To introduce the procedures of two-step fundus fluorescein angiography (FFA) and evaluate its utility in the management of pediatric retinal vasculopathy.In this retrospective study, medical records of 12 patients who received two-step FFA were studied. The two-step FFA consisted of step 1 [low-dose (LD)] FFA at an intravenous dose of 1.5 mg/kg fluorescein, followed by step 2 [reduced dose (RD)] FFA at a dose of 6.2 mg/kg fluorescein. Demographic data, including age, gender, diagnosis, weight, gestational age, birth weight, and weight on the examination day were taken, were collected. The results of two-step FFA and treatment were recorded.A total of 20 eyes were studied. The top 5 common FFA changes in RD-FFA included peripheral avascular zone (15 eyes), fluorescein leakage (10 eyes), supernumerous vascular branching (10 eyes), neovascularization (NV) (8 eyes), and absence of the foveal avascular zone (6 eyes). LD-FFA was efficient to show all the NV without severe vitreous dye in 8/8 (100.0%) eyes with NV, partial peripheral avascular zone in 11/15 (73.3%) eyes, while RD-FFA always offered more information in all the eyes. Thirteen eyes had laser photocoagulation under the guidance of LD-FFA. In 4 (30.8%) eyes, RD-FFA revealed more lesions and an immediate relaser was performed. Laser photocoagulation was successfully performed in all the 13 eyes in one session without being rearranged. After a median follow-up of 28.1 months, all the eyes were in a stable status.Step-one LD-FFA acted as a pre-FFA to show the NV, and step-two RD-FFA acted as a double-check. The modified strategy may be a helpful clinical adjuvant in the laser photocoagulation of pediatric retinal disorders, especially for young ophthalmologists. Widefield fundus fluorescein angiography (WF-FFA) offers more details of microvascular abnormalities, retinal non-perfusion areas, neovascularization, vascular fluorescein leakage , and vasTraditional FFA was performed with an intravenous injection of 5% fluorescein dye dosed by weight 7.7 mg/kg) , 7. For .7 mg/kg Occasionally, we found 1.5 mg/kg fluorescein that could help to show the retinal NV without severe vitreous dye. To avoid secondary interference caused by dye leakage, we came up with this two-step FFA procedure and evaluated its utilization in a real-world setting. The two-step FFA consisted of step 1 [low-dose (LD)] FFA at an intravenous dose of 1.5 mg/kg fluorescein, followed by step 2 (reduced dose of 6.2 mg/kg) FFA to ensure the observation and treatment in pediatric patients. In this pilot study, we postulated that two-step FFA may be increasing as a meaningful clinical adjuvant in the management of pediatric retinal disorders.With Institutional Review Board approval by Xinhua Hospital Affiliated to Shanghai Jiao Tong University (XHEC-D-2022-071), a retrospective study was conducted in our department from 6 December 2019 to 15 May 2022. It adhered to the tenets of the Declaration of Helsinki . WrittenDemographic data, including age, gender, diagnosis, weight, gestational age, birth weight, and weight on the examination day were collected. All the FFA and fundus color images were acquired under general anesthesia with RetCam III . Scleral indentation was performed as needed to show the far periphery of the retina.After proper general anesthesia and mydriasis, color images were initially taken before step 1 FFA (LD-FFA). Intravenous sodium fluorescein (10%) was injected into the peripheral vein followed by a saline flush. Contact high-resolution WF-FFA images were obtained. To avoid possible neglect of lesions of both eyes, speculums were placed on both eyes simultaneously. FFA images of posterior pole and four quadrants were first taken on the eye with severer abnormalities or possible NV and the camera was shifted to the fellow eye quickly to take the early venous-stage images. After the LD-FFA, we also checked the skin and vital signs for any allergic reactions. Only when no severe allergic reactions occurred, the further procedures performed. LD-FFA-guided laser photocoagulation was carried out wherever required immediately. After laser photocoagulation, step 2 FFA with a dose of 6.2 mg/kg sodium fluorescein FFA [reduced dose FFA (RD-FFA)] was performed to confirm the possible residual lesions and proper laser spots. Relaser treatment can be applied to the skipped areas accordingly. For eyes with massive NV or severe fluorescein leakage, intravitreal injection of ranibizumab could be applied after the laser photocoagulation.Statistical analysis was performed using Statistical Package for the Social Sciences (SPSS) version 26 . Data were expressed as the mean ± SD or as median and range.Twelve patients (6 boys and 6 girls) with a median age of 6.70  months, and a mean birth weight of 3070.83 ± 751.50 g received two-step FFA were studied. The clinical diagnoses included FEVR , IP , Coats disease , and retinal detachment (RD) with unknown reason . Eight patients underwent bilateral FFA and four patients underwent unilateral FFA due to simultaneous surgery on the fellow eye. As a result, a total of 20 eyes were studied.The results are given in Low-dose fundus fluorescein angiography was efficient to show all the NV with fluorescein leakage in 8/8 (100.0%) eyes. In 14 eyes with different kinds of abnormal vessels, LD-FFA always showed less information and neglected some information in 14 (100%) eyes. Among 15 eyes with peripheral avascular zone, LD-FFA showed partial peripheral avascular zone in 11 eyes frontier to abnormal vessels or NV, while LD-FFA was unable to show peripheral avascular zone anterior to normal or fine vessels, and not efficient to show all the peripheral avascular zone in all the 15 eyes as shown in RD-FFA. As a double-edged sword, low-dose (1.5 mg/kg) fluorescein could show NV in LD-FFA without obvious disturbance in the color fundus images in all the 8/8 eyes. With a higher dose, RD-FFA shows more distinct vascular details about peripheral retinal anatomy, vascular network, and non-perfusion areas, and necessary clinical information was obtained in all the cases. As laser spots were performed after the LD-FFA, laser spots were not recorded as an FFA change.In total, 13 (65.00%) eyes had laser photocoagulation under the guidance of LD-FFA. In 4  eyes of them, RD-FFA revealed more lesions asking for immediate relaser . The reaAll the patients tolerated the procedures without adverse effects during or after the procedures, such as rash, respiratory distress, tachycardia, fever, and local injection site reactions as reported in adults . The medFundus fluorescein angiography is helpful in clinical situations for a variety of pediatric retinal diseases. In patients with darkly pigmented fundi, such as Asians, the border of the vascularized zone and the demarcation line are barely detectable with an ophthalmoscope. Intraoperative FFA-guided laser photocoagulation in children offered good anatomic outcomes, with preservation of visual acuity in most cases of retinal vascular disease , 12, 15.Fundus fluorescein angiography was commonly performed with intravenous injection of fluorescein at a dose of 7.7 mg/kg , 7 or 3.The LD-FFA was able to show all the NV in all the eyes (100.00%), which is helpful in clinical applications. It showed the retinal NVs without strong vitreous staining, making it easy to evaluate the retinal vessels and laser spots under the indirect ophthalmoscope and the camera. A clear view is very important for the doctor to give precise treatment. Inappropriate laser photocoagulation may end up with disease progression, including rhegmatogenous RD, tractional RD, or a combination of both . With thFor pediatric patients with poor compliance, the FFA and laser photocoagulation are always performed under general anesthesia in the spine position. The laser ablations are conducted with binocular laser indirect ophthalmoscope, which needs a longer learning curve. The procedures are even harder with obscure media. Step-one LD-FFA acted as a pre-FFA to show the NV without obvious vitreous opacity. As a double-edged sword, LD-FFA was surely unable to show all the vascular anomalies, especially for subtle lesions, such as microaneurysms, vessels fluorescence staining, absence of the FAZ, bulbous vascular terminals, distinct pruning of vessels, PFV, and peripheral vessels in normal eyes. Hence, RD-FFA can serve as a good complement and confirmation on this basis. Step-two RD-FFA reveals more detailed information about retinal vessels and new laser spots, which is the double-check and guides supplementary laser photocoagulation when needed. On the basis of former laser ablation, the residual supplementary laser photocoagulation required less time before excessive fluorescein extravasation blurred the view, which is friendly to surgeons with comfort, especially for young ophthalmologists in their early career.As a result, in our opinion, we highly recommend this technique to be applied in cases with retinal exudates and suspected NV. An LD-FFA will show whether and where the NV existed without severe media opacity, making the laser treatment easier. With the same pharmacokinetics, the circulation time is the same as traditional ones. So, the NV is always shown within 1 min. If no NVs were noted 1 min after the fluorescein injection, the RD-FFA could be performed immediately to save time. The step-one LD-FFA could be performed as a pretest for NV.Besides, lesions of the fellow eye could be sometimes neglected since the quality of the angiogram of the fellow eye was suboptimal when performed at a late stage of the angiogram sometimes . In caseFurthermore, the LD-FFA could also be considered an allergic test for fluorescein. After the LD-FFA, we also checked the skin and vital signs for any allergic reactions. Only when no severe allergic reactions occurred, the further procedures performed. The LD-FFA instead of a full dose may evoke less severe FFA-associated complications, which could also make the procedure a safer one.With no doubt, there are drawbacks of this two-step FFA and limitations of the pilot study. First, the modified technique with two steps is more time-consuming than traditional ones, and with more risks for corneal or conjunctival abrasions. Besides, the doses for each step may not be the optimal ones. There was only one dose without a gradient method to test the most suitable dose strategies. The perfect dose should always be one effective enough to show all the abnormal vessels without severe vitreous dye, which is most optimal when personalized. In our opinion, due to disease heterogeneity, the ideal dose should be decided in multiple FFA in the same group of patients in different concentration gradients and choose the best one. However, it is impossible in a clinical set. Furthermore, with LD-FFA, the vessels are commonly obscure; it was harder to fetch good images with the right position and perfect focus, which may rely on an experienced photographer. Other limitations included those biases intrinsic to a retrospective study. The study sample was small, so it was only a pilot study. It was impossible to evaluate all the vascular lesions in this study. At last, ideally, a comparison group with traditional FFA with a regular dose should be arranged to evaluate this two-step FFA’s feasibility. However, in this pilot study, we merely want to introduce the concept and first attempts. A further study with a larger scale, gradient doses, and comparison groups is needed.In summary, we postulated a modified strategy with two-step widefield FFA. The step-one FFA is effective to show retinal NV without severe fluorescein leakage. The step-two FFA could also be a double-check to prevent possible skip areas. The strategy may be a helpful clinical adjuvant in the laser photocoagulation of pediatric retinal disorders, especially for young ophthalmologists early in their careers in laser photocoagulation.The original contributions presented in this study are included in the article/The studies involving human participants were reviewed and approved by the Institutional Review Board approval by Xinhua Hospital Affiliated to Shanghai Jiao Tong University. Written informed consent to participate in this study was provided by the participants’ legal guardian/next of kin.JP, JR, XZ, YY, YZ, and HX collected, analyzed, and interpreted the data. JP drafted the manuscript. JR, XZ, YY, YZ, and HX carefully revised the manuscript. YX and PZ contributed to the conception of the study. All authors have read and approved the final version of the manuscript.


The ankle is a crucial joint that supports the human body weight. An ankle sprain will adversely affect the patient’s daily life, so it is of great significance to ensure its strength. To help patients with ankle dysfunction to carry out effective rehabilitation training, the bone structure and motion mechanism of the ankle were analyzed in this paper. Referring to the configuration of the lower-mobility parallel mechanism, a 3-RRS  parallel ankle rehabilitation robot (PARR) was proposed. The robot can realize both single and compound ankle rehabilitation training. The structure of the robot was introduced, and the kinematics model was established. The freedom of movement of the robot was analyzed using the screw theory, and the robot kinematics were analyzed using spherical analytics theory. A circular composite rehabilitation trajectory was planned, and the accuracy of the kinematics model was verified by virtual prototype simulation. The Multibody simulation results show that the trajectory of the target point is basically the same as the expected trajectory. The maximum trajectory error is about 2.5 mm in the simulation process, which is within the controllable range. The experimental results of the virtual prototype simulation show that the maximum angular deflection error of the three motors is 2° when running a circular trajectory, which meets the experimental requirements. Finally, a control strategy for passive rehabilitation training was designed, and the effectiveness of this control strategy was verified by a prototype experiment. Ankle sprains are a common type of musculoskeletal system injury, accounting for about 7% to 10% of patients with sports injuries ,3. If paAnkle rehabilitation robots have been a research hotspot in medical rehabilitation in the past few decades. According to the interactive human–machine spectrum, ankle rehabilitation robots can be divided into the wearable type and the pedal type ,9. AnkleIn conclusion, many institutions have conducted research into ankle rehabilitation robots and have achieved diverse results. The ankle rehabilitation robot has many existing problems, such as a complex structural design, insufficient or redundant degrees of freedom, and non-existent rotational centering. Therefore, improvements should be made in the aspects of structural design and human-machine compatibility.In terms of lower-mobility parallel mechanisms, a parallel ankle rehabilitation robot (PARR) is proposed in this paper. Essentially, PARR is a 3-RRS parallel mechanism. This mechanism can rotate around the rotation center to match the ankle motion mechanism. As a parallel mechanism, PARR has the characteristics of a typical parallel mechanism. In addition, PARR offers other properties, such as a simple and compact structure and high human–machine compatibility, so it is suitable for ankle rehabilitation training.The rest of this article is organized as follows. In the second part, the bone structure and motion mechanism of the ankle are analyzed, and the mechanical structure of the PARR is introduced. In the third part, the kinematics model and the kinematic analysis are explained. In the fourth part, we show a simulation model that was built using Multibody. Kinematic simulation analysis was conducted to verify the safety and feasibility of the PARR. The passive rehabilitation training control strategy was designed and the prototype experiment was carried out to compare and analyze the feasibility of the robot to achieve rehabilitation training. Finally, our conclusions are presented and further work is forecasted.The ankle joint is the most heavily loaded joint in the human body and is involved in the majority of movement postures of the lower limbs. Its bone structure consists of the tibia, the fibula’s distal end, and the trochlea talus see a. The coThe ankle joint has three motion forms: dorsiflexion/plantar flexion, adduction/abduction, and varus/valgus. The instep and the shank form a right angle when they are in a normal state. When the toe moves downward, the angle between the shank and the instep increases gradually, which is plantar flexion. The opposite movement is dorsiflexion. The outward rotation of the foot around the shank is known as abduction, and the opposite movement is known as adduction. The movement of raising the internal margin of the foot and lowering the external margin is varus, and the opposite movement is valgus. The motion model is constructed based on the form of ankle motion, as shown in The motion range of the ankle joint is affected by many factors, such as the joint surface area, the strength of the ligaments, and the volume and elasticity of muscles ,30. The Rehabilitation training after ankle damage can improve the strength of the muscles related to ankle movement, stimulate the activation of related neural mechanisms, and promote the recovery of normal ankle function. To achieve comprehensive ankle rehabilitation training, the ankle rehabilitation robot needs to be designed to work in two modes: single-DOF (degree of freedom) rehabilitation training with only one form of exercise, and multi-DOF compound rehabilitation training with two or more forms of exercise simultaneously.Compared to the 6-DOF parallel mechanism, the lower-mobility parallel mechanism has a concise configuration and a simple control strategy. This lower-mobility parallel mechanism is acceptable for ankle rehabilitation training ,32. BaseThe 3-RRS PARR has three degrees of rotational freedom, and its rotation center is fixed in the space. Through the design of the robot structure, the rotation center can coincide with the patient’s ankle joint during the rehabilitation training process. Except for the rotation of three degrees of freedom, the ankle does not have axial movement during rehabilitation. The 3D model of the 3-RRS PARR was shown in The static platform and the moving platform of the 3-RRS PARR are connected by three branch chains. Each branch chain has three kinematic pairs. The branch chain is an RRS configuration, and kinematic pairs of the branch chains are the same. The ends of the branch chains are connected to the moving platform through a spherical joint. The rotation axes of the base shafts of three branch chains coincide on a common axis. The rotation axes of middle revolute pairs are perpendicular to each other in space. The common axis of the branch chains at the base revolute pair and the middle rotation axis intersect at a fixed point in space. The moving platform has a 3-DOF to rotate around the fixed point. The PARR is driven by a low-speed torque motor. Compared with a high-speed DC motor, a low-speed torque motor can work continuously in a locked-rotor state. Besides the motor, an adjustable pedal is installed on the moving platform. By adjusting the height and the length of the adjustable pedal, the patient’s ankle joint coincides with the rotation center. This structural design ensures the patient’s safety in the process of rehabilitation training, avoids secondary injury, and realizes human–machine compatibility.The composition of the RRS branch chain is shown in iu, iv, iw . The static coordinate system is O-XYZ, the origin of which is the rotation center O of the mechanism. The z-axis is perpendicular to the static platform, and the x-axis is in the plane formed by the vector w1 and the z-axis. The y-axis is determined according to the x-axis and z-axis with the right-handed spiral rule. The moving coordinate system is O-xyz, the origin of coordinates is rotation center O. The axes of Ox, Oy, Oz are respectively along the direction of iw. The angles between the axis of the middle revolute pair and the vertical direction in the moving coordinate system are both 54.74°. Therefore, the axes of the direction vectors iw and iv are perpendicular to each other in space and intersect at rotation point O.According to the D-H matrix, the kinematics model of the 3-RRS parallel mechanism is established see . The dirThe DOF of the 3-RRS parallel mechanism is analyzed using the screw theory, which determines the common and virtual constraints of the mechanism . The twiO. Therefore, $i1, $i2, $i3 is linearly independent. According to the twist system, the basic screw system of the reverse screw can be obtained as:The three kinematic pairs of each branch chain have different axes in space and intersect at point x-axis, y-axis, and z-axis, without the force couple. Therefore, each branch chain has three rotational DOF, and there is no moving DOF. There is a spherical joint in the branch chain. Therefore, $i3 =  = , where a1, a2, a3 is not 0. The twist of the spherical joint can be converted into:According to the relationship between the wrench and twist in the screw theory, it can be established from the reverse screw that the branch chain is subject to forces in the directions of the Due to the existence of the spherical joint, according to the analysis of the branch chain’s DOF and the spherical joint, it can be found that the branch chain has two local DOF. Therefore, the motion of the branch chain can be simplified to the motion of the 3R structure. Since the whole mechanism consists of three branch chains, the general wrench consists of three branches of the wrench. The wrench of the 3-RRS parallel mechanism can be reduced to a base wrench system.x-axis, y-axis, and z-axis, and is not affected by the force couple. The inverse wrench of Equation (4) is deduced as:Thus, the general constraint of the mechanism is 3. According to Equation (4), it is established that the mechanism is subject to forces in the direction of the Equation (5) establishes the twist corresponding to the general wrench. It can be seen that the mechanism has three mutually perpendicular rotational degrees of freedom in space .According to the modified Kutzbach–Gruble equation, the DOF of the mechanism can be calculated as:According to Equation (6), According to the screw theory analysis results, the number of DOF corresponds to the rotation around the three axes. There are no redundant DOF in other directions. That is to say, the rotation center of the PARR is consistent with that of the ankle joint, which meets the requirements of ankle rehabilitation.α, β, and γ represent the attitude angles of the moving platform rotating around the static coordinate system’s z-axis, y-axis, and x-axis, respectively. The process of solving the three input angles iθ  of the mechanism through the attitude change angle α, β, γ of the moving platform is by the inverse kinematics solution of the PARR [iu and iv is δ1, the angle between iv and iw is δ2, the angle between iw and the z-axis of the static coordinate system is ε1, and the angle between plane YOZ and plane iOZw is iε.Here, the PARR . When thWhen the PARR is in the initial pose, the direction vector connecting the rotation center of the mechanism and the center of the spherical joint can be expressed as:When the pose of the moving platform changes, the direction vector can be expressed as:R = RRR.The Euler rotation matrix is v1 in the initial pose can be expressed as:According to the spherical analytic theory, the direction’s cosine of v2 and v3 can be obtained by rotating v1 in the negative direction about the Z-axis, where ijε is the angle between plane iOZw and plane jOZw. The general expression of the cosine of the vector of the middle revolute pair direction is as follows:According to the structural features of the PARR, the vectors of the middle revolute pairs of each branch chain are distributed uniformly; then, iv and iw form a fixed angle, and the constraint equation is constructed based on this condition:According to the structural feature of the linkage, the two vectors Substituting and simplifying the constraint equation can be obtained with:iA, iB, and iC are expressions containing structural parameters, input angles, and output angles, assuming When the attitude angle of the moving platform is known, input angles of the branch chains can be obtained:α, β, and γ of the moving platform through three input angles via iθ  is the forward kinematics solution of the PARR. The angle between plane v1Ow1 and plane u1Ov1 is ψ1, and the angle between plane v1Ow1 and plane w1Ow2 is ψ2. According to the spherical analytic theory, the motion chain of the spherical polygon  can be simplified as follows:The cosine of the direction vector of iD, iE, and iF are expressions containing the structural parameters, input angle, external angle, and parameters ξ1 and ξ2. The unknown quantity iψ can be solved using a trigonometric function, as follows:D1E2 − D2E1≠ 0:When In order to solve this efficiently, assuming ψ1 and ψ2 into iw. According to Equation (8), the following equation can be obtained:Analyzing Equation (18) shows that there are eight sets of solutions for y. According to the structural features of the PARR, it can be seen that there is a unique solution for the mechanism, by substituting the solved According to the matrix element correspondence principle, the corresponding attitude angle of the moving platform is:δ1 at 54.74°, and the mechanical structure angles δ2 at 40°, based on the inverse kinematics model, the relationship between iθ  and α, β, and γ are established, as shown in iθ  and γ is as shown in iθ  and β is as shown in iθ  and α is as shown in Setting the mechanical structure angles There is no obvious mutation in the curve. It is evident that if stabilized input is given to the motor, the PARR can move smoothly. Compared with the motion range of the ankle given in To verify the accuracy of the kinematics model and the feasibility of achieving ankle rehabilitation training, the simulation model of the PARR was established with the MATLAB Simulink toolbox see a.O-XYZ. The inverse kinematics model module can calculate the motion law of each motor. The PARR model module was used for the PARR simulation model. The transform sensor module is used to extract simulation data. The kinematics simulation results are compared and analyzed with the theoretical values. The picture of the PARR module is shown in Signal alpha, signal beta, and signal gamma represent the rotational angle of the moving platform around the static coordinate system YOZ projection plane was planned for this paper. This trajectory can realize compound rehabilitation training of ankle dorsiflexion/plantarflexion and adduction/abduction. The target point is 200 mm along the positive direction of the OX coordinate axis, as shown in The feasibility of single-DOF rehabilitation training is shown in YOZ surface is shown in The motion trajectory of the target point projected on the This verifies the feasibility of the PARR model designed in this paper for rehabilitation training.In the early stage of the rehabilitation process, the passive rehabilitation training mode is mainly used to train the range of motion of the patient’s ankle joint. Since the ankle joint is relatively stiff during this period, the patient cannot carry out activities by him- or herself; therefore, passive rehabilitation training should be carried out with the help of external forces. Passive rehabilitation training requires the following error to be as small as possible, so the position-speed double closed-loop control strategy was adopted, as shown in The PARR experimental prototype was designed to test the circular rehabilitation motion track planned above. The experimental device is shown in The circular trajectory motion period was set to 7 s, and the rehabilitation trajectory motion experiment was carried out. As shown in To help patients with ankle dysfunction in rehabilitation training, based on the lower-mobility parallel mechanism, a novel 3-RRS parallel ankle rehabilitation robot was proposed in this paper. To obtain better kinematic characteristics in the process of rehabilitation training, the bone structure and motion mechanism of the ankle were analyzed, and the motion range of the ankle joint was detailed. Then, the structure and the kinematics model of the PARR were introduced. The DOF of the PARR was calculated using the screw theory, and the kinematics analysis of the PARR was analyzed with the spherical analytic theory. The feasibility of single-DOF rehabilitation training was proved with relevant numerical examples. The PARR simulation model was established by the MATLAB Simulink toolbox. A circular trajectory was planned and simulated. The Multibody simulation results show that the trajectory of the target point is basically the same as the expected trajectory. The maximum trajectory error is about 2.5 mm in the simulation process, which is within the controllable range. The feasibility of multi-DOF rehabilitation training was verified through a simulation analysis of the circular trajectory. A passive rehabilitation training control strategy was designed according to the rehabilitation training requirements. The experimental results of the virtual prototype simulation show that the maximum angular deflection error of the three motors is 2° when running a circular trajectory, which meets the experimental requirements. Based on the above research, the feasibility of the 3-RRS PARR proposed in this paper was proved, and the foundation for the human-machine experiments was laid.Regarding the existing research on ankle rehabilitation robots, comfort during rehabilitation is one of the urgent issues to be addressed. In follow-up research, the structural improvement and kinetic analysis of PARR should be combined with rehabilitation medicine to further improve human–machine compatibility. Further human–machine experiments will be conducted in the future to evaluate the reliability of the robot system.


This work presents a foundation for thedetection of circulating mRNA in PCa on a non-invasive lab-on-chipdevice for use at the point-of-care. This technique was implementedonto a lab-on-chip platform integrating an array of chemical sensors(ion-sensitive field-effect transistors) for real-time detection ofRNA. Detection of RNA presence was achieved through the translationof chemical signals into electrical readouts. Validation of this techniquewas conducted with rapid detection (<15 min) of extracted RNA fromprostate cancer cell lines 22Rv1s and DU145s.Prostate cancer (PCa) is the second most common causeof male cancer-relateddeath worldwide. The gold standard of treatment for advanced PCa isandrogen deprivation therapy (ADT). However, eventual failure of ADTis common and leads to lethal metastatic castration-resistant PCa.As such, the detection of relevant biomarkers in the blood for drugresistance in metastatic castration-resistant PCa patients could leadto personalized treatment options. mRNA detection is often limitedby the low specificity of qPCR assays which are restricted to specializedlaboratories. Here, we present a novel reverse-transcription loop-mediatedisothermal amplification assay and have demonstrated its capabilityfor sensitive detection of AR-V7 and YAP1 RNA (3 × 10 Current clinical diagnosis for PCa relies on multi-parametricMRI, PSA testing, and trans-rectal ultrasound-guided biopsy.4 PSA screening in the UK is not currently implementedbased on the limited benefits at diagnosing PCa on account of falsenegatives and false positives.5 Currenttesting for PCa is very limited prognostically and often leads toovertreatment of patients with clinically insignificant PCa. Anotherurgent biomarker requirement is for the accurate and early detectionof resistance to hormonal therapies, that is, the development of castrationresistance. This would facilitate the prompt discontinuation of ineffectivetherapies (with their significant side effects) and potential adoptionof new approaches.One in eight men are expected to be diagnosedwith prostate cancer(PCa) within their lifetime.9 However, RNA and DNA detection is often compounded by the limitedspecificity of qPCR assays.11 In addition, the relativelow abundance in circulating biofluids of mRNA and its inherent labilitycan make this species a challenging yet potentially valuable dynamicbiomarker for PCa prognosis. Detection of mRNA biomarkers at the point-of-care(PoC) could provide rapid in situ responses to directtreatment options for PCa patients. Previous work has establishedseveral mRNAs of interest for PCa prognostics, including both androgenreceptor (AR) variant 7 (AR-V7) and Yes-associated protein 1 (YAP1)mRNA.13 AR-V7 is deficient of the ligand bindingdomain (LBD) which normally makes the AR a ligand-activated transcriptionfactor, as a result it is constitutively active. As such, AR-V7 presencein PCa patients is often associated with resistance to androgen deprivationtherapy (ADT), the gold standard treatment for disseminated diseasewhich targets the AR LBD.14 Across dataextracted from 12 clinical trials, the proportion of mCRPC patientswith detectable circulating AR-V7 mRNA is 18.3%.15 Detection of circulating AR-V7 mRNA in mCRPC patients treatedwith ADT, corresponded to reduced overall survival and progressionfree survival in these patients, supporting AR-V7 as clinically actionablemRNA for detection in the blood.12 YAP1has multiple roles, including as a mechanosensor. Stiff matrices resultin nuclear localization of YAP1 where transcriptional regulation forcell survival and proliferation can take place.16 As such, YAP1 is commonly associated with the epithelialto mesenchymal transition in several types of cancers.20 YAP1 upregulation in the nucleus is correlated with reduced overalland disease-free survival in various cancers.23 In multiplePCa cell lines, YAP1 knockdown is associated with reduction in cellularmotility, invasion, and progression to metastatic phenotypes.26 However, the YAP1 gene is downregulated by latestage PCa-associated miR, miR-375-3p in mCRPC samples.20 Therefore, YAP1 potentially presents a temporal biomarker for progressionfrom locally advanced PCa to mCRPC. Because the miR-375-3p - YAP1pathway is implicated in docetaxel resistance, it could also directtreatment for mCRPC patients.13Recent research has indicated that detectionof circulating biomarkersincluding cell-free DNA, microRNAs, mRNAs, and circulating tumor cellspresent a minimally invasive alternative to current testing methods.27 As a result, qPCR experimentation is furthercompounded by transfer times to a laboratory. Alternative solutionsfor amplification tests are therefore required for PoC prognosticand diagnostic tests. Loop-mediated isothermal amplification (LAMP),developed and optimized by Notomi et al. and Nagamine et al., respectively,is a rapid (<30 min) and sensitive DNA amplification technique.29 LAMP utilizes six primers targeting eight specific DNA regions forexponential and isothermal amplification resulting in a high-yieldingDNA assay.28 Reverse transcriptase LAMP(RT-LAMP) allows application of the technique to mRNA and has previouslybeen used to detect mRNA in various diseases, including distinguishingdengue serotypes, prostate cancer antigen 3 for PCa diagnosis, andmore recently the N gene for SARS-CoV-2 virus detection.32 Integration of LAMP assays with ion-sensitive field-effect transistors(ISFETs) and unmodified complementary metal oxide semiconductor technologyfor lab-on-chip (LoC) detection of biomarkers has previously beensuccessful.36 RT-LAMP can be adjusted to result in a pH readout (RT-pHLAMP) duringamplification events , which allows for compatibilitywith the pH-sensing ISFET for use in a microfluidic PoC device.38 Double-stranded DNA synthesis, which occurs in the RT-pHLAMP amplificationevent, releases a proton per nucleotide addition to the DNA strand.40qPCRis commonly referred to as the “gold standard”for nucleic acid amplification tests, on account of its high accuracyand sensitivity. However, thermal cycling equipment crucial to qPCRexperimentation is expensive and limited to use in specialized laboratories.de novo development of RT-LAMP assays forthe detection of AR-V7 and YAP1 mRNA. Adaptation of this assay forISFET compatibility resulted in an accurate, sensitive (3 × 101 copies per reaction), and rapid (<15 min) test for YAP1and AR-V7 synthetic RNA presence. The assays were successfully testedon the ISFET LoC device presenting use of this device for PoC. Validationof this assay and the LoC device was confirmed with detection of AR-V7and YAP1 mRNA extracted from PCa cell lines. The development of thisbiosensor and these assays present the potential for PoC prognostics,where clinicians can rapidly adjust treatment options for PCa patients values were achieved. The AR-V7primers specifically targeted a region in cryptic exon 3 to avoidamplification of the full-length androgen receptor (AR-FL) mRNA.42 Recent evidence has suggested that AR-V7 crypticexon mRNA in the blood is more abundant than mRNA across splice boundaries,further supporting the target region for primer design.43 Because mRNAs present in the blood are oftenfragmented, synthetic RNA fragments of both AR-V7 and YAP1 targetregions  were synthesized forinitial assay development.45 Both the AR-V7 andYAP1 RT-qLAMP assays achieved linear detection of 3 × 107 to 3 × 102 copies of synthetic RNA per reactionin under 18 min  of 0.997 and 0.988 for the AR-V7and YAP1 RT-pHLAMP assays, respectively, which indicates the potentialof these assays for accurate quantification of RNA per sample. TTPvalues for the pH sensitive reactions were significantly reduced:the average TTP for detection of 3 × 102 copies ofsynthetic AR-V7 RNA was 17.6 min in RT-qLAMP and 9.5 min in RT-pHLAMP.This is likely due to the increased optimization of the RT-pHLAMPassay, allowing for faster TTP values. Detection from 3 × 107 to 3 × 101 copies of RNA was achieved inunder 12 min for both RT-pHLAMP assays.In order to generate a pH readout for ISFET compatibility,theRT-qLAMP assays were adjusted as previously described to omit tris(hydroxymethyl)-aminomethane(tris), the pH buffering agent present in Isothermal AmplificationBuffer (New England Biolabs).reaction2. The stSupporting Information, Figure S4). Primersdetecting this AR-FL region were developed to confirm its presencein these spiked assays . No amplification occurred between the AR-FL syntheticRNA and AR-V7 primers after the reaction was terminated at 35 min.A serial dilution experiment for AR-V7 detection spiked with AR-FLthen took place. These results indicate that amplification of theAR-V7 RT-pHLAMP assay only occurred with the presence of AR-V7 mRNA.In this instance, the sensitivity of the reaction was reduced to 3× 102 copies, indicating that the presence of off-targetRNA decreased the efficiency of the RT-pHLAMP assay.Specificity of the AR-V7RT-pHLAMP reaction was confirmed by spikingthe assays with a synthetic RNA fragment present in the AR-FL LBD. The limit of detection in these experimentswas 1 × 102 copies per reaction although TTP valueswere marginally increased. Sensitive and expeditious detection ofboth YAP1 and AR-V7 mRNA was therefore achieved in human plasma samples.However, pH values for these reactions indicated that no pH changetook place, likely due to the carbonic acid/bicarbonate buffer systempresent in the blood.47 Integration ofplasma samples directly onto the LoC platform would subsequently requirefurther optimization, outside of the scope of this study.To indicate if both the RT-pHLAMP assays for AR-V7and YAP1 mRNAwere feasible for detection of circulating mRNA in the blood, assaysincluding citrated human plasma were conducted . RT-qPCR assays  confirmed the high concentration of YAP1 mRNAs inDU145s and lower concentration in 22Rv1s . A negative cell line for YAP1 mRNA was alsointroduced to confirm the specificity of the RT-pHLAMP reaction. Becauseendogenous expression of YAP1 is present in many human cell lines,Schneider 2 (S2) cell RNA from Drosophila melanogaster was utilized. Supporting Information, Figure S9). From thestandard curve generated DU145s contain 5.5 × 103 copiesof YAP1 per 1 ng of RNA. However, because the TTP for 22Rv1 is outsideof the quantitative range of the standard curve, YAP1 copy numberwas not ascertained for this cell line.YAP1mRNA presence was also tested in RNA extracted from 22Rv1and DU145 cell lines. High expression of YAP1 mRNA concentration haspreviously been recorded in DU145 cells.Supporting Information, Figure S6). This indicates that the RT-pHLAMP assay data correspondwell with the gold standard of nucleic acid amplification tests.As expected, no amplificationcurves were seen in DU145s with theAR-V7 RT-qPCR assay, whereas fast amplification was observed in the22Rv1 cell line , releasesa proton per each nucleotide addition.6 copies per reaction. TTP values were slightly increasedon the LoC platform, likely due to non-optimal conditions for theRT-pHLAMP assay in the acrylic reaction chamber. These increased valuesare still significantly reduced (indicating more rapid detection)relative to the PCR gold standard for nucleic acid amplification tests.The averaged TTP value across triplicate experiments for detectionof YAP1 and AR-V7 synthetic RNA at 3 × 106 copiesper reaction was 7.25 ± 0.62 min and 7.11 ± 0.65 min, respectively. 48 The voltage outputis converted to proton count and sigmoidal fitting is then carriedout to return the amplification curve illustrated in Supporting Information, eqs S1 and S2.Synthetic YAP1 and AR-V7RNA samples were successfully detected at a concentration of 3 ×106 copiesof both AR-V7and YAP1 synthetic RNA had taken place with the LoC device, the limitof detection was tested at 3 × 101 copies. For bothof the RT-pHLAMP assays, the pH change at 3 × 106 and3 × 101 copies were similar, likely due to the DNAproduction being the same at both concentrations. 1 copies of AR-V7 synthetic mRNA on the LoCdevice. Here, the TTP values were 10.88 ± 0.95 min for the AR-V7RT-pHLAMP assay and 11.50 ± 0.98 for the YAP1 RT-pHLAMP assay.This illustrates that the sensitivity of the LoC device is comparableto the benchtop RT-pHLAMP assays.Once the detection of 3 × 1046 Contrastingly,detection of 1 ng of AR-V7 mRNA per reaction was achieved in 8.48± 1.43 min in extracted RNA from 22Rv1s on the LoC device. Once it had been determined thatthese assays were compatible with the ISFET biosensor, detection ofAR-V7 and YAP1 mRNA present in RNA extracted from 22Rv1 and DU145cells was assessed. As confirmed in the previous benchtop RT-pHLAMPand RT-qPCR assays, 22Rv1 cells are AR-V7 positive and DU145s containhigh levels of YAP1. 1 copies per reactionfor both markers. The RT-pHLAMP reactions, on account of their isothermalnature, remove the necessity of specialized and expensive thermalcycling equipment required for RT-qPCR experiments. Further developmentof these assays for the detection of circulating mRNA directly inserum would further increase their potential for rapid prognostics.This paper presents a foundation for theLoC detection of circulatingmRNA in PCa. The two novel assays judiciously developed for this workare, to the authors’ knowledge, the first RT-qLAMP experimentsfor the detection of AR-V7 and YAP1 mRNA. Authentication of AR-V7and YAP1 detection was confirmed with extracted RNA from PCa celllines and RT-qPCR. These RT-pHLAMP assays produced a suitable pH changefor use with complementary metal oxide semiconductor technology containingan array of ISFET sensors. This compatibility resulted in a LoC devicewith potential for direct PoC usage. Detection of synthetic RNA wasachieved at a sensitivity of 3 × 1012 This illustrates thatthe presence of circulating AR-V7 mRNA could be used to monitor mCRPCpatients and direct treatment options. Therefore, PoC detection ofAR-V7 through this novel assay could show clinical benefit to mCRPCpatients.The PROPHECY study, completed in 2019, has confirmed that the presenceof circulating AR-V7 mRNA associates with a lower progression-freesurvival and overall survival in mCRPC patients treated with enzalutamideand abiraterone.YAP1 concentration can be distinguished in the quantitativeRT-pHLAMPassay and detected using the LoC device with varying TTP values intwo PCa cell lines. High YAP1 concentration can be illustrative ofPCa tumors progressing to EMT, whereas low YAP1 concentration couldindicate advancement of mCRPC toward docetaxel resistance. In conjunction,AR-V7 and YAP1 mRNA detection on a LoC device could result in clinicallyactionable information, obtainable rapidly (<20 min), sensitively,and directly in the clinic. Further evaluation utilizing blood samplesfrom PCa patients will be required to confirm the validity of theseassays for use directly in hospitals. Progression in sample preparationallowing for direct detection of circulating markers in the bloodin RT-pHLAMP reactions will expedite the time taken from biofluidextraction to a prognosis using this PoC device. Optimization of plasma-basedreactions on the ISFET biosensor will be crucial to determine appropriateloading parameters for clinical samples. Alternatively, a rapid RNAextraction technique coupled to the LoC device could remove the necessityof direct testing in plasma.Further detection of a larger rangeof circulating nucleic acidbiomarkers could create a multiplex LoC device to serve as a prognostictest to personalize medication for PCa patients. The development ofmore RT-pHLAMP assays in conjunction with the ISFET LoC device couldresult in a robust handheld device for rapid, reliable, and simultaneousdetection of multiple circulating prognostic PCa biomarkers.10 copies per μL and stored at −80 °C inpreparation for experiments.RNA fragments ofAR-V7 and YAP1 sequences were synthesized from DNA gBlocks (IntegratedDNA Technologies) utilizing the HiScribe T7 Quick High Yield RNA SynthesisKit (NEB) according to the manufacturer’s instructions includingthe DNase step. Stock concentrations were maintained at 3 × 104 (100 mM stock), 1.4 μL of dNTPs (10 mM stock of eachnucleotide), 0.6 μL of BSA (20 mg/mL stock), 0.8 μL ofbetaine (5 M stock), 0.25 μL of SYTO 9 green (20 μL stock),0.25 μL of NaOH (0.2 M stock), 0.042 μL of Bst 2.0 DNApolymerase , 0.1 μL of RiboLock RNAseInhibitor , 0.3 μL of WarmStartRTx reverse transcriptase , 1 μL of 10×LAMP primer mix , 1 μL of RNA sample, and the remaining solutionwas topped up to 10 μL with nuclease-free water. Reactions wereconducted at 63 °C for 35 min. One melting curve from 63 to 97°C was conducted to confirm the specific amplification of thereaction at a ramp of 0.2 °C/s. Reactions were conducted witha LightCycler 96 instrument (Roche Diagnostics) in 96-well plates.All reactions were completed intriplicate. Each 10 μL experiment contained: 1 μL 10×isothermal buffer [New England Biolabs (NEB)], 0.6 μL of MgSO4 (100mM stock), 1.4 μL of dNTPs (10 mM stock of each nucleotide),0.6 μL of BSA (20 mg/mL stock), 0.25 μL of SYTO 9 green(20 μM stock), 0.25 μL of NaOH (0.2 M stock), 0.042 μLof Bst 2.0 WarmStart DNA polymerase , 0.3μL of WarmStart RTx reverse transcriptase , 1 μL of 10× LAMP primer mix , 1 μL ofRNA sample, and the remaining solution was topped up to 10 μLwith nuclease-free water. For plasma experiments, the RNA sample wasserially diluted in TE buffer. 10 μL of citrated human plasma(TCS Biosciences) was spiked with 1 μL of the RNA sample. 1μL of this solution was then added to each reaction. Reactionswere conducted at 63 °C for 35 min. One melting curve from 63to 97 °C was conducted to confirm the specific amplificationof the reaction at a ramp of 0.2 °C/s. Reactions were conductedwith a LightCycler 96 instrument (Roche Diagnostics) in 96-well plates.Reactions were scaled up to either 12 or 20 μL reactions forimplementation onto the LoC device and proportions of each reagentwere kept the same.All reactions were completedin triplicate. Each 10 μL experiment contained: 1 μL ofcustomized isothermal buffer, 0.5 μL of MgSOAll reactions were completed intriplicate. RT-qPCR reactions were completed in two steps. 50 ng ofmRNA samples were initially converted to cDNA with a RevertAid FirstStrand cDNA synthesis kit (Thermo Fisher Scientific) as per the manufacturer’sinstructions including the optional step for GC rich regions. cDNAwas used immediately for qPCR assays. qPCR experiments were conductedin 10 μL quantities and contained the following: 5 μLof Fast SYBR Green Master Mix (Applied Biosystems), 2 μL ofcDNA sample, 0.5 μL of forward primer ,0.5 μL of reverse primer , and nuclease-freewater was added to make the reaction volume up to 10 μL. Reactionswere aliquoted into a 96-well plate for analysis with a StepOnePlusReal-Time PCR system (Applied Biosystems). Reactions were initiallyheated to 95 °C for 20 s. The cycling stage included heatingat 95 °C for 3 s followed by 60 °C for 30 s. The cyclingstage was repeated for 40 cycles. Melting curves were conducted withheating to 95 °C for 15 s followed by 60 °C for 1 min.3N4). The ISFET array is composed of 56 × 78ISFET pixels .49 Temperature was maintained at 63 °C witha Peltier heating module contacting the underside of the cartridge.The LoC device was battery-powered and data were sent to an androidphone through a Bluetooth connection. Data extracted from the mobilephone was run through a MATLAB (R2021b) algorithm designed to spotfor amplification events. The RT-pHLAMP assay solutions were housedin an acrylic manifold with either 12 or 20 μL sized chambers.Adhesive gaskets is composed of Tesa double-sided smooth laminationfilmic tape that sealed the acrylic manifold to the cartridge. A 0.03mm chlorodized silver wire served as the Ag/AgCl reference electrode.This electrode was in contact with the assay solution and was placedbetween the adhesive gasket and the microchip’s surface. Nuclease-freewater was added to the chamber of the manifold for the first 700 sto equilibrate the system and set a common voltage across the ISFETarray. The water was then extracted and the RT-pHLAMP reaction mixturewas added. All samples that contained synthetic RNA or extracted RNAwere run for 30 min after the addition of the RT-pHLAMP reaction.Negative controls contained nuclease-free water instead of RNA andwere run for 20 min after the addition of the RT-pHLAMP reaction.All reactions were completed in triplicate. Confirmation of amplificationpresence in these reactions was confirmed with a Qubit 3.0 fluorometer(Invitrogen). Measured pH values post-reaction were conducted witha microFET pH probe (Sentron).The LoC system detects changes in proton concentration on the interfaceof the RT-pHLAMP assay solution with the passivation layer (Sil-glutamine (5 mM). Cells were passaged at70% confluency to maintain optimal growth and kept to under 10 passagespost-thawing. For RNA extraction, cells were harvested at 70% confluencyand spun down to remove media. For the 22Rv1 cells, RNA extractionwas performed using the Total RNA Miniprep Kit (Monarch) as per manufacturer’sinstructions including the DNase I digestion step. For DU145 cells,RNA extraction was performed using the RNeasy Mini Extraction kit(Qiagen) as per the manufacturer’s instructions. In both cases,RNA was eluted in 50 μL and RNA quantity/quality was measuredusing a Nanodrop D1000. Extracted RNA was stored at −80 °Cuntil use.22Rv1and DU145 cell lines were cultured in T-75 flasks with RPMI-1640 containingFBS (10%) and Drosophila Schneider 2 cells were grown in Schneider’sInsect Medium with FBS (10%) and penicillin–streptomycin (1%)in T-75 flasks at room temperature. RNA extraction was performed usingTRIzol LS Reagent (Thermo Fisher) according to the manufacturer’sinstructions. RNA quantity/quality was measured using a Nanodrop D1000.Extracted RNA was stored at −80 °C until use.t-test was chosen to determine the statistical significance of YAP1RT-pHLAMP TTP values and YAP1 qPCR Cq values in the 22Rv1 and DU145cell lines experiments. This test is commonly utilized in scenarioswhere the two compared data sets have different variance or differentsample sizes.50 The null hypothesis ineach case was that the mean TTP or Cq valuewas the same between the 22Rv1 and DU145 prostate cancer cell lines.Welch’s v) forWelch’s t-test is shown below (s1 and s2 are the standard deviationsof the two data sets and N1 and N2 are the number of samples per data set.The calculation for degrees of freedom (wn below 1, where t value for Welch’s t-test of unequal varianceis shown below (The equation for wn below 2,where p < 0.05.The null hypothesis was rejected when


By reducing procedure-related problems, advancements in computer-assisted surgery (CAS) and surgical training aim to boost operative precision and enhance patient safety. Orthopaedic training and practice have started to change as a result of the incorporation of reality technologies like virtual reality (VR), augmented reality (AR), and mixed reality (MR) into CAS. Today's trainees can engage in realistic and highly involved operational simulations without supervision. With the coronavirus disease 2019 (COVID-19) pandemic, there is a greater need for breakthrough technology adoption. VR is an interactive technology that enables personalised care and could support successful patient-centered rehabilitation. It is a valid and trustworthy evaluation method for determining joint range of motion, function, and balance in physical rehabilitation. It may make it possible to customise care, encourage patients, boost compliance, and track their advancement. AR supplementation in orthopaedic surgery has shown promising results in pre-clinical settings, with improvements in surgical accuracy and reproducibility, decreased operating times, and less radiation exposure. As little patient observation is needed, this may lessen the workload clinicians must bear. The ability to use it for home-based therapy is often available commercially as well.The objectives of this review are to evaluate the technology available, comprehend the available evidence regarding the benefit, and take into account implementation problems in clinical practice. The use of this technology, its practical and moral ramifications, and how it will affect orthopaedic doctors and their patients are also covered. This review offers a current and thorough analysis of the reality technologies and their uses in orthopaedic surgery. Over the past century, the world's health system has undergone a transformation thanks to the exponential expansion of processing power and imaging technologies, which have enhanced patient care from diagnosis to treatment. The most cutting-edge imaging and computing technologies have come together to create reality technologies, which have the potential to revolutionise many facets of the healthcare sector, particularly surgery. In contrast to virtual reality (VR), which immerses the user in a wholly artificial world, augmented reality (AR) adds information to real items in real-time . Using tNumerous experts contend that pilot training, which makes use of simulation before exposing students to real-world scenarios, is a good model for surgical training . Some coFundamental building blocks of VR and ARVR exists as a method that saturates the viewer visually in a wholly unnatural, computer-generated environment. Mobile headsets are the most common type of standalone VR equipment. However, even in a simulated environment, the most cutting-edge VR experiences can allow for freedom of movement. The computer may potentially supply VR visual components in addition to fabricating false audio and other inputs. The VR experience can also be enhanced with specialised hand-operated controllers . ContrarThe most recent development in reality-machine technology is mixed reality (MR). A stereographic picture is created in an MR system by fusing a real-world surface with a three-dimensional (3D) virtual replica created through diagnostic imaging scans such as computed tomography (CT) scans or magnetic resonance imaging (MRI) taken prior to surgery. Virtual objects aren't as clearly projected onto real-world surroundings when employing MR technology as they are with AR. However, the software helps the MR user to interact with both the real environment and the added digital material. The 3D picture (hologram) may be altered from the user's point of view by using webcams in collaboration with the viewfinder . OrthopaThe Implantation of Device or ImplantThe 3D orientation of the orthopaedic surgeon is frequently used for placing instruments or implants. Two-dimensional (2D) information is provided by intraoperative fluoroscopy. In order to map the 2D radiographs to the 3D anatomy, the surgeon must do it mentally. By placing preoperative planning into the surgeon's field of vision or even by displaying the proper implant placement trajectories with overlays, AR systems reduce the reliance of the outcome on the surgeon's parameters. Based on preoperative CT data, Wu et al. projected the spinal bone structure at the back of a sufferer with approaching locations for vertebroplasty using a camera-projector AR system .In a phantom investigation, Abe and colleagues simulated the insertion of needles into vertebral bodies . The plaGains and advantages of VR and ARPrecision and AccuracyThis was attributable to enhanced, logical visual aids that made sure the operator placed the surgical tools precisely. As an illustration, consider a head-mounted gadget that displays a hologram of the precise location where a spinal needle should be inserted. Through an ocular see-through HMD, Gibby et al. superimposed the virtual path of needle implantation using computed CT data .Efficiency in Terms of TimeThe analyzer might have categorised it as proper because the needle spike was on the inside the joint capsule. The implementation of such technology thus seems to have the potential to considerably improve the accuracy and safety of orthopaedic surgery . AccordiDecreased Unnecessary RadiationsThe reduction of needless radiation exposure towards operating room employees was a frequent goal of development in AR technology. When compared to conventional intraoperative fluoroscopy, the use of AR to just provide 3D surface reformation via digitally recovered radiographs showed significant decrease in radiation exposure while enabling real-time visual display with no need for consistent fluoroscopy .Pauly et al. employed a ceiling-mounted C-arm including an AR surgical navigation system to measure how much radioactive operating room staff in a hybrid operating room were exposed to. The staff members had 20 surgeries to have pedicle screws inserted while wearing an active personal radiation detector on their breast. These findings were compared to a benchmark radiation exposure level, or "worst case," as measured by a dosimeter mounted on a C-arm. The personnel-to-standard dosage ratio amounted to 0.05% for each procedure, and after several procedures, it further decreased to less than 0.01%, illustrating once more how technology advances with use [Benefits in EducationThe usage of AR/HUD as a teaching aid for orthopaedic surgery has a lot of potential to grow. The use of a HUD  for perioperative instruction and interactions between a junior surgical team wearing the headset and a senior remote surgeon viewing the operator's surgical site via a computer or mobile device using the Google Hangouts app (Google LLC) was demonstrated by Armstrong et al. . The stuApplication and use in orthopaedic diagnosisIt is no longer possible to fully master all the abilities necessary to become fully competent as an orthopedist in a clinical setting alone. Due to rising resident training costs, declining resident working time, and ethical concerns about patient care, teaching trainees outside of the operating room is important . VR can Sometimes, it becomes vital to visualise the inner workings of a joint to help with diagnosis or to support the surgeon during surgery. An answer to this issue is arthroscopy. By putting a tiny camera into the articular cavity of a synovial joint, it involves a minimally invasive investigation of interior structures like cartilage or ligaments. Results are shown on a screen outside the computer. A number of businesses created arthroscopy simulators that incorporate virtual reality and mannequins for training. The two most important ones at the moment in this field are ArthroS™  and TraumaVision® , which have been supported by numerous study publications . In ordeUsability of ARStudies that visualised AR in orthopaedic surgery used head-mounted gadgets like Google Glass, but these methods had some drawbacks. First of all, after using the headset for an extended period of time, numerous consumers complained of visual discomfort. As the computer changed the deepest portion of the image, the consumer had to adapt one‘s eyes to a fixed focus distance of 2 m away, which led to vergence and visual fatigue- accommodation issues . Even thIn darker settings, it can be difficult to see some AR graphics, that is holographic displays and overlaying systems, which might prove problematic in surgical units. To ensure that these real-world and AR pictures are readily visible or through head-mounted devices, more contrast and other colour designations should be established. Furthermore, issues like colour blindness need to be addressed ,28. LastThe rehabilitation programmeThe advantages of AR and VR may include an increase in the surgeon's focus on the operating room, a decrease in the risk of radiation exposure, through outpatient therapies including rehabilitation and a decrease in surgical field blockage ,32. A reFuture aspects of ARDespite the popularity of AR for surgical navigation, there is still some debate regarding the intraoperative usage of HMDs and their place in clinical care. Although few clinical research and pilot studies have shown its usefulness in bench and cadaver models, surgeons still face a number of logistical and moral dilemmas when integrating this technology into patient treatment. HMDs could soon be utilised to display essential information, photographs, and even fluoroscopy images in the surgeon's field of view if bigger clinical studies indicate improvements over current practice. More sophisticated tracking software needs to be developed in order to maximise the usefulness of these devices in the operating room. While optical tracking is an accurate method of measuring position during surgery, it does have the drawback of requiring .Reality technologies hold considerable promise as an improvement over conventional computer-assisted surgical techniques for orthopaedic training and practice. The ability to visualise patient data in real-time, better preoperative planning, and increase the accuracy and precision of interventions lead to an improvement in the calibre of care and patient outcomes. Reality technologies offer a fresh approach to live evaluation and remote coaching from the standpoint of education. On the other hand, AR is more equipped to help surgeons. Although they both have great potential, they haven't been thoroughly studied to entirely replace current practises. For these platforms to be finally implemented in a real-time surgical scenario, new, more potent, and accurate instruments must be developed.Additionally, since organ features may be approximated and genuine surgical instruments can be used, AR with 3D projections may be a better training tool than VR simulators. Due to this feature, this technology is currently popular in medicine. In fact, it is feasible that the invention and application of breakthrough technologies will quicken given the rapidity of change seen during the coronavirus disease 2019 (COVID-19) pandemic. Even so, it will be crucial to make sure that any such acceptance of innovation is grounded in safety and better results, and that the rate of change is moderated with the goal of achieving those results.


Very few research about atrioventricular blocks (AVB) and use of antipsychotic drugs has been made, although it may play an important role in the outcome of any patient affected by psychosis and AVB.To describe a case and review clinical data about AVB progression and neuroleptic treatment.1. No structural pathology was assessed by ecocardiography. Holter EKG showed also episodes of third degree AV block. Electrophysiology studies were performed showing a supra-hisian AV Block.We describe a 37 years old inmate male patient who suffered from a first degree AVB and Schizophrenia, being long term treated with neuroleptics . Our patient presented very mild symptoms of asthenia and dizziness. An EKG was performed, showing AVB progression to Mobitz Type ILower doses of Paliperidone were used (6mg) and maintened until nowadays. Control EKG showed regression to a known first degree AVB.Being asymptomatic and studies revealing a supra-hisian AVB, no pacemaker was needed.There is only a few cases described in scientific literature, and very limited data about AVB and neuroleptic drugs, although it is described as possible side effect using risperidone at higher doses. We suggest monitoring EKG to patients affected by AVB, using high doses of neuroleptic drugs. There is no data available about paliperidone metabolites and a possible progression of AVB.We suggest more studies are needed to better understand and prevent side effects of neuroleptic drugs.No significant relationships.


Allergic rhinitis (AR) is a prevalent allergic disease affecting individuals of all ages, especially children and adolescents. Patients with AR develop a specific immunoglobulin E antibody response to allergens, including pollen, mold, dust mites, and animal dander. The main symptoms of AR patients include nasal congestion, rhinorrhea, sneezing, and nasal itching. Symptoms such as these can interfere with the patient's sleep and quality of life when they become severe. Moreover, AR contributes too many complications, can exacerbate asthma, and has a negative impact on productivity and social costs. Therefore, the current review focuses on how to treat AR effectively. This review discusses the correlation between AR and constitution from the perspective of the Traditional Chinese Medicine (TCM) Constitution Theory in light of increasing research on AR in TCM. The constitution is adjustable, and adjusting the patient's biased constitution can improve the diagnosis, treatment, and prognosis of the disease. TCM constitution is critical in AR pathogenesis, and both are closely linked. TCM constitution theory can be applied to treat AR with positive effects, which merits popularization and application, and provides a new approach to the treatment of AR. Allergic rhinitis (AR) is a chronic noninfectious inflammatory disease of the nasal mucosa primarily caused by immunoglobulin E (IgE)-mediated allergic reactions to allergens, including pollen, mold, dust mites, and animal dander . AR causThroughout history, TCM theory has been widely used in clinical practice, and much experience has been accumulated in treating AR. Using TCM constitution theory to differentiate and treat AR has the advantages of low treatment costs, no toxic and/or side effects, and obvious therapeutic effects. Additionally, it reduces the number of allergens present in the body of AR patients, reduces the intensity of allergic reactions, and significantly improves their clinical symptoms. Modern clinical studies have demonstrated that allergic diseases are primarily caused by an allergic constitution . TherefoPhysical constitution is an internal factor determining the human body's vital-qi strength in disease development.  In contrBook of Rites, Yueling,” ..41]. incTCM treatment of AR based on the TCM constitution theory is currently being investigated in clinical trials and is primarily divided into two categories: TCM internal treatment and TCM external treatment. Chinese herbal decoction, acupuncture, moxibustion, massage, and acupoint application are among the most common methods of treatment. Moreover, the principles of TCM treatment of AR can be seen in p < 0.05) higher than that of the control group (75%). The total IgE levels of AR patients decreased to the lowest level in the intervention group after four weeks of treatment. The recurrence rate of AR in the intervention group was also lower at three and six months after treatment. These findings indicate that Fuzheng Qufeng Decoction is effective in treating AR patients with a qi-deficiency constitution, which provides relief from clinical symptoms and improves the quality of life for AR patients. The effectiveness of Fuzheng Qufeng Decoction is superior to that of budesonide nasal spray treatment, and the recurrence rate is low. Taking into account that patients with AR due to lung-qi deficiency and cold are characterized by a qi-deficiency constitution, the principle of TCM treatment is primarily to replenish qi and strengthen the exterior defenses. Therefore, Zhizhong et al. [Zhizhong et al.  treated g et al.  treated p < 0.05) higher than that of the control group (70%). In both groups after treatment, the scores and total scores of TCM syndromes decreased, and the scores and total scores of TCM syndromes in the intervention group decreased more significantly (p < 0.01) than those in the control group. These findings indicate that the above five acupoints can be acupunctured to reinforce qi and strengthen the spleen in TCM treatment to regulate the phlegm-dampness constitution of the patient and to treat AR.Using the random number table method, Xingang et al.  divided n = 105), while oral montelukast sodium was administered to the control group (n = 105). Taking into account AR patients with yang-deficiency constitutions, the TCM syndrome differentiation was performed, and the selected acupoints included Dazhui (GV14), Fengmen (BL12), Mingmen (GV4), Feishu (BL13), and Shenshu (BL23). The intervention group was treated with ginger-separated moxibustion at the five acupoints listed above. A total clinical effective rate, a clinical symptom score, and a clinical sign score were used to evaluate the efficacy of the treatment. A higher score indicates more severe clinical symptoms. The two groups of AR patients were also scored for clinical signs before and after a 3-month follow-up. A lower score indicates fewer clinical signs. A total effective rate of 89.8% was observed in the intervention group after four weeks of treatment, which was significantly (p < 0.05) higher than that of the control group (75.3%). In both groups after treatment for three months, the scores of clinical symptoms and signs decreased in both groups were lower than those before treatment, and the scores of clinical symptoms and signs in the intervention group decreased more significantly (p < 0.01) than those in the control group. These findings indicate that ginger-partitioned moxibustion can warm the meridians and activate the yang in AR patients with a yang-deficiency constitution. The ginger-partitioned moxibustion treatment is effective in improving the clinical symptoms of AR patients and can have an excellent long-term curative effect and prevent the recurrence of AR.Xiaoyong et al.  investigThe current review suggests that constitution is an intrinsic factor in the pathogenesis of AR. To some extent, constitution determines the strength of the human body's vital-qi, which will directly affect the body's ability to resist pathogens . TherefoAmong the advantages of the current review are the following: I) Explicitly exploring the relationship between AR and physical constitution can provide a strong theoretical basis and new treatment ideas for the clinical treatment of AR;  II) The Based on the TCM constitution theory, the current review concludes that the TCM constitution plays an intrinsic role in AR pathogenesis. Additionally, treating AR by intervening and adjusting the constitution of TCM is beneficial and worthy of clinical application. More importantly, it provides clinicians with a favorable evidence-based basis to treat AR and opens up new treatment options.


Arctostaphylos uva-ursi” (Ar. uu) to modulate the aflatoxinB1 (AFB1) adverse effects in broiler embryos. As a result, 0.1 g of Ar. uu in ovo injection alleviates AFB1 embryotoxicity by enhancing chicks’ physiological responses and optimizing antioxidant status, which is reflected in a low mortality rate and heavier relative weight of the hatched chicks. The in ovo injection of AFB1 + Ar. uu significantly declined AFB1-induced toxicity in embryos and increased broiler chicks embryo’s survival by 62.5%, increased relative embryo weight by ∼65.25% compared to eggs injected with AFB1 alone. Regarding immune responses, the in ovo injection of Ar. uu enhances the embryo’s humoral immune responses and regulates oxidative stress biomarkers. In general, the in ovo injection of Ar. uu modulates broiler chicks’ embryotoxicity caused by AFB1.The poultry industry is of great importance worldwide; however, mycotoxins contamination, especially aflatoxins, has harmful effects on poultry production. Therefore, finding out new strategies to alleviate aflatoxin contamination is an important trend of research. In our study, we focused on the in ovo injection techniques using the methanolic extract of brae beery “Arctostaphylos uva-ursi (Ar. uu.) methanolic extract. The total polyphenols, total flavonoids, total antioxidant capacity, and GC-MS analysis were all assessed in the Ar. uu. methanolic extract. A total of 180 ten-day-old embryonated eggs were distributed into six groups of 30 replicates each. The first group was used as a control (non-injected), and the second, third, fourth, fifth, and sixth groups were injected with 10 µ double-distilled water (DDW), 500 µL methanol, 0.01 g Ar. uu./500 µL methanol, 50 ng AFB1/10 µL DDW, and 50 ng AFB1 in 10 µ DDW + 0.01 g Ar. uu./500 µL methanol, respectively. The relative embryo weight, residual yolk sac weight, tibia length and weight, and survival were recorded. Total and differential leukocytes, oxidative stress, and humoral immune responses were observed. The residual yolk sac was lower (p < 0.05) in the Ar. uu. group than other groups. The embryonic growth (tibia weight and length) was enhanced in AFB1 + Ar. uu.-injected embryos compared with those injected with AFB1 alone. In conclusion, in ovo injection of Arctostaphylos uva-ursi could modulate AFB1-induced toxicity in chicken embryos.In ovo injection of nutrients can modulate the embryo’s physiological responses against aflatoxin B1 (AFB1) embryotoxicity. This hypothesis was tested using in ovo injection of  Contamination of poultry feed with mycotoxins, particularly aflatoxin B1 (AFB1), is a major problem for the poultry business; aflatoxicosis causes economic losses attributable to inefficient feed utilization, reduced egg production, and greatly reduced body weight growth as well as higher mortality . MycotoxArctostaphylos uva-ursi L. (Ar. uu.), is a member of the evergreen heath family. The astringent leaves have long been used to treat bladder infections and other infections of the urinary system [Ar. uu. are most likely due to the glycoside arbutin fraction [Ar. uu. extract has antibacterial properties against microorganisms found in food. The glycosides arbutin (5–15%) and methyl-arbutin (up to 4%), as well as small amounts of free aglycones, are the most important components of Ar. uu. leaves [p-coumaric acid, ursolic acid, and flavonoids, including glycosides of quercetin, myricetin, and kaempferol [Arctostaphylos uva-ursi is a rich source of natural antioxidants. However, the effects of Ar. uu. fruit methanolic extract against AFB1 embryotoxicity has not yet been reported. Thus, in this study, we determine the efficacy of methanolic extract of Arctostaphylos uva-ursi fruit in protecting chicken embryos from AFB1 toxicity by in ovo injection of AFB1 with or without Arctostaphylos uva-ursi fruit methanolic extract into embryonated eggs.Detoxification tools of AF-contaminated feed that are both feasible and cost-effective are in high demand. In addition to preventive maintenance, physical, chemical, and biological procedures have been used to disinfect aflatoxins (Afs) from contaminated feeds and feedstuffs ,17. The y system . The antfraction ,34. Dykefraction  reported. leaves . Other iempferol . ArctostThe experimental technique used in this study followed Egyptian animal welfare requirements and was approved by the Animal Care and Use Committee of Minia University, Faculty of Agriculture, Department of Animal and Poultry Production (APRIL24-2021), El-Minya, Egypt.Arctostaphylos uva-ursi were dried at 40–60 °C for 12 h and then soaked in MeOH at room temperature for 24 h. After filtration through filter paper (Whatman No. 1), the residue was re-extracted three times with MeOH under the same conditions, and to obtain a crude methanolic extract, the solvent was evaporated in a rotary evaporator at 40 °C [Fifty grams of at 40 °C .2CO3 solution (0.5 mL). The absorbance at 740 nm was measured after 1 h of incubation at 25 °C in the dark. Each trial was carried out in triplicate. The phenolic content was calculated using an external gallic acid curve as a standard, and the results were expressed as mg gallic acid equivalent per gram dry weight (mg GAE/g DW).Total phenolic content was predicted spectrophotometrically using the Folin-Ciocalteau reagent, as previously mentioned by Limmongkon et al. . The rea3·6H2O solution, 0.2 mL of 1 M potassium acetate, and finally 2 mL of distilled water were added to 0.5 mL of MeOH extract. The absorbance at 430 nm was measured after 30 min of incubation at room temperature. Each trial was performed in triplicate. The measurement was calibrated using a standard curve of the prepared quercetin dihydrate solution, and the results were expressed in mg quercetin equivalent per gram dry weight (mg QE/g DW).The aluminum chloride colorimetric method previously stated by Munhoz et al.  was modiArctostaphylos uva-ursi was determined in vitro using the DPPH radical-scavenging assay. The assay was performed in triplicates and average values were considered. The free radical scavenging ability of the methanol extract against 2,2-biphenyl1-picrylhydrazyl (DPPH) was evaluated using the method declared by Dawood et al. [The antioxidant activity assessment of methanol extract of d et al. . In brieArctostaphylos uva-ursi. The chromatography conditions were as follows: column flow rate of 1 mL/min, injection mode split, and carrier gas Helium 99.999%. GC-MS spectra with mass library search  and relative retention indices were used to identify the components [Gas chromatography  equipped with an HP-5MS column  mass spectrometer programmed at temperature 30–280/300 °C with a hold time of 5 min and rate of 10 °C/min was used to investigate bioactive compounds in methanol extract of dried mponents .n = 180) were then injected or not injected with 50 ng AFB1/10 µL or/and 500 µL of 0.1 g Ar. uu. fruit methanolic extract. On day 21 of incubation (hatching day), all rested eggs/groups were broken, and samples of residual yolk and tissues  were taken. Fertile broiler (Ross 308) eggs were obtained from a commercial breeder flock aged 38 weeks of age. Eggs were incubated in an incubator  under optimal incubation temperature (37.8 °C) and 65% relative humidity, and automatically turned every 2 h; then, after 10 days of incubation the eggs were candled with a lamp, and those containing dead embryos were removed from the incubator. The live embryos (n = 180) 58.6601 ± 2.6105 were randomly divided into six groups (30 eggs/group) using the YMC scale . A 21-gauge needle with a small, beveled tip was used to inject the eggs into the air cell, reaching for the amnion. Pilot testing using a visible dye before the injection indicated the solutions were safely delivered into the amnion. The 1st group served as a control (non-injected eggs), the 2nd group was only injected with 10 μL double-distilled water (DDW), the 3rd group was injected with 500 µL Methanol, the 4th group was injected with 500 µL of a freshly prepared solution of 0.01 g of Ar. uu., the 5th group was injected with 10 μL Methanol 20 containing 50 ng AFB1 (AFB1 group), and the 6th group was injected with 50 ng AFB1 + 500 µL of 0.01 g Ar. uu (Pure Aflatoxin B1 (AFB1) brushed from Sangon Biotech, Shanghai Co., Ltd. ,  was dissolved with methanol (20%) to obtain final concentrations of 50 ng AFB1/10 μL. The AFB1 level was determined using an AFB1 ELISA Kit, product number: MM-1911O1, Romer Labs, Union, CN. At day 10 of incubation, eggs , the hatched embryos from each group were individually weighed. Then relative yolk sac weight and mean relative embryo weight of each group were calculated.The tibia from each embryo at hatching day was removed, weighed, and expressed as relative to embryo weight. Tibia length was measured using a digital caliper.g) at room temperature to separate the serum that was stored in Eppendorf tubes (1500 µL) at −80 °C until analyzed. Immunoglobulins A, M, and G were detected using ELISA kits according to the instructions of the kits .At hatching time, fifteen blood samples from each treatment were randomly collected from the jugular vein before slaughtering and then centrifuged for 10 min , glutathione peroxidase (GSH-Px), total antioxidant capacity (TAOC), superoxide dismutase (SOD), malondialdehyde (MDA), and catalase (CAT) in tissues and serum in five samples, each group using Nanjing Jiancheng Bioengineering Institute  reagent kits.The tissue samples  were stored at −80 °C, then homogenized, after being diluted ten times (0.1 g per mL) using isotonic physiological saline; they were then centrifuged at 1295× The samples were fixed in 4% paraformaldehyde (PFA) and routinely processed in paraffin. Thin sections (5 μm) of each tissue were sliced and mounted on glass. Slides were stained with hematoxylin and eosin Y. Rabbit anti-AFB1 antibody was used in the immunohistochemical procedure. Apoptosis examination, using TUNEL assay techniques, was used as described in our previous work . The tisp ≤ 0.05. A post-hoc test (Tukey–Kramer) was performed following ANOVA. Data are presented as means and pooled SEM. Graphpad prism 7 was used to draw graphs. Treatment-dependent alterations were analyzed using the general linear model approach (GLM). SAS version 9.4  was used for all analyses. The normality of distribution assumptions was checked using Shapiro Wilk followed by Levene’s test for homoscedasticity. Statistical differences among means were considered significant at Ar. uu. were determined and illustrated in Ar. uu. methanol extract. The DPPH radicals were found to be inhibited by 60.25%.The levels of total polyphenol, total flavonoids, and total antioxidant capacity of the methanolic extract of dried Ar. uu. fruit methanol extract, the GC-MS technique was used. More than 12 phytochemicals were found in the methanolic extract of Ar. uu. separated at different retention times (RT) (To obtain information on the major metabolites present in the dried mes (RT) . Based oArctostaphylos uva-ursi fruit methanolic extract and/or AFB1 in ovo injection on the survival rate of broiler chicks is illustrated in Ar. uu. plus, AFB1 augmented the survival rate of broiler embryos by 62.51% compared with those injected with AFB1 alone. However, the in ovo injection of 500 µL of 0.1 g Ar. uu. into the broiler enhanced embryos’ survival rate by 3.56% compared to the control. Additionally, the in ovo injection of 50 ng AFB1 into the broiler embryos declined the survival rate by 39.64% compared with negative controls. There were no differences among negative controls  in the survival rate.The effect of Ar. uu. alone or with AFB1 increased (p = 0.01) embryos’ relative weights at hatching day by 29.34% compared to those in ovo injected with AFB1 alone; at the same time, 50 ng AFB1 in ovo injection had the lowest (p = 0.01) relative embryo weight (41.09%) and highest residual yolk sac relative weight (61.42%). Moreover, the relative residual yolk sac weight of embryos injected with AFB1 (50 ng/egg) was found to be augmented (p = 0.01) compared to other groups. In addition, 0.01 g Ar. uu. significantly lessened the relative residual yolk sac weight of embryos injected with AFB1 (50 ng/egg). Nevertheless, there were no differences in relative yolk sac weights among embryos in negative controls  and non-injected groups (control) (The relative yolk sac weight and relative embryo weight at hatching day (d21) were calculated as the percentage of the whole eggs each time, and the results of all groups are illustrated in control) B.Arctostaphylos uva-ursi fruit methanolic extract and/or AFB1 in ovo injection into broiler embryos on the tibia bone relative weight and length on 21 days of incubation are presented in Ar. uu. plus AFB1 increased tibia bone relative weight and length compared with those of the AFB1 group. Additionally, the in ovo injection with Ar. uu. alone augmented the tibia bone length and relative weight on the 21 days of incubation. However, there was no difference in tibia length and relative tibia weight among embryos in negative controls (DDW and Methanol) and the non-injected group (control) (The effect of control) A,B.Ar. uu. in ovo injection on serum total protein profiles, enzymatic activities of GOT, GPT, AKP, GGT, and creatinine kinase uric acid and urea in the serum of newly hatched chicks is illustrated in Ar. uu. (500 µL) plus AFB1 (50 ng/egg) alleviated AFB1-induced liver damage by significantly lowering albumin and the Alb/Glo ratio. However, the activity of GGT, AKP, GPT, GOT, levels of uric acid, urea nitrogen, and creatinine kinase activity were declined compared to AFB1 alone. Nevertheless, no significant difference in total protein profile, enzymatic activities of GOT, GPT, AKP, GGT, uric acid, and urea nitrogen, and creatinine kinase activity was observed among negative controls (DDW and Methanol) and the non-injected group.The influence of AFB1 and/or Ar. uu. recovered the adverse influences of AFB1 in ovo injection by reducing bad cholesterol levels and increasing HDL levels as good cholesterol. Serum lipid profiles  of new hatched broilers chicks exposed to AFB1 during incubation are displayed in Arctostaphylos uva-ursi fruit methanolic extract and/or AFB1 during incubation is shown in Ar. uu. + AFB1 enhanced thyroid function by 29.19, 71.18, and 32.88%, respectively, compared with AFB1 alone. Serum thyroid activity of newly hatched broilers chicks exposed to Ar. uu. + AFB1 recovered the harmful effects of AFB1 to be nearest the control group and better than the AFB1 group alone by 81.68, 51.16, 100.81, and 135.71% of total immunoglobulins, IgG, IgM, and IgA, respectively. Moreover, there were no significant differences among the control, DDW, Methanol and Ar. uu. groups. Total immunoglobulins and immunoglobulins fractions determined in the serum were shown in p = 0.01) total antioxidants capacity (TAOC) and the activities of enzymatic oxidative stress biomarkers  in the serum by 55.96, 35.122, 33.76, 25.73, and 59.57, respectively, and the same trend was observed in tested tissues . Serum MDA had the opposite direction of other oxidative stress biomarkers, which was increased by 139.21%. The in ovo injection of Ar. uu. partially recovered the harmful influences of AFB1 by elevating the levels of TAOC, GSH, SOD, GSH-px, and CAT  and decreasing MDA activity by 45.75% compared with AFB1 alone. Concerning the activities of dehydrogenase enzymes , results revealed that the in ovo injection of 50 ng AFB1 augmented the activity of LDH and SDH by 71.86 and 273.96% compared with the control group and decreased the activity of GluDH by 34.32% in the serum, exhibiting the same trend in tissues. However, the in ovo injection of Ar. uu. plus, AFB1 leads to recovering the activity of dehydrogenase enzymes as partially to be nearest of the control. Data in Ar. uu. plus AFB1 , while lymphocyte density was mostly reduced (thin arrows) in the white pulp in the AFB1 group H1–H6. Imlus AFB1 I6 declinlus AFB1 A1–A6. TUlus AFB1 A5. FurthB1 alone A5. Polyphenols are characteristically composed of flavonoids, phenolic acids, coumarins, and tannins . Herbs’ Arctostaphylos uva-ursi was 102.11 mg GAE/g DW, is nearly two-fold higher. Dragana et al. [Ar. uu., was 73.46 mg/g rutin equivalent, which was lower than the levels found in our study. The disparity between their findings and ours could be explained by different factors, including the regions and seasons in which the sample material was collected, the nature of phenolic components, extraction techniques, and the solubility of these compounds in different solvents with different polarities [The total polyphenol results in our study appeared to differ from those detailed by Azman et al. , who staa et al.  discoverlarities . Commonllarities ,50. Ar. uu. in protecting chicken embryos from AFB1 toxicity, and the results clarified that the dose of AFB1 (50 ng/egg) caused significant embryonic mortality at hatching day, resulting in 44.67% mortality. Additionally, the presence of 500 µL of Ar. uu., significantly reduced the mortality rate to 13.33% when embryos were injected with AFB1 (50 ng/egg). One of the major effects of aflatoxicosis on birds is a decrease in bodyweight, which directly affects the success of the poultry sector. Aflatoxin B1 inhibits the synthesis of DNA and RNA, thereby consequently decreasing the synthesis of protein that ultimately lessens the growth [Ar. uu., enhanced the relative embryo weight despite exposure to AFB1, signifying the potential protective impact of Ar. uu. to AFB1-injected embryos. Moreover, the residual yolk supplies more than 90% of the total energy requirements of the embryo via yolk lipids oxidation [Ar. uu., in the presence of 50 ng AFB1/egg significantly improved the tibia length as compared with that of embryos treated with AFB1 alone; this might be related to normal embryonic development due to nutritional active constituents in Ar. uu.Aflatoxins cause aflatoxicosis in birds and lead to increased susceptibility to infectious diseases and the reduction of growth performance. The residual AFs in the egg can adversely affect hatchability, embryonic survival, and organ malfunctions . Jelineke growth . In the xidation . Yolk coxidation . In the xidation ,58. Chauxidation  reportedAr. uu. injection reduced the AFB1 negative effects on the development of the embryo. The current study demonstrated that the mortality rates were augmented in the groups treated with AFB1 compared to the control group. The hatchability rate in this study was decreased in the groups treated with AFB1. Khan et al.  determin2O2), and superoxide anion radicals (O2), which cause oxidative injury in biomolecules due to secondary metabolites with antioxidant properties [Ar. uu., active compounds which are almost characterized as phenols, polyphenols, or phenolic acids. Our results show the TFC, TPC, and antioxidative activity of Ar. uu., were 91.28, 212.88, and 60.253, respectively. Five-Hydroxymethylfurfural (5-HMF) was the most active constituent (43.83%) in our study. Recently, 5-HMF was found to have antioxidant activity by scavenging ABTS and DPPH free radicals [P-coumaric acid is a member of the hydroxycinnamic acid group. Polyphenols’ positive benefits are mostly attributed to their ability to alleviate the oxidative stress conditions that accompany these illnesses. Several polyphenols have been shown to have strong antioxidant activities in vitro since they can function as chain breakers or radical scavengers depending on their chemical structures [Bioactive compounds derived from medicinal plants, such as phenolic, anthocyanin, and flavonoid chemicals, have been employed as alternative therapeutic tools for treating different ailments throughout history . Numerouoperties . Furtheroperties . As a reoperties . The modradicals . Accordiradicals . Hexadecradicals . Also, Pructures ,67. Polyructures ; utilizeructures ; be assoructures ; interacructures ; increasructures ; affect ructures ; possiblructures ; and intructures . Polypheructures . They maructures ; the cheructures . Aflatoxructures . During ructures . The latructures ,80,81. Aructures . After atuent 43.% in our ructures . These mructures ; neverthructures .Concerning blood biochemistry, Kubena et al.  revealedThyroid hormones are necessary to maintain the systemic physiological balance in organisms . Our resThe intake of aflatoxin-contaminated diets leads to several undesirable influences in poultry; for instance, altered morphology of the liver , kidneysThe AFB1-induced immunotoxin influences have been well-recognized in the literature, including humoral response cell-mediated and innate immunity . AFB1 haAFs decline in serum proteins, owing to low α, β, and γ globulins, with IgG being more sensitive than IgM , may cauAr. uu., extract plus AFB1 into eggs boosted the formation of aflatoxin antibodies and lowered reprogrammed apoptotic cells. This might be due to Ar. uu., extracts that contain active substances that work synergistically to inhibit the accumulative effects of AFB1. Aflatoxin B1 can prevent the migration of macrophages and intervention in the complement hemolytic activity [The current findings clarified that injection of AFB1 significantly declined TIg, IgG, IgM, and IgA. The immunosuppressive influences of AFs owing to the direct inhibition of protein synthesis including IgA and IgG have been stated ,109. Addactivity .Aflatoxins encourage the formation of free radicals and therefore cause liver peroxidation, leading to antioxidant depletion, apoptosis, and oxidative stress. All of these contribute to the development of malabsorption . Aflatox2O2, alkoxyl radicals, and hydroxyl radicals [The main route of AFB1 detoxification is through GST enzymes that conjugate AFBO with GSH. The basic determinant of species sensitivity to AFB1 is the efficiency and rate of GST activity . Cellularadicals ; there iradicals . Arctostaphylos uva-ursi extract, when injected in ovo, modulates the adverse effects of AFB1 in broiler embryos contaminated with AFB1, as it improves oxidative stress biomarkers, liver and kidney function, and immunological responses in broiler chicks. In addition, histological data revealed that Ar. uu., reduced the embryotoxicity induced by 50 ng AFB1/10 µL. As a result, Ar. uu., reduced the antioxidant enzymes’ capacity to counteract oxidative damage generated by AFB1. The recent findings will add to our understanding of the 0.01 g Ar. uu./500 µL of Ar. uu., in ovo injection as a therapeutic agent against AFB1-induced embryotoxicity.


Traumatic aortic regurgitation (AR) is a rare complication of blunt chest trauma. We described the case of a 35-year-old male who presented to our hospital with shortness of breath 7 years after sustaining blunt chest trauma associated with a motorcycle accident. Transthoracic and transesophageal echocardiogram detected severe AR with two separate jets. The patient was diagnosed with congestive heart failure due to severe AR, and surgical aortic valve replacement was performed. A large perforation of the right coronary cusp likely sustained during the initial blunt chest trauma injury was confirmed surgically. As AR caused by blunt chest trauma can gradually worsen, it is necessary to confirm if there is a history of trauma in patients with severe AR of unknown origin.