Mycotoxins are considered the most threating natural contaminants in food. Among these mycotoxins, aflatoxin B1 (AFB1) and fumonisin B1 (FB1) are the most prominent fungal metabolites that represent high food safety risks, due to their widespread co-occurrence in several food commodities, and their profound toxic effects on humans. Considering the ethical and more humane animal research, the 3Rs  principle has been promoted in the last few years. Therefore, this review aims to summarize the research studies conducted up to date on the toxicological effects that AFB1 and FB1 can induce on human health, through the examination of a selected number of in vitro studies. Although the impact of both toxins, as well as their combination, were investigated in different cell lines, the majority of the work was carried out in hepatic cell lines, especially HepG2, owing to the contaminants’ liver toxicity. In all the reviewed studies, AFB1 and FB1 could invoke, after short-term exposure, cell apoptosis, by inducing several pathways . Among these pathways, mitochondria are the primary target of both toxins. The interaction of AFB1 and FB1, whether additive, synergistic, or antagonistic, depends to great extent on FB1/AFB1 ratio. However, it is generally manifested synergistically, via the induction of oxidative stress and mitochondria dysfunction, through the expression of the Bcl-2 family and p53 proteins. Therefore, AFB1 and FB1 mixture may enhance more in vitro toxic effects, and carry a higher significant risk factor, than the individual presence of each toxin. Aspergillus, Fusarium, Penicillium, and Claviceps purpurea genera [Mycotoxin contamination in food represents serious threats toward public health . Mycotoxa genera . Among ta genera ,5.Aspergillus flavus and Aspergillus parasiticus species, and it contaminates different crops, such as nuts, dried fruits, oilseeds, and maize and other cereals. Since the discovery of AFB1 in 1960, after the famous incidence where it killed 100,000 young turkeys in the UK, which was called, at that time, Turkey X disease, several fatal outbreaks have been associated with the consumption of AFB1-contaminated food, as reported in India (the states of Gujrat and Rajasthan in 1974) and in Kenya  and corn-based foods, but also in other cereals, such rice, oat, rye, barley, and wheat [FB1, a sphingosine analogue compound , was thend wheat ,13, and nd wheat . The IARnd wheat . It was nd wheat ,17. The nd wheat . Recentlnd wheat ,18.Both AFB1 and FB1 can co-occur in a variety of agricultural commodities, especially maize ,20. TherThe toxicities of AFB1 and FB1 have been studied by many scientists in laboratory animals, as well as in in vitro cell lines and models. However, considering ethical and more humane animal research, the 3Rs  principle has been implemented by international legislation and regulations. The main objective of the 3Rs is to change traditional animal testing practices, in order to minimize animal testing as much as possible . In addiMost of the available toxicological knowledge on aflatoxins is related to AFB1. Once it is absorbed by the small intestine, AFB1 is metabolized in hepatic cells by cytochrome CYP450s enzymes, predominantly liver-localized enzymes, to the ultimate carcinogen AFB1-exo-8,9-epoxide . This inn = 14) investigated the effect of FB1 in liver and intestine in which HepG2 and Caco-2 cells were the in vitro models of choice, accounting for 100% and 60%, respectively.FB1 is a water-soluble molecule, and typically has a low bioavailability (3–6%). It is rapidly distributed in liver and kidney, extensively biotransformed, and rapidly excreted, mostly in feces . It is rAround 57% of the presented 14 studies indicated that FB1 toxicity was related to the biosynthesis of sphingolipids, which are fundamental components of eukaryotic cells . In addiThe combined exposure to AFB1 and FB1 is of concern to public health. It has been reported that a synergistic interaction between AFB1 and FB1 is present via the induction of cell apoptosis ,92. Du eAs human exposure to AFB1 and FB1 results in several serious toxicological effects, mitigating both mycotoxins is a prerequisite. Several compounds with antioxidant properties, food components, and medicinal herbs and plant extracts have been proposed based on their potential efficacious effects to alleviate AFB1 and/or FB1 toxicity in vitro. As shown in Amaranthus hybridus, Resveratrol, and Momordica charantia have been reported the mitigation capability of both toxicity in different ways  extract was reported a protective effect against AFB1 and FB1 that induced cytotoxicity and DNA damage and induced genotoxicity in hepatoma cells [In fact, some food components not only keep the body’s systems functioning properly, but also mitigate the toxic effects of mycotoxins including AFB1 and FB1. Among these components, ent ways . Amarantma cells ,107. Resma cells . Besidesma cells .Medicinal herbs and plants also contain many natural components that were used in the prevention, treatment, diagnosis, rehabilitation and health care of diseases, including the capability to counteract the AFB1 and FB1 toxicity . NaturalIn the last few decades, several efforts have been made to minimize exposure to mycotoxins, especially AFB1 and FB1, from food. Despite all the attempts to control their content in food, the research has not fully succeeded in solving this major problem. In addition, several underlying toxic mechanisms have not been completely unraveled. Therefore, there is a need to deeply investigate their toxicities by implementing state-of-the art methodology, such as Omic technologies. The review shows that AFB1 and FB1 could invoke, after short-term exposure, cell apoptosis, by inducing several pathways  in different cell models (mainly in HepG2 cells and Caco-2 cells). The combination of AFB1 and FB1 is mainly manifested as a synergistic effect, and their interaction is mainly related to the FB1/AFB1 ratio and the organs. However, the in vitro toxicity work was performed using two-dimensional (2D) models, or the cell monolayer. More advanced three-dimensional (3D) in vitro models, such as organoids and spheroids, which exhibit features that are closer to the complex in vivo conditions, have not been adequately used in mycotoxin field. The 3D culture models have proven to be more realistic for translating the study findings for in vivo applications. To better understand the toxicity of AFB1 and FB1 in vitro, the use of 3D models should be increased, to study various aspects of cell physiology and pathology in the future. Additionally, investigations on the effects of long-term exposure to low doses of AFB1 and FB1 should receive more attention, as humans are more likely to be exposed to low doses on a daily basis from food. Finally, due to insufficient data, the mechanisms of interaction still need to be elucidated. In the future, more combined ratios of AFB1 and FB1, and more pathways proposed and target proteins for their combined toxicity, should be observed, to support this synergistic interaction between AFB1 and FB1. Moreover, focusing on the numerous organ models for their combined toxicity would fill the knowledge gaps around the currently uncertain hazards for human health.


Electroacupuncture has been used as a treatment; however, a visual bibliometric analysis has not yet been performed in this field. In this study, we aimed to suggest future research topics and directions related to the field by examining the last 20 years of research trends and hotspots of electroacupuncture in stroke.We searched the Web of Science database on electroacupuncture as a treatment for stroke published from 2003 to 2022. We analyzed the papers by annual publication, research fields, nations, affiliations, authors, journals, and keywords. VOSviewer software was used to visualize the bibliometric analysis and results. A total of 440 papers were included in the analysis.The number of publications has gradually increased every year, and neuroscience has become the most actively studied field. Neural Regeneration Research journal and China had the most publications. Fujian University of Traditional Chinese Medicine, as an affiliated institute, published the most articles. Chen Lidian and Tao Jing presented the largest number of papers, making them the leading contributors in this field. Four clusters were created by analyzing keywords, such as “neuroprotection,” “clinical rehabilitation,” “neuroplasticity,” and “pretreatment-induced tolerance”.This study is the first to analyze the research trends in electroacupuncture as a treatment for stroke using the VOSviewer. It shows the current state of research in the field by visualizing research trends and hotspots. This will help offer reference data for future studies. Despite recent improvements in medicine, stroke is one of the leading causes of mortality worldwide . AdditioTraditionally in East Asia, Traditional Chinese Medicine (TCM) has been used for stroke care . In the The bibliometric analysis method uses mathematical and statistical tools, thereby providing an overview of academic research publications and a quantitative analysis of research trends. This method can identify influential publications, authors, journals, affiliations, and countries and analyze and present topics, methods, and influential keywords . There a2.2.1.All data were extracted from WOSCC. Research papers published between January 01, 2003, and December 31, 2022, were collected, and data was extracted from articles published between February 20, 2023, and February 27, 2023, based on WOSCC using TS:  as search queries. The results were then converted into text files and organized for further data analysis on March 01, 2023.A total of 661 papers were extracted during the first data search. Articles and reviews were excluded, and 636 works of literature were selected. Two independent researchers screened the remaining papers by examining each title and abstract. Criteria of inclusion were: (1) Original article (2) Time range: published from 2003 to 2022 (3) research regarding EA as treatment for stroke. Criteria of exclusion were: (1) research regarding encephalopathy other than stroke  (2) EA intervention without penetration of skin  (3) article without abstract, book chapters, and editorial meetings (4) retracted or overlapped research. Papers with incomplete text were also excluded, while those with a full text were reviewed. In total, 440 papers were selected for this study .Keywords were unified into a single form to enhance the accuracy of our analysis. Full names were used in the author analysis to enhance accuracy . The affiliation name was unified .2.2.The screened publications were categorized and analyzed according to the publication year, nation, journal, research institution, author, and affiliation. Data on nation, affiliation, keywords, and authors were analyzed using VOSviewer to visualize the links between categories. Research areas were categorized using the WOSCC algorithm, which considers the characteristics of the journal. Each article was sorted into one to six research areas . To visu3.3.1.In 2003, four papers were published, but in 2022, 53 papers were published. This result shows that the number of publications has increased from 27 in the year 2019 to 55 in 2020. The number of papers published doubled, and highest number of publications, 55, were in 2020, constituting 12.5% of all papers over 20 years .3.2.Most papers were published in the field of neuroscience (36.6%), followed by Complementary Medicine (26.8%), Cell Biology (14.5%), and Medicine Research Experimental (14.31%) . Among p3.3.Among the 440 papers, Neural Regeneration Research had the highest number of publications (10.9%). Evidence-Based Complementary and Alternative Medicine had the second most published papers (9.5%), followed by Acupuncture in Medicine (4.1%) and Medicine (3.2%) .3.4.China published 374 papers, accounting for 85% of the 440 papers, and published the most number of research articles. The following nations were South Korea (8.4%), the United States (6.1%), and Taiwan (4.1%) .First, countries that published more than five papers were analyzed using the VOSviewer program, and three clusters were defined. The first cluster comprises China and Australia, the second cluster comprises USA and Taiwan, and third cluster includes South Korea .Subsequently, research papers from each country were distinguished based on the average publication year. As the average publication year increased, nodes were visualized in blue, and as they became more recent, they were visualized in red .Finally, the average citation count of the publications from each country was analyzed. Fewer cited country nodes were visualized in blue and more cited nodes were visualized in red .3.5.The analysis by affiliation showed that Fujian University of Traditional Chinese Medicine produced the highest number of studies (9.5%), followed by Guangzhou University of Chinese Medicine (7.7%), Air Force Military Medical University (5.9%), Tianjin University of Traditional Chinese Medicine (5.2%), and Fudan University (4.8%) .A total of 43 institutions published more than five research papers, which were categorized into nine clusters using the VOSviewer program . ClusterSecond, the published research papers of each affiliation were distinguished based on the average publication year. As the average publication year increased, it was visualized in blue; if it became more recent, it was visualized in yellow . Anhui UFinally, the average number of citations of publications from each affiliation was analyzed. The more blue a node, the less frequently it is cited. The more yellow a node, the more frequently it was cited . The leaAnalysis by cluster showed that Cluster 5 had the earliest average publication year, and Cluster 8 had the latest. Cluster 9 had the largest average number of citations, and Cluster 3 was the least cited .3.6.The authors’ analysis revealed that Chen Lidian (7.7%) and Tao Jing (from Fujian University) published the most papers, followed by Liu Weilin (4.5%), Huang Jia (4.3%), and Xiong Lize (3.9%) from Tongji University .First, those who published more than five papers (65 authors) were analyzed using the VOSviewer program, and 18 clusters were defined. Cluster 1 included 12 authors: Chen Lidian, Tao Jing, and etc. Eight authors, including Xiong Lize and Wang Qiang, were categorized in Cluster 2. Cluster 3 included five authors: Lin Ruhui, Chen Bin, and etc. In Cluster 4, there were five authors: Xu Nenggui, Yi Wei, and etc. Further clusters are visualized in The research papers published by each author were then distinguished by average publication year. As the average publication year increased, the authors cluster turned blue. The more recent the average publication year, the more yellow the author cluster . Cheng aFinally, the average number of publications by each author was determined. Blue indicates that the average number of citations is small. As the node color approached yellow, the average number of citations increased . Chen anAnalysis of each cluster showed that Cluster 12 had the earliest average publication year (2009.02) and Cluster 18 had the most recent average publication year (2019.06). Cluster 16 had the lowest average number of citations (3.9) and Cluster 7 had the highest average number of citations (32.62) .3.7.First, 1,616 keywords were mentioned in 440 papers, and 52 keywords that were found more than 15 times were analyzed. Four keyword clusters were defined using the VOSviewer .The keywords were categorized according to the average publication year. As the average publication year increases, the results were visualized in blue. Because the average publication year was recent, it is visualized in yellow . AnalysiFinally, the keywords were sorted by the average number of citations. The smaller the average number of citations, the closer the color is to blue. The larger the average citation, the closer it is to yellow . The resComparison analysis between each keyword cluster showed that keywords from Cluster 4 had the oldest average publication year but were the most cited. Keywords from the second cluster were the most recent but were the least cited .4.To conduct a bibliometric analysis of EA in patients with stroke, 440 papers published over the last 20 years were reviewed. The number of publications has gradually increased annually. In the first 10 years, starting from 2003, 89 papers were published, and in the last 10 years, 351 papers were published. By 2020, more than 45 papers had been published annually. We believe that this is because EA has been extensively studied and is actively used clinically to treat stroke.The field of Neurosciences had the most research, followed by the fields of Integrative Complementary Medicine, and Cell Biology. In the field of Neuroscience, research focusing on this mechanism has been actively conducted because of objective and quantitative measurements of the frequency and strength of EA stimulation .Neural Regeneration Research journal had the most numerous publications, followed by Evidence-Based Complementary and Alternative Medicine, and Acupuncture in Medicine. The multiplication of studies in neuroscience is reflected by the published journals. EA treatment is a complementary medical method that has been actively studied.China published the highest number of studies, followed by Korea and the United StatesIt appears that this is proportionally related to the fact that China is the center of worldwide TCM research. Analysis of clusters showed China and Australia, which recently conducted research in this field actively, was included in the same cluster. Australia conducted studies on the use of EA for the sequelae of stroke. In the early stages, the United States and Taiwan conducted highly cited and influential studies. United States has the highest medical expenditure of post stroke care per patient in the world , and in Keyword analysis was performed to identify the research trends and contents of the published papers. The three oldest keywords by average publication year were “neural regeneration, cerebral ischemia, and artery occlusion.” In a previous study, an animal model of ischemic stroke created through the middle cerebral artery and internal carotid artery occlusion and reperfusion was used to verify the nerve regeneration effect of EA . The thrThe published research keyword clusters were categorized according to “neuroprotection,” “clinical rehabilitation,” “neuroplasticity,” and “pretreatment-induced tolerance.” The first cluster included “rat,” “apoptosis,” and “mechanism” and was categorized as a “neuroprotection” topic. Neuroprotection focuses on minimizing additional damage of nerve cells after acute brain injury  and EA sComparing through average publish year, cluster 4 “EA pretreatment induced rapid tolerance” studies were conducted in the past than the other clusters and also showed high average citation. Cluster 2, a “clinical rehabilitation study,” had a more recent average publication year, and the average citation rate was lower than that of the other clusters. Overall, animal model studies have mainly been conducted in the past, and we found that human-subject clinical studies have recently increased in animal experiments.This study has several limitations. First, only the WOSCC was used as the extraction source for the research papers. WOSCC was used in this study because the database provides high quality and globally influential research papers and details of the research papers needed for bibliometric analysis. If other databases from East Asian countries that were actively studying the field were included as research sources, the results would have been more significant, but this study focus on database which is used worldwide. Second, the number and name of each cluster were discussed between two individual researchers and defined because individual subjectivity can intervene in the process of defining its properties, hindering objectivity; however, researchers may have different views. Considering the research trend on EA about stroke, mechanism of EA on neuroplasticity and its consequent motor recovery might be of interest of future research. Despite major advances in understanding pathophysiology and acute phase stroke intervention, not much progress has been made especially in chronic phase. Because timely intervention is crucial in stroke rehabilitation, EA can be a relatively convenient and cost-efficient intervention especially where Conventionally therapy is not readily available. To achieve this, large scale, high-quality, multicenter or multinational clinical trials must be further conducted to adequately measure the proper effect size. Additionally, analysis of the research trends in various TCM treatment methods, such as manual acupuncture, herbal medicine, and pharmacopucture for stroke could be conducted to provide insight into stroke treatment and rehabilitation.5.Using WOSCC as a source, the research trends of 440 papers on EA as a treatment for stroke were analyzed using a bibliometric method, and the results were as follows:In the last 20 years, published studies have gradually increased, and especially between 2019 and 2020, the number has more than doubled, indicating that research has been actively conducted until recently.Studies have been conducted in the fields of neuroscience and complementary medicine.China had the largest number of publications, followed by the United States and other East Asian countries, such as South Korea and Taiwan.Fujian University of Traditional Chinese Medicine published the most research papers, whereas Chen Lidian and Tan Jing published the largest number of studies.The keyword analysis indicated topics related to “neuroprotection,” “clinical rehabilitation,” “neuroplasticity,” and “pretreatment-induced tolerance.”The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author.HC: Conceptualization, Data curation, Formal analysis, Investigation, Writing – original draft. W-CS: Resources, Writing – review & editing. J-mK: Investigation, Writing – original draft. HK: Methodology, Software, Writing – review & editing. J-HC: Supervision, Validation, Writing – review & editing. M-YS: Supervision, Validation, Writing – review & editing. W-SC: Conceptualization, Project administration, Supervision, Writing – review & editing.


This study aimed to investigate the value of a machine learning-based magnetic resonance imaging (MRI) radiomics model in predicting the risk of recurrence within 1 year following an acute ischemic stroke (AIS).The MRI and clinical data of 612 patients diagnosed with AIS at the Second Affiliated Hospital of Nanchang University from March 1, 2019, to March 5, 2021, were obtained. The patients were divided into recurrence and non-recurrence groups according to whether they had a recurrent stroke within 1 year after discharge. Randomized splitting was used to divide the data into training and validation sets using a ratio of 7:3. Two radiologists used the 3D-slicer software to label the lesions on brain diffusion-weighted (DWI) MRI sequences. Radiomics features were extracted from the annotated images using the pyradiomics software package, and the features were filtered using the Least Absolute Shrinkage and Selection Operator (LASSO) regression analysis. Four machine learning algorithms, logistic regression (LR), Support Vector Classification (SVC), LightGBM, and Random forest (RF), were used to construct a recurrence prediction model. For each algorithm, three models were constructed based on the MRI radiomics features, clinical features, and combined MRI radiomics and clinical features. The sensitivity, specificity, and area under the receiver operating characteristic (ROC) curve (AUC) were used to compare the predictive efficacy of the models.Twenty features were selected from 1,037 radiomics features extracted from DWI images. The LightGBM model based on data with three different features achieved the best prediction accuracy from all 4 models in the validation set. The LightGBM model based solely on radiomics features achieved a sensitivity, specificity, and AUC of 0.65, 0.671, and 0.647, respectively, and the model based on clinical data achieved a sensitivity, specificity, and AUC of 0.7, 0.799, 0.735, respectively. The sensitivity, specificity, and AUC of the LightGBM model base on both radiomics and clinical features achieved the best performance with a sensitivity, specificity, and AUC of 0.85, 0.805, 0.789, respectively.The ischemic stroke recurrence prediction model based on LightGBM achieved the best prediction of recurrence within 1 year following an AIS. The combination of MRI radiomics features and clinical data improved the prediction performance of the model. Stroke is a major chronic non-communicable disease that poses a serious health risk to the population. The disease is characterized by high morbidity, disability, mortality, recurrence, and economic burden. According to the global burden of disease (GBD), the lifetime risk of stroke in China is 39.3% for people over 25 years of age . The 1-yCurrently, AIS are diagnosed using computed tomography (CT) and MRI . MRI seqTherefore in this study, we aimed to use DWI radiomics and clinical features to develop different machine-learning algorithms for predicting recurrence within 1 year following an AIS. The optimal predictive model could be used to facilitate the early diagnosis and treatment of AIS and hence improve survival while reducing the disease burden on the patients, carers, and society.This study was evaluated and granted by the Medical Research Ethics Committee of the Second Affiliated Hospital of Nanchang University (2018-05). Patients with MRI-diagnosed AIS were recruited from the Second Affiliated Hospital of Nanchang University from March 1, 2019, to March 5, 2021. All patients aged between 18–85 years who had DWI imaging within 48 h of admission for AIS and a National Institute of Health stroke scale (NIHSS) score of 15 or less were included in the study. Cancer patients and those with severe cardiac, pulmonary, and hepatic system diseases were excluded. In addition, the patients with major artifacts on the DWI were also excluded. A total of 612 patients were eventually included, all were followed up for 1 year to track whether the patients experienced a recurrence of stroke. The patients were divided into training and validation sets using a 7:3 randomized grouping ratio.The patient’s demographic characteristics (age and gender), history of previous diseases , and alcohol and smoking history were extracted from the clinical medical records. In addition, the physical examination data  and laboratory test results  were also extracted. Finally, the DWIs and the recurrence outcome variable within 1 year after hospital discharge were also collected.b-value of 0, 1,000 s/mm2, and a duration of 30 s.All patients underwent a DWI brain imaging within 48 h of admission. All images were acquired on a GE 3.0 T MRI scanner, using a repetition time (TR) of 4,090 ms, an echo time (TE) of 98 ms, a flip angle of 180°, a scan field of view of 230 mm × 230 mm, a matrix of 192 × 192, 20 layers of a thickness of 6 mm and layer spacing of 1.3 mm, a 1 to annotate the ischemic lesions on the DWI, respectively. The MDice, MIOU, and Hausdorff metrics were used to verify the consistency of the annotations. The consistency of the annotated volumes was evaluated using the Bland–Altman method and the intraclass correlation coefficient (ICC). If the ICC was less than 0.75, a third imaging specialist with a senior title was asked to label and verify the annotation. Subsequently, the first-order, morphological, and texture radiomics features were extracted from the annotated DWI images using the Pyradiomics package (Two radiologists with 5 years of experience used the 3D slicer software (Version 4.13.0) package . The firThe Pearson intergroup correlation coefficient was used for feature redundancy analysis. Features with correlation coefficients greater than 0.9 were excluded. The optimal radiomics features for the prediction of AIS were screened using LASSO regression with 10-fold cross-validation.Four machine learning algorithm models, including LR, SVC, LightGBM, and RF, were used to develop the prediction model for recurrent AIS. For each algorithm, three prediction models were constructed using the radiomics features, the clinical features, and the radiomics features combined with the clinical features. Training model using 10-fold cross-validation. The sensitivity, specificity, receiver operating characteristic (ROC) curve, and area under the curve (AUC) were used to compare the performance of the models. The clinical application value of the prediction models was evaluated using a calibration curve and a decision curve analysis (DCA).t-test. Conversely, the non-normally distributed data were described using median and upper and lower quartiles [M ], and comparisons between groups were made using the Mann–Whitney U test. For all statistical tests, a p-value below 0.05 was deemed statistically significant.The statistical package for social science (SPSS) software version 22.0 was used to analyze the data. The categorical data were summarized as frequency (%), and Fisher’s exact test was used to evaluate the differences between the recurrent AIS group and the non-recurrent AIS group. The continuous data were tested for normality using Kolmogorov–Smirnov test. The normally distributed data were summarized as mean ± standard deviation (x ± S), and the differences between the two groups were compared using the P < 0.05), as shown in A total of 612 patients, including 337 males and 275 females, were enrolled in this study. The average age of the patients was 63.9 ± 11.16 years. 67 (10.95%) patients were found to have had a recurrence of stroke at the 1-year follow-up. Univariate analysis showed that smoking history, ischemic heart disease history, stroke history, alkaline phosphatase, creatinine, prothrombin time, fibrinogen concentration, age, absolute neutrophil value, total protein, albumin, white blood cell count, and international labeling ratio were associated with stroke recurrence (n = 592) of the cases. The delineated volume was outside the limits of agreement (mean ± 1.96 SD) in 3.3% (n = 20) of cases. The mean ICC between the lesion volumes annotated by the two radiologists was 0.99 , indicating a good agreement.The mean MDice, MIOU, and Hausdorff distances for the annotations made by the two radiologists were 0.87, 0.77, and 4.36 mm, respectively. These results indicate a good agreement between the observers. A total of 1,037 radiomics features were extracted, including 198 histogram features, 14 morphological factor features, and 825 texture features. The texture features included 264 grayscale co-generation matrices (GLCM), 176 grayscale tour matrices (GLRLM), 176 grayscale size region matrices (GLSZM), 154 grayscale dependence matrices (GLDM), and 55 neighborhood grayscale difference matrices (NGTGM). The feature redundancy analysis  resulted2The interpretation of the imaging histology features can be retrieved from.The training and validation datasets consisted of 428 and 184 patients, respectively. In the validation set, the prediction accuracy of the 4 machine learning models for the radiomics features, clinical features, and the radiomics and clinical features combined are summarized in The calibration curves showed that all four models had a high calibration. The LightGBM model has the best discrimination. The specific performance is shown in The LightGBM algorithm achieved the best performance. The top 20 influencing factors in the LightGBM model are shown in Recurrence following an AIS leads to poor survival and quality of life. As a result, there is a need to develop models to predict the onset of AIS and hence minimize the morbidity and mortality of this disease. In this study, we developed four machine learning algorithms  to predict recurrence following an AIS. Each algorithm was trained using clinical treatment data, MRI radiomics data, and combined clinical and MRI radiomics data.The DWI sequence in MRI reflects the random Brownian motion of water molecules in the tissue. AIS causes water molecules to move from outside the cell to the inside of the cells. The DWI signal increases as the extracellular water volume decreases . ChangesPrevious studies have extracted radiomics features from FLAIR and ADC, which can significantly improve the predictive performance of functional outcomes in AIS patients compared to clinical features only . Ramos eIn our study, the LightGBM model achieved the best prediction performance. LightGBM is a fast, distributed, high-performance gradient-boosting framework based on a decision tree algorithm . CompareThe LightGBM algorithm identified 10 important radiomics features for the prediction of recurrence in AIS. The imaging risk factors showed that higher stroke voxel differences and higher voxel kurtosis indicate a more severe disease and an increased risk of developing recurrent AIS. In addition, a lower texture characteristic skewness coefficient, lower HighGrayLevelZoneEmphasis, and higher Gray Level Non-Uniformity also increased the risk of recurrence.Several clinical factors can influence the risk of developing recurrence after an AIS. In this study, we found that platelet large cell ratio (PLCR), smoking history, age, fibrinogen concentration, hemoglobin, albumin, creatinine, white blood cell count, absolute neutrophil value, and discharge the modified Rankin scale (mRS) score as important risk factors for the development of recurrence. Previous studies  also ideThis study has some limitations that have to be acknowledged. All the patients in this study were recruited from a single center, potentially introducing selection bias. Therefore larger multicenter studies are required to validate the model. The radiomics model was trained on 2-dimensional (2D) images instead of volumetric images. As a result, useful radiomics features between the 3D layers may have been omitted. Previous studies have shown that the 3D stereoscopic shape of AIS lesions could be used to predict recurrence . Future The LightGBM model based on radiomics features extracted from DWI and clinical data achieved the best performance for predicting recurrence following an AIS. This model could provide a non-invasive tool for clinicians to assess the risk of recurrence following an AIS and hence improve the monitoring of high-risk patients. However, although this model was trained and validated on a relatively large dataset, further multicenter studies are required to validate the performance of this model.yyp66@126.com.The datasets presented in this article are not readily available because raw data involving sensitive personal information. Requests to access the datasets should be directed to YY, The studies involving human participants were reviewed and approved by the Ethics Committee of the Second Affiliated Hospital of Nanchang University. The patients/participants provided their written informed consent to participate in this study.JLiu and YW carried out the research, drafted the original manuscript, analyzed the data, and built the prediction models. YW, WJ, MH, YC, JLi, BW, SY, XZ, and JC collected the data. PY and HL provided technical support. JT, FZ, and XC provide clinical guidance. YY contributed to the design of the experiment and revised the manuscript. All authors contributed to the article and approved the submitted version.


Vitis vinifera L.). Caused by the oomycete Plasmopara viticola (Berk. and Curt.) Berl. and de Toni, it can appear anywhere where vines are cultivated. It is habitually controlled by the application of phytosanitary agents (copper-based or systemic) at different stages of the vine growth cycle. This, however, is costly, can lead to reduced yields, has a considerable environmental impact, and its overuse close to harvest can cause fermentation problems. All grapevines are susceptible to this disease, although the degree of susceptibility differs between varieties. Market demands and European legislation on viticulture and the use of phytosanitary agents (art. 14 of Directive 128/2009/EC) now make it important to know the sensitivity of all available varieties, including minority varieties. Such knowledge allows for a more appropriate use of phytosanitary agents, fosters the commercial use of these varieties and thus increases the offer of wines associated with different terroirs, and helps identify material for use in crop improvement programmes via crossing or genetic transformation, etc. Over 2020–2021, the susceptibility to P. viticola of 63 minority vine varieties from different regions of Spain was examined in the laboratory using the leaf disc technique. Some 87% of these varieties were highly susceptible and 11% moderately susceptible; just 2% showed low susceptibility. The least susceptible of all was the variety Morate . Those showing intermediate susceptibility included the varieties Sanguina , Planta Mula , Rayada Melonera , Zamarrica , Cariñena Roja , Mandrègue  and Bastardo Blanco . The highly susceptible varieties could be differentiated into three subgroups depending on sporulation severity and density.Downy mildew is one of the most destructive diseases affecting grapevines ( Vitis vinifera L.) [Plasmopara viticola (Berk. and Curt.) Berl. and de Toni. Its control largely relies on the use of phytosanitary agents. Copper-based contact-acting products or systemic agents are commonly applied at different times during the vine growth cycle. Indeed, successful modern viticulture is dependent on the repeated application of large quantities of fungicides. Their use in Europe is particularly heavy. Not only does this have a negative environmental impact, it reduces the profitability of viticulture compared to the raising of other crops, calling into question the sustainability of wine production . The continued (and excessive) use of fungicides could also provoke the development of resistance to them, and the appearance of more aggressive, more virulent ‘races’ of the causal pathogen.Downy mildew is, on a global level, one of the diseases that most affects grapevines (fera L.) . It is cterroirs. Material that could be used in crop improvement programmes  designed to produce plants resistant to disease, might also be identified. Such programmes, however, have so far only involved wild American  and Far Eastern  species. These can confer partial or even total resistance to downy mildew (such is the case of V. rotundifolia and V. piasezkii) [Muscadinia rotundifolia Michaux, Rpv3 and Rpv19 in Vitis rupestris Scheele, Rpv4, Rpv7, Rpv11, Rpv17, Rpv18, Rpv20 and Rpv21 in unspecified American species, and Rpv5, Rpv6, Rpv9 and Rpv13 in V. riparia. Recently, three loci—Rpv29, Rpv30 and Rpv31—have been identified in V. vinifera (Georgian germplasm) that confer resistance.As far as we know, all grapevine varieties are susceptible to downy mildew, although not all to the same degree ; differeasezkii) ,15. To dasezkii) ,18,19,20The aim of the present work was to determine the susceptibility to downy mildew of minority, pre-phylloxera vine varieties from different regions of Spain.V. berlandieri cv. Rességuier n°2 × V. rupestris cv. Martin) and SO4  were planted to provide controls.Sixty-three minority varieties from 13 regions of Spain  were stuPlasmopara viticola for inoculations was obtained from natural infections of vines at the MBG-CSIC research vineyard, following the method of Rumbolz et al. [z et al. .P. viticola using a 50 μL of suspension of sporangia  and left to incubate for six days . On the 6th day, each disc was visually inspected for signs of infection, measuring sporulation incidence (percentage of discs showing sporulation), sporulation severity (surface area affected by sporulation), and sporulation density (concentration of sporangia). The latter two variables were quantified using a visual scale . Inoculations were performed twice, once in June and once in July, using the material produced in the two rounds of bud break (see above).The leaf disc technique described by Rumbolz et al.  and Staup < 0.05, 0.01 and 0.001). The F test was then used to compare each fixed factor against its error. The means of those variables that returned a significant F value were then subjected to Fisher’s least significant difference (LSD) test. Principal components analysis was used to confirm the existence of groups of varieties with different levels of susceptibility. All analyses were made using SAS System v.8.1 software [Differences in the recorded variables between the varieties were analysed by two-way ANOVA (significance was recorded at software .p < 0.01) among the varieties and years for all three variables. The interaction variety × year had no significant impact on the variables recorded; thus, the plants of each variety behaved similarly in both years. The control varieties (110-Richter and SO4) showed resistance to the disease, returning sporulation incidence values of <15% and sporulation density values of <20%, confirming what was reported by other authors [4 being the more resistant of the two —the least susceptible, with sporulation incidence, severity and density values all <40%. The second comprises the varieties showing moderate susceptibility ; within this latter group, the varieties Sanguina , Planta Mula , Rayada Melonera , Zamarrica , Cariñena Roja , Mandrègue  and Bastardo Blanco  were the most resistant. Note that the last variety clustered with this group despite returning a sporulation density of 59% , sporulation severity and density had the greatest weight, while in the second component (Prin 2) the incidence of sporulation had the greatest weight . With rey of 59% . Finallyy of 59% .Subgroup A: varieties with an incidence value of >75% plus severity and density values of >50%, thus including Riera 2, Albana and Trobat Negre , Rufete Serrano, Hebén and Zurieles , Albillo del Pozo, Zurieles and Terriza , Diega 1 and Tortozona Tinta , Tinto Jeromo and Gajo Arroba , Jarrosuelto and Tortozona Tinta , Rayada Melonera and Indiana , and Forcallat and Planta Nova .Subgroup B: varieties with an incidence value of >75%, plus a severity value of <50%, but a density value of >50%, thus including Sanguina and Riera 46 , Santa Fe, Castellana Blanca, Jarrosuelto and Diega 2 , Montonera del Casar, Jarrosuelto and Maquías , Albarín Tinto and Xafardán , Tortozona Tinta and Cagarrizo , Greta and Santa Fe , Rufete Serrano and Estaladiña , Corchera , Arcos , and Giro Negre .Subgroup C: varieties with an incidence value of >75%, plus severity and density values of <50%, thus including Moscatel de Grano Menudo, Castellana Blanca, Tinto Fragoso, Tortozona Tinta and Tinto Jeromo , Ratiño, Albariño Tinto and Albillo do Avia , Cenicienta , Evena 1 , Cagarrizo , Albana , Terriza, Hebén and Castellana Blanca , and Riera 43 .The present results thus show that the majority of the tested varieties were either highly or moderately susceptible to downy mildew, although it should be noted that the severity of sporulation  was often <50% . Thus, oErysiphe necator) varying between low and moderate depending on the material’s origin. This phenomenon might be explained by clonal differences, agronomic- or climate-linked factors, etc. Some authors attribute host–pathogen interactions to the adaptation of plants to the damage caused to the former by the latter [Plants of the variety Rayada Melonera from the IFAPA were more susceptible  compared to those from the IMIDRA. Similarly, Hebén material from Extremadura was more susceptible than the same from the IMIDRA, and significant differences were seen in terms of incidence between Castellana Blanca material from the IVICAM and both the IMIDRA and EVENA. In a further difference between these latter materials, the plants from the EVENA returned significantly higher sporulation density values. Other authors  have repe latter . Thus, pBotrytis cinerea [The clustering of the present varieties in terms of their susceptibility was independent of berry colour. It might be thought that red varieties, with their higher contents in phenolic compounds such as resveratrol ,35, migh cinerea ,38. Howe cinerea .P. viticola and agronomic or ampelographic factors such as earliness or the density of reclining or erect hairs (which provide a barrier to zoospores trying to enter the plant via the stomata) (at press). Other reports [Colleagues working in tandem within the same major project (RTI 2018-101085-RC32—see Acknowledgements) detected no relationship between susceptibility to  reports ,40 also Importantly, 2% of the tested varieties—in particular Morate—showed low susceptibility to the pathogen. If cultivated, they might require fewer fungicide treatments than other varieties, reducing costs as well as the environmental impact associated with the use of these agents. They might also be good material for the production of resistant varieties. Their cultivation would not infringe current viticultural legislation, and might improve the selection of wines available to consumers.


Background (1): Football is the most popular sport among men, associated with a certain risk of injury, which leads to short- and long-term health consequences. While the injury profile of professional footballers is known, little is known about the injury profile of amateur footballers; amateur football is a major and diverse area, the development of which should be a priority for football associations around the world and UEFA. The aim of this study was to perform a systematic review of epidemiological literature data on injuries in professional and amateur football players belonging to certain leagues. Methods (2): A systematic review according to the PRISMA guidelines was performed until June 2023 in the databases PubMed, Web of Science, Google Academic, Google Scholar, and Diva portal. Forty-six studies reporting injury incidence in professional and amateur men’s football were selected and analyzed. Two reviewers independently extracted data and assessed study quality using an adapted version of the Strengthening the Reporting of Observational Studies in Epidemiology (STROBE) statement and the Newcastle Ottawa Scale (NOS) to assess risk of bias for the quality of external validity. Results (3): The overall incidence of injuries in professional male football players was 7.75 ± 2.28, 95% confidence interval, injuries/1000 h of exposure and that of amateur football players was 7.98 ± 2.95, 95% confidence interval, injuries/1000 h of exposure. The incidence of match injuries  was 7.71 times higher than the training injury incidence rate  in professional football players and 5.45 times higher in amateurs . Aggregate lower extremity injuries had the highest prevalence in both categories of footballers, being 83.32 ± 4.85% in professional footballers and 80.4 ± 7.04% in amateur footballers: thigh, ankle, and knee injuries predominated. Conclusions (4): Professional and amateur football players are at substantial risk of injury, especially during matches that require the highest level of performance. Injury rates have implications for players, coaches, and sports medicine practitioners. Therefore, information on football injuries can help develop personalized injury risk mitigation strategies that could make football safer for both categories of football players. The current findings have implications for the management, monitoring, and design of training, competition, injury prevention, especially severe injury, and education programs for amateur football players. Football is a team sport played between two teams. Currently, football is played by about 270 million players from over 200 countries, which makes it the most popular sport in the world ,2,3. AccInjuries to adult football players during both training and matches are widely documented, although methodological inconsistencies make comparisons difficult.The incidence, pattern, and characteristics of football injuries, such as the type and nature of the injury, anatomical sites of injury, severity, mechanisms, and biomechanical, anthropometric, and physiological aspects of football players, have been investigated and described in previous studies.In professional football players, the incidence of injuries during matches ranged from 12 to 66.0/1000 h and from 1.5 to 7.6/1000 h of training exposure ,14,15. MA wealth of research has focused on injuries in professional football, but millions of athletes play football at a non-professional level. Football injuries affect the quality of life of the player and constitute a serious threat to a career . ExpertiThe primary aim of our study was to conduct a systematic review of the literature quantifying the incidence of injuries in male professional and amateur football. Secondarily, we sought to highlight general effects regarding location, type, severity, mechanism of injuries, and type of incident.Our review was designed in accordance with the Guidelines of Protocols for Systemic Revisions of Observational Studies in Epidemiology, PRISMA  ,32,33. TA review of a relatively large number of studies identified in the electronic databases PubMed, Medline, Google Scholar, and Diva Portal was performed up to June 2023. Keywords entered were the following: epidemiology football injuries, male football injuries, professional football, amateur football, incidence, location, type, and severity of injuries.The search and selection of studies was performed by 2 co-authors. Disagreements between the 2 reviewers were resolved by discussion, without the need for the intervention of a third reviewer to reach a consensus. To be considered eligible for inclusion, studies had to meet some criteria.Inclusion criteria for the selected articles considered were as follows: prospective and retrospective longitudinal research studies, original research studies, systematic reviews, and meta-analyses; studies that included professional or amateur football players  as participants aged 18 years or older in professional or 17 years or older in amateur football players, participating in a senior men’s football league (elite club or club by amateurs)  or the iAdditional criteria: studies had to be full-text articles or abstracts published in peer-reviewed journals before June 2023, published in English.Exclusion criteria: studies that provided hypotheses, reasoning, and methodologies and not study results (protocol studies); studies that included women, children, and adolescents under 17 years of age; studies that presented incomplete data or data from a study already included; studies that used definition of injury other than time loss; and studies related to sports other than football. Literature reviews, non-conclusive abstracts, editorial comments, and letters to the editor were also excluded.A data extraction sheet specially created in Microsoft Excel was used to extract data from each eligible study. Data extraction was performed by two reviewers (AB and CD) independently. A third rater (EF) was consulted to resolve disagreements between these raters and facilitate consensus. The data extraction sheet included the following items: (a) general descriptors of the study, (b) characteristics of the studied population, (c) epidemiological results , and (d) data on exposure . OperatiThe reporting quality of included studies was assessed using an adapted version of the Strengthening the Reporting of Observational Studies in Epidemiology (STROBE) statement by . SupplemIn addition, an adapted version of the Newcastle Ottawa Scale (NOS) was used to assess risk of bias for the quality of external validity. The criteria proposed by  compriseAll statistical analyses were performed with Windows Excel 2010. Descriptive data such as percentage lesion characteristics were calculated as mean values and standard deviations (±SD). Where incidence rates were not reported but the required elements were presented, they were calculated using the following formula:Confidence intervals (95% CI) were calculated as follows :Lower boCorrelations between overall, training, and match incidence were determined by calculating the Pearson coefficient (Correl), which indicates the relationship between two variables. The significance level was set at 0.05.n = 214; 14.0%); in children, adolescents, and young athletes under the age of 17 ; in women’s football ; and other subjects . The remaining full-text articles and some conclusive abstracts  were screened for eligibility, referring to adult professional and amateur football players. Of these, n = 55; 3.6% of studies did not report injury incidence ; they defined injury differently from injuries/1000 h of exposure; and they did not express the severity, location, and type of injuries as a percentage, nor did they provide sufficient data to allow their calculation. The search process resulted in 46 articles  that met the inclusion criteria. These studies were conducted between 1990 and June 2023 for professional athletes and between 2012 and June 2023 for amateurs. Eligible studies included professional and amateur football players and participants in world tournaments, continental tournaments, and professional leagues from different countries . A number of 1525 references were identified by using the search strategies, of which 720 were excluded at the first check as duplicates (47.2%). Of the remaining 805 (52.8%), 704  were eliminated after reading the titles and abstracts. They referred to injuries in other sports , moderate associations between overall and match incidence (0.603), and 0.822 between training and match incidence, indicating a strong association at a level of statistical significance Professional football. Thirty studies reported lesions location as percentage values, which varied widely by anatomical region and study  in male professional football players and 80.49% (69.8–91.0%) in amateurs. These values are close to the data reported by , which iIn our study, the four most common locations of lesions were thigh (26.07 ± 5.53%), ankle (15.84 ± 3.93%), knee (14.7 ± 2.84%), and hip/groin (12.96 ± 2.15%) in professional football players and (23.4 ± 7.69%), (17.99 ± 3.63%), (17.28 ± 5.26%) and (11.5 ± 4.97%), respectively, in amateur football players. Our results differ from the data reported by , which iThe thigh muscles involved in football injuries are usually bi-articular with a complex architecture, with a high percentage of fast-twitch fibers and which undergo eccentric contractions and are capable of producing high forces . The injThe biceps femoris (53%) was the most common muscle of the hamstring complex exposed to injury. This finding is supported by other studies. The anatomy of the biceps femoris may help explain its higher injury rate. First, it has a long head and a short head, both with separate nervous systems . This duKnee and ankle injuries are the most common injuries suffered by football players as well as handball, basketball, and tennis players, and alpine skiers. They generate the biggest consequences in terms of absence from the game; the rehabilitation of a knee injury takes an average of 45 days. The group mean values established in our review of 14.7 ± 2.84% in professional football players and 17.28 ± 5.26% in amateur football players are different from those reported by , who indAnkle injuries are common in both professional and amateur footballers, especially lateral ligament injuries, as football is a high-impact sport that puts a lot of stress on the ankle. They can be caused by the explosive and shifting movements in football, combined with the constant contact between players. It usually happens when the foot is rolled inward, either from a misstep or from an attack. Some authors reported different values for ankle injuries: 11%; 15%; 22%; 32%; 23% (for both men and women) ,95,96,97The hip/Groin is a complex anatomical region halfway between the abdomen and the thigh, traversed by a multitude of anatomical structures. The anatomical complexity of the groin area makes hip/groin injuries often a diagnostic challenge for the physician. Groin injuries are common in high-intensity team sports   and accoIn our review, several studies identified differences between elite, sub-elite, and amateur adult male players in match and training incidences. Also, the variation of these indicators was noted depending on the country of origin of the football team (climate); type of competition, such as friendly matches or competitive matches ; season; and lawn type.In European male professional football, Waldén et al.  reportedWhalan et al.  reportedBetween South American teams and European teams, Bengtsson et al.  found noAsian professional football is characterized by an overall injury incidence similar to that reported in Europe but with a high rate of ACL tears and hamstring injuries (54.4%) . IncidenThe incidence and characteristics of football injuries during matches in high-level international tournaments such as the Olympic Games, European Championships, and World Cups are well documented ,106,107.Several studies have reported injury characteristics during playing on artificial turf compared to playing on natural grass in both male and female elite players ,110,111,Compared to professional footballers, the incidence of injuries during training was higher among amateur footballers (3.9 vs. 2.1/1000 h), in contrast to the incidence of injuries among professionals, which was higher during matches (31.8 vs. 20.4 injuries/1000 h of exposure) . These vOur study, conducted according to international guidelines for systematic reviews, has some limitations. They are related, in part, to the limited availability of some databases and the use of only articles published in English, which do not exclude the possibility that some valuable studies were not selected by us. The lack of uniform data collection methods can be sources of inconsistency in results, as can differences between national leagues in terms of climatic regions, peak match periods, number of matches and breaks, and level of professionalism. Also, the large variation in sample sizes, the limited geographical areas of the studies , and the different durations of investigation  could partially explain the heterogeneous estimates obtained in our systematic review. Furthermore, it is not excluded that some studies may have reported not only “time loss” injuries, but all injuries, without specifying this. This discrepancy in injury definitions between studies would partially account for the different incidences of injuries observed. We did not include in the analysis injuries that required medical attention, which might have led to higher incidence rates, nor was it possible to report injury burden due to a lack of data on the number of days lost per injury in some of the studies included. In both professional and amateur footballers, our data extraction template allowed the documentation of only the data related to the location of the injury and the type and severity of injuries by main groups and not stratified by subgroups, also motivated by the fact that not all studies eligible showed a complex stratification. We did not allow space for interactive effects within risk factors such as pitch type , playing position, assessment of protective equipment used by players, violation of competition rules, and quality of referees. Exposure of amateur athletes to additional interventions outside of game protocols as part of their normal routine is not excluded, but their monitoring was not recorded or feasible in some studies. The small number of eligible studies in amateur football players did not allow us to make a satisfactory comparison with professional football players. The life of a professional footballer is totally different from the life of an amateur player. Professional football players benefit from customized training programs with better equipment and a variety of training programs. Retrospective study designs may have increased bias in assessing accurate injury history.The main findings of this study were as follows: the overall injury incidence rate was higher in amateur compared to professional footballers, but the training and match incidence rates were higher in professionals than in amateurs. For both categories of footballers, the incidence at a match was more than five times higher than the incidence at training, the highest values being observed during the European and world championships. Lower extremity injuries, thigh, ankle, and knee injuries predominated. Professional and amateur football players remain athletes at substantial risk of injury, especially during matches that require the highest level of performance. The current findings have implications for the management, monitoring, and design of training, competition, injury prevention , and education programs for amateur football players.


The primary aim of this research was to establish the importance of risk factors in the development of medication-related osteonecrosis of the jaw in cancer patients receiving zoledronic acid therapy for bone metastases. The present study is an observational retrospective investigation conducted at two university centers, namely, Craiova and Constanța, and included cancer patients treated with zoledronic acid. The medical records of the patients were obtained over a four-year timeframe spanning from June 2018 to June 2022. The data analysis was carried out between January 2021 and October 2022. Patients were treated for cancer, bone metastases, and MRONJ according to the international guidelines. The research investigated a cohort of 174 cancer patients  aged between 22 and 84 years (with a mean age 64.65 ± 10.72 years) seeking treatment at oncology clinics situated in Craiova and Constanța. The study conducted a binomial logistic regression to analyze ten predictor variables, namely, gender, age, smoking status, treatment duration, chemotherapy, radiotherapy, endocrine therapy, presence of diabetes mellitus (DM), obesity, and hypertension (HT). The results of the analysis revealed that only five of the ten predictor variables were statistically significant for MRONJ occurrence: duration of treatment ( The condition known as BRONJ (Bisphosphonate-Related Osteonecrosis of the Jaw) ,2, MRONJBisphosphonates (BF), drugs with antiresorptive properties, significantly reduce fracture risk in patients with benign bone disease and have demonstrated effectiveness in addressing skeletal-related events (SREs) in individuals with bone metastases (BM) . BisphosZoledronic acid is a bisphosphonate administered intravenously (IV) to cancer patients, usually at a dosage of 4 mg at 4-week intervals for the purpose of preventing bone metastases ,16. It hThe main risk factor in the occurrence of MRONJ with zoledronic acid is represented by the treatment duration . The risThe local factors that contribute to the occurrence of MRONJ are tooth extraction in a large percentage (70%) and mandibular localization in 75% .According to the findings of a systematic literature review, the factors contributing to systemic risk for medication-related osteonecrosis of the jaw are age and gender ,24,25, aThe objective of the current study was to determine the systemic and local risk factors for MRONJ in a group of cancer patients treated with zoledronic acid for bone metastases and to establish the risk for MRONJ through a binomial regression analysis.The retrospective study used databases from two hospitals, County Clinical Emergency Hospital of Craiova and County Clinical Emergency Hospital of Constanta, and a cancer ambulatory treating center, Oncolab Craiova, all from Romania. The database contains information on patients’ demographic data , cancer diagnosis and treatment, comorbidities, complications, inpatient and outpatient care in hospital, and ambulatory services. Data from the study were collected from March 2019 to December 2022. The study was approved by the Ethics Committee of the University of Medicine and Pharmacy of Craiova, no. 59/22.03.2019.Patients included in the study were cancer patients treated with bisphosphonates approved in Romania for bone metastases from June 2018 to June 2022 in two geographical regions from Romania, South-West Oltenia, and South-East Dobrogea. The inclusion criteria were the following: patients previously diagnosed with various types of neoplasms and treated with 4 mg IV zoledronic acid administered once a month. The exclusion criteria were patients younger than 20 years, patients with only oral bisphosphonate treatment, patients treated with radiotherapy in the maxillo-facial area, patients treated with bisphosphonates for osteoporosis, and patients with oral cancers. Data analysis was performed between January 2021 and October 2022 and the results are in compliance with STROBE guidelines .All participants in this study signed an informed consent form prior to their medical admission for treatment.The retrospective study compared risk factors encountered in cancer patients with MRONJ (the study group) to risk factors in cancer patients without MRONJ (control group).Current or previous treatment based on antiresorptive or antiangiogenic agents;The presence of exposed bone or an intraoral or extraoral fistula in the maxillofacial region that has persisted for more than eight weeks;Patients with no history of radiotherapy to the jaws or obvious metastatic disease of the jaws.The criteria used to define MRONJ published by AAOMS in a position paper from 2022  were as Patients diagnosed with MRONJ were identified according to the above-mentioned criteria as established by position papers of AAOMS in 2014 and 2022 ,4 and toThe data collected pertaining to the enrolled patients comprised the interval between the first administration of bisphosphonate to the end of the observation period or to the culmination of the study (June 2022), whichever came first.The sample size was computed using G*Power 3.1.9.7, Heinrich Heine University Düsseldorf, Germany. There were several parameters considered: a significance level α of 0.05, a power 1-β equal to 0.85, and a medium effect size value of 0.3. Consequently, the analysis led to a minimum enrolment of 160 patients for the study.In the study, demographic data, comorbidities, and oncological data were extracted from the clinical records for each patient individually. The demographic data included the medical center, gender, age, residency, and smoking status. Comorbidities were assessed in terms of the presence or absence of bone metastasis, cardiovascular diseases, hypertension, diabetes mellitus, obesity, anemia, and renal diseases. With regard to oncological data, the primary diagnosis of the underlying disease was determined, along with the associated treatment, which may have involved chemotherapy, endocrine therapy, immunotherapy, radiotherapy, or corticotherapy. Furthermore, specific BF-related data such as the administration of zoledronic acid or other resorptive treatments, the type of administration, the duration of BF administration, and the presence or absence of osteonecrosis were recorded.The data collected for all the subjects with MRONJ included the following: MRONJ detailed location , stage  ,4, the tp < 0.003846. The α threshold was set to 5%, and the value p < 0.05 was considered statistically significant.The data collected from the patients’ medical charts were initially processed using Microsoft Excel 365 . Consequently, this led to a basic distribution of the study group into subgroups. Continuous variables were presented as mean ± standard deviation and were compared using Kendall’s tau-b or Mann–Whitney U test for non-gaussian distributions. The categorical variables were expressed as numerical values and percentages, and their association was evaluated with either the Chi-square test or the Fisher Exact test. All statistical tests were employed using Statistical Package for Social Sciences (SPSS), version 20 . The acquired information was incorporated into a binomial logistic regression model to assess the likelihood of developing osteonecrosis in relation to the following variables: gender, age, smoking status, previous or current chemotherapy, endocrine therapy or cortico-therapy, duration of BF treatment, and smoking status. Linearity of the continuous variables with respect to the logit of the dependent variable was assessed via the Box–Tidwell (1962) procedure. A Bonferroni correction was applied using all thirteen terms in the model resulting in statistical significance being accepted when The initial study group comprised 178 patients .After the exclusion criteria were applied, in the study group remained 174 patients , 109 femMore than half of the patients were from Constanta , while 73 patients (41.9%) were from Craiova. Patients were distributed in age groups, expressed as the following: 22–54 (young adults), 55–64 (mature adults), 65–71 (young old), and 72–84 years old (old old) . For ourAll 174 patients were previously diagnosed with neoplasms, of which breast and prostate cancers were predominant in our study group. Overall, 82 females had breast cancer (47.2%), 46 males had prostate cancer (26.4%), while the rest of the 46 patients had other neoplasms: pulmonary, myeloma, genital, digestive, renal, cerebral, bladder, spinal cord, pharynx, or thyroid. Bone metastases were diagnosed in 154 patients . Neoplasms’ general distribution is presented in Among the cohort of patients who received chemotherapy, 133 had bone metastasis, 68 had cardiovascular diseases, from which 52 had hypertension, 49 had nutritional diseases , 24 had renal diseases, and 17 were anemic. Endocrine therapy was recommended mostly for more than half of all males with prostate cancer and with few other comorbidities, the most common being cardiovascular and renal diseases. Patients with radiotherapy had a similar distribution regarding the neoplasm type; however, in Craiova, this type of therapy was mostly recommended for females with breast cancer. All 10 patients who underwent corticotherapy were from Craiova, mostly females aged less than 64 years old, with very few comorbidities except bone metastasis . Immunotherapy was used only in two cases, one in Craiova, and one in Constanta, for elderly females, both with cardiovascular diseases and other comorbidities.The entire cohort within the study was subjected to the therapeutic intervention of BF-zoledronic acid, administered intravenously every month with a dosage of 4 mg. Regarding the duration of treatment, 29.9% had less than 12 months of BF treatment, 34.5% had between 12 and 24 months of treatment, 19.5% had between 2 and 3 years of treatment, while 16.1% received BF treatment for more than 3 years. The average treatment duration for each age group is presented in 2(1) = 21.9, p < 0.0005.Approximately half of all patients  who developed osteonecrosis of the jaw had an average treatment duration with zoledronic acid of 29.0 ± 12.1 months; the rest of the 84 patients (48.3%) who did not develop MRONJ had an average treatment duration of 19.6 ± 17.9 months). Of the 90 patients with MRONJ, 53 were from Craiova (58.9%), and only 37 were from Constanta (41.1%). A Chi-square test for homogeneity was conducted between the medical center and MRONJ development. There was a statistically significant difference regarding the proportion of patients with MRONJ from Craiova compared to patients with MRONJ from Constanta, χb = 0.055, p = 0.382. In addition, a Chi-square test of homogeneity was conducted between the two groups with and without MRONJ in relation to the patients’ age group, with an adequate sample size established according to Cochran (1954). There were no differences in proportions between the four age groups, χ2(3) = 4.985, p = 0.173. Observed frequencies and percentages of age groups for each group are defined in Kendall’s tau-b correlation was run to determine the relationship between age and MRONJ presence amongst all 174 participants. There was a moderate, positive association between age and osteonecrosis development , which was not statistically significant, τ2(1) = 0.079, p = 0.778). Similar results were obtained for males and prostate cancer, with 52.2% of males having jaw osteonecrosis (χ2(1) = 0.545, p = 0.460). The overall analysis of neoplasm types  did not reveal any statistically significant differences regarding MRONJ development (χ2(2) = 0.075, p = 0.963).Approximately half of all females with breast cancer developed jaw osteonecrosis (52.4%); however, this cancer type does not seem to be a risk factor for developing MRONJ in females , and corticotherapy. In this case, all treated patients developed MRONJ. Endocrine therapy seems a protective factor.2(1) = 30.044, p < 0.0005), with more than three-quarters of patients having stage 2 MRONJ.Of the 90 patients with MRONJ, 71 patients (representing 78.9%) had stage 2 MRONJ, and 19 patients (21.1%) had stage 3 MRONJ . A chi-sb = −0.025, p = 0.778. Similarly, there were no statistical differences in the MRONJ stage regarding residency, gender, smoking status, or the medical center.Kendall’s tau-b correlation was run to determine the relationship between age and MRONJ stage amongst all 90 participants. There was a very weak, negative association between age and stage , which was not statistically significant, τBisphosphonates treatment duration was recorded in months, and the following categories were identified: 1–12 months , 13–24 months , 25–36 months , and >36 months  . The cump < 0.0005. Treatment duration was not related to the MRONJ stage .A Mann–Whitney U test was run to determine if there were differences in treatment duration between MRONJ and non-MRONJ groups. Distributions of duration were not similar, as assessed by visual inspection. Median treatment duration was statistically significantly higher for the MRONJ group (24 months) than for the non-MRONJ group (12 months), U = 5638, z = 5.620, 2(2) = 24.867, p < 0.0005), with more than half of patients having extraction as a trigger factor.The following trigger factors were identified in MRONJ development: tooth extraction , periapical disease , and periodontal disease . A chi-square goodness-of-fit test was conducted to determine whether an equal number of patients for the three trigger factors were represented in the study. The minimum expected frequency was 45. The chi-square goodness-of-fit test indicated that the number of patients with various trigger factors was statistically significantly different (χ2(6) = 15.752, p = 0.015.For patients from the first age group, the main trigger factor was periodontal disease (with 50% from this group), compared to the other age groups, where it was the least representative factor. Periapical disease was predominant for groups 55–64 and 72–84 years old, while extraction was the main factor for patients with ages above 54 years old. Overall, the differences between age groups regarding the trigger factor were statistically significant, χ2(2) = 48.800, p < 0.005), with approximately two-thirds of the patients presenting mandibular osteonecrosis. Age group analysis revealed significant differences between patients with ages below 55 years old, with predominant upper jaw MRONJ (60% from the entire group), and the other three age groups, with predominant lower jaw MRONJ , χ2(6) = 13.348, p = 0.038.Of the 90 patients with MRONJ, 58 patients (representing 64.4%) developed MRONJ in the lower jaw, compared to only 31.2% (28 patients) in the upper jaw, while 4 patients (4.4%) presented osteonecrosis in both jaws. A chi-square goodness-of-fit test indicated that the number of patients for each location was statistically significantly different (χ2(1) = 78.400, p < 0.0005). Similar results were obtained regarding hypoesthesia, as it was identified for only 16 patients (17.8%), compared to 74 patients (82.2%), and this difference was statistically significant (χ2(1) = 37.378, p < 0.0005).The presence of denudated bone was recorded for 87 patients, representing 96.7%, compared to only 3 patients without denudated bone (3.3%). According to the result of the chi-square goodness-of-fit test, the difference was statistically significant (χ2(1) = 4.003, p < 0. 0.045).Surgical treatment for patients with osteonecrosis of the jaw was represented by sequestrectomy , resection , or curettage . Among this study lot, sequestrectomy was the election surgical procedure for more than two-thirds of patients; for statistical purposes, these patients have been grouped with patients with curettage, with the differences between the number of patients with and without these procedures being statistically significant (χ2(1) = 25.600, p < 0.0005).Resection was performed for less than a quarter of patients, reflecting statistically significant differences between the number of patients with and without resection (χ2(2) = 77.400, p < 0.0005), with around three-quarters of patients being the subject of a single surgical procedure.The majority of MRONJ patients underwent one surgical procedure , eight patients suffered a relapse and underwent a second surgical intervention (8.9%), while six patients (6.7%) were not surgically treated. With a minimum expected frequency of 30 for each category, a chi-square goodness-of-fit test reflected statistically significant differences between the number of patients with one or more surgical procedures (χ2(1) = 9.443 and p = 0.002. The analysis of the MRONJ stage in relation to comorbidities revealed similar results.Several common comorbidities have been analyzed for this study group. Following a Chi-Square test, only cardiovascular diseases (mostly hypertension) may be considered a risk factor for developing MRONJ , with χ22(10) = 62.406, p < 0.0005. The model explained 40.2% of the variance in osteonecrosis development and correctly classified 76.4% of cases. Sensitivity was 81.1%, specificity was 71.4%, positive predictive value was 75.26%, and negative predictive value was 77.92%. The area under the ROC curve was 0.824 , which is an excellent level of discrimination. Of the ten predictor variables, only five were statistically significant: duration of treatment, chemotherapy, endocrine therapy, obesity, and hypertension . There were three standardized residuals with values around three standard deviations, which were kept in the analysis. The logistic regression model was statistically significant, χrtension .Cancer patients undergoing chemotherapy had 7.53 times higher odds to develop osteonecrosis of the jaw, while endocrine therapy was associated with a reduced frequency of MRONJ. Patients with hypertension had 3.79 times higher odds to develop osteonecrosis of the jaw than the patients with normal blood pressure values, while obesity was associated with a reduction in the likelihood of developing osteonecrosis. Longer zoledronic acid treatment duration was associated with an increased risk for MRONJ.Since MRONJ is a complication with a major negative impact on the quality of life of cancer patients ,37,38,39Like the study by Marciano et al. conducted in an Italian population , the curThe incidence of MRONJ in zoledronic acid-treated cancer patients varies between 0.2% and 9.9%, reaching 15–20% in some case studies ,38,43. TThe results of the binomial logistic regression performed on ten predictor variables  pertaining to the likelihood of MRONJ occurrence, showed that only five were statistically significant, namely, chemotherapy, hypertension, duration of treatment, endocrine therapy, and obesity.Romania is among the top 10 European countries with the highest mortality rates due to cancer ,44. The p < 0.05. This can be correlated with the fact that the highest prevalence of cancer in Romania in 2019 was recorded in the S-W Oltenia region  followed by the prevalence in the S-E region (Dobrogea)  [Although more than half of the patients were from the S-E Dobrogea region , compared to 73 patients (42.0%) from the S-W Oltenia region, out of 90 patients with MRONJ, the majority  were from S-W Oltenia and only 37 were from S-E Dobrogea (41.1%) /10,000) ,47, with/10,000) ,46,47. IThe majority of the patients in the studied groups were women (over 62%), over 55 years old (over 82%), more from the region of S-E Dobrogea, Constanta (over 58%), and more from the urban environment (over 71%). Most of the population studied (over 73%) were diagnosed with breast or prostate cancer. Another study published in 2020 regarding the incidence of MRONJ in the period 2009–2018 in patients from Craiova (S-W Oltenia) treated with bisphosphonates showed that most patients with MRONJ were women; the age of the patients was higher in men compared to women and the origin was urban for most patients .In a study conducted by Ishimaru et al., in 2021, middle-advanced age (65 to 74 years old) was correlated with the occurrence of MRONJ in cancer patients, especially in men . RodriguMost of the patients in our study group were from an urban environment, which is consistent with another previous study . StatistOne identified risk factor that has been linked to a higher incidence of MRONJ is the type of cancer, namely, breast and prostate cancers ,22. BreaIn a review regarding MRONJ, Anastasilakis et al.  showed tOverall, these studies emphasize the importance of dental prophylaxis and maintenance of good oral hygiene for the prevention of MRONJ in patients treated with bisphosphonates and denosumab and highlight important factors in the risk of development and re-occurrence of this condition ,54. A stSeveral studies have suggested that comorbidities may constitute potential risk factors for the development of MRONJ ,34. ComoIn Romania, campaigns have been carried out with the aim of involving authorities and the media in increasing awareness regarding diabetes prevention and control . In the In the study conducted by Ishimaru et al., dementia and renal diseases related to renal cancer, which was the most common form of cancer associated with MRONJ in the patients studied, were identified as significant comorbidities . In the Anemia is found in all four groups of patients  .Several studies have identified chemotherapy as an important risk factor for MRONJ ,30,31,59Among the other cancer therapies, radiotherapy was used more frequently, while endocrine therapy was used less often as it is reserved especially for less serious cases of breast and prostate cancer. Immunotherapy was very rarely encountered ,60,61,62p < 0.05. A duration of more than 12 months  doubles the risk of MRONJ development, while a duration of more than 24 months  triples it. The relationship between the risk of MRONJ development and the duration of treatment with antiresorptive agents in cancer patients was also analyzed in other studies [In the binomial regression analysis from the present study, a longer zoledronic acid treatment duration was associated with an increased risk for MRONJ. The average duration of treatment with bisphosphonates was longer in Craiova than in Constanta, longer in patients with prostate cancer, followed by breast cancer, and then by the other types of cancer. MRONJ appeared in a significantly higher percentage of patients from Craiova, who had a longer duration of treatment with BF (2 months longer on average). The duration of treatment with bisphosphonates is presented as the main risk factor for MRONJ in several studies ,39,61,62 studies ,63,64, rTreatment duration was not related to the MRONJ stage. In other studies, the average duration of treatment with BF was between 12 and 55 months . AccordiAccording to Kemp et al., among the risk factors for medication-related osteonecrosis of the jaw in oncological patients treated with zoledronic acid is the monthly administration rate, as well as the lack of dental control, surgical procedures , and smoking. In Kemp’s study, the localization of MRONJ in the upper jaw predominates; unlike in our study, where most patients had MRONJ in the lower jaw .Studies have also been carried out regarding the association of risk factors with the characteristics of MRONJ, such as the stage of MRONJ, the trigger factors, the location, the presence of exposed bone, the presence of hypoesthesia, and the type of surgical treatment.p < 0.0005). Age groups were correlated with the trigger factor, and the differences between age groups regarding the trigger factor were statistically significant (p = 0.015): extraction was the main risk factor for patients with ages above 54 years old, periapical disease was predominant for groups 55–64 and 72–84 years old, and periodontal disease was the main trigger factor for patients from the first age group.In the present study, the most important trigger factor for MRONJ was tooth extraction, followed by periapical disease and periodontal disease. More than half of the patients had extraction as a trigger factor  of patients had osteonecrosis in both jaws. Similar rates regarding the localization of MRONJ were also recorded in the study by Kawahara et al. in 2021 . Some stIn our study, the most frequent MRONJ localization was in the posterior area both in the maxilla and in the mandible, without any statistical significance. The same was observed in the study by Feng et al. .A correlation between the location of osteonecrosis and the age of the patients was noticed. Mandible osteonecrosis was encountered for patients over 55 years old while maxillary osteonecrosis was predominant (60%) in patients with ages below 55 years old.One of the clinical characteristics of MRONJ with a significant statistical value was the exposed bone, in a proportion of 96.67%. Hypoesthesia was present in only 17.78% of MRONJ patients. Other studies showed that focal and diffuse bone sclerosis and the occurrence of bone sequestrations could be observed more frequently in patients with exposed bone, compared to patients without exposed bone .Most of the patients analyzed in this study (78.90%) had MRONJ stage 2, and only 10% were diagnosed with MRONJ stage 3. The MRONJ stage determines the choice of treatment method. Although in 2014, the AAOMS considered that surgical treatment is not recommended for patients with stage 1 and 2 MRONJ and should be limited to patients with stage 3 lesions or stage 2 lesions unresponsive to non-surgical treatment; other studies suggest that the surgical removal of necrotic bone may be an effective treatment for all stages of MRONJ ,72. AnotA surgical approach may be considered for any necrotic bone exposed when conservative treatment has failed. A conservative surgical approach can be achieved through sequestrectomy associated with the administration of antibiotics and rinsing with antiseptic solutions ,71. The Although the treatment of patients with an established diagnosis of drug-induced osteonecrosis of the jaw (MRONJ) should be approached with a pragmatic multidisciplinary treatment, prioritizing the patient’s quality of life and the management of their skeletal disease, sometimes a complete resection of the necrotic bone  is necessary to achieve complete healing ,72.More recent studies have shown that stages 2 and 3 of MRONJ could be treated both surgically with or without adjuvant therapies, as well as conservatively ,74,75,76p < 0.05). More than three-quarters of patients underwent only one surgical procedure, while 8.9% of patients underwent two different surgical procedures, and 6.70% of patients were not surgically treated (p < 0.05). Although in stage 3 MRONJ, the treatment of choice was most often surgical, the general rate of complications was high, with relapses being recorded [In the present study, resection was performed for less than a quarter of patients, reflecting statistically significant differences between the number of patients with and without resection (recorded . Surgicarecorded .p < 0.005).The strengths of this study are reflected by a sufficient follow-up time (of 4 years) for all 174 patients residing in the two geographical regions, allowing the analysis of possible risk factors regarding demographic factors, cancer type and cancer treatment, co-morbidities, period duration of bisphosphonate treatment use, and also the MRONJ stage, trigger factor, overall lesion location, denudated bone presence, hypoesthesia presence, and surgical treatment type. Binomial regression analysis performed for ten predictor variables indicated the statistically significant factors: chemotherapy, hypertension, duration of treatment, endocrine therapy, and obesity. Chemotherapy increased 7.53 times the odds of developing osteonecrosis of the jaw of the cancer patients, while endocrine therapy was associated with a reduction in the likelihood of developing osteonecrosis. Hypertension increased 3.79 times the odds of developing MRONJ in the cancer patients, while obesity was associated with a reduction in the likelihood of developing osteonecrosis. Increasing treatment duration was associated with an increased likelihood of developing MRONJ (The limitations concern the lack of data regarding the oral health of the subjects before BF treatment initiation and regular oral monitoring during the treatment.Risk factors identified for MRONJ correlated with zoledronic acid were chemotherapy, hypertension, and duration of treatment. Cancer patients with chemotherapy or hypertension had higher odds of developing MRONJ, while endocrine therapy and obesity were associated with a reduced frequency of MRONJ. Increased treatment duration with zoledronic acid was associated with an increased risk of developing MRONJ.