Osteosarcoma is the most common malignant bone tumor in children, with a 5-year survival rate ranging from 70% to 20% depending on the aggressiveness of the disease. The current treatments have not evolved over the past four decades due in part to the genetic complexity of the disease and its heterogeneity. This review will summarize the current knowledge of OS origin, diagnosis and therapies.Osteosarcoma (OS) is the most frequent primary bone tumor, mainly affecting children and young adults. Despite therapeutic advances, the 5-year survival rate is 70% but drastically decreases to 20–30% for poor responders to therapies or for patients with metastasis. No real evolution of the survival rates has been observed for four decades, explained by poor knowledge of the origin, difficulties related to diagnosis and the lack of targeted therapies for this pediatric tumor. This review will describe a non-exhaustive overview of osteosarcoma disease from a clinical and biological point of view, describing the origin, diagnosis and therapies. Osteosarcoma (OS) is the most common primary malignant bone tumor. It is also the third most common type of cancer affecting children and adolescents after lymphomas and brain tumors . OS cellOS mainly affects children or young adults. The incidence of OS increases from two to three million per year in the general population to eight to eleven million per year when only the 15–19-year-old age group is considered. OS represents 3–6% of childhood cancers  and 1% of adult cancers. After the peak of incidence at 15–19 years of age, another peak of incidence occurs in elderly populations (over 65 years of age) and occurs as a result of pre-existing bone pathology or fractures. Young males are 1.4 times more likely to develop the disease than young females. However, the risks are equivalent between the sexes in people older than 65 years [The causes of OS are still poorly understood. Its appearance in young populations during growth and its location at the ends of the long bones suggest the involvement of rapid bone production . This hyOS is often diagnosed after persistent local pains, often mistakenly attributed to growth in young people, or to physical activity. Patients may also exhibit a palpable mass and reduced joint range of motion. Systemic symptoms  may also be present. The tumor will most frequently be located in the metaphysis of long bones , with 50% of cases located near the knee ,11,12. TDiagnosis is made after palpation to determine the presence of a soft mass near the bone. Radiographically, calcifications resulting from ectopic bone formation may be seen within the soft mass, as well as a “sunburst” appearance. Ectopic bone formation is often associated with osteolysis . IncreasBecause biopsy can identify OS with an accuracy level of 90%, histological analysis is often performed to confirm the diagnosis. It allows to determine the level of proliferation of the tumor cells, to classify the tumor according to the stage of severity.OS is classified into secondary or primary OS. Secondary OS occurs after pre-existing events, such as diseases  or irradiation ,17. ConvThere are other subtypes of OS, including sclerosing osteoblastic, chondromyxoid fibroma-like, chondroblastoma-like, clear cell, giant cell, epithelioid, osteoblastoma-like or malignant fibrous histiocytoma-like OS. However, these types of OS are considered subtypes of the conventional type due to their similarities and behaviors to the conventional type .-The Musculoskeletal Tumor Society (MSTS) or Enneking System ,21:The grade, used to characterize OS, takes into account the severity of the disease and the presence of metastases. Grade of OS helps to adjust treatment, estimate patient prognosis, assess treatment outcomes and facilitate communication between professionals ,20. SeveThree main pieces of information are given by this system: grade, extension of tumor and presence or absence of metastasis .Grade (G): indicates the potential of the tumor to grow and spread. It is based on the histological appearance of the tumors. The G1 grade refers to low-grade tumors . They are less likely to grow quickly and metastasize. The G2 grade refers to high-grade tumors that are abnormal in morphology, rapidly dividing and likely to metastasize.-Extent of the primary tumor (T): T1 refers to a tumor that is confined to the bone , and T2 refers to a tumor that affects surrounding structures .-Metastasis (M): M1 indicates that a tumor has spread to nearby lymph nodes. M0 indicates no spread to lymph nodes.-The TNM  System ,22:The TNM classification system was created by the AJCC (American Joint Committee on Cancer): the T parameter describes the size of the primary tumor as well as its presence or absence in different regions of the bone; the N parameter describes the spread to nearby lymph nodes; the M parameter indicates whether the cancer has metastasized to other organs in the body .The AJCC classification contains 7 stages , which are subdivided into about 30 stages, each of which accurately describes the tumor. A simplified version of this classification is shown in Patients with OS are treated with a multidisciplinary approach established by Rosen et al. in the 1970s . This prFurthermore, radiotherapy could be used to delay tumor growth and to reduce symptoms, such as pain, in the case that surgery is not possible, and could lead to possible side effects . The used radioactive drugs are radium-233 or samarium153-EDTMP (ethylenediaminetetramethylene phosphonic acid), which have high affinity for bone tissue and, thus, selectively deliver radiation to osteoblastic lesions ,29. SamaChemotherapy is used before surgery to reduce the size of the tumor and create the best conditions for limb salvage surgery and after surgery to eliminate residual lesions and metastases . The durSurgery for OS aims for the complete removal of the tumor, with a focus on large resection margins that include the tumor and the surrounding healthy tissue. However, the local recurrence rate could be up to 25% if the tumor removal is not complete . There iAfter preoperative chemotherapy, patients are classified as good or poor responders based on the number of viable cells remaining in the resection specimen . The metDespite treatment, 30 to 40% of patients will have a recurrence within 2 to 3 years after the end of treatment. At the time of recurrence, the metastases, mostly pulmonary (90%), must be surgically removed in order to increase patients’ survival. However, the 5-year survival after the second diagnosis is about 20%. The current treatments cause severe side effects that diminish patients’ quality of life ,41. WhilDespite the fact that conventional therapies have lowered the frequency of amputations, the most commonly used drugs date from the 1970s, and the survival rates have remained relatively constant since then. This lack of progress might be attributed to the disease’s rarity as well as its wide variability.OS is a complex and heterogeneous tumor characterized by a high level of genomic instability, aneuploidy and genomic rearrangements, with gains of portions of chromosomes  or losses of portions of chromosomes  in conventional disease, generally corresponding to regions where oncogenes and tumor suppressor genes are located, respectively . OS can fl/fl mouse model, in which neither OS development nor skeletal abnormalities were observed, concluding that Rb mutation is not sufficient to induce OS development [Tumor suppressor genes inhibit tumor growth, for example, by regulating the cell cycle. Many genes belonging to this category are affected in OS. For example, the TP53 gene is the most mutated gene in human tumors , encodinelopment .The functions of p53 and Rb can also be affected by the mutation of CDKN2A (cyclin-dependent kinase Inhibitor 2A). This gene encodes two proteins, p14Arf and p16INK4, which activate p53 and Rb, respectively. P14 prevents the degradation of p53 mediated by the E3 ubiquitin ligase MDM2 (mouse double minute 2 homolog). P16 inhibits CDK4 (Cyclin-dependent kinase 4), a protein capable to inactivate Rb by phosphorylation. The CDKN2A locus (9p21) is altered in 5–21% of OS ,83,84,85ATRX is another protein frequently mutated in OS (29% of tumors). It is part of a multiprotein complex regulating chromatin remodeling and telomere maintenance . ATRX isOncogenes confer a proliferative advantage to tumor cells. Their expression or activity is increased in cancers. In OS, gain-of-function mutations are observed, for example, in E2F3 (60% of tumors), a transcription factor of the E2F family involved in many cellular processes, including replication, DNA repair and apoptosis. Its inhibition in cancers notably decreases tumor growth by disrupting the cell cycle . CDK4  gene coding for the MTAP, a key enzyme in the salvage of cellular adenine and methionine synthesis, is frequently observed in OS patients. Indeed, deletion of at least one exon of MTAP gene was observed in 37.5% of the patients in a cohort of 96 patients with high-grade OS . No exprBeyond proteins, other cellular actors are involved in genetic abnormalities in OS. Thus, miRNAs (microRNAs), lncRNAs (long non-coding RNAs) and non-protein coding ribonucleotide sequences also have deregulated expression in OS. For example, Pasic and colleagues have shown that the lncRNA loc285194, known to be a p53-controlled tumor suppressor, has its locus deleted in OS. This deletion is associated with poor prognosis in patients ,124. StuThus, OS is highly heterogeneous genomically, genetically and epigenetically. As an example, Poos et al. identified 911 proteins and 81 miRNAs described as associated with OS . This tuThe use of experimental models makes it possible to understand the effects of new therapeutic strategies before their use in humans. Several types of models are currently used to mimic the pathology. Cellular models from patients with OS have been used routinely in the laboratory for decades . They arThe mouse (Mus musculus) is used as an in vivo model in many pathologies. One of the oldest in vivo xenograft models used in cancer is the transplantation of human OS cell lines to mice ,145,146.Other OS animal models exist, such as dogs (Canis familiaris), especially large ones, which spontaneously develop OS with a higher incidence than in humans, although the disease remains rare ,148. As The zebrafish model (Danio rerio) is a model used to study tumor growth, migration and invasion of tumor cells in cancers . After iAnimal models, although imperfect or restrictive, are essential for the development of new and effective treatments.It has been shown that the tumor is able to hijack the bone remodeling mechanism to ensure its growth. This phenomenon, called a vicious cycle, is found in OS but also in other tumors metastasizing to bone, such as breast or prostate cancer ,153,154.Immunomodulation is the modification of immunity or the immune response, with the use of interferons (IFN) as an example. These are glycoproteins of the cytokine family that are produced by immune cells in response to viral infections, showing some efficacy in cancers . In OS, One of the immunotherapy strategies is to target the cell surface proteins using monoclonal antibodies, which bind specific antigens on the surface of tumor cells, and then activate NK cells and macrophages . A phaseMonoclonal antibodies have also been used to target the growth hormone receptor insulin-like growth factor-1 (IGF-1). A first phase II clinical trial including 38 OS patients showed that R1507, a recombinant human monoclonal antibody, is well tolerated but has limited efficacy in patients with relapsed or refractory bone and soft tissue sarcomas (NCT00642941) . The effDisialoganglioside (GD2) is highly expressed in more than 95% of OS and is involved in cell proliferation, motility, migration, adhesion, and invasion, amounting tumor development and malignant phenotypes ,180. TheLeucine rich repeat containing 15 (LRRC15) is a membrane protein highly expressed on the cell surface of stromal fibroblasts in many solid tumors including OS and its expression is induced by TGFß. This protein has a role in cell–cell and cell–extracellular matrix interaction. It is a novel mesenchymal protein and stromal target for monoclonal antibody-drug conjugates ,186. A pB7 Homolog 3 (B7-H3), also known as CD276, is a protein whose main role is to inhibit adaptive immunity by suppressing T cell activation and proliferation . A phaseOther immunoconjugates have been tested in OS using toxin or radionuclide as a linker joined with a carrier antibody and have shown promising results . Indeed,CD146, overexpressed in OS, was used to develop an anti-CD-146 murine antibody named OI-3 coupled with Iodine-125 or Lutetium-177 and was evaluated in an OS xenograft model, showing promising results with therapeutically relevant biodistribution of the radionuclide . InsulinCheckpoints restrict overly aggressive immune responses and, in some cases, block T cells from killing tumor cells . T lymphPD-1, also known as CD279, is a transmembrane member of the immunoglobulin family expressed on activated cytotoxic CD8+ T cells and natural killer cells . PD-1 liHowever, the clinical evaluation of the checkpoint inhibitors in sarcoma was not a success, as observed in melanoma as an example, probably due to the immunosuppressive role of the microenvironment ,214.Some therapeutic strategies are based on the inhibition of osteoclasts . By doinOther bone resorption inhibitors have shown promising results. Indeed, bisphosphonates  are molecules capable of inhibiting bone resorption. They are pyrophosphate analogues capable of binding to the hydroxyapatite constituting the bone matrix and of inducing apoptosis of osteoclasts . They haReceptor tyrosine kinases (RTKs) are transmembrane receptors with an extracellular domain that binds the ligand and an intracellular domain with kinase activity . RTKs are involved in cell growth, proliferation and survival, in particular through their involvement in signaling pathways, such as the PI3K (phosphoinositide 3-kinase) pathway or the MAPK pathway. About 30% of RTKs are mutated or overexpressed in cancer, including osteosarcoma. As a result, the expression of certain RTKs is associated with poor prognosis for patients . Six subSince the FDA approved imatinib for the treatment of chronic myeloid leukemia in 2001, many potent and well-tolerated tyrosine kinase inhibitors have been developed and have contributed significantly to advances in cancer treatment . TherefoThe intertwining of different signaling pathways downstream of RTKs is responsible for the rapid emergence of compensatory mechanisms that negate the effect of molecules targeting a limited number of RTKs. Thus, multi-tyrosine kinase inhibitors (mTKIs) have emerged and have shown effects on patients in relapse and/or with unresectable OS. Five molecules stood out for their major effects: Sorafenib, Regorafenib, Cabozantib, Lenvatinib and Pazopanib. Sorafenib, an inhibitor of VEGFR, PDGFR, RET and c-Kit, has shown antiangiogenic and anti-metastatic effects in preclinical models . In the Inhibition of RTKs is promising and actively studied, but other approaches aim to inhibit intracellular signaling downstream of RTKs . Indeed,Inhibitors of mTOR have also been developed. mTOR is a serine/threonine kinase involved in the deregulated PI3K/Akt pathway in most cancers ,280. mTOThe YAP/TAZ  signaling pathway was recently described to be involved in OS with aberrations in the Hippo signaling pathway ,289. IndThus, many therapeutic targets are currently being studied for the treatment of OS . Often vDevelopment of therapeutic targets in OS is very complex due to the heterogeneity of this pathology, which limits the effectiveness of treatments and favors tumor recurrence and the emergence of drug resistance. Indeed, in the last few decades, only a few new therapies have shown a clinically significant impact for patients with OS. To supplement the current knowledge and uncover possible ways to improve patient outcomes, fundamental, translational and clinical research must cooperate, thus allowing the development of new prognostic markers and new therapeutic targets in OS.


Glyphosate is one of the most widely used non-selective herbicides, and the creation of glyphosate-resistant cultivars solves the problem of limited spraying area. Therefore, it is of great significance to quickly identify resistant cultivars without destruction during the development of superior cultivars. This work took maize seedlings as the experimental object, and the spectral indices of leaves were calculated to construct a model with good robustness that could be used in different experiments. Compared with no transfer strategies, transferability of support vector machine learning model was improved by randomly selecting 14% of source domain from target domain to train and applying transfer component analysis algorithm, the accuracy on target domain reached 83% (increased by 71%), recall increased from 10 to 100%, and F1-score increased from 0.17 to 0.86. The overall results showed that both transfer component analysis algorithm and updating source domain could improve the transferability of model among experiments, and these two transfer strategies could complement each other’s advantages to achieve the best classification performance. Therefore, this work is beneficial to timely understanding of the physiological status of plants, identifying glyphosate resistant cultivars, and ultimately provides theoretical basis and technical support for new cultivar creation and high-throughput selection. High efficiency and low cost make herbicides become an important means in weed management . Among tin vitro culture and field screening verifications are often required in the process of new glyphosate resistant cultivars creation. Common screening methods including visual observation and bioassays, take 10–14 days from spraying glyphosate to resistant identification  technology can obtain the images and spectra of samples simultaneously . Images Generally speaking, an optimal machine learning model can achieve satisfactory results based on specific data sets . But it Therefore, HSI technology is a powerful tool to rapidly screen target cultivars and accelerate the breeding process. The spectral index can be used to evaluate the state of plant growth. Machine learning can fully mine spectral information to improve model performance on the test set. And emerging transfer learning can further improve model performance in terms of universality on various datasets. However, there are few researches on the detection of glyphosate-tolerant cultivars based on spectral indices of leaves of maize seedling, not to mention the transferability of machine learning model between different experiments.In this study, we aimed to propose a high-throughput rapid non-destructive model for identifying glyphosate-resistant cultivars which could be used to screen new samples from different times. Specifically, the following questions were discussed: (1) what was the difference in spectral index between glyphosate resistant and sensitive cultivars? (2) how to build a robustness model for identifying glyphosate resistant cultivar? (3) could the transfer strategy improve the classification model? By responding to the above questions, this research could help breeders timely understand the physiological conditions of plant stress, complete the detection of glyphosate-tolerant cultivars, and ultimately provide theoretical basis and technical support for new cultivar creation.Two maize cultivars, glyphosate-resistant and glyphosate-sensitive, were designated as R and S, respectively. Glyphosate resistance of R maize was obtained by expression of mutant 5-enolpyruvylshikimate-3-phosphate synthase enzyme. All the seeds were provided by the Institute of Insect Science, Zhejiang University, Hangzhou, China. The detailed information on these two seeds was introduced in our previous study . Three iFirst of all, it is worth noting that the treatment group of R, the control group of R, the treatment group of S, and the control group of S are designated as RT, RW, ST, and SW, respectively. Time-series visible/near-infrared hyperspectral images of alive maize plants were collected at 2, 4, 6, and 8 days after treatment (DAT) by a line-scan HSI system in the visible/near-infrared range , which was reported in detail in previous study . Over thA general processing workflow of hyperspectral data in plant science includes pre-processing, machine learning preparation and model building . TherefoTo eliminate the impact of ambient light, original hyperspectral images needed correcting with a black (selected the camera lens cap with reflectance close to 0) and white reference image (selected the pure white Teflon board with reflectance close to 1). Then, in order to focus on the spectral features of regions of interest and facilitate further analysis, it was essential to identify and segment each leaf in each hyperspectral image and then extract the spectrum of the leaf. This process was divided into two main phases. First, the threshold segmentation method was used to extract the plant region . Second, the stem and leaf were separated by manually selecting the stem region with several rectangles. Based on the shape and reflectance of leaf spectral curve, the abnormal samples caused by measurement errors were rejected. As the study reported , the heap = 0.05) was used to study the feasibility of 16 spectral indices in identifying glyphosate-tolerant cultivar.Leaf surface reflectance provides a wide perspective for plant growth conditions . As a deTo prepare the data for modeling, the dataset was divided into two subgroups (training set and test set) with the same feature distribution. The Kennard-Stone (KS) algorithm was used to divide the dataset. KS algorithm selects training dataset samples based on Euclidean distance between variables, and ensures uniform distribution of training dataset samples according to spatial distance . SpecifiTo investigate the transferability of machine learning model in the case of the training set and test set coming from different data distribution, a total of 24 transfer tasks were designed . In detaConsidering the data distribution differences between source domain dataset and target domain dataset, in order to further improve the model performance and transferability, two transfer strategies of transfer component analysis (TCA) and source domain updating were used before modeling. On the one hand, as a typical transfer learning algorithm, TCA generally performs the role of preprocessing in data analysis, and its input and output are two large matrices and two small matrices, respectively. TCA maps the source domain dataset and target domain dataset with different distribution to a reproducing kernel Hilbert space, and then continuously reduce the distance between the two domain datasets and retain their internal attributes as many as possible . Specifi•x + b = 0, where ω is an n-dimensional vector (n is the number of the features), x is the data of sample, and b is a constant. More detailed theories of SVM algorithm are available in the literature , compared with no parameter optimization, automatic parameter optimization greatly increased the training time (685–1304 times), and did not improve model performance (0.86–1.10 times for accuracy) significantly. This result supports the opinion that linear SVM model is not very sensitive to its hyperparameter  algorithm is one of the most robust and accurate discrimination methods. From the geometric point of view, the merit of SVM is reflected in the maximum margin needed when constructing hyperplane decision boundaries, so there is sufficient space between interval boundaries to contain test samples. For linear SVM, the general function of the decision boundary is ωterature . In thisarameter . TherefoTo quantitatively evaluate classification model performance, the statistical indices in Stem and leaf segmentation, model construction, and model performance calculation were processed in MATLAB R2016a . All of the graphs were designed by using Origin 2021b  and Microsoft PowerPoint 2016.In order to investigate the feasibility of identifying glyphosate-resistant cultivars with 16 spectral indices selected in this work, ANOVA was used to compare each spectral index among four groups  at each sampling time point  in each experiment . p > 0.05) between RT and ST, while at 8 DAT, the difference in most spectral indices of RT and ST were more pronounced (p < 0.05).It can be seen from the descriptive statistics of 16 spectral indices at 2, 4, 6 and 8 DAT in Exp.1 , at 2 DAp < 0.05). At 8 DAT, EVI (enhanced vegetation index), NRI (nitrogen reflectance index), RDVI  and TCARI/OSAVI (the ratio of transformed chlorophyll absorption in reflectance index to optimized soil-adjusted vegetation index) of RT and ST exhibited pronounced differences for the first time. Although TVI (triangular vegetation index) of RT and ST showed no significant difference up to 8 DAT, it showed significant difference at 6 DAT , SVM algorithm was used to evaluate the model performance of each experiment at each sampling time point. For each dataset, confusion maps of classification results on training set and test set were shown in When the treatment days were not distinguished, the average of accuracy, precision, recall, F1-score, and FPR of SVM models on the test set of three experiments were 0.83, 0.76, 0.86, 0.80, and 0.20, respectively. Among them, three experiments showed significant difference in the recall values, varied from 0.69 to 1. When the dataset obtained on each sampling day was modeled separately, the difference between experiments were even more pronounced. Specifically, for Exp.1, RT was identified correctly at 6 DAT without misjudging ST as RT (FPR = 0 in test set), and the accuracy was 100% at 8 DAT; for Exp.2, the accuracy in test set as early as 6 DAT was 100%; for Exp.3, the SVM model was able to accurately classify RT as early as 4 DAT (accuracy = 1 in test set). The results demonstrated that the earliest accurate identification time of the SVM model may vary with different experiments. It was worth noting that both in Exp.2 and Exp.3, the performance of SVM model at 8 DAT inferior to that at 6 DAT, which was mainly reflected in the misjudgment of ST as RT. Those results may be attributed it to small size of dataset and the fact that some old leaves came close to death no matter what cultivar on 8 DAT. In conclusion, the combination of the selected 16 spectral indices and SVM algorithm could rapidly identify glyphosate resistant and sensitive cultivar in a non-destructive manner.According to the results in As the benchmark for evaluating the performance of transfer strategies, the SVM algorithm was also conducted in 24 transfer learning tasks represented in Compared to For the three transfer tasks  with poor classification accuracy in section “The performances of SVM models on transfer learning tasks,” TCA algorithm was applied in an attempt to improve the transferability of SVM models. After narrowing the data distribution distance difference between source domain and target domain, the SVM algorithm was applied to develop models using the five transformed features of source domain, and the results were showed in After TCA applied, although the classification accuracy of the transfer tasks (Exp.2→Exp.1 and Exp.3→Exp.1) improved, it was still failed to reach the level of SVM models based on a single experiment. Therefore, did transfer learning strategy_2 (update source domain) performed better in improvement of SVM transferability?In this work, five source domain dataset updating levels were set, namely 10, 20, 30, 40, and 50% of target domain. Compared with direct transfer , TCA algSince the performance of TCA_SVM and Update_SVM model in the transfer learning task Exp.3→Exp.1 was quite different, this section explores the question of whether the two transfer strategies could complement each other’s advantages to further improve the performance of the SVM model.in vitro leaves reported in previous study , recall increased from 10 to 100%, and F1-score increased from 0.17 to 0.86. The overall results indicated that compared with direct model transfer, both transfer learning strategies improved model transferability between different experiments although the prediction results varied with different added number of new samples from source domain, and these two strategies could complement each other’s advantages. Inspired by the distinct positive contribution of the transfer learning strategy, future work will be concentrated on experiments with more cultivars, growing conditions and spectral devices to investigate the universality of the transfer learning strategies. Ideally, these results could someday be validated and optimized enough that ultra-portable instrument combined with the transfer learning strategy could be employed to screen glyphosate resistant cultivars on large scale in a rapid, non-destructive and high-throughput way, which could help breeders improve work efficiency.The original contributions presented in this study are included in the article/MT and XF conceived, designed the experiments, analyzed the data, and wrote the manuscript. MT, XB, and XC performed the experiments. XF, YH, YW, and CP made critical comments and revisions. All authors contributed to the article and approved the submitted version.


Objective: The study aimed to assess the efficacy and safety of clinical trials of biologics in improving the salivary gland (SG) function in primary Sjögren’s syndrome (pSS), which has not been analyzed critically and systematically.Methods: PubMed, Web of Science, ClinicalTrials.gov, the EU Clinical Trials Register, and the Cochrane Library were searched for clinical trials that reported effects of biological treatment on the SG function and safety in pSS patients. Inclusion criteria were defined following participants, interventions, comparisons, outcome, and study design (PICOS) recommendations. The objective index  flow) and the serious adverse event (SAE) were assessed as main outcome measures. A meta-analysis of the efficacy and safety of the treatment was conducted. Quality assessment, sensitivity analysis, and publication bias were assessed. The effect size together with a 95% confidence interval was used to estimate the efficacy and safety of biological treatment and was plotted as a forest plot.Results: The literature search yielded 6,678 studies, nine of which fulfilled the inclusion criteria, with seven randomized controlled trials (RCTs) and two non-RCT clinical studies. Generally, biologics do not significantly increase UWS from the baseline of pSS patients compared to the control group at a matched time point . However, pSS patients with shorter disease duration  were prone to have a better response to biological treatment by showing higher increased UWS than patients with longer disease duration  (p = 0.03). For the meta-analysis of the safety of biological treatment, the SAEs in the biologics group were significantly higher than those of the control group .Conclusion: Biological intervention during the early course of the disease may benefit pSS patients better than that during the late course. Significantly, more SAEs in the biologics group indicate that the safety of biologics needs to be addressed for future biological clinical trials and treatment. Primary Sjögren’s syndrome (pSS) is a systematic and chronic autoimmune disease with a strong organ bias, which is clinically manifested with exocrine gland dysfunction (particularly dry mouth and dry eyes) and various extra-glandular manifestations. Serological and histopathological assessments show an increased level of serum autoantibodies and lymphocytic infiltration in exocrine glandular tissues, respectively . pSS is + T cells play a more important role  pathology. In the earlier phase of disease progression, the innate immune response and CD4ant role . In the ant role  and, theant role .Hyposalivation significantly affects the life quality of pSS patients, who suffer from sicca (dry mouth) symptoms with various complaints, including dental caries, change of taste, and difficulties in eating, sleeping, and speaking . Saliva The mechanism behind deteriorated SG function remains elusive. Thus, current treatment for pSS mainly focuses on symptom alleviation, such as using artificial saliva and secretagogues . Debates exist as to whether chronic inflammation results in hyposalivation in pSS patients and whether the depletion of inflammation contributes to the alleviation of a dry mouth . Recent Biologics are medical products produced through the biological process, working as a new type of drug that suppresses immune responses and reduces inflammation . BiologiClinicalTrials.gov, the EU Clinical Trials Register, and the Cochrane Library were searched for this systematic review and meta-analysis (updated until October 2022). Studies about the biological treatment of pSS with endpoints of the alleviation of hyposalivation and the safety of drugs were searched. Studies being searched were limited to those published in English. The keywords used were primary Sjögren’s syndrome, biologics, saliva, and safety or serious adverse event (SAE) and additional terms such as rituximab, abatacept, seletalisib, iscalimab (CFZ533), infliximab, etanercept, BMS-931699/Lulizumab, and epratuzumab. The title, abstract, and full text were downloaded and read for being assessed for relevance. The reference list of included articles was examined to include the possible missing studies. Published or registered clinical trials with results were considered as the source of this systematic analysis.Electronic databases of PubMed, Web of Science, The selection of studies was conducted according to the participants, interventions, comparisons, outcome, and study design (PICOS) recommendations.Study type: Original research. Participants (P): pSS patients. Interventions (I): Biological treatment. Comparisons (I): No intervention and interventions without biological treatment, standard treatment, or placebo. Outcome (O): The change of saliva secretion and the occurrence of SAE during/after the biological treatment. Study design (S): Well-designed clinical trials.Study type: Review articles, case reports, comments, and articles with the abstract only. Incomplete data or data impossible to be extracted were excluded. Replicative studies or studies using the same group of patients were excluded. Participants (P): Non-pSS patients  and non-human animals. Interventions (I): Non-biological treatment. Comparisons (I): Absence of comparisons. Outcome (O): No outcome related to the change of saliva secretion or SAE during/after the biological treatment. Study design (S): Non-clinical trials.All searched articles were screened by two independent authors for determining whether the inclusion and exclusion criteria were met. The discrepancy was resolved by discussion with the third author. Data on the author; year of publication; country/region where the trial was conducted; sample size; type and dosage of biological intervention; age, gender, and disease duration of participated patients; the method of measurement of saliva secretion, outcomes for the biological treatment, and control groups were extracted. The change of unstimulated whole saliva (UWS) secretion from baseline was used for the evaluation of salivary gland function. Total SAEs and SAEs specified into different system disorders were employed for the safety assessment of biologics.The quality of RCT studies was assessed by using the Cochrane risk of bias tool by two independent authors and illustrated by Review Manager 5.3 . When inThe sensitivity analysis of the UWS response and SAEs in the biological treatment and control groups was conducted by omitting the included studies one by one. The publication bias is shown by the funnel plot and also quantified by the regression-based Begg’s test for small-study effects.p- and I2 values were calculated. When p ≥ 0.10 and I2 ≤ 50%, suggesting the homogeneity was appropriate for meta-analysis, the fixed effects model was employed. Otherwise, a random effects model was used. In addition to the overall assessments, these included studies were also subjected to two groups by the mean disease duration (less or more than 3 years) for subgroup analysis to assess the effect of biologics and their safety.The extracted data were imported into Stata 16.0 software and subjected to statistical analysis. For dichotomous variables, a log odds ratio (OR) with 95% confidence interval (CI) was calculated. For continuous outcomes, standard mean differences (SMDs), as indicated, with 95% CI were calculated. To assess the heterogeneity among studies, From database searching, 6,678 studies were identified. Of them, 5,689 review articles, case reports, comments, and articles with only the abstract were excluded. A total of 922 irrelevant studies were excluded from this systematic analysis after title and abstract reading. A total of 35 studies were excluded because of duplication. The full text of the remaining 32 studies was read, and 23 of them were found to contain incomplete data or data that were impossible to be extracted or were studies that did not contain the control group. Finally, nine studies were included in this systematic meta-analysis  Mariett.In these nine clinical trials, with seven RCTs  and the outcome of the SAE (p = 1.971) of these included studies was found for this meta-analysis. There was also no significant bias for SAEs specified into different system disorders , showing no significant effect on UWS of pSS patients  = 0.05; 95% CI: −0.11 and 0.21). During these biological clinical trials, early intervention was recommended to expect a better response on pSS treatment (p = 0.38 and I2 = 0.00%). A significant group difference was found (p = 0.03). The increase of UWS flow after biological treatment was much greater in patients with shorter baseline disease duration  than those with longer baseline disease duration .The meta-analysis of the overall effect of biological treatments on the UWS flow in pSS patients is shown in reatment . Accordireatment . As the reatment , the autp = 0.0021, OR = 1.03, and 95% CI: 0.37 and 1.69), with an acceptable heterogeneity (p = 0.96 and I2 = 0.00%). The disease duration of pSS patients did not cause a difference in the incidence of SAEs between the biologics and control groups . Reported SAEs in the biological treatment group included five neoplasms, six immune system disorders, five infections and infestations, five gastrointestinal disorders, three general disorders, three hepatobiliary disorders, two reproductive system issues, three musculoskeletal and connective tissue disorders, two cardiac disorders, one blood and lymphatic system disorder, and one nervous system disorder  is still controversial. It was commonly accepted that infiltrated lymphocytes occupy and destroy the parenchyma SG and result in the decrease of saliva production . HoweverInterestingly, in the trial with the biological intervention of pSS patients with shorter disease duration, the increased secretion of the UWS flow of patients was significantly higher than that in patients with longer disease duration. Early diagnosis to recognize pSS, in addition to providing this potential benefit, may also prolong the treatment window available and should be prioritized in future endeavors. However, only two biological trials were performed in early pSS patients; more studies are needed to test the efficacy of biologics in the alleviation of hyposalivation in early pSS.As introduced earlier, the homeostasis of SG is maintained by the cellular duplication and differentiation of SG progenitor cells . Our preIn this meta-analysis, the safety of biological intervention was also considered. Significant, more frequently occurring SAEs remind us that drug safety needs to be carefully considered before intervention. Interestingly, the individual system SAEs were not significantly different between biological treatment and the control groups, indicating the increased SAEs in the biologics group were not confined to one system or one type of disease. Influence factors of the safety of biologics may include the type of biologics, the dose, the administration route, and the administration frequency . The safBlinding of the outcome assessment is a common item that may cause a high risk of bias (five out of seven RCTs). Moreover, the limitation to this study is that only nine trials fulfilled the inclusion criteria and were included for the systematic analysis. If more outcomes are reported with the completion of current ongoing biological trials, the conclusion of this systematic review could be more convincing.To conclude, although clinical trials of biologics show limited efficacy in the restoration of SG function in pSS patients, the better response of SG function to biologics in pSS patients with shorter disease duration reminds us to administrate timely biological interventions in the early course of the disease before the function of SG becomes irreversible. Additionally, the safety of administered drugs needs to be always kept in mind. Future biological clinical trials need to concentrate on trials that have better SG response and higher safety, such as interventions that can topically, rather than systemically, alleviate the inflammatory microenvironment.


The morbidity and fatality rates of non-small-cell lung cancer (NSCLC) were high, although a combination of multiple treatments was used. Fisetin, a small flavonoid compound, had shown anticancer activities. Thus, we aimed at exploring the mechanisms of Fisetin in the treatment of NSCLC.  TCMSP and Swiss target tools were used to screen the targets of Fisetin, and GeneCards was used to collect the genes related to NSCLC. The genes common to Fisetin and NSCLC were obtained by Venn analysis, whose possible functions were further annotated. A “Compound-Target-Disease” network was then constructed and hub genes were filtered. Also, molecular docking was performed to predict the binding abilities between Fisetin and the hub genes. Then, the effects of Fisetin on the expression of hub genes in lung adenocarcinoma cells were preliminarily evaluated in vitro.  A total of 131 genes common to Fisetin and NSCLC were filtered out, which might be enriched in several biological processes including antioxidation, cell proliferation, and various signaling pathways, such as PI3K-Akt and IL-17 signaling pathways. Among them, PIK3R1, CTNNB1, JUN, EGFR, and APP might be the hub genes. Molecular docking indicated the close bond between Fisetin and them. Experiments implied a possible effect of Fisetin on the expression of hub genes in A549 cells.  The present study found a series of novel targets and pathways for Fisetin treating NSCLC. Multiple angles, targets, and pathways were involved in the biological processes, which need to be verified in further experiments. Lung cancer is a malignant tumor with the leading incidence and mortality rates in the world. Its morbidity and fatality rate is the first among men and the second among women. Non-small-cell lung cancer (NSCLC) accounts for the vast majority of all lung cancers, about 80–85%. Although it has been explored continuously, the mechanisms of its occurrence and development have not been clearly understood . At presTraditional Chinese medicine has unique advantages in tumor treatment, such as low toxicity, minor side effects, and strong anticancer activity. It can clinically reduce the toxic and side effects of radiotherapy and chemotherapy and improve the immunity, the quality of life, and even the prognosis of patients by “supporting righteousness and removing evil” . FisetinTraditional studies often focus on a single gene or target, ignoring the complexity and systematicness of biological processes. In recent years, the network pharmacology has developed, which integrates the system biology and pharmacology, integrates the biological network and pharmacology, changes the traditional search for a single target to comprehensive network analysis, emphasizes the interaction mode of multicomponents, multitargets, and multipathways, and is especially suitable for predicting the action target and possible mechanism of natural compounds from traditional Chinese medicines or different plants .Therefore, we intended to analyze the possible anticancer mechanisms of Fisetin on NSCLC from multiple levels and angles through network pharmacology and further explore the pharmacological effect and pharmacodynamic material basis of Fisetin from numerous aspects.The traditional Chinese medicine systems pharmacology (TCMSP) database was a public pharmacological analysis platform for studying traditional Chinese medicine, which comprised 499 kinds of Chinese herbs registered in the Chinese pharmacopeia with 29,384 ingredients, 3,311 targets, and 837 associated diseases . Moreovehttp://www.swisstargetprediction.ch) for predicting the target genes. The cutoff (probability >0) was set as the screening criteria. After the target genes/proteins were obtained, the protein names were transformed to the corresponding gene names through the UniProt database (http://www.uniprot.org) if necessary. Finally, the target genes obtained in the above two ways were combined to form a gene set.The target genes of Fisetin were obtained in two ways. First, the target genes/proteins of Fisetin were directly obtained from the TCMSP platform; second, the 2D or 3D structure of Fisetin was downloaded from the PubChem website  and was http://www.genecards.org) [In order to obtain NSCLC-related genes, we had a search in the GeneCards tool (rds.org) . The keyIn order to obtain the common target genes of Fisetin and NSCLC, we took the intersection of Fisetin-targeted genes and NSCLC-related genes obtained from the above steps. Through the Venn diagram, the common gene targets are obtained in the intersection, which could be considered as the potential target genes of Fisetin in the treatment of NSCLC.http://www.genemania.org) [Homo sapiens” was selected to construct a PPI network.A PPI network may help us understand the relationship among the common genes. There may be many interactions between the genes/proteins. The GeneMANIA database (nia.org)  was usedThe CytoHubba plug-in of the Cytoscape tool was used to calculate the parameters regarding the gene network. The hub genes were identified and sorted by the betweenness values.In order to understand the biological functions of the common target genes, we conducted Gene Ontology (GO) analysis. At the same time, in order to understand the possible enrichment signaling pathways of these genes, we analyzed the pathway enrichment through the Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis.During the GO analysis, we first downloaded the c5.go.bp.v7.4.symbols.gmt subset from the Molecular Signatures Database as the background, mapped the genes into the background set, and used the R software package clusterProfiler (version 3.14.3) for enrichment analysis to obtain the gene set enrichment results. The minimum gene set was 5, and the maximum gene set was 5000. The false discovery rate (FDR) < 0.05 was considered to be statistically significant.During KEGG analysis, we used the KEGG rest API to obtain the latest gene annotation of the KEGG pathway as the background, mapped the genes into the background set, and also used the R software package clusterProfiler (version 3.14.3) for enrichment analysis. Similarly, the minimum gene set was set as 5, and the maximum gene set was 5000. FDR < 0.05 was considered statistically significant.To clearly show the possible relationship among Fisetin, NSCLC, and the gene targets, the “Compound-Target-Disease” network interaction model was constructed by using Cytoscape software, in which “node” represented Fisetin or NSCLC or the targets, and “edge” represented the interaction relationships.http://www.rcsb.org) [Molecular docking between Fisetin and the hub genes/proteins was conducted using the SwissDock tool . The 2D csb.org) , and the2 at 37°C. The cells were divided into two groups: the control group (DMEM-treated) and the Fisetin-treated group (10 μM Fisetin). After intervention for 24 hours, the supernatant was discarded, and the mRNAs of the top hub genes  were detected by the RT-PCR assay. The total RNA of cells was extracted and then reversely transcribed to generate cDNA. Real-time PCR was conducted with a PikoReal Real-Time PCR system . PCRs were conducted. Each sample was repeated in triplicate. GAPDH was used as the internal reference. The primer sequences were listed as follows:  PIK3R1   CTNNB1   JUN   GAPDH: The human lung adenocarcinoma cell line, the A549 cell line, was purchased from the American Type Culture Collection and conserved in our laboratory. The use of A549 cell lines was approved by our institutional research ethics committee. The cells were cultured in Dulbecco's modified Eagle's medium (DMEM) containing 5% fetal bovine serum in a humidified atmosphere with 5% COA Western blot assay was performed to test the protein expressions. In brief, cells were harvested, washed with ice-cold PBS, and lysed with RIPA buffer supplemented with a protease inhibitor. Proteins were running on a 10% SDS-polyacrylamide gel electrophoresis technique and transferred to polyvinylidene difluoride membranes (Roche). Blots were then incubated in fresh blocking solution with an appropriate dilution of the primary antibody at 4°C for 24 h. The sources of antibodies were as follows: c-Jun (JUN) rabbit polyclonal, Beta-Catenin (CTNNB1) rabbit polyclonal, PI 3 Kinase p85 alpha (PIK3R1) mouse monoclonal, and GAPDH rabbit polyclonal .P < 0.05 was considered statistically significant.Differences between the groups were analyzed by using Analysis of Variances (ANOVA), and the Student–Newman–Keuls method was used for comparison between the groups. These analyses were performed by using MedCalc software . Fisetin is a small flavonoid compound that is rich in herbal medicines, vegetables, and fruits. The anticancer activities of Fisetin have attracted much attention. The present study explored the molecular targets and possible mechanisms for Fisetin in the treatment of NSCLC . A total15H10O6, relative molecular weight 286.25, the ratio of lipid water partition coefficient (AlogP) 1.77, oral bioavailability (OB) 52.60, blood-brain barrier (BBB) −0.69, and drug-likeness (DL) 0.24.Through the TCMSP database, we obtained the pharmacokinetic parameters of Fisetin. The 2D and 3D structures of Fisetin are shown in Among them, OB ≥ 30 and DL ≥ 0.18 were used as the criteria for screening bioactive components in other studies , 17. TheFirst, the targets of Fisetin were screened. Forty-three targets were obtained from the TCMSP database, and 100 targets from the Swiss target database. Then, a total of 134 target genes were obtained after merging these gene sets. Second, a total of 14837 NSCLC-related genes were obtained through the GeneCards tool. Third, 131 genes common to Fisetin and NSCLC were obtained by Venn analysis .In the process of tumor development, various proteins may interact with each other, thus forming a complex regulatory network and jointly completing various biological processes. The roles of different proteins were varied. Some proteins that have more relationships with other proteins may occupy a greater weight. The genes encoding these proteins are called hub genes.The network contains 115 nodes, with each node representing a gene/protein and each edge representing the interaction between proteins .To explore the exact relationships among the genes in the network, Cytoscape software was used to calculate the parameters. The top five genes were listed in the order of betweenness value in Go enrichment analysis mainly includes BP , CC (cellular component), and MF (molecular function). In this study, we selected the most commonly used BP for analysis.The GO enrichment results showed that these genes were enriched in 1000 GO terms according to the standard of FDR < 0.05. The top 10 GO terms are listed in KEGG pathway enrichment analysis showed that these genes were enriched in 139 pathways . The topA network model was constructed for visualization. Docking between the top hub proteins  and Fisetin, respectively, was carried out. If the binding energy was less than zero, it usually indicated that the molecules and ligands could effectively bind with each other. The delta energy for these proteins ranged from −7.61 to −6.31 , indicatP < 0.05), while the expression of CTNNB1 was decreased  is one of the most important pharmacokinetic parameters and an important index to evaluate the effectiveness of drugs entering human circulation. Good OB is the basic premise for compounds to have pharmacological activity. An OB value of greater than 30 is often used as a standard for compound screening. The present study found that the OB value of Fisetin was 52.6, indicating that Fisetin may be easily absorbed into the blood after oral administration, and it thus can conveniently exert biological activity. Drug-likeness (DL) is a physicochemical property and biological characteristic related to good clinical efficacy. The greater the DL value, the safer the compound during absorption and metabolism, suggesting that it is prone to be used as a drug. A DL value of greater than 0.18 is often used as a safety index for drug screening. The DL value of Fisetin was 0.24, indicating that the drug-likeness property of Fisetin was good. Since Fisetin exists in a large number of all kinds of vegetables and fruits in addition to some traditional Chinese medicine, it might be used as a potential dietary agent for disease prevention and treatment.β-catenin, NF-κB, and TRAIL/TRAIL-R [Fisetin has a variety of biological activities, such as antitumor, anti-inflammatory, and antioxidative stress . For ins/TRAIL-R . In addi/TRAIL-R . Thus, tSome reports have concerned the effect of Fisetin on lung cancer cells. For example, Fisetin induces apoptosis of the A549 cell line by reducing the expression of c-myc, cyclin-D1, cyclooxygenase-2, Bcl-2, and CXC chemokine receptor 4 . AdminisThrough the network pharmacology research, we found more gene targets of Fisetin treating NSCLC. The hub genes, including PIK3R1, CTNNB1, JUN, EGFR, and APP, might play an important role in tumor development. For example, PIK3R1 was overexpressed in hepatocellular carcinoma, which promotes cell proliferation and migration capabilities . TP53 isThe results of GO analysis suggested that the common target genes were mainly enriched in antioxidation, cell proliferation and apoptosis, and signaling pathway regulation. Pathway enrichment analysis shows that these genes may be involved in various signaling pathways, such as PI3K-Akt signaling pathway, IL-17 signaling pathway, and endocrine resistance. AKT1 is an important signal pathway factor, which participates in all stages of lung cancer occurrence and development ; IL-17 cIt is worth noting that some hub genes were downregulated, while others were upregulated after Fisetin-treated lung cancer cells in the process of experimental verification. Because these hub genes have been proved to play a very complex biological role in the process of tumorigenesis and development, hub genes may interact with each other. Also, they may play an antitumor effect by regulating the expressions of other genes. Therefore, the results deserve careful interpretation. For example, the experimental verification part only shows that the administration of Fisetin may change the expression level of these genes in A549 cells. Although molecular docking prediction shows that Fisetin can directly bind to the spatial structure of these hub proteins; in fact, the regulation of Fisetin on them is not necessarily a direct relationship, but it is very likely to be a complex indirect regulation process. Therefore, it is very necessary and important to clarify their relationship in future experimental verification. However, as a prediction tool based on big data, network pharmacology has very important practical significance in guiding the experimental direction, finding the experimental molecular mechanism, and reducing the blindness of exploration.In the present study, the TCMSP and Swiss target were used to predict the possible targets of Fisetin, while the GeneCards tool was used to obtain the relevant genes of NSCLC. Given that different databases may have different prediction targets due to different algorithms, some target genes may be inevitably missed in the screening process. Thus, the reliability of the obtained genes was inconsistent, which might cause some bias in the results. However, most of the gene targets were obtained due to the use of the mainstream databases for retrieval. It is noted that the low water solubility and rapid metabolism of Fisetin may limit its application in medicine, although it has good oral bioavailability and drug-likeness properties. Thus, novel strategies such as nanocarriers need to be designed and investigated for improving the bioavailability of Fisetin .The network pharmacology technology used in this study analyzed the potential molecular mechanisms of Fisetin in the treatment of NSCLC from multiple angles. The data suggest that Fisetin may play various roles in treating NSCLC through multiple targets, pathways, and angles. The results also need to be verified in future experiments to further uncover the molecular mechanism of Fisetin in anticancer fields.


Metronidazole is a 5-nitroimidazole antibiotic effective against anaerobic bacterial and parasitic infections. Long-term use may cause side effects in the central nervous system, although the occurrence of encephalopathy is rare.A 73-year-old man was diagnosed with acute pyelonephritis and received antibiotic treatment. During the treatment, the patient complained of back pain. Lumbar spinal magnetic resonance imaging (MRI) revealed infective spondylitis, and metronidazole (1.5 g) was administered daily for approximately 160 days. The patient developed cognitive dysfunction and gait disorder after antibiotic treatment, and brain MRI showed acute infarction in both cerebellar lobes. Secondary prevention with antiplatelet and physiotherapy was prescribed; however, functional recovery was not achieved.After 1 month, a follow-up brain MRI showed high signal intensity and diffusion restriction in the corpus callosum on diffusion-weighted images and high signal intensity in the dentate nucleus on T2-weighted images. Therefore, metronidazole-induced encephalopathy was suspected.Metronidazole was discontinued, and ceftriaxone (2 g/day) was administered to manage the infective spondylitis.One week after the discontinuation of the drug, the patient’s cognition improved to the extent that communication was possible. Thus, even if other neurological deficits, such as cerebellar infarction, are found in patients with long-term disability, the possibility of metronidazole-induced encephalopathy should be considered when metronidazole is used for a long time. Long-term use of metronidazole may cause adverse effects on the nervous system, such as metronidazole-induced encephalopathy (MIE). Because MIE is not a commonly reported case, its diagnosis could be easily overlooked. If other illnesses are present simultaneously, diagnosis can be delayed, making it challenging to take necessary action. To the best of our knowledge, there are no cases in which the diagnosis of MIE was delayed due to a preceding stroke. Here, we report the case of a patient initially diagnosed with cerebellar infarction and later diagnosed with MIE due to the long-term use of metronidazole for infective spondylitis, along with a literature review.Metronidazole is a 5-nitroimidazole antibiotic effective against anaerobic bacterial and parasitic infections. Its common side effects include nausea (12%–64%), and it has a bitter taste (65%–72%).A 73-year-old man presented to the emergency room with a fever and acute pyelonephritis. The fever subsided after antibiotic treatment; however, the patient complained of severe back pain. Magnetic resonance imaging (MRI) of the spine with contrast was performed, and the patient was diagnosed with infective spondylitis. Surgical treatment  was performed. Subsequently, he was transferred to the Department of Rehabilitation Medicine. At the time of transfer, the strength of the upper and lower extremities was generally evaluated as grade 4 on the medical research council scale. The patient was able to walk independently using a walking aid. Antibiotic treatment was continued, and metronidazole was taken at 1.5 g/day for approximately 160 days Fig. A–D. HoweIn this report, we present a case in which MIE was found because of the persistent decline in cognition and motor functions of the patient after a stroke. Stroke is common in the elderly and may cause deterioration in cognitive and motor functions; however, it is not uncommon to show deterioration of these functions continuously after the first onset. Although this continued deterioration of function was shown by overlapping MIEs, it is rare for the diagnosis to be delayed because of the effects of a previous stroke. In 138 cases of MIE reported in 2020, men  were more affected than women , and the average age of adults (130/138) was 56.8 years. The duration and indication for metronidazole use varied depending on each patient and report. However, the average period was 101.6 days, ranging from 2 days to almost 8 years, the cumulative dose varied from 5 g to 2000 g, and an average of 125.7 g was used as the cumulative dose. In our case, the patient was prescribed metronidazole 1.5 g/day for approximately 160 days, and the total cumulative dose was 240 g , gait instability (55%), limb dyscoordination (53%), altered mental status (41%), and dizziness (18%). However, no clear pathognomonic signs or symptoms were observed in the current case. In addition, vague symptoms and the chronic progressive nature of the disease could be the cause of delayed diagnosis. The findings of the bilateral cerebellar infarction on the first brain MRI appear to be incidental. Furthermore, in our case, the diagnosis was delayed mainly because of the presence of mild cerebellar stroke, which also presented with mild dysarthria and gait disturbance.The pathophysiology of MIE has not been elucidated; however, several mechanisms have been proposed, suggesting that metronidazole may reduce the catecholamine neurotransmitter, which may lead to the formation of free radicals in the process, resulting in neurotoxicity.,6,7 In addition, among several MIE cases, high signal intensity on the DWI of these structures indicates metronidazole-induced cytotoxicity and angioedema. Due to the persistent neurological deterioration in our case, we performed a follow-up MRI, which showed high signal intensity on T2 FLAIR images in the corpus callosum and dentate nucleus, and further diffusion restriction on DWI in the corpus callosum was observed, leading to the final diagnosis of MIE.MRI is the most useful diagnostic tool for these patients who have neurological changes The most characteristic imaging findings of MIE are symmetric hyperintensity of the cerebellar dentate nuclei, splenium of the mesocerebrum, dorsal pontine, and corpus callosum on T2-weighted and FLAIR images. In most cases, symptoms and follow-up imaging findings were recovered from the initial images. However, some patients had persistent neurological sequelae, especially when cystic necrotic degeneration developed on follow-up MRI.,5,7,9 In our patient, cystic necrotic degeneration appeared in the corpus callosum, with a poor prognosis. The cause of the poor prognosis could be irreversible damage to necrotic brain tissues, mostly due to late detection and prolonged high-dose metronidazole use.The treatment of MIE involves the discontinuation of metronidazole and conservative supportive management. Most patients with MIE have a favorable prognosis after the discontinuation of metronidazole treatment. Most patients either fully recover (29%) or improve their symptoms (65%).Herein, we present a case report of a late diagnosis of MIE due to mild cerebellar infarction. Suspicion from the physician and close patient monitoring could be the key to diagnosing MIE. Moreover, in patients using metronidazole with chronic progressive functional loss, even with proper care and rehabilitation therapy, MIE should be suspected, and a brain MRI should be performed.www.editage.co.kr) for editing and reviewing this manuscript in English.The authors would like to thank the patient for his contributions to this work and Editage (Conceptualization: Sang Ha Cha, Byung Chan Lee, Chang Won Moon.Data curation: Sang Ha Cha, Byung Chan Lee, Chang Won Moon.Supervision: Kang Hee Cho.Writing – original draft: Sang Ha Cha.Writing – review & editing: Sang Ha Cha, Byung Chan Lee, Chang Won Moon.


Happiness is one variable of subjective well-being, which has been increasingly shown to have protective effects on health. Although the association between happiness and cognition has been established, the mechanism by which happiness leads to cognition remains unclear. Since happiness, depression, and physical activity may all be related to cognition, and happiness is related to depression and physical activity, this study explored the effect of depression and physical activity on the relationship between happiness and cognition among middle and old-aged individuals in China.Data on 14,344 participants above 45 years of age were obtained from the 2018 China Family Panel Studies survey. A multiple linear regression analysis was performed to identify the correlation factors of cognition. The conditional process analysis was used to assess the mediatory effect of depression and physical activity on the relationship between happiness and cognition.Residence, age, sex, income level, social status, smoking, napping, reading, education, exercise times, satisfaction, happiness, and depression had associations with cognition. When other variables were held constant, cognition score increased by 0.029 standard deviation(SD) for every 1 SD increased in happiness. Mediation analysis showed that happiness had a significant positive total effect on cognition. The direct effect of happiness was significant and accounted for 57.86% of the total effect. The mediatory effect of depression (path of happiness→depression→cognition) accounted for 38.31% of the total effect, whereas that of physical activity (path of happiness→exercise times→cognition) accounted for 3.02% of the total effect.Happiness has a positive correlation with cognitive function, and depression and physical activity play mediatory roles in this association. Effective interventions to improve happiness levels of middle and old-aged population will not only improve their subjective well-being but also improve their cognitive function, which carries great potential for reducing public health burdens related to cognitive aging. Cognition is the most complex function of the brain. Cognitive decline is a significant concern among middle and old-aged individuals, particularly in China, a country with rapidly aging population. Age-related cognitive changes are cumulative irreversible changes occurring in a long-term process . AccordiStudies have shown that besides age, which is an irreversible factor, many factors are related to changes in human cognitive function. Individuals are often concurrently exposed to both risk and protective factors, which may interact with each other during the life course of individuals, and the net effect of these interactions determines the overall risk of cognitive impairment. Educational level , sleep , physicaHappiness, a variable of subjective well-being, is defined as “the overall appreciation of one’s life-as-a-whole” . HappineOlder individuals with higher levels of happiness are more likely to be physically active  and liveSince happiness, depression, and physical activity may all be related to cognition, and happiness is related to depression and physical activity, we hypothesized that happiness may have an indirect effect on cognition through depression and physical activity aside its direct effect on cognition. In other words, depression and physical activity may play mediatory roles between happiness and cognition. To the best of our knowledge, no study has explored the possible association between happiness and cognition with depression and physical activity as mediatory factors. Therefore, this retrospective cross-sectional study was conducted to assess the direct effect of happiness on cognition and assess the mediatory effects of depression and physical activity on the relationship between happiness and cognition.Briefly, the effect of happiness on cognition is considered as a direct effect, whereas the effect of happiness on cognition through depression or physical activity is considered as an indirect effect. The overall effect of happiness on cognition is considered as the total effect; where total effect = direct effect + indirect effect. Therefore, we proposed four hypotheses to be tested.Hypothesis 1 (H1): The total effect of happiness on cognition is positively significant.Hypothesis 2 (H2): The indirect effect of happiness on cognition through depression is significant. In other words, depression plays a mediatory role in the process of happiness and cognition.Hypothesis 3 (H3): The indirect effect of happiness on cognition through physical activity is significant. In other words, physical activity plays a mediatory role in the process of happiness and cognition.Hypothesis 4 (H4): The indirect effect of happiness on cognition through depression and physical activity is significant. In other words, depression and physical activity play a joint mediatory role in the process of happiness and cognition.http://www.isss.pku.edu.cn/cfps/), which was organized and conducted by the Institute of Social Sciences of Peking University. The CFPS sample was drawn from 25 provinces/municipalities/autonomous regions in China, and they account for 95% of the total Chinese population. Therefore, CPFS can be regarded as a nationally representative sample (The survey data were obtained from the China Family Tracking Survey (CFPS)  to 10 (very happy).The first mediatory variable was depression, which was assessed using the Center for Epidemiological Research Depression (CES-D) scale. The CES-D scale was developed by the National Institute of Mental Health in 1977, was widely used in epidemiological surveys to screen people with depressive symptoms for further examination and diagnosis. Compared with other self-rating depression scales, CES-D focuses more on individual emotional experience and less on somatic symptoms during depression. The CES-D scale  consistsPhysical activity was the second mediatory variable. In this study, it was measured by the number of exercises. Respondents recalled the number of exercises in the past week. The exercises included walking, long-distance running, jogging, mountaineering, dancing, yoga, various ball games, water sports (swimming), ice and snow sports, and physical contact sports (judo and boxing).Since the residence, age, sex, education, smoke, napping, reading, satisfaction of life, income status, and social status of study participants were potential confounders of cognition, they were used as control variables. The definition of control variables was shown in Descriptive and multiple regression analyses of the data were performed using STATA/MP version 16.0. Continuous variables were described using mean ± standard deviation (SD), and categorical variables were described using counts and percentages.p < 0.05.We performed multiple regression analysis using cognitive score as the dependent variable to examine possible associations with various participant demographics and cognitive score. Independent variables were residence, age, sex, marriage, education, smoking, drinking, napping, reading, exercise times, life satisfaction, income status, health status, social status, depression, and happiness. Variables of happiness, depression, life satisfaction, income status, and social status were standardized prior to conducting regression analyses. All independent variables had no multicollinearity (VIF <2). The degree of statistical significance was set at In this study, conditional process analysis was used to analyze mediatory and moderation effects. It is a method used to test hypotheses about “how” and “under what conditions” a certain factor works . A theorThe average age of respondents was 59.46 years. Approximately half of the respondents were female (50.08%) and resided in rural areas (52.84%). Most participants had no education (35.63%) or completed primary school (24.85%). Most participants were married (87.62%). More than half of them reported no smoking (69.39%) and drinking habits (82.29%). Most participants had a habit of napping (60.73%). Few participants had recently read books (13.7%). The mean happiness score was 7.51 on a 10-point Likert scale. The average exercise time was 3.08 times in the past week. The average depression score was 33.58, and the average score on the cognitive test was 22.16 .p < 0.05). When other variables were held constant, cognition increased by 0.009 points for each increase in exercise times. Cognition increased by 0.029 SD for every 1 SD increased in happiness and decreased by 0.066 SD for every 1 SD increased in depression . Therefore, Hypothesis 1 was accepted.The mediatory effect of depression and physical activity were tested by path analysis. After eliminating the influence of control variables, the direct effect of happiness was statistically significant. The estimate value was 0.0287, which accounted for 57.86% of the total effect of happiness on cognition. The indirect effect of mediators was statistically significant, and the estimate value was 0.0209, which accounted for 42.14% of the total effect. For two mediatory variables, the mediatory effect of depression (path of happiness →depression →cognition) accounted for 38.31% of the total effect. The mediatory effect of physical activity (path of happiness →exercise times →cognition) accounted for 3.02%, and the joint mediatory effect of depression and physical activity (path of happiness →depression →exercise times →cognition) accounted for 0.81%. These results indicated that depression and physical activity had mediatory effects, indicating that happiness had an indirect effect on cognition through depression and physical activity. Therefore, Hypotheses 2, 3, and 4 were all accepted. Mediation analysis results were shown in The main objective of this study was to explore the association between happiness, depression, physical activity and cognition of the middle and old-aged Chinese individuals. Using CFPS data from 2018, the findings supported our hypothesis that study participants who were happier had higher scores on cognitive tests after controlling for relevant covariates. Moreover, depression and physical activity played mediatory roles between happiness and cognition. Direct effects accounted for 57.86% of the total effects of happiness on cognition, and indirect effects accounted for the remaining 42.14% through depression and physical activity.Mental factors are widely acknowledged to affect physical functioning. Moreover, well-established evidence suggest that mental distress, such as depression, has negative effects on physical health, whereas positive mental state, such as happiness, has beneficial effects on physical health . HappineAdditionally, our study sought to investigate whether happiness can predict cognitive decline in older adults. We found that happiness had a significant positive total effect on cognition. Consistent with our findings, those from studies in the United States and Singapore have shown that individuals with high levels of happiness had a reduced risk of mild cognitive impairment and dementia . ContrarRegarding the total effect of happiness on cognition, the indirect effect of depression and physical activity on cognition cannot be overemphasized. Evidence suggest that the associations between happiness and depression were complex and may coexist . ConsistIn this study, happy individuals had more physical activity levels. Similar to some retrospective studies, this study found physical activity to be one of the protective factors in reducing the risk of cognitive decline . AdditioTherefore, ensuring that middle and old-aged individuals in China are happy should not be of utmost importance to society. Strategies to ensure happiness in this population with favorable effects on their mental and physical conditions need to be recognized and developed. Although happiness has a genetic underpinning , multiplOur study had limitations. First, this study was a cross-sectional study with limited power to draw causal inferences from findings. Second, the medical diagnosis of cognitive dysfunction often relied on comprehensive clinical evaluation, and the use of cognitive tests in our study had its limitations. Future studies could consider collecting more clinical data. Despite these limitations, this study, for the first time, used the mediation model to explore the association between happiness and cognitive function among middle and old-aged individuals in China. The large sample size, multivariate analyses controlling for many possible confounds, and the analysis method of conditional process analysis were strengths of the study making it a unique contribution.This cross-sectional study in a representative population sample suggested that happiness was positively correlated with cognitive function. Mediation analysis further found significant association of depression and physical activity on the relationship between happiness and cognition. This finding provided a better understanding of how happiness has an effect on cognition and provided clues for future research concerned with links between happiness and cognitive function. Effective interventions for happiness of middle and old-aged individuals in China carries great potential for reducing public health burdens related to cognitive aging.


The results show that Slovak and Hungarian apricots can be differentiated from the Wachau apricots, a well-known regional Austrian brand, and those from other regions in Austria, even though the differentiation from the latter is only partial. 87Sr/86Sr investigations can be a very potent tool; however, its success depends on the exact question that needs to be answered.Consumers prefer food commodities of certain origins over the same products of other provenances and are willing to pay higher prices for them. Thus, it is possible to increase profit simply by giving an incorrect geographic origin to a product. To effectively control the declared geographic origin of food, the product itself has to be investigated to discover whether it actually originates from the declared origin, or if it has been mislabeled. Conventionally, control of a geographic origin is conducted by stable isotope analysis of the main elements, which has proven to be successful in numerous cases, but often reference data have to be produced anew for every harvest to control, resulting in additional costs and delays. Applying entirely geogenic parameters for the control of provenance requires reference data to be produced only once. As they do not vary between years and harvests, they can often be used for different (food) commodities. Here, we investigate whether the geographic origin of apricot samples can be controlled by their  Food authenticity describes the sum of good practices and the absence of bad practices. It means that the food commodity is pure, without unwanted additions, not adulterated, produced according to the respective codes of practice, and correctly labelled. Our study deals with one of the labelled parameters: the provenance, or geographic origin.The indication of geographic origin has become an important parameter for food. On the one hand, consumers are increasingly aware of the relevance of geographic origin, as this might be a parameter for food quality , an important socio-economic factor , a safety issue (backtracing in the case of unconformities), or a legal requirement .87Sr/86Sr signal has an advantage in that the accurate reference values from the reference samples might not be required, as the Sr signal is incorporated from the geologic bedrock into the soil, and from the soil into plants  without fractionation , isotope analysis of heavy elements such as Sr or Pb, element concentrations, molecular markers, organic compound concentrations, and screening methods such as FTIR, NMR, untargeted metabolomics, etc. . In . In 87Sronation  a,17 a87Sronation . It shos . Usually and traditionally, apricots are regional products, as they are perishable, and transport can further shorten their shelf-life as a consumable product. Thus, apricots are usually consumed regionally and, therefore, regional brands have been established. Some of these brands have obtained national and international relevance. However, in today’s world, many perishable goods are transported and are available globally. Therefore, control of the correct declaration of geographic origin needs to be established for delicate food commodities such as apricots, as consumers are willing to pay significantly higher prices for an especially valued provenance. Control of the declared geographic origin ensures protection for consumers against deception, for producers against dishonest competitors and for markets/traders from being liable for selling products under incorrect claims and labels. In Austria, the “Wachauer Marille” (Marille = apricot) is a regional, high-value apricot brand connected to the Wachau Valley, which is a part of the Danube River Valley that cuts through the Bohemian Metamorphic Massif. Besides being the origin of the Wachauer Marille, it is also well known as a wine region. The Wachauer Marille is an EU-registered product of protected origin (PDO: protected denomination of origin), and only apricots from 21 communities  in and around the Wachau Valley may be sold under this brand. Only certain apricot cultivars may be sold as Wachauer Marille ; the ma87Sr/86Sr analysis enables the differentiation of various geographic origins and unequivocal determination of the certain provenance on apricots from Austria, Slovakia, and other production regions. Furthermore, we compared our results with the published results of other commodities, to evaluate whether or not the results can be used for other foods. In the present study, we tested whether 87Sr/86Sr signal between vintages, 5 apricot samples from 2020 were analyzed: one commercial sample from Italy, one sample from Slovakia, two samples from the Wachau region and one sample from Weinviertel. A sample consists of at least 10 apricots, with a weight between 0.5 and 1 kg. The samples were either directly transferred or frozen until transfer to the laboratory, where the samples were stored in a freezer prior to sample preparation. The apricots (at least 10) from each sample were de-stoned, homogenized in a kitchen blender, and freeze-dried, after the supernatant juice was decanted. In 2019, 69 authentic apricot samples were collected directly from the apricot orchards of producers located in Austria, Slovakia and Hungary, where the apricots were collected from the trees. One commercial sample labelled as originating from “Wachau” , and two commercial samples of foreign origin were bought from supermarkets in Austria . For the3 and centrifugation, the Sr was purified and accumulated in vacuum columns filled with 0.05 mL of Sr-specific crown-ether resin . Strontium (estimated: 50 to 100 ng) was loaded on single-band Tungsten filaments  in a mixture of TaCl5, HF, HNO3, H3PO4 and H2O. All relevant analytical steps were performed under clean room conditions using ultra-clean and blank-controlled reagents  and ultra-clean devices . The total analytical blank was included in the uncertainty consideration. Isotope ratios were analyzed in static mode  with a thermal ionization mass spectrometer  at the Ries Crater Museum in Nördlingen, Germany. Extant Rb was evaporated completely from the loaded filament by controlled pre-heating before the isotopic Sr composition was measured. Possible traces of 87Rb were corrected over the measured 85Rb/86Sr (<0.0001) and were included in the uncertainty consideration. The measured isotope values were normalized for mass fractionation using the naturally invariant value for 88Sr/86Sr of 8.37521 and the exponential fractionation law. Precision of the mass spectrometer runs was controlled by measuring the reference material SrCO3 NIST SRM 987. In the analysis period, 9 standard measurements resulted in a mean value of 0.710210 for 87Sr/86Sr, with a standard deviation (1SD) of 0.000024, 2 standard deviations of the mean (2SD_M) of 0.000016 and a 99% confidence interval of 0.000021. According to replicate analyses of natural samples and in-house standard materials, the accuracy of 87Sr/86Sr is estimated to be better than 0.0035% of the value.For Sr analysis, about 200 mg of the freeze-dried dry matter was ashed for two hours at 850 °C in quartz vessels. After dissolution of the ash in 6.5 N HNOpost-hoc” pairwise comparisons based on the Fisher’s least significant difference (LSD) procedure at the 95.0% confidence level. Additionally, a box plot displaying the dataset of 87Sr/86Sr values for each geographic origin and a frequency histogram showing a comparison of two samples were constructed using the software Statgraphics x64 . Similarly, this software was used to calculate a t-test to compare means, an F-test to compare standard deviations, a Mann–Whitney (Wilcoxon) W-test to compare medians, and a Kolmogorov–Smirnov (K-S) test to compare the distributions of the two samples.Statistically significant differences among the geographic origins were tested using analysis of variance (ANOVA) and subsequently by using the “87Sr/86Sr ratio of 0.71169 with maximum and minimum values of 0.70925 and 0.71683, respectively; the Weinviertel samples show an average of 0.700995 with minimum and maximum values of 0.70917 and 0.71073, respectively; the Mostviertel samples show an average of 0.70992 with minimum and maximum values of 0.70896 and 0.71028, respectively; the Burgenland apricot samples have an average of 0.70972 with minimum and maximum values of 0.70896 and 0.71028, respectively; the Styrian samples show an average of 0.71172 with minimum and maximum values of 0.70944 and 0.71301, respectively; and one Carinthian sample has the value of 0.70872. The Slovakian samples show an average of 0.70863 with minimum and maximum values of 0.70868 and 0.71051, respectively, and the Hungarian samples show an average of 0.70884 with minimum and maximum values of 0.70848 and 0.70927, respectively. One commercial Italian sample has the value 0.70872, one commercial Spanish sample has the value of 0.70805, and the commercial Wachau apricot sample has the value of 0.70955 , even though the groups of Styrian and Wachau apricot samples also include individuals possessing similar ratios as the group mentioned before. A comparison of the results indicates that the Carinthian sample only overlaps with the Styrian and Wachau area results. The Hungarian samples are almost completely separated from the Wachau and Weinviertel samples. The Spanish sample only overlaps with the Slovakian results. The Italian sample only overlaps with the Slovakian, Styrian, Mostviertel and Hungarian samples. The commercial Wachau apricot sample is right at the limit of the standard deviation of the authentic Wachau samples. The samples from Burgenland, Mostviertel, Slovakia, Hungary and Weinviertel cluster together to form a group (Group 1) and possess a similar range of 87Sr/86Sr ratios of the vintage 2020 apricot samples show excellent agreement with the results from 2019  from 0.704 to more than 0.720  expected in a specific area is only possible if the geological and geographical conditions in the wider environment are thoroughly reviewed and taken into account.Furthermore, in 2022, Horacek  noted t., 2011 , who in87Sr/86Sr ratio. This is most likely due to the co-existence of some siliciclastic material, as the 87Sr/86Sr ratio range exceeds marine carbonate values . The Carinthian sample has a high 87Sr/86Sr ratio. In Carinthia, there are only very few apricot orchards  have values beyond the marine range. One Italian sample and one Spanish sample both show 87Sr/86Sr ratios within the marine range, enabling a very good differentiation for the Spanish sample from all regions except Slovakia, and a good one for the Italian sample, which only overlaps with Mostviertel, Slovakia, Hungary, and Styria. However, Italy and Spain are both large countries, with each possessing a heterogeneous and diverse geology; therefore, these single values do not allow the conclusion that differentiation will be possible. This is evidenced, e.g., by Aguzzoni et al., 2020 .As the result of the Wachau commercial apricot sample lies well within the range of the authentic Wachau samples, there is no indication of an incorrect declaration of the geographic origin. Our results are in excellent agreement with the data of Swoboda et al., 2007 , who in87Sr/86Sr ratio of apricot samples from different regions in Austria and neighboring countries, our study achieved a differentiation of apricots from the Wachau region, which represents a well-known regional brand, and apricots from Slovakia and Hungary. Besides this, the apricot samples of most of the other regions overlap with regards to their 87Sr/86Sr ratios, as is to be expected due to the heterogeneous bedrock geology. Considering the range of individual regions defined by the standard deviation, further regions can be differentiated, for example, the Weinviertel apricot samples from the Slovak and Hungarian apricot samples. Still, some overlap occurs for many of the individual regions. Thus, the use of the 87Sr/86Sr ratio analysis for the control of the geographic origin of apricots from Austria/Central Europe is, in most cases, a challenge and does not enable a complete differentiation. Therefore, additional markers and methods are required for a complete and unequivocal differentiation/identification of the geographic origin. The 87Sr/86Sr ratio analysis can be a very potent tool for the discrimination or identification of the geographic origin; however, its success depends on the exact problem to be addressed and the question that needs to be answered. Heterogeneous bedrock geology in individual regions can be a problem for the differentiation of goods/samples originating from these regions by the 87Sr/86Sr ratio analysis, as it results in large variations within individual regions and, thus, overlapping results between the regions. Nevertheless, the successful separation of the apricot samples from the Wachau region with the apricots from Hungary and Slovakia demonstrates and evidences the relevance and potential of this method in well-defined and suitable settings. As the 87Sr/86Sr ratio patterns are usually independent of the commodity, which was confirmed by the results published for asparagus samples, the results should also be applicable for other (food) commodities .By investigating the