Cervical cancer is the most commonly diagnosed gynecological malignant carcinoma worldwide. It is crucial to develop reliable prognostic models to predict clinical outcomes and identify patients who will benefit from different treatment strategies. In current study, we identified a reliable metabolism-related signature composed of ALOX12B, CA9, FAR2, F5 and TDO2 for the prognosis and anti-tumor immunity in cervical cancer. Patients with high-risk score underwent apparently worse prognosis and displayed lower infiltration of tumor infiltrating lymphocytes. Additionally, the metabolism-based risk score could also predict the prognosis of patients with cervical cancer based on the expression of immune checkpoints. Since this risk score signature achieves a good performance in predicting clinical outcome, we genuinely expect that our study could provide an effective prognostic tool for guidance of personalized treatment of cervical cancer patients.n = 304, p < 0.0001), validation cohort  and pan-cancer cohorts (including nine TCGA tumors). Using a gene set enrichment analysis (GSEA), we observed that the model was correlated with various immune-regulation-related pathways. Furthermore, pan-cancer cohorts and immunohistochemical analysis showed that the infiltration of tumor infiltrating lymphocytes (TILs) was lower in the high-score group. Additionally, the model could also predict the prognosis of patients with cervical cancer based on the expression of immune checkpoints (ICPs) in both the discovery and validation cohorts. Our study established and validated a metabolism-related prognostic model, which might improve the accuracy of predicting the clinical outcome of patients with cervical cancer and provide guidance for personalized treatment.Cervical cancer is the most frequently diagnosed malignancy in the female reproductive system. Conventional stratification of patients based on clinicopathological characters has gradually been outpaced by a molecular profiling strategy. Our study aimed to identify a reliable metabolism-related predictive signature for the prognosis and anti-tumor immunity in cervical cancer. In this study, we extracted five metabolism-related hub genes, including ALOX12B, CA9, FAR2, F5 and TDO2, for the establishment of the risk score model. The Kaplan-Meier curve suggested that patients with a high-risk score apparently had a worse prognosis in the cervical cancer training cohort (TCGA,  Cervical cancer is the most commonly diagnosed gynecological malignant carcinoma and accounts for an estimated 604,000 new cases and 342,000 deaths annually worldwide . During Metabolic reprogramming in cells and changes in energy metabolism levels have been identified as an emerging hallmark of cancer ,3. IncreIt is increasingly clear that crosstalk between abnormal metabolism and immune escape assumes a key role in the process of tumor progression ,12. For In the current study, we established a novel prognostic metabolism-related risk score based on cancer genomics, bioinformatics and immunohistochemical analysis. The association between the risk score and infiltration of tumor infiltrating lymphocytes (TILs) was explored in a cervical cancer training cohort, a validation cohort and pan-cancer cohorts. Moreover, we also utilized the risk score to predict the prognosis of cervical cancer patients in the context of different expression of immune checkpoints.http://software.broadinstitute.org/gsea/index.jsp (accessed on 3 May 2021)). The RNA sequencing transcriptomics data and corresponding clinical information were retrieved for a total of 304 cervical cancer tissues and another 32 cancer types from the TCGA database (https://tcga-data.nci.nih.gov/tcga/ (accessed on 3 November 2020)). We excluded samples whose overall survival (OS) or survival status were not available. We also obtained GTF files from Ensembl (http://asia.ensembl.org (accessed on 3 November 2020)) for annotation of the mRNA. Besides, 24 normal and 28 cervical cancer samples (GSE63514) based on the GPL570 (Affymetrix Human Genome U133 Plus 2.0 Array) platform, as well as 300 early cervical cancer tissues (GSE44001) based on the GPL14951 (Illumina HumanHT-12 WG-DASL V4.0 R2 expression beadchip) platform were, respectively, collected from the GEO database (http://www.ncbi.nlm.nih.gov/geo (accessed on 3 May 2021)).The metabolism-related gene sets were downloaded from the Molecular Signature Database (MSigDB) via the Gene Set Enrichment Analysis tool ).The differentially expressed genes (DEGs) were identified using the clusterProfiler based on the DEGs between high- and low-score groups to speculate the possible function terms. The p values were adjusted via the BH method and an adjusted p value < 0.05 was considered statistically significant. Herein, we displayed some significant enrichment outcomes in the aspect of the biological process (BP). Furthermore, we also evaluate the enrichment levels of 50 hallmark pathways using R packages GSVA and msigdbr. Based on the GSVA score, we conducted the differential analysis for these hallmark pathways with the R package limma between high- and low-score groups. In addition, we also resorted to the online tool EMTome (www.emtome.org (accessed on 19 May 2021)) to obtain the enriched networks of five metabolism-related hub genes.Gene ontology (GO) analysis was performed using the R package p value less than 0.05 were selected for further multivariate Cox regression analysis. In this manner, we identified five hub genes that had independent prognostic values. In this step, the R packages plyr and survival were applied. Then we developed the risk score signature consisting of the five hub genes, and the total risk score of this biosignature was calculated as the following formula: Risk Score = N is the number of hub metabolism-related genes, iβ refers to the regression coefficient and iGene represents the expression level of each gene identified by multivariate Cox analysis. The cut-off for this risk model was defined as its median value. Subsequently, the patients were classified into high-risk and low-risk groups according to the median threshold. The ROC curve was plotted via the R package timeROC to assess the predictive potential of this signature for overall survival. The Kaplan-Meier survival curve was also conducted to evaluate the difference in patient prognosis of two subgroups using the R packages survival and survminer.The association of the 211 overlapped genes with clinical outcomes of cervical cancer patients was analyzed via univariate Cox regression analysis. Those genes with a R package rms. Furthermore, the C-index of this model was measured and the result suggested that the signature had reliable predictive power. Moreover, the calibration plots of this nomogram were presented to measure its predictive accuracy in comparison to the actual curve of the survival time.The predictive performance of the risk score derived from the set of five metabolic genes was evaluated in the training cohort, as well as the validation cohorts. The multivariate Cox regression analysis demonstrated that the risk score served as an independent prognostic factor and, subsequently, we constructed an OS-related nomogram with this score using the R package timeROC. Moreover, we validated the predictive power of this model in other cancer types. We performed a pan-cancer analysis in 33 cancer types from the TCGA project by univariate Cox regression analysis, taking advantage of this risk score. The R packages survival and plyr were applied in this step.The expression profile of early cervical cancer patients in GSE44001 was retrieved as a testing cohort to validate the model externally. The Kaplan-Meier analysis was used to explore the universality of this signature, while the ROC curve was also plotted via the R package GSVA, which could measure the normalized enrichment score of multiple immune cell types. The tumor abundance of these immune cells between high-and low-risk groups was also plotted using the ggplot2 package. In the meantime, we downloaded the enriched results from ImmuCellAI, which estimated the abundance of 24 immune cells in the TCGA-CESC cohort [R packages reshape2, corrplot and igraph. In this Circos plot, we shed light on the correlation among these cell types and their survival impact by Spearman correlation analyses and univariate Cox regression analyses. Correlation between the established signature and these immune cells was also calculated by Spearman analyses using the R software.An integrated list of representative marker genes of tumor-infiltrating immune cell types was acquired from Charoentong’s research, which involved a total of 366 microarrays of immune cells summarized from 37 previous studies . To evalC cohort . The immR packages survival and survminer were utilized to draw the plot and conduct multiple comparisons between different survival curves.Kaplan-Meier analysis was carried out to clarify the relationship between the prognosis of patients with similar expression levels of immune checkpoints and the established signature. The n = 32) and normal cervix (n = 32) tissue microarrays were purchased from Shanghai Zuocheng Biotech . We performed the IHC analysis as previously described [+ cells, CD8+ cells, CD57+ cells and CD68+ cells at the tumor site were counted under five randomly selected microscopic fields.The study was approved by the Institutional Ethics Committee of Shanghai General Hospital. Cervical cancer  according to the methods used in the previous study [We performed the scoring of immune cell infiltration (CD4us study . BrieflyR software (version 4.0.4). The Wilcoxon test was applied to compare between two groups, while the Kruskal-Wallis test was used to compare among more than two groups. The Kaplan-Meier plot was performed to present survival curves for different groups, and the log-rank test was employed to evaluate the significance of statistical differences. Spearman analysis was carried out to determine the correlation coefficient. For all the analyses above, a two-tailed p < 0.05 was considered as statistically significant.All of the statistical process was completed within the The whole flow diagram of this study is presented in p < 0.05 subsequently underwent a multivariate Cox regression. Eventually, five genes  with significant regression coefficients were incorporated to set up a prognostic risk model , whereas there was no significant association between the signature and the histological grade of TCGA-CESC patients  was used to validate the aforementioned risk score. Three hundred early cervical cancer patients were categorized into low- and high-risk groups based on the median cut-off value, and the heatmap of expression levels of the five hub genes validated their differential distribution between the two subgroups B. SimilaMeanwhile, the pan-cancer validation analysis in the other 32 cancer types of the TCGA project revealed that the risk score composed of the five metabolism-related genes could successfully discriminate between patients with better or worse clinical outcomes in the other eight cancer types C, such aAccording to the estimated infiltration levels of 24 immune cells from the ImmuCellAI database, we visualized the immune landscape of patients in the TCGA-CESC cohort via an immune network A. Among GO and GSEA analyses were conducted to shed light on the possible function of these DEGs and the underlying mechanism. As shown in Furthermore, we also obtained the enrichment terms of the five metabolism-related hub genes via the online tool EMTome and the enriched networks are shown in The normalized enrichment scores (NES) of different immune cells in the TCGA-CESC project were calculated based on the gene expression profiles via the ssGSEA algorithm E. We fouTo verify the difference in infiltration levels of immune cells, we stained histology sections for immune markers including CD4, CD8, CD57 and CD68, and scored for the extent of infiltration both in the epithelial and stromal tumor compartments based on the density of positive staining for immune cell populations A. The IHIn the pan-cancer cohorts, our conclusion was also validated in a number of cancer types, as shown in Meanwhile, we retrieved the list of genes encoding immunostimulators, immunoinhibitors, chemokines and receptors from the TISIDB website . Notablyp < 0.001). Similar survival patterns could be also observed using the risk score and PD-L1, CTLA-4, CD47, CD38, CD28 and BTLA , and found that a higher risk score indicated a worse prognosis in patients with similar levels of ICPs . Consistand BTLA . These oPrevious studies have established metabolism-related signatures for the survival prediction of patients with several types of cancer ,22. Here+ T cell exhaustion, giving rise to post-chemotherapy evasion of immune surveillance [The TME is a complicated system which undergoes dynamic changes . The comeillance ,30. Metaeillance . As a reBiomarker-based patient stratification has gained much attention, which calls for more accurate evaluation of these molecular properties. Especially for those patients who receive immunotherapy, the comprehensive analysis of the risk score, as well as immune checkpoint expression, could hopefully foretell the reactivity of these patients and therefore screen out the appropriate patients for immunotherapy ,33,34. IAs a member of our risk score model, tryptophan 2,3-dioxygenase 2 (TDO2) catalyzes the commitment step of the KYN metabolic process, which subsequently activates the AhR and contributes to an immunosuppressive TME, and supports the cancer immune escape ,41. FAR2On the other side, high expression of coagulation factor 5 (F5) was found to be associated with improved overall survival of patients with breast cancers . TherefoOur study also has its limitations due to objective reasons. We analyzed the expression levels of metabolism-related hub genes in tumor specimens of the TCGA database, whereas we could not distinguish the cell origin of these hub genes. This means that our conclusion only represents the immunity pattern on the macro level. Further, single-cell sequencing data might better illustrate the complex association between tumor cells and the surrounding microenvironment. In the meantime, it has been a controversial issue whether the relatively small piece of tissues in TMAs could fully represent the characteristics of original tissues due to tumor heterogeneity, which might give rise to disparities in diagnosis. Since we affirmed our conclusion with an independent TMA cohort, the experimental results might also show some technical limitations to a certain extent.In summary, our constructed metabolism-related prognostic model allows for a more accurate categorization of patients at different risk levels of cervical cancer. We also determined the infiltration of TILs, the expression pattern of immune-related molecules and the prognostic value of our signature for immunotherapies. Since this risk score signature achieves a good performance in predicting clinical outcomes, we genuinely expect that our study could provide an effective prognostic tool for the guidance of personalized treatment for cervical cancer patients.


The Pacific Island country of Vanuatu is considering strategies to remove border restrictions implemented during 2020 to prevent imported coronavirus disease. We performed mathematical modeling to estimate the number of infectious travelers who had different entry scenarios and testing strategies. Travel bubbles and testing on entry have the greatest importation risk reduction. Many Pacific Island Countries and Territories (PICTs) implemented border entry restrictions and mandatory quarantine in 2020 to prevent imported coronavirus disease (COVID-19). Although some PICTs have experienced large-scale community transmission of COVID-19 , many PICTs have not (as of January 2022) experienced community transmission, including Vanuatu. Since March 2020, Vanuatu  has restricted entry to citizens and residents and required all incoming travelers to a complete 14-day quarantine period . We simulated 10,000 infected travelers stochastically and used 1,000 bootstrap samples to estimate uncertainty intervals. We applied the model to 40,000 passengers  . We did The number of infectious persons in the community decreased by 98%–99% when travel was restricted entry to persons from low point-prevalence countries, compared with no restrictions on the country of departure for travelers . The numOur analysis highlights that the scenario with the greatest importation risk reduction for Vanuatu is travel bubbles with low point-prevalence countries. The risk for case importation through quarantine-free travel with low COVID-19 incidence countries is <3.2 cases/40,000 travelers, an importation risk reduction of ≈100-fold compared with open borders. Several countries in the Pacific region have a low or zero COVID-19 point prevalence . Our results confirm the usefulness of this strategy.A limitation of our study is that the model does not estimate the number of secondary cases. Assumptions for parameters were based on published evidence for the original variant; these parameters might differ with new and emerging variants. In summary, as Vanuatu and other PICTs move toward removing border restrictions and importation prevention measures, on-arrival testing and restricting entry to travelers from low point-prevalence settings are essential strategies to limit COVID-19 cases.Additional information on mathematical modeling for removing border entry and quarantine requirements for COVID-19, Vanuatu.


Several authors have reported the formation of slit valves as the underlying pathomechanism of space-occupying tumor bed cysts. Iatrogenic slit valves following the resection of high-grade gliomas have been linked to certain risk factors such as intraoperative opening of the ventricles and attempts to seal these. The best therapeutic management of such cystic lesions remains elusive. Several treatment options such as cyst fenestration or cystoperitoneal shunting have been employed but remain associated with high rates of recurrence. With the given complications of the above-described treatment options, the objective was to devise a new therapy option that is safe and effective and treats the slit valve itself rather than its symptoms.Between the years of 2010 and 2020, we successfully treated four patients with high-pressure tumor bed cysts following glioma resection by implantation of synthetic ringed vascular grafts into the slit valve.Postoperatively, the tumor bed cysts were regressive in all patients. Moreover, none of the treatment patients developed any complications associated with the implanted vascular grafts. Revision-free survival was 10, 12, 53, and 126 months, respectively.The use of synthetic vascular grafts as a means of stenting slit valves is a safe and effective novel treatment option for high-pressure tumor bed cysts.The online version contains supplementary material available at 10.1007/s00701-022-05123-y. Radical resection with adjuvant radiochemotherapy has been established as the modern standard treatment regime for patients with high-grade glioma (HGG) , 20. A mIn this study, we present the retrospective analysis of four patients treated by a novel surgical intervention for treating tumor bed cysts. All patients were treated in our university hospital between the years of 2010 and 2020. Patients were considered for the implantation of synthetic vascular grafts if they developed high-pressure tumor bed cysts following glioma resection. Elevated pressure in the tumor bed cysts was determined by clinical and radiographic findings that were indicative of pressure higher than that inside the physiological cerebrospinal fluid (CSF) spaces. Such findings comprised progressive cyst growth on consecutive imaging that extends over the boarders of the initial resection cavity; midline-shift and/or displacement of brain parenchyma/ ventricles; pericystic edema; major and progressive subcutaneous CSF collections  in 02/2010. As the tumor infiltrated the posterior horn of the left lateral ventricle, the ventricle was opened during the resection. Three weeks postoperatively, the patient presented to our department with symptoms of raised ICP. CCT imaging showed a space-occupying cyst in the former tumor bed causing significant midline-shift and perifocal edema. In a first endoscopic revision, a slit valve between the ventricle’s posterior horn and the cyst could be observed. Endoscopically, the wall separating the cyst and the ventricle was then fenestrated. However, postoperative control imaging soon revealed the persistence of the high-pressure cyst. In order to establish long-term communication between the tumor bed and the lateral ventricle, a 12-mm GORE-TEX vascular graft was implanted into the lumen of the slit valve as a means of keeping it open and establishing drainage. Revision-free survival following implantation of the synthetic vascular graft is 126 months for this patient.Patient 2 is a 55-year-old male who underwent resection of a right temporo-occipital glioblastoma (WHO IV) in 08/2015 and re-resection in 01/16. Six months after removal of the tumor, the patient developed a tumor bed cyst that was initially treated by cystoperitoneal (CP) shunting. After three consecutive revisions of the shunt system due to blockage, the patient gave informed consent for the implantation of a GORE-TEX vascular graft. The implantation was conducted in a similar manner as was described for patient 1. After implantation, the CP shunt was left in situ. The patients’ symptoms are managed with both vascular graft and VP shunt and revision-free survival in this patient is 53 months.Patient 3 is a 46-year-old woman who underwent resection of a left frontal anaplastic astrocytoma (WHO III) in 12/2014. After re-resection in 05/18, the histological diagnosis of the recurrent tumor was adjusted to a glioblastoma (WHO IV). Seven months after re-resection, the patient developed symptoms of elevated ICP and underwent cCT imaging. The cCT revealed a large high-pressure cyst occupying the former tumor bed. Intraoperatively, a slit valve between the tumor bed cyst and the frontal horn of the lateral ventricle could be observed. In this case, due to our experience with the recurrence rates of slit valve cysts when treated with fenestration or shunting, the patient was directly treated by implantation of a ring-walled GORE-TEX vascular graft. Following implantation of the vascular graft, the cyst was successfully managed for 10 months until the patient died in 09/19 due to tumor progression.Patient 4 is a 57-year-old male who was operated on a left parieto-occipital oligodendroglioma (WHO II) in 02/18. After resection of recurrent tumor mass in 10/19, the histological diagnosis was adjusted to an anaplastic glioma (WHO III). Following the second operation, the patient developed a sizeable subcutaneous collection of CSF. CMRI imaging revealed a tumor bed cyst that communicated with the subcutaneous CSF collection . A multitude of medical devices made out of PTFE/Teflon have been approved for medical use by the FDA. Therefore, we considered its use safe for this study. In addition to that, GORE-TEX dura substitutes  consisting of the same fabric are FDA-approved for neurosurgical interventions and enjoy widespread use in dural plasty or replacement , 16, 21.This study has several limitations. We were only able to retrospectively report the clinical history of four patients that have been treated with this novel procedure in an off-label and compassionate use setting. In order to properly evaluate the procedure, prospective studies with a bigger quantity of patients must be conducted. Moreover, the details of the technique remain purely experimental. Points such as the sort of vascular graft used, having it fixed or just laid in or long-term complications, must be addressed in future studies.In conclusion, we believe that the evidence published on slit valves is sufficient to establish their existence as a possible pathomechanism of high-pressure cyst formation. In case of cyst formation in such a manner, we deem the use of synthetic ringed vascular grafts as a means of stenting the iatrogenic slit valve a promising new treatment option.ESM 1Video The intraoperative video shows how recommunication of the tumor bed and ventricular system was established by implanting two synthetic ringed vascular grafts. The mechanism tumor bed filling due to a slit valve is demonstrated by use of the Valsalva maneuver (MP4 49478 kb)


Background: Ubiquitination-related genes (URGs) are important biomarkers and therapeutic targets in cancer. However, URG prognostic prediction models have not been established in triple-negative breast cancer (TNBC) before. Our study aimed to explore the roles of URGs in TNBC.Methods: The Molecular Taxonomy of Breast Cancer International Consortium (METABRIC) and the Gene Expression Omnibus (GEO) databases were used to identify URG expression patterns in TNBC. Non-negative matrix factorization (NMF) analysis was used to cluster TNBC patients. The least absolute shrinkage and selection operator (LASSO) analysis was used to construct the multi-URG signature in the training set (METABRIC). Next, we evaluated and validated the signature in the test set (GSE58812). Finally, we evaluated the immune-related characteristics to explore the mechanism.Results: We identified four clusters with significantly different immune signatures in TNBC based on URGs. Then, we developed an 11-URG signature with good performance for patients with TNBC. According to the 11-URG signature, TNBC patients can be classified into a high-risk group and a low-risk group with significantly different overall survival. The predictive ability of this 11-URG signature was favorable in the test set. Moreover, we constructed a nomogram comprising the risk score and clinicopathological characteristics with favorable predictive ability. All of the immune cells and immune-related pathways were higher in the low-risk group than in the high-risk group.Conclusion: Our study indicated URGs might interact with the immune phenotype to influence the development of TNBC, which contributes to a further understanding of molecular mechanisms and the development of novel therapeutic targets for TNBC. Breast cancer ranks first in terms of incidence among all cancers according to statistics from the International Agency for Research on Cancer (IARC) . Triple-Ubiquitination is one of the most common and important post-translational modifications (PTMs). The ubiquitin–proteasome system is a highly specific, ATP-dependent pathway regulating specific protein degradation in eukaryotes. Ubiquitination is a reversible process that is mediated by three types of enzymes, namely, E1 ubiquitin-activating enzyme, E2 ubiquitin-conjugating enzyme, and E3 ubiquitin ligase . E1 actiIn the present study, we used the Molecular Taxonomy of Breast Cancer International Consortium (METABRIC) and Gene Expression Omnibus (GEO) databases to screen prognostic URGs. Based on these prognostic URGs, we identified a novel URG-based molecular classification of TNBC. Moreover, we constructed the 11-URG signature with good performance for patients with TNBC. Our analysis suggests that URGs play important roles in TNBC and are potential prognostic biomarkers and therapeutic targets.http://www.METABRIC.org/) and the GEO database (http://www.ncbi.nlm.nih.gov/geo/). We excluded patients with a survival time of less than 30 days. The gene expression profiles included 297 TNBC patients in METABRIC and 106 TNBC patients in GSE58812. We combined the METABRIC and GSE58812 and removed the batch effects using the ComBat function in the “sva” package (http://uucd.biocuckoo.org). We merged the URGs and gene expression profiles of TNBC patients to acquire URG expression in both the METABRIC database and the GSE58812 dataset. As a result, 403 TNBC patients with 525 URG expression data and baseline data were included for subsequent analysis.The gene expression quantification data (HTSeq-FPKM) and corresponding clinic data of patients with TNBC were retrieved from the METABRIC database ( package . We appl package  (UUCD)  analysis to cluster the 297 METABRIC patients and 106 GSE58812 patients. The clustering number K was set as 2–10. We determined the average profile width of a common member matrix by using the NMF package  and OS time. Those URGs with  package  in R wit package , the imm package , we perf package . The enr package  (MCP-cou package  (CIBERSO1*Gene1Exp + β2* Gene2Exp + β3* Gene3Exp + ⋯ + βn* GenenExp). In this formula, β represents the coefficients in the LASSO Cox regression analysis. Then, we calculated the risk score for each TNBC patient and classified TNBC patients into a high-risk group and a low-risk group according to the median risk score. The Kaplan–Meier curve and log-rank method were performed to evaluate the OS difference between the high-risk and low-risk groups. The distribution of risk scores, survival statuses of TNBC patients, and expression profiles of prognostic URG were exhibited using R software. We used a time-dependent receiver operating characteristic (ROC) curve to assess the sensitivity and specificity of the URG signature by calculating the area under the curve (AUC) (The prognostic URGs were entered into the least absolute shrinkage and selection operator (LASSO) method analysis to identify the prognostic multi-gene signature by using the glmnet  package ve (AUC) . We applWe used the GDSC database and the pRRophetic package  to estimp-value <.05 in the univariate Cox regression analysis were selected to construct the nomogram model. We used the concordance index (C-index) of 1,000-sample bootstrap and ROC curve to evaluate the prognostic prediction ability of the nomogram model. We also applied calibration curves to further validate the nomogram model.To verify the independence of the prognostic value of the URG signature and clinicopathological factors , cellularity, tumor mutation burden (TMB), menopause status, and breast surgery procedure), we performed univariate and multivariate Cox regression analysis to explore their associations with OS of TNBC patients. Factors with In order to reveal the heterogeneity between high-risk and low-risk group patients, we performed gene set enrichment analysis (GSEA). Gene Ontology (GO) items of biological process (BP), cellular component (CC), molecular function (MF), the reactome pathway, and the hallmark gene set were selected as the reference gene sets. The results of GSEA were visualized using the enrichplot package in R language.To further explore the immune phenotype between high-risk and low-risk group patients, we conducted immune infiltration and tumor immune microenvironment analyses. The infiltrating score of 16 immune cells and the activity of 13 immune-related pathways were determined by the ssGSEA function of the “gsva” package in R. Based on the ESTIMATE algorithm, we calculated the tumor purity, stromal score, immune score, and ESTIMATE score between high-risk and low-risk group patients. In addition, we performed Spearman’s analysis to explore the correlation of the risk score with the tumor microenvironment.p-value <.05 was considered statistically significant.All statistical analyses were performed by R software (version 3.6.1). The Wilcoxon rank-sum test was used to compare the difference between the two groups, and the Kruskal–Wallis test was performed to compare the difference among the four groups. The Kaplan–Meier curve and log-rank method were performed to evaluate the OS difference between groups. The ROC curves were plotted to assess the sensitivity and specificity of the URG signature and nomogram. The correlation between two sets of quantitative data was estimated by Spearman’s correlation test. A two-tailed The workflow of the present study is presented in p < .0001, A total of 17 prognostic URGs were used as variables of consensus clustering by using the NMF package. n = 148) and a low-risk group (n = 149) according to the median risk score. The Kaplan–Meier curve showed that patients in the high-risk group have significantly worse OS than patients in the low-risk group  and a low-risk group (n = 60). As presented in p = .002). The AUC of the 11-URG signature for predicting 3-, 5-, and 8-year survival in the test set was 0.662, 0.738, and 0.720, respectively . We identified 11 URGs  using LASSO Cox analysis and constructed a prognostic signature by integrating the 11 URG expression profiles and corresponding Cox regression coefficients . We calcectively . The resectively . The disUnivariate Cox regression analysis suggested that the 11-URG signature-based risk score, lymph node status, NPI, menopausal status, and breast conserving surgery were significantly associated with patients’ survival . We furtAs shown in To further identify the potential mechanism of the heterogeneity between high-risk and low-risk group patients, we conducted tumor immune microenvironment and immune infiltration analyses. All of the immune cells and immune-related pathways were higher in the low-risk group than in the high-risk group . CompareIn the present study, we identified 17 prognostic URGs and constructed four molecular classifications of TNBC. The immune signatures were significantly different among distinct TNBC clusters. Then, we developed the 11-URG signature with good performance for patients with TNBC. According to the 11-URG signature, TNBC patients can be classified into a high-risk group and a low-risk group with a significantly different OS. The predictive ability of the 11-URG signature was validated in the test set (GSE58812). Univariate and multivariate Cox regression analyses showed that the 11-URG signature was an independent risk factor for TNBC patients. Moreover, we constructed a nomogram comprising the risk score and clinicopathological characteristics with favorable predictive ability. GSEA showed that enriched GO terms, hallmark gene sets, and reactome pathways were evidently different between the high-risk and low-risk groups. In addition, tumor immune microenvironment and immune infiltration analyses also exhibited a significant difference.TNBC is a heterogeneous cancer. Tailored treatment based on molecular subtypes is meaningful for improving the outcomes of TNBC. In our study, we constructed four clusters of TNBC patients, including 41 patients in cluster 1, 154 patients in cluster 2, 164 patients in cluster 3, and 44 patients in cluster 4. As cluster 2 patients had the worst OS and cluster 4 patients had the best OS among all clusters, we conducted further analysis to reveal the mechanism. GSVA of the pathway showed that glycolysis, cholesterol homeostasis, hypoxia, and DNA repair pathways were the mainly upregulated pathways in cluster 2, while interferon response and inflammatory response were the mainly upregulated pathways in cluster 4. As is known, metabolic reprogramming is one of the hallmarks of cancer. Metabolic reprogramming is used by TNBC to fulfill bioenergetic and biosynthetic demands; maintain the redox balance; and further promote oncogenic signaling, cell proliferation, and metastasis . Metabolvia deubiquitination, further promoting breast cancer cell resistance to cisplatin. Moreover, STAMBPL1 could regulate snail stability by deubiquitination mechanisms in breast cancer (To the best of our knowledge, there are numerous specific gene-based prognostic prediction models for TNBC such as the hypoxia-related gene signature , immune-t cancer . The afoAccording to the 11-URG signature, TNBC patients can be classified into a high-risk group and a low-risk group with significantly different OS. We first analyzed the sensitivity of TNBC patients to chemotherapy drugs. Low-risk group patients are more sensitive to the majority of the chemotherapy drugs than patients in the high-risk group, which may partially explain the preferable OS of low-risk group patients. Then, we conducted GSEA to explore the mechanism. What stands out in the GSEA is the pathway difference between the high-risk and low-risk groups. Cell cycle- and glycolysis-related pathways were the mainly enriched pathways in the high-risk group, while chemokine receptors that bind chemokines, the inflammatory response, and complement cascades were the mainly enriched pathways in the low-risk group. As discussed earlier, the glycolytic phenotype promotes the proliferation, migration, and invasion of TNBC cells. Deregulation of the cell cycle is also a hallmark of cancer that enables limitless cell division and is frequently observed in breast cancer . TherefoConventional clinicopathological predictors such as age, gender, and the TNM staging system are insufficient to predict the prognosis of breast patients due to molecule complexity and biological heterogeneity of breast cancer. To provide a quantitative tool for predicting the survival rate of TNBC patients, we constructed a nomogram comprising the 11-URG signature-based risk score and clinicopathological characteristics. The ROC curve and calibration curve suggested that the nomogram is a stable and reliable predictor for OS of TNBC patients.in vitro and in vivo studies because of their strong relevance to the prognosis of TNBC. In conclusion, we identified four novel URGs based on the molecular classification of TNBC and constructed the 11-URG signature with good performance for patients with TNBC. Our analysis suggests that URGs play important roles in TNBC and are potential prognostic biomarkers and therapeutic targets.Admittedly, our study has some limitations because it was based only on high-throughput RNA-sequencing, array profiles, and data analysis. The roles of these prognostic URGs require further


Response criteria were based on (i) HDRS-17-sum, (ii) the sum score of the HDRS-6 subscale (HDRS-6-sum) and (iii) the depressed mood item. The separation between SSRI and placebo with respect to response rates increased when HDRS-17-sum was replaced by HDRS-6-sum or depressed mood as effect parameter and was markedly dependent on SSRI dose. With the exception of extreme cutoff values, differences in response rates were largely similar regardless of where the cutoff was placed, and also not markedly changed by the exclusion of subjects close to the selected cutoff . The observation of similar response rate differences between active drugs and placebo for different cutoffs was corroborated by the analysis of duloxetine data. In conclusion, the suggestion that using a cutoff close to the median when defining response has markedly overestimated the separation between antidepressants and placebo may be discarded.Response defined as a 50% reduction in the sum score of the Hamilton Depression Rating Scale (HDRS-17-sum) is often used to assess the efficacy of antidepressants. Critics have, however, argued that dichotomising ratings with a cutoff close to the median may lead to scores clustering on either side, the result being inflation of miniscule drug-placebo differences. Using pooled patient-level data sets from trials of three selective serotonin reuptake inhibitors (SSRIs)  ( A 50% reduction in the total sum of the 17 items comprising the Hamilton Depression Rating Scale (HDRS-17-sum) has been a common definition of response in trials assessing the efficacy of selective serotonin reuptake inhibitors (SSRIs) and other antidepressants. Critics have, however, argued that selecting a cutoff close to the median of the endpoint score distribution curve may result in significant differences in response rates between groups also when the actual differences in mean rating are miniscule and clinically irrelevant –4.Using pooled patient-level data from 28 trials comparing an SSRI with placebo, we explored the impact of the placement of dichotomised cutoffs on the SSRI versus placebo separation. To this end, three different outcome measures were used: HDRS-17-sum, the sum score of the unidimensional HDRS-6 subscale including six core symptoms of depression from the HDRS (HDRS-6-sum) , and thePatient-level data from 28 industry-sponsored, placebo-controlled, acute phase trials for adults with major depression using the HDRS-17 as symptom inventory were obtained for citalopram , paroxetine  and sertraline . In two of the paroxetine studies and one sertraline study, fluoxetine was used as active control; while these patients were also included, those treated with non-SSRI comparators were not. To corroborate results obtained using data from the SSRI trials, we also analysed patient-level data from 13 trials comparing duloxetine to placebo . SSRI comparators  were excluded. Both these data sets have been previously described in greater detail [To visualise the separation of SSRIs and placebo at different cutoffs for percentage reduction from baseline, we plotted the cumulative proportions of SSRI- and placebo-treated patients whose remaining symptoms at endpoint corresponded to every 5% fraction of their baseline score for HDRS-17-sum and HDRS-6-sum, respectively. For depressed mood, comprising only four possible levels, change scores rather than % reduction were plotted. For simplified visualisation, patients who were unchanged or had deteriorated during treatment were all included in one data point as if they had displayed 100% of baseline scores at endpoint for HDRS-17-sum and HDRS-6-sum or had displayed no change for depressed mood.Corresponding visualisations were produced for endpoint scores with respect to the HDRS-17-sum, HDRS-6-sum and the depressed mood item; for visualisation purposes, maximum scores were capped at 40 points for HDRS-17-sum (range 0–52) and at 20 points for HDRS-6-sum (range 0–22); subjects displaying higher scores being included in the highest visualised score.All visualisations were performed on patients with at least one pre- and post-baseline HDRS-17 measure in both the intention-to-treat (ITT) last observation carried forward (LOCF) population and in the observed cases (OC) population. Week 6 was used as the primary endpoint since most studies had an evaluation at that time. For studies with no week 6 observation, the closest observation was used  for response (SSRI versus placebo) for all possible 10% intervals of HDRS-17-sum and HDRS-6-sum reductions from baseline, and also for all possible change scores for depressed mood. Similar analyses were undertaken with respect to endpoint scores in the range of 0–10 points for HDRS-17-sum and HDRS-6-sum and 0–4 points for depressed mood, hence including cutoffs commonly used to define remission –15. ThisTo explore whether differences between-treatment groups with respect to response rates might be explained by patients clustering close to, but on either side of, any particular cutoff, we also used the SSRI data to model ORs for established response or remission criteria , 10 afteFinally, to assess the impact of SSRI dose on dichotomous definitions of response and remission, we pooled data from those trials that had assessed fixed SSRI doses, hence excluding flexible-dose trials . For the duloxetine data, remote desktop access to the Clinical Trial Data Transparency environment was provided through SAS Solutions OnDemand by the Clinical Study Data Request website. All The Regional Ethics Review Board of Gothenburg, Sweden, issued an advisory opinion stating no objection to the conduct of post hoc analyses of clinical trial data. As data were anonymised, informed patient consent was waived.In total, 7909 patients from SSRI versus placebo trials with a post-baseline HDRS-17 observation were included in the analyses. Inspection of the cumulative plots for relative score reductions in SSRI- and placebo-treated patients, respectively, revealed largely parallel lines across a wide range of possible outcomes  and are displayed in Supplementary figs. The modelled analyses confirmed that the separation between SSRIs and placebo on relative measures of improvement was largely independent of the cutoff used except for at very high and very low values Table . ORs werFor all tested cutoffs, drug versus placebo differences were larger for optimal SSRI doses than for suboptimal ones Table . DiffereIn this report, we rebut the claim  that difThe lack of impact of the placement of the cutoff is visually illustrated by the largely parallel lines over most of the range with respect to the cumulative distribution of symptom reduction scores Fig. –3. In liWe , 8 and oOf note is that the separation between active drug and placebo with respect to the proportion of subjects displaying very low remaining endpoint scores as well as proportions of baseline scores was substantial when assessed using the shorter and unidimensional HDRS-6 subscale but less so when using HDRS-17-sum. Since healthy volunteers on average score about three points on the HDRS , since pA factor possibly impacting efficacy is the dose of active treatment. While the dose–response curve for the SSRIs have often been described as flat , 24, andThis study has some limitations. First, HDRS scores might be inflated at baseline , which mIn conclusion, this report rebuts the previous claim that differences between antidepressants and placebo with respect to response rates be inflated by the common use of a cutoff (50%) close to the median with subjects clustering on either side. Instead, we report response rate differences consistently larger for higher SSRI doses and for outcomes based on the unidimensional HDRS-6 subscale or the single item depressed mood, indicating that previous meta-analyses in this field may have, on the contrary, deflated the differences between SSRIs and placebo with respect to response rates by including suboptimal doses and applying an insensitive outcome measure.Supplementary information


Immune checkpoint inhibitor (ICI) combined with chemotherapy has exhibited promising results in small sample studies of pancreatic cancer patients. The efficacy of toripalimab, a programmed cell death protein 1 (PD-1) monoclonal antibody has been explored in the previous studies and it was established that immune-related adverse events (irAEs) associated with administration of this drug deserve proper attention and adequate management.A 43-year-old female patient with advanced pancreatic ductal adenocarcinoma (PDAC) was treated with toripalimab in combination with gemcitabine and nab-paclitaxel (T-GA) as the first-line treatment. She developed immune-related encephalopathy with stuttering as the main clinical symptom and Magnetic resonance imaging (MRI) showed multiple cerebral white matter demyelination changes, concomitant with asymptomatic cardiac enzyme elevation and hypothyroidism. The symptoms resolved after the discontinuation of toripalimab and corticosteroid treatment.Stuttering might be an early sign of neurotoxicity which can be easily neglected during the treatment. These findings provide guidance for the identification of these rare and occult neurological irAEs (n-irAEs) in the clinical practice. The advent of various immune checkpoint inhibitors (ICIs) have revolutionized the treatment of solid tumors and hematological malignancies in recent years . The appToripalimab, a humanized monoclonal anti-PD-1 antibody, has been approved by FDA for the treatment of advanced melanoma , nasopha2 and nab-paclitaxel 125 mg/m2 on day 1 and day 8 and toripalimab 240 mg on day 1, administered every 3 weeks) as the first-line therapy in May 2019, despite the fact that genetic analysis of the tumor tissues indicated a low tumor mutation burden, microsatellite stability, and negative PD-L1 expression. After completion of 4 cycles of the treatment, the levels of asymptomatic myocardial markers were increased, but the thyroid function was found to be substantially decreased  were all positive, while CA125 and DPC-4 were found to be negative. However, shortly after the surgery, new metastatic lesions in the liver and peripancreatic lymph nodes appeared, and she began receiving T-GA  that have been implicated in the neurological paraneoplastic syndromes was found to be negative. Magnetic resonance imaging (MRI) revealed the presence of the cerebral white matter demyelinating polyneuropathy but no evidence of encephalitis or cancer metastasis was found  due to the increase in the liver metastases. However, later once the multiple metastases appeared in the gastric antrum and abdominal cavity, the patient was treated with nimotuzumab in combination with oxaliplatin and nab-paclitaxel based on the results of mini patient-derived xenograft drug sensitivity test, which has the lowest relative proliferative power of tumor cells, but died after 3.3 months of progression-free survival (PFS). Overall, from the start of first-line treatment to death, the overall survival (OS) of the patient was 21 months  can occur in approximately 1% of patients receiving ICIs as monotherapy and in 2-3% of patients receiving it as a combination therapy . The mosHere, we report a female patient with advanced PDAC who presented after 5 cycles of treatment with T-GA with symptoms of stuttering as well as slow thinking as the main clinical manifestation. The neurotoxicity of GA chemotherapy has been reported primarily in the periphery . AlthougThe lack of specificity in the diagnosis of n-irAEs often requires recourse to exclusionary diagnostic thinking, but in this case antibodies elicited in response to paraneoplastic neurological syndrome were not detected. However, subsequent MRI imaging showed a T2 Flair hyperintense signal in the multiple areas of the cerebral white matter, primarily in the parietal lobes but also affecting the posterior frontal lobes, corpus callosum, and right brachium pontis. In addition, none of these lesions were observed to be enhanced following the contrast administration and no restricted diffusion was present. Moreover, no significant mass effect or midline shift was identified. These findings were indicative of acute demyelinating encephalomyelitis, and she was thought to be suffering from immunotherapy-induced demyelination. Among the patients in whom MRI changes can be observed for ICI-induced neurotoxicity, manifestation of the central demyelination abnormalities has been reported to be the most common . It can Considering the possible further progression of the patient's n-irAEs and the combined increase in the level of cardiac enzymes in grade 3, it was reasonable to discontinue toripalimab. However, due to the high malignancy of pancreatic cancer, chemotherapy was not suspended completely. Current treatment guidelines usually recommend starting corticosteroids therapy as soon as the diagnosis of n-irAEs is confirmed. In general, oral corticosteroids are used for the management of the mild to moderate disease, whereas intravenous methylprednisolone infusions are mainly used for the more severe symptoms. Intravenous immunoglobulin or plasma exchange are employed for more severe diseases or for patients who are refractory to the steroids alone. Antibody-positive autoimmune encephalopathy can also be treated with rituximab to induce massive T-cell depletion , 28. TheHere, we report a case of advanced PDAC patient treated with toripalimab-induced encephalopathy, exhibiting stuttering as the first clinical signal, followed by slowness in thought, and confirmed the presence of multiple cerebral white matter demyelinating imaging changes. The symptom remission was achieved upon discontinuation of toripalimab and corticosteroid therapy. Overall, stuttering might serve as an early symptom of neurotoxicity, which can provide optimal guidance for recognizing this early occult ICI-related n-irAEs that is easily overlooked currently in the clinical practice.


N = 46,617 from 27 countries) and accelerometer-based average acceleration and intensity gradient (N = 855 from 10 countries) were analyzed. Mixed-model regressions with two levels  were used for analyses. Between countries differences accounted for relatively small portions of the variability in self-reported physical activity , average acceleration (ICC = 3.5%), and intensity gradient (ICC = 1.9%). There were more associations between individual- and country-level factors and self-reported physical activity than with accelerometer-based physical activity. The association between individual-level variables and accelerometer-based physical activity did not differ between countries. Cross-level interactions suggested that associations between some personality traits and self-reported physical activity were stronger in countries with lower GDP. Both individual- and country-level factors are related to participation in more intensive physical activities. Adults with less resilient personality traits living in countries with lower resources are at the highest risk for physical inactivity.This study aimed to investigate associations between individual-level  and country-level  factors with self-reported and accelerometer-based physical activity and cross-level interactions among European countries. Based on the Survey of Health, Ageing and Retirement in Europe (SHARE) from 2019–2020, self-reported physical activity (The online version contains supplementary material available at 10.1007/s10433-022-00737-8. Engaging in physical activity is one of the most promising ways to prevent non-communicable diseases and age-related loss in functional capacity  or demographic factors and neighborhood environmental level   the association between individual-level  and country-level  factors and self-reported and accelerometer-based physical activity, and 2) whether country-level factors moderate the association between individual-level factors and physical activity. Based on previous literature, we hypothesized that all individual- and country-level factors are associated with self-reported physical activity but the association with accelerometer-based physical activity may be weaker. We also hypothesized the association between individual-level factors and physical activity will be stronger in countries with lower GDP.The data from the Survey of Health, Ageing and Retirement in Europe (SHARE) and its accelerometer sub-study were used. SHARE is a cross-national multidisciplinary database from samples of community-based adults aged 50 or older , two southern , three eastern , and three central  European countries. The sampling strategy targeted a net sample of 200 participants from each country. The gross sample was a stratified sample selected from each country’s longitudinal sample stratified by age group and self-reported physical activity levels in previous waves  and moderate physical activity . The response options were 1 = More than once a week, 2 = Once a week, 3 = One to three times a month, and 4 = Hardly ever, or never. The response scale was reversed so that higher values indicated more physical activity and the mean for two questions was calculated (range 1–4)  on their upper thigh for eight consecutive days all day and night  was used to measure neuroticism, extraversion, openness, agreeableness, and conscientiousness . To facilitate interpretation of model estimates, GDP/10,000 was used in the analyses. The number of national policies and action plans was collected from the World Health Organization latest physical activity country factsheets (WHO Country-level indicators. Gross domestic product (GDP) per capita and the number of national policies or action plans for the promotion of physical activity for health were used as country-level indicators. GDP from the year 2020 was collected from the World Bank (eets WHO . For Swi2). The number of chronic diseases was calculated from a list of 21 common diseases or conditions . Participants reported conditions that a doctor had diagnosed them with, and they were currently either treated for or bothered by the condition. As some  of the accelerometer measurements were taken after the onset of the COVID-19 pandemic, the country-specific situation during the measurement period was captured , sex , education, body mass index (BMI), chronic diseases, and the pandemic restrictions. The 1997 International Standard Classification of Education with seven categories (from 0 = none to 6 = second stage of tertiary education) was used in SHARE to harmonize education categories across European countries (UNESCO Institute for Statistics n = 69) of the sample. Romania was excluded from the analysis on quality of life and Israel was excluded from analyses on the number of policies as this information was unavailable. Due to missing values in predictors and covariates, the analytic sample varied between 39,750–44,858 for self-reported physical activity and between 821–851 for accelerometer-based physical activity. Missing values were expected to be missing at random.All analyses were conducted using SPSS 26.0 software  and figures were created in RStudio 2022.02.0 . The Netherlands was excluded from analyses with personality traits because personality was available only from 3.6%  nested within countries (level 2). The same set of analyses was performed for each outcome: average acceleration, intensity gradient, and self-reported physical activity. Because the interest was in the associations between each individual predictor and outcomes, and to avoid suppression effects, all factors predicting physical activity outcomes were analyzed in separate models.First, the intraclass correlation coefficient (ICC) that assessed the proportion of variance in physical activity between countries was examined in a null model with no other variables. Likelihood ratio tests that compared a null multilevel model (including a random intercept) and a null single-level model were used to test the significance of country effects.p > 0.05) indicated that the random slope did not improve model fit or model convergence problems indicated the lack of random effects in the data, the random-intercept-only model was used as a final model (Meteyard and Davies p < 0.05), the random-intercept-random-slope model was chosen as the final model. Additional models that included health-related covariates were estimated to test whether the results were similar after adjusting for chronic diseases and BMI (Model 2). A model including all personality traits and covariates  simultaneously (Model 3) and a model including all predictors  and covariates simultaneously (Model 4) were also estimated for each outcome. These models evaluate whether each trait association with the outcomes is independent of the other traits (Model 3) and whether the associations hold when accounting for quality of life and country-level factors (Model 4).Second, the associations between predictors and physical activity outcomes were tested in models that included the predictor variable and demographic covariates  (Model 1). To test whether these associations varied between countries, both a random-intercept-only model and a random-intercept-random-slope model were estimated. In the latter, a random slope allowed the association between an individual-level predictor and physical activity outcome to vary between countries and an unstructured covariance matrix was used. Likelihood ratio test model comparisons were used to test whether the random slope improved the model fit. If the likelihood ratio test  to the model. Covariates were added in two steps as described above (Model 1 and 2) and an additional model including all interaction terms between personality traits and a country-level factor simultaneously was estimated (Model 3).n = 856) to examine whether the results in the large sample can be replicated in the accelerometer sample. Second, the analyses for accelerometer-based outcomes were repeated after the exclusion of participants (n = 73) who had at least some COVID-19 pandemic measurements in their country during the measurement period.Two sets of sensitivity analyses were done. First, the analyses for self-reported physical activity were repeated restricting the sample to those who also had accelerometer data .In the null model without predictors, the ICC indicated that 7.5% of the total variance in self-reported physical activity, 3.5% in average acceleration, and 1.9% in intensity gradient was attributable to between-country differences. The corresponding value for self-reported physical activity was 3.7% in the accelerometer sample. Based on the likelihood ratio tests, the effect of country was statistically significant for all outcomes (p < 0.01). This suggests that the effects of personality traits and quality of life on self-reported physical activity varied across countries. In the accelerometer sample, the random-intercept-only models were better for all outcomes suggesting similar associations between individual-level predictors and accelerometer-based physical activity in all ten countries. The random-intercepts-random-slope models did not achieve convergence for average acceleration with all predictors, and for intensity gradient with some predictors . The intercepts, variances of intercepts and slopes, covariances between intercepts and slopes, and -2 loglikelihoods for models are presented in supplementary materials (Tables S1 and S2).The random-intercept-random slope models were better than the random-intercept-only models when predicting self-reported physical activity in the whole sample  (Model 1). After adjusting the models for health-related variables (Model 2), the associations between GDP and the number of policies and intensity gradient were not statistically significant; the association with conscientiousness and quality of life remained significant. When all personality traits were estimated simultaneously (Model 3), the association of conscientiousness was not statistically significant, and when all predictors were estimated simultaneously, only quality of life was associated with intensity gradient (Model 4). The supplementary analysis showed that the results were the same after the exclusion of participants who had at least some COVID-19 pandemic measurements in their country during the measurement period (Supplementary Table S3).Because models for the accelerometer sample with 10 countries did not support the inclusion of the random slope in the models, the interaction models were estimated only for self-reported physical activity in the whole sample.Some cross-level interactions were found Table : The assThe purpose of this study was to test individual-level and country-level predictors of physical activity in a sample of older adults from European countries. The results showed that there were more associations between individual- and country-level factors and self-reported physical activity than with accelerometer-based physical activity. Furthermore, cross-level interactions suggested that associations between some personality traits and self-reported physical activity were stronger in countries with lower GDP.r = 0.27, p < 0.001) between intensity distribution and self-reported physical activity in the present study indicates that intensity distribution may be related more to participation in purposeful intensive physical activities, whereas average acceleration was weakly related to self-reported frequency of moderate and vigorous physical activities . Average acceleration may better reflect the amount of habitual light daily activities and individual- and country-level factors assessed in the present study were not related to average acceleration. Further studies focusing on environmental level predictors may shed light on determinants beyond overall physical activity volume.All significant associations found in the present study were either with self-reported physical activity or intensity gradient; none of the individual- or country-level predictors were associated with average acceleration. While average acceleration captures all step-based activity across the measurement period and indicates volume of physical activity, it does not provide any information about intensity distribution. Individuals who spend a lot of time being sedentary but engage in a few vigorous exercise sessions may have the same average acceleration as individuals who spend most of their time in light physical activities  and quality of life (CASP-12) were assessed with shortened versions of original questionnaires that may produce smaller effect sizes than longer questionnaires. Self-reported physical activity was based on only two questions on the frequency of participation in moderate or vigorous physical activities. Country-level indicators also have their limitations. GDP is a general indicator of a country´s economic situation and for example, does not reveal regional differences within a country. The number of policies and action plans for physical activity indicates only the existence of these policies and not their implementation. In addition, some policies may not be relevant for older adults . Future studies including also low-income countries are needed to strengthen the understanding of country-level differences.Despite these limitations, this study makes an important contribution to the research field by investigating how relatively stable and persistent individual-level factors and country-level factors are associated with physical activity. The results suggest that both individual- and country-level factors are related to participation in more intensive physical activities and the role of personality traits seems to be stronger in countries with relatively poorer economic conditions. This implies that persons with less resilient personality traits in terms of physical activity living in less advantageous countries are at the highest risk for physical inactivity and could be targeted in interventions.Supplementary file1 (DOCX 39 KB)Below is the link to the electronic supplementary material.


AHP) simultaneously in most piriform cortex layer II pyramidal neurons. Such sIAHP reduction is expressed in attenuation of the post-burst afterhyperpolarization (AHP) potential and thus in enhanced repetitive action potential firing. Previous studies have shown that a causal relationship exists between long-lasting post-burst AHP reduction and rule learning. A specific channel through which the sIAHP flows has not been identified. The sIAHP in pyramidal cells is critically dependent on membrane phosphatidylinositol 4,5-bisphosphate [PtdInsP(2)]. PtdInsP(2) regulates the calcium sensitivity of the sIAHP by acting downstream from the rise in intracellular calcium. These findings led to the interesting hypothesis that PtdInsP(2) activates a variety of potassium channels. Thus, the sIAHP would not represent a unitary ionic current but the embodiment of a generalized potassium channel gating mechanism. We thus hypothesized that the learning-induced increase in intrinsic excitability is mediated by reduced conductance of one or more of the currents that contribute to the sIAHP. Here we first show, using current-clamp recordings, that the post-burst AHP in piriform cortex pyramidal neurons is also mediated by the Ih, and the contribution of this current to the post-burst AHP is also affected by learning. We also show, using whole-cell patch-clamp recordings, that the sIAHP in neurons from trained rats is not sensitive to blocking membrane phosphatidylinositol 4,5-bisphosphate [PtdInsP(2)], and to the blocking of the current mediated by the cholinergic muscarinic acetylcholine receptor (M-current). Further current-clamp recordings also show that blocking PtdInsP(2) synthesis and application of a specific IKCa blocker have no effect on the post-burst AHP in neurons from trained as well as control rats. Taken together with results from our previous studies, these data suggest that rule learning-induced long-lasting enhancement in intrinsic neuronal excitability results from reduced conductance of the M-current and thus the slow potassium currents, which control repetitive spike firing.Training rats in a particularly difficult olfactory discrimination task initiates a period of accelerated learning, manifested as a dramatic increase in the rats' capacity to discriminate between pairs of odors once they have learned the discrimination task, implying that rule learning has taken place. At the cellular biophysical level, rule learning is maintained by reduction in the conductance of the slow current (sI Training rats in a particularly difficult olfactory discrimination (OD) task initiates a period of accelerated learning of other odors, manifested as a dramatic increase in the rats' capacity to acquire memories for new odors once they have learned the first discrimination task, implying that rule learning has taken place , in both the piriform cortex  remains intact  processed in the network composed of these neurons. In particular, enhancement of intrinsic neuronal excitability results in enhanced learning abilities P(2)]. Moreover, PtdInsP(2) regulates the calcium sensitivity of sIAHP by acting downstream from the rise in intracellular calcium  male Sprague Dawley rats were used. Before training, they were maintained on a 23.5-h water deprivation schedule, with food available The olfactory discrimination protocol was performed in a 4-arm radial maze, with commercial synthetic odors that are regularly used in the cosmetics and food industry purchased from Value Fragrances & Flavors. Odors were diluted by a factor of 1,000 before use in the olfactory maze. Odors were composed of natural and naturally derived materials in their formulations. The odors used in this study were as follows: almond, gardenia, wintergreen, and lavender .Olfactory training consisted of 20 trials per day as previously described . Brain slices were taken from the posterior piriform cortex. The 400-μm coronal rat brain slices were used for intracellular sharp recordings, and 300-μm slices for whole-cell patch-clamp recordings were prepared as previously described  amplitude of 80 mV]. For post-burst AHP measurement, neurons were depolarized in the current-clamp mode to a holding potential of −60 mV with direct current injection ke train . The AHPhreshold .AHP was recorded in a TTX (1 μM) and TEA (5 mM) containing aCSF solution . To generate a pure sIAHP, apamin (50 nM), the SK channel blocker was added to the solution , and We previously showed that rule learning-induced post-burst AHP reduction is mediated by metabotropic activation of GluK2 (kainate receptor), and that GluK2 is both necessary and sufficient for OD rule learning  was significantly higher  than that in neurons from the naive  and pseudo-trained  rats  rats .n = 60) was significantly lower  than that in neurons from the naive  and pseudo-trained  rats  rats .The strong correlation between the post-burst AHP and the number of intrinsically evoked action potentials shown in AHP. Specifically, this notion is supported by three main findings: a. The difference between neurons from the trained and control mice in the post-burst AHP amplitudes is abolished in the presence of an intrinsically applied calcium chelator , on the post-burst AHP amplitude. The Ih blocker significantly reduced the amplitude of the average post-burst AHP in neurons for control rats . Such an effect was not observed in neurons from trained rats . Notably, the difference in the average post-burst AHP between neurons from trained rats and controls remained significant in the presence of the blocker P(2); facilitation of PtdInsP(2) biosynthesis protects against sIAHP rundown, and inhibition of its biosynthesis accelerates the sIAHP rundown P(2) biosynthesis affects this rundown differently.We thus examined if the rundown of the sIAHP is similar in neurons from the trained rats and controls (n = 15) for neurons from controls and 0.36 + 0.19 (n = 11) for neurons from trained rats (P = 0.39) (n = 15) for neurons from controls and 0.37 + 0.24 (n = 11) for neurons from trained rats (p = 0.44). Notably, while wortmannin significantly reduced the normalized value in neurons from controls .Our results show that in control conditions (with only apamin in the aCSF solution), the rundown of the sIcontrols . The ave = 0.39) . When re = 0.39) , the norThus, while blocking PtdInsP(2) biosynthesis in piriform cortex pyramidal neurons has a similar effect to that described for cortical neurons  channel blocker which is specific to the cholinergic M current. XE991 (10 μM) had a profound effect on the rundown of the sIAHP in neurons from the controls (n = 19). This value is significantly lower than the averaged values in apamin and wortmannin (p < 0.05) (n = 21); it remained similar to that obtained in the presence of apamin only (p = 0.30), and in the presence of apamin+wortmannin (p = 0.45). Notably, in the presence of XE991, the averaged values in neurons from the trained rats were significantly higher than those in controls (p < 0.001)  . In shar< 0.001) .Figure 7), these results suggest that the shutdown of the M receptor-mediated current enables prolonged OD learning-induced reduction of the post-burst AHP current.Taken together with our previous findings P(2) activity. To test this hypothesis, we measured the effect of the PtdInsP(2) biosynthesis inhibitor, wortmannin, on the post-burst AHP amplitude recorded under current-clamp conditions on neurons from the control and trained rats. We predicted that wortmannin would reduce AHP in neurons from the control rats only.p = 0.12). In six neurons from the trained rats, the averaged post-burst AHP amplitude was 4.9 ± 1.0 mV before and 5.4 ± 1.3 mV after 20 min of wortmannin application (p = 0.51). Consequently, as shown in p = 0.013, t = 2.78) remained after wortmannin application .We find that wortmannin had no effect on the post-burst AHP in piriform cortex pyramidal neurons. As shown in These data do not support a role for modulation of the PtdInsP(2)-mediated process in maintaining long-lasting AHP reduction in piriform cortex neurons after complex olfactory learning.Previous work suggests that the IKCa channels have a role in generating the sAHP of CA1 hippocampal pyramidal cells . The lack of effect of TRAM-34 on the post-burst AHP was apparent in all recorded neurons, regardless of the initial post-burst AHP measured in each neuron (We found that TRAM-34 (1 μM) had no effect on the post-burst AHP in piriform cortex pyramidal neurons. As shown in h neuron .These data do not support a role for IKCa channels in generating the post-burst AHP in piriform cortex neurons.In this study, we show that while the post-burst AHP appears to be mediated by a complex combination of overlapping, calcium-dependent, and voltage-dependent currents, the shutdown of the M-receptor-mediated current is the principal mechanism underlying the learning-induced enhancement of neuronal excitability.Learning-induced enhancement in neuronal excitability has been shown in hippocampal neurons following classical conditioning of the trace eyeblink response , generated by potassium currents, which develop following a burst of action potentials ] and that neuronal calcium sensors are mediators for the sIAHP. The sIAHP is carried by different potassium channel types in different cell types P(2), and Ca(2+) appears to gate this current by increasing PtdInsP(2) levels, which in turn activates a variety of potassium channels. Thus, the sIAHP does not represent a single current but is the embodiment of a generalized potassium channel gating mechanism.The inability to identify the sIAHP during recording piriform cortex pyramidal neurons with whole-cell patch-clamp electrodes. We found that blocking PtdInsP(2) biosynthesis significantly enhances the decay time course of the sIAHP in neurons from the control rats only, but it did not affect neurons from the trained rats. However, application of the same blocker had no effect on the post-burst AHP amplitude, recorded with sharp intracellular electrodes, in neurons from the controls, as well as from the trained rats. Thus, while leaning-induced modifications in the sIAHP are accompanied by changes in its sensitivity to PtdInsP(2), the post-burst AHP in the same neurons shows no response to the same blocker. A similar lack of effect on the AHP was found for the intermediate conductance calcium-activated potassium channels (IKCa).Based on their model, we first examined if inhibiting PtdInsP(2) biosynthesis would have a different effect on the decay rate of the sIAHP in neurons from the control rats only, it had no effect on neurons from the trained rats. These data complete well the results from our previous study (Saar et al., We also found that while blocking the M-current significantly enhanced the decay of the sITo conclude, our data show that OD learning-induced reduction in the post-burst AHP in piriform cortex pyramidal neurons is strongly correlated with the ability of these neurons to sustain repetitive spike firing, as expected. While the post-burst AHP is a complex phenomenon, composed of several currents, and sensitive to different neuromodulators and neuropeptides, rule learning-induced long-lasting reduction of the post-burst AHP is mediated, at least mostly, by reduced conductance of the cholinergic M-current.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The animal study was reviewed and approved by University of Haifa Ethics Committee.RA and NC performed the experiments and analyzed the data. EB planned the experiments and wrote the manuscript. All authors contributed to the article and approved the submitted version.This study was supported by a grant from the Israel Science Foundation to EB.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


The proposed process could be used not only for SIW-based devices, but also for various millimeter-wave applications where a glass substrate with TGV structures is required.A millimeter-wave substrate-integrated waveguide (SIW) was firstly demonstrated using the micromachining of photoetchable glass (PEG) for 5G applications. A PEG substrate was used as a dielectric material of the SIW, and its photoetchable properties were used to fabricate through glass via (TGV) holes. Instead of the conventional metallic through glass via (TGV) array structures that are typically used for the SIW, two continuous empty TGV holes with metallized sidewalls connecting the top metal layer to the bottom ground plane were used as waveguide walls. The proposed TGV walls were fabricated by using optical exposure, heat development and anisotropic HF (hydrofluoric acid) etching of the PEG substrate, followed by a metal sputtering technique. The SIW was fed by microstrip lines connected to the waveguide through tapered microstrip-to-waveguide transitions. The top metal layer, including these feedlines and transitions, was fabricated by selective metal sputtering through a silicon shadow mask, which was prefabricated by a silicon deep-reactive ion-etching (DRIE) technique. The developed PEG-based process provides a relatively simple, wafer-level manufacturing method to fabricate the SIW in a low-cost glass dielectric substrate, without the formation of individual of TGV holes, complex time-consuming TGV filling processes and repeated photolithographic steps. The fabricated SIW had a dimension of 6 × 10 × 0.42 mm The advent of 5G communications has been accelerating the development of radio frequency (RF) devices and components working at millimeter-wave frequencies. For many millimeter-wave applications, such as wireless communications, automotive radars, high-resolution imaging systems and gigabit backbone networks, demand for small-footprint, high-density, low-loss and low-cost system integration is continuously increasing ,2. Packa (LTCCs) , as well (LTCCs)  and poly (LTCCs) . LTCC isAs an alternative to resolve these challenges, glass substrate with through glass vias (TGVs) has recently been used for the packaging substrate or interposers for high-frequency applications ,24,25,26Photoetchable glass (PEG) is a special type of glass showing a large anisotropy of etch rates when exposed to ultraviolet (UV) light and heat treatment . The pho3+ ions absorb photons and release one electron to enter the stable Ce4+ state, and this electron is absorbed by Ag+ ions reduced to Ag atoms. The exposed glass is then heated to higher temperatures, typically between 580~680 °C. During this “heat development” process, Ag atoms are clustered into bigger nuclei around which the glass crystallizes to form lithium metasilicate (Li2SiO3), producing the latent image of the photomask [PEGs, which are normally known by their trademark names, such as FORTURAN  or APEX , are lithium aluminum silicates with some well-defined impurities of metal oxides that significantly contribute to photosensitive properties. These impurities include photosensitive metals, with silver (Ag) being used more than others, and optical sensitizers, among which cerium (Ce) is the most important. Typical procedures to fabricate microstructures in PEG are described in the literature ,38. The hotomask . The exphotomask .Q values. The split cylinder uses the TE011 mode of resonance, where the electric flux circulates in the sample plane, eliminating the negative effects of ends and allowing accurate measurements. PEG plate samples were fabricated by dicing the substrate into a rectangular plate of 30 mm × 50 mm, followed by polishing them down to the thickness of 154 μm. Two different samples were prepared, one of which is an original unprocessed PEG, while the other is thermally treated at a temperature of 585 °C for 2 h to emulate the heat development conditions. The prepared samples were loaded in the split cylinder resonator  and measured using a vector network analyzer . The measured results are summarized in To design the SIW using PEG, the electrical properties of the PEG substrate need to be evaluated. In our study, the dielectric constant and loss tangent of the PEG substrate  at 28 GHz were experimentally measured using a split cylinder resonator method , where tIn our work, SIW was selected to demonstrate the potential of PEG in the millimeter-wave regimes. The entire three-dimensional schematic view of the proposed SIW is illustrated in 10 mode is given by the following well-known equations [10 mode, 10 mode in the waveguide [When the width of the waveguide is larger than its thickness, the cutoff frequency of the dominant TEquations :(1)fc,10The simulated S-parameters of the designed SIW are shown in 2 using a soda-lime glass photomask  and a vector network analyzer . The experimental setup is shown in The measured S-parameters of the fabricated SIW were compared with the simulation results in The measured insertion loss of the fabricated PEG SIW is higher than that of the reported SIWs fabricated on the borosilicate glass ,21,22. OTo confirm the effect of surface roughness on the RF performances, the S-parameters of the two SIWs fabricated on the PEG substrate with or without the CMP process are compared in As mentioned before, very few works about microwave applications of PEG are reported, especially in the regime of millimeter-wave frequencies. Since we could not find the SIW realized in the PEG substrates, the performances of our fabricated SIW on the PEG substrate were compared with some other glass-based SIWs, including our previous results, which were mostly manufactured on the borosilicate glass substrates, as presented in In this study, we proposed a manufacturing method to fabricate SIWs based on the micromachining of photoetchable glass (PEG). Empty TGV holes were fabricated by the UV exposure, heat development and HF etching of PEG, and the sidewalls of the TGV holes were metallized with selective sputtering processes using a silicon shadow mask to connect the top metal and bottom ground plane. By using this process, the wafer-level manufacturing of SIWs in a low-cost glass substrate can be carried out in a relatively simple way without further photolithographic steps or time-consuming via metallization processes. An SIW operating at the Ka-band was designed and fabricated by the developed process. The measured average insertion loss of the SIW was 2.53 ± 0.55 dB with a return loss better than 13.86 dB, from 26.5 to 40 GHz. A microstrip line fabricated on the PEG substrate showed an average insertion loss of 2.26 ± 0.37 dB in the same frequency range. Due to the higher dielectric loss factor of PEG, the fabricated SIW on the PEG substrate tends to show a somewhat higher insertion loss than the SIW on the borosilicate glass. The PEG substrate, however, still has the potential to be applied to millimeter-wave passives or packages for 5G applications because of its simple, wafer-level machineability to fabricate fine-pitched micro TGVs in the glass dielectric material. The process developed in this work could be applied to demonstrate millimeter-wave devices such as SIW-based resonators, filters, 3D inductors and helical antennas, or interposers for integrated packaging where conductive TGVs are required.