration, which was 2.7 times that of the BRT eye drops, and the AUC(0-t) (1397.08 mg·min/L) was 3.4 times that of the BRT eye drops. The optimal prescription can prolong the retention time of BRT in front of the cornea and significantly improve the bioavailability of BRT in the eye. Combined with the results of in vitro release, permeation and pre-corneal retention studies, the improvement of BRT-ISG bioavailability in rabbit eyes was found to be mainly due to the retention effect after the mixture of ISG and tears. Brimonidine tartrate  were HPLC-grade. Sodium chloride, sodium bicarbonate and citric acid were obtained from Chengdu Kelon Chemical Reagent Factory . 0.2% brimonidine tartrate eye drops were provided by Allergan, Inc., . Other reagents and chemicals were of analytical grade.Brimonidine Tartrate Reference Substance  was purchased from Hangzhou Deli Chemical Co., Ltd. . Gellan gum was procured from Zhejiang Tianwei Biotechnology Co., Ltd. . Hydroxypropyl methylcellulose  and tromethamine were supplied by Aladdin Reagent Co., Ltd. . Mannitol and triethylamine were purchased from Sinopharm Chemical Reagent Co., Ltd. . Benzalkonium chloride was provided by Fluka Company. Methanol  and acetonitrile  2013-0055; Zhejiang Academy of Medical Sciences Laboratory Animal Center license SCXK (Zhejiang) 2014-0008. They were housed in individual cages and maintained on a 12 h light/dark cycle with the room temperature of 24 ± 2 °C in the breeding facility. They were raised following standard protocols of animal care and use guidelines established by the Zhejiang Academy of Medical Sciences, with free access to standard laboratory food and water. All animal experiments were approved by the animal ethics/use committee of Zhejiang Academy of Medical Sciences .The gellan gum solution was prepared as reported in the literature . The speGelation ability and viscosity were the two most important factors for ISG formulation screening.The gelation ability of the gel formulation was to be evaluated to select a composition suitable for ISG. The viscosity was an important factor in determining the retention time of the drug on the ocular surface. Gelation ability measurement: 2 mL STF was taken in a test tube with a stopper in a water bath at 34 °C, then 100 μL of ISG was added to the test tube. The gel formation with or without shaking was observed and recorded .After the ISG was gelled in STF, it was measured with the No. 52 rotor of the viscometer  . Since t18 column ; mobile phase, acetonitrile: 0.017 μg/mL citrate buffer (triethylamine was added until the pH = 3) = 0.08:0.92; flow rate of mobile phase, 1.0 mL/min; column temperature, 30 °C; the UV detection wavelength was set at 248 nm; injection volume, 20 µL.In vitro drug release experiments were carried out via the dialysis bag method ,32. PrecIn order to better simulate the pre-corneal release behavior of the drug, the in vitro dialysis membrane permeation study was performed by using the dialysis membrane method to compare the BRT-ISG preparation and the commercially available BRT eye drops. The pretreated dialysis membrane  was embedded between the supply and the receiving pool of the flat-mouth Franz diffusion cell a, and 3 Ex vivo transcorneal and transscleral permeation studies were carried out to evaluate the permeation of BRT-ISG and commercially available BRT eye drops in the rabbit cornea and sclera ,33. New The cumulative drug release rate (Q%) is calculated using the following equation:n—The cumulative drug release or permeability rate, %;QV—sampling volume, mL;0—Volume of the medium in the receiving chamber, mL;Vn—Drug concentration at sampling points, μg/mL;Ci—Drug concentration before sampling points, μg/mL;Cdrug—Total amount of drug BRT, μg.mThe permeability coefficients (Papp) was calculated by formula (Equation (2)) .(2)PappV—buffer volume in the donor compartment (3 mL);2);A—diffusional area of the membrane ; mobile phase, acetonitrile (A) and 0.017 μg/mL citrate buffer (triethylamine was added until the pH = 3) (B)—gradient eluted was programmed as follows: 0.01–1.00 min 8% eluent A, 1.01–5.00 min 8–20% eluent A, 5.01–13 min 20% eluent A, 13.01–22.00 min 8% eluent A; flow rate of mobile phase, 1.0 mL/min; column temperature, 30 °C; the UV detection wavelength was set as follows: 0.01–6.50 min 248 nm, 6.51–16.00 min 266 nm, 16.01–22.00 min 248 nm; injection volume, 20 µL.The BRT-ISG contents were determined using HPLC by the method described in the section above, “In vitro release study”. The chromatographic conditions of impurities concentration determined by HPLC were as follows: chromatographic column, Diamonsil CBRT-ISG stability tests were evaluated using an influencing factor experiment including high temperature and strong light . The preparations were divided among light blue high-density polyester eye drop bottles , sealed and kept for samples. The samples were placed in a constant temperature oven at 40 °C and 60 °C for 10 days. Samples were taken on the 5th and 10th days to investigate various indicators of the preparations. The samples were placed in a light incubator equipped with a fluorescent lamp under the condition of 4500 lx ± 500 lx for 10 d, and samples were taken on the 5th and 10th days to investigate the various indicators of the samples.The accelerated stability tests were conducted at a temperature of 40 ± 2 °C and relative humidity of 25 ± 5% (saturated potassium acetate solution) for 6 months. Samples were taken at 1st, 2nd, 3rd, and 6th months to investigate the various indicators of the samples.The chromatographic conditions were as follows: chromatographic column ZORBAX SB-C18 ; mobile phase, 0.1% formic acid water (A), acetonitrile (B), isocratic elution program: A:B = 50:50; flow rate, 0.4 mL/min; injection volume, 5 μL; column temperature, 40 °C. The MS conditions were as follows: positive (ESI+) electrospray ionization mode: multiple reaction monitoring (MRM) mode; ion source parameters, gas temperature 300 °C, gas flow 5 L/min, nebulizer 45 psi, sheath gas temperature 250 °C, sheath gas flow rate 11 L/min, capillary voltage 3500 V, nozzle voltage 500 V. The detailed method of HPLC-MS/MS analysis of BRT can be seen in our previous publication [m) to the recovery rate , Cr = Cm/Rdial [The in vivo pharmacokinetics study was performed by employing the microdialysis technique which was modified slightly from the previous literature ,41. SimpCm/Rdial . The samThe gelling ability of each formulation was investigated, and the results are shown in The effect of ion intensity on the viscosity of different formulations is shown in p > 0.05). The results showed that both the BRT-ISG group and commercial group gave a rapid release of the BRT, which could take effect quickly after intraocularly delivered. Origin8.0 software was used to fit the data of the self-prepared BRT-ISG cumulative release rate in the range of 0–120 min of mathematical models. The results are shown in The dialysis bag method was used for the formulations’ in vitro drug release study, and the commercially available eye drops were used as the control group. The in vitro release results of the dialysis bag are shown in p < 0.05), and very significant difference after 60 min (p < 0.01). The self-prepared BRT-ISG had a cumulative release rate of 85% at 120 min, while the commercial group has a cumulative release rate of 52% at 45 min, which tends to be stable, indicating that the self-prepared BRT-ISG had retention characteristics on the flat-mouth Franz diffusion cell. The reason for this phenomenon may be that BRT-ISG was concentrated in the middle part of the flat-mouth Franz diffusion cell due to its viscosity while the position of the eye drop solution group could easily lead to loss from the mouth of the Franz diffusion cell. The self-prepared BRT-ISG cumulative release rate was fitted with mathematical models, and the results are shown in n = 0.564. Since 0.5 < n <1, this showed that the in vitro release mechanism of the preparation dialysis membrane method was combined erosion with diffusion.The dialysis membrane was also used to study the formulations in vitro using a flat-mouth Franz diffusion cell, with the commercially available eye drops as a control. The results for self-prepared BRT-ISG are shown in p < 0.05). The cumulative permeability of the self-prepared BRT-ISG was significantly lower than that of the commercial group, indicating that the self-prepared BRT-ISG had a certain slow-release effect, and the cumulative permeability of 300 min was more than 80%. The sclera cumulative permeability of the self-prepared BRT-ISG sclera was fitted by mathematical models and the results are shown in The ex vivo permeation study of formulations were carried out using the isolated sclera, and the commercially available eye drops were used as a control. The ex vivo permeation results of self-prepared BRT-ISG are shown in p > 0.05). Mathematical models were applied to fit the results of the self-prepared BRT-ISG corneal cumulative permeability as shown in −3 (cm/h), and that of in situ gel was 36.24 × 10−3 (cm/h). The permeability coefficient of eye drops in the cornea was 32.11 × 10−3 (cm/h), and that of in situ gel was 35.36 × 10−3 (cm/h). There was no significant difference in the permeability efficiency of the two preparations on the sclera and cornea calculated by software GraphPad Prism 8.0. A pH responsive in-situ gel prepared with carbomer was reported [−3 (cm/h). In another study, a scholar used polyamidoamine (PAMAM) to prepare an ophthalmic polymer hydrogel to realize the co-delivery of bromonidine tartrate and timolol maleate [−5 cm/s. The permeability coefficient is 35.36 × 10−3 (cm/h) of the in-situ gel in this study, equivalent to 0.98 × 10−6 cm/s. Compared with the other two preparations, the permeation efficiency is similar. The permeability coefficient of the in-situ gel in this study is higher than that of pH responsive in-situ gel and lower than that of polymer gel. To a certain extent, the in-situ gel of this study achieved the effect of sustained release. However, ionic in-situ gel is more convenient for storage and transportation than polymer gel. Compared with pH responsive in-situ gel, it is less affected by the environment, and the ions in tears can complete the phase transition. The cumulative drug permeation ex vivo was similar for the two preparations  on the sclera and cornea, because the permeation cell was an upper and lower arc structure, the cornea and sclera were stretched on the cell mouth, and the upper and lower cell mouths fit tightly; there was no fluid loss of commercially available eye drop from cell, so there was more permeation, which cannot reflect the better retention effect of ISG than solution. Therefore, in vivo pharmacokinetic experiments were needed for further verification.Drugs were absorbed through the eye and generally needed to pass through ocular tissues such as cornea, sclera and conjunctiva. Therefore, the ex vivo corneal permeability was investigated using cornea. The results are shown in reported . Its cor maleate . BromoniIt has been reported that gellan gum was used as the gel matrix of BRT, with the best concentration of 0.6%, which had a sustained-release effect in an in vitro experiment only studying cellophane membrane using a Franz diffusion apparatus . In our The change of ocular surface fluorescence with time after the instillation of Fls-gel and Fls-solu is shown in The results of the high temperature test and intense light test are shown in The results of intense light test showed that compared with the initial condition, after being placed for 10 days under the condition of 4500 lx ± 500 lx, the appearance, pH value and content of the preparation were basically unchanged, but the impurity content increased, indicating that the BRT preparation should be stored in the dark.The results of the accelerated test are shown in 1-bromoquinoxalinyl-6-thiourea was selected as the most representative related impurity for detection ,47. The max (3.06 mg/L) about 60 min after administration, while the self-prepared BRT-ISG group reached Cmax (8.16 mg/L) about 93 min after administration. The results showed that the Cmax of BRT-ISG was significantly higher than that of the commercially available eye drops. The MRT(0-t) of self-prepared BRT-ISG group was 1.3 times later than the commercial group, indicating that as the retention time of the drug in front of the cornea was prolonged, the drug could continue to penetrate the cornea into the aqueous humor. The AUC(0-t) of the self-prepared BRT-ISG group was 1397.08 ± 444.57 mg·min/L, which was 3.4 times higher than that of the commercial group. The two groups had extremely significant differences (p < 0.01). The bioavailability of BRT-ISG eye drops group in the eye was significantly improved.The drug concentration-time curves of the two preparations in the rabbit aqueous humor are shown in ® eye drops, its concentration in aqueous humor was lower than the results of this research; these differences could be due to different administered doses [® (0.2% brimonidine tartrate) in the eyes in their experiment, while 50 μL drops were given in this study. Mathematically, the results of AUC and Cmax would be close to our study if dose influence were removed. This meant that, the results obtained by the two different sampling methods were consistent. Combined with the results of in vitro release and permeation experiments, the improvement of BRT-ISG bioavailability in rabbit eyes was mainly due to the retention effect after the mixture of ISG and tears.Microdialysis sampling has been used in pharmacokinetic studies of BRT in aqueous humor after topical administration of commercially available BRT solution. It was a well known traditional sampling method requiring sacrificing a large number of animals. The sample pretreatment procedure, which involved solid-phase extraction, was tedious and subject to error and time-consuming. According to literature reports on the pharmacokinetics of aqueous humor after local instillation of Alphaganed doses ,49. RabbThe current commercially available BRT eye drops have the disadvantages of fast loss, short effective time, and poor safety. In this study, an ion-sensitive ophthalmic BRT in situ gel was optimized. This preparation is simple and convenient to prepare, has good fluidity at room temperature, is suitable for industrial production, and has good stability. BRT-ISG can be released quickly, which means it can take effect quickly after intraocular administration. In vivo experiments provided evidence that the BRT-ISG formulation can prolong the retention time in the eye and significantly improve the bioavailability of BRT. The optimal prescription had the dual characteristics of rapid release and continuous release. In the future, it is hoped that the new BRT-ISG preparation can be developed into a new drug to market, providing safer, more effective and convenient dosage forms for the majority of patients. It has the characteristics of rapid release and continuous release, and its intraocular bioavailability is significantly higher than that of commercial ordinary eye drops.


A simple and rapid coupling reaction of Pauly’s reagents and BPA not only solved the problem of poor affinity between BPA and noble metals, but also provided the SERRS activity of BPA azo products. The distribution of hot spots and the influence of incremental introduction of noble metals on the performance of SERRS were analyzed by a finite-difference time-domain (FDTD) algorithm. The abundance of hot spots generated by core–shell–satellite structure and outstanding SERRS performance of Au@Ag nanocrystals were responsible for excellent SERRS sensitivity of CSSN NCs in the results. The limit of detection (LOD) of CSSN NCs for BPA azo products was as low as 10−10 M. In addition, the saturation magnetization (Ms) value of CSSN NCs was 53.6 emu·g−1, which could be rapidly enriched and collected under the condition of external magnetic field. These magnetic core–shell–satellite NCs provide inspiration idea for the tailored design of ultra-sensitive SERRS substrates, and thus exhibit limitless application prospects in terms of pollutant detection, environmental monitoring, and food safety.As a typical representative of endocrine-disrupting chemicals (EDCs), bisphenol A (BPA) is a common persistent organic pollutant in the environment that can induce various diseases even at low concentrations. Herein, the magnetic Fe Endocrine-disrupting chemicals (EDCs), also known as environmental hormones, are exogenous substances that affect mammalian reproduction by interfering with the endocrine system of organisms ,2,3. Bis4 to 1011, while the CE mechanism only contributes to 10 to 103 enhancement [At present, direct detection and indirect detection are two commonly used methods for BPA detection. The direct detection method of BPA mainly includes high-performance liquid chromatography (HPLC), gas chromatography/mass spectrometry (GC-MS) and liquid chromatography/mass spectrometry (LC-MS) ,12,13. Nancement . Therefoancement . Given tancement ,26,27.Inducing aggregation and constructing interlayers are two commonly used methods for introducing high- density hot spots. Nonetheless, the introduction of aggregation will inevitably bring about non-uniform distribution of noble metals, which is not conducive to the purpose of increasing hot spots . In comp3O4-Au@Ag@(Au@Ag) nanocomposites (CSSN NCs), which could serve as ultra-sensitive SERRS substrates for BPA detection. A simple and rapid coupling reaction of Pauly’s reagents and BPA was used not only to solve the problem of poor affinity between BPA and noble metals, but also to advance SERRS enhancement performance. BPA azo products were selected as target molecules to discuss the effect of the incremental introduction of noble metals on SERRS activity. The distribution of hot spots was obtained by a finite-difference time-domain (FDTD) theoretical method, and the relevant SERRS enhancement mechanism was discussed. Given that Fe3O4 hollow spheres with large specific surface area have a good superparamagnetic property, CSSN NCs could be easily recovered through an external magnet, which provided potential possibilities of recycling and reuse in complex liquid environments [Herein, we propose magnetic-core–shell–satellite Feronments . Our worThe details of this section can be found in 3O4 hollow spheres, Au seeds, and Au@Ag nanocrystals were obtained on the basis of our previous work [PEI-DTC aqueous solution, Feous work .3O4 hollow spheres were added to 20 mL of methanol, followed by dropwise addition of 25 mL of PEI-DTC aqueous solution. After the mixture stood for 60 min, the product was separated and gathered by a magnet. Then 20 mL of Au seeds were added under sonication. Finally, FA NCs were obtained after continuous sonication for 1 h and repeated washing.An amount of 20 mg of Fe3  and reductant NH2OH·HCl  were added. FA@Ag NCs were obtained after sonication for 2 h and several wash cycles with ethanol.As illustrated in The second step was the synthesis of CSSN NCs. The previously obtained FA@Ag NCs were blended with 20 mL of methanol, and 25 mL of PEI-DTC was dropwise-added. The mixture was stored for about 1 h. Then, FA@Ag@PEI-DTC NCs were obtained through washing and drying. After 10 mg of FA@Ag@PEI-DTC NCs was put into 5 mL of deionized water, 40 mL of Au@Ag nanocrystals was poured into the mixture and sonicated for about 2 h. Finally, the product was washed and dried to obtain CSSN NCs.p-aminobenzenesulfonic acid (4.5 g), HCl solution  and deionized water (500 mL). Reagent B was 5% NaNO2, and reagent C was 10% Na2CO3.Three kinds of reagents were prepared and kept at 4 °C for further use. Reagent A was a mixture of Coupling reaction: Reagents A + B + C + BPA ethanol solution in a volume ratio of 1:1:1:2.Details of FDTD algorithm method can be found in p-aminobenzenesulfonic acid through electrophilic aromatic substitution [−10 to 10−4 M and CSSN (1 mg) NCs were mixed in the aluminum pan, respectively. SERRS spectroscopy was performed under 514.5 nm laser and acquisition time was about 10 s.Before SERRS measurements, BPA was changed into azo dyes through a coupling reaction of BPA and diazonium ions to bind noble metals with high affinity and thus achieve highly sensitive detection of BPA. In this work, the phenol group of BPA could be converted into azo dyes with titution ,42. Afte3O4 hollow spheres, FA, FA@Ag and CSSN NCs. As shown in 3O4 hollow spheres located at 30.4°, 35.5°, 43.4°, 53.4°, 57.3°, and 62.8° refer to the (112), (211), (220), (024), (303), and (224) planes of Fe3O4, respectively, which can be indexed to the cubic inverse spinel structure of Fe3O4 (JCPDS 19-0629) [3O4 hollow spheres have high phase purity and good crystallization. However, owing to the fact that Fe3O4 as well as γ-Fe2O3 have the identical spinel structure, it is not sufficient to identify them only by XRD results [3O4 hollow spheres was further verified by Mössbauer spectroscopy. As depicted in 3O4 hollow spheres can be fitted into two sextets, and the magnetic sextets lines illustrate the typical double six peak structure of Fe3O4 [3O4 hollow spheres are presented in 2+ and Fe3+ at octahedral interstitial sites and Fe3+ at tetrahedral interstitial sites. After Au seeds were loaded on surfaces of Fe3O4 hollow spheres, four new XRD diffraction peaks emerged at 38.2°, 44.3°, and 64.5°, which were assigned to (111), (200), and (220) planes of Au (JCPDS 04-0784) [3O4 characteristic peaks than that of Fe3O4 hollow spheres, FA, and FA@Ag NCs. This may be attributed to the declining proportion of Fe3O4 contents caused by the successful modification of the large amount of Au seeds and Au@Ag nanocrystals. Consequently, the information obtained from above XRD and Mössbauer analysis preliminary confirm the successful construction of Fe3O4 hollow spheres, FA, FA@Ag, and CSSN NCs.XRD technology was used to study the structure and phase purity of Fe19-0629) . In addi results . Therefoof Fe3O4 ,47. The 04-0784) . It shou04-0784) . Since t04-0784) ,51, the 3O4 hollow spheres, FA, FA@Ag, and CSSN NCs, their magnetic hysteresis (M-H) loops were tested via vibrating sample magnetometer (VSM), as displayed in M-H loops show that Fe3O4 hollow spheres, FA, FA@Ag, and CSSN NCs have superparamagnetic property. The saturation magnetization (Ms) values of Fe3O4 hollow spheres, FA, FA@Ag, and CSSN NCs were 89.1, 74.5, 63.7, and 53.6 emu·g−1, respectively. It was found that the Ms value gradually reduces with the incremental introduction of noble metal due to the diamagnetism of noble metal nanocrystals [For researching the magnetic properties of Fecrystals . As depi3O4 nanocrystals show uniform spherical shapes with size of about 600 nm, and the dark edges and shallow cores of Fe3O4 nanocrystals suggest the formation of hollow structures. It can be seen from the TEM image of FA NCs  was chosen as the target molecule and FA, FA@Ag, and CSSN NCs served as SERRS substrates to explore their SERRS-enhancing capabilities, respectively. The detailed band assignments of BPA azo products are exhibited in In order to directly evaluate the SERRS performance of various substrates, BPA , which is well below the safety limit of the European Union (0.6 mg/kg), as well as China (10 ng/mL) [−1 is reflected in 2) is up to 0.96, which further proves that CSSN NCs are high performance SERRS sensors and can realize quantification of BPA down to 10−10 M.In order to assess the practicability of CSSN NCs as SERRS substrate for quantitative and sensitive detection of BPA, BPA azo products with different concentration from 100 ng/mL) . More im0 ng/mL) ,73,74,75−10 to 10−4 M, SERRS intensities followed linear relationship versus the logarithm of the concentrations, and LOD was as low as 10−10 M. In addition, the Ms value of superparamagnetic CSSN NCs was 53.6 emu·g−1, which gave CSSN NCs the function of rapid separation and detection in complex liquid environments by an external magnetic field. This study not only provides a novel ultra-sensitive SERRS substrate, but also shows enormous potential for the field of food safety and environmental pollution control.In conclusion, magnetic core–shell–satellite CSSN NCs for ultra-sensitive SERRS detection of BPA have been successfully developed. The coupling reactions between BPA and Pauly’s reagents not only improved the affinity between BPA and substrates, but also amplified the SERRS signals due to the SERRS effect generated by the combination of resonance of BPA azo products and plasma resonance of noble metals. BPA azo products were chosen as target molecules to investigate the effect of incremental introduction of noble metals on SERRS activity. The distribution of electromagnetic field of CSSN NCs was studied through FDTD theoretical algorithm. The results revealed that a considerable number of hot spots were produced on the core–shell–satellite structure. The excellent SERRS activity of CSSN NCs was attributed to abundant hot spots of core–shell–satellite structure as well as outstanding SERRS activity of Au@Ag nanocrystals. BPA azo products were used to evaluate the practicability of CSSN NCs as SERRS substrate. When the concentrations of BPA azo products ranged from 10