Influenza represents a profoundly transmissible viral ailment primarily afflicting the respiratory system. Neuraminidase inhibitors constitute a class of antiviral therapeutics employed in the management of influenza. These inhibitors impede the liberation of the viral neuraminidase protein, thereby impeding viral dissemination from the infected cell to host cells. As such, neuraminidase has emerged as a pivotal target for mitigating influenza and its associated complications. Here, we apply a de novo hybridization approach based on a breed-centric methodology to elucidate novel neuraminidase inhibitors. The breed technique amalgamates established ligand frameworks with the shared target, neuraminidase, resulting in innovative inhibitor constructs. Molecular docking analysis revealed that the seven synthesized breed molecules (designated Breeds 1–7) formed more robust complexes with the neuraminidase receptor than conventional clinical neuraminidase inhibitors such as zanamivir, oseltamivir, and peramivir. Pharmacokinetic evaluations of the seven breed molecules (Breeds 1–7) demonstrated favorable bioavailability and optimal permeability, all falling within the specified parameters for human application. Molecular dynamics simulations spanning 100 nanoseconds corroborated the stability of these breed molecules within the active site of neuraminidase, shedding light on their structural dynamics. Binding energy assessments, which were conducted through MM-PBSA analysis, substantiated the enduring complexes formed by the seven types of molecules and the neuraminidase receptor. Last, the investigation employed a reaction-based enumeration technique to ascertain the synthetic pathways for the synthesis of the seven breed molecules. Influenza virus infection, commonly known as the flu virus, is an acute respiratory ailment. It frequently manifests in global outbreaks and epidemics, primarily coinciding with the winter season. Substantial quantities of influenza viral particles are discernible within the respiratory excretions of afflicted individuals, thereby facilitating transmission through mechanisms such as sneezing and coughing, which facilitate the dispersion of larger particle-laden droplets ,2. InfluA breed-based de novo strategy was employed in the current investigation to develop new neuraminidase inhibitors. A total of 282 different fragments were generated from thirty potent neuraminidase inhibitors using the Schrödinger PowerShell command “run.\fragment_molecule.py”. We initially docked all the generated fragments (282) in the active site of neuraminidase using SP docking. Antivirals are essential to the control and prevention of influenza. Oseltamivir and zanamivir are the only neuraminidase inhibitor medications that are now licensed globally. Recently, laninamivir and peramivir gained permission in Japan . To knowThe physicochemical characteristics of a compound have a major effect on its pharmacokinetics in the body, and a firm understanding and accurate prediction of these properties are critical for successful drug discovery ,24. We eThe seven breed molecules had molecular masses below 410 g/mol, which facilitated the intestinal absorption of these molecules by oral administration. Second, the partition coefficients (logP) of the breed molecules were between −2.28 and 0.77. These results clearly show the affinity of these molecules toward biological membranes and confirm the good distribution of these substances in the body. For aqueous solubility, all the breed molecules were soluble in aqueous media (including the intracellular medium), and the Log S (ESOL) values were between −0.74 and 0.24. Because of this aqueous solubility, the breed molecules would be easily eliminated through the kidneys. The bioavailability score for Breed 1 was 0.55, while the rest of the breed molecules had the same bioavailability score (0.17). The seven breed molecules did not inhibit the vital function of hepatic enzymes (cytochrome P450), such as CYP 1A2, CYP 2C19, CYP 2C9, CYP 2D6, and CYP 3A4, and they also had Log Kp (skin permeation) values between −8.41 and −11.38. Ultimately, the synthesis of these seven breed molecules in the chemical laboratory is quite simple for in vitro and in vivo research.The results obtained by ProToxII  show thaMolecular dynamic simulation (MDS)  was emplThe RMSD is a factor used to determine the equilibration, protein flexibility, and average distance between backbone atoms of a protein . The RMSThe rest of the complexes were very stable during the simulation period. The average RMSD values for the NA and its complexes with Breed 1, Breed 2, Breed 3, Breed 4, Breed 5, Breed 6, and Breed 7 were 0.148, 0.149, 0.131, 0.135, 0.166, 0.172, 0.172, and 0.148 nm, respectively. Finally, the RMSD analysis indicated that the MD trajectories were generally stable during the 100 ns of simulation and were able to help in the development of new influenza virus inhibitors. In this research, we calculated the root-mean-square fluctuation (RMSF) of the neuraminidase (NA) and its complexes with Breeds 1–7, as shown in We estimated the radius of gyration (Rg) of each system to assess the stability of the neuraminidase and its complexes (NA_Breed 1–7) during the 100 ns simulation . In geneWe analyzed the hydrogen bond profiles of the seven complexes to obtain a better understanding of the interaction between the breed molecules and neuraminidase, as shown in A greater SASA value shows that the protein volume is expanding, and a low fluctuation is observed over the simulation duration . The aveThe binding free energy of the seven complexes (Neuraminidase_Breed 1–7) was estimated using the MM-PBSA approach applied in Gromacs using MD trajectories. The van der Waals interactions (ΔEVDW), electrostatic interactions (ΔEEEL), nonpolar interactions in a solvated system (ΔEPB), nonpolar contribution of repulsive solute–solvent interactions to the solvation energy (ΔENPOLAR), nonpolar contribution of attractive solute–solvent interactions to the solvation energy (ΔEDISPER), total gas-phase molecular mechanics energy (ΔGGAS), and total solvation energy (ΔGSOLV) are all included in the total binding free energy. The total binding energies of the seven complexes were found to be within an acceptable range of between −76.06 and −34.96 KJ/mol. Gibbs free-energy landscapes were also produced using the first two PCs to differentiate the conformational modes of the neuraminidase and its complexes (NA Breeds 1–7). The Gibbs free-energy landscapes examined the orientation of the backbone atom fluctuation in neuraminidase and neuraminidase complexes (NA Breeds 7–1) from the MD trajectory. The Gibbs energy landscape plot following 100 ns of simulation, with the extracted structures from low-energy regions for each system, is shown in The blue, cyan, and green areas of the plot represent low-energy states with extremely stable protein conformations, whereas the red area represents a high-energy conformation. The energy landscape had numerous distinct minima that represented metastable structural states separated by a modest energy barrier. The binding of all breed molecules produced the most metastable conformational designs, with regional minima dispersed to approximately two to three areas of the energy landscape. In comparison to neuraminidase alone, the smaller and more concentrated blue minimal-energy regions in NA_Breed 1, NA_Breed 2, and NA_Breed 3 imply extremely stable complexes. Furthermore, the structures (neuraminidase conformations) were found to be similar for all systems. The results demonstrate that the seven types of molecules formed very stable complexes with neuraminidase.At this step, we used reaction-based enumeration to predict the synthetic pathways of the breed molecules (Breeds 1–7). It is another Schrödinger tool for predicting the synthetic pathway of every molecule using a retro-synthetic methodology. The reaction enumeration tool showed that the amide coupling, ether, and Mitsunobu reactions could be utilized to synthesize all of the breed molecules, as described in Neuraminidase is a key target in virology and the development of potent anti-influenza medicines. By inhibiting the biological activity of neuraminidase, the influenza virus is prevented from moving to further uninfected host cells and is eliminated. The primary objective was to create prospective novel compounds with anti-influenza activity that is more effective than existing anti-influenza drugs . Compared with the inhibitory power of clinical neuraminidase inhibitors, all seven designed molecules bound effectively to the neuraminidase receptor, where the proposed molecules attached to other amino acids forming the active site, such as Asp152 and Arg372, which explains the good inhibitory activity of the proposed molecules. The contribution of 2-amino-1-(2-methylthiazolidin-3-yl) ethanone, 2-methoxybutane, 2-methoxypropane, methoxycyclopentane, 1-methoxy-3-methylbutane, 1-methoxybutane, and 1-methoxypropane groups via the interaction of hydrogen bonds can again explain the good inhibitory activity of the developed molecules against neuraminidase. However, these chemical groups had different functions, and they interacted effectively with the essential amino acids that composed the active site, showing that the suggested compounds had a high affinity for the neuraminidase receptor. As pharmacokinetic profiles, the proposed molecules had good permeability toward the membrane bilayer (LogP between −1.55 and 0.24) and good aqueous solubility, which allowed the solubilization of these molecules in the intracellular medium. The hepatic metabolism of the designed compounds was quick and did not produce toxic substances, and no inhibition of cytochrome P450, such as CYP 3A4, was observed. Because of their high water solubility (LogS between −2.28 and 0.77), all of the designed compounds would be quickly removed by the kidneys at the nephron level. In terms of potential toxicity, we found no evidence of any toxicity produced by any of the proposed compounds. All of the aforementioned results indicate that their pharmacokinetic profile is optimal. When we examined the stability of the designed molecules within the neuraminidase receptor using molecular dynamics simulations, we clearly saw insignificant atomic mobility and the continuation of structural stability. During 100 ns of simulation, the seven proposed compounds had average RMSD values ranging from 0.131 to 0.172 nm. In addition, the average RMDF was between 0.1 and 0.123 nm. For the validation of the molecular docking results, the MM-PBSA calculations were in line with the molecular docking study, indicating that all of the molecules proposed formed more stable complexes with the neuraminidase active site, with binding free energies between −76.06 and −34.96 KJ/mol. At the molecular level, Gibbs free-energy landscapes again showed the small dynamic shift of the neuraminidase and its complexes, which confirms the extent of structural stability and the preservation of the initial configuration of all these complexes. Through the experimental examination, our outcomes indicate that the proposed compounds can be synthesized in a chemical laboratory, and it is worth noting that all of the proposed molecules are simple to manufacture using traditional mechanisms. This will make it easier to evaluate and estimate the effectiveness of the designed compounds to inhibit the biological function of neuraminidase in vitro and in vivo. The results of this study will possibly help researchers in the development of anti-influenza medications and virology.In this step, we used Maestro software . In breed-based de novo drug design, cocrystalized compounds from diverse PDB structures or well-known inhibitors against a particular target must be used ,31. The The crystal structure of neuraminidase with zanamivir was downloaded from the Protein Data Bank (PDB ID: 5L17) with a resolution of 2.40 Å and zero mutations . The “prThe process of energy minimization was used to create a protein with a lower energy state with the help of the OPLS3e force field  . The griFinally, we docked two clinical neuraminidase inhibitors (peramivir and oseltamivir) as supplementary reference ligands in the active site of neuraminidase to estimate the inhibitory activity of the breed molecules.50) was determined for both active and inactive cell types [ADMET properties are a very helpful drug discovery approach . The expll types .In this phase, we conducted molecular dynamic (MD) simulations for all breed molecules. The first process consisted of identifying compounds with high binding affinities for neuraminidase and good ADMET properties. The Gromacs-2022.4 package and the CHARMM27 force field were used to execute the production MD simulation for 100 ns . The SwiThe commands gmx rms, gmx rmsf, gmx gyrate, gmx hbond, and gmx sasa were used to determine the various parameters for each system. The root-mean-square deviation (RMSD) was used to quantify the structural stability of the protein-ligand complex, and the flexibility of the protein residues was assessed using the root-mean-square fluctuation (RMSF) . During For the molecular mechanics Poisson–Boltzmann surface area (MM-PBSA), the protocol implemented in the g_mmpbsa package was used to perform the analysis of the binding free energy (ΔG bind) . The MM-Reaction-based enumeration is another Schrödinger tool that predicts the synthetic pathway of any compound using a retro-synthetic approach . The curDeveloping new neuraminidase inhibitors is vital for combating evolving flu strains, enhancing treatment options, reducing resistance, and safeguarding global public health against influenza outbreaks. The objective of this research was to identify novel inhibitor molecules against the neuraminidase of influenza. To develop novel neuraminidase inhibitors, we used a breed-based de novo approach. According to the docking studies, seven breed molecules (Breeds 1–7) demonstrated high stability within the neuraminidase receptor compared to the clinical neuraminidase inhibitors . On the other hand, to reach the pharmacological target, the pharmacokinetics of the seven breed compounds were investigated, and the results show that they have excellent pharmacokinetic profiles, such as bioavailability and permeability toward biological membranes. The molecular dynamics simulations for 100 ns revealed that the seven breed molecules (Breeds 1–7) were particularly stable in the active site of neuraminidase. In addition, MM-PBSA computations demonstrated that the complexes were very stable throughout the duration of the simulation. This research can contribute to the development of novel and potent neuraminidase inhibitor drugs for the treatment of influenza and could give researchers the opportunity to examine these breed molecules for the treatment of influenza and its symptoms. Finally, the future of neuraminidase inhibitors for the flu involves improved efficacy, reduced resistance, and personalized treatments, aiding in better management and prevention of influenza outbreaks.


The integration of wearable sensor technology and machine learning algorithms has significantly transformed the field of intelligent medical rehabilitation. These innovative technologies enable the collection of valuable movement, muscle, or nerve data during the rehabilitation process, empowering medical professionals to evaluate patient recovery and predict disease development more efficiently. This systematic review aims to study the application of wearable sensor technology and machine learning algorithms in different disease rehabilitation training programs, obtain the best sensors and algorithms that meet different disease rehabilitation conditions, and provide ideas for future research and development. A total of 1490 studies were retrieved from two databases, the Web of Science and IEEE Xplore, and finally 32 articles were selected. In this review, the selected papers employ different wearable sensors and machine learning algorithms to address different disease rehabilitation problems. Our analysis focuses on the types of wearable sensors employed, the application of machine learning algorithms, and the approach to rehabilitation training for different medical conditions. It summarizes the usage of different sensors and compares different machine learning algorithms. It can be observed that the combination of these two technologies can optimize the disease rehabilitation process and provide more possibilities for future home rehabilitation scenarios. Finally, the present limitations and suggestions for future developments are presented in the study. With the rapid development of information technology, traditional medical rehabilitation methods combined with various disciplines and technologies, such as wearable sensors and machine learning algorithms, are widely used in clinical diagnosis, rehabilitation medicine, and other fields ,2. CerviWearable sensors first appeared in the mid-20th century. As a hardware device, they can perform data interactions. According to different needs, users wear devices with specific functions to collect behavior or health records . WearablMachine learning is a mechanism that uses computers to simulate human learning activities, enabling machines to learn autonomously without explicit programming, or researching how to effectively use information to obtain hidden and effective knowledge from big data . MachineThe review of wearable sensors and the machine learning algorithms in the literature mainly focuses on stroke rehabilitation , gait moIt outlines the application of wearable sensors and machine learning technology in rehabilitation training;It specifically analyzes the sensor type, sensor location, and feature extraction applied in the recovery process of different diseases;It evaluates the type and accuracy of machine learning algorithms applied in different rehabilitation exercises;It discusses the limitations, trends, and directions of sensors and machine learning algorithms in rehabilitation applications.At present, some reviews have summarized the latest research progress of wearable sensors and machine learning technology; however, a summary of disease rehabilitation training is lacking in the research. Many studies in the literature discuss the application of various sensors and machine learning techniques in the treatment and rehabilitation of certain diseases. For example, force sensors and bending sensors are added to stroke rehabilitation gloves to measure the grip strength and bending degree of the hand, and use machine learning technology to recognize gestures to promote the completion of the rehabilitation training process for patients . FacciorThe purpose of this study is to review the application of wearable sensor technology and machine learning algorithms in rehabilitation training for different diseases. The research results include the best sensors and ML algorithms that meet the rehabilitation conditions of different diseases, providing researchers with a choice of research directions and ideas for future research and development purposes.This review used the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) for the paper selection .The literature search used the Web of Science database and IEEE Xplore to retrieve all the literature published during the ten-year period from 1 January 2013 to 4 July 2023.Firstly, the basic keywords used for the literature search were “wearable sensor”, “machine learning”, and “rehabilitation training”. Then, more relevant keywords were selected. The search formats of the two databases are shown in Through the search, potentially relevant articles published between 1 January 2013 and 4 July 2023 were identified. The paper should include research conducted on wearable sensors, machine learning, and disease rehabilitation;The paper should provide a detailed analysis of the performance characteristics, where the sensor is worn, and the accuracy of the wearable sensor;The paper should elaborate on the application of the machine learning algorithm involved in data processing;The paper should include research conducted on the treatment and rehabilitation of one or more diseases.This review only included peer-reviewed journals or conference papers written in English between 1 January 2013 and 4 July 2023. The article research content was required to meet all of the following criteria:Exclude all review papers, review articles, and papers that lack specific research results;If the research exists in both academic journals and conference papers, select the former;Exclude papers that briefly mention wearable sensors or machine learning or disease recovery.In addition, the abovementioned criteria should be followed for paper selection, the exclusion criteria were as follows:The investigator (WSY) entered the data exported from the two databases into a table, which included information on the author, title, keywords, abstract, DOI number, etc. After excluding duplicate records according to the title and DOI number of the article, the investigator (WSY) then excluded the papers that did not meet the requirements according to the screening criteria based on the title, keywords, and abstract. Finally, the investigators (WSY and WZH) checked whether the specific content of the paper met all the screening requirements. The final screening was conducted and the selected papers were summarized.A total of 1490 documents were retrieved from the database, including 527 from Web of Science and 963 from IEEE Xplore. First, after removing 111 duplicates, 1379 papers were retained. Then, 1064 articles were excluded according to the title, keywords, and abstract. Then, 57 reviews were excluded. Subsequently, the full-text content was reviewed according to the screening criteria, 226 articles were excluded, and finally 32 articles were obtained. The whole process of document retrieval shown in We sorted and summarized the 32 selected papers by year, and the results are shown in Wearable sensors have the characteristics of light weight, flexibility, stability, and comfort. They are widely used for pulse and heartbeat monitoring purposes, gait analysis, and other health monitoring systems, disease diagnosis, and rehabilitation fields . In the Among the 32 selected papers we examined, a total of 24 diseases were addressed. As shown in It can be observed in In the process of disease rehabilitation training, different training actions are adopted to achieve the effect of assisting the recovery of different diseases in patients. Rehabilitation training actions are determined by the type of disease and the site requiring rehabilitation. The analysis of the training movements in the rehabilitation process collected from the selected papers helps us to understand the research hotspots of disease rehabilitation. Among them, 19 articles addressed upper-limb movement, accounting for approximately 60% of the total; 13 articles addressed lower-limb movement, accounting for approximately 40%. The studies conducted on upper-limb movement mainly addressed the movements of hands , arms, shoulders, and other parts; the studies conducted on lower-limb movement primarily concerned the movements of the thighs, calves, and feet.Taking strokes as an example, different parts of the brain can cause different degrees of limb dysfunction, such as hemiplegia, impaired mobility, and a loss of hand function . For infFeature engineering includes feature construction, extraction, and selection; the generation of features can be used as the input data for machine learning algorithms. Feature extraction is the optimization of a subset of features used to extract new features from the original features as the input . The feaThe machine learning algorithm is an application of artificial intelligence. It is based on data-trained algorithms that can automatically learn to continuously improve, make predictions, or act on data without being explicitly programmed ,41. MachWe summarized the machine learning algorithms that could be obtained from each article from the selected 32 documents and created statistics on all types of algorithms; the results are presented in This systematic review included 32 papers based on wearable sensors and machine learning algorithms used to assess the degree of recovery of patients and assist rehabilitation training. On the one hand, this review summarized the relevant research results and determined that wearable sensors and machine learning algorithms can be better applied in the course of disease rehabilitation, helping doctors to keep abreast of patients’ recovery status and relieve social medical pressure. On the other hand, for the patients themselves, the application of wearable sensors facilitated their recovery at home, which could greatly reduce the factor of psychological burden. Therefore, it is necessary to summarize the application results of wearable sensors and machine learning algorithms in the field of disease rehabilitation, explore the limitations of the research, and propose the possibility of future studies.We searched and screened papers for our analysis using the IEEE and the Web of Science core databases. The following section discusses (1) the selection of wearable sensor types in rehabilitation training; (2) the application of machine learning algorithms; (3) the analysis of the rehabilitation training process; and (4) the suggestions for future research.This review determined that wearable sensors are more frequently used for upper-limb than lower-limb rehabilitation purposes. During the rehabilitation process of stroke patients, the recovery speed of the upper limbs was slower than that of the lower limbs. During the recovery process of the upper limbs, certain complications, such as shoulder pain, shoulder–hand syndrome, and upper-limb flexor spasms often occurred. Therefore, additional studies in the field are focusing on the upper-limb recovery of stroke patients . He et aParkinson’s disease is a common neurodegenerative disorder characterized by tremors, stiffness, and slowness of movement . For theIt has become a trend in the research to apply wearable sensors for neck disease detection and rehabilitation purposes. For oropharyngeal dysphagia, Lee et al.  designedMost of the problems targeted by lower limb rehabilitation focus on lower limb dysfunction caused by spinal cord injuries, and diseases of the knee, hip, and other joints. In their study, Amir et al.  installeAccording to the statistical results of this review, 11 of the 32 papers used IMUs, which was the most frequently used sensor. IMU sensors not only have good wearability features and can be worn on any part of the wrist, arm, shoulder, and leg, but also collect kinematic parameters, such as body position, acceleration, and speed of motion with higher-accuracy results . TherefoIn A total of 17 papers used SVM to perform the disease rehabilitation evaluation, rehabilitation exercise classification, rehabilitation action recognition, and disease prediction steps. As a result of their high-accuracy characteristics, SVMs have a wide range of applications, and the number of SVMs used in the past decade in the field has been high compared to other ML algorithms . He et aDeep learning methods also belong to machine learning methods, and their applications in the field of disease rehabilitation are gradually increasing in the field. CNN is an important neural network in the field of deep learning, which can be applied to many different scenarios and has an excellent learning ability . Chae etIn addition to SVM and CNN algorithms, there are a variety of machine learning algorithms applied in the field of disease rehabilitation. Amir et al.  studied This review aimed to study the application of wearable sensors and machine learning algorithms in the field of disease rehabilitation. It was necessary to discuss the training required for various disease rehabilitation techniques.According to the research, it can be observed that wearable sensors are most widely used in the rehabilitation of stroke diseases. The purpose of stroke rehabilitation training is to improve the patient’s ability to control their muscles, enhance the coordination of muscle groups, and improve the coordination ability for daily activities and body balance . The mosPatients with spinal cord injuries must experience a long-term rehabilitation phase, which has a considerable impact on body motor functions . Amir etBased on the analysis of the selected articles, this review summarized some possible future development directions and some limitations of previous studies.In terms of the selection of experimental subjects, the experimental participants presented in some papers ,73,74,75From the types of wearable sensors summarized in A robot-assisted rehabilitation system is an emerging intelligent rehabilitation training system in the field. The main function of the robot is to help patients train by simulating normal activities. It can also be worn on the patient to force them to perform various rehabilitation exercises, continuously stimulating their brains, improving the ability of their motor organs, and achieving an early recovery . Robot-aIn the study of joint rehabilitation training such as those on the knee joint, wearing the sensor at the joint position affects the accuracy of motion detection during the movement of the joint ,99. The The combination of VR and disease rehabilitation training has become a development trend in the field. From the perspective of interaction, increasingly more researchers have proposed that future rehabilitation training can use virtual scenes to improve the interest and autonomy of patients. At present, some studies in the literature combine VR and sensors to assist patients in effective exercise rehabilitation techniques ,102,103.Machine learning, including deep learning, is developing very rapidly, and it involves a wide range of applications ,105. ForThis paper reviewed the research of wearable sensors and machine learning algorithms in disease rehabilitation training. It can be observed that using machine learning algorithms to process data obtained from wearable sensors is helpful for rehabilitation training for different diseases. Based on the results obtained by this review, it is concluded that IMUs are the most used sensors during rehabilitation. Most of the sensors used in disease rehabilitation are non-invasive, and the research on sensors in the field of disease rehabilitation should also pay more attention to other types of sensors. Machine learning algorithms such as SVM have a good auxiliary effect on data analysis and prediction in the process of disease recovery. In order to find the optimal solution, more algorithms should be used in experiments. In the future, other approaches can be tested to compensate for our deficiencies and complete a more comprehensive review of wearable sensors and machine learning algorithms in the field of medical rehabilitation. In the future, with the development of wearable sensor technology, characteristic data can be collected for additional diseases, so as to facilitate the understanding of the recovery status of diseases. At the same time, machine learning algorithms are transforming the field of healthcare. Smarter machine learning algorithms are being developed to help healthcare professionals improve diagnostic accuracy, predict the progression of a patient’s disease, and make personalized treatment recommendations. Combining the two methods increases the possibility of remote disease diagnosis and home rehabilitation, which may change the shortage of medical resources at present due to the aging population, to a certain extent. This review may not have included some relevant papers as the data were only collected from the Web of Science and IEEE Xplore. In addition, some recent high-quality papers may not have received enough citations.


Acute rejection (AR) continues to be a significant obstacle for short- and long-term graft survival in kidney transplant recipients. Herein, we aimed to examine urinary exosomal microRNAs with the objective of identifying novel biomarkers of AR.Candidate microRNAs were selected using NanoString-based urinary exosomal microRNA profiling, meta-analysis of web-based, public microRNA database, and literature review. The expression levels of these selected microRNAs were measured in the urinary exosomes of 108 recipients of the discovery cohort using quantitative real-time polymerase chain reaction (qPCR). Based on the differential microRNA expressions, AR signatures were generated, and their diagnostic powers were determined by assessing the urinary exosomes of 260 recipients in an independent validation cohort.We identified 29 urinary exosomal microRNAs as candidate biomarkers of AR, of which 7 microRNAs were differentially expressed in recipients with AR, as confirmed by qPCR analysis. A three-microRNA AR signature, composed of hsa-miR-21-5p, hsa-miR-31-5p, and hsa-miR-4532, could discriminate recipients with AR from those maintaining stable graft function (area under the curve [AUC] = 0.85). This signature exhibited a fair discriminative power in the identification of AR in the validation cohort (AUC = 0.77).We have successfully demonstrated that urinary exosomal microRNA signatures may form potential biomarkers for the diagnosis of AR in kidney transplantation recipients. Although renal allograft survival has improved substantially over the past decades, acute rejection (AR) continues to be an important cause of both early and late graft loss . GeneralExosomes are specialized subsets of extracellular vesicles, 30 – 100 nm in size; they are derived from the inward budding of endosomal membranes, and they play crucial roles in the cell-to-cell communication system as well as in modulation of immune functions . All humMicroRNAs are small, non-coding RNA molecules that can modulate the translational activity of target messenger RNAs (mRNAs), thereby playing a vital role in the maintenance of cellular homeostasis . InteresThis study aimed to determine the molecular profiles of urinary exosomal microRNAs in kidney transplant recipients, such that novel biomarkers necessary for non-invasive diagnosis of rejection may be established. We observed that the urinary exosomal microRNA expression was significantly altered in kidney transplant recipients with biopsy-proven AR. These distinctive expression signatures of urinary exosomal microRNAs might be used to distinguish AR from other allograft status. Finally, we validated the diagnostic ability of the urinary exosomal microRNA signatures in an independent cohort of kidney transplant recipients. Overall, our data demonstrate the potential of urinary exosomal microRNAs as novel, non-invasive biomarkers for diagnosing AR after kidney transplantation.22.1This study was conducted in three sequential stages, as illustrated in 2.2Study participants were recruited from the Assessment of Immunologic Risk and Tolerance in Kidney Transplantation (ARTKT-1), a multicenter cross-sectional study, which collected transplant-related information as well as blood and urine samples from six different hospitals in Korea. The 368 kidney transplant recipients, who had been enrolled in this study, were divided into a discovery set (n = 108) and a validation set (n = 260) , 25, andThe age, sex, duration after kidney transplantation, serum creatinine, urinary protein/creatinine ratio, and use of induction and maintenance immunosuppressive drugs, as well as donor type, ABO incompatibility, number of human leukocyte antigen (HLA) mismatches, and other necessary donor information was recorded for each patient at the time of graft biopsy for the biopsy cohort or at the time of visit to the outpatient clinic for the LTGS group. Graft function was assessed based on the estimated glomerular filtration rate (eGFR) using the Chronic Kidney Disease Epidemiology Collaboration (CKD-EPI) formula . This st2.3g for 20 minutes at room temperature, and the supernatant was transferred into RNA later solution  and stored at -80°C until further use. Urinary exosomal RNA was isolated from 1 mL of urinary supernatant using spin column-based exoRNeasy serum/plasma midi kits , as described previously . Sample processing and microRNA expression analysis were performed according to the manufacturer’s instructions. Each target microRNA was normalized with internal positive controls, and the geometric means of the top 100 expressed targets were calculated using nSolver software in a digital analyzer. Normalized microRNAs were expressed as fold change (FC) values among the groups, and microRNAs fulfilling the criteria of |FC value| ≥ 2 and 2.5https://bio.tools/mirtarbase), TargetScan (http://www.targetscan.org/vert_72), and miRbase (http://www.mirbase.org). The mRNAs were selected using GeneMeta R package and categorized in four data sets, according to the approach outlined by Choi et al.  was synthesized from the extracted RNA using TaqMan advanced miRNA cDNA synthesis kit (Thermo Scientific), according to the manufacturer’s protocol. Initially, each microRNA was subjected to universal 3′ poly-A tailing and 5′ ligation of adaptor sequences, followed by amplification using universal primers to increase the amount of cDNA. This method is advantageous as compared to the traditional reverse transcription PCR with step-loop primers because multiple microRNA targets can be assessed from a single amplified sample.Commercially available, gene-specific oligonucleotide primers and TaqMan probes were used to measure the levels of each candidate microRNA. The hsa-miR-16-5p was used as an endogenous control for normalization of the microRNAs, and the expression levels were calculated using the comparative cycle threshold (Ct) method , 29. Two2.72 tests were performed to compare the baseline characteristics and laboratory observations, as appropriate. Urinary exosomal microRNA expressions were compared among the subgroups using Kruskal–Wallis test, while Dunn’s test was performed for comparisons within each group, since these data are non-normally distributed. Receiver operating characteristic (ROC) curves and areas under the curve (AUC) were constructed to assess the abilities of urinary exosomal microRNAs with respect to prediction of AR in recipients. Differences with p-values < 0.05 were considered as statistically significant.All statistical analyses were performed using SPSS for Windows, version 20.0 . Baseline clinical data are expressed as mean ± standard deviation (SD) or as the number of patients and corresponding percentages. Independent t-tests, analysis of variance (ANOVA), and χ33.1de novo donor specific antibody (DSA), respectively.The baseline demographics and clinical characteristics of the enrolled patients are shown in 3.2To screen and identify microRNA candidates using a transcriptome-based approach, we isolated urinary exosomal RNAs from urine samples of patients in the discovery set. The median quantity of total RNA was 0.342 μg , and the median purity of total RNA was 1.485 . All samples qualified in the quality control tests and were processed for NanoString analysis. Of the 798 microRNAs that were assessed, 14 fulfilled the selection criteria and were selected as candidate microRNAs for predicting AR  as candidate biomarkers of AR  in the urinary exosomal fractions of the enrolled patients. qPCR analysis revealed that eight microRNAs exhibited significant differences between their urinary exosomal expressions in the AR and STA groups  to validate the performance of the AR-specific microRNA signature. The fixed cut-off value set in the discovery cohort was applied in the assessment process of the validation cohort. The expressions of the eight microRNAs are displayed in 4In the present study, we extensively investigated the urinary exosomal microRNA profiles of kidney transplant recipients and attempted to determine their clinical utility as non-invasive biomarkers of AR. We identified differentially expressed urinary exosomal microRNAs in recipients with AR using NanoString-based transcriptomics, public databases, as well as previously published literature and successfully validated a urinary exosomal microRNA signature that could effectively discriminate AR from STA as well as OGIs, including BK virus-associated nephropathy, acute tubular necrosis, or calcineurin inhibitor toxicity. Altogether, these data suggest that urinary exosomal microRNAs may be a useful source of novel, non-invasive biomarkers for renal allograft rejection.Renal allograft biopsy is currently considered the gold standard for diagnosing acute rejection in kidney transplant recipients. However, graft biopsy possesses several complications such as bleeding, infection, and fistula formation. However, this procedure can lead to complications such as bleeding, infection, and fistula formation. Additionally, since biopsy tissue reveals only a small portion of the kidney, it can hinder the accurate diagnosis of transplant pathology. A recent study compared pathologic scores of whole kidney tissues and biopsy specimens that were obtained from the same kidneys. The examinations of biopsy specimens consistently resulted in lower pathologic scores, thus underestimating the severity of the entire kidney tissues, particularly in the case of tubulointerstitial fibrosis . To overPrevious studies have consistently demonstrated that microRNAs have the potential to function as biomarkers of transplant pathologies such as acute and chronic rejection , 34–37, Urinary exosomes have several unique advantages over urinary cell pellets or supernatant as a source of biomarkers. These urinary exosomes reflect the local pathophysiological status of renal allografts, since they are mostly derived from renal epithelial cells , 44, 45.In a previous study, we had demonstrated that the spin column-based enrichment of urinary exosomes can substitute the traditional ultracentrifugation-based method, which is labor-intensive and difficult to implement in clinical applications . HoweverMajority of the studies that have investigated biomarkers for rejection with respect to kidney transplantation have used different threshold values for the discovery and validation stages, thereby limiting their application in clinical practice . In factThe differentially expressed urinary exosomal microRNAs have various pathophysiological characteristics –70, althThis study has certain limitations. We observed that the discriminative power of the AR prediction models developed in the discovery set decreased slightly during the assessment of the validation cohort , 75. FurIn conclusion, this study demonstrated that urinary exosomal microRNA profiles could serve as potential biomarkers for the non-invasive diagnosis of AR in kidney transplant recipients. We expect that future prospective trials will clarify the clinical relevance of our AR-prediction signature.The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found below: GSE230858 (GEO).The studies involving human participants were reviewed and approved by #2012-01-030, KHNMC. The patients/participants provided their written informed consent to participate in this study.J-WS: formal analysis, methodology, software, and writing original draft. YL: funding acquisition, formal analysis, methodology, software, and writing original draft. DT: formal analysis. YK, J-YM, SJ, JK, HH, K-HJ, HJ, S-YL: supervision. BC, C-DK, JP, YHK: sample and data collection, supervision. JS: methodology, software, and formal analysis. S-HL: funding acquisition, conceptualization, methodology, formal analysis, and writing original draft. All authors contributed to the article and approved the submitted version.


This review article explores the foundation of laypeople’s understanding of the physical world rooted in perceptual experience. Beginning with a concise historical overview of the study of intuitive physics, the article presents the hypothesis that laypeople possess accurate internalized representations of physical laws. A key aspect of this hypothesis is the contention that correct representations of physical laws emerge in ecological experimental conditions, where the scenario being examined resembles everyday life experiences. The article critically examines empirical evidence both supporting and challenging this claim, revealing that despite everyday-life-like conditions, fundamental misconceptions often persist. Many of these misconceptions can be attributed to a domain-general heuristic that arises from the overgeneralization of perceptual-motor experiences with physical objects. To conclude, the article delves into ongoing controversies and highlights promising future avenues in the field of intuitive physics, including action–judgment dissociations, insights from developmental psychology, and computational models integrating artificial intelligence. Scientific physics relies on precise measurement instruments, mathematical tools, and the minds of skilled scientists. These resources are unavailable to those who lack formal training yet seek to comprehend the underlying rules governing the behavior of physical objects in their environment. Intuitive physics, also known as naïve physics, refers to the understanding of the physical world held by individuals without formal instruction in physics . Here, IAfter a brief historical introduction to the study of intuitive physics , the preA) would initiate movement towards the initially stationary square (B) at a certain point in time. Upon contact, B would start moving in the same direction as A had been moving, while A came to a halt. Under specific conditions, observers reported an overwhelming visual impression that A had caused the motion of B, known as the launching effect. Michotte extensively manipulated various parameters of the animation, such as the relative speeds and trajectories of A and B, and even introduced delays during their contact, to explore how such manipulations influenced observers’ impressions. Occasionally, Michotte also referenced Newtonian laws of collisions to demonstrate that the launching effect is governed by inherent laws of the visual system, distinct from physical laws. However, as pointed out by Albert Michotte’s ground breaking work on the perception of causality  can be cThe first investigations into what later on has been called intuitive physics were conducted by Paolo Bozzi, who examined the visual perception of pendulum motion and motion along inclined planes  from A increases and to decrease as B’s resistance increases. As the impetus gradually dissipates, B is expected to decelerate until it eventually halts. Thus, there exists a strong parallel between how laypeople perceive first-person interactions with physical objects and how they conceptualize mechanical interactions involving inanimate objects. In other words, it appears that perceptual-motor experiences during personal interactions serve as an interpretative framework for comprehending mechanical interactions between physical, non-living objects .Many misconceptions regarding the motion of objects seem to stem from an ramework . For exaRelying on phenomenal experience leads laypeople to believe that when an object applies or experiences a force in a particular direction, it will move in that direction with a speed directly proportional to the force  and an outcome event Y . Normatively, determining the existence or absence of a causal link between X and Y requires applying Bayes’ theorem, which involves combining the relative frequencies of different events . However, research has consistently shown that laypeople tend to rely on simplified heuristics in such cases. One common heuristic is to count the relative frequency of the outcome event Y, disregarding the frequencies of ¬X and Y, as well as X and ¬Y. Although this heuristic may yield satisfactory results in certain contexts, it is not a universally valid strategy. Consequently, its usage can lead to a significant overestimation of the strength of the causal link between the source and outcome events, known as a causality bias  as more likely than the occurrence of a single event  alone. As a result, these findings contradict the predictions made by Bayesian models, revealing that probabilistic reasoning in the context of simple mechanical events appears to violate the normative laws of probability (see also In addition to the previous point, it is worth highlighting that Another intriguing debate concerns the role of intuitive physics in everyday motor interactions with physical objects. Some researchers propose that the cognitive system and the motor-action-oriented system employ distinct representations of physical phenomena . They suD/√H rule when manually propelling a tennis ball along a horizontal surface, to make the ball land on a target positioned on the ground at a vertical height H from the surface and at a horizontal distance D from the edge of the surface. However, when asked to predict the unseen velocity of the ball, they failed to consider the influence of height. The same disconnect between motor and cognitive abilities was observed in other areas, such as time, space, and velocity integration (Supporting the dissociation between action and cognition in intuitive physics, egration . At a moegration , Neupärtegration , and Kriegration  conceptuegration  had partThe hypothesis that motor interactions with physical objects are guided by internalized representations of physical laws and constraints is undeniably intriguing. It offers an explanation for the efficacy of these interactions, even when cognitive processes rely on heuristics. In simpler terms, one does not necessarily need precise cognitive and perceptual representations of physical phenomena to successfully adapt to the environment, as long as actions are guided by accurate internalized representations of physical laws. Nonetheless, due to the limited number of empirical studies examining motor interactions with physical objects, further investigation is necessary to delve deeper into this hypothesis. It is worth mentioning that a dissociation between action and cognition was not observed in a developmental study that employed a slingshot throw paradigm , as wellThis review primarily focused on studies involving adult participants, but it is worth noting that there is a vast and valuable field of research dedicated to the development of intuitive physics in infancy and childhood. Across a wide range of physical scenarios, studies have demonstrated that children gradually acquire the ability to predict outcomes by integrating multiple physical variables using additive, multiplicative, and averaging rules . Some noIn a noteworthy developmental study conducted by A new frontier in research on children’s intuitive understanding of the physical world involves the construction of neural networks that mimic children’s knowledge of physics. Notably, studies have shown that neural networks trained with thousands of videos of real physical motion can learn to predict the future positions of moving objects, even through unsupervised learning processes . RecentlComprehending how the cognitive system constructs a representation of the physical world is a valuable yet challenging endeavor. It advances our theoretical grasp of human representation of the external world and holds practical implications, particularly for enhancing physics education.This review centered on the hypothesis that intuitive physics relies on simulations rooted in internalized physical laws, yielding precise solutions in real-life scenarios. I examined empirical evidence both supporting and refuting this notion. Notably, misconceptions often persist, even in realistic situations involving perceptual judgment or active interactions with tangible objects. A domain-general heuristic, stemming from the overgeneralization of perceptual-motor experience with physical objects, was proposed as a potential explanation for many such misconceptions. However, the precise conditions under which accurate task-specific knowledge may override this general heuristic remains a contentious subject necessitating future investigation.Finally, I underscored the significance of action–judgment dissociations and developmental models based on neural networks as promising research avenues within the realm of intuitive physics.


Hyperglycemia is recognized as a common adverse event for patients receiving alpelisib but has been little studied outside of clinical trials. We report the frequency of alpelisib-associated hyperglycemia in a real-world setting and evaluate proposed risk factors.We retrospectively identified patients with PIK3CA-mutated, hormone receptor-positive, metastatic breast cancer who initiated treatment with alpelisib plus fulvestrant between August 2019 and December 2021. Ordinal logistic regression evaluated 5 characteristics  as independent risk factors for ALP-associated hyperglycemia grades 2-4. Risk of error from multiple hypothesis testing was controlled using the false discovery rate method.n = 92 subjects, all but 1 female, mean age 59.9 (+11.9) years with 50% non-Hispanic White, 15% Hispanic/Latino, 13% Asian, 9% African/Black, and 13% other/unknown. In total 34% of patients had diabetes, 10% had pre-diabetes, and 56% had normoglycemia. Thirty-six percent were obese, 32% were overweight, 25% were normal weight, and 7% were lean. Frequency of grades 1-4 hyperglycemia in current subjects (64.1%) was similar to hyperglycemia reported in the SOLAR-1 trial (63.7%). Our subjects’ risk of grades 2-4 hyperglycemia was independently increased by pre-existing diabetes , pre-diabetes , Asian ancestry , and each unit of BMI above 20 .The study included While receiving alpelisib, patients of Asian ancestry, as well as patients with pre-existing hyperglycemia and/or BMI above 20, should be closely monitored for hyperglycemia. The mechanism underlying the current association of alpelisib-associated hyperglycemia with Asian ancestry is independent of BMI and merits further study. The high incidence of hyperglycemia resulted in a change in practice to include consultation with a diabetes nurse educator or endocrinologist at the start of alpelisib. Hyperglycemia is a common adverse event for patients receiving alpelisib. This article reports the frequency of alpelisib-associated hyperglycemia in a real-world setting and evaluates proposed risk factors. Findings from the current study suggest that patients with breast cancer with one or more risk factors  should be closely monitored for hyperglycemia while receiving alpelisib. Neither age, hypertension, nor hyperlipidemia increases the risk of this adverse reaction.1 The inhibition of PI3Kα induces insulin resistance and results in hyperglycemia.2 The SOLAR-1 trial excluded patients with type 1 diabetes and those with uncontrolled type 2 defined as a fasting glucose of >140 mg/dL or glycosylated hemoglobin level of > 6.4%. The most common side effect in this trial was hyperglycemia which occurred in 63.7% of alpelisib-treated patients. Additional toxicities included diarrhea (57.7%), nausea (44.7%), decreased appetite (35.6%), and rash (35.6%).3 We observed the similar toxicity profile in Bylieve study.4In May 2019, alpelisib  was given FDA approval in combination with fulvestrant for postmenopausal women and men with recurrent/metastatic hormone receptor positive HER2-negative breast cancer patients who had a phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha (PIK3CA) mutation. The approval was based on the SOLAR-1 trial which demonstrated that the addition of alpelisib to fulvestrant improved progression-free survival from 5.7 to 11 months.1 We undertook a retrospective chart review study of women with metastatic hormone receptor positive breast cancer treated with fulvestrant and alpelisib to determine the incidence of hyperglycemia and to identify risk factors for its development.The study inclusion for SOLAR-1 was not overly restrictive but may not have represented the real-world population of women with breast cancer. In the developing world, 16% of women with metastatic breast cancer with diabetes were considered ineligible for SOLAR-1.With a waiver of consent from the Institutional Review Board, data for the current study were abstracted retrospectively from the subjects’ medical charts. Subjects were patients at our institution who began treatment with alpelisib for advanced breast cancer between August 2019 and December 2021. Excluded were patients who dissented from to the use of their medical data in research.6Most BMI values available from the patient chart were measured after the start of treatment with alpelisib. Because the drug is known to cause decrease in body weight and because our risk factor analysis required BMI at pre-alpelisib baseline, we sought to estimate pre-alpelisib BMI from the patient’s post-alpelisib value. To do so, we modeled the extent to which recorded BMI varied by its timing after initiation of alpelisib. That generalized linear regression model specified a Poisson distribution, and was adjusted for all covariates that improved the model’s fit to the observed data and explored whether BMI’s slope over time was modified by any covariate. Using the slopes identified by the model, all BMI values obtained after alpelisib initiation were converted to their model-estimated pre-alpelisib value using the following formula. Specifically, for patients with prior diabetes or pre-diabetes, pre-alpelisib BMI was calculated as exp[log(BMI from chart) − log], while for patients with normoglycemic history, pre-alpelisib BMI was calculated as exp[log(BMI from chart) − log]. Subjects were classified by their pre-alpelisib BMI values into categories of lean, normal, overweight, and obese at baseline using the appropriate ancestry-specific cut-point standards .1 using the Jonckheere-Terpstra test7 .The distribution of hyperglycemia among current subjects was compared to that reported in the SOLAR-1 trial8 was used to limit to below 5% the overall risk of error from evaluating 5 hypothesized risk factors for hyperglycemia on alpelisib.The hypothesized risk factors for hyperglycemia were evaluated using ordinal logistic regression, a method appropriate for the ranked outcome under study. For that model, the Akaike Information Criterion was used to define the outcome categories. Specifically, it was most efficient to define the highest ranked outcome by combining the grade 3 cases with the few grade 4 cases. In addition, analyzing the few grade 1 cases as a separate outcome worsened the model’s fit. For clarity, the grade 1 cases were set aside rather than combined with normoglycemia. As a result, the model considered only mid- (grade 2) and high-level (grades 3-4) hyperglycemia in comparison to normoglycemia (the lowest-ranked outcome). For that regression model, it was found optimal to enter BMI as continuous variable having a minimum threshold of 20. No minimum threshold, categorization, or transformation of age was found to change the evident lack of association between age and hyperglycemia. The false discovery rate methodn = 92 subjects, all but 1 female, mean age of 59.9 (+11.9) years, with 50% non-Hispanic White, 15% Hispanic/Latino, 13% Asian, 9% African/Black, and 13% other/unknown. In total 34% of patients had diabetes, 10% had pre-diabetes, and 56% had normoglycemia, 36% were obese, 32% were overweight, 25% were normal weight, and 7% were lean (n = 22), 2020 (n = 32), or 2021 (n = 37). At the time of chart review, 55/92 (60%) subjects had discontinued alpelisib  and 37/92 (40%) were still receiving alpelisib .The study included ere lean . With a P < .05) . According to a Poisson regression model of recorded BMI , the relationship between BMI and its post-alpelisib timing depended on the patient’s glycemic history prior to alpelisib (P < .05) . Specifin = 64 patients, all female) were converted to their estimated pre-alpelisib value using the slopes from the model in n = 27) to be median +4.52  BMI units higher than the post-alpelisib observation, by which time the patients had lost an estimated −13.9%  of their pre-alpelisib BMI. In contrast, previously normoglycemic patients had model-estimated pre-alpelisib BMI values (n = 37) that were scarcely higher  than their post-alpelisib observation, corresponding to an estimated −1.4%  decrease in BMI on alpelisib. By thus imputing pre-alpelisib BMI values for the n = 64 subjects whose BMI had been measured post-alpelisib, it was found that 16/64 (25%) patients shifted BMI category after initiating alpelisib: These patients included 1 of 23 normal weight pre-alpelisib who became lean, 8 of 29 overweight pre-alpelisib who became normal weight, and 9 of 33 obese pre-alpelisib who became overweight (n = 8) or normal weight (n = 1) after initiating alpelisib.Post-initiation BMI values .While receiving alpelisib, 59/92 (64.1%) current subjects developed hyperglycemia of any grade, similar to 181/284  observed among alpelisib recipients in the SOLAR-1 trial.glycemia  was not P = .56). The association with BMI was independent of and unmodified by Asian ancestry. Exploratory analysis detected no univariable or multivariable association with preexisting hypertension or hyperlipidemia (data not shown).Turning to the risk factor analysis, multivariable modeling  showed tn = 8) or Filipino (n = 4) surnames. Their ages ranged from 36.5 to 82.1 years, their pre-alpelisib BMI ranged between 19.5 and 28.8, and 6 of the 12 had pre-existing diabetes. Grades 2-4 hyperglycemia developed in 9 patients of Asian ancestry, specifically the 6 patients with pre-existing diabetes, and 3 normoglycemic patients who were under age 65 and normal-weight, overweight, or obese, respectively.All patients who reported Asian ancestry were female and had Chinese , whereas grade 2+ gastrointestinal toxicity can begin at any time during treatment. The risk of hyperglycemia on alpelisib  was associated with baseline glycemic status (diabetes > prediabetes > normoglycemia), body mass index (BMI) , and advanced age (above age 75 years versus below). However, whether glycemic history, BMI, and age are independent risk factors for this AE remains to be determined. In contrast to hyperglycemia, rash on alpelisib was associated solely with prophylactic use of antihistamine, and gastrointestinal toxicity was possibly associated with advanced age.10-12This study is the first to identify Asian ancestry as a novel risk factor for alpelisib-associated hyperglycemia. Other chemotherapeutic agents have been found to cause greater toxicity in patients with East Asian ancestry compared to their Western patients.6 However, our multivariable analysis makes clear that the current association between Asian ancestry and alpelisib-associated hyperglycemia is independent of BMI, and thus cannot be explained by it. Moreover, our analysis detected no interaction between ancestry and BMI, indicating that the association between hyperglycemia and BMI does not differ for persons of Asian versus non-Asian ancestry. A potential explanation for the current association with Asian ancestry may involve genetic polymorphism in a cytochrome P450 enzyme. Investigating this possibility will require a larger multi-institutional sample of cancer patients.The possible biological mechanism underlying the current association between Asian ancestry and alpelisib-associated hyperglycemia remains to be elucidated. A heightened susceptibility to hyperglycemia has been observed among patients of Asian ancestry. In addition, an elevated prevalence of diabetes is present among patients of Filipino, Vietnamese, Korean, South Asian, or Japanese descent whose BMI is classified as normal or overweight using standard cut-points but actually corresponds to overweight or obese under the Asian-specific cut-points recommended by the World Health Organization.13) is both time-dependent and modified by pre-existing glycemic status. This finding suggests that only those patients with pre-existing hyperglycemia will lose weight while on alpelisib.An incidental finding of the current study is the novel observation that the decrease in BMI on alpelisib  inhibitors have been used in combination with metformin. The SGLT2 inhibitors prevent reabsorption of glucose in the kidney and preclinical data have found these agents to be superior to metformin in managing alpelisib hyperglycemia.The findings from this study led us to change the order set within the electronic record so that every patient who is started on alpelisib is also evaluated by a diabetes nurse educator or an endocrinologist.The chief limitations of the current study are its limited sample size and retrospective design. Current sample size precluded our investigating whether ancestries other than Asian are associated with hyperglycemia and whether Asian ancestry is associated with other classes of AEs on alpelisib. The retrospective design precluded uniform assessment of BMI prior to initiation of alpelisib. However, our analysis was able to convert post-initiation BMI values to their estimated pre-alpelisib value. Future studies of alpelisib-associated hyperglycemia should be prospective in order to record the time to onset of each AE. The analysis of SOLAR-1 did plot time to hyperglycemia, and future studies should address whether current risk factors are associated with an accelerated onset.Breast cancer patients of Asian ancestry as well as patients with pre-existing hyperglycemia and/or BMI above 20 should be closely monitored for hyperglycemia while receiving alpelisib. Given the predictably high incidence of hyperglycemia with alpelisib, we routinely include consultations with a diabetic educator or endocrinologist.oyad024_suppl_Supplementary_Table_S1Click here for additional data file.


Androgen deprivation therapy (ADT) has been the mainstay of prostate cancer (PCa) treatment, with success in developing more effective inhibitors of androgen synthesis and antiandrogens in clinical practice. However, hormone deprivation and AR ablation have caused an increase in ADT-insensitive PCas associated with a poor prognosis. Resistance to ADT arises through various mechanisms, and most castration-resistant PCas still rely on the androgen axis, while others become truly androgen receptor (AR)-independent. Our research identified the human tousled-like kinase 1 (TLK1) as a crucial early mediator of PCa cell adaptation to ADT, promoting androgen-independent growth, inhibiting apoptosis, and facilitating cell motility and metastasis. Although explicit, the growing role of TLK1 biology in PCa has remained underrepresented and elusive. In this review, we aim to highlight the diverse functions of TLK1 in PCa, shed light on the molecular mechanisms underlying the transition from androgen-sensitive (AS) to an androgen-insensitive (AI) disease mediated by TLK1, and explore potential strategies to counteract this process. Targeting TLK1 and its associated signaling could prevent PCa progression to the incurable metastatic castration-resistant PCa (mCRPC) stage and provide a promising approach to treating PCa. Androgen steroid hormones play a crucial role in PCa by binding to the AR and triggering a specific oncogenic transcriptional program . This unRecent more potent inhibitors of AR signaling  demonstrate some benefits for castration-resistant PCa (CRPC) patients, highlighting the critical role of AR signaling . CRPC ceIn recent years, our laboratory has discovered that the DNA damage and response (DDR) kinase TLK1 plays a vital role in facilitating the adaptation of PCa cells to ADT . InitialRecognizing TLK1 as a pivotal contributor to PCa’s ability to adapt to ADT, promote growth with minimal androgen stimulation, evade apoptosis, and facilitate cell motility and metastasis offers a hopeful foundation for the ongoing quest for potential treatments. By focusing on TLK1 and its related pathways, we can develop innovative strategies to delay or halt the progression of PCa to the incurable mCRPC stage. Therefore, our endeavor to comprehend the biology of TLK1 represents a particular initiative to comprehensively understand PCa and identify appropriate therapeutic options for clinical trials.Recent advancements in inhibiting AR signaling, mainly through potent AR-targeted therapies, have resulted in a concerning trend of metastatic PCas transforming into AR-negative diseases. This change renders these tumors unresponsive to inhibition of the AR signaling pathway. While AR-negative disease is uncommon in untreated patients, its incidence has been rising in individuals receiving AR-targeted therapies , a trendTreatment options for AR-negative PCa, specifically the subtype with neuroendocrine differentiation, are still uncertain due to a lack of consensus. Diagnosing therapy-induced AR-negative disease is challenging as it requires assessing current tumor tissue. The recommended approach for confirmed AR-negative disease with neuroendocrine differentiation is a platinum-based regimen, similar to treatments for other neuroendocrine small cell carcinomas. However, the response rates to combinations of cisplatin and carboplatin with docetaxel or etoposide are relatively high but not long-lasting ,33,34. ADuring the progression of PCa, the loss of AR expression is accompanied by significant alterations in cellular differentiation, forming part of a more extensive cellular rewiring process . This shNeuroendocrine PCa (NEPC), also known as small cell PCa, is a significant subtype among AR-negative tumors ,39. NeurNewer functional analyses have demonstrated that NEPC typically originates from an AR-positive adenocarcinoma through transdifferentiation. Zou et al., in lineage-tracing mouse model studies, showed that the neuroendocrine features arise from the transdifferentiation of luminal cells . InactivRecent findings have revealed a connection between the activation of Wnt/β-Catenin signaling and the presence of AR-negative disease. This connection is established through the involvement of specific genes regulated by Wnt/β-Catenin, namely FOXA2, and MYCN, which promote the process of neuroendocrine transdifferentiation ,52,53,54A recently identified subtype of AR-negative PCa, the double-negative subtype, has become more prevalent in recent years. This subtype, which lacks AR and neuroendocrine markers, has shown a strong association with the use of newer AR pathway inhibitors . Its occA newly discovered subtype of mCRPC has been linked to the loss of chromodomain helicase DNA-binding protein 1 (CHD1). When CHD1 is deficient, cancer cells undergo significant changes in their chromatin structure, making them resistant to enzalutamide . These rLabrecque et al. recently identified five mCRPC subtypes based on AR and neuroendocrine marker RNA expression patterns. These subtypes include tumors with high AR expression (ARPC), low AR expression (ARLPC), tumors expressing both AR and neuroendocrine markers (AMPC), tumors lacking both AR and neuroendocrine markers (DNPC), and tumors exhibiting small cell and neuroendocrine features without AR expression (SCNPC) . AlthougThe subtypes mentioned earlier are only sometimes distinct from each other and can change over time . They liTousled-like kinases (TLKs) are mammalian homologs of plant-tousled kinases, first discovered in 1999 . They plOne specific isoform of TLK1 called TLK1B has been very well characterized. TLK1B is a spliced variant with a truncated N-terminal region, a long GC-rich 5’ untranslated region (UTR), and two short upstream open reading frames (ORFs) inhibitory for translation. However, TLK1B retains the intact catalytic domain of TLK1 and exhibits similar substrate specificity ,66,67. TTLK1B plays a role in phosphorylating histone H3 at the Serine-10 residue . This phOur research group characterized one such proteome target of TLK1 and identified the NIMA-related kinase NEK1 as a significant substrate , a critiPCa is diagnosed in ~200,000 US men annually, resulting in approximately 30,000 deaths. ADT with anti-androgens is the standard of care for patients with locally advanced prostate cancer, metastatic prostate cancer, and biochemically recurrent disease after failure of localized treatments. ADT is known to provide remission of the disease, as best evidenced by a decline in prostate-specific antigen (PSA) in about 90% of patients. However, after a mean time of 2–3 years, the disease progresses despite continuous hormonal manipulation . This tyhttps://ualcan.path.uab.edu/cgi-bin/TCGA-survival1.pl?genenam=TLK1&ctype=PRAD; accessed 12 October 2022). However, since the expression of TLK1B is controlled mainly at the post-transcriptional level, it may have been primarily underestimated in transcriptomics analyses of PCa. TLK1 activates NEK1 by phosphorylating T141, which is adjacent to the activation loop [We found that androgen deprivation in LNCaP cells results in a rapid, mTOR-dependent, rapamycin-suppressible, substantial increase in the translation of TLK1B, whereas its mRNA does not change . Similarion loop , and we ion loop . We reasion loop . It is tion loop . We alsoion loop . Moreoveion loop  in xenogion loop .Extensive studies also suggest the critical importance of the Hippo/YAP pathway in various solid tumors and in mCRPC . HoweverOur study revealed that the TLK1-NEK1 pathway also significantly supports PCa cells’ survival by maintaining mitochondrial membrane integrity . SpecifiTLK1 promotes cell survival through various other pathways independent of NEK1. One such pathway involves TLK1’s interaction with AKTIP, a protein that facilitates anti-apoptotic responses. TLK1 phosphorylates AKTIP at specific sites, enhancing its ability to anchor AKT and PDK1 to the plasma membrane, where AKT is subsequently fully activated through phosphorylation by the mTORC2 complex . Once acAdditionally, we have discovered a new interaction between TLK1 and MK5, which promotes the motility and invasion of PCa cells . MK5 is Our 2013 research paper highlighted the importance of identifying antipsychotic PTH compounds with similar structures that can inhibit TLKs in laboratory settings . These sC. elegans [Our goal was to create new PTHs that effectively treat PCa while avoiding the harmful side effects of THD. By employing in vitro kinase assays and conducting docking studies, we identified a novel PTH derivative, J54, with selective TLK inhibition and seemingly devoid of toxicity. Through our collaboration with a medicinal chemist and structural modelers, we have synthesized, modeled, and tested several custom-made PTH inhibitors of TLK1 ,111. Tho elegans . The hombind = −39.7 kcal/mol, computed by the MM/GBSA method [A 100 ns MD simulation of this TLK1-J54 complex indicated the pose was stable, further evident from a favorable total binding free energy ∆GA method . THD wasA method . We noteA method . In addiA method ). Such iA method  (better A method ). While A method , we are We tested J54 with the panel of PCa cells in the androgen-containing medium by proliferation assays . The effC. elegans which has a simple but complete nervous system and has been well characterized for its responses to antipsychotic drugs, including actions at serotonin or dopamine receptors [C. elegans on plates containing dopamine results in their immobility , concomitant inclusion of DR2 antagonists like risperidone or TFP suppressed their immobility. In contrast, J54 had a very weak effect in suppressing immobility [We also performed a general assessment of mice following injections of J54 up to 30 mg/kg-IP . We notieceptors ,120. In mobility .Early-stage, organ-confined PCa is generally well controlled with standard therapy, and even more invasive disease can be managed significantly with current ADT modalities. Unfortunately, once cancer has become resistant to these regimens (mCRPC), there is no effective therapy beyond a few additional months. Even though there is a substantial amount of research aimed at understanding the process of conversion to AI growth of PCa cells, there is still limited knowledge of this process, and most advances in the field have been made, regardless, on developing better androgen receptor-targeted therapeutics. Substantial work has been carried out in studying and targeting the DDR during conversion to CRPC ,121, butTLKs play vital roles in various essential cellular processes, including chromatin formation, DNA replication, repair, and transcription ,123,124.Early studies suggested Asf1 as the principal target of TLKs and implicated them in chromatin assembly roles . HoweverPreviously, TLK2 kinase inhibitors were discovered through a screening process involving commercially available small molecules ,132. HowWhile we aimed to demonstrate that J54 operates by inhibiting the ADT-activated TLK1-NEK1-ATR-Chk1 DDR pathway, we cannot entirely dismiss the possibility that the observed tumor regression effects may result from the inhibition of alternative targets. Another PTH, similar to J54 but lacking DR2 antagonism, was found to exhibit antitumor activity by activating PP2A. J54 might inhibit essential kinases such as ATR, ATM, and mTOR, which were not assessed in the SCANMAX panel. In our experiments, we observed ATM activation in cells treated with BIC+THD or J54, while THD has been reported to indirectly or directly inhibit mTOR ,136. TheTogether, these findings substantiate the potential use of PTH derivatives as alternative treatments for cancer by pharmacologically targeting the TLK1/2-mediated DDR pathways. Moreover, alongside other studies, our research adds to the comprehensive understanding of the molecular mechanisms of TLKs and positions them as attractive kinase targets for therapeutic interventions against cancer.For more than 50 years, the focus of research and treatment for PCa has revolved around ADT and developing therapies that target AR through various mechanisms . HoweverInterestingly, one of the most significant findings in initial in vitro studies, later confirmed in an LNCaP xenograft model, was the combination of a specific TLK1 inhibitor called J54 with the first-generation non-steroidal antiandrogen, BIC, or the second-generation antiandrogen Enzalutamide (ENZ). When BIC alone was used, it induced a cell cycle arrest in the G1 phase in PCa cell lines LNCaP or TRAMP-C2. However, when combined with J54, this arrest was bypassed due to impairment of the TLK1-NEK1-ATR-Chk1 axis. Instead, the cells temporarily accumulated in the G2/M phase (the next possible checkpoint after DNA damage and response activation) and then underwent apoptosis (sub-G1 cells) due to a catastrophic attempt at mitosis (®) compared to ENZ (Xtandi®) in combination with J54, at least in vitro. Neither BIC nor ENZ alone induced apoptosis in LNCaP cells; instead, they induced quiescence, as commonly observed [Treatment with J54, BIC, or ENZ showed similar induction of apoptosis in androgen-sensitive (AS) cells (observed . The mecobserved . The byp®) is a more cost-effective option for PCa patients than ENZ (Xtandi®) or Apalutamide (Erleada®) in the United States. However, when considering the modest improvements in overall survival (OS) and progression-free survival (PFS) observed in ENZ-treated patients compared to those treated with BIC, these benefits seem limited to white patients only [Based on our observations, it must be pointed out that, from an economic perspective, BIC  in treating PCa with BRCAness characteristics reveals novel insights into PCa biology. Intriguingly, PARPi has demonstrated efficacy in cases with BRCAness ,147 and,In response to these insights, numerous researchers, including our team, are actively developing alternative treatment approaches involving combination therapy utilizing cisplatin and a novel DNA repair inhibitor (J54 in our case). Such an approach holds promise for circumventing conventional ADT or using antiandrogens, which often involve chemical castration and are generally undesirable for most men. Additionally, it may offer an alternative to late-stage chemotherapy relying on taxanes. Our research is centered around investigating the role of TLK1 in the DDR process, its anti-apoptotic function mediated through the NEK1-VDAC1 axis and AKT, and its influence on the activity and stabilization of YAP. Significantly, these various mechanisms converge on actionable targets that can be effectively addressed by introducing TLK inhibitors.


Aortic root dilatation, aortic valve prolapse and commissure commitment to a perimembranous VSD are associated to aortic regurgitation.Ventricular septal defects (VSD) are the most common congenital heart diseases in children. Among them, perimembranous VSD (pm-VSD) have a higher risk of complications, including aortic valve prolapse and aortic regurgitation (AR). The aim of our study was to assess echocardiographic criteria associated with AR during follow-up of pm-VSD. Forty children with restrictive pm-VSD, followed-up in our unit and who underwent a workable echocardiographic evaluation between 2015 and 2019 were included and retrospectively analyzed. The propensity score was used to match 15 patients with AR to 15 patients without AR. Median age was 2.2 year [1.4–5.7]. Median weight was 14 kg [9.9–20.3]. Aortic annulus z-score, Valsalva sinus z-score, sinotubular junction z-score, valve prolapse and commissure commitment were significantly different between the two groups ( Perimembranous VSD (pm-VSD), is deemed to be less associated with AR. In this type of VSD, the mechanism of AR seems to implicate several factors.In doubly committed juxta-arterial defects and perimembranous defects, the anatomical position of the defect frequently affects the mobility and function of the aortic leaflets, resulting in aortic incompetenceThe aim of this study was to assess echocardiographic criteria associated with aortic regurgitation in restrictive pm-VSD.Between January 2015 and January 2019, a total of 158 children with pm-VSD were admitted to our department for medical or surgical treatment.All patients < 18 year old with perimembranous VSD and for who a workable and recent (less than three months) echocardiographic evaluation was available, were considered for inclusion. Patients with non-perimembranous VSD and patients with associated congenital heart anomalies other than atrial septal defect were excluded.Direction de Recherche en Santé) and the local ethic committee (Commission d'Accès aux Données de Santé du CHU) approved the study protocol (PADS22-135). Local ethical approval was given with waiver of informed consent for retrospective, anonymized data. Sixty five patients were eligible and thirty children were included after propensity score matching Non-AR group: patient without AR (n = 15)The cohort was divided into 2 groups:Demographic, clinical and TTE data were collected from each patient’s medical record. The clinical parameters collected were those recorded on the day of the echocardiography.All the echocardiographic studies were analyzed by the same observer (FS).Images analyzing the VSD in 2D, color Doppler and continuous Doppler were reviewed. The size of the pm- VSD and the maximum flow velocity through the defect were recorded.5. The ratio between the VSD diameter measured in parasternal short axis (mm) and the aortic annulus diameter measured in parasternal long axis (mm) was calculated. The anatomy of the aortic valve (AV) was described and each leaflet size was measured.The cusp imbalance index was defined as: [width of right (R) or non- (N) coronary cusp/width of left coronary cusp (L)]6.Parasternal long axis, short axis and four chamber views of the aortic valve were analyzed. Data collection included diameter of the aortic valve annulus, the Valsalva sinus and the ascending aorta Fig. . Respect7.The coaptation height (CH) and the effective height (EH) were measured Fig. . The aorLong axis and 5 chamber views were used to assess cusp (leaflet) prolapse. Commissure commitment was assessed in short axis view Fig. .Figure 3Propensity score was performed to match pm-VSD with AR patients (AR group) with pm-VSD without AR patients (Non AR group) according to age and weight. Patients were 1:1 matched using the nearest neighbour method without replacement and with a calliper of width equal to 0.25. Table Continuous variables are expressed as mean (with standard deviation) or median (with IQR), where appropriate. Discrete or binary variables are presented as number (percent).2 test or Fisher’s exact test (if appropriate) were used for categorical variables and Mann Whitney test was used for continuous variables to assessed influencing factors Associations between echocardiographic parameters and AR were determined with univariate binary logistic regression analysis.The χp value of 5%. Statistical analysis was performed using SPSS software, version 22.0 .All p values were bilateral and significance was pronounced for a The median age was 2.2 year [1.4–5.7] and the median weight 14 kg [9.9–20.3]. There was no significant difference concerning demographic parameters between the two groups  patients had a LV z-score above + 2. In this study, 25 (83.3%) patients presents restrictive VSD.Review of previous echocardiograms of patients in the AR group shows that: two patients had a stable minimal AR. Three patients had mild to moderate AR of recent worsening and the other patients had mild AR of recent onset. In AR group, 13 (86%) patients were operated on within a year and the procedure was VSD closure only. In non AR group, 8 (53%) were operated. In the last follow up, non-operated patients are stable without AR (or AR worsening).p = 0.001).In the AR group, the size of the aortic root, especially the median Valsalva sinus size, was significantly larger than in the non-AR group .In AR group, there was significantly more AVP and commissure commitment .No significant difference was found for the AEH index, ACH index and cusp imbalance index  and AR is shown in Table Several echocardiographic parameters differed significantly between the 2 groups: , and in particular AVP have been widely studied. The incidence of AVP, reaches 73% in some studies, with progression to AR in 52 to 78% of the patients13.Complications of Several mechanisms have been are suggested to explain the association of pm-VSD and aortic valve regurgitation. Beside aortic valve prolapse, which remains the main cause, dilatation of the ascending aorta, decrease of the coaptation height and aortic cusp imbalance have been involved.14. In our study, dilatation of the ascending aorta was associated with AR.Momma et al. showed that dilation of the ascending aorta was often associated with aortic valve distortion7.We used, as recommended in their publication7, a ratio to avoid variations in relation with patient’s weight and height difference. We did not find any statistically significant difference between the two groups.Iwashima et al., suggested a decreased coaptation height as a non-invasive marker for the assessment of severity of AVP in the presence of a VSD6.In the study of Salih and al, concerning 41 consecutive patients, operated for VSD (36 pm-VSD) with prolapsed AV, with or without AR, the presence of cusp imbalance did not impact post-operative AR improvement15.Tomita et al., showed that an imbalance in the width of a cusp could predict a possible progressive worsening of ARIn our study, this parameter was not significantly different between the two groups.17. AR has a tendency to worsen as soon as it appears and may not disappear in post-operative course. Even minor prolapse can cause AR18 and eccentric AR can reveal AVP of the right coronary cusp that is very likely to progress19. In our population, AVP was found to be associated with AR.Several studies have shown that the presence of AVP should indicate regular ultrasound monitoring for AR, which appears in more than half of the cases4 proposed a differentiation of the mechanism of aortic regurgitation according to the position of defect. In infundibular defect (corresponding to Outlet defect with hypoplastic or absent muscular outlet septum3), the mechanism seems to be a lack of support of the leaflet resulting in a gradual prolapse through the defect4. In pm-VSDs, infundibular septum is conserved but the defect is potentially located in the intercommissural zone of the right coronary leaflet and non-coronary leaflet (corresponding to interleaflet triangle) and incriminates both right and non-coronary leaflet21. This hypothesis is corroborated by recent publications on the relationship of the septum with the aortic valve in the context of assessment and management of transcatheter implantation of the aortic valve22. Indeed, due to variations in rotation of aortic root, the membranous septum can have variable relationship with its components23. Membranous septum can be located below aortic annulus, it can crossed the annulus projecting into interleaflet triangle or it can projected into the noncoronary sinus deep to the aortic leaflet22.Our study is the first to show a significant association between the commissure commitment into the VSD and the AR. Azcarate et al.4. Our findings support this mechanism.In the case where the membranous septum is located into interleaflet triangle and pm-VSD, aortic incompetence is located in the commissure due to poor coaptation of the two leaflet margins (non-coronary and right coronary leaflet)-dependent and observer-dependent procedure. The operator-dependency has been limited by eliminating all non-workable and incomplete ultrasound studies. The observer-dependency has been limited by proceeding to a methodical rereading of all ultrasound examinations by one single observer.Our study has 2 main limitations. : First of all, a technical limitation since echocardiography is a highly operatorThe second limitation is statistical related to the small size of our population and the retrospective nature of the study. This work, however, provides a basis for a prospective pilot study to further investigate echocardiographic parameters association with AR.Increased Valsalva sinus size as well as aortic valve prolapse and commissural commitment are clearly implicated and associated with AR in pm-VSD. Our study reinforces the idea that anatomical variables are directly implicated in AR. We hypothesize that commissural commitment to the VSD Fig.  must be