Silent brain infarction (SBI) is a special type of stroke with no definitive time of onset, which can be found on pre-thrombolysis imaging examination in some patients with acute ischemic stroke (AIS). However, the significance of SBI on intracranial hemorrhage transformation (HT) and clinical outcomes after intravenous thrombolysis therapy (IVT) is uncertain. We aimed to explore the effects of SBI on intracranial HT and the 3-month clinical outcome in patients with AIS after IVT.We consecutive collected patients who were diagnosed with ischemic stroke and received IVT from August 2016 to August 2022, and conducted a retrospective analysis in this study. The clinical and laboratory data were obtained from hospitalization data. Patients were divided into SBI and Non-SBI groups based on clinical and neuroimaging data. We use Cohen’s Kappa to assess the interrater reliability between the two evaluators, and multivariate logistic regression analysis was used to further assess the association between SBI, HT and clinical outcomes at 3 months after IVT.p = 0.560) and favorable outcome  between patients with SBI and Non-SBI. However, patients with SBI had a lower incidence of excellent outcome than the patients with Non-SBI . After adjustment for major covariates, multivariate logistic regression analysis disclosed that SBI was independently associated with the increased risk of worse outcome .Of the 541 patients, 231 (46.1%) had SBI, 49 (9.1%) had HT, 438 (81%) had favorable outcome, 361 (66.7%) had excellent outcome. There was no significant difference in the incidence of HT (8.2 vs. 9.7%, We found that SBI was no effect for HT after thrombolysis in ischemic stroke patients, and no effect on favorable functional outcome at 3 months. Nevertheless, SBI remained an independent risk factor for non-excellent functional outcomes at 3 months. Silent brain infarction (SBI) refers to a patient who has no history of stroke or transient ischemic attacks (TIA), but cerebral infarction or encephalomalacia lesions are found on computed tomography (CT) or magnetic resonance imaging (MRI), with no corresponding symptoms and signs of neurological impairment . CompareIn acute ischemic stroke (AIS) patients without intravenous thrombolysis therapy (IVT), studies have shown that SBI was independently associated with lower stroke severity at admission and good function outcome at discharge . AnotherIn this study, we retrospectively analyzed 541 patients with first-ever ischemic stroke who underwent intravenous thrombolysis therapy. In particular, we examined the HT and clinical outcome after intravenous thrombolysis therapy in the SBI and Non-SBI groups. We further determined the influence of SBI on the risk of worse outcome for patients with AIS following intravenous thrombolysis therapy.Totally 909 patients who suffered from an ischemic stroke within 4.5 h of onset and received recombinant tissue plasminogen activator (rt-PA) thrombolytic therapy in the emergency green channel of the First Affiliated Hospital of Soochow University between August 2016 and August 2022 were enrolled in this study. Eligible patients were further included if they had: (1) no history of stroke or TIA, (2) completed intravenous thrombolysis therapy, (3) completed a cranial CT scan within 24 h after intravenous thrombolysis therapy, or (4) completed head MRI examination within 1 week. On the other hand, patients were excluded if they had: (1) history of stroke or TIA, (2) not completed intravenous thrombolysis therapy, (3) intravenous thrombolysis combined with thrombectomy, (4) lacking imaging data，or (5) lacking laboratory data. The flow chart of the study is shown in The standard dosage was 0.9 mg per kilogram of body weight . The low dosage was 0.6 mg per kilogram of body weight . All patients received only one dose.We recorded the following baseline information of patients: gender, age, NIHSS score on admission, systolic and diastolic blood pressure on admission, head CT and MRI imaging examinations, and TOAST classification. We also collected previous medical history of patients including hypertension, diabetes mellitus, hyperlipidemia, atrial fibrillation, smoking and drinking history, and antithrombotic medication history . Consequently, we estimated the 3-month outcome using the modified Rankin Scale (mRS). We defined mRS scores ≥3 as poor outcome and mRS scores ≤2 as favorable outcome, mRS scores ≥2 as worse outcome and mRS scores 0 or 1 as excellent outcome. The 3-month follow-up data was obtained by trained nurse through outpatient visits or telephone contact with patients or relatives.MRI data were acquired using a clinical 3.0 Tesla MR scanner, whereas follow-up CT imaging was performed using a clinical 64 slice CT scanner. Specifically, structural MRI comprised of transversal diffusion-weighted imaging (DWI), fluid-attenuated inversion recovery sequence (FLAIR), T1WI, and T2WI. Silent brain infarction on MRI lesions ≥3 mm, were characterized as hypointense on T1WI and hyperintense on T2WI  of ≤0.1 corresponds to no agreement, 0.1 < k ≤ 0.4 weak agreement, 0.4 < k ≤ 0.6 good agreement, 0.6 < k ≤ 0.8 strong agreement and 0.8 < k ≤ 1 complete agreement. Statistical analyses were performed using the statistical package for social sciences, version 22.0 .Kolmogorov–Smirnov test was used to test normal distribution of the continuous variables. Normally distributed data were presented as mean and standard deviation (SD) and Non-normally distributed data as median with interquartile range (IQR) or counts and percentages. And categorical variables are presented as frequencies and percentages. Moreover, we used student’s k = 0.893, 95%CI: 0.855–0.929, p = 0.000), 49 (9.1%) patients experienced HT , which 18 (3.3%) had sICH . Results for the interrater reliability correspond to a complete agreement. There were 438 (81%) patients with MRS 0–2 and 361 (66.7%) MRS 0–1 at 90 days.A total of 909 patients with AIS who received IVT treatment were screened for eligibility. Of these patients, 172 patients were first excluded because of a previous history of stroke or TIA. 100 patients did not receive cranial MR examination after thrombolysis, and 91 patients received incomplete laboratory data, 5 patients were lost for follow-up. Finally, 541 patients were included in our analysis , and thep = 0.000; 178 (77.1%) vs. 200 (64.5%), p = 0.020; 70 (30.3%) vs. 70 (22.6%), p = 0.040; 77 (33.3%) vs. 77 (24.8%), p = 0.030]. The TOAST subtype distribution also showed statistically significant differences between the two groups (p = 0.008). Furthermore, patients with SBI had higher serum homocysteine levels and shorter activated partial thromboplastin time than those without SBI  and sICH  between the two groups. Although there was no significant difference in the incidence of favorable outcome between the two groups , the incidence of excellent outcome was lower in the SBI group than the Non-SBI group [139 (60.2%) vs. 222 (71.6%), p = 0.005, p < 0.05). In the laboratory test results, glucose level, admission neutrophil and lymphocyte count, serum HDL-C level, serum TG level, serum HCY level, and International Normalized Ratio also significant differences (p < 0.05). The rate of SBI in patients with worse function outcome was higher than that in patients with excellent function outcome (p = 0.005).p < 0.1 in the univariate analysis and the Age- and sex-adjusted analysis were included in the multivariate logistic regression model. After age, sex, and multivariate adjustment, only initial NIHSS , GLU , and admission lymphocyte count , SBI  were significantly related to worse outcomes at 3 months after discharge , but not an independent risk factor for HT and favorable outcome after IVT.To our knowledge, this is the first research on the effect of SBI on hemorrhagic transformation and clinical outcomes after thrombolysis in patients with acute ischemic stroke. We found that SBI was relatively common in patients with a first-ever ischemic stroke, and SBI was an independent risk factor for worse outcome  and eGFR (<60 mL/min/1.73m2) are useful for risk stratification (Silent brain infarction (SBI) refers to lesions discovered via neuroimaging that lack associated clinical symptomology . The prefication . Nacafalfication . In the Studies have shown that SBI is not completely asymptomatic, and oldish people with SBI have an increased risk of dementia and a faster decline in cognitive function than those without such lesions , 17. In Admission glucose level and neutrophil-to-lymphocyte ratio can predict 3-month functional outcome in AIS patients , 22. OurWhen symptomatic cerebral infarction occurs, intravenous thrombolytic therapy with rt-PA within 4.5 h can effectively improve the clinical prognosis of patients after the removal of thrombolytic contraindications . HemorrhHowever, there are some limitations in this study. Firstly, the present study was a retrospective analysis with limited sample size. Second, the findings of this study are limited by selection bias, as patients who did not complete a cranial MRI examination were not included in the MRI-based study, and these excluded patients may be associated with more severe intracranial hemorrhage and poorer clinical outcomes. Third, acute large cerebral infarction occurred in lobes with previous small SBI lesions in the cortex or medulla of these lobes. SBI lesions may be compressed by edema in acute cerebral infarction and cannot be detected by MRI. This hypothesis has not been proven, but it could lead to information bias. Last, we did not further explore the relationship between SBI and post-stroke cognitive impairment and post-stroke depression. Further multi-faceted exploration should be carried out in multi-center study with a large sample size.We found that SBI was no effect for HT after thrombolysis in ischemic stroke patients, and no effect on favorable functional outcome at 3 months. Nevertheless, SBI remained an independent risk factor for non-excellent functional outcomes at 3 months.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by the Ethics Committee of the First Affiliated Hospital of Soochow University. The ethics committee waived the requirement of written informed consent for participation. No potentially identifiable human images or data is presented in this study.YZ, DD, and QF contributed to conception and design of the study. LZ wrote the first draft of the manuscript. SW organized the database. QF contributed the patient follow-up. JiJ and JuJ performed the statistical analysis. YZ and DD revised the manuscript. All authors contributed to manuscript revision, read, and approved the submitted version.This work was supported by the National Key Research and Development Program of China (2017YFE0103700), the National Natural Science Foundation of China (No. 82001219 to DD), the Natural Science Foundation of Jiangsu Province (BK20190183), and the Advanced Research Project of Suzhou University of Natural Science Foundation (SDY2012B25).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


The abundant and rare bacterial and fungal β-diversities changed significantly during secondary forest succession. Network analysis showed no obvious changes in the topological properties  of abundant microbial networks during secondary forest succession. In contrast, these properties of the rare microbial networks in the secondary forest were higher than those in the grassland and shrubland, indicating that rare microbial networks are more responsive to secondary forest succession than abundant microorganisms. Additionally, rare microbial networks revealed more microbial interactions and greater network complexity than abundant microbial networks due to their higher numbers of nodes and links. The keystone species differed between the abundant and rare microbial networks and consisted of 1 and 48 keystone taxa in the abundant and rare microbial networks, respectively. Soil TP was the most important influencing factor of abundant and rare bacterial communities. Successional stages and plant richness had the most important influences on abundant and rare fungal communities, respectively. C:P, SM and N:P were mainly related to abundant and rare microbial network topological properties. Our study indicates that abundant and rare microbial communities, interactions and driving factors respond differently to secondary forest succession.Knowledge of variations in abundant and rare soil microbial communities and interactions during secondary forest succession is lacking. Soil samples were gathered from different secondary successional stages  to study the responses of abundant and rare bacterial and fungal communities, interactions and driving factors to secondary forest succession by Illumina sequencing of the 16S and ITS rRNA genes. The results showed that the  Secondary succession refers to a sequential replacement of plant species over time or due to a natural or human forces disturbance . In receαdiversity and βdiversity , available phosphorus (AP) and land-use type had extremely significant influences on abundant bacteria, while soil pH, EC, soil water content (SM), AP and land-use type had extremely significant influences on rare bacteria in reforestation succession soil. In addition, abiotic factors have also been reported to have important influences on microbial interactions. For example, soil bulk density, altitude, soil moisture  and abiotic factors . For insmoisture , SOC  and is dominated by Quercus aquifolioides, followed by Picea asperata, Sorbus koehneana and Betula albosinensis; the secondary forest represents the late stage of forest recovery (approximately 60 years) and is dominated by Picea asperata, followed by Quercus aquifolioides and Betula platyphylla. The three selected successional stages were similar in climate, slope and geology.The study site is located at the Miyaluo Nature Reserve of Sichuan Province . The region is characterized by a warm summer and a dry winter with a snowy climate. With the annual rainfall precipitation ranging from 600 mm/y to 1,100 mm/y, the mean annual temperature is shown to be 8.7°C. In addition, the soil type in this area is mountain brown soil. This area gradually formed a natural recovery sequence (from grassland to secondary forest) after being extensively deforested from 1918 to 1998 . In thisIn August 2018, we set up approximately 100 m × 100 m sampling areas in grassland, shrubland and secondary forest. For sampling rhizosphere and bulk soil, this study established five plots of grassland (1 m × 1 m), shrubland (5 m × 5 m) and secondary forest (10 m × 10 m). In order to avoid edge effects and spatial autocorrelation, the plots of each successional stage were more than 20 m apart . Detaile4+-N) and NO3−-N was performed using the indophenol blue method as well as the phenol disulfonic acid method following extraction with 2 M KCl, respectively. Total phosphorus (TP) was calculated by molybdenum antimony blue colorimetry after digestion with HClO4-H2SO4. Using the ratio of SOC and TN, the soil C:N was calculated; C:P was calculated by the ratio of SOC and TP, and N:P was calculated by the ratio of TN and TP. All of the soil properties were previously published in Soil EC and pH were determined with a soil-to-water ratio of 1:2.5 by a DDS307 conductivity meter and Mettler-Toledo FE28 digital pH meter, respectively. We calculated SM by drying soil samples for 72 h at 65°C. SOC and TN were measured with a 2400II CHN elemental analyser . The measurement of ammonium (NHw/v) agarose gel electrophoresis. In addition, with a NanoDrop NC 2000 spectrophotometer (Thermo Scientific), the quality of the DNA extracts was calculated. To amplify the ITS2 region of fungi , total DNA was extracted from each soil sample. We evaluated the integrity of the extracted DNA by 1.0% p < 0.05) in plant richness and soil properties among different successional stages by SPSS software . The Wilcoxon test was used to identify the differences in abundant and rare microbial α-diversities among different successional stages. Principal coordinate analysis (PCoA) based on Bray–Curtis distance was conducted to compare the abundant and rare microbial communities among different successional stages. One-way analysis of similarity (ANOSIM) was used to identify the differences in abundant and rare microbial communities between different successional stages. The partial Mantel test with 999 permutations was used to calculate the relationships between abundant and rare microbial communities and environmental factors based on Pearson correlation (p < 0.05) with the “vegan” package. The Pearson correlation analysis was used to analyse the correlations between environmental factors and microbial network topological properties. Partial least squares path modeling (PLS-PM) was conducted to identify the complex relationships between forest succession, plant richness, soil properties and microbial communities with SmartPLS 4 software.One-way analysis of variance (ANOVA) and Tukey’s HSD test were conducted to analyse the differences (p > 0.05), while the rare bacterial Shannon index of shrubland and secondary forest was significantly higher than that in the grassland (p < 0.05) (p > 0.05), while the abundant fungal Chao 1 index of shrubland and secondary forest was significantly higher than that in the grassland (p < 0.05) (p < 0.05)  and rare bacteria and fungi  changed significantly with secondary forest succession  ,C. The a < 0.05) ,D. Moreo < 0.05) . The β-dccession .A total of 7,263 bacterial ASVs were detected in all samples, and abundant and rare bacterial taxa included 166 ASVs and 5,671 ASVs, respectively. The numbers of abundant and rare bacterial phyla were 10 and 29 at the three successional stages, respectively . The abuAccording to the network analysis results, the modularity of abundant and rare microbial networks was >0.4 . The nodGemmatimonas). The rare microbial networks consisted of 49 keystone taxa, including 5 in Acidobacteria , 4 in Actinobacteria , 2 in Bacteroidetes (Microscillaceae and uncultured Flexibacter sp.), 1 in Chloroflexi , 1 in Gemmatimonadetes (Gemmatimonadaceae), 2 in Planctomycetes (Gemmataceae and Zavarzinella), 10 in Proteobacteria , 1 in Verrucomicrobia (Opitutus), 17 in Ascomycota , 2 in Basidiomycota (Hymenogaster and Mycena) and 4 king taxa  had significant influences on the abundant and rare bacterial communities (p < 0.05) (p < 0.05) but no significant influence on the abundant bacterial community (p > 0.05). Among them, soil TP was the most important factor affecting abundant  and rare  bacterial communities (p < 0.05)  and plant richness  were the most vital influencing factors of the abundant and rare fungal communities, respectively  and abundant and rare bacterial and fungal communities. As shown in the results, all the soil factors (except for NO < 0.05) . Plant rmunities . For the < 0.05) . NH4+-N,ectively .β-diversities, respectively , but had a negative effect on the rare fungal β-diversity (path coefficient = −0.578)   . Soil pr= 0.487) .p < 0.05) (p < 0.05) (p < 0.05) (p < 0.05)  . SOC was < 0.05) . C:P, SM < 0.05) . Plant r < 0.05) .β-diversities during secondary forest succession (p = 0.001). This was in line with previous studies in different ecosystems  . Moreover, the α-diversities of the abundant bacterial and fungal communities were significantly lower than those of the rare communities, which was consistent with previous studies in reforestation succession   and soilosystems . This coccession , salt maccession  and glacccession . This reccession . In thisccession , thus shccession ; they caccession  and playNetwork analysis has been extensively adopted for elucidating microbial interactions as well as their responses to environmental changes . In the in vitro culturing experiments. In this study, the abundant keystone taxon Gemmatimonas, which plays a major role in nitrous oxide reduction due to product-specific nitrous oxide reductases  have rarely been studied. Thus, the ecological functions of these microbes are still unknown and need to be focused on in the future. Overall, our study explores the keystone species of abundant and rare microbial communities, providing a theoretical basis for an in-depth understanding of microbial community composition and interactions. Notably, network analysis does not always represent actual microbial interactions, so the keystone species identified by network analysis are statistical. The ecological function and importance of keystone taxa still require further experimental verification   (p < 0.001)   ,D. Plant< 0.001)  and dive< 0.001)  for soil< 0.001) ,D, and t< 0.001) . The PLS< 0.001) . Soil TP= 0.001) . This coudy area . Previouudy area  also fouudy area . The difudy area  and partudy area . The chaβ-diversities of abundant and rare bacterial and fungal communities changed significantly during secondary forest succession. The α-diversities of rare bacterial and fungal communities were significantly higher than those of the abundant communities, indicating that rare taxa might be the main contributors to microbial α-diversities. In addition, rare microbial networks showed more microbial interactions and greater network complexity, and were more responsive to secondary forest succession than abundant microbial communities, indicating that abundant and rare microbial co-occurrence networks responded differently to secondary forest succession. The keystone species varied in abundant and rare microorganisms at the three successional stages, and the numbers of keystone taxa of rare microorganisms were higher than those of abundant microorganisms. These keystone species might participate in plant growth and nutrient cycle during secondary forest succession. Moreover, plant richness and soil properties jointly drove changes in abundant and rare microbial communities and co-occurrence networks during secondary forest succession. However, the main influencing factors of abundant and rare microbial communities and co-occurrence networks were different. This finding provides theoretical knowledge for deeply understanding the mechanism driving microbial dynamics in the process of secondary forest succession.In conclusion, our results showed that both the The original contributions presented in the study are included in the article/QL, YK, and WZ designed this study. QL, YK, WZ, YL, and HH collected the soil samples. HH carried out the soil property measurement. XZ performed the data analysis and wrote the manuscript. QL, YK, and KF revised the manuscript. All authors contributed to the article and approved the submitted version.This work was financially supported by the National Natural Science Foundation of China (grant numbers 41930645 and 32171550); the Second Tibetan Plateau Scientific Expedition and Research Program (2019QZKK0303); the Youth Innovation Promotion Association of the Chinese Academy of Sciences (grant numbers 2021371); the Open Fund of Ecological Restoration and Biodiversity Conservation Key Laboratory of Sichuan Province, Chengdu Institute of Biology, Chinese Academy of Sciences (KXYSWS2202).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Three-dimensional (3D) cell culture models such as spheroids and organoids are widely used in the field of experimental biology. To analyze these 3D experimental models, formalin-fixed paraffin-embedded (FFPE) sections are superior to whole-mount imaging for some experimental purposes, such as exploring samples with a depth limitation of primary antibody penetration immunohistochemically. However, tiny 3D cell culture samples are difficult to embed in paraffin and acquire appropriate sections. In this report, we optimized a protocol of paraffin embedding for spheroids and organoids. In addition, we compared FFPE sections with frozen sections in ratio of sample collection and section condition after staining, and could reproduce improved results reliably. The protocol we established could be widely used in many laboratories and become a useful technique for analyzing spheroids and organoids. Summary: Three-dimensional cell culture models such as spheroids and organoids are used in the field of experimental biology. This paper presents an optimized protocol of paraffin embedding for spheroids and organoids. In the 3D experimental models, spheroid and organoid culture models are widely used  cell culture models has enabled various physiological and pathological experiments resembling erformed . It is wely used . To analParaffin embedding is a widely used technique in clinical pathology and experimental histopathology. Formalin-fixed paraffin-embedded (FFPE) samples are easy to handle when we want to slice serial sections. In this report, we describe an optimized a protocol of paraffin embedding for spheroids and organoids. Moreover, we compared FFPE sections with frozen sections in ratio of samples collection and section condition.g for 5 min. Remove the medium and wash the organoids with ice-cold, sterile 1× phosphate-buffered saline (PBS). Centrifuge at 400×g for 5 min to collect the organoids. Discard the supernatant. Visually inspect the samples and carefully pipette the supernatant in each step. If the size of the samples is >100 μm, there is less chance of the samples being lost. (2) Add 1 ml 10% neutral buffered formalin  fixative, and fix organoids for 30 min at room temperature. Centrifuge at 400×g for 5 min to remove fixatives  Collect organoids in 1.5 ml centrifuge tube by centrifugation at 400×ixatives A. (3) Thixatives A. These n blocks A. In thin blocks B. (5) MoThe advantages of our FFPE method are that the process is completed in a single 1.5 ml centrifuge tube, samples are easily stored, and serial sections can be easily made from a small organoid. Serial sections are available for Hematoxylin and Eosin (HE) staining, special staining such as Periodic acid–Schiff (PAS) staining and immunostaining C. AnalysP=0.0226, The frozen section procedure is also widely used in many laboratories for microscopic analysis of a specimen. We compared the quality of FFPE sections with frozen sections in HE staining. In frozen sections of spheroids, some crushing of the samples was observed and details of individual cells were not clear. Whereas almost all spheroids retained their round shape, and cell–cell contact and the shape of each cell were maintained in FFPE sections A. We nexIn this paper, we present a protocol of paraffin embedding for spheroids and organoids. Generally, an automated tissue processor is used for paraffin embedding in the pathology laboratory. However, spheroids and organoids are too small to be processed in this machine. Our paraffin embedding protocol using centrifuge tubes could process small samples without loss. Moreover, the equipment needed for this protocol is widely available in many laboratories.One advantage of FFPE sections is their compatibility with HE staining. HE staining is suitable for morphological observation. The frozen section technique is commonly used in clinical pathology for rapid microscopic diagnosis. However, it is well known that the quality of the slides of frozen sections is lower than that of FFPE sections. In our observation, frozen spheroids showed mildly collapsed, rough and shaggy shapes in the sections A. Thus, Our verification showed that the rate of sample collection, caused by that the samples are in the same plane and appear in the same section, was higher with the FFPE method than with the frozen method B. Our pr4 cells in 200 μl Dulbecco's Modified Eagle's Medium  with 10% FBS were seeded in each well of 96-well Nunclon Sphera plate . 10% neutral buffered formalin or 4% paraformaldehyde -fixed and paraffin-embedded organoids tissue blocks were cut into 4-μm-thick sections and mounted on adhesive glass slides  for HE and immunohistochemical staining. Frozen sections were prepared by embedding samples in Tissue-Tek optimal-cutting-temperature (OCT) compound  and cryosectioning. The images of HE and immunohistochemical staining were captured using an AXIO Vert.A1 microscope .Methods of salivary gland organoid culture have been previously described . This stThe primary antibodies; AQP5 , CK18 , p63 , α-Smooth Muscle Actin , CK19 , α-amylase , SOX9 , SOX10 , β-Catenin , Ki67 . The secondary antibodies; horseradish peroxidase (HRP)-conjugated polymer anti-rabbit and -mouse antibodies were purchased from DAKO-Agilent Technologies.t-test was used for statistical evaluations. Statistical significance was set as *P<0.05.All data were expressed as the mean±standard error of the mean (s.e.m.). A two-tailed Student's


Many human diseases, including metabolic diseases, are intertwined with the immune system. The understanding of how the human immune system interacts with pharmaceutical drugs is still limited, and epidemiological studies only start to emerge. As the metabolomics technology matures, both drug metabolites and biological responses can be measured in the same global profiling data. Therefore, a new opportunity presents itself to study the interactions between pharmaceutical drugs and immune system in the high-resolution mass spectrometry data. We report here a double-blinded pilot study of seasonal influenza vaccination, where half of the participants received daily metformin administration. Global metabolomics was measured in the plasma samples at six timepoints. Metformin signatures were successfully identified in the metabolomics data. Statistically significant metabolite features were found both for the vaccination effect and for the drug-vaccine interactions. This study demonstrates the concept of using metabolomics to investigate drug interaction with the immune response in human samples directly at molecular levels. The dysfunction of immune system often underlies a large number of autoimmune, metabolic and inflammatory conditions9. The health burden is significantly greater in the ageing population10, which commonly live under pharmaceutical medications, and respond poorly to many vaccines11. It is clearly important to understand how pharmaceutical drugs interact with the immune system, but it is not an easy task given that human immunology is still a rapidly evolving science13. In recent years, much progress in human immunology was achieved via vaccine studies, as vaccines are an excellent tool to probe human immune system providing an opportunity to learn about immunological molecular perturbations over time from days to years14.The human immune system is our defense against infectious agents and malignancy, but also susceptible to many signaling cues17. Using hospital records, Kidd et al. showed that a number of small molecular drugs influence the numbers of white blood cell subsets18. These hospital records are not easy to match to immunological studies, and they are not meant to be comprehensive. Furthermore, direct measurement of concentration of small molecular drugs and their metabolites, similar to the tests on professional athletes for prohibited drugs, is the most important information. Because it reflects the ongoing biological state and varying metabolic rate among individuals. In this regard, the advancement of mass spectrometry based metabolomics is potentially a game changer.Limited epidemiological studies have reported evidence of small molecular drugs impacting human immune responses, e.g., administration of statins has a minor detrimental but significant effect on influenza vaccination22. Liu et al. recently showed successful identification of nicotine (tobacco), naphthol sulfate , omeprazole (medication) and piperine (food) and their derivatives in routine metabolomics analysis of human samples23. With both the environmental factors and biological responses in the same data, a new paradigm of gene-metabolome-environment interaction is emerging25. Thus, in a controlled study of vaccine induced immune responses, metabolomics offers the opportunity to examine both the drug response and vaccine response, and test for potential interactions.Metabolomics is the global measurement of small molecules in a biological system, which includes biological metabolites, dietary intake, microbial contributions, environmental pollutants, and often pharmaceutical drugs26 and by herpes zoster vaccine27, with broad interactions with cellular and gene programs. A significant observation in the integrative analysis of human transcriptome and metabolome in Li et al. (2017)27 was that baseline SREBF1 (Sterol regulatory element-binding protein 1) activity was associated with B cell responses to vaccination, and the finding was recently confirmed in a detailed mouse study28.The application of metabolomics to vaccinology is still in a nascent stage. Previous metabolomic analyses showed that systemic metabolites were perturbed by seasonal influenza vaccine29, and targeting multiple hallmarks of aging30. Given the prominent role of metformin in aging studies and its geroprotective potential at molecular level31, it becomes an important question if metformin enhances the immune responses to vaccines. Research in this direction is urgent because better vaccine efficacy is needed for the older population and how to optimize their immunity has broad health impacts. In this report, we focus on how the metabolomics data manifest the biological responses to metformin, IIV vaccine and their interaction.Here, we have conducted a pilot study of seasonal inactivated influenza vaccine (IIV) in older adults, where half of the participants received metformin, a common drug for controlling diabetes, for a total of 20 weeks. The study was double-blinded and placebo-controlled, enrolling nondiabetic/nonprediabetic men and women over the age of 65 years. Ultrahigh-resolution metabolomics was performed on the plasma samples from 15 participants over six timepoints. The study was motivated by the effects of metformin in improving mitochondrial functions, reducing chronic pro-inflammatory signalingn = 7) or metformin (n = 8) treatment. The cohort was between 67 to 89 years old, with 8 males and 7 females and no significant differences in basic characteristics at baseline . The metformin group received Metformin Hydrochloride Extended-Release, 1500 mg/day . High dose trivalent inactivated influenza vaccine  was administered via intramuscular injection to all participants at approximately day 70  methods to increase the coverage of assays: hydrophilic interaction chromatography (HILIC) with positive electrospray ionization (ESI + ) and with negative electrospray ionization (ESI-), reversed phase (RP) chromatography with ESI+ and ESI−. The numbers of metabolite features in each method are reported in Table 13C isotopologue . With FDR < 0.05 and fold change >1.5, the numbers of significant features are shown in Table To analyze the effect of IIV vaccine administration, we focused on the two timepoints before and after vaccination . Our feature 147.0847@65.03 was matched to 147.0843@354.85 in the Broad study, which has a highly significant association pattern to a cluster of compounds at 556 s . Interest are also growing in its anti-aging, anti-inflammatory and anti-proliferative roles51. Metformin reduces pro-inflammatory cytokines and inhibits NF-κB signaling53, both contributing to increased basal inflammation with aging. Overall, metformin regulates several aspects of nutrient sensing and energy homeostasis in various metabolically active organs leading to improved blood glucose and lipid profiles55. Our data revealed that several lipids and amino acids related pathways were altered by metformin administration. De novo fatty acid biosynthesis was the most significantly altered pathway  and ACC1/2 (a rate-limiting enzyme for fatty acid synthesis) inhibits hepatic de-novo lipogenesis60. Moreover, ACC1/2 phosphorylation leads to a decreased production of malonyl-CoA ) and subsequent enhanced hepatic fatty acid oxidation45. Our results showed a decrease of abundance in several metabolites in pathways responsible for synthesis of lipids and their derivatives including de novo fatty acid biosynthesis pathway, arachadonic acid metabolism, glycerophosopholipid metabolism and prostaglandin formation from arachidonate. These results are in line with the previous reports where many lipids and lipid derivatives including poly unsaturated fatty acids (PUFAs), eiconsaids, glycerophospholipds were observed to show decreased plasma abundance upon metformin administration in healthy volunteers34, and alterations in de-novo fatty acids synthesis and inflammatory lipid derivatives in different pathological conditions66.The metabolic responses to metformin here are consistent with previous reports. The glucose lowering potential of metformin has been largely attributed to its ability to suppress hepatic gluconeogenesis through both AMPK dependent and independent pathways  and plasma cells (between days 7–11) corresponding to the induction of innate and adaptive responses, respectively. Many of these studies have highlighted the age dependent differences in vaccine immunogenicity, and our cohort is considered as older adults. Our data revealed several perturbed pathways on carbohydrate and amino acid metabolism 1 week post vaccination. They are in line with previous studies where serine metabolism was shown to be associated with vaccine response39. Mitochondrial biogenesis and oxidative phosphorylation processes were observed to be impacted by immune responses induced by influenza vaccine in ours and previous reports41.So far, metabolomics has been applied to only few vaccine studiesIn this study, we have focused on day 7 after vaccination. In future opportunities, it will be informative to analyze more time points, including the early ones. The field of immunometabolomics is in its infancy, and it will take time to gain fuller understanding of the vaccine responses in terms of metabolic phenotypes. More and more immunological and vaccine studies take a systems or holistic approach, by collection high throughput multi-omics data, which shall contribute to useful insights on how small molecules, biological or abiological, interact with the immune system. Advantages of metabolomics also include that it’s easy to use biobanked materials, and that it can become very economical in the near future.73. Metabolite identification, however, is still challenging in metabolomics, especially for low-abundance or less common metabolites. This is similar to the early days of genomics, when genes were deposited as unknown sequence but annotation improved over time. The ultrahigh mass resolution of our data also means that people can reuse the data from public repository, and the unknown metabolites, like unknown genes, can gain annotation in the future. This is already demonstrated in our reuse of the Broad dataset44: the MWAS of our feature 147.0847@65.03, significant in the interaction between IIV and metformin, is reproduced in the larger study in Fig. We reported the annotation confidence according to the MSI standardsThis pilot study was designed to test the interaction between metformin and IIV in a small cohort. Nonetheless, with highly stringent statistical analysis, ultrahigh-resolution metabolomics prove to be powerful to identify (i) metformin and its metabolic signatures in untargeted metabolomics data, (ii) significant metabolic responses to IIV, and (iii) significant metabolites as a result of the interaction between metformin and vaccination. The proof-of-principle is important, demonstrating the feasibility of studying the interaction of drugs and immune responses in human populations.n = 8 metformin, n = 7 placebo) were randomized and completed the study on treatment with no differences in basic characteristics at baseline .This pilot study is a double-blinded placebo-controlled trial in men and women over the age of 65 years. Subjects were screened rigorously for eligibility. Study exclusion criteria included the following: any unstable medical conditions or severe co-morbidities , contraindications for metformin , contraindication for flu vaccine , immunosuppressive disorders, immunosuppressive medications, and active cancer. Importantly, participants were excluded if they were prediabetic or diabetic (HbA1c ≥ 5.7%) to avoid any confounding impact of metformin on diabetes status. Eligible participants were randomized to metformin /day) or placebo treatment. To limit gastrointestinal issues per current metformin label recommendations, participants started with 1 tablet a day for week 1 (500 mg metformin ER/day or placebo), then 2 tablets a day for week 2 (1000 mg metformin ER or placebo), and finally 3 tablets a day for week 3 until the completion of the study (1500 mg metformin ER or placebo). Fifteen subjects  after ~70 days of treatment. Blood was drawn via standard venipuncture into EDTA-treated vacutainers prior to treatment (Day 0), prior to vaccination (~day 35 and ~day 70), and 7, ~35, and ~70 days post vaccination. The study protocol was approved by the Institutional Review Board at the University of Connecticut Health Center (UCHC) and registered at ClinicalTrials.gov (NCT03996538). All study participants provided written informed consent to participate in the study.EDTA-treated whole blood was immediately centrifuged and the resultant plasma was stored at −80 °C until analyses. Plasma metabolites extraction was carried out by protein precipitation technique using extraction solvent, acetonitrile:methanol  containing 0.1% formic acid and isotope labelled [Trimethyl-13C3]-caffeine, [13C5]-L-glutamic acid, [15 N2]-Uracil, -L-methionine, [13C6]-D-glucose and [15N]-L-tyrosine as spike-in controls. 30 μl of plasma was taken and 60 μl of extraction solvent was added. Extraction blanks were also prepared to remove features of non-biological origins. All samples were vortexed and incubated with shaking at 1000 rpm for 10 min at 4 °C followed by centrifugation at 4 °C for 15 min at 20,817 × g. The supernatant was transferred into mass spec vials and 2 μl injected into UHPLC-MS.TM TranscendTM Duo LX-2 UHPLC system interfaced with high resolution Thermo ScientificTM Orbitrap ID-XTM TribidTM mass spectrometer with a HESI ionization source, using positive and negative ionization modes. All samples were maintained at 4 °C in the autosampler. Data were acquired using hydrophilic interaction liquid chromatography (HILIC) and reversed phase (RP) column in parallel both in positive and negative polarities in full scan mode with mass resolution of 120,000. An AccucoreTM−150-Amide HILIC column  and a Hypersil GOLDTM RP column  maintained at 45 °C were used for chromatographic separation. 10 mM ammonium acetate in acetonitrile:water  with 0.1% acetic acid as mobile phase A and 10 mM ammonium acetate in acetonitrile:water  with 0.1% acetic acid as mobile phase B were used for HILIC method. 0.1% formic acid in water and 0.1% formic acid in acetonitrile were used as mobile phase A and B respectively for RP acquisition. For HILIC acquisition, following gradient was applied at a flow rate of 0.55 ml/min: 0–0.1 min: 0% B, 0.10–5.0 min: 98% B, and 5 min for cleaning and equilibration of column. For RP column, following gradient was applied at a flow rate of 0.4 ml/min: 0–0.1 min: 0% B, 0.10–1.9 min: 60% B, 1.9–5.0 min: 98% B, and 5 min cleaning and column equilibration. This way the mass spec data for each sample was collected consecutively, carrying only one (either HILIC or RP) eluent to the MS for 5 min, while the other eluent was directed to the waste during washing and re-equilibration.The chromatographic separations were performed using Thermo ScientificMass spectrometry data were collected with the following MS settings: mass range, 80–1000 m/z; spray voltage, 3500 V (ESI + ), 2800 V (ESI−); sheath gas, 45 Arb; auxiliary gas, 20 Arb; sweep gas, 1 Arb; ion transfer tube temperature, 325 °C; vaporizer temperature, 325 °C; full scan mass resolution, 120,000 (MS1); normalized AGC target (%), 25; maximum injection time, 100 ms. Data dependent fragmentation (dd-MS/MS) parameters for each polarity as follows: isolation window (m/z), 1.2; stepped HCD collision energy (%), 20,40,80; dd-MS/MS resolution, 30,000; normalized AGC target (%), 20; maximum injection time (ms), 54; microscan, 1; cycle time (sec), 1.2. A full scan data-dependent MS2 (ddMS2) method was utilized to collect MS2 spectra for identification of compounds.74. Asari (version 1.9.2), an open source Python software was used for m/z and retention time (rt) alignment, peak detection, feature quantification, and empirical compound based putative identification (level 4 annotation using HMDB)75 using the default parameters. Level 1 annotation of compounds were obtained by matching retention times and accurate masses from in-house authentic compound libraries. Level 2 annotation of compounds were obtained by matching acquired MS2 spectra of accurate precursor masses from pooled plasma sample against public spectral databases 76 using R package Spectra with >=0.7 cosine similarity score. Matching of MS1 features with precursor ion’s accurate masses and retention times was performed using within 10 ppm tolerance and 10 s, respectively. All the metabolite annotation levels adhere to Metabolomics Standard Initiative (MSI) guidelines73.All samples were analyzed in a single batch after randomization. Raw LC-MS data was converted to mzML format using ThermoRawFileParserasari is defined by the mean of all non-peak data points in an extracted ion chromatogram). Two different quality control samples  were used to verify the chromatography and signal reproducibility. Visual inspection of outliers through PCA plots and Total ion count (TIC) was conducted. Data were log2 transformed and mean normalized using top ten percent of high abundance features. Features below detection limit were imputed using half of the minimum intensity value. After QC filtering, 5987, 4245, 3284, 8546 features were retained for downstream data analysis, in each HILIC ESI+, HILIC ESI−, RP ESI+ and RP ESI− modes respectively.Features were filtered using two criteria. First, the features with three times greater intensity in biological samples than in blanks samples were retained. Second, the features with signal to noise ratio (SNR) greater than 100 were retained  controls for repeated measurements on the same participant. The significance of timepoints was assessed with ANOVA and Model 2: In order to quantify the vaccine response and the interaction of metformin and vaccine, we constructed a mixed effect model utilizing a pre-vaccination time point  and a post-vaccination time point (day 77) as follows:p value of coefficient term of the variable ‘day‘. The interaction term day*metformin was used to quantify statistically the response as a function of both vaccine and drug administration. The term (1|participant) controls for repeated measurements on the same participant. P values assessed using ANOVA were adjusted for multiple-testing based on Storey FDR method79.The ‘day‘ and ‘metformin‘ are used as proxy for vaccine treatment and drug treatment, respectively and modeled as fixed effects. We assessed the differentially abundant metabolite features between pre- (day 70) and post-vaccination (day 77) visits using the 80 using top metabolite features with p value < 0.05.All the above statistical analyses were performed using R version 4.2.0. Pathway enrichment analysis was performed using mummichog (version 2.6.1)p-values for MWAS. FDR was calculated using the Benjamini-Hochberg method. The sample number in this study was 90, in the Broad study 1172. The Broad dataset was retrieved from Metabolomics Workbench (accession number ST001237). The m/z and retention time of identified compounds in the Broad study were obtained from the authors. Known compounds from both studies were compared, then the uniquely matched compounds (same name and m/z within 5 ppm) were used for realigning the retention time.Spearman correlation was performed between the features of interest and the remaining features in the same dataset, generating Further information on research design is available in the Supplementary FiguresREPORTING SUMMARY


Introduction: This study aimed to assess the prognostic role of visual evoked potentials (VEPs) of the non-neuritic eye at the diagnosis of multiple sclerosis (MS). Patients and methods: We enrolled 181 MS patients  at the time of the first diagnostic work-up, including VEPs. We collected P100 latency and N75-P100 amplitude of non-neuritic eyes at diagnosis, and then we calculated the mean values in 127 patients with no history of optic neuritis (ON) or considered the unaffected eye in the remaining. At last follow-up (minimum: one year), disability was evaluated according to MS Severity Score or MSSS . Statistical analysis included Mann–Whitney descriptive analysis, Spearman correlation for independent samples, and linear regression for significant predictors of MSSS. Results: 38/181 patients had P100 latency >115 ms, and 63/181 showed N75-P100 amplitude < 5 microV in the unaffected eyes at MS diagnosis. At last follow-up, MSSS correlated with P100 latency  and N75-P100 amplitude  collected at diagnosis. P100 latency (not N75-P100 amplitude) resulted in a predictor for disability over time (MSSS) in the regression model . Conclusions: Our study showed a prognostic value of VEPs in clinically unaffected eyes at MS diagnosis to predict future disability, independently from a history of ON. Multiple sclerosis (MS) is an inflammatory autoimmune disease of the nervous system in which neurodegeneration occurs at early stages and relates with the accumulation of disability. The visual system emerged as a model for detecting both inflammation and axonal loss in the subclinical phase . DespiteOur study aims to evaluate the prognostic role of subclinical abnormalities of the visual pathway at disease onset. We thus related VEP components, derived from non-ON eyes at the diagnosis, to disability over time. We collected data from 181 MS patients , who performed VEPs at their first diagnostic work-up. At that time, mean disease duration from clinical onset was 1.4 years (±standard error or ES 0.3). Patients were subsequently recruited from January 2016 to December 2021 at the University of Piemonte Orientale “Maggiore della Carità” Hospital in Novara and at the “San Andrea” Hospital in Vercelli. Patients with other inflammatory and neurodegenerative diseases, psychiatric or ophthalmological comorbidities were excluded. Minimum follow-up from diagnosis was one year (mean 2.6 years ± ES 0.2). At last clinical evaluation, we evaluated their outcome, according to MS Severity Score (MSSS), and collected disease-modifying treatments (DMTs), grouped as follows: none, low–moderate , or highly effective . Pattern reversal VEPs were recorded according to the International Society for Clinical Electrophysiology . Full-fip < 0.05.Statistical Analysis was performed using SPSS 25.0 for Windows . We verified whether the data were distributed according to the Gaussian model with the Kolmogorov–Smirnov test and consequently used non-parametric tests: Mann–Whitney U for comparison between continuous variables; Fisher test for categorical variables. The Spearman’s rank correlation coefficient test was used for the correlation between continuous variables. Linear regression analysis was performed for disability according to MSSS (dependent variables are discussed in the Results section). All tests were two-sided and the significance threshold was set to We collected 166 relapsing-remitting (RR) MS, and 15 progressive patients . Clinical–demographic data at the diagnostic work-up are shown in p < 0.001). Of note, a history of ON did not relate with abnormal VEP responses in the fellow eye. Males (p = 0.004) and patients with progressive MS (p = 0.01) showed more frequently pathological VEPs recorded from non-neuritic eyes.Considering VEP responses collected at diagnosis in non-ON eyes, 38/181 patients presented a P100 latency > 115 ms, and 63/181 a N75-P100 amplitude < 5microV  and progressive MS course at diagnosis (rho = 0.31 p < 0.001), both established MS prognostic factors [p = 0.004) and amplitude , that were recorded at the unaffected eye at diagnostic work-up, correlated with MSSS. Disability measures were collected at last follow-up and varied largely among our cohort . MSSS correlated with age at onset (rho = 0.30 p = 0.002). Age at onset and disease course were also confirmed as disability predictors  at diagnosis, the history of previous ON (yes/no), and VEP measures at baseline as dependent variables. P100 latency (not N75-P100 amplitude), collected at diagnosis in the non-ON eye, emerged as a mild predictor for disability over time (MSSS)  , and confirmed previous results for P100 latency at last follow-up  B. In thi= 0.005) B. Of notOur study suggested a prognostic role for VEPs at MS diagnosis independently from the clinical history of visual involvement. In fact, abnormal P100 latency in the unaffected eyes emerged as a marker for future disability. Moreover, age at onset, MS course, and DMTs were confirmed as predictors for MSSS in our cohort. To prevent damage accumulation, an early personalized DMT should be discussed since MS onset and according to risk factor evaluations. Patients with more severe RRMS need to start highly effective therapies at diagnosis. To identify those cases, conventional magnetic resonance imaging (MRI) is considered the most effective tool to evaluate disease burden, whereas it is not that sensitive for visual pathways . On the We decided to focus on the optic pathway, since it was not affected by spinal damage. Regarding the visual system, Leocani et al.  reviewedAnother strength of our study was to focus on subclinical visual damage. In fact, 21% of our 181 patients at diagnosis showed a prolonged P100 latency in non–ON eyes, and irrespectively from a history of contralateral ON. Seven (18%) patients with abnormal VEPs in clinically unaffected eyes showed a progressive MS course at diagnosis, according to previous data . A role We decided to investigate the disability outcome in the short term using MSSS, to correct for disease duration. In our multivariate model, a delayed visual response in clinically unaffected eyes resulted as a mild predictor of disability. For this reason, we suggested that P100 latency at baseline should be included in predictors of disability over time.(1)We employed traditional full-field VEPs as an easily repeatable and largely used marker. Some authors evaluated multifocal (mf) VEPs in smaller groups and found some correlations with disability. Blanco et al. showed delayed and reduced visual responses in more than 70% of non-ON cases among a group of 28 patients. They also described lower wave amplitudes with higher disability scores .Our study has some limitations.(2)Since our study included a large cohort of 181 MS patients at diagnosis, our results could not reflect long-term treatment-related prognosis. The choice of starting highly effective DMTs could be considered as a marker of disease severity at diagnosis in our cohort.(3)Pivotal data on optical coherence tomography we performed in a limited subgroup with no definite results. Nonetheless, VEPs are currently diffusely employed in MS neurological assessments at first MS diagnostic work-up.Generally, mfVEPs are more specific and sensitive in detecting dysfunctions of specific parts of the visual field, but this technique is not standardized among centers and time-consuming to be performed .(2)SincAdding other biomarkers, such as neurofilaments, to our model could improve the prognostic value of VEP latency at diagnosis.Our study showed a prognostic value of VEPs at MS diagnosis in predicting short-term disability, independently from a previous history of ON. P100 latency in non-ON eyes could be considered as a prognostic factor over time.


Black/purple rice is a pigmented rice variety that contains high levels of anthocyanins, flavonoids, and other valuable bioactive compounds. Owing to its robust anti-inflammatory and antioxidant properties, black/purple rice exerts a beneficial effect on human health. Extrusion puffing technology has emerged as a promising means of improving rice flavor with lesser effect on nutrient content. In this study, metabolomics approach was used to conduct comprehensive metabolomics analyses aimed at examining the impact of extrusion puffing on black/purple rice nutritional value and flavor.Firstly, the basic nutrient composition contents and extrudate characteristics of black/purple rice and Extrusion puffed black/purple rice were conducted. Then metabolomics profiling analyses of black/purple rice samples were performed to explore the impact of the extrusion puffing process on nutrient content and bioactive properties, in which we quantitatively determined the flavonoids and evaluated relative contents of volatile compounds.These analyses revealed that following extrusion puffing, black/purple rice exhibited significant improvements in the content of nutrients including flavonoids, minerals, and proteins together. Extrusion puffing additionally increased the diversity of volatile compounds within black/purple rice.These results suggest that extrusion puffing represents an effective means of substantially improving the functional and nutritional properties of black/purple rice, offering beneficial effects on consumer health. Overall, these data provide novel insights into the quality of extrusion puffed black/purple rice that will guide future efforts to establish how extrusion puffing can alter the nutrient content in a range of foods, thereby supporting the further development of a range of healthy food products. Oryza sativa L.) is a staple crop throughout the world that is rich in minerals, vitamins, fiber, protein, unsaturated fatty acids, polysaccharides, and flavonoids found in the pericarp, seed coat, aleurone, germ, and endosperm. Historically, however, culinary preferences have favored polished rice, which exhibits a ~ 80% drop in nutrient content relative to bran  in living organisms . Based oConsidering flavonoids, volatile metabolites, and their changes during extrusion puffing, ultra-performance liquid chromatography with a tandem mass spectrometry (UPLC-MS/MS) and gas chromatography-ion mass spectrometry (GC-IMS) was employed to identify metabolites, respectively. The significantly different metabolites were identified to clarify the impact of extrusion puffing on the enhancement of the nutrition, flavor, and antioxidant activities of black/purple rice. As such, these findings provide a robust theoretical foundation for further efforts to apply extrusion puffing as a means of preparing healthy food.The Zhongzi 4 variety of black/purple rice (designated as BR) was obtained from Peking University and used as the experimental focus in this study.Extrusion puffed black/purple rice (EBR) was generated using an SLG40-A twin-screw extruder  , the barMoisture, protein, fat, and carbohydrate levels were assessed as per the respective AACC methods 44-15A, 46-11A, 30–10.01, and 12–21. AACC method 40–71.01 was used to determine levels of minerals . All analyses were repeated in triplicate.The bulk density (BD) was assessed as reported previously by Hashemi et al. . BD was 3. In total, the BD of sample was expressed as the average of 3 determinations.Where M is mass (g) and V is the volume of the beaker in cm0) was mixed with 25 mL of dH2O in a centrifuge tube (W1), shaking until fully dispersed. This solution was then incubated for 30 min at 30°C in a water bath, stirring gently every 10 min. Samples were then spun for 15 min at 5,180 × g after which supernatants were transferred to a beaker that had been weighed (W2) and heated at 105°C until reaching a constant weight (W3). Precipitates and tubes were then weighed at the same time (W4). Testing was performed in triplicate.Using an approach detailed previously by Yang et al. , a 1.6–2Relative WAI and WSI proportions were determined with the following equations:A CR-400 Chroma Meter  with a measuring area 8 mm in diameter and a 0° viewing angle was used for the measuring of sample flour color. Prior to use, instrument calibration  was performed using a white calibration plate . Four color parameters were analyzed including L* , a* (−a* indicates greenness and + a* indicates redness), b* (−b* indicates blue and + b* indicates yellow) and the total color difference (△E=g under 4°C for 5 min. The supernatant was filtered through a 0.22 μm membrane filter for further Ultra Performance Liquid Chromatography-Mass spectrometer (UPLC-MS/MS) analysis.Freeze-dried BR and EBR were ground into powder  using a Grinding mill . The sample was stored at –80°C before further analysis. The sample powder (20 mg) was weighed and dissolved in 0.5 mL methanol  and 0.01 mL internal standard solution . The extract was sonicated for 30 min and centrifuged at 12, 000 × The extraction of flavonoids metabolites in two samples was conducted by using a UPLC-MS/MS referring to the established method with minor modifications . The flaThe identification and quantification of flavonoids metabolites were operating in positive and negative ion mode and controlled by Analyst 1.6.3 software (Sciex). Temperatures of the Electrospray Ionization (ESI) was set as 550°C; the spray voltage was 5,500 kV for positive ion and the spray voltage was −4,500 kV for negative ion. Curtain gas (CUR) was set at 35 psi.The mass spectrometric data were processed in Analyst 1.6.3 and MultiQuant 3.0.3 software. According to the retention time and peak type information of the standards, the chromatographic peaks detected in different samples were corrected for integration to ensure the accuracy of qualitative and quantitative analysis. The actual metabolite contents were calculated using the peak areas obtained and brought into the calibration curve.2FC (fold change). VIP values were extracted from orthogonal partial least squares discriminant analysis (OPLS-DA) result, which also contain score plots and volcano plots. The data was log transform (log2) and mean centering before OPLS-DA. The hierarchical cluster analysis (HCA) results of differential metabolites were present as heatmaps with dendrograms.Unsupervised principal component analysis (PCA) was performed by statistics function prcomp within R. The data was unit variance scaled before unsupervised PCA. Significantly regulated metabolites between groups were determined by variable importance projection (VIP) and absolute LogA GC-IMS (FlavourSpec®) instrument from the G.A.S. Department of Shandong Haineng Scientific Instrument Co., Ltd. was used for analyses of volatile compounds (VCs) using a slightly modified version of a previously reported protocol . Brieflyp < 0.05) between the composition of BR and EBR samples. The 2022 Origin software  was used for figure generation.Data are means ± standard deviation. Student’s t-tests followed by Duncan’s multiple range test were used to detect significant differences  in BR and EBR samples were thus evaluated via GC-IMS . RelativExtrusion puffing technology is one of the most widely used processing methods in the food industry due to its low processing cost compared to other cooking and processing methods . At the Extrusion puffing process is a highly-promising technique for the production of the rice and rice-based products, which greatly impacts the physicochemical properties of rice extrudates . In termIn terms of the basic nutritional compositions of rice, starch and protein are the primary components, and their contents increased significantly after processing. While proteins are generally heat-labile and profoundly affected by the extrusion-mediated cooking process , the degBioactive compounds like flavonoids, alkaloids, and phenolic acid are the focus of growing interest due to their reported anti-inflammatory, antioxidant, and anticancer properties , 8. HighThe OPLS-DA model was further used to analyze differentially abundant metabolites in EBR and BR samples. Remarkably, the identified upregulated differentially abundant metabolites comprised a variety of pharmacologically active compounds including quercetin, hydroxygenkwanin, isorhamnetin, and (−)-catechin, which have been reported to exhibit anticancer, anti-inflammatory, antioxidant, and gut probiotic effects –36. Thes2u-globulin nephropathy in male rats (Flavor and appearance are key determinants of food quality, and volatile aromatic compounds play a central role in shaping the flavor of a given food . Aromatiale rats , and itsIn conclusion, the identification of fundamental nutritional compounds, differential flavonoid and volatile metabolites lays the groundwork for future research on the functional and nutritional value of various extrusion puffed products.Here, metabolomics profiling analyses of black/purple rice samples were performed to explore the impact of the extrusion puffing process on nutrient content and bioactive properties. Overall, extrusion puffed black/purple rice exhibited pronounced increases in protein, carbohydrate, and mineral content together with reductions in moisture and fat content. Additionally, extrusion puffing causes flavonoids and volatile compound content and variety rose markedly in black/purple rice samples. This suggests that extrusion puffing represents an effective means of substantially improving the functional and nutritional properties of black/purple rice, offering beneficial effects on consumer health. These data provide novel insights into the quality of extrusion puffed black/purple rice that will guide future efforts to establish how extrusion puffing can alter the nutrient content in a range of foods, thereby supporting the further development of a range of healthy food products.The original contributions presented in the study are included in the article/YM: investigation, formal analysis, writing – original draft, and review and editing. JL: investigation and formal analysis. YaX and YuX: writing – review and editing. CL and DS: supervision, project administration, and writing – review and editing. All authors contributed to the article and approved the submitted version.


Key messagePlasma exchange is important in lupus for some rare immunological assaults non-responsive to immunosuppressive treatments.Dear Editor, A 22-year-old female presented with complaints of fever on and off for 6 months, altered sensorium and limb weakness for 1 week. The weakness started with a sensation of heaviness in both feet, progressing rapidly to both upper and lower limbs in the next 5 days, leading to quadriparesis and a bed-bound state. Bowel and bladder sensations were intact. She had a history of malar rash, oral ulcers, photosensitivity and polyarthralgia. There was no history of recent flu-like symptoms, gastroenteritis, travel or immunization. There was no history of autoimmune diseases in family members. Neurological examinations revealed lateral rectus palsy of the left eye with left facial nerve palsy, power of 1/5  in bilateral upper and lower limbs, with generalized hypotonia and generalized areflexia. She had truncal and respiratory muscle weakness, with wide fluctuations in pulse and blood pressure and increased sweating. Routine investigations, including a haemogram, liver and kidney function tests and serum electrolytes, were normal. Further work-up revealed positivity of ANA IF , an anti-dsDNA level of 105 IU/ml (reference <27 IU/ml), low complement components (low C3 and C4), negative aPL and proteinuria, with quantitative assessment of urine protein being 7 g/24 h. HIV, Hepatitis B and C were negative. Cerebrospinal fluid revealed albuminocytological dissociation, with absence of cells, a protein level of 140 mg/dl (reference range 18–58 mg/dl) and normal cerebrospinal fluid glucose. Nerve conduction revealed sensorimotor axonal polyneuropathy. A magnetic resonance angiogram of the brain revealed features of moyamoya syndrome (syndrome . Renal bAfter 2 weeks, the patient showed no improvement in neurological status, and tracheostomy was done in anticipation of prolonged mechanical ventilation. Plasma exchange was initiated. The patient showed significant motor improvement after six cycles of plasma exchange, with weaning of mechanical ventilation and improvement in the motor power in the extremities to 3/5. Injection of CYC was continued monthly for 6 months. She had complete motor recovery at 3 months and angiographic resolution of moyamoya  with comSLE is an autoimmune disease that can cause widespread immunological damage involving virtually any organ. GBS is the peripheral nervous system manifestation of neuropsychiatric lupus, with a reported prevalence of 0.6–1.7% . Both ceMoyamoya syndrome is a cerebrovascular disorder characterized by progressive stenosis of intracranial internal carotid arteries and their proximal branches, leading to formation of collaterals and a ‘puff-of-smoke’ appearance on angiography . MoyamoyIsolated cases of lupus with GBS or lupus with moyamoya have been reported in the literature, with good treatment outcome. However, lupus presenting with GBS and moyamoya syndrome has never been reported. This is the first case of lupus with widespread immunological insult in the form of GBS, moyamoya and lupus nephritis, which was managed successfully with immunosuppressants and plasma exchange.


MMP8 gene polymorphism and CRC risk in the Chinese Han population.Colorectal cancer (CRC) is one of the most common malignant tumors, influenced by several genetic loci in its clinical phenotypes. The aim of this study was to determine the relationship between the MMP8 polymorphism and CRC susceptibility was assessed by calculating the odds ratio (OR) and 95% confidence interval (CI) after stratifying by age, gender, body mass index (BMI), smoking, and alcohol consumption under a multi-genetic model.This study recruited 688 CRC patients and 690 healthy controls. The relationship between MMP8 rs3740938 was associated with increased CRC predisposition , and this association was detected particularly in subjects aged > 60 years, females, people with BMI > 24 kg/m2, smokers, and drinkers. Moreover, rs3740938 was found to be associated with the pathological type of rectal cancer.MMP8 was a risk factor for CRC predisposition. This finding may provide a new biological perspective for understanding the role of the MMP8 gene in CRC pathogenesis.Our results first displayed that rs3740938 in The online version contains supplementary material available at 10.1186/s12885-023-11662-z. Colorectal cancer (CRC) is the third most diagnosed cancer in the world and the second leading cause of cancer death, with a 10.0% incidence and 9.4% mortality . In receMMP8 , a member of the MMP family, is an endopeptidase primarily produced by neutrophils. It plays a crucial role in degrading extracellular matrix proteins, growth factors, and cytokines [MMP8 are associated with the risk of a variety of cancers, including breast cancer [MMP8 rs11225395 and CRC susceptibility has been reported [MMP8 and the risk of CRC has not been reported yet.ytokines . For insytokines . High seytokines . The enhytokines . Relevant cancer , thyroidt cancer , and lart cancer . Previoureported . The impreported . The relMMP8 rs3740938 with the risk of breast cancer has been explored [MMP8 rs1940475 is associated with the risk of breast cancer [MMP8 rs3765620 is related to ischemic stroke susceptibility [MMP8 gene to explore their role in CRC occurrence in the Chinese Han population.In previous studies, the association of explored . MMP8 rst cancer  and gasthttps://stats.idre.ucla.edu/other/gpower/) to estimate the sample size. The specific parameters we set are as follows: effect size d = 0.2; α error probability = 0.05; power (1-β err prob) = 95%. This calculation yielded a sample consisting of at least 651 cases and 651 controls. Here, we enrolled a total of 1378 subjects  from Hainan Cancer Hospital from 2020 to 2023. The inclusion criteria for CRC cases are: newly diagnosed and histologically confirmed by rectoscopy, endorectal ultrasonography, magnetic resonance imaging (MRI), computed tomography, and histopathological results based on the American Joint Committee on Cancer (AJCC) classification. Patients with a history of cancer or severe chronic diseases were excluded. Prior to any treatment, blood samples were collected from the patient. For the control group, we selected healthy individuals without malignant tumors or digestive diseases from the same hospital as cases, ensuring they were genetically unrelated to CRC patients. All subjects belonged to the Chinese Han ethnicity, and no minors or illiterates were involved in our study. Questionnaires surveys and medical records were used to obtain epidemiological characteristics and pathological data. This research plan was implemented in accordance with the Helsinki Declaration and approved by the Ethics Committee of Hainan Cancer Hospital (No. ZDKJ202008). All subjects provided written informed consent before registration in this study.To ensure the accuracy and credibility of the research results, before we plan to conduct this study, we used G*power 3.1.9.7 software  the dbSNP database with minor allele frequency (MAFs) ≥ 5%; (2) with MassARRAY primer design, a call rate > 99%; (3) and previous association studies [https://www.ncbi.nlm.nih.gov/snp/), HaploReg v4.1 , RegulomeDB (https://regulome.stanford.edu/regulome-search/), and QTLbase (http://www.mulinlab.org/qtlbase/index.html) were employed to identify the potentially functional SNPs.Three SNPs  in  studies –20. Bioihttps://www.agenabio.com/). In addition, this study also set up double wells for each sample to ensure the accuracy of the results. For quality control, about 10% of the total samples were chosen randomly and re-genotyped, and the concordance rate reached 100%.Peripheral whole blood samples (5 mL) were obtained from each participant and stored in tubes containing Ethylene Diamine Tetraacetic Acid (EDTA) anticoagulant. Within 24 h, genomic DNA was isolated using the GoldMag DNA Purification Kit . The DNA samples were quantified using NanoDrop 2000  and stored at − 20 °C. DNA samples with low concentrations or poor quality were excluded from subsequent studies. The MassARRAY platform is based on MALDI-TOF (matrix-assisted laser desorption/ionization—time of flight) mass spectrometry , 22. The2 test for continuous or categorical variables, respectively. The Hardy Weinberg balance (HWE) of the control group was assessed by a goodness-of-fit χ2 test. The relationship of MMP8 polymorphisms with CRC risk was determined by calculating odd ratios (ORs) and 95% confidence intervals (CIs) using a multi-genetic model adjusted for age, sex, BMI, smoking, and alcohol consumption. SNPstats (https://www.snpstats.net/start.htm) was utilized for this analysis. The subgroup analyses were completed within specific subpopulations stratified by age, sex, BMI, smoking, and drinking status. In addition, the impact of genotypes on different pathological types was also evaluated. The false positive reporting probability (FPRP) threshold was set at 0.2 with a prior probability of 0.1, which is used to evaluate the significant association of significant findings [p value < 0.05, and a Bonferroni-corrected p < 0.05/3 was considered significance.The demographic data between two groups were tested by student t-test or χfindings . The optp = 0.774), gender (p = 0.964), smoking (p = 0.624), and drinking (p = 0.828). There was a significant difference in BMI between the two groups (p < 0.001). Among the patient cohort, there were 320 (46.5%) individuals diagnosed with colon cancer and 368 (53.5%) with rectal cancer. Within this group, 183 (26.6%) patients experienced lymph node metastasis, while 263 (38.2%) patients were classified as stage III-IV based on their cancer staging.The case group (59.78 ± 11.29 years) included 402 males and 286 females, and the control group (59.62 ± 9.55 years) consisted of 404 males and 286 females  binding/chromatin accessibility peak. HaploReg v4.1 database displayed that these SNPs might be associated with the regulation of promoter/ enhancer histone marks, DNAse, and /or motif changes. Moreover, the genotypes of rs3740938 (p = 0.027), rs1940475 (p = 2.720e-13), and rs3765620 (p = 1.620e-12) were associated with the expression of MMP8 in blood. In the allele model, rs3740938 was associated with the higher CRC risk .Three SNPs  in MMP8 rs3740938 contributed to an increased predisposition to CRC under the codominant , dominant  and log-additive  models. The risk-increasing significance of rs3740938 for CRC occurrence still existed after Bonferroni multiple correction (p < 0.05/3).Genetic model analysis between selected variants and CRC risk was shown in Table p = 0.045, OR = 1.49; and dominant: p = 0.018, OR = 1.44) might contribute to the increasing CRC risk. After stratification by gender, an association between rs3740938 and an increased CRC susceptibility was observed among females under the dominant  and log-additive  models, and this SNP could be identified as a potential risk marker with a marginal p value in codominant model . In subjects with BMI > 24 kg/m2, rs3740938 conferred to the higher susceptibility to CRC . In smokers, rs3740938 might be a -risk-increasing factor for CRC under the codominant , dominant  and log-additive  models. Among drinkers, rs3740938 was associated with the increased CRC susceptibility . After Bonferroni multiple correction, the relationships of rs3740938 with CRC susceptibility in females, subjects with BMI > 24 kg/m2, smokers and drinkers were also remarkable.Stratification analyses by age, gender, BMI, tobacco use, and alcohol were displayed in Fig. p = 0.022, OR = 1.35; and log-additive: p = 0.018, OR = 1.34). Due to lack of information, the correlation of MMP8 variants with stage and lymph node metastasis in CRC patients has not been explored.We also explored the correlation of selected SNPs with the pathological types of CRC Table . Stratif2, smokers, and drinkers. Furthermore, the significant association of rs3740938 with susceptibility to rectal cancer remained prominent.Table p = 0.012, testing accuracy = 0.5336, cross–validation consistency, 10/10) with the information gain of 0.34%.The interaction between these SNPs was evaluated using MDR analysis, and the results were shown in Table MMP8 rs3740938 with an increased CRC predisposition in the Chinese Han population. This relationship was particularly significant in subjects aged > 60 years, females, people with BMI > 24 kg/m2, smokers and drinkers. Moreover, MMP8 rs3740938 was related to the pathological type of rectal cancer. These findings contributed valuable data that could potentially be utilized in constructing a genetic panel for predicting CRC risk.In this study, we first examined the association of MMP8 is known to be expressed in various cancer types and may be associated with cancer cell invasion, proliferation, metastasis, and the poor prognosis of cancer patients [MMP8 rs11225395 was connected to the higher CRC risk in a Chinese Han population [MMP8 gene to CRC occurrence has not been reported. Here, we examined these three SNPs in the MMP8 gene. Our data displayed a significant association between MMP8 rs3740938 and an increased CRC predisposition in the Chinese Han population. Bioinformatics analysis using HaploReg v4.1 demonstrated that rs3740938 was related to promoter histone marks, enhancer histone marks and motifs changed. Furthermore, according to the QTLbase database, the genotypes of rs3740938 (p = 0.027) were found to be negatively associated with the expression of MMP8 in blood. Compared with rs3740938-GG and -GA genotypes, AA genotype may be associated with the lower expression of MMP8 mRNA. These findings suggested that the role of rs3740938 in CRC may be through affecting gene expression of MMP8, thereby affecting CRC occurrence. However, further experimental confirmation is needed to validate this hypothesis.patients . MMP8 inpatients . Proteinpatients . In CRC patients . MMP8. Mpulation . HoweverMMP8 polymorphisms with CRC occurrence under the stratified analysis by age, sex and BMI was explored. MMP8 rs3740938 might contribute to an increased susceptibility to CRC in participants aged > 60 years, females, and people with BMI > 24 kg/m2, indicating that the effects of rs3740938 on CRC occurrence are specific to age, gender, and BMI. As is well known, smoking increases the risk of various cancers, such as lung cancer, head and neck cancer, stomach cancer, etc [The incidence and mortality rates related to CRC have shown a steady increase . The inccer, etc . It is acer, etc . For a lcer, etc . We alsoMMP8 levels withMMP8 SNPs have not been fully elucidated. In subsequent researches, we will design detailed experiments to explore the expression data of MMP8 and the potential mechanisms and functions of these SNPs in CRC. Fourth, our study only assesses the correlation between three SNPs in the exon region of the MMP8 gene and CRC risk, and a large number of exonic or intronic variants remain to be studied. In subsequent studies, we will further explore the association of other loci in MMP8 with CRC susceptibility. Despite the above limitations, this is the first study that has reported MMP8 rs3740938 was associated with the increased CRC predisposition in a Chinese Han population, and this variant could serve as potential biomarkers of CRC susceptibility. These findings increased our knowledge regarding the effect of MMP8 on the process of CRC occurrence, provided some data for future explorations of the relationship between MMP8 and CRC risk in different populations, and also helped to establish new warning and treatment methods for CRC in futures studies. In the next step, we will further explore the functions of these SNPs based on the results of this study, in order to provide new theoretical basis and targets for the diagnosis and treatment of CRC.There are also potential limitations to our research. First, the sample size of our study was limited, and all participants were Chinese Han people. Future studies will involve a larger and more diverse sample population, along with validation experiments to confirm our results. Second, the lack of comprehensive information on various factors such as environmental exposure, lifestyle choices, and clinicopathological data  hinders a thorough understanding of their role in the association between the selected SNPs and CRC risk. Therefore, further studies is required to investigate these aspects and collected complete clinicopathological data to evaluate the relationship accurately. Third, the potential mechanisms and functions of these SNPs in relation to CRC risk, including association of high serum MMP8 rs3740938 might be a risk-increasing factor for CRC, revealing for the first time the role of rs3740938 in MMP8 in CRC risk among the Chinese Han population. Our findings might provide new biological insights into the role of MMP8 gene in the formation and progression of CRC.Our results show that Below is the link to the electronic supplementary material.Supplementary Table 1. Stratification for the effect of MMP8 rs1940475 and rs3765620 variants on CRC susceptibility.Supplementary Material 2


Upper limb motor recovery is one of the important goals of stroke rehabilitation. Intermittent theta burst stimulation (iTBS), a new type of repetitive transcranial magnetic stimulation (rTMS), is considered a potential therapy. However, there is still no consensus on the efficacy of iTBS for upper limb motor dysfunction after stroke. Stimulus dose may be an important factor affecting the efficacy of iTBS. Therefore, we aim to investigate and compare the effects and neural mechanisms of three doses of iTBS on upper limb motor recovery in stroke patients, and our hypothesis is that the higher the dose of iTBS, the greater the improvement in upper limb motor function.This prospective, randomized, controlled trial will recruit 56 stroke patients with upper limb motor dysfunction. All participants will be randomized in a 1:1:1:1 ratio to receive 21 sessions of 600 pulses active iTBS, 1,200 pulses active iTBS, 1,800 pulses active iTBS, or 1,800 pulses sham iTBS in addition to conventional rehabilitation training. The primary outcome is the Fugl-Meyer Assessment of the Upper Extremity (FMA-UE) score from baseline to end of intervention, and the secondary outcomes are the Wolf Motor Function Test (WMFT), Grip Strength (GS), Modified Barthel Index (MBI), and Stroke Impact Scale (SIS). The FMA-UE, MBI, and SIS are assessed pre-treatment, post-treatment, and at the 3-weeks follow-up. The WMFT, GS, and resting-state functional magnetic resonance imaging (rs-fMRI) data will be obtained pre- and post-treatment.The iTBS intervention in this study protocol is expected to be a potential method to promote upper limb motor recovery after stroke, and the results may provide supportive evidence for the optimal dose of iTBS intervention. Stroke In recent years, repetitive transcranial magnetic stimulation (rTMS) has been increasingly used as a safe and non-invasive neuromodulation technique for functional rehabilitation of the upper limb after stroke . rTMS plOne of the most important factors influencing the effectiveness of iTBS is the stimulus dose . When stTherefore, the objectives of this randomized controlled clinical trial are: (1) to investigate the safety and efficacy of one block of iTBS (600 pulses-iTBS), two blocks of iTBS , or three blocks of iTBS  in the treatment of upper limb motor dysfunction in stroke patients; (2) to compare the efficacy of three doses of iTBS; (3) to explore the neural mechanism of upper limb motor recovery after stroke using resting-state functional magnetic resonance imaging (rs-fMRI). Our hypothesis is that the higher the dose of iTBS, the greater the improvement in upper limb motor function in stroke patients. This study may provide new insights into the efficacy and underlying neuroplastic mechanisms of iTBS for treating poststroke upper limb motor dysfunction.2.2.1.www.chictr.org.cn under the registration number ChiCTR2300068177.This is a prospective, single-center, randomized, parallel, sham-controlled clinical trial. This study will be conducted at Beijing Bo’ai Hospital, China Rehabilitation Research Center . Hospitalized patients will be screened in accordance with strict inclusion and exclusion criteria. After written informed consent is obtained, demographic information collection and baseline assessments are scheduled for each patient. All participants will be randomly assigned with a ratio of 1:1:1:1 to one of the following four groups: Group A, Group B, Group C, and Group D. Details of the study design and data collection are presented in 2.2.2.2.1.Participants are considered eligible if they meet the following criteria: (1) have a unilateral ischemic or hemorrhagic subcortical stroke; (2) first or previous stroke without residual disability ; (3) 1–6 months post-stroke; (4) are 35–75 years old; (5) presented with a unilateral upper limb motor dysfunction with a Brunnstrom stage score between 2 and 5 in the upper limb or hand; (6) can sign an informed consent form.2.2.2.Participants will be excluded from the study if they meet the following criteria: (1) have a neurological impairment, upper limb limitation, amputation, joint swelling, contracture, severe pain, or other hand condition caused by a subtentorial stroke, brain tumor, brain trauma, fracture, or other disease; (2) the modified Ashworth scores (MAS) for the shoulder, elbow, wrist, and fingers of the affected upper extremity are all in the 3–4 range; (3) contraindications involving rTMS and rs-fMRI ; (4) have a disturbance of consciousness  1(a) ≥1); (5) have a malignant hypertension; (6) have a malignant tumor or any cause other than a stroke that results in a life expectancy of less than 1 year; (7) have severe aphasia (NIHSS language item ≥2), dysarthria (NIHSS dysarthria item ≥2), cognitive impairment  score ≤20), deafness, etc., such that they are unable to communicate, understand or follow instructions, and cannot cooperate with treatment and assessment; (8) have a major depressive disorder or anxiety disorder -17 score ≥18; Hamilton Anxiety Scale (HAMA)-score ≥21) or have been diagnosed with another mental disorder; (9) medication of antidepressants or benzodiazepines; (10) have severe sensory impairment (NIHSS sensory items = 2) or severe neglect (NIHSS neglect items = 2); (11) received neuromodulation therapy such as rTMS, transcranial electrical stimulation, and transcranial focused ultrasound within 3 months prior to enrollment; (12) have a history of alcohol or drug abuse; (13) are ineligible for participation in this trial due to other examination abnormalities; (14) are pregnant, or plan to get pregnant; (15) are participating in other clinical trials.2.2.3.Participants will be asked to withdraw from this trial if they meet the following criteria: (1) other neuromodulation therapies similar to iTBS are added during the study period; (2) miss iTBS treatment for 3 or more consecutive days or not complete treatment within 26 days; (3) have a sudden worsening of a stroke or other serious medical condition during the study period; (4) refuse to continue treatment; (5) the iTBS protocol is arbitrarily changed; (6) lost visits.2.3.According to a previously published meta-analysis comparing iTBS and sham stimulation , the exp2.4.Block randomization method will be used in this study. Once informed consent and baseline data have been obtained, all eligible participants will be randomly assigned to four groups using a random number sequence generated by SPSS 25.0 software. Random assignment is carried out by an independent researcher and kept in a sealed opaque envelope. The researchers responsible for iTBS will not be involved in the study design, recruitment, randomization, assessment, or data analysis. Participants and other researchers (such as outcome assessors or statisticians) will be blinded to the group allocation.2.5.A transcranial magnetic stimulator  equipped with a figure-of-eight coil (7 cm in diameter) is used to perform the iTBS procedure. An active stimulation coil is used to deliver the active stimulation, and a sham stimulation coil is used to deliver the sham stimulation. Prior to intervention, resting motor threshold (RMT) is determined by applying a single pulse of TMS over the contralateral M1 . RMT is Following daily iTBS intervention, each participant will undergo conventional occupational therapy and physical therapy for 1 h. The daily rehabilitation programs consist of exercises designed to promote voluntary motor recovery, including muscle stretching, passive and passive-assisted mobilization, progressive neuromuscular facilitation training, and task-oriented training. All exercises are performed by the same experienced therapists, who are unaware of the group allocation.2.6.The Fugl-Meyer Assessment of the Upper Extremity (FMA-UE) score from baseline (T0) to end of intervention (T1) is defined as the primary outcome. The FMA-UE is a standardized scale of upper limb motor impairment ranging from 0 to a maximum of 66 points . Seconda2, Flip angle = 90°, matrix = 64 × 64, Slice number = 32, voxel size = 3.5 × 3.5 × 4.35 mm3. Anatomical images are acquired using a high-resolution T1-weighted sequence .The magnetic resonance imaging (MRI) data will be collected by using a Philips Ingenia 3.0 T Magnetic Resonance System at the China Rehabilitation Research Center. Three rs-fMRI sessions of 8 min 6 s each are acquired using a gradient echo planar imaging sequence with the following parameters: TR = 2,000 ms, TE = 30 ms, FOV = 224 × 224 mm2.7.2 tests  will be used to compare demographic and baseline characteristics. A mixed-design ANOVA  will be used to detect any significant differences in the change of outcomes. Any factor with a significant difference between the groups at the baseline will be included as a covariate in the ANOVAs. If a significant time-by-group interaction effect is found, comparisons using pairwise t-tests will be performed by separately comparing the changes from baseline. Bonferroni correction is applied for post hoc tests. Before entering the data into the ANOVAs, the Shapiro–Wilk test will be used to test whether the data are normally distributed. For non-normally distributed measures, the non-parametric Kruskal-Wallis H test is performed to compare across conditions. The level of significance is defined as p < 0.05 (2-tailed). For missing data, intention-to-treat (ITT) analysis is used  or χ is used .p < 0.05 at the voxel level. A false discovery rate (FDR) cluster corrected p < 0.05 is applied.The pre-processing of the MRI data will be in accordance with the previously described pipeline . After p2.8.Any adverse effects occurring during MRI scan will be reported within 24 h. Participants will be provided with earplugs to protect their hearing from noise. iTBS treatment may cause headaches, scalp sensations or nociception, dizziness, or fatigue in some patients, which may resolve on their own after stimulation stops without special treatment. In particular, seizures, which are the most serious TMS-related adverse effect with a risk of about 0.02%, are expected to occur only during or immediately after stimulation . To mini3.This randomized controlled clinical trial aims to investigate and compare the efficacy of three doses of iTBS on the recovery of upper limb motor function in stroke patients. Furthermore, we hope to provide a theoretical basis for clinical application by investigating the neural effects of iTBS intervention on upper limb motor dysfunction. Our results may provide supportive evidence for the optimal dose of excitatory iTBS intervention in the treatment of upper limb motor dysfunction after stroke.In this study, we hypothesize that iTBS can promote upper limb motor recovery in stroke patients by modulating functional connectivity within the motor network. We also hypothesize that the higher the stimulus dose, the greater the improvement in motor function. The following evidence supports this hypothesis. Increasingly, it is thought that increasing the dose of rehabilitation may lead to better motor function outcomes in stroke patients . rTMS isIn terms of experiment design, our study raises some methodological concerns worth discussing. We set up four groups  corresponding to four conditions  in this study. It is important to note that, for statistical reasons, the iTBS intervention protocol for both Groups A and B includes sham stimulation so that the total stimulation time per day is the same for each group, thus ensuring successful blinding. Referring to a previous similar study , and conM1 plays an important role in motor recovery after stroke. The researchers examined the brain activation during movement of the upper limb in stroke patients and described the altered activity in the ipsilateral and contralateral M1 . PrevioufMRI, a non-invasive brain imaging technique with high spatial resolution, has been widely used to explore the brain. Some previous studies have used fMRI to investigate the neural mechanisms by which low-frequency rTMS or high-frequency rTMS improves motor recovery after stroke , but fewiTBS has the advantages of easy operation and short stimulation time, and its efficacy may be influenced by the stimulus dose. We hope that our protocol and results can elucidate this potential effect and provide guidance for the treatment of patients with upper limb motor dysfunction after stroke.This study involving human subjects was reviewed and approved by the Medical Ethics Committee of China Rehabilitation Research Center. Patients/participants will provide written informed consent to participate in this study.ZT: Supervision, Writing – original draft, Writing – review & editing. TL: Investigation, Resources, Writing – review & editing. YL: Investigation, Resources, Writing – review & editing. KH: Methodology, Writing – review & editing. WS: Visualization, Resources, Writing – review & editing. JZ: Resources, Writing – review & editing. QC: Funding acquisition, Investigation, Writing – review & editing. XZ: Writing – review & editing. HZ: Conceptualization, Writing – review & editing.