The possible correlation between melanoma and Parkinson’s disease (PD) has been intensively studied. In this work, we aimed to assess the coincidence of skin malignancies and PD at a dermato-oncological university centre in Central-Eastern Europe, Hungary.From 2004 to 2017, a retrospective analysis of the centre’s database was performed based on International Statistical Classification of Diseases-10 codes.Out of the patients who visited the clinic during the study period, 20,658 were treated for malignant skin tumours. Over the 14 years, 205 dermatological patients had PD simultaneously, 111 (54%) of whom had at least one type of skin malignancy: melanoma (n=22), basal cell carcinoma (BCC) (n=82), or squamous cell carcinoma (SCC) (n=36) . Compared to the age- and sex-matched control group, patients with PD had a significantly lower risk for basal cell carcinoma  and for all skin tumours  but not for melanoma.We found a decreased risk of all skin tumours and basal cell carcinoma and an unchanged risk of melanoma among patients with PD. However, it should be kept in mind that some large-scale meta-analyses suggest a higher incidence of melanoma after a diagnosis of PD, indicating the importance of skin examination in this vulnerable population. The theThe mechanism by which melanoma develops more frequently in PD patients and why patients with melanoma more frequently have PD than the general population have not yet been clarified. Although several mechanisms have been proposed, the data are contradictory. The background of both diseases seems to be multifactorial, and both genetic and environmental factors may play a role in their pathomechanisms , 16–24.Parkin, DJ-1, LRRK2, α-synuclein, PLA2G6, EIF4G1, and SYNJ1 gene/protein can contribute to the association between the two diseases. Parkin is a tumour suppressor gene that encodes a ubiquitin ligase found in several cancers and up to half of patients with early-onset familial PD. Loss or mutation of the gene interferes with the degradation of cyclin E; consequently, its level increases and forces the cell to divide faster  together with environmental factors  can equally contribute to the association between the two diseases  found that PD patients had decreased risks for urogenital, gastrointestinal, lung, and haematological malignancies but increased risks for melanoma and brain cancer compared to controls . Similarobserved . A meta-observed . The resobserved . Another1.2The connection between PD and non-melanoma skin cancer (NMSC) is contradictory. Some authors have reported that patients suffering from PD have a higher risk for NMSCs , 18, 41,Various studies have investigated the connection between PD and extracutaneous malignancies. Many authors found a significantly lower risk for all malignancies among patients with PD, including colorectal, lung, and larynx cancer, while breast, prostate, renal, and thyroid cancer risk was reported to be higher than expected , 50, 51.Since studies focusing on the association of PD and skin tumours were mainly carried out in the USA , 54–58 a22.1A retrospective analysis of the database of the Department of Dermatology, Venereology, and Dermatooncology at Semmelweis University was performed between 1 January 2004 and 31 December 2017. The total number of patients who visited the department during the 14-year examination because of any skin malignancy was 20,658. To identify patients with melanoma, NMSC, and PD, we used International Statistical Classification of Diseases-10 (ICD-10) codes; patients with melanoma were selected by the c4300–c4390 codes, squamous cell carcinoma by the c4401–c4491 codes, and basal cell carcinoma by the c4402–c4492 codes. The patients with PD were collected based on the ICD-10 code G20. Combining the patient populations of the four different diagnoses, we selected those patients who had melanoma or NMSC associated with PD. Data on skin tumours and PD were obtained by reviewing the patient’s medical history.Control group. Because the mean age of the patients diagnosed with PD and skin tumours was different, in addition to these diseases affecting the two sexes differently, for each patient with PD, five age- and sex-matched controls without PD were selected randomly from the patients of the department from 2004 to 2017. These five control patients had the same sex and were born in the same year as the studied patient. Consequently, 205 patients with PD associated with skin disease were matched with 1,025 age- and sex-matched control patients with skin disease but no PD.2.2First, descriptive statistics were performed. The association between PD and melanoma and basal and squamous cell carcinoma was assessed separately. To examine the odds of having skin malignancy in PD patients, we used GraphPad Prism 9.3.1 (147) software. Odds ratios (ORs) and their 95% confidence intervals (CIs) were calculated for each group from contingency tables. An OR >1 indicated a positive association, and an OR <1 indicated a negative association between a given skin tumour and PD. Comparing the ORs of different tumour types, we used the case numbers (number of skin tumours) instead of the number of patients because, contrary to most other malignant disorders, tumours of the skin are often multiple. Consequently, in the case of calculation with the number of patients instead of the number of skin neoplasms, cases were lost. Since we had a small sample size, to calculate the correlation between our categorical variables, we used Fisher’s exact tests to determine whether the difference was statistically significant. A probability value of <0.05 was considered statistically significant.33.1From 2004 to 2017 at the Department of Dermatology, Venereology, and Dermatooncology at Semmelweis University, 20,658 patients were treated for skin malignancy. A total of 6,300 patients had melanoma, 11,983 had basal cell carcinoma, and 2,375 had squamous cell carcinoma.3.1.1Over the 14 years, 205 patients visited the department due to a skin disease and PD simultaneously. The ratio of male to female patients was 61.5% to 38.5%. Of the 205 patients, 94 had no skin malignancy, while 111 (54%) patients had malignant skin tumours. The mean age of the studied patients was 78 ± 8.4 years, with a median age of 79 years. The youngest patient was 47 years old, and the oldest patient was 96 years old. The mean age at PD onset was 72.2 ± 9.5 years, while the mean age at the diagnosis of the first skin malignancy at the department was 77.9 ± 8 years Table 1.Multiple skin tumours were frequent among these 111 patients; 65 of them had only one skin malignancy, and in the remaining 46 patients, multiple tumours occurred. Altogether, 140 skin malignancies (positive events), 22 melanomas, 82 basal cell carcinomas, and 36 squamous cell carcinomas were diagnosed in the 111 patients. In addition, we found some rare skin tumours simultaneously with PD: two trichilemmal carcinomas, a malignant fibrous histiocytoma, an atypical fibroxanthoma, and a Merkel cell carcinoma. There were 27 patients who had different types of skin malignancies simultaneously, and one patient had 35 malignant skin tumours, including melanomas, trichilemmal carcinomas, and basal and squamous cell carcinomas.3.1.2We randomly coupled each of the 205 patients with PD with five age- and sex-matched patients who did not have PD from the department from 2004 to 2017. Consequently, 205 patients with PD and 1,025 control patients were matched. A total of 560 patients were negative for skin tumours, while 465 patients had a total of 1,127 skin malignancies : 132 melanomas, 752 basal cell carcinomas, and 243 squamous cell carcinomas .3.2Assessing the odds of all skin malignancies, it proved to be significantly decreased among the patients with PD . The odds proved to be significantly decreased as well in cases of basal cell carcinoma and PD . On the other hand, the results did not show a significant correlation between PD and melanoma or PD and squamous cell carcinoma Table 2.3.3Single and multiple skin malignancies equally developed more frequently in men than in women, which was noticeable for every type of skin tumour. Seven of 19 patients suffering from melanoma had additional basal cell carcinomas, and two had additional squamous cell carcinomas as well Table 3.The average age at melanoma diagnosis was 73±11.5 years . The most frequent localisation was the head–neck area (41% of the melanomas). All of these patients were treated orally with anti-Parkinson’s agents. A total of 10 patients received levodopa/benserazide, six received amantadine, five received ropinirole, five received selegiline, three received rasagiline, two received levadopa/carbidopa/entacapone, and two received pramipexole. One patient was treated with rotigotine, biperiden hydrochloride, procyclidine, and carbidopa/levodopa.in situ melanoma (n=3). We had a male patient who had 35 different malignant skin tumours , most of which developed in sun-exposed areas. The patient’s PD was diagnosed 8 years earlier than the melanoma, and he was treated with amantadine and levodopa/carbidopa/entacapone followed by levodopa/benserazide.The average Breslow thickness of all melanomas among male patients was 8.27±10.9 mm. Out of the 13 male patients with melanoma, six had advanced, thick tumours with a high average thickness of 12.6±12.5 mm. Two of these six patients also developed second primary In two of the six female patients, the histological data of the primary melanoma remained unknown. The other four melanomas were thin and belonged to the IA or IB tumour stage.4During the 14 years examined, we found a significant association between PD and skin malignancies, with lower odds of having all skin malignancies or basal cell carcinoma among the PD patients of our department.Some data suggest that the total cancer risk is reduced in patients with PD , 62, 64.LDOC1L, leucine zipper, downregulated in cancer 1-like gene) may have played a pathogenetic role in the development of these malignancies , and the number of squamous cell carcinomas was approximately one-third (10 vs. 26) in women than in men. A higher number of basal and squamous cell carcinomas was also reported in men in the general population, where the risk factors for multiple NMSCs are age, history of ≥10 sunburns, male sex, and family history of melanoma –69. Amongnancies . Our patOur data showed no association between PD and squamous cell carcinoma or melanoma, which contradicts the results of some large-scale meta-analyses –38.LRRK2 G2019S gene mutation carrier PD patients (Some explanations for this contradiction may be raised. For example, Lee et al. did not find increased melanoma risk in their meta-analysis among patients . Geneticpatients , 39, 40.The sex distribution of our patients was in accordance with the literature that reports a higher incidence rate of PD among men than women . In the In conclusion, this is the first study that analysed the association between skin tumours and PD in Central-Eastern Europe. While the majority of the studies focused on the risk of melanoma in PD patients, our examination was extended to other skin malignancies, such as basal and squamous cell carcinoma. The risk of all skin malignancies was significantly lower, which may be related to the similarly decreased risk of basal cell carcinoma in patients with PD. Despite this encouraging result, the OR of melanoma was not influenced. Moreover, we noticed a large proportion of high-risk melanomas among men, mostly in sun-exposed areas, while women had incipient melanomas. In conclusion, UV protection and regular oncodermatological screening remain highly important, especially for male patients treated for PD, to prevent or to notice potentially lethal melanoma tumours earlier.The original contributions presented in the study are included in the article/supplementary material. Further inquiries can be directed to the corresponding author.The study plan was reviewed and approved by Semmelweis University Regional and Institutional Committee of Science and Research Ethics. Approval number: 209/2021.Conceptualization: BS and VT; methodology: BT; investigation: DK; software: KL and JS; original draft preparation: SD; review and editing: EK; supervision: SK and PH. All authors contributed to the article and approved the submitted version.


Patients with non-muscle invasive bladder cancer (NMIBC) have a high possibility of recurrence after surgery. We aimed to assess the factors associated with tumor recurrence and to construct a nomogram model that can contribute to personalized treatment plans of each patient.496 patients with primary bladder cancer (BC) from 2 centers were retrospectively analyzed. Preoperative neutrophil/lymphocyte ratio (NLR), platelet/lymphocyte ratio (PLR), systemic immune-inflammation index (SII), and traditional clinical parameters were collected, then using univariate and multivariate Cox regression analysis to find out the independent risk factors associated with tumor recurrence among them, and then these independent factors were incorporated into the nomogram model. The internal calibration curves and the external calibration curves were used to verify their usefulness.In the training cohort, 150 patients (43.1%) experienced recurrence. After Cox regression analysis, the independent risk factors affecting recurrence-free survival (RFS) were tumor grade, immediate postoperative instillation therapy (IPPIT), NLR, and SII. These factors were used to construct a model to predict RFS 1, 2, 3, and 5 years of NMIBC patients after surgery. And then, we found that the constructed model outperforms the conventional model in terms of accuracy and predictability, the results were verified by statistical tests.Preoperative inflammatory response markers have a predictive value for postoperative recurrence in patients with NMIBC. The constructed nomogram model can be helpful in guiding personalized clinical evaluation and subsequent treatment. Worldwide, its incidence ranks 10th among malignant tumors, and the incidence has steadily increased recently, causing a serious impact on patient survival . Accordiin situ (CIS). Although these traditional clinical parameters have been used to predict recurrence and progression in patients with BC, their accuracy still needs to be improved  with or without intravesical instillation, depending on the patient’s risk stratification. Despite complete resection and adjuvant intravesical instillation, disease recurrence occurs in up to 70% of these patients . Therefoimproved , 5. Receimproved , 7. In fRecently, the interaction between the inflammatory response of the body and the development of cancer has received increasing attention, as the inflammatory response can stimulate cellular oxidative damage and gene mutation and provide a favorable microenvironment for the growth of tumor cells, which in turn accelerates tumor cell proliferation, invasion, and metastasis , 9. Simi22.1yongchuan hospital of Chongqing Medical University, and the second affiliated hospital of Chongqing Medical University, who were treated by TURBT from 2012 to 2022. The inclusion criteria were as follows: 1. histopathological diagnosis of NMIBC; 2. no history of preoperative or postoperative pelvic radiotherapy; and 3. patients with complete clinical, laboratory, and follow-up data. The exclusion criteria were as follows: 1. the surgery did not correspond to the standard TURBT procedure, or patients received two or more bladder tumor resections or total or subtotal bladder resections; 2. concomitant systemic autoimmune diseases, blood diseases, and other diseases affecting blood inflammatory markers; 3. bladder carcinoma in situ (CIS): excluded due to poor differentiation, high risk of muscle invasion and high-grade aggression; 4. Types with variant histologies (VHs) include squamous, glandular, micropapillary, nested, and so on; they affect the patient’s disease-free survival (DFS) and cancer-specific survival (CSS)  ; 5. thos2.2Clinical, histopathological, and demographic characteristics of the patients and hematological indicators within 2 weeks before surgery were obtained from the electronic medical record system. NLR was defined as the ratio of neutrophil count to lymphocyte count, PLR was the ratio of platelet count to lymphocyte count, and SII was the ratio of the product of neutrophil count and platelet count to lymphocyte count. According to previously published studies, the threshold values for high NLR, PLR, and SII were set at 2.5, 150, and 525, respectively , 16. The2.3All patients received complete TURBT. The postoperative chemotherapy regimens were determined according to international guidelines, the patient’s condition, and what was observed intraoperatively. The chemotherapy regimen included immediate postoperative instillation therapy (IPPIT) (within 24 hours of surgery), early postoperative instillation chemotherapy, which was conducted once a week (4–8 weeks after surgery), and continuous bladder instillation chemotherapy, which was conducted once a month (lasting for 6–12 months) immediately after the completion of early instillation chemotherapy. According to international guidelines, all patients in the low-risk group completed only early postoperative instillation chemotherapy, and the middle–high risk patients completed early postoperative instillation chemotherapy and subsequent continuous instillation chemotherapy . Accordi2.4The statistical analysis was performed using SPSS 27.0 and R 4.2.1 software. t tests and rank sum tests were employed to analyze continuous variables, while Fisher exact tests and chi-square tests were applied to categorical variables. To examine the risk factors affecting the prognosis, the Cox regression model (SPSS software) was applied. Differences between groups were assessed with the log-rank test. All of the above tests were considered statistically significant with differences at P < 0.05.Patients from Yongchuan Hospital of Chongqing Medical University were designated as the training cohort, and the factors associated with recurrence were included in the univariate Cox regression for analysis. Then, the statistically significant indicators were included in the multivariate Cox regression analysis, and finally, the prognostic independent risk factors were obtained. These prognostic independent risk factors were constructed into the nomogram model using the R software to predict patients’ RFS at 1, 2, 3, and 5 years postoperatively. Calibration curves were then used for internal and external validation. The optimal threshold of 1-year RFS for patients was determined using the receiver operating characteristics (ROC) curve and Youden index ([sensitivity + specificity] − 1), and the patients were divided into high and low RFS groups based on this threshold. The survival curves of the two groups were plotted using the Kaplan–Meier curve, and the nomogram was validated using Harrell’s concordance index (C-index). To further validate the predictive efficiency of this model, we will compare the C-index of the different models by comparing this model with the traditional model.33.1yongchuan hospital of Chongqing Medical University as the training cohort and 148 patients from the second affiliated hospital of Chongqing Medical University as the external validation cohort. Overall, the median age of the patients was 69 years (range: 23–90 years), and the median follow-up time was 55 months (range: 3–125 months). Twenty-four patients (4.8%) died from recurrence or metastasis of bladder cancer. In the training cohort, 150 (43.1%) patients experienced recurrence, with a median follow-up of 55 (4–125) months and a median RFS of 25 (4–125) months; in the validation cohort, 58 (39.2%) patients experienced recurrence, with a median follow-up of 60 (4–118) months and a median RFS of 30 (4–116) months. The patients’ basic clinical information from the training group and validation group are listed in This study finally included 348 patients from 3.2The collected potential factors that may be associated with recurrence were included in the univariate regression analysis, which ultimately showed that smoking, tumor stage, pathological grade, number, IPPIT, NLR, PLR, and SII were associated with RFS . These factors were then included in the multivariate analysis, which showed that tumor grade, IPPIT, NLR, and SII were independent risk factors for RFS Table 2.3.3Independent risk factors affecting RFS in inpatients were included in the plotting of the nomogram, and a nomogram model for predicting RFS in patients with NMIBC at 1, 2, 3, and 5 years was obtained Figure 13.4In the training cohort, the ROC curve and Youden index indicated that the optimal threshold for the model to predict 1-year RFS was 84.5%  , vascular endothelial growth factor (VEGF), elastase, and matrix metalloproteinases , 20. LymIn this context, some inflammatory markers based on the above two or three types of cells have been proposed to predict tumor prognosis, such as NLR, PLR, and SII. These inflammatory markers can better reflect the body’s inflammatory response and the dynamic balance of antitumor effects than single inflammatory cells, so they have a certain predictive effect on the prognosis of tumors.In addition to TURBT, bladder infusion chemotherapy is also an important adjuvant treatment for NMIBC patients. Previously, several studies have reported that postoperative instillation chemotherapy is effective in reducing local tumor recurrence, especially IPPIT, which was more effective in reducing the 5-year recurrence rate of patients based on its ability to promptly kill intraoperatively disseminated tumor cells and residual tumor cells at the trauma site , 25. TypPathological grade, IPPIT, NLR, and SII were independently linked with RFS in this study when inflammatory factors were combined with conventional clinicopathological factors. In the end, the combination of the aforementioned elements results in the construction of a nomogram model for predicting postoperative recurrence in NMIBC patients, which may roughly predict the recurrence of patients after surgery. Previous studies have found that NMIBC patients tended to experience recurrence one year after surgery; in this study, the optimal cutoff value of 1-year RFS for patients was calculated using the ROC curve; and the results showed that the 1-year RFS rate was significantly higher in the high RFS group than in the low RFS group . TherefoIn recent years, variant histology BC is an emerging topic and several studies have reported their impact on the prognosis of both NMIBC and MIBC. A study analyzed VHs of high-grade T1 (HGT1) bladder urothelial carcinoma and found that it was identified as a significant predictor of DFS. They considered patients with micropapillary, nested, or basaloid morphology or glandular divergent differentiation carcinoma as high-risk HGT1, while the presence of divergent squamous differentiation, inverted growth, microcystic, and villouslike or lymphoepithelioma-like carcinoma was considered as low-risk HGT1 . FrancesOf course, this study had certain limitations. First, some of our limitations are inherent in retrospective and multicenter studies, with surgeon experience and treatment strategies varying from institution to institution. Second, due to the presence of telephone follow-up, the specific situation of recurrence or progression is not yet known, so this study does not distinguish between tumor recurrence and progression. Additionally, we did not collect some nutrition-related markers and biomarkers, and there are currently no standardized cutoff points for inflammatory markers. Despite our exclusion criteria for patients with VHs, the fact remains that some pathologists do not recognize or report about one-half of cases with variant histology in their practice . Therefo5In this study, a nomogram model constructed with inflammatory markers and traditional clinical parameters effectively predicted RFS after TURBT in patients with NMIBC. Based on this model, clinicians can provide patients with more ideal personalized treatment plans after surgery to improve patients’ quality of life.The original contributions presented in the study are included in the article/supplementary material. Further inquiries can be directed to the corresponding author.The studies involving human participants were reviewed and approved by Medical Ethics Committee of Yongchuan Hospital of Chongqing Medical University. Written informed consent for participation was not required for this study in accordance with the national legislation and the institutional requirements.Conceptualization, XY and TZ. Formal analysis, YX and WF. Methodology, XY, JP, JL and TZ. Resources, JL and LW. Software, XY and JP. Validation, JP and CL. Writing – original draft, All authors; Writing – review and editing, CL and TZ. All authors contributed to the article and approved the submitted version.


Objective: The objective of this research is to construct a method to alleviate the problem of sample imbalance in classification, especially for arrhythmia classification. This approach can improve the performance of the model without using data enhancement.Methods: In this study, we have developed a new Multi-layer Perceptron (MLP) block and have used a Weight Capsule (WCapsule) network with MLP combined with sequence-to-sequence (Seq2Seq) network to classify arrhythmias. Our work is based on the MIT-BIH arrhythmia database, the original electrocardiogram (ECG) data is classified according to the criteria recommended by the American Association for Medical Instrumentation (AAMI). Also, our method’s performance is further evaluated.Results: The proposed model is evaluated using the inter-patient paradigm. Our proposed method shows an accuracy (ACC) of 99.88% under sample imbalance. For Class N, sensitivity (SEN) is 99.79%, positive predictive value (PPV) is 99.90%, and specificity (SPEC) is 99.19%. For Class S, SEN is 97.66%, PPV is 96.14%, and SPEC is 99.85%. For Class V, SEN is 99.97%, PPV is 99.07%, and SPEC is 99.94%. For Class F, SEN is 97.94%, PPV is 98.70%, and SPEC is 99.99%. When using only half of the training sample, our method shows that the SEN of Class N and V is 0.97% and 5.27% higher than the traditional machine learning algorithm.Conclusion: The proposed method combines MLP, weight capsule network with Seq2seq network, effectively addresses the problem of sample imbalance in arrhythmia classification, and produces good performance. Our method also shows promising potential in less samples. Electrocardiogram plays an important role in the clinical diagnosis and treatment of cardiovascular diseases, but traditional methods only rely on professional physicians to analyze the electrocardiogram, which is not only time-consuming, but also requires high professional knowledge of doctors. Therefore, many researchers have been working on computer-aided diagnosis to improve the efficiency of ECG analysis .Traditional machine learning arrhythmia classification methods rely on well-designed feature extraction methods and classification models to show acceptable arrhythmia classification performance. Compared with traditional machine learning algorithms, which require complex feature extraction, deep learning end-to-end ECG classification has attracted more and more attention . Among tAlthough the combined method of convolutional network and cyclic neural network has improved the classification performance of arrhythmia, it is susceptible to the influence of sample imbalance, so its performance in the class with a small sample size is not ideal. At present, sampling and weighting are the main methods used by deep learning to solve the problem of sample imbalance in beat classification . MousaviTo this end, in the past we used the method of combining weight capsules  with SeqMulti-layer Perceptron (MLP) is a forward structured artificial neural network ANN. MLP is mainly used as classifier or feature extraction in arrhythmia classification, and has achieved good performance . RecentlCapsule network has great potential for feature extraction and classification of time series data. But at the same time, the common capsule network still has some problems. Some scholars have put forward the capsule variable model successively and achieved good results , but the(1) We propose an arrhythmia classification method combining MWCapsuleNet and Seq2seq, which not only improves the performance of the algorithm model in arrhythmia classification, but also provides a new scheme for alleviating sample imbalance.(2) In order to better exploit the potential of weight capsules, we propose an improved MLP block structure and construct a weight capsule network with MLP. As far as we know, this is the first time that MLP and weight capsule network have been combined and have been applied to arrhythmia classification, which adds a new idea for further exploring the potential of weight capsules.This paper aims to alleviate the problem of sample imbalance in arrhythmia classification and construct an accurate and effective cardiac beat classification method without SMOTE. Inspired by the existing work , we propThe design process of this study is shown in In this work, we use the publicly accessible MIT-Beth Israel Hospital (MIT-BIH) arrhythmia database  to evaluBased on the recommendations of existing studies , we evalThe interpatient assessment model divides the MIT-BIH database into two groups of records: DS1 = {101, 106, 108, 109, 112, 114, 115, 116, 118, 119, 122, 124, 201, 203, 205, 207, 208, 209, 215, 220, 223, 230} and DS2 = {100, 103, 105, 111, 113, 117, 121, 123, 200, 202, 210, 212, 213, 214, 219, 221, 222, 228, 231, 232, 233, 234}. In addition, in order to explore the potential of this work, we divide DS1 (training set) into DS1_2 = {101, 106, 108, 109, 112, 114, 115, 116, 118, 119, 122}, DS1_3 = {124, 201, 203, 205, 207, 208, 209, 215, 220, 223, 230}. Detailed data sample distribution is shown in The MLP block structure designed in this work is shown in p × p, which can be divided into p × p, that is, expand into a one-dimensional vector, and obtain a vector with length p2. S of these vectors are put together to form a tensor of dimension , and the tensor is mapped linearly to the size of the second dimension C, which is called hidden dimension. Then we have a tensor with dimensions of  = patches × channels. It’s made up of S 1 × C vectors. This tensor X ∈ RS×C). Finally, the input sequence for each beat is associated with the vector C ∈ Rd. α is a hyperparameter, α = 0.1.When the length of the capsule module is large, it will lead to the saturation problem of the compression function. In order to alleviate this problem, we proposed a new variant compression function S-S (Sigmoid-squash) , as showThe low-level weight capsule kj  and each element of p represents the probability of each class in the category.The Seq2seq model in this work is the same as that used in the past work, where the encoder encodes the input sequence and the decoder calculates the category of each beat of the input sequence. The encoder is actually composed of long-and-short-term memory (LSTM) units, which are also known as many-to-one LSTM. As shown in In order to better evaluate our approach, we compared different heartbeat feature extraction models under the same equipment and environment and the same Seq2seq classification model as in this work. These model structures are all different combinations of MLP blocks, convolutional layers, ordinary capsules and weight capsules. It mainly includes: Baseline , MLP block, McNets(MLP block + conv), M-Baseline(MLP block + Baseline), CapsuleNets(conv + Capsule), MCapsuleNets(MLP block + Capsule), WCapsuleNets(conv + WCapsule), and CWCapsuleNets(conv block + WCapsule).The structure diagram for each specific model is shown in (1) Normalize the given ECG signal to be between 0 and 1;(2) Search the R-wave set of ECG by the corresponding annotation file in MIT-BIH arrhythmia database;(3) Segment the continuous ECG signal into a series of heartbeats according to the extracted R-wave, and assign a label to each heartbeat according to the annotation file;(4) Resize each heartbeat to a predefined fixed length (280 samples).The input of the model in this work is a series of heart beats. In order to extract heart beats from a given ECG signal, according to the recommendations of existing studies , we use These preprocessing steps for beat extraction are very simple and do not involve any form of filtering or noise removal methods. The source data preprocessing code used in this work is available here.We build the model based on TensorFlow 2.4, using Adam optimizer and cross entropy loss function, with a learning rate of 0.001 and a maximum training period of 500. The initial LSTM hiding and cell state of the Seq2Seq model are set to 0. The drop rate of MLP is set to 0.8.In order to compare with existing studies, four indexes are mainly used in the evaluation: sensitivity (SEN), specificity (SPEC), positive predictive value (PPV) and accuracy (ACC).Here, TP (true positive), TN (true negative), FP , and FN  represent the number of heartbeats correctly labeled, correctly identified as non-corresponding heartbeats, incorrectly labeled, and not identified as expected heartbeats, respectively.Because the sample size of class Q is too small, and the reference methods did not take it into account. Therefore, we do not list it in the comparison results table. When comparing with a wide range of earlier publications, we use the results published in their original papers as a reference. The differences calculated in this work are all differences in percentage points.In order to evaluate the effectiveness of our innovative elements, we evaluate the model performance using DS1 as the training set and DS2 as the test set. It is worth noting that the number of parameters of the model proposed in this work is less than that of the baseline model (252980Three-classification task: As shown in Five-classification task: In order to further evaluate our method, we increase the difficulty of classification. As shown in To better evaluate the performance of MLP block and MWCapsuleNets in our innovative work, we augment the data with SMOTE method, as shown in In order to better tap the potential of our method, we conduct less-sample exploration on the model. Because DS1_2 has a worse sample distribution than DS1 (as shown in In order to alleviate the problem of unbalanced distribution of heartbeat class samples in arrhythmia classification, this work innovatively proposes MWCapsuleNets + Seq2seq, which combines the improved MLP and weight capsule network. It not only has excellent performance on classes with a large sample size, but also obtains satisfactory performance on classes with a small sample size. The performance on less-samples is also expected.Traditional machine learning arrhythmia classification methods achieve acceptable performance through preprocessing, segmentation, feature extraction and classification steps . HoweverCompared with traditional machine learning algorithms, which require complex feature extraction, deep learning end-to-end ECG classification has attracted more and more attention from scholars . As showPrevious studies have shown that capsules can classify rare ECG beats , which hIn order to better alleviate the problem of sample imbalance in arrhythmia classification, we also consider other innovative elements. The results are shown in Although the MLP + WCapsule + Seq2seq model shows excellent performance, it fails to achieve the expected performance in the classification of class S. Especially in less-sample experiments, as shown in Although our model has achieved good results in arrhythmia classification, it also has the limitations of small sample size and single lead data set. We will further evaluate the dependence of the model on leads. Furthermore, we will also consider combining with the other leads or a random lead. In the future, we will verify our model on a larger 12-lead data set. We will also consider the combination of transformer in tasks with more data or more leads.The MLP + WCapsule + Seq2seq method proposed in this work can effectively alleviate the problem of sample imbalance in arrhythmia classification, and obtain good performance. At the same time, this method also shows a potential performance with less sample, which also provides a new reference for ECG classification to solve the problem of sample imbalance.


Lespedeza cuneata extract in the management of the aging male syndrome.Aging male syndrome is a clinical biochemical syndrome characterized by typical aging symptoms and serum testosterone deficiency. Although it is accompanied by various health problems, directly affects life satisfaction, and requires proper management, no clear prevention or treatment other than hormone replacement therapy is currently available for this syndrome. Here, we aimed to determine the efficacy and safety of the Males aged 43-70 years who provided consent for participation and had a total Aging Males’ Symptom questionnaire score ≥ 37 and testosterone level ≤ 500 ng/dL were enrolled in this study. This study was conducted in a randomized, double-blind manner. Participants were randomly assigned to either the experimental or control groups and orally administered the assigned product twice a day. Efficacy was evaluated by measuring changes in Aging Males’ Symptom score, Androgen Deficiency in the Aging Male questionnaire score, International Index of Erectile Function score, International Prostatic Symptom Score, blood test results, and body mass index at 8 weeks.After 8 weeks, the experimental group had significantly improved symptom scores compared to the control group on the Aging Males’ Symptom and Androgen Deficiency in the Aging Male questionnaires. However, no significant differences in the International Index of Erectile Function score, International Prostatic Symptom Score score, blood test results, and body mass index were observed between the experimental and control groups.Lespedeza cuneata extract safely alleviates andropause symptoms without any significant side effects, suggesting its potential for the treatment of the aging male syndrome. Lespedeza cuneata extract (LCE) had significantly improved symptom scores on the Aging Males’ Symptom and Androgen Deficiency in the Aging Male questionnaires.Significant changes had not been observed in the safety assessment between the control group and the experimental group.This study concludes that LCE has the potential for the treatment of the aging male syndrome without any significant side effects.2 The prevalence of symptomatic androgen deficiency is 5.6% in men aged 30-79 years and increases substantially with age.3Andropause syndrome is a clinical and biochemical syndrome characterized by typical symptoms such as decreased cognitive ability, spatial orientation, fatigue, depression, and mood changes. It occurs due to old age and serum testosterone deficiency.4Andropause, a condition characterized by decreased serum testosterone levels, can be partially managed via lifestyle changes such as maintaining a healthy diet, getting sufficient rest, and doing regular exercise and strength training. Testosterone replacement therapy (TRT) is an important treatment option for this syndrome that can be administered via injection, oral preparations, and transdermal gels. Injections should be administered slowly to prevent adverse side effects such as acne, facial flushing, depression, anxiety, and abnormal liver function tests. Oral preparations need to be administered multiple times daily, which may cause jaundice, gynecomastia, and edema. Transdermal administration of testosterone is another treatment option that requires regular monitoring of serum testosterone levels to ensure proper dosage and frequency of the gel. Despite its potential side effects, TRT is the primary treatment for andropause, as no other prevention or treatment options are currently available for this condition.Lespedeza cuneata are used as functional raw materials for andropause treatment as they alleviate andropause symptoms and improve overall male health without any side effects. These natural products improve testosterone levels, enhance sexual function, reduce fatigue and depression, and promote the overall well-being of the affected males.7 Therefore, further exploration of natural products can aid in the development of safe and effective treatment strategies for andropause.Natural products have gained increasing interest as alternative therapeutic agents for andropause owing to the potential adverse effects of TRT. The MR-10 complex extract (dandelion and fermented leaves of rooibos) and a mixed extract of fenugreek seeds and L.cuneata G. Don, a vascular plant belonging to the Fabaceae family, is a perennial herb with shrub properties that typically grows in lowland grasslands and fields.8 The nourishing effects of L. cuneata on the liver and lungs and its alleviation of symptoms related to male menopause have been detailed in Oriental medical literature.10L.cuneata contains many physiologically active substances, such as pinitol, flavonoids, phenol compounds, and β-sitosterol. Among the flavonoids, quercetin, kaempferol, vitexin, and desmodin have been reported in previous studies.11 A preclinical experiment reported that the oral administration of the L. cuneata extract (LCE) not only alleviated the symptoms of aging but also increased the serum levels of testosterone in aged rats (34-weeks-old).12 Here, we designed a clinical study to confirm the safety and efficacy of LCE in alleviating the andropause syndrome.This study was approved by the Institutional Review Board (IRB) of Dankook University Hospital (IRB Number: DKUH 2020-08-018) and conducted in accordance with the International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use guidelines and ethical principles of the Declaration of Helsinki. Informed consent was confirmed by the IRB.This study was a double-blind, randomized, placebo-controlled, and parallel-group study. Patients were prospectively and consecutively selected between August 2020 and August 2021. Only the eligible patients after the screening test were randomly assigned to either the experimental or control (placebo) group at the first visit (visit 1) and instructed to take their respective products orally for 8 weeks. After the ingestion of the experimental and control products, the patients were evaluated for any changes at the end of the eighth week.Male subjects were eligible if they were aged between 43 and 70 years and had a total score ≥ 37 on the Aging Males’ Symptoms (AMS) questionnaire during screening and a total testosterone level ≤ 500 ng/dL in the blood test. Before randomization, the qualified subjects who voluntarily agreed to participate in the study and provided written informed consent to comply with the clinical trial guidelines were included in the study.2, prostate-specific antigen (PSA) levels ≥ 4.0 ng/mL, and alcoholism. Additionally, patients who had taken phosphodiesterase-5 inhibitors, donated whole blood, or received blood transfusions, androgen or anti-androgen medications, or systemic corticosteroids within the past 2 months were excluded from the study. Patients with a blood pressure ≥ 160/100 mmHg or fasting blood sugar level ≥180 mg/dL were also excluded from the study. Those who had started new medications for diabetes within the past 3 months had a total score ≥ 20 on the International Prostate Symptom Score (IPSS) questionnaire, or had participated in other clinical trials within the past 2 months were also excluded from the study.Exclusion criteria for this study included the following: serum alanine aminotransferase or aspartate aminotransferase levels > 100 IU/L, gamma-glutamyltransferase levels > 100 IU/L, serum creatinine levels > 1.4 mg/dL, uncontrolled psychiatric disorders, body mass index (BMI) ≥ 35 kg/mExperimental and placebo products were orally administered twice daily to the subjects. A single tablet of the experimental product was 800 mg containing 625 mg LCE powder (LCEP), and the daily requirement was 2 tablets containing 1250 mg of LCEP. In the placebo group, dextrin was administered instead of the LCEP. Each product was consumed for 8 weeks, starting from visit 1 (baseline week 0).Before starting the medication, several baseline measurements, including age, blood pressure, comorbidities, IPSS, International Index of Erectile Function (IIEF) score, AMS score, Androgen Deficiency in the Aging Male (ADAM) questionnaire score, homeostatic model assessment of insulin resistance (HOMA-IR) values, serum testosterone, sex hormone-binding globulin (SHBG), serum fasting glucose, total cholesterol, low-density lipoprotein (LDL) cholesterol, high-density lipoprotein (HDL) cholesterol, triglycerides, and insulin levels, were taken. The same measurements were repeated after 4 and 8 weeks of treatment to evaluate the efficacy of the experimental product.Primary efficacy assessment measured any changes in the AMS, ADAM, IIEF, and IPSS questionnaire scores from baseline. Secondary efficacy assessment measured the changes in the serum testosterone, SHBG, serum fasting glucose, serum cholesterol , triglyceride, and insulin levels and HOMA-IR values from the baseline. For safety evaluation, changes in blood urea nitrogen/creatinine (BUN/Cr) and PSA levels were measured, and urine analysis, complete blood count (CBC), and liver function tests were performed. t-tests, chi-square tests, and repeated-measures analysis of variance. Statistical significance was set at P < .05.All values are represented as the mean ± standard deviation. Statistical analyses were conducted using the R statistical program version 3.4.4 . Differences among groups were evaluated usingA total of 103 men aged 43-70 years who scored ≥37 on the AMS questionnaire and had a testosterone level ≤ 500 ng/dL at a single institution participated in this study after providing written consent. Baseline characteristics of the participants did not differ significantly between the experimental and control groups .No significant differences in the baseline total AMS scores were observed between the experimental and control groups. The control group did not show significant differences in the total AMS scores 4 and 8 weeks after placebo administration. In contrast, the experimental group showed significant differences in the AMS scores 4 and 8 weeks after administration of the experimental product. Moreover, significant differences were observed between the 2 groups 4 and 8 weeks after administration of each product ; Table 2No differences in the psychological, somatic, and sexual scores were observed between the experimental and control groups at baseline. However, significant differences were observed between the 2 groups 4 and 8 weeks after treatment ; Table 2The ADAM questionnaire score in the experimental group was 7.0 ± 2.1 at baseline, which decreased significantly to 4.8 ± 3.3 at 4 weeks and 3.8 ± 3.3 at 8 weeks after administration. No significant difference was observed in the control group. The differences between the 2 groups were significant 4 and 8 weeks after the administration of the experimental product ; Table 3In the sexual questions (items 1 and 7) of the ADAM questionnaire, no statistically significant differences were observed in the average number of “yes” responses between the experimental and control groups at baseline and 4 weeks after treatment. However, a significant difference between the 2 groups was observed 8 weeks after treatment ; Table 3IIEF scores significantly increased at 4 and 8 weeks compared to the baseline in the experimental group, whereas no significant change was observed in the control group at 8 weeks. Moreover, no significant differences were observed in the IIEF scores between the two groups at 8 weeks .Both the experimental and control groups showed a significant decrease in IPSS and quality of life scores at 4 and 8 weeks. However, no significant difference was observed between the 2 groups at 8 weeks .No statistically significant differences in the laboratory test results, including changes in SHBG, serum testosterone, serum glucose, and insulin levels, serum lipid profile, and HOMA-IR values, were observed between the experimental and control groups at 8 weeks .No statistically significant differences in the BMI were observed between the experimental and control groups during either period or group comparisons. Additionally, no changes were observed in the CBC, BUN/Cr, PSA levels, and factors in urine analysis between the 2 groups (data not shown).13Aging male syndrome  is a clinical and biochemical syndrome characterized by the symptoms of aging and serum testosterone deficiency. Typical symptoms include decreased libido, impaired sexual function, depression, and lethargy. However, unlike menopause in women, symptoms do not appear rapidly in men and do not affect all men. Excluding testicular aging, factors that can contribute to a decline in testosterone levels include excessive drinking and smoking, chronic stress, poor nutritional status, obesity, hypertension, and various chronic conditions such as diabetes, hyperlipidemia, and liver disease. Unhealthy dietary habits and lifestyle are also important causes of andropause.7Restoring the decline in testosterone levels is the primary goal of treating andropathic symptoms. However, testosterone is mostly administered via injection, with only a small portion available in transdermal or oral forms. This can be challenging for individuals who fear needles. In addition, long-term treatment is necessary to achieve sustained benefits. When taking testosterone over a prolonged period, it is important to monitor for potential risks, such as prostate cancer and thrombosis, and cost, as it can be a concern. Therefore, andropause treatment should prioritize improving the overall quality of life rather than solely treating the condition. Therefore, safer and more convenient treatment options are needed to ensure continued patient satisfaction and improved quality of life. One treatment option is to take natural products that can delay or prevent the development of andropause or increase the serum levels of testosterone.15 Self-diagnostic methods are also used for andropause; in clinical studies, AMS is used to screen patients with andropathy. Park et al6 and Lee et al16 enrolled subjects with a total AMS score of 27. Although diagnostic methods for andropause include biochemical tests to measure testosterone levels and screen for hyperlipidemia, it is difficult to diagnose andropathy. This is because of 2 reasons: the accepted lower limit for serum testosterone levels and the diagnostic methods for andropause that are influenced by factors such as lifestyle and stress.14 Previous study suggested that testosterone levels > 500 ng/dL are effective in healthy males.17 Another study showed that males with >500 ng/dL of testosterone do not require any treatment, but patients with 250-350 ng/dL testosterone need testosterone therapy, and periodically measuring serum testosterone levels is recommended in aged males with 300-500 ng/dL testosterone.18 Therefore, we enrolled subjects by screening them with a total score of more than 37 on the AMS, which indicates moderate or severe symptoms, rather than with a testosterone level of <350 ng/dL. This clinical study included participants aged 43-70 years who had a total score ≥ 37 on the AMS questionnaire and testosterone level ≤ 500 ng/dL.Here, we performed a double-blind, randomized, placebo-controlled, and parallel-group study. The AMS and ADAM questionnaires are internationally well-adopted for andropause diagnosis.Eurycoma longifolia extract (Physta®), a mixed extract of Trigonella foenum-graecum seed and L. cuneata, and a complex of Korean dandelion and rooibos on andropause-related symptoms by presenting the decreased scores of ADAM and AMS.19 Consistent with the results of previous studies, the current study showed the significantly decreased total AMS score and the mean of positive number of all questions in ADAM in the experimental group compared to those in the control group, indicating that LCEP has the ability to improve the health of aged males with andropause. Contrary to the results for AMS and ADAM, no significant differences were observed in the IIEF scores between the control and experimental groups. However, while the control group did not show any significant changes in IIEF scores, the experimental group showed improved performance from with baseline. These findings suggest that the duration of the study may have been too short to observe changes in the IIEF score. Therefore, long-term clinical trials are needed to investigate the effects of LCEP on these parameters.Primary efficacy assessment measured the changes in the AMS, ADAM, IIEF, and IPSS scores from the baseline. In the AMS and ADAM questionnaires, the experimental group had significantly improved symptom scores compared to the control group after 8 weeks; however, no significant improvement was observed in the IIEF questionnaire score. The AMS and ADAM scores revealed that all patients in the experimental group reported satisfaction with the treatment compared to the control group, even within a short period of 2 months. Previous studies suggested the alleviating effects of 12 no significant differences were observed in the serum testosterone levels in this clinical study. However, LCEP decreased the total scores of AMS and ADAM compared to those in control groups at 8 weeks. These discrepancies may be due to the androgen receptor and dosage of the extract. LCEPmay increase the expression of androgen receptors in the testes of subjects. Androgen receptors are nuclear receptors that regulate physiological phenomena, such as spermatogenesis and Leydig cell proliferation, by binding to testosterone, translating in the nucleus, and regulating the transcription of DNA.20 Androgen receptor in the testes are used as biomarkers that positively regulate the symptoms of andropause. Several studies have examined the changes in the expression levels of androgen receptors in animals and checked the sequence of androgen receptors in humans.23 In addition, some pharmaceutical companies have developed androgen receptor regulators.24 Oral administration of LCE to rats at 150 and 300 mg/kg/day increased the RNA expression levels of the androgen receptors,20 suggesting that LCEP regulates the androgen receptor expression to improve the andropause symptoms in affected patients. The second possible explanation may be the dosage. The dosage of 1.25 g/day of LCEP used in this study is equivalent to approximately 150 mg/kg/day of LCE in rats, according to the calculation method suggested by Shin et al25 Previous studies had presented the result that, in terms of increasing testosterone, significant effect was shown on the high dosage of LCE at 250 and 300 mg/kg/day to rats, but the low dosage of LCE at 125 and 150 mg/kg/day was not effective to rats.20 Thus, further clinical trials are necessary to determine whether LCEP regulates the serum levels of testosterone at dosages >2.5 g/day.Secondary efficacy assessment measured the changes in serum testosterone, SHBG, serum fasting glucose, and serum cholesterol  levels from the baseline. Additionally, the safety assessment measured the changes in CBC, BUN/Cr, urinalysis, PSA levels, serum glucose levels, and serum lipid profile from the baseline. Changes in major variables, such as total cholesterol, HDL cholesterol, LDL cholesterol, and fasting blood glucose levels, after LCEP ingestion did not differ between the experimental and control groups. Although LCE alleviated aging symptoms and increased serum testosterone levels in aged rats,L. cuneata has many physiologically active substances, such as quercetin, kaempferol, vitexin, pinitol, β-sitosterol, daidzein, and desmosin.26 Phytochemicals exert positive effects on male menopause. For instance, daidzein increases the levels of dehydroepiandrosterone, a testosterone derivative, in middle-aged male Wistar rats.27 Quercetin increases the activities of 3β- and 17β-hydroxysteroid dehydrogenase, which are key enzymes in the synthesis of testosterone.28 β-Sitosterol acts as an inhibitor of 5α-reductase, blocking the degradation of testosterone to dihydrotestosterone in the prostate of hamsters.29 Li et al30 reported that vitexin alleviates sexual dysfunction and fertility impairment in streptozotocin-treated male mice by regulating the hypothalamus-pituitary-gonadal axis. Each compound has its own molecular mechanisms affecting spermatogenesis, testosterone content, and fertility impairment. Thus, the phytochemical complex in LCEP exerts the major positive effects in alleviating the andropause symptoms in aged males.In conclusion, LCEP improved the symptoms of andropause with minimal side effects in this study, making it a promising option for andropause treatment. However, additional clinical trials of longer duration are needed to validate our findings and evaluate the long-term safety and efficacy of LCEP in a larger population.


This article aims at exploring the role of hypoxia-related genes and immune cells in spinal tuberculosis and tuberculosis involving other organs.In this study, label-free quantitative proteomics analysis was performed on the intervertebral discs (fibrous cartilaginous tissues) obtained from five spinal tuberculosis (TB) patients. Key proteins associated with hypoxia were identified using molecular complex detection (MCODE), weighted gene co-expression network analysis(WGCNA), least absolute shrinkage and selection operator (LASSO), and support vector machine recursive feature Elimination (SVM-REF) methods, and their diagnostic and predictive values were assessed. Immune cell correlation analysis was then performed using the Single Sample Gene Set Enrichment Analysis (ssGSEA) method. In addition, a pharmaco-transcriptomic analysis was also performed to identify targets for treatment.The three genes, namely proteasome 20 S subunit beta 9 (PSMB9), signal transducer and activator of transcription 1 (STAT1), and transporter 1 (TAP1), were identified in the present study. The expression of these genes was found to be particularly high in patients with spinal TB and other extrapulmonary TB, as well as in TB and multidrug-resistant TB . They revealed high diagnostic and predictive values and were closely related to the expression of multiple immune cells . It was inferred that the expression of PSMB9, STAT 1, and TAP1 could be regulated by different medicinal chemicals.PSMB9, STAT1, and TAP1, might play a key role in the pathogenesis of TB, including spinal TB, and the protein product of the genes can be served as diagnostic markers and potential therapeutic target for TB.The online version contains supplementary material available at 10.1186/s12920-023-01566-z. Mycobacterium tuberculosis infects about 1/4 of the global population, of which approximately 10 million develop active TB and 1.6 million die from it , and acetyl [protein N-term] modifications were used. Carbamidomethyl [C] was set as the fixed modification (maximum of three variable modifications). Peptides without variable modifications were used for quantification, with an FDR of 0.01. Ten samples were standardized, and missing values were imputed using Perseus software. Protein groups with fewer non-missing values than biological replicates were removed. LFQ quantification results were log-transformed.To identify differentially expressed proteins between spinal TB and controls, we performed differential analysis of the normalized quantitative results using the “limma” package. | logfc | > 1 and p-value < 0.05 were set as the conditions for screening differentially expressed proteins , 24. To To further explore the biological functions of these differential proteins, we used the “clusterprofiler” package for gene ontology (GO) and Kyoto encyclopedia of genes and genomes (KEGG) enrichment analyses –27. In aWeighted gene co-expression network analysis (WGCNA) is a system biology method used to describe the gene association pattern between different samples. It can be used to identify the gene set with highly synergistic changes and identify the gene set with the strongest correlation with the disease according to the interconnection of the gene set and the association between a gene set and phenotype. It is widely used in the research of diseases and other traits and gene association studies . In thisIn this study, we investigated the role of hypoxia-related proteins in spinal TB by intersecting the two most disease-related modules in WGCNA with a set of all hypoxia-related genes in humans downloaded from the Molecular Signatures Database (version 7.5.1) and differential proteins . Later, https://www.ncbi.nlm.nih.gov/geo/). The mRNA expression levels of these hypoxia-related proteins in spinal TB and other extrapulmonary TB from the GSE144127 dataset were extracted for differential analysis. Finally, 11 hypoxia-related genes with consistent changes at the transcriptional and protein levels were obtained. We utilized two machine learning methods, LASSO and SVM-REF, to screen these 11 hypoxia-related genes further. LASSO is a regression analysis method that performs variable selection and regularization while fitting a generalized linear model and selects the best variable by the smallestλvalue [In order to investigate the transcriptome expression level of hypoxia-related proteins closely related to spinal TB in TB, the GSE144127 dataset, GSE83456 dataset, and GSE147690 dataset related to TB were downloaded from the GEO database . DrugBank database integrates the chemical structure and pharmacological action of drugs, as well as the sequence, structure, and physiological pathway of drug action targets . It is aIn this study, 5 cases of intervertebral disc tissue resected during surgery for spinal tuberculosis diagnosed at First Affiliated Clinical Hospital of Guangxi Medical University were taken as a test group, and 5 cases of intervertebral disc tissue resected during surgery for lumbar intervertebral protrusion were taken as the control group. The differences in expression of PSMB9, STAT1, and TAP1 between experimental and control groups were compared by immunohistochemistry. After separating the disc tissue, we immersed it in formalin solution and preserved it within 10 min. We then made immunohistochemical sections and done staining after laboratory operations such as wax sealing, sectioning, antigen repair, antibody hybridization, color development, and tissue sealing. The specimens were observed under the inverted microscope, and the experimental and control group images were collected, respectively. We used Image J software to evaluate the positive rate of all immunohistochemical images and used an independent samples t-test to statistically analyze the positive rate of PSMB9, STAT1, and TAP1 in the experimental group and the control group, respectively, through IBM SPSS Statistics 26.0.Following label-free quantitative proteomic analysis, we obtained 1965 quantifiable proteins. The quantitative repeatability analysis between samples revealed that the quantitative experiment had good sensitivity and reliability . The difference in transcriptional level was significant, in extrapulmonary TB and the control group Fig. A. These Finally, the five machine learning methods of logistic regression, Bayesian logistic regression, decision tree, random forest, and extreme gradient boosting were used to build a prediction model based on these three genes. In the GSE144127 dataset, the accuracies in extrapulmonary TB and the control group were 0.764, 0.764, 0.758, 0.701 and 0.783, respectively  Fig. A-C. AccoIn the GSE147690 dataset, we found that PSMB9, STAT1, and TAP1 were also highly expressed in the multidrug-resistant TB group, and the difference was very statistically significant  . Ribosomal stability is very important for the persistence and latent infection of mycobacteria. Under hypoxic conditions, ribosome-associated factor during hypoxia (Rafh) is the primary factor leading to the hypoxic survival of mycobacteria mediated by response regulator dose . All theIn this study, we screened out three key hypoxia-related genes, PSMB9, STAT1, and TAP1, which were highly expressed at the protein and transcriptional levels in spinal TB. Notably, previous studies have shown that PSMB9, STAT1, and TAP1 are all associated with TB. A meta-analysis integrating the transcriptional expression dataset of whole blood of multiple hosts and integrating and comparing different data through the network method found that there is a highly active core gene group in TB, which is composed of 380 genes, of which STAT1 and PSMB9 are the key hubs of the gene group . PSMB9 iIn addition, PSMB9, STAT1, and TAP1 were also significantly upregulated in the multidrug-resistant TB group. Pharmaco-transcriptomic analysis showed that estradiol, cyclosporine, cisplatin, and other drug compounds could upregulate the expression of PSMB9, while acetaminophen and calcitriol can down-regulate PSMB9 expression. Cyclosporine, dactinomycin, diethylstilbestrol, and other drug compounds can upregulate the expression of STAT1, while 14 kinds of drug compounds, such as afimoxifene, azathioprine, and diclofenac can down-regulate the expression of STAT1, and acetaminophen, estradiol, and methotrexate have effects on the up and down regulation of STAT1. Dactinomycin, daunorubicin, camptothecin, and other drug compounds can upregulate the expression of TAP1, while arsenic trioxide can down-regulate the expression of TAP1. Cyclosporine is an important immunosuppressant. Its main mechanism is to inhibit the activity of the immune system by inhibiting the activity and growth of T cells . DelayedM. tuberculosis antibodies are not completely clear [M. tuberculosis [M. tuberculosis antigens, respond to the BCG vaccine, inhibit the growth of mycobacteria, and are potential vaccine targets against TB [TB is closely related to the immune response in the body but the immune mechanism of anti-ly clear . By anally clear , 66. Denrculosis . Gamma drculosis –70. In tainst TB . We alsoSimilar to other studies, our study also had limitations. First, the sample size was inadequate. Taking into account the analysis of large samples, we only used five pairs of 10 samples for the protein park test, which was insufficient. Second, there are limitations in using routine blood data to check differential immunocyte analysis; tissue-based flow cytometry should be used for further verification. In addition, we do not have more laboratory analysis to verify our results, and this study should be further verified through cell and animal experiments.PSMB9, STAT1, and TAP1, might play a key role in the pathogenesis of TB, including spinal TB, and the protein product of the genes can be served as diagnostic markers and potential therapeutic target for TB.Below is the link to the electronic supplementary material.Supplementary Material 1Supplementary Material 2Supplementary Material 3


The initiation of puberty is a crucial timepoint of development, with its disruptions being associated with multiple physical and psychological complications. Idiopathic Central Precocious Puberty (iCPP) has been correlated with Single-Nucleotide Polymorphisms (SNPs) of certain genes that are implicated in various steps of the process of pubertal onset. The aim of this review was to gather current knowledge on SNPs of genes associated with iCPP. We searched articles published on the PubMed, EMBASE and Google Scholar platforms and gathered current literature. KISS1, KISS1R, PLCB1, PRKCA, ITPR1, MKRN3, HPG axis genes, NPVF/NPFFR1, DLK1, KCNK9Q, LIN28B, PROK2R, IGF-1, IGF2, IGF-1R, IGF-2R, IGFBP-3, insulin, IRS-1, LEP/LEPR, PPARγ2, TAC3, TACR3, Estrogen receptors, CYP3A4 and CYP19A1 were studied for implication in the development of precocious puberty. SNPs discovered in genes KISS1, KISS1R, PLCB1, MKRN3, NPVF, LIN28B, PROK2R, IRS-1 TAC3, and CYP3A4 were significantly correlated with CPP, triggering or protecting from CPP. Haplotype (TTTA)13 in CYP19A1 was a significant contributor to CPP. Further investigation of the mechanisms implicated in the pathogenesis of CPP is required to broaden the understanding of these genes’ roles in CPP and possibly initiate targeted therapies. The timing of pubertal onset has been a topic that has not yet been fully elucidated, despite the extent of research conducted. It is well known that puberty constitutes an important time during one’s life since it is the era of physical and psychological changes that result in the acquisition of reproductive capacity ,2. PuberClinically, the onset of puberty is defined as reaching Tanner and Marshall stage 2 in subjects of both sexes. In girls, thelarche is considered as the landmark of pubertal onset. Puberty is defined as precocious when it starts during a time earlier than that of 95% the individuals belonging to the same population. Thus, even though recent data show a decrease in the mean age of pubertal onset in Western countries, puberty in girls is typically considered precocious when it begins before the age of 6 in black girls and 7–8 in girls of different ethnicities, according to various sources ,5.Much research has focused on the possible implications of precocious puberty and menarche. Pubertal precocity is quite often responsible for low final height due to premature bone maturation . Early mPrecocious puberty can be distinguished as central (CPP) or peripheral (PPP) ,9. CPP, The human genome is known to be 99.5% identical among different individuals. Diversity in phenotypes occurs because of deviations in the remaining 0.5% along with epigenetic modifications . Single-The aim of this study was to record the SNPs among different genes related to pubertal precocity and elucidate the mode of their functions. We attempted to gather elements concerning the effect of SNPs on precocious puberty phenotypes. We analyzed the mechanism of function of these genes and studied the role of the genetic deviations found to be statistically related to CPP in the creation of CPP phenotypes, wherever that was possible.The study was conducted according to the instructions of the Preferred Reporting Items for Systematic Reviews and Meta-analyses (PRISMA) statement. The study protocol included the following steps: (i) original systematic literature search; and (ii) selection of appropriate studies to be included. KT and GP conducted a search of the appropriate literature simultaneously and independently. After thoroughly examining all titles and abstracts that appeared during the research, the two researchers selected articles that were relevant to the topic of interest and cooperated for a more detailed study of relevant literature. The time limit set during this process included studies that had been published until 25 May 2021.The first database used was PubMed. The search was conducted using two groups of keywords. The Medical Subject Headings (MeSH) database was used for the identification of synonyms. These two groups were combined by the Boolean “AND” and the terms utilized within these search categories were combined by the Boolean “OR”. The full search strategy used for Pubmed was:  OR (precocious puberty)” [All Fields] . The initial search revealed 47 studies. After reading all titles and abstracts, 8 articles were excluded because of an evident lack of relevance. Subsequently, 8 more articles were excluded from this study since incompatibility with the aims of the study was detected. More precisely, articles containing male-only populations, research on genes based exclusively on animal models, and articles examining the role of specific SNPs on specific endocrinopathies, other than CPP, were excluded, leaving 31 articles eligible for review. Expansion of the research in Google Scholar and EMBASE revealed 4 more eligible articles that were included in our research. Thus, in total, 35 articles examining the association of SNPs and haplotypes with the occurrence of CPP were finally included in our study. For every gene with an established association to CPP, after genetic and statistical analysis, brief research for the mode of function was performed, followed by search of additional original articles indicating specific genetic polymorphisms’ correlation to CPP. Only original research articles were included in this systematic review.The method of study selection is more thoroughly described in KISS 1: The KISS1 gene is located in the long arm of the first chromosome (1q32) . It consTwo studies, one conducted in Chinese Han girls and one in Korean girls discovered a possible implication of the same SNP in the position 54650055 (G > T) with CPP ,20. LuanKISS1R: KISS1R, formerly known as GPR54 is a gene of 3 kb, situated in the 19q13.3 region. It is the gene that codes a G-protein coupled receptor, consisting of 398 aminoacids and going through the cellular membrane 7 times. It is a peptide expressed in many tissues, including the placenta, pituitary, pancreas, and spinal cord. There seems to be a gradual increase in the levels of GPR54 in animal models during the pubertal years  (p = 0.013), and [IGF-2(3580) + IGFBP-3] (p = 0.036) [IGF-1, IGF2, IGF-1R, IGF-2R, IGFBP-3, insulin: The IGF pathway consists of IGF-1, IGF-2, IGF receptors  and 6 IGF-binding proteins (IGFBPs) that interfere with IGF transfer and protect from protein degradation in the periphery . IGF-1 i= 0.036) .IRS-1: Insulin receptor substrate 1 is implicated in insulin metabolic functions. The hormone binds to its transmembranic receptor (IR), which in turn is activated by autophosphorylation and phosphorylates IRS. Thus, after a long molecular pathway RNA, protein and glycogen synthesis are induced . IRS-1 9PPARγ2: The PPARγ2 genetic polymorphisms were examined considering their relation to the IGF pathway. The Pro12Ala SNP, however, did not seem to exert any effect on the timing of adrenarche, even though the Ala12 variant was associated with lower growth potential .TAC3 and TACR3: TAC3 is the gene responsible for preprotachykinin B production, which is then cleaved to neurokinin B (NKB). TACR3 codes the NK3R receptor, belonging in the rhodopsin family of receptors, which is coupled with a Gq-protein. A Turkish study examining the genetic base of familiar IHH highlighted the contribution of NKB and NK3R in regulation of the reproductive system . The NKBEstrogen receptors: Estrogenic stimulation of hormone-sensitive tissues constitutes the final step in the molecular cascade of events, leading to pubertal development. Estrogens exert their actions by binding to intracellular estrogenic receptors in their target tissues. Two estrogen receptors, ERα and ERβ, have been discovered. The function of these receptors might be affected by their genetic structure, as shown by Lee et al. . Their sXbal (351 A/G) and Pvull (397 T/C) are among the most-investigated polymorphisms of the ERα gene. These are intronic sequences, residing in intron 1 . StavrouCYP3A4: Cytochrome P450 (CYP) enzymes are proteins that contain heme and participate, among others, in metabolic pathways of various hormones . CYP3A4,13 was possibly linked to CPP and higher concentration of estrogens, whereas SNPs that were found did not have any statistical significance [CYP19A1: This gene encodes aromatase, an enzyme that is responsible for androgen to estrogen conversion. Thus, aromatase activity might affect the levels of estrogens. Further genetic analysis revealed polymorphisms in the repeat number of the (TTTA) sequence in intron 4. More precisely, the number of repeats varied between 7 and 13. (TTTA)ificance .LEP/LEPR: LEP codes leptin, while LEPR codes leptin receptor . A leptiThe most important SNPs and haplotypes that have been linked to CPP, in an either triggering or protective manner, are shown in Given the physiologic functions of the abovementioned genes, mutations leading to gain of function in the KISS1/KISS1R pathway genes along with loss of function mutations in MKRN3 and DLK1 were extensively studied for their potential causative effects in the presentation of CPP.As discussed above, numerous polymorphisms have been found to play a statistically significant role in the pathogenesis of CPP. Polymorphisms in the KISS1/KISS1R pathway, the MKRN3, as well as the genes of the HPG axis are among the most commonly involved as statistically significant contributors of CPP, having either triggering or protective functions . HaplotyMKRN3, DLK1, KCNK9, and LIN28b are recognized as imprinted genes, and have all been implicated in CPP investigation. This means that gene expression is epigenetically permitted or prohibited, depending on parental origin. Epigenetic modifications utilise DNA methylation and demethylation mechanisms to modify genetic expression in a dynamic pattern throughout mammalian development ,73,74. TAnother keystone of our study is the great number of SNPs with significant correlations with CPP that are situated in regions that are not coded during the translation process. Interestingly, this is a common finding, as genome-Wide association studies (GWAS) have indicated that most SNPs with some correlations with complex diseases reside in untranslated regions of the human genome ,76. ThisAnother notable finding is that the frequency of diverse polymorphisms is different among patients with different ethnic backgrounds. This reinforces the need for extended research and sequencing among females all over the world to discover more related polymorphisms and clarify the pathophysiology of pubertal precocity.A final point of interest is the deviation in CPP incidence among the two sexes. As discussed above, ICPP is known to be significantly more common among females ,78,79. TIn conclusion, the genetic basis of CPP has been discussed extensively in the past. Multiple genetic polymorphisms among diverse genes have been studied, with some genetic polymorphisms appearing as correlated with precocious puberty phenotypes. Evaluation of family history in individuals affected by CPP is a prerequisite, especially when idiopathic precocious puberty is suspected. In this direction, some even suggest systematic genetic testing in the presence of a positive family history . In any


Ghrelin is an orexigenic neuropeptide that is known for stimulating the release of growth hormone (GH) and appetite. In addition, ghrelin has been implicated in addiction to drugs such as nicotine. Nicotine is the principal psychoactive component in tobacco and is responsible for the reward sensation produced by smoking. In our previous in vitro superfusion studies, it was demonstrated that ghrelin and nicotine stimulate equally the dopamine release in the rat amygdala, and ghrelin amplifies the nicotine-induced dopamine release in the rat striatum. However, less attention was paid to the actions of ghrelin and nicotine in the bed nucleus of the stria terminalis (BNST). Therefore, in the present study, nicotine and ghrelin were superfused to the BNST of male Wistar rats, and the dopamine release from the BNST was measured in vitro. In order to determine which receptors mediate these effects, mecamylamine, a non-selective nicotinic acetylcholine receptor (nAchR) antagonist, and GHRP-6, a selective growth hormone secretagogue receptor (GHS-R1A) antagonist, were also superfused to the rat BNST. Nicotine significantly increased the release of dopamine, and this effect was significantly inhibited by mecamylamine. Ghrelin increased dopamine release even more significantly than nicotine did, and this effect was significantly inhibited by GHRP-6. Moreover, when administered together, ghrelin significantly amplified the nicotine-induced release of dopamine in the BNST, and this additive effect was reversed partly by mecamylamine and partly by GHRP-6. Therefore, the present study provides a new base of evidence for the involvement of ghrelin in dopamine signaling implicated in nicotine addiction. Nicotine is the principal psychoactive component in tobacco that is responsible for the reward sensation produced by smoking . NicotinGhrelin is an orexigenic neuropeptide that is known for stimulating the release of growth hormone (GH) and appetite ,18. IsolAs regards the interaction between ghrelin and nicotine, previous studies focused mainly on their actions on the typical dopaminergic areas, such as the VTA, SNc, striatum, and amygdala, in which ghrelin and nicotine had a similar stimulatory effect on the extracellular dopamine output ,32,34,35Therefore, in the present study, nicotine and ghrelin were superfused to the BNST of male Wistar rats, and the dopamine release from the BNST was measured in vitro. In order to determine which receptors mediate these effects, mecamylamine, a non-selective nAchR antagonist, and GHRP-6, a selective GHS-R1A antagonist, were also superfused to the rat BNST.Male Wistar rats weighing 150–250 g (N = 6) were used for each in vitro experiment. The rats were treated in accordance with the ARRIVE guidelines, and the experiments were carried out in accordance with the EU Directive 2010/63/EU for animal experiments. They were housed together and kept in their home cages at a constant temperature on a standard illumination schedule with 12 h light and 12 h dark periods (lights on at 6:00). Commercial food and tap water were available ad libitum. After all, the rats were decapitated; however, every effort was made to limit the number of animals used and minimize animal suffering. 4, 25 mM NaHCO3, 11.5 mM glucose, 1.2 mM KH2PO4, and 2.5 mM CaCl2, solutions that were provided by Reanal Ltd., Budapest, Hungary. The tritium-labeled dopamine ([3H]DA) and the Ultima Gold scintillation fluid were ordered from Perkin-Elmer Inc., Waltham, MA, USA. The superfusion system itself was purchased from MDE Ltd., Heidelberg, Germany, and the method itself was originally described by Gaddum DA with a concentration of 1 mCi/mL and a specific activity of 60 Ci/mmol was added to the incubation medium with a Hamilton microliter syringe. The BNST slices obtained were incubated in 8 mL of Krebs solution, submerged in a water bath at 37 °C, and gassed through a single-use needle with a mixture of 5% COSix BNST slices were transferred to each of the cylindrical chambers of the superfusion system and superfused with Krebs solution for 30 min. A multichannel peristaltic pump  was used to maintain the superfusion rate constant at 300 μL/min. After 30 min of superfusion, the BNST slices were superfused for 32 min more, and during this time the fractions were collected in Eppendorf tubes by a multichannel fraction collector . The BNST slices were treated with 100 μM nicotine and/or 1 μM ghrelin 20 min after the superfusion had started and eventually pre-treated with 100 μM mecamylamine and/or with 1 μM GHRP-6, a selective GHS-R1A antagonist, 10 min after the superfusion had started. The doses of the agonists (nicotine and ghrelin) and antagonists (mecamylamine and GHRP-6) were based on previous in vitro superfusion studies ,35. After 32 min of superfusion , electrical stimulation was carried out. The electrical stimulation consisted of square wave impulses with a voltage of 100 V, a pulse length of 5 ms, and a frequency of 10 Hz. The electrical impulses were delivered to each of the four chambers for 2 min, as golden electrodes were previously attached to both halves of the superfusion chambers and connected to an ST-02 electrical stimulator. After the next 30 min of superfusion, the remaining BNST slices were removed and solubilized in 200 mL of Krebs solution using an ultrasonic homogenizer . The homogenized slices and the collected samples were mixed with 3 mL of Ultima Gold scintillation fluid in glass vials. The glass vials were transferred to a liquid scintillation spectrometer  that measured the radioactivity emitted from the vials. The fractional [3H]DA release was calculated as a percentage of the radioactivity present in the collected samples relative to that of the remaining slices, which were expressed in counts per minute (CPM). p ≤ 0.05) was accepted as indicating a statistically significant difference. Statistical analysis of the results was performed by repeated measures of analysis of variance followed by Tukey’s post-hoc test for pair-wise comparison . A probability level of 0.05 or less (p < 0.001), an effect that was inhibited significantly by mecamylamine  = 19.78; p < 0.001) (p < 0.001), an effect that was inhibited significantly by GHRP-6  = 16.58; p < 0.001)  = 19.78; < 0.001) . Ghrelin< 0.001) .p < 0.001) (p < 0.001) and GHRP-6  = 13.5; p < 0.05)  = 13.19; < 0.001) , and thi < 0.05) .Nicotine significantly increased the release of dopamine from the rat BNST after electrical stimulation, and this effect was inhibited significantly by mecamylamine, a non-selective nAchR antagonist. This finding is concordant with previous in vivo and in vitro studies, which indicated that nicotine stimulates dopamine release in several subcortical and cortical brain regions ,48,49,50Ghrelin increased even more significantly the release of dopamine from the rat BNST after electrical stimulation than nicotine did, and this effect was inhibited significantly by GHRP-6, a selective GHS-R1A antagonist. This finding is supported by in vivo studies, which reported that the administration of ghrelin induces the concomitant release of ventral tegmental acetylcholine and accumbal dopamine, which is associated with the locomotor hyperactivity induced by ghrelin ,29,30,32Moreover, when administered together, ghrelin significantly amplified the nicotine-induced release of dopamine from the rat BNST after electrical stimulation, and this effect was partly reversed by mecamylamine and partly by GHRP-6 .Our previous in vitro superfusion studies led to similar results, as ghrelin and nicotine stimulated equally the dopamine release in the rat amygdala, and ghrelin amplified even more the nicotine-induced dopamine release in the rat striatum ,31. DespThe present study provides a new base of evidence for the contribution of ghrelin to dopamine signaling implicated in nicotine addiction. In addition, the amygdala and the striatum, a possible place of interaction between ghrelin and nicotine could be the BNST, and the mechanism would be the activation of cholinergic, GABA-ergic, or glutamatergic neurons with an impact on the dopaminergic terminations found at the level of the BNST. However, the intimate mechanism of how ghrelin interacts with nicotine remains to be elucidated. Both animal experiments and human studies indicate the existence of an interaction between ghrelin and addictive drugs, such as nicotine. Taken together, these studies indicate a positive association between smoking and ghrelin level, an association that is reversed during abstinence, particularly among those likely to remain abstinent. Therefore, our preclinical study may have clinical implications, as it suggests that ghrelin may be used as a prognostic tool and the ghrelin receptor may serve as a pharmacological target in the treatment of nicotine addiction.