Supplemental Digital Content is available in the text. Although the 99th percentile is the recommended diagnostic threshold for myocardial infarction, some guidelines also advocate the use of higher troponin thresholds to rule in myocardial infarction at presentation. It is unclear whether the magnitude or change in troponin concentration can differentiate causes of myocardial injury and infarction in practice.In a secondary analysis of a multicenter randomized controlled trial, we identified 46 092 consecutive patients presenting with suspected acute coronary syndrome without ST-segment–elevation myocardial infarction. High-sensitivity cardiac troponin I concentrations at presentation and on serial testing were compared between patients with myocardial injury and infarction. The positive predictive value and specificity were determined at the sex-specific 99th percentile upper reference limit and rule-in thresholds of 64 ng/L and 5-fold of the upper reference limit for a diagnosis of type 1 myocardial infarction.P<0.001 for all). Discrimination improved when troponin concentration and change in troponin were combined compared with troponin concentration at presentation alone .Troponin was above the 99th percentile in 8188 patients (18%). The diagnosis was type 1 or type 2 myocardial infarction in 50% and 14% and acute or chronic myocardial injury in 20% and 16%, respectively. Troponin concentrations were similar at presentation in type 1 (median [25th–75th percentile] 91 [30–493] ng/L) and type 2 (50 [22–147] ng/L) myocardial infarction and in acute (50 [26–134] ng/L) and chronic (51 [31–130] ng/L) myocardial injury. The 99th percentile and rule-in thresholds of 64 ng/L and 5-fold upper reference limit gave a positive predictive value of 57% , 59% (58%–61%), and 62% (60%–64%) and a specificity of 96% (96%–96%), 96% (96%–96%), and 98% (97%–98%), respectively. The absolute, relative, and rate of change in troponin concentration were highest in patients with type 1 myocardial infarction (Although we observed important differences in the kinetics, cardiac troponin concentrations at presentation are insufficient to distinguish type 1 myocardial infarction from other causes of myocardial injury or infarction in practice and should not guide management decisions in isolation.https://www.clinicaltrials.gov; Unique identifier: NCT01852123.URL:  In 46 092 consecutive patients with suspected acute coronary syndrome, we evaluated the performance of the 99th percentile rule-in threshold and thresholds of 64 ng/L and 5 times the upper reference limit for the diagnosis of type 1 myocardial infarction.Troponin concentrations at presentation have a low positive predictive value for type 1 myocardial infarction, and a threshold 50 times the upper reference limit is required to achieve a positive predictive value ≥70%.Change in troponin on serial testing only marginally improves positive predictive value for type 1 myocardial infarction over presenting troponin alone .Troponin concentrations at presentation are insufficient to distinguish type 1 myocardial infarction from other causes of myocardial injury or infarction and should not be used in isolation to guide management decisions in patients with suspected acute coronary syndrome.1 In practice, there are many causes of troponin elevation, with about half of all increases attributable to conditions other than type 1 myocardial infarction.9 Nonetheless, the early differentiation between types of myocardial infarction and acute or chronic nonischemic myocardial injury is important because the immediate management of these conditions differs.11 Prompt treatment with antiplatelet agents, anticoagulation, and coronary revascularization is recommended in type 1 myocardial infarction, whereas these may not be indicated in type 2 myocardial infarction or myocardial injury and may be contraindicated.12To promote the adoption of common standards, the Universal Definition of Myocardial Infarction recommends cardiac troponin testing and the 99th percentile upper reference limit (URL) as the diagnostic threshold for myocardial infarction.16 The European Society of Cardiology guidelines propose the use of rule-in thresholds above the 99th percentile to guide admission to cardiology and coronary angiography.11 These rule-in thresholds and those 5 times the URL are purported to give a PPV of at least 70% and 90%, respectively.11 They were derived in selected patients with chest pain, but, in practice, troponin testing is applied more widely to evaluate patients with suspected acute coronary syndrome presenting with a broader range of symptoms.17 Guidelines also recommend serial testing with a rise or fall in cardiac troponin needed to confirm the diagnosis of myocardial infarction.18 However, patients with type 2 myocardial infarction and acute nonischemic myocardial injury also have dynamic changes in troponin concentration on serial testing.21 It is unclear whether rule-in thresholds or troponin kinetics can reliably differentiate between types of myocardial infarction or between myocardial injury and infarction in clinical practice.22Alternative thresholds above the 99th percentile have been proposed to improve the positive predictive value (PPV) and specificity of troponin for type 1 myocardial infarction.23We aimed to evaluate the performance of recommended cardiac troponin thresholds to rule in the diagnosis of type 1 myocardial infarction at presentation. We also aimed to determine whether the kinetics of cardiac troponin differs sufficiently to discriminate between myocardial injury and infarction.The High-STEACS trial  makes use of multiple routine electronic health care data sources that are linked, deidentified, and held in a national safe haven that is accessible by approved individuals who have undertaken the necessary governance training. Summary data and the analysis code can be made available on request from the corresponding author.2 but in summary, all patients attending the emergency department were screened for suspected acute coronary syndrome by the attending clinician at the time cardiac troponin was requested with the use of an electronic form integrated into the clinical care pathway. Patients were eligible for inclusion if they presented with suspected acute coronary syndrome and had paired cardiac troponin measurements from the standard care and trial assay. Patients were excluded if they had been admitted previously during the trial period or were not residents of Scotland. In this analysis, we excluded patients with ST-segment–elevation myocardial infarction,24 those for whom troponin concentration at presentation was missing, or patients for whom the adjudicators were unable to arrive at a consensus for the final diagnosis.High-STEACS is a stepped-wedge cluster randomized controlled trial that evaluated the implementation of a high-sensitivity cardiac troponin I assay in consecutive patients with suspected acute coronary syndrome across 10 secondary and tertiary care hospitals in Scotland. A detailed description of this trial has been reported elsewhere,26 All patients had troponin measured with a high-sensitivity cardiac troponin I assay . This assay has an interassay coefficient of variation of <10% at 4.7 ng/L and a limit of detection of 1.2 and 1.9 ng/L. For consistency with prior studies, we defined the limit of detection as any concentration <2 ng/L, and for the purpose of this analysis, we assigned concentrations below the limit of detection a value of 1.0 ng/L.28 The assay has a 99th percentile URL of 26 ng/L, with sex-specific thresholds of 34 and 16 ng/L in men and women, respectively.30Cardiac troponin testing was performed at presentation to hospital and was repeated 6 or 12 hours after the onset of symptoms at the discretion of the attending physician in accordance with national guidelines.1 Two physicians independently reviewed all clinical information, with discordant diagnoses resolved by a third physician. Type 1 myocardial infarction was defined as myocardial necrosis  in the context of a presentation with suspected acute coronary syndrome and symptoms or signs of myocardial ischemia on the ECG. Patients with myocardial necrosis, symptoms or signs of myocardial ischemia, and evidence of myocardial oxygen supply-demand imbalance secondary to an alternative condition without evidence of acute atherothrombosis were classified as having type 2 myocardial infarction.21 Patients with elevated troponin concentrations without symptoms or signs of myocardial ischemia were classified as having nonischemic myocardial injury. All nonischemic myocardial injury was classified as acute unless troponin concentrations changed ≤20% on serial testing in accordance with the universal definition or if the adjudicated diagnosis was chronic heart failure or chronic renal failure, for which the classification was chronic myocardial injury. A detailed summary of the adjudication procedures is provided in the Data Supplement.All patients with any high-sensitivity cardiac troponin I concentration above the sex-specific 99th percentile were adjudicated and classified according to the Fourth Universal Definition of Myocardial Infarction.The study was approved by the Scotland A Research Ethics Committee, by the Public Benefit and Privacy Panel for Health and Social Care, and by each National Health Service Health Board. Individual patient consent was not required, and data from consecutive patients were collected prospectively from the electronic record, deidentified, and linked within secure National Health Service Safe Havens.Patients and lay representatives were members of the steering committee for the trial and all related studies and were involved in the design, conduct, and approval of the High-STEACS trial.2, Kruskal-Wallis, or 1-way ANOVA tests as appropriate. We constructed confusion matrices and calculated the PPV and specificity for type 1 myocardial infarction of a high-sensitivity cardiac troponin I concentration at presentation above the uniform 99th percentile (26 ng/L), sex-specific 99th percentile , guideline-recommended rule-in threshold of 64 ng/L, and 3-fold and 5-fold URL thresholds . From prior literature, we also determined the cardiac troponin concentration at presentation that met a prespecified PPV of 75%.31 We calculated the 95% CI using a bayesian approach by sampling from a binomial likelihood with noninformative Jeffreys prior . In a sensitivity analysis, we evaluated the PPV and specificity in patients for whom the primary presenting symptom recorded by the attending clinician was chest pain.Baseline characteristics were summarized for the study population and in groups according to the diagnostic classification: type 1 myocardial infarction, type 2 myocardial infarction, acute myocardial injury, chronic myocardial injury, and no myocardial injury. Group-wise comparisons were performed with χ11 All analyses were performed in R (version 3.5.1).In patients with serial sampling within 12 hours of presentation, we used linear mixed-effects modeling with random slopes and intercepts to evaluate the relationship among symptom onset, troponin, and change in troponin concentration. Nonlinear associations were evaluated by adding a second-order polynomial term for time to the model. We have compared the models with and without a quadratic term for time, and the final model was chosen according to the lowest Akaike information criteria. To illustrate the kinetics of cardiac troponin across the groups, we developed additional models for each diagnostic classification. In each of these models, we included type 2 myocardial infarction or acute or chronic myocardial injury as a fixed effect, with type 1 myocardial infarction as the reference group. To evaluate whether relative or absolute change in troponin on serial testing improves discrimination for type 1 myocardial infarction over troponin concentration at presentation alone, we used logistic regression and compared the area under the receiver-operating characteristic curve. We evaluated models that incorporated relative and absolute changes as continuous measures and absolute and relative delta values of 15 ng/L and 20% as recommended in international guidelines.Figure I in the Data Supplement).The analysis population comprised 46 092 of the 48 242 patients enrolled in the trial after exclusion of those with ST-segment–elevation myocardial infarction (n=925), those for whom the diagnosis could not be adjudicated (n=1241), and those with missing troponin concentrations at presentation  patients. The adjudicated diagnosis was type 1 myocardial infarction in 50% (n=4064), type 2 myocardial infarction in 14% (n=1116), acute myocardial injury in 20% (n=1676), and chronic myocardial injury in 16% (n=1287) of patients  and type 2 (50 [22–147] ng/L) myocardial infarction and in acute (50 [26–134] ng/L) and chronic (51 [31–130] ng/L) myocardial injury , the PPV and specificity for type 1 myocardial infarction at the rule-in threshold of 64 ng/L were 72% and 98%, respectively  of patients in whom the primary presenting symptom was chest pain .Serial troponin testing within 12 hours of presentation was performed in 4187 patients (51%) with concentrations above the sex-specific 99th percentile. The time from symptom onset to initial troponin sampling was similar in patients with type 1 and type 2 myocardial infarction and acute myocardial injury (240 [180–420] minutes) but was longer in patients with chronic myocardial injury (300 [180–780] minutes). The rate of change in troponin within 12 hours of presentation was highest in patients with type 1 myocardial infarction compared with those with type 2 myocardial infarction and acute or chronic myocardial injury .Combining troponin concentration at presentation with an absolute change in troponin on serial testing of ≥15 ng/L or relative change of ≥20% improved discrimination for type 1 myocardial infarction compared with troponin concentration at presentation alone  in which just 29% of patients with an elevated cardiac troponin T concentration had a diagnosis of type 1 myocardial infarction.1 We observed differences in the rate of troponin release with a higher rate of change, as well as larger absolute and relative changes on serial sampling, in patients with type 1 myocardial infarction compared with those with type 2 myocardial infarction or acute and chronic myocardial injury. Despite these differences, the use of relative or absolute delta change criteria only marginally improved discrimination compared with the troponin concentration alone. This is perhaps not surprising given the observed changes in troponin concentration on serial testing in both type 2 myocardial infarction and acute myocardial injury. Although previous studies using a contemporary sensitive troponin assay in a small cohort of 66 and 188 patients with type 1 and type 2 myocardial infarction, respectively, suggested no improvement in discrimination when change in troponin at 3 or 6 hours was combined with the absolute concentration,45 we observed a modest improvement. It would seem unlikely that the shorter time intervals between serial testing would improve discrimination, but this should be evaluated in future studies.Although our observations highlight the limitations of using single troponin measurements to triage patients, confirmation of the diagnosis of myocardial infarction requires serial testing and a rise or fall in cardiac troponin.47 These approaches recognize the limitations of applying fixed thresholds to triage a heterogeneous population of patients and the challenge of performing serial testing at precise intervals in clinical practice. The Troponin Only–Manchester Acute Coronary Syndrome rule uses logistic regression to provide individual patient risk estimates for non–ST-segment–elevation myocardial infarction by incorporating age, sex, clinical variables, and a measure of high-sensitivity cardiac troponin T at presentation. This rule performs well but does not discriminate type 1 myocardial infarction from other causes of troponin elevation or take into account serial testing.47 In a collaborative analysis that pooled data from multiple cohorts, the COMPASS-MI  investigators highlight that a more flexible approach is required and demonstrate proof of concept that the negative predictive value and PPV for type 1 myocardial infarction vary across a range of thresholds and delta change in troponin values.46 Finally, the myocardial ischemic injury index uses a gradient-boosting machine learning algorithm to combine age, sex, and paired high-sensitivity cardiac troponin I values to compute a value (0–100) that reflects the likelihood of type 1 myocardial infarction for an individual patient.31 Serial testing can be performed at any time point, and the algorithm incorporates a measure of rate of change in troponin. Although each of these approaches shows considerable promise, it is unclear at present whether the use of these probabilistic scores in practice improves clinical decisions compared with existing guideline-recommended pathways using fixed thresholds.In recent years, a number of approaches have been proposed that could enable clinicians to use cardiac troponin more flexibly.We recognize some strengths and limitations of this study. First, we enrolled consecutive patients in whom the attending clinician suspected acute coronary syndrome by embedding our screening tool into the electronic health care system. This avoided selection bias and ensured that our study population was representative. Second, all diagnoses were adjudicated according to the Fourth Universal Definition of Myocardial Infarction, ensuring that our findings are relevant to contemporary practice. Although few patients had missing troponin values for the evaluation of rule-in thresholds (0.1%), serial testing was performed at the discretion of the attending clinician and was performed in only 53% of patients with myocardial injury or infarction. It is likely that those patients undergoing serial testing differed from those who had a single test performed; however, our comparison between patients with type 1 and type 2 myocardial infarction and those with myocardial injury was limited to the subgroup of patients with ≥2 tests performed within 12 hours of presentation. Cardiac troponin was measured with a single high-sensitivity cardiac troponin I assay, and we recognize that the performance of rule-in thresholds for myocardial infarction is likely to differ for other assays. Last, the approach to patient selection for cardiac troponin testing will vary across health care systems, and we recommend some caution in extrapolating the performance of rule-in thresholds to sites where testing is performed more widely.Although we observed important differences in the kinetics, cardiac troponin concentrations at presentation are insufficient to distinguish type 1 myocardial infarction from other causes of myocardial injury or infarction in practice. Clinical context may be more helpful than any rule-in threshold for guiding initial triage and management decisions.R.W., F.S.A., P.O.C., D.M.K., A.R.C., and N.L.M. conceived the study and its design. R.W., D.M.K., and A.R.C. had access to the data and performed the analysis. R.W., D.M.K., A.R.C., and N.L.M. interpreted the data and drafted the article. All authors revised the article critically for important intellectual content and provided their final approval of the version to be published. All authors are accountable for the work. The authors thank the High-STEACS Investigators for their contributions to the conception or design of the work, or the acquisition, analysis, or interpretation of data for the work.The High-STEACS trial was funded by a Special Project Grant from the British Heart Foundation (SP/12/10/29922). Dr Wereski, Dr Bularga, and D. Doudesis are supported by Clinical Research Training Fellowships  and a PhD studentship (MR/N013166/1) from the Medical Research Council. Dr Lee is supported by a Clinical Research Training Fellowship (FS/18/25/33454) from the British Heart Foundation. Dr Kimenai is supported by a grant from Health Data Research UK, which receives its funding from Health Data Research UK Ltd (HDR-5012) funded by the UK Medical Research Council, Engineering and Physical Sciences Research Council, Economic and Social Research Council, Department of Health and Social Care (England), Chief Scientist Office of the Scottish Government Health and Social Care Directorates, Health and Social Care Research and Development Division (Welsh Government), Public Health Agency (Northern Ireland), British Heart Foundation, and Wellcome Trust. Dr Chapman receives support from a Starter Grant for Clinical Lecturers by the Academy of Medical Sciences (SGL021/1075). Dr Mills is supported by the Butler British Heart Foundation Senior Clinical Research Fellowship (FS/16/14/32023) and a Program Grant (RG/20/10/34966) and Research Excellence Award (RE/18/5/34216) from the British Heart Foundation. Abbott Laboratories provided cardiac troponin assay reagents, calibrators, and controls without charge. The funders played no role in the design and conduct of the study; collection, management, analysis, and interpretation of the data; preparation, review, or approval of the manuscript; and decision to submit the manuscript for publication.Dr Apple reports research grants awarded to the Minneapolis Medical Research Foundation from Abbott Diagnostics, Siemens Diagnostics, Ortho-Clinical Diagnostics, and Beckman Coulter outside the submitted work and personal fees from HyTest Ltd. Dr Mills has received honoraria from Abbott Diagnostics, Siemens Healthineers, Roche Diagnostics, and LumiraDx, and the University of Edinburgh has received research grants from Abbott Diagnostics and Siemens Healthineers. The other authors report no conflicts.Expanded MethodsSupplemental Figures I and IISupplemental Table I


Oligomycin, an OXPHOS inhibitor, substantially attenuated the migration and invasion potential of breast cancer cells. Our study provides new insights into the pathogenesis of BCBMs.There is an urgent need to improve our understanding of breast cancer brain metastases (BCBMs). Thus, we obtained transcriptome data of BCBMs, primary breast cancers (BCs), and extracranial metastases (BCEMs) from the Gene Expression Omnibus (GEO) database, including GSE43837, GSE14017, and GSE14018, for immune and metabolic analysis. Firstly, we performed immune and metabolic analysis on BCBMs and primary breast cancers of GSE43837 using RNA sequence. We identified significant immunosuppression and gene signatures associated with immune infiltration in BCBMs; the lower the expression of the signatures, the worse the prognosis of breast cancer patients in the Kaplan–Meier (KM) plotter [Breast cancer] database. We also identified increased oxidative phosphorylation (OXPHOS) utilization in BCBMs compared with BCs and gene signatures associated with increased OXPHOS utilization in BCBMs; the higher the expression of the signatures, the worse the prognosis of breast cancer patients in the KM plotter [Breast cancer] database, which can predict the prognosis of breast cancer patients better, as it can also predict the prognosis of patients with different breast cancer subtypes. In addition, we performed immune and metabolic analysis on BCBMs and extracranial metastases of GSE14017 and GSE14018 using RNA sequence. Compared with extracranial metastases, we identified more significant immunosuppression but no difference in OXPHOS utilization in BCBMs, which may be because OXPHOS was also involved in extracranial metastases. We have proven that OXPHOS was functionally significant in metastasis Our study reports the most comprehensive gene expression analysis of BCBMs, BCs and extracranial metastases to date. We identified immunosuppression and OXPHOS enrichment in BCBMs compared with BCs, which provide new insights into the pathogenesis of BCBMs and will facilitate the development of new therapeutic strategies for patients with BCBMs. Breast cancer is one of the most common causes of brain metastases , 2. BraiIn the past, the brain was considered an organ with immune privilege. However, many studies have shown that this immune privilege is not absolute, but relative to the immune privilege of other organs . The desThere is growing evidence that BCBMs possess different molecular characteristics compared with primary tumors and extracranial metastases. Other investigators’ whole exome sequencing study has detected the mutational signatures indicative of HRD scores increased in BCBMs compared with patient-matched primary tumors . PreviouHowever, there is no comprehensive immune and metabolic analysis on BCBMs, primary tumors, and extracranial metastases. This may be the reason why no significantly enriched pathways have been identified. In general, the mechanism of BCBMs is still unclear and needs to be further explored.To address this urgent need, we collected gene expression profiles of BCBMs, BCs, and extracranial metastases from the GEO database: GSE43837 contained 19 BCBMs and 19 patient-unmatched BCs, GSE14017 contained 15 BCBMs and 14 extracranial metastases, and GSE14018 contained 7 BCBMs and 29 extracranial metastases. Together with functional assays on human breast cancer cell line (MDA-MB-231 cells), our study identified unique immune and metabolic features of BCBMs, which may contribute to develop new rational therapeutic strategies.https://www.ncbi.nlm.nih.gov/geo/), including GSE43837, GSE14017, and GSE14018  and Microenvironment Cell Populations-Counter (MCP-counter) R package to characterize immune infiltration in samples. ESTIMATE can infer the proportion of immune cells and stromal cells in tumor samples using gene expression . Howeverβ = 6. Then, the gene set was divided into several modules with similar expression patterns. Module–trait associations referred to the correlation between the module eigengene and the immune infiltration.WGCNA R package were used to construct a weight co-expression network associated with immune infiltration . First, p-values or false discovery rate (FDR) was determined by the default Benjamini–Hochberg (BH) correction in edgeR. For selecting the top features in a dataset, FDR < 0.05 and fold change (FC) > 1.5 were set as the cutoff criteria.Differentially expressed genes (DEGs) in different groups were identified using edgeR package . Specifihttps://david.ncifcrf.gov/summary.jsp) database integrates biological data and functional annotation tools to provide systematic and comprehensive biological function annotations for large-scale gene or protein lists. It was used to identify enriched Gene Ontology (GO) terms and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways of DEGs  v6.8 . Specific genes were indicated in a priori defined set of genes has statistically significant difference in expression under two different biological conditions (http://software.broadinstitude.org/gsea/msigdb/index.jsp). The normalized enrichment score (NES) represented the degree of enriched KEGG pathways in cluster of interest. p-values corresponding to each NES were determined by the Fisher’s exact test  in GSEA. Adjusted p-values were determined by BH correction in GSEA. For selecting significant pathways, FDR < 0.25 was set as the cutoff criteria.GSEA determines whether an nditions . GSEA soWe crossed core genes in OXPHOS enrichment in interested cluster determined by GSEA with DEGs to obtain signatures related to OXPHOS enrichment in the cluster of interest. The gene expression values for those signatures were then averaged to form the OXPHOS metagene (OP-metagene). Specific genes were indicated in http://kmplot.com/analysis/index.php?p=background)  were also displayed on all KM plots.Kaplan–Meier (KM) plotter [Breast cancer] is an online survival analysis tool that can assess the prognostic function of 22,277 genes in breast cancer patients using microarray data (kground) . All KM MDA-MB-231 (human breast cancer cell line) cells were purchased from Procell Life Science & Technology Co. Ltd. MDA-MB-231 cells were cultured in Dulbecco’s modified Eagle’s medium  supplemented with 10% fetal bovine serum  and 1% penicillin–streptomycin  in a 95% humidified incubator containing 5% CO2 at 37°C.3) were seeded on 96-well plates. After the cells adhered to the wall, the cells were treated with 1.0 µM oligomycin [Oligo(1.0)] and incubated in a 5% CO2 incubator at 37°C; 10 µl Cell Counting Kit-8  solution was then added into each well at 0 h, 12 h, 24 h, and 48 h, respectively, and cultured for 2 h. Next, the 96-well plates were put on the enzyme-linked immunoassay instrument and shaken for 2 s. The absorbance was measured at 460 nm. The growth rate was calculated as follows: Growth rate of Control = ABS)/mean, Growth rate of Oligo(1.0) = ABS − mean)/mean.Cell proliferation assay. MDA-MB-231 cells (4 × 10−/PI− cells was used to represent the cell viability.Cell apoptosis assay. Annexin V and propidium iodide (PI)  were used to stain the cells cultured in medium. FSC-H and SSC-H of flow cytometry were used to detect single cells. The percentage of annexin V6) were seeded on six-well plates. When the cell confluence reached 95%, the fused cells were scratched along the pore diameter with a sterile 200-µl pipette tip and then washed five times with PBS to remove floating cells and debris. The medium in each well was replaced with serum-free medium containing 1.0 µM oligomycin. The wound healing was observed at 0 and 48 h, and photos were taken under a microscope.Scratch assay. MDA-MB-231 cells . After 24 h, the invaded cells in the middle and four surrounding fields were counted and photographed under a microscope. The average number of cells in the five fields was used as the number of migrated and invaded cells.After starvation in serum-free medium for 6 h, cells were digested with 0.25% trypsin. The cell density was then adjusted to 2 × 10We used edgeR package to identify DEGs between BCBMs and BCs of GSE43837. A total of 539 DEGs were identified, of which 394 protein-coding genes were upregulated and 145 protein-coding genes were downregulated in BCBMs compared with BCs, respectively . ESTIMATE is a tool used to infer tumor purity and immune infiltration from gene-expression data that were originally validated in 11 cancer types . Howeverβ = 6  as the soft-thresholding power  R package to search for genes related to the immune infiltration of GSE43837. WGCNA R package is an effective tool that can be used to mine hub modules with similar expression patterns related to clinical traits . To builp = 5.3e−06), OS , and DMFS  with lower expression of IM-metagene in patients with breast cancer  directly, some gene information will be lost, which may result in missing significant biological and metabolic pathways. Therefore, we performed GSEA in the GSE43837 dataset. GSEA does not require a fixed threshold to filter genes. It is a method based on all-gene expression analysis and avoids the shortcomings of traditional enrichment analysis methods. Because there were not many DEGs in BCBMs compared with BCs, if the threshold was set to FDR < 0.05, a lot of GO terms will be missed. Therefore, we set the screening conditions as To obtain the core signatures related to OXPHOS enrichment of BCBMs, we crossed DEGs with 49 core genes in OXPHOS enrichment in BCBMs compared with BCs determined by GSEA (p < 1e−16), OS , and DMFS  with higher expression of OP-metagene in breast cancers (p = 2.7e−10), luminal B , basal-like , and HER2+   , we compp = 4.064e−05; p = 0.0202; p < 0.05), Cytotoxic lymphocytes (p < 0.05), Monocytic lineage (p < 0.01), and Neutrophils (p < 0.05) infiltration decreased significantly in BCBMs compared with BCEMs of GSE14017 (p < 0.01), Cytotoxic lymphocytes (p < 0.001), Myeloid dendritic cells (p < 0.01), and Neutrophils (p < 0.001) infiltration in BCBMs compared with BCEMs of GSE14018 F(o)ATPase, to inhibit OXPHOS in MDA-MB-231 cells. Schematic diagram of experimental plan for determining the effect of oligomycin treatment on MDA-MB-231 cells was presented in Although the treatments in BCs have been greatly improved, its outcome is not ideal. The drug resistance of BCs and the incidence of brain metastasis are gradually increasing . TherefoWe found significant immunosuppression in BCBMs compared with primary tumors using RNA sequence, a finding also observed by other investigators using IHC . We idenin situ to lung metastasis . However, we did not detect significant difference in the RNA expression of PGC1A between BCBMs and BCs, which has been shown to mediate mitochondrial biosynthesis and OXPHOS in cancer cells to promote metastasis . That retastasis . It has tastasis . Some drtastasis ; CSC acqtastasis . Human etastasis . In supptastasis  and had OXPHOS can promote metastatic seeding in a variety of ways. OXPHOS may induce epithelial–mesenchymal transition progression of cancer cells , 44. Thein vitro assays. Together, we identified immunosuppression and enrichment of OXPHOS in BCBMs compared with BCs, which provides ideas for the development of more reasonable treatment strategies for patients with BCBMs. Our results suggest that immunotherapy combined with chemotherapy, radiotherapy, and/or OXPHOS inhibitors may improve the prognosis of patients with BCBMs.In conclusion, our study identified immunosuppression in BCBMs compared with BCs and extracranial metastases using RNA sequence and an IM-metagene that can be used as a prognostic indicator of breast cancer patients in the KM plotter [Breast cancer] database. We also identified OXPHOS enrichment in BCBMs compared with nonmetastatic primary tumors using RNA sequence and an OP-metagene that can better predict the prognosis of patients with breast cancer than IM-metagene, as it can predict the prognosis of patients with various subtypes of breast cancer in the KM plotter [Breast cancer] database. However, we did not identify a significant difference in OXPHOS utilization in BCBMs compared with extracranial metastases, which may be because the increased utilization of OXPHOS not only is unique to BCBMs, but also plays an important role in extracranial metastases , 39, 45.However, our study has some limitations. We did not verify our findings in animal experiments. We did not further link our findings to DNA alterations, which play a pivotal role in the clinical administration of BC patients, because we are unable to collect valid DNA sequence at present. We did not clarify whether immunosuppression simply represents a response to the brain microenvironment or is involved in the whole cascade process of brain metastases. We did not clarify the relationship between OXPHOS and immunosuppression in BCBMs. That will be the focus of our future efforts.The authors declare that all data supporting the findings of this study are available within the article and its LX: Conceptualization, methodology, data curation, writing—original draft preparation, visualization, investigation, supervision, software, validation, and writing—reviewing and editing. JZ: Co-designing and performing the experiments. HL, YZ, and WC: Reagents and materials. YLZ and WC: Project administration and funding acquisition. All authors contributed to the article and approved the submitted version.Natural Science Foundation of China: 81770294.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 determination of Human Chorionic Gonadotropin (hCG) and Alpha Fetoprotein (AFP) levels on serum and amniotic fluid plays a fundamental role in the diagnosis and follow-up of specific physiological or pathological conditions . Recently, the quantification of hCG and AFP in other biological fluids has gained great attention to support the diagnosis, prognosis and follow-up of neoplastic diseases deriving from trophoblastic cells, such as germinomas. Most of the commercial kits for hCG and AFP assays are developed to be used on biological fluids such as serum/plasma and/or urine by manufacturing companies. The aim of this work was to evaluate the suitability of the analytical method certified for the use on serum, and/or amniotic fluid for the quantification of hCG and AFP in cerebrospinal fluid, carrying out an internal validation protocol. The data reported here show that the automated immunochemical method is fit for quantification of hCG and AFP in cerebrospinal fluid (CSF), allowing selective and specific diagnosis of secreting germ cell tumors. This is confirmed by the positive correlation between elevated levels of hCG or AFP and the diagnosis of brain tumors. The determination of the levels of alpha-fetoprotein (AFP) and Human Chorionic Gonadotropin (hCG) on serum and amniotic fluid plays a fundamental role in the diagnosis, prognosis and follow-up of specific physiological or pathological conditions such as pregnancy, threat of abortion and germ cell tumors (GCTs). Recently, there has been great interest in the possibility of determining the values of hCG and AFP in other organic fluids such as cerebrospinal (CSF) ,2.GCTs are a heterogeneous group of cancers in terms of age of onset, location, clinical presentation and prognosis, and represent 2–3% of pediatric malignancies [Germ cell tumors (GCTs) of the central nervous system (CNS) are classified according to the World Health Organization (WHO) into pure germinomas and non-germinomatous germ cell tumors (NGGCTs) . The preWhen the suspicion of an intracranial GCT arises, the increase of serum or CSF markers could precede the radiological finding by several months, resulting in a high predictive/diagnostic power . The relMost of the commercial kits for hCG and AFP assays are developed to be used on biological fluids such as serum and/or amniotic liquid by manufacturing companies. The aim of this work was to evaluate whether the analytical method already used in the Clinical Biochemistry Laboratory of the Bambino Gesù Children’s Hospital for the determination of hCG, an AFP on established biological fluids, could also be used on CSF. As matrix effects can influence test results when alternative sample types are used, and alternative sample types should always be validated before clinical use, we carried out the CSF assay of both AFP and hCG validation using an internal validation protocol according to the Eurachem Guide .® XPT Immunoassay System  for AFP and hCG determinations in CSF samples and serum. The diagnostic kits used in this study to determine the concentrations of AFP and hCG were a kind gift from the Siemens Healthineers Diagnostics . The AFP and hCG assays are a two-site sandwich immunoassay based on direct chemiluminometric technology, which uses constant amounts of two antibodies. The first antibody for the AFP assay is a rabbit polyclonal anti-AFP affinity purified antibody labeled with acridinium ester, and the second is a mouse monoclonal anti-AFP antibody covalently conjugated to paramagnetic particles. For hCG assay, the first antibody is an anti-hCG goat polyclonal purified and labeled with acridinium ester, and the second is a purified mouse monoclonal anti-hCG antibody, conjugated covalently to paramagnetic particles. These two antibodies are specific for different epitopes present on the free ß-subunit and on the ß-subunit of the intact hCG.We used the ADVIA CentaurThe validation study was performed using 370 leftover CSF samples sent to the Clinical Biochemistry Laboratory of the Bambino Gesù Children’s Hospital for physical and chemical evaluation. In particular, the CFS samples used in this study were selected based on medical records showing no documented history of blood–brain breakdown or CNS infection. Clear and colorless CSF samples with a leukocyte count ≤5 and normal values of proteins, glucose and chloride, and levels of AFP and hCG comparable with those found in HPLC-grade water  were pooled and used as blank matrix. We collected the leftover CSF samples obtained from 41 patients aged from 3 months to 20 years presented to the Bambino Gesù Children’s Hospital with radiological diagnosis of brain lesion suspected for GCT, and we analyze both hCG and AFP. We performed hCG and AFP assays on the serum from the same patients before and after the CSF collection, in order to compare the results for diagnostic and prognostic–therapeutic purposes. In addition, the medical records of each patient were examined for the evaluation of the cytological/histological or radiological data that could be useful for patient diagnosis and follow-up.® XPT Immunoassay System, previously validated only for serum and/or amniotic fluid specimens. In this study, we validated its use for CSF samples. CSF hCG and AFP assays were validated through evaluation of linearity [hCG and AFP concentration were measured using ADVIA Centaurinearity , limit oinearity .Linearity was studied according to IUPAC guidelines . One insntration  and chect-test (95% confidence level) for n − 1 degree of freedom [Limit of detection (LOD) and limit of quantification (LOQ) were calculated by analysing ten experimental blanks. LOD and LOQ were calculated with the following formula: freedom ,12. The For upper-end sensitivity studies, pooled CSF samples were spiked by serially diluted serum samples containing high amounts of AFP, obtaining CSF concentrations of 15, 45, and 125 ng/mL for AFP and 35, 125, and 600 mIU/mL for hCG.Precision was calculated in terms of repeatability (intraday precision) as relative standard deviation (RSD%) .Trueness was assessed from the recovery assay. In order to verify possible matrix effects as constant and proportional systematic errors, the recovery function over the entire working range was calculated . RecoverWhen both constant and proportional systematic errors are negligible, the calculated intercept (b0f) and slope (b1f) of recovery function should not significantly differ from 0 and 1, respectively, at a defined confidence level.The homogeneity of the variance was evaluated using the Levene test. The significance of the differences for CSF levels of AFP and hCG among the study groups was assessed by one way analysis of the variance (ANOVA) followed by Fisher’s least significant difference (LSD) post hoc test or Kruskal–Wallis test followed by multiple comparison of mean ranks. After mean centering, unit variance scaling, and log transformation of AFP and hCG concentrations, unsupervised hierarchical cluster analysis and supervised multivariate partial least squares-discriminant analysis (PLS-DA) were performed to explore possible clustering of the subjects based on CSF levels of AFP and hCG. Receiver operating characteristic (ROC) analysis and the calculation of the area under curve (AUC) were performed to assess the sensitivity and the specificity of AFP and hCG levels for the diagnosis of NGGCT. Statistical analysis was performed using Statistica 6.0  and MetaboAnalyst 5.0 module .p < 0.050, 95% confidence level), weighted least squares fitting was applied to calculate the calibration functions over the investigated concentration ranges. We used the following weighting factors: 1/X and 1/X2 where X is the concentration of the calibration standard. For both AFP and hCG, the variance test scores indicated that the weight 1/X2 gives the smallest spread of the weighted variances compared with 1/X (data not shown). Accordingly, the calculated RE percentage was lower when 1/X2 is used for both analytes compared with 1/X (data not shown). However, the weighted calibration functions calculated weighting for 1/X2 showed significant deviation from linearity for both AFP and hCG at 95% confidence level . Therefore, 1/X was chosen as the weighting factor. As shown in p < 0.001 for both AFP and hCG). Therefore, the Kruskal–Wallis test followed by multiple comparison of mean ranks was applied to evaluate the significance in the differences of AFP and hCG CSF levels between the study groups. n = 16) when compared to the other GCT patients  and patients affected by other cancer types . Furthermore, we found significantly higher levels of CSF hCG in NGGCT patients  when compared with the other study groups  of the central nervous system (CNS) are classified according to the World Health Organization (WHO) into pure germinomas and non-germinomatous germ cell tumors (NGGCTs) . CNS gerThe onset symptomatology of GCT is related to the localization of the tumor itself  . SometimDespite neuroradiological characteristics, in the absence of a clear bifocal localization and histological confirmation, diagnosis can be difficult and the assessment of both hCG and AFP CSF values would be fundamental. The presence of hCG and AFP in serum and/or CSF at levels above the maximum attended for age is diagnostic for NGGCTs and biopsy can be avoided. Yolk sac tumors can secrete AFP and choriocarcinomas can release hCG. Moreover, elevated AFP and hCG can sometimes occur with immature teratomas. Germinomas may be associated with mild increases in total hCG and may show elevated placental alkaline phosphatase (PLAP) . AFP levDifferentiating pure germinomas from NGGCTs is crucial because treatments and prognoses are quite different. Without a positive tumor marker result, a biopsy is required for a definitive diagnosis .The relative increase in hCG and AFP values at the serum and/or CSF level are also of fundamental importance not only for the diagnosis, but also for monitoring the response to treatments and the follow-up, even in the absence of histological data . This waIn our observational study aimed at evaluating the predictive value of AFP and hCG, we demonstrated that AFP provides a sensitivity and specificity of 90%, suggesting this biomarker is suitable for the diagnosis of NGGCT. Regarding hCG, we found a sensitivity of 50% and a specificity of 100%, indicating that this marker was able to correctly identify the subject not affected by NGGCT. The combination of the two biomarkers in a multivariate discriminant model could provide better sensitivity and specificity compared with that obtained using AFP or hCG alone, thus demonstrating how the combined use of hCG and AFP can represent valid support in the diagnosis and follow-up of NGGCT neoplastic diseases.The determination of markers in non-standard samples represents, from an analytical point of view, a critical issue for the laboratory and requires a complex process of validation of the analytical method in order to certify its suitability. The results should be evaluated together with the patient’s clinical history and other diagnostic tools to obtain the most appropriate diagnostic framework.


Our results suggest that controlling oxidation may be utilized to improve thermoelectric performance in nanostructures, and PO can be a promising candidate for low-dimensional thermoelectric devices.We performed density functional theory calculations to investigate the thermoelectric properties of phosphorene oxide (PO) expected to form by spontaneous oxidation of phosphorene. Since thermoelectric features by nature arise from the consequences of the electron-phonon interaction, we computed the phonon-mediated electron relaxation time, which was fed into the semiclassical Boltzmann transport equation to be solved for various thermoelectric-related quantities. It was found that PO exhibits superior thermoelectric performance compared with its pristine counterpart, which has been proposed to be a candidate for the use of future thermoelectric applications. We revealed that spontaneous oxidation of phosphorene leads to a significant enhancement in the thermoelectric properties of  Their thermoelectric efficiencies can be assessed at temperature T by a dimensionless figure of meritS, ZT value should concurrently possess incompatible characteristics: high electrical conduction with a high 1, antimony telluride 5, and copper selenide  materials have drawn much attention since, in general, they tend to have high values of Seebeck coefficient originating from the effect of the low-dimensionality, such as an abrupt change in the density of states (DOS)14, there have been several theoretical studies proposing that phosphorene would be a candidate material for thermoelectric applications18. Based on numerical calculations performed within the constant relaxation time approximation using the deformation potential theory, they reported that phosphorene, which is environmentally friendly or non-toxic, has not only a large Seebeck coefficient originating from its semiconductivity, but also highly anisotropic transport behaviors of high electrical and low thermal conductivities along a certain direction caused by its intrinsic puckered structure16. An explicit inclusion of electron-phonon interactions, however, revealed that those reported room-temperature ZT values were an order of magnitude overestimated19, implying that phosphorene may not be a suitable choice for thermoelectric applications. To make it worse, phosphorene is known to be structurally unstable under the atmospheric conditions and thus can be easily oxidized due to the lone pair electrons of each phosphorus atom24.After the discovery of a single layer of black phosphorus or phosphoreneZT values accurately, we explicitly evaluated the electron relaxation times n and electron wave vectors 24, we determined their thermoelectric-related characteristics. It was found that the p-type phosphorene exhibits better thermoelectricity along the armchair direction but worse along the zigzag direction than PO, which shows a much smaller directional dependence. Furthermore, we found that the n-type PO is thermoelectrically superior not only to the n-type phosphorene regardless of their directions but also to the p-type phosphorene. This result is attributed to a significant reduction in 24 albeit a small decrease in power factor ZT values of PO were estimated to range from 0.1 to 0.5 at n-doped PO would be a promising and eco-friendly thermoelectric material, and controlling the degree of oxidation may be a useful strategy to improve the thermoelectric properties of the material.Here we propose that spontaneous oxidation of phosphorene may be a resolution of the issues in phosphorene for a thermoelectric purpose. To correctly describe the thermoelectric properties of both phosphorene and PO or to calculate their mentclass2pt{minim24. Our previous studies24 showed that fully-oxidized phosphorene or PO possesses highly-anisotopic configuration similar to its pristine counterpart, as shown in Fig. 23. It was also shown that the flexible bonds between phosphorus and oxygen atoms in PO lead to a significant decrease in the lattice thermal conductivity 24. This reduction implies that PO would provide a favorable condition for thermoelectric performance.Various oxidation processes of phosphorene were reported earliern-type PO would be a better choice for a thermoelectric material than p-type PO, because the carrier relaxation time is closely associated with the spectral conductivity tensor To consider the effect of the electron–phonon interaction on transport properties such as entclass1pt{minimaentclass1pt{minimad in Eq. , and thup-type) and electron (n-type) mobilities, T = 300 K). These results are in good agreement with previously evaluated values25. For PO, on the other hand, the hole mobility We used the calculated relaxation time to evaluate the carrier mobility tensor ture T = 00 K. Theentclass1pt{minimature T = 00 K. TheZT given in Eq. . T. T\docummentclass2pt{minimZT. For example, phosphorene has 24, as shown in Fig. ZT value, due to a significant reduction of 24. Figure n-type PO reached T24. It is, thus, inevitable to consider the electronic contribution to the thermal conductivity for an accurate estimation of the ZT value of PO.In most semiconductors, the electronic contribution to the thermal conductivity ZT of PO and phosphorene for comparison. Figure ZT of p- and n-type phosphorene and PO along the zigzag and armchair transport directions computed with each particular value of hole and electron carrier density, with which their ZT values reached to its maximum at p-type phosphorene exhibits a better thermoelectric performance along the armchair direction than not only along the zigzag direction but also along any directions for the n-type counterpart, due mainly to its higher power factors shown in Fig. ZT value reaches ZT value of 24. Furthermore, the optimal electron doping concentration  was sampled using a k-points mesh for the integration over the BZ. Then, the phonon dispersion was calculated using the density functional perturbation theory (DFPT)34 with a q-points grid.To investigate thermoelectric properties of phosphorene and PO, we first calculated the electronic structures by carrying out first-principles calculations based on the density functional theoryn and 36m and T. 37. It is related to the probability amplitude of phonon-mediated scattering process from a state k- and q-points meshes and then using maximally localized Wannier functions40 through the EPW package35 to interpolate over k- and q-points grids.As a next step, we explicitly evaluated the energy- and temperature-dependent relaxation time 42. The computed electron relaxation times T defined byFor various transport-related quantities, we solved the semi-classical Boltzmann transport equationThese moments are directly related to various transport coefficients, such as electrical conductivity, Seebeck coefficient, and electronic thermal conductivity tensors, asSupplementary Information.


Spinal imaging is essential to identify and localize cerebrospinal fluid (CSF) leaks in spontaneous intracranial hypotension (SIH) patients when targeted treatment is necessary.Provide an in-depth presentation of the conventional dynamic myelography (CDM) technique for localizing spinal CSF leaks in SIH patients.Consecutive SIH patients with a CSF leak confirmed on CDM and postmyelography computed tomography (CT) investigated at our institution between 2013 and 2019 were retrospectively analyzed. Intraoperative reports were reviewed to confirm the accuracy of CDM.In total, 62 patients (mean age 45 years) were included; 48 with a ventral dural tear, 12 with a meningeal diverticulum, and in 2 patients positive for spinal longitudinal extradural CSF collection the site remained unclear. The leak was identified during the first and the second CDM in 43 and 17 patients, respectively. The use of CDM correctly identified the site of the CSF leak in all but one patient undergoing surgical closure . The mean fluoroscopy time was 7.8 min (range 1.8–14.4 min) with a radiation dose for a single examination of 310 mGy (range 28–1237 mGy).The CDM procedure has a high accuracy for spinal CSF leak localization including dural tears and spinal nerve diverticula. It is the technique with the highest temporal resolution, is robust to breathing artifacts, allows great flexibility regarding patient positioning, compares favorably to other dynamic examinations with respect to the radiation dose and does not require general anesthesia. For CSF venous fistulas, however, other dynamic examinations, such as digital subtraction myelography, seem more appropriate.The online version of this article contains 4 videos. The article and the videos are online available (10.1007/s00062-020-00943-w). The videos can be found in the article back matter as “Electronic Supplementary Material”. Spontaneous intracranial hypotension (SIH) is a well-recognized disorder usually presenting with disabling orthostatic headache that manifests within minutes after assuming the upright position and subsides after lying down. Schievink et al. proposed three types of spontaneous spinal CSF leaks: type 1) the dural tear, type 2) the meningeal diverticulum and type 3) the CSF-venous fistula [To overcome this difficulty methods with high temporal resolution, which can pinpoint the level of spinal dural breach, such as conventional dynamic myelography (CDM) , digitalThe goal of our study was therefore to review the role of CDM in SIH patients and provide a detailed description of the application of this technique to confirm and localize a spinal CSF leak.This study was granted institutional review board approval, and the need for informed consent was waived owing to its retrospective nature. The registry was approved by the local ethics committee.All consecutive SIH patients investigated at our institution between February 2013 and January 2019 with positive spinal imaging (CSF leak identified on CDM and PMCT) were retrospectively reviewed. No additional exclusion criteria were applied.Subgroups of patients have previously been included in other studies, which have investigated different outcome measures, including optic nerve sheath ultrasonography, surgical details of dural closure, CSF dynamics, and brain MRI, spine MRI, but which did not report on the imaging aspects of CDM in particular , 10–15.A detailed medical history was obtained, then physical examination, optic nerve sheath ultrasonography, and lumbar infusion testing were performed , 11. ForThe CDM was performed on a monoplane high-resolution angiographic system  equipped with a flat panel detector (30 × 40 cm) by two board certified neuroradiologists . To facilitate orientation during CDM and postinterventional image analysis, a small radiopaque skin marker was taped to the patient’s back just off the midsagittal plane, at the level of the most suspicious lesion , considered by the operator as potentially responsible for the leak.Lumbar puncture was performed with the patient in a sitting position and, after intrathecal injection of 20 ml Iopamiro 300 , the needle was withdrawn. Since its specific gravity is higher than that of CSF, layering of contrast material in the dependent portion of the thecal sac occurs. To prevent false positive results due to contrast leakage at the level of the lumbar puncture, which may occur during needle withdrawal  and both arms were extended above the head. The table was progressively tilted into the Trendelenburg position (approximately 30–45°) to facilitate intrathecal caudocranial flow of the injected contrast medium  was suspected, a Valsalva maneuver was performed .  to identify possible causative pathology at the level of dural dehiscence. If no epidural contrast was identified in the first PMCT, a late phase PMCT was performed in general 4–12 h after initial intrathecal injection to exclude low flow leaks.If epidural contrast accumulation was demonstrated on PMCT, but after review of all fluoroscopic sequences the precise site of the leak remained undetermined, the procedure was repeated if the performing neuroradiologist considered that a specific area could be better assessed on a second run. The re-examination was usually performed on the following day (>24 h) to allow for resorption of intrathecal contrast material. The technique was adjusted according to the results of the previous examination . If the neuroradiologist believed that owing to overprojection of bony structures usually present at the cervicothoracic junction this specific area should be reassessed, a DCTM was performed as DSM was not part of our routine diagnostic work-up during the study period.For all patients with a confirmed leak on CDM/PMCT undergoing microsurgical closure of the dural dehiscence, intraoperative reports were reviewed. This was done to confirm the presence of epidural CSF and verify the level of dural dehiscence, since operative exploration remains the gold standard. In patients managed conservatively or with epidural blood patching, two neuroradiologists (E.I.P and T.D) established the standard of reference through the combination of multimodal imaging , since the sensitivity of each individual method is not clear. When a disagreement between different imaging modalities was identified, the finding was reviewed together to reach a consensus.2). Intraoperative reports were reviewed to confirm the presence of epidural CSF and verify the level of dural dehiscence. Descriptive statistics were calculated using Microsoft Excel 2016. Continuous variables were described as a mean ± standard deviation (SD) and categorical variables as frequencies (percentage).All imaging studies were reviewed independently on a picture archiving and communication system (PACS) station  by two board-certified neuroradiologists (E.I.P and K.P). Precise location of the CSF leak as demonstrated on CDM, and the underlying pathology as seen on PMCT, were recorded. Conflicts were resolved by consensus. Demographic data including patient age, and sex were recorded. Fluoroscopy-specific data reported included patient positioning, number of CDM examinations, and periprocedural complications. Patient radiation dose was approximated by indirect measures of radiation output from the angiographic system including fluoroscopy time (FT), cumulative air kerma (CAK in mGy), and kerma area product . The mean brain SIH score was 6.6 (SD ± 2.23), indicating a high probability of a spinal CSF leak. The mean number of CDM examinations per patient was 2 (range 1–4) with a mean FT of 7.8 min (range 1.8–14.4 min). The radiation dose for a single examination was 310 mGy (range 28–1237 mGy). The leak was identified during the first CDM examination in 43, and during the second CDM in 17 patients.The site of CSF leakage was at the cervical, thoracic, and lumbar levels in 4, 49, and 7 patients, respectively. In two patients positive for spinal longitudinal extradural CSF collection on PMCT, the level of leakage remained unclear. These were investigated at the beginning of the study period and were not repeated due to symptom improvement. A ventral dural tear was identified in 48 patients  and a leaking meningeal diverticulum in 12 patients. In 45 out of 46 patients undergoing microsurgical closure, CDM correctly identified the leakage site, in 1 patient the leakage site was one level off. One patient who underwent two CDM examinations on the following day (>24 h) had transient aphasia after the second examination, most likely an adverse reaction to intrathecal contrast medium administration: the brain MRI did not reveal any suspicious findings. No other serious periprocedural complications were reported.In 19 other patients not included in this study, in whom CDM was insufficient for precise localization of the CSF leak either due to superimposed bony structures of the shoulder and rib cage or low-flow leaks, dynamic CT myelography was performed for leak localization and have been previously published .A 54-year-old female patient presented with orthostatic headache that she had suffered for 20 weeks. Sagittal T2-weighted fat-suppressed spine MRI demonstrated a CSF collection in the epidural space spanning several levels T1–T10). The CDM was performed with the patient in the prone position, focusing on these levels. During progressive table tilting, immediate contrast spillage into the ventral epidural compartment at the level of T7/8 occurred, confirming a type 1 leak according to Schievink et al.  or meningeal diverticula (type 2), contrast leakage into the epidural space occurs almost instantaneously, spanning several vertebral levels within a few seconds [ or meninConcerning other underlying mechanisms of SIH, as reported by Kranz et al. CSF-venous fistulas type 3) can be detected with both CTM and dynamic myelography  can be d; howeverWhen reporting PMCT and GdM, knowledge about the level of the lumbar puncture is essential to prevent false positive results, which may lead to confusion and unnecessary follow-up examinations. Extrathecal contrast in the epidural space confined to the level of the puncture site and the adjacent 1–2 vertebral levels should be considered iatrogenic. In patients referred for persisting post-dural puncture headache , lumbar puncture should be performed below the level of the previous puncture in order to distinguish between them Fig. .Fig. 7MiThe major strength of our study is a meticulous description of the CDM technique, including useful illustrations of the workflow in patients with epidural CSF collection due to a dural breach. In addition, typical case examples are presented, which are accompanied by images and videos. In the majority of patients the leakage site was confirmed by microsurgical exploration, which underlines the accuracy of CDM. The limitation of this technique is the fact that is not reliable to demonstrate CSF-venous fistulas (type 3) as well as the retrospective and single center design which are inherent to the study design.The conventional dynamic myelography provides a real-time demonstration of contrast flow necessary to identify dural leaks and leaking meningeal diverticula, is robust to breathing artifacts, and does not require general anesthesia. When performed meticulously it is a valuable technique with a high accuracy for prospective identification of the site of cerebrospinal fluid leakage, and the radiation dose is lower than for digital subtraction myelography and dynamic CT myelography.Video 1: middle-aged female with known migraine (same as in Fig. Video 2: middle-aged patient with sudden onset of orthostatic headache (same as in Fig. Video 3: middle-aged female with occipital headache and vertigo she has been suffering for 1 year (same as in Fig. Video 4: middle-aged female with sudden onset of orthostatic headache after a mild trauma (same as in Fig.


Traumatic optic neuropathy (TON) may cause severe visual loss following direct or indirect head trauma which may result in optic nerve injuries and therefore contribute to the subsequent loss of retinal ganglion cells by inflammatory mediators and reactive oxygen species (ROS). Granulocyte colony-stimulating factor (G-CSF) provides the anti-inflammatory and anti-oxidative actions but has a short half-life and also induces leukocytosis upon typical systemic administration. The purpose of the present study was to investigate the relationship between the anti-oxidative response and neuroprotective effects of long-acting pegylated human G-CSF (PEG-G-CSF) in a rat model of optic nerve crush (ONC). Adult male Wistar rats (150–180 g) were chosen to have a sham operation in one eye and have ONC in the other. PEG-G-CSF or phosphate-buffered saline (PBS control) was immediately administered after ONC by intravitreal injection (IVI). We found the IVI of PEG-G-CSF does not induce systemic leukocytosis, but increases survival of RGCs and preserves the visual function after ONC. TUNEL assays showed fewer apoptotic cells in the retina in the PEG-G-CSF-treated eyes. The number of sorely ED1-positive cells was attenuated at the lesion site in the PEG-G-CSF-treated eyes. Immunoblotting showed up-regulation of p-Akt1, Nrf2, Sirt3, and HO-1 in the ON of the PEG-G-CSF-treated eyes. Our results demonstrated that one IVI of long-acting PEG-G-CSF is neuroprotective in the rONC. PEG-G-CSF activates the p-Akt1/Nrf2/Sirt3 and the p-Akt1/Nrf2/HO-1 axes to provide the antioxidative action and further attenuated RGC apoptosis and neuroinflammation. This provides crucial preclinical information for the development of alternative therapy with IVI of PEG-G-CSF in TON. Traumatic optic neuropathy (TON) refers to any insult to the optic nerve secondary to direct or indirect head or ocular injury . TON is The reactive oxygen species (ROS) produced mainly by mitochondria are required to maintain normal physiological functions, including gene expression, signal transduction, cell proliferation, and host defense . ExcessiWe previously demonstrated that systemic administration of granulocyte colony-stimulating factor (G-CSF), an old drug used to recruit the hematopoietic progenitors from bone marrow to peripheral blood, has neuroprotective effects not only in a rat model of anterior ischemic optic neuropathy (rAION) ,18,19,20The systemic administration of human recombinant G-CSF needs repeated injections for its’ short circular half-life (3.5–3.8 h) , and sysOur current study aimed to investigate the safety of IVI of PEG-G-CSF and its effect on prolonged neuroprotection. Furthermore, we wanted to examine the role of PEG-G-CSF in oxidative stress in the rat ONC model.All animal care and surgical procedures were approved by the Institutional Animal Care and Use Committee at Tzu Chi Medical Center and managed in accordance with the Association for Research in Vision and Ophthalmology Statement for the Use of Animals in Ophthalmic and Vision Research. Male Wistar rats  used in this study were around 150–180 g in weight. The rats were maintained under a controlled 12-h shift of the light-dark cycles and had free access to food and water in a controlled environment with a constant temperature of 23 °C and constant humidity of 55%. All operations were performed with the animals under general anesthesia, which was achieved by intramuscular administration of a mixture of ketamine (100 mg/kg body weight) and xylazine .A total of 108 rats (6/group/time point) were used in this study. 36 rats received sham operations. 72 rats were randomized into two equal groups after ONC in the right eye. The first group received IVI of 5 µL phosphate-buffered saline (PBS), and the second group received IVI of 5 µL PEG-G-CSF alone in the operated eyes one hour after ONC. All animals survived until the end of the experiment without developing any complications. Post-operation day 3, the optic nerve samples were collected for Western blot analysis. Post-operation day 7, peripheral blood samples were collected for white blood cell (WBC) counts. Post-operation day 14, visual function was assessed by performing flash visual-evoked potential (fVEP) analysis; the RGC density was measured by retrograde FluoroGold labeling. In situ TdT-dUTP nick end-labeling (TUNEL) assays in the RGC layer and immunohistochemistry of injured sites in the ON were also conducted. A brief summary of the study is illustrated in ONC injury was induced as described in our previous reports ,21,22,30Pegfilgrastim  is a pegylated human colony-stimulating factor (PEG-G-CSF) produced by genetic recombination technology . It work2, a single flash with a flash rate of 1.02 Hz, and a test average of 64 sweeps. The first positive wavelet was defined as the P1 component, and the first negative wavelet was defined as the N1 component. The peak-to-peak amplitudes of P1-N2 waves were measured and compared among the groups (n = 6 in each group) to evaluate visual function.The procedure has been described in detail in our previously published reports ,23,30,31Blood was drawn from cardiac puncture into the heparinized coated tubes by using a 23-gauge needle. The blood samples were collected for complete blood count (CBC) analysis on day 0 and day 7 later after different treatments. All manipulations were performed with animals under general anesthesia. Total WBCs were counted by the Cellometer K2 automated cell counter  .2) in the central region of each retina were obtained to retrieve the mean RGC densities in the central retina (n = 6 in each group).The detailed steps have been described in our previous reports ,23,30,31The TUNEL assays were performed under the protocol provided by the manufacturer  to detect apoptotic cells. All the frozen retinal sections were collected at 1–2 mm anterior to the optic nerve head for further comparison. Ten high-powered fields  were selected randomly to count the TUNEL-positive cells in the RGC layer of each sample. Then the average was calculated for further comparison.The ectodysplasin 1 (ED1) antibody reacted against extrinsic macrophages ,32,33, wThe ON samples were collected on day 3 from each group. The protein extracts of the ON were separated by using a 4 to 12% NuPAGE Bis-Tris gel . The separated proteins were transferred onto polyvinylidene difluoride membranes. The membranes were blocked by using 5% nonfat milk in Tris-buffered saline/Tween-20 solution containing 20 mM Tris-HCl (pH 7.5), 0.5 M NaCl, and 0.5% Tween-20. Subsequently, the membranes were blotted with either anti-p-AKT , anti-nuclear factor erythroid 2–related factor 2 , anti-sirtuin 3 , or anti-heme oxygenase 1  antibody overnight at 4 °C. The membranes were incubated with a secondary antibody conjugated to horseradish peroxidase against the appropriate host species for one hour at room temperature. The developing reaction was performed by using an enhanced chemiluminescent substrate . Thermofisher’s iBright Analysis software was used for the analysis. Precise protein bands were processed for intensity (background subtracted). The results obtained were normalized against that of GAPDH. The statistical analysis was carried out using Mann–Whitney U test.p-values less than 0.05 were considered statistically significant.Every measurement was conducted in a single-blind fashion in this study. The Mann–Whitney U test was used to evaluate the differences among groups in all experiments. Data are presented as the mean ± standard deviation (SD). All statistical analyses were performed using the SPSS statistical software program . p = 0.72).We first measured the change of fVEP to evaluate visual function two weeks after IVI of PEG-G-CSF. The amplitude of the P1–N2 wavelet can indicate the RGC function in vivo. In this study, we determined the amplitude of the P1–N2 wavelet in the normal Wistar rat, which was 68.61 ± 7.19 μV. The amplitude of the P1–N2 waves in the PEG-G-CSF-treated group was 64.85 ± 9.43 μV . There w3/mL in the sham group on day 0, while it was 7.5 ± 1.5 × 103/mL seven days later after sham operation. The WBC counts were slightly elevated in the PEG-GCSF-treated sham group (9.4 ± 0.2 × 103/mL), in the ONC group with no treatment (6.8 ± 2.3 × 103/mL), and the PEG-GCSF-treated ONC group (8.9 ± 2.4 × 103/mL). However, there was no significant difference in peripheral WBC counts between ONC groups with or without IVI of PEG-GCSF . IVI of PEG-G-CSF did not induce significant leukocytosis in the injured animals.The peripheral WBC count was checked on day 0 and day 7 after treatment . The nump < 0.05; fVEP was arranged to evaluate the visual function of these experimental rats in the sham group, the PBS-treated, and the PEG-G-CSF-treated ONC groups A. The P12, 686.4 ± 245.5/mm2, and 1181.5 ± 167.5/mm2, respectively (p < 0.05). These apoptotic cells in the RGC layers in the sham group, the PBS- and the PEG-G-CSF- treated groups were 0.8 ± 0.3/HPF, 22.4 ± 4.1/HPF, and 7.3 ± 2.6/HPF, respectively (p < 0.05) when compared with treatment with PBS.The PEG-G-CSF-treated group preserved a higher density of RGCs in the central retinas than the PBS-treated group A. Two weectively B. The nuectively A,B. Treap < 0.05). The optic nerve is composed of retinal ganglion cell axons and glial cells which are critical to its functional integrity and homeostasis through maintenance of functional interactions among glia and the axons [p < 0.05)  C.p < 0.01) (p < 0.05)  B upper.  < 0.05) B bottom.Our previous studies showed that G-CSF has neuroprotective effects on RGCs survival after ONC, in both subcutaneous administration and IVI approach . The subG-CSF is a cytokine and hormone that stimulates the bone marrow to make granulocytes and progenitor cells and recruit them into the peripheral blood . It is aG-CSF was proved to have neuroprotective effects not only in the rat model of anterior ischemic optic neuropathy ,18,19,20Neuroinflammation is a double-edged sword with both neurotoxic and neuroprotective effects in animal models of traumatic spinal cord injury (SCI) ,54,55,56Sutherland et al. reported that the higher proportion and numbers of classically activated macrophages and microglia in the rat SCI model may contribute to a more inflammatory microenvironment and lead to further injury . These pFurthermore, an animal study of SCI demonstrated that subcutaneous administration of G-CSF could recruit microglia to the injury site in the first 72 h after spinal cord injury, inhibit the expression of pro-inflammatory factors and promote the expression of neurotrophic factors. G-CSF also increases the expression of markers of M2 macrophage and inhibits the expression of markers of M1 macrophage in vitro, favoring the M2 polarization via the NFκB signal pathway . Many puCansler and Evanson made a brief commentary on the roles of endoplasmic reticulum (ER) and oxidative stress for the degeneration seen in retinal degeneration, traumatic brain injury (TBI), and TON recently . NeutropFurthermore, the inflammatory process begins within hours of the insult after CNS damage and lasts for weeks . RecombiHowever, the main limitation of this study was that we did not evaluate nuclear-fractionated samples to better evaluate the activation of the Nrf2 pathway. The subcellular fractionation approach is an indispensable tool to facilitate the study of specific intracellular events and the characterization of protein functions . Nrf2 isIn summary, we found that IVI of PEG-G-CSF has neuroprotection in a rat model of ONC via anti-inflammation, anti-apoptosis, and anti-oxidation, especially the anti-oxidative stress reaction system involving the p-Akt1/Nrf2/HO-1 and the p-Akt1/Nrf2/Sirt3 axes.


The resulting accumulation of dermatan and heparan sulfate induces intellectual disabilities and pre-mature death, and only a few treatment options are available. In a previous study, we demonstrated the feasibility, safety, and efficacy of gene therapy by injecting recombinant adeno-associated viral vector serotype (AAV)2/5-IDUA into the brain of a canine model of MPS I. We report on a quantitative proteomic analysis of control dogs and untreated dogs with MPS I cerebrospinal fluid (CSF) that had been collected throughout the study in the MPS I dogs. Mass spectrometry (MS) analysis identified numerous proteins present at altered levels in MPS I CSF samples. Quantitative immunoblotting, performed on CSF from healthy controls, untreated MPS I dogs, and MPS I dogs early treated and late treated by gene therapy, confirmed the MS data for a subset of proteins with higher abundance . Scoring of the results shows that the expression levels of these proteins are close to those of the control group for dogs that underwent gene therapy early in life but not for older treated animals. Our results disclose four novel predictive biomarker candidates that might be valuable in monitoring the course of the neurological disease in MPS patients at diagnosis, during clinical follow-up, and after treatment.Mucopolysaccharidosis type I (MPS I) is caused by a deficiency of the lysosomal hydroxylase alpha- The disorder is usually diagnosed before the age of 5 years. The most severely affected children show profound intellectual disabilities before the age of 10, and the progression of MPS I results in multiple disabilities. When performed in early childhood, bone marrow transplantation and hematopoietic stem cell transplantation are somewhat effective in patients with MPS I or II  biomarkers of disease severity, disease progression/regression, and response to treatment. Although neurological diseases are most prevalent in the elderly, they also affect children with inherited metabolic diseases. In particular, lysosomal storage disorders (LSDs) result in intellectual disabilities and pre-mature death . The muc IDUA, EC.2.1.76, www.clinicaltrials.gov). However, reliable brain-related biomarkers are still lacking for MPS I and other types of MPS but would be very helpful for monitoring the efficacy of these novel therapies.These promising results prompted us to test this treatment in patients with MPS IIIB in a phase I/II study . Indeed,In this context, the primary objective of the present study was to identify individual proteins or specific combinations thereof in the CSF, the presence or concentration of which would specifically reflect the MPS I disease stage. We addressed this goal by analyzing CSF samples that had been collected throughout the study in the MPS I dogs. Samples from healthy control dogs and untreated MPS I dogs underwent an unbiased proteomic analysis. Quantitative mass spectrometry (MS) revealed several potential biomarkers of disease progression. These candidates might be useful in monitoring brain lesions in patients with MPS I and other types of MPS at diagnosis, during clinical follow-up, and—as a guide to therapeutic effectiveness—after treatment.CSF samples used in this work had been collected throughout the study in the MPS I dogs, and all animal experiments have been described and published previously . Full de2/day) and a weekly-adjusted dose of cyclosporine to maintain cyclosporinemia above 300 ng/ml) and the pathology marker are summarized in The investigated dog groups, the individual dog reference, the age at treatment, the immunosuppressive treatment (combination of mycophenolate mofetil (800 mg/mSamples of CSF (0.5–1 ml) from healthy control dogs and untreated, early-treated, and late-treated MPS I were collected, centrifuged within one hour, and stored at −80°C in polypropylene tubes , in accordance with the international biobanking consensus guidelines optimized for CSF proteomics .The proteomic analysis has been described previously , 8. BrieWhole gel lanes were cut into slices of equal size (1 mm), and the excised gel slices were subjected to protein digestion for subsequent MS. During in-gel tryptic digestion in the presence of either 18O1 and 18O2 isotope labeling were considered possible modifications. Based on decoy database searches, the false-positive rate was estimated to be below 1%. Protein quantifications were accomplished using the Mascot Distiller Quantitation Toolbox , based on calculations of isotope intensity ratios of at least two tryptic peptides with individual MASCOT scores indicating homology. Relative protein ratios were calculated from the intensity-weighted average of all peptide ratios.The nanoLC-MS/MS analyses were performed on a LTQ-Orbitrap XL mass spectrometer  equipped with a reverse-phase capillary liquid chromatography system . The MASCOT server  and the Proteome Discoverer 2.4 (Thermo Scientific) were used to search the Swiss-Prot /TrEMBL (dog) database (release 2020_02). A maximum of two missed cleavages was allowed, and mass tolerance of precursor and fragment ions was set to 10 ppm and 0.5 Da, respectively. Acrylamide or carbamidomethyl modification of cysteine, methionine oxidation, acetylation , and C-terminal The level of IDUA activity was assayed as described previously ; the methttps://imagej.nih.gov/ij/index.html, National Institutes of Health, Bethesda, MD/USA).As suggested for CSF , total pn = 3) from each of the four groups: healthy controls, untreated MPS I dogs, and early-treated and late-treated MPS I dogs. As expected, lack of IDUA activity was detected in CSF from MPS I dogs, whereas CSF from healthy controls exhibited high levels of IDUA activity  MPS I dogs were analyzed to confirm the higher abundance of these proteins and to check whether gene therapy normalized the respective protein levels in the CSF of MPS I-treated dogs. Special consideration was given to the exact determination of each sample's protein concentration, a pre-condition for the use of total protein as a protein standard for CSF  Supplem. All selUsing the immunoblot of each individual dog's CSF sample, the relative concentrations of NP1, Ch3L1, CD14, and IGFBP2 proteins were quantified , and an www.clinicaltrials.gov).Biomarker research is an expanding, emerging field which constitutes an important aspect in the development of 3P  medicine . HoweverCerebrospinal fluid is close to the disease site in neurodegenerative disorders, providing several advantages as a source of biomarkers compared with more distal fluids such as plasma , 15. TheN-acetylglucosaminidase enzymatic activity in CSF was 15–20% of that in unaffected children  at D30 (in the early-treated dogs), while the overall score for the four proteins at D30 is 1.4—much closer to control values. These observations confirm the higher adequacy of a set of biomarkers for assessing the course of the disease.Considering the lack of reliable brain-related biomarkers of MPS and ongoing clinical trials, this study demonstrates that the determination of the CSF levels of NP1, Ch3L1, CD14, IGFBP2, possibly also of other upregulated proteins identified here could be helpful for assessing the course of the disease in infants having received gene therapy. Further validation of these biomarker candidates, e.g., by investigating the correlation between CSF and plasma/serum levels in MPS patients, may offer a less-invasive means of routine monitoring.Datasets are available on request: data supporting the conclusions of this article will be made available by the authors (RH), without undue reservation.The animal study was reviewed and approved by The Institutional Animal Care and Use Committee of the National Veterinary School of Nantes and the University of Nantes approved experiments, which were performed by authorized investigators.RB and CG performed the biochemical experiments. EK and MS performed the mass spectrometry experiments. MS and RH analyzed MS data. J-MH and IB conceived and designed the study. RH, ST, and JA conceived and designed the experiments, interpreted the data, and wrote the manuscript. All authors read and approved the final manuscript.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.


Orchiectomy is the gold standard for managing testicular tumors. According to the American Urological Association guideline, patients with a suspected malignant testicular lesion and a normal contralateral testis should undergo radical inguinal orchiectomy, instead of testis‐sparing surgery (TSS).Sertoliform cystadenoma is a very rare benign tumor originating from the rete testis. Ultrasound findings show a cystic mass in many cases, but sometimes a solid mass is observed with an epididymal cyst.In this case, the tumor was judged to be benign because there was no change over a long period, and the authors successfully performed TSS.The author declares no conflict of interest.