Cerebral injury during donation after brain death may induce systemic damage affecting long-term kidney function posttransplantation. Conventional evaluation of donor organ quality as a triage for transplantation is of limited utility.We compared donor kidneys yielding opposing extremes of the continuum of posttransplantation outcomes by several common kidney biopsy evaluation techniques, including Kidney Donor Profile Index and Remuzzi scoring, and analyzed tissue from a minimal sample cohort using label-free quantitation mass spectrometry. Further assessment of the proteomic results was performed by orthogonal quantitative comparisons of selected key proteins by immunoblotting.We show that common evaluation techniques of kidney biopsies were not predictive for posttransplantation outcomes. In contrast, despite the limited cohort size, the proteomic analysis was able to clearly differentiate between kidneys yielding extreme posttransplantation outcome differences. Pathway analysis of the proteomic data suggested that outcome-related variance in protein abundance associated with profibrotic, apoptosis, and antioxidant proteins. Immunoblotting confirmation further supported this observation.We present preliminary data indicating that there is scope for existing evaluation approaches to be supplemented by the analysis of proteomic differences. Furthermore, the observed outcome-related variance in a limited cohort was supported by immunoblotting and is consistent with mechanisms previously implicated in the development of injury and cytoprotection in kidney transplantation. Proteomic analyses using mass spectrometry and immunoblot of a small cohort of kidneys from brain death donors is superior to predict graft function (estimated glomerular filtration rate at 3 and 12 months) versus clinical and histological standard classifications. Supplemental digital content is available in the text. In response, the deceased donor pool has been expanded to include higher-risk donors who are older or have comorbidities.4 Donors older than 60 years now represent almost 40% of the donation after brain death (DBD) donors in the United Kingdom.5 Kidneys obtained from this “extended criteria” category have suboptimal transplant outcomes when compared with those from “standard criteria” DBD or living donors.7Organ transplantation is a life-saving and life-transforming treatment of patients with end-stage organ disease. There is a persistent shortage of deceased donor organs; in the United Kingdom, between April 2016 and March 2017, 457 patients on the waiting list died and 875 became too ill to receive a transplant.9 Improving the discriminatory power of the diagnostic tools available to clinicians could help increase deceased donor organ utilization. This is a major goal of the UK Quality in Organ Donation (QUOD) biobank, a recently established nationwide project to collect longitudinal blood, urine, and biopsy samples from deceased donors and link these samples to demographic and clinical data for both donor and recipient.10The decision to use or reject a donor organ is made at the clinical level based on an evaluation of organ quality. Current methods of kidney assessment combine surrogate markers of kidney function during donor management, known risk factors such as donor age, and histological evaluation. These assessments have a high degree of subjectivity and are imperfect predictors of posttransplantation organ performance. Clinicians are thus conservative in accepting organs from “high-risk” donors. In consequence, many useable kidneys may be declined as transplants.14 In this study, we applied proteomic analysis to a minimal cohort of QUOD biobank samples to investigate whether is feasible to identify a proteomic profile of donor kidneys that may add predictive value to current assessment methods of donor kidney quality with regard to posttransplantation outcome, which would justify a subsequent large-cohort study using many tissue samples. To this end, we compared the kidney proteomes of donor groups with clearly defined extreme posttransplantation outcomes , matching as many clinical and demographic parameters as possible, to assess whether observable proteomic differences were able to discriminate between outcomes. The observed differences in this limited-cohort comparison were verified for the biological relevance of implicated pathways by selective immunoblotting.The evolution and application of mass spectrometry (MS) techniques in medical research, including in the field of transplantation, has allowed for monitoring and near simultaneous analysis of thousands of proteins and has led to clinically relevant findings.The QUOD biobank is a national bioresource that houses an extensive repository of samples from deceased donors obtained at specific time points according to predefined collection protocols during donor management and organ procurement throughout the United Kingdom. Donor samples are linked to corresponding donor and recipient demographic and clinical data. Appropriate informed consent by the donor families precedes sample collection. All kidney biopsy samples analyzed in this study were procured from donors after brain death and obtained from the UK QUOD biobank, under the ethical approval of the QUOD project 13/NW/0017. Kidney biopsies were obtained ex situ from the upper pole of kidney cortex during preparation at the back table using a 23 mm needle biopsy gun. Each biopsy specimen was divided in two; one half was stored in RNAlater followed by subsequent storage in liquid nitrogen and the other half in formalin.15 The Kidney Donor Profile Index (KDPI) was calculated using donor age, height, weight, serum creatinine at retrieval, hepatitis C virus status, history of hypertension, diabetes, cause of death and Donation after Circulatory Death category using an online calculator16 . The incidence of acute kidney injury in the donors was also assessed using Acute Kidney Injury Network (AKIN) scoring, by calculating the fold change between the levels of terminal and the baseline serum creatinine at donor admission in intensive care17. A fold change of less than 1.5 was defined as no AKIN, a fold change of 1.5 to 2 as AKIN-1, a fold change of 2 to 3 as AKIN-2, and a fold change of more than 3 as AKIN-3.Estimated glomerular filtration rate (eGFR) was calculated using the four-variable Modification of Diet in Renal Disease formula and is expressed in milliliters per minute, adjusted for body surface area.Figure S1A, SDC, http://links.lww.com/TP/B599). To reduce the impact of recipient-related variation on outcomes, only instances where both kidneys from a donor yielded similar transplant outcomes were considered. On this basis, two experimental groups were formed; suboptimal (SO) and good outcome (GO) . To select the eligible donors per group, we set an upper eGFR limit for SO (eGFR ≤39 mL/min/1.73 m2) and lower eGFR limit for GO (eGFR ≥ 50 mL/min per 1.73 m2) using data from the UK Transplant Registry, published yearly, as a guide.18 Good outcome donor kidneys functioned immediately after transplantation and had a mean 3-month eGFR of 65.2 ± 8 mL/min per 1.73 m2 . The SO donor kidneys developed delayed graft function  after transplantation and had a mean 3-month eGFR of 29.8 ± 7 mL/min per 1.73 m2 .Biopsy samples were selected from donors on the basis of the 3-month posttransplantation outcomes of the pairs of kidney recipients from the same donor   was significant different  were dewaxed, rehydrated, and stained with 0.5% periodic acid for 5 minutes, rinsed with distilled water, and then placed in Schiff reagent for 15 minutes followed by 5 minutes washing in tap water. The slides were counterstained with Mayer hematoxylin and washed in tap water for 5 minutes.Baseline donor chronic kidney injury was assessed retrospectively using the formalin stored, paraffin-embedded part of the core needle biopsy. Biopsies were evaluated by an experienced histopathologist (blinded to the donor characteristics) and scored for chronic histopathological changes according to the classification system introduced by Remuzzi and colleagues.Figure S1B, SDC, http://links.lww.com/TP/B599). Sample size was selected such that relatively substantial changes (twofold or more) were predicted be measured reliably (setting power at 0.8 and confidence threshold at 0.05), assuming a combined technical and biological variation of 50%, whereas minimizing the number of biological replicates and thus tissue sample usage. The selected minimum cohort subset size of five per condition was informed by previously published estimation of experimental power in quantitative proteomics.20 The rest of the donor samples  were used for immunoblotting analysis  that was performed to verify observed outcome-related variance between the two groups SO versus GO .A subgroup of donor samples was randomly selected from the overall cohort of 38 donor kidney biopsies for proteomic analysis  followed by alkylation with 200 mM iodoacetamide for 30 minutes at room temperature. Trypsin digestion was performed overnight at 37°C with gentle mixing using a 1:50 trypsin:protein ratio. Samples were acidified with 1% formic acid or trifluoroacetic acid. Peptide digests were then desalted using Sep-Pak C18 cartridges (Waters) and dried by Speed Vac centrifugation. Pellets were re suspended in 30 μL of buffer A  before MS analysis. Peptides were analyzed in duplicates using a C18 column on a Dionex Ultimate 3000 nano-ultrahigh-performance liquid chromatography system  coupled to a Q Exactive mass spectrometer . The MS data were processed and identified proteins quantified using the Central Proteomics Facilities Pipeline.22 Normalized Spectral INdex Quantitation values, were calculated for each protein.23Kidney cortical biopsy samples from 10 donors were lysed in 300 μL of RIPA buffer  containing protease  and phosphatase inhibitor cocktails . Homogenization was performed on a bead beater at 6500 rpm for 3 cycles of 40 seconds each with intermediate 1 minute on ice between cycles. Label-free quantitation MS analysis was performed as previously described.2 or Fisher exact test for discrete variables and to define whether the association of donor organs to the transplantation outcomes is independent of the listed variables as listed in Table Differences in demographic and clinical characteristics of donors and their corresponding kidney recipients between SO and GO groups were examined by Mann-Whitney test for continuous variables (mean ± standard deviation [SD]) and by χP < = 0.05, t test, no multiple-testing correction) and identification in at least 3 of 5 analyzed samples per group were further analyzed in a supervised manner using hierarchical clustering using the PermutMatrix software.24 Missing abundance values were imputed for each protein by the mean abundance of that protein within each condition. Dissimilarity between the donors was assessed by single linkage (closest neighbor linkage) for columns and rows.A nonsupervised analysis of proteomic changes by Principle Component Analysis was performed and the first two principle component dimensions visualized using R (v3.4.2). Proteins with differential levels of abundance between the nonmissing values in the two subgroups  29.8 ± 7 ml/min/1.73 mP = 0.65, Mann-Whitney test). The 25th percentile KDPI values were 58% and 47.7% for SO and GO, respectively, while the 75th percentile KDPI values were identical for both experimental cohorts at 87.5%  . The low P value does suggest that the Remuzzi comparison lacked power, resulting in a false negative, although this would still imply that the scoring system is insufficiently sensitive. Assessing the onset of acute kidney injury during donor management using AKIN classification revealed that the majority of donors did not show indications of acute kidney injury with 85% of the donor kidneys with SO and 74% with GO classified as no AKIN; again, these results did not associate with outcome . We identified and quantified 1743 unique proteins at a false discovery rate (FDR) of <=1%. Unsupervised principle component analysis of this data set demonstrated that the two groups were clearly resolved by the first principle component , demonstrating substantial merit for prediction of transplant outcome by proteomic methods  from the full cohort for proteomic profiling of major fold changes  and presence in at least 3 of 5 samples per group gave a subset of 214 proteins. Hierarchical clustering analysis using this subset easily segregated the donor kidneys by transplant outcome. Comparing this clustering to long-term outcome as measured by 12-month eGFR illustrated the close association between the proteins with significant differences and long-term kidney function . Thresholding by 25 of this 214-protein subset suggested enrichment of cellular metabolic processes that included cellular response to stress  and cell surface receptor signaling pathways  in SO versus GO, and enrichment of reactive oxygen species (ROS) detoxification  in GO versus SO. Among the predominant pathways (via Kyoto Encyclopedia of Genes and Genomes26 database) were metabolic dysregulation  and tight junction molecules .We examined the set of proteins with significant statistical differences between the two outcomes to verify the overarching trends. Pathway analysis via STRINGFigure S1B, SDC, http://links.lww.com/TP/B599). In SO versus GO, we were able to reproduce the observation of significantly increased expression of the apoptotic signal transduction protein STAT-1, observed in proteomics data with sevenfold SO/GO (P = 0.03) and by immunoblot with P = 0.02 . The proteomics data also suggested a role for a profibrotic response, with TGF-β1 . We were unable to quantify TGF-β1 confidently due to technical noise; however, immunoblotting of PDGFRα (which has a synergetic role in the onset and propagation of fibrosis) did indicate elevation (P = 0.01) . In GO versus SO, we were able to observe enrichment of several notable antioxidant and cytoprotective proteins by Western blot, including thioredoxin-1 (P = 0.005), glutathione S-transferase (P = 0.02), and peroxiredoxin-3 (P = 0.04) . Interestingly, catalase was not observed to be enriched by either the proteomic analysis (P > 0.05) or immunoblotting (P > 0.05) .As a further assessment of the proteomic results, we performed orthogonal quantitative comparisons of selected key proteins by immunoblotting  were consistent with follow-up immunoblotting and are biologically plausible in the context of previously reported studies. As we and others have previously reported, brain death leads to dysregulation of metabolic pathways and mitochondrial dysfunction that results in ROS formation, causing cellular injury in the kidney.Our pilot study results indicate that the current assessment methods of donor kidney quality may be augmented by proteomic profiles that indicate how the balance between injury and cytoprotection is disrupted and how this is associated with long-term allograft function. Now that feasibility has been established, future work to identify a predictive protein profile can be performed in a much larger cohort with a broader continuum of outcomes.


The nucleic acid mutation status in intracranial metastasis is markedly significant clinically. The goal of the current study was to explore whether the tumor-associated mutations can be detected by different next-generation sequencing (NGS) pipelines in paired cerebrospinal fluid (CSF) and plasma samples from lung adenocarcinoma (LAC) patients with leptomeningeal metastases (LM).Paired CSF cell free DNA (cfDNA), CSF cells, plasma and formalin-fixed and paraffin-embedded (FFPE) samples of primary tumors were collected from 29 LAC patients with LM to detect the mutations by different NGS pipelines.DNA libraries were generated successfully for 79 various samples in total for NGS sequencing, of which mutations were detected in 7 plasma samples (24.14%), 12 CSF cfDNA samples (66.67%), and 10 CSF cells (76.9%) samples. For the 26 patients with detected mutations, 8/26(30.77%) had mutations in plasma, which was significantly lower than that those from CSF cfDNA , CSF cells  and FFPE samples . When the input DNA of CSF cells was less than 20 ng, the cHOPE pipeline of NGS identified the most mutations for epidermal growth factor receptor (EGFR).NGS-based detection of mutations in cfDNA or cells from CSF provided more information than from plasma samples from LAC patients with LM. In addition, the cHOPE pipeline performed better than the other three NGS pipelines when input DNA from CSF cells was low.The online version of this article (10.1186/s12885-019-5348-3) contains supplementary material, which is available to authorized users. Lung cancer is one of the most common malignant tumor in the world. Leptomeningeal metastasis which predicts poor prognosis occurs in about 5% lung cancer patients . GenomicIt was reported that tumor-associated DNA could be detected in the CSF samples of cancer patients with central nervous system (CNS) tumor using NGS sequencing , 7. PrevIn this study, different detection pipelines were utilized for CSF cfDNA, cells, plasma and FFPE samples of primary lung cancer to identify sequence mutations. It aimed to find out whether CSF as liquid biopsy samples in a clinical setting can be substantiated and can be considered as an excellent substitute to primary cancer tissue.Twenty-nine pathologically confirmed lung cancer patients with leptomeningeal metastases were enrolled in this cohort Table  between About 10 ml of CSF, 10 ml blood and FFPE primary tumor tissues were collected from the enrolled patients. Two ml of CSF was used for cytological examination, and the remainder was centrifuged at 1600 g for 15 min at 4 °C to separate the supernatant and cell pellets. Blood samples were immediately centrifuged at 1600 g for 15 min at 4 °C after being sampled from patients. The supernatant was collected into a new tube for another centrifuge at 16,000 g for 10 min at 4 °C, and the second supernatant was collected as plasma samples. The supernatant of CSF (CSF cfDNA), the cell pellets of CSF (CSF cells), and the plasma were stored at − 80 °C until DNA extractions.cfDNA from CSF supernatant and plasma were extracted using the QIAamp Circulating Nucleic Acid Kit . DNA from the cell pellets of CSF were extracted using the QIAamp DNA Mini Kit . DNA from the FFPE tissues were extracted using the QIAamp DNA FFPE Tissue Kit . All the DNA extraction experiments were handled strictly in accordance with the corresponding manufacturer’s instructions. DNA was quantified using the Qubit Fluorometer .For NGS sequencing, DNA input should not be less than 30 ng and the majority of fragments were located between 150 and 180 bp for DNA from the CSF supernatant and the plasma; while DNA input should not be less than 20 ng and the majority of fragments were located above 500 bp for DNA from CSF cell pellets and the FFPE tissues.Different pipelines of NGS library preparation and sequencing were supplied by Singlera Genomics, Inc. Shanghai, China, for different types of samples. ddCAP pipeline was used for libraries prepared from plasma samples and CSF supernatant samples. OncoAim, cHOPE, or ddCAP-on-tissue pipelines were used for CSF cell pellets samples. OncoAim pipeline was used for FFPE samples. DNA library preparation and NGS sequencing were performed according to manufacturer’s recommended protocols. Generally, target regions were enriched by either targeted captureor multiple PCR 150 bp paired-end sequencing was performed on Illumina platforms including NextSeq 500, Hiseq 4000, MiSeq, or MiniSeq . Features of these NGS pipelines were showed in Table The CSF cells sample in patient #12 was analyzed by drops digital PCR (ddPCR) for the mutation of EGFR E746_A750del, which showed conflicting statuses between the results of cHOPE and ddCAP-on-tissue pipelines. The ddPCR experiment was conducted using the QX200TM ddPCR system , and ddPCR Supermix and primer/probes for EGFR from BioRad were used.Bioinformatical analyses were performed for results from each pipeline. Briefly, sequencing reads were quality-filtered, assembled and aligned against the reference genome hg19/GRCh37. Unique reads derived from GATK were used for variant calling. The minimum confidence threshold for variant and insertion/deletion (indel) calling was set to 0.05 (5%). Variation annotation, effect prediction and clinical practice guidance were integrated into the pipelines through vcf files.p-value of less than 0.05. Column graphs were generated using Graphpad Prism (version 5.0a) software.Comparisons among the detection rates of plasma, CSF cfDNA, CSF cells, and FFPE samples were performed by Fisher’s exact test using SPSS v19.0 software. Significance was assumed for a Twenty-nine patients with an average age of 55 (range from 35 to 73) were enrolled in our study, of which 18 were female and 11 were male. All the enrolled patients had LAC. Patients’ information was shown in Table A total of 29 plasma samples were collected, and the input DNA for library preparation ranged from 13 ng to 150 ng. Mutations were detected in only 7/29 (24.14%) plasma samples. NGS library of CSF cfDNA were generated for 18 patients with input DNA ranging from 9.5 ng to 50.5 ng. Mutations were detected in 12 of 18 (66.67%) CSF cfDNA samples.We used different panels based on the quantity of DNA we extracted from the 13 CFS cell samples, and in 10/13(76.9%) samples we identified positive mutations. Samples having over 50 ng extracted DNA could be sequenced using all available pipelines, including ddCAP-on-Tissue, which was specialized for FFPEs sample in this study.When the input DNA was less than 20 ng, the cHOPE pipeline was capable of identifying the largest amount of mutations. Indeed, seven individuals’ CSF-cell samples were analyzed using both cHOPE and a non-cHOPE pipeline. Among them 4 individuals  had more mutations detected by cHOPE than the non-cHOPE pipelines. Two individuals (#2 and #9) had identical mutations identified by the two pipelines. The remainder (#6) was shown to have two mutations in EGFR, E746Valfs and P753Rfs, based on cHOPE pipeline, whereas a complex deletion was identified by OncoAim. In summary, mutation discoveries in CFS cells samples may yield different results due to different detection panels.In the CSF-cell sample from patient #12, conflicting results were obtained from 2 different NGS pipelines Table . EGFR E7Most mutations detected in this study were located in the genes EGFR and TP53. Mutations detected in the plasma and CSF samples were also detected in the FFPE samples except the ALK G689R  and KRAS Q61L (CSF cfDNA of #9).P < 0.05, Fig. P = 0.622).In all 29 patients, 12 (41.38%) patients showed same results between at least two different types of samples. In the 16 patients with 3–4 types of samples, only 4 (25%) showed identical results among various samples . No mutation was detected in the plasma, CSF or FFPE samples of patient #3, #8 and #16  without matching CSF cfDNA sample had a mutation of EGFR E746_A750del, which was in agreement to the EGFR 19del identified in the primary tumor by the ARMS technology  of different sample types in an ascending order were plasma, CSF cells, FFPE tissues and CSF cfDNA, which were 13.19% , 20.18% , 42.72%  and 49.62% , respectively. , CSF cfDNA (2.5~12 ml), CSF cells (0.2~2.6 ml) and FFPE sample. The input DNA for cHOPE pipeline was as low as about 5 ng. Both cHOPE and OncoAim pipelines utilized multiple PCR as enrich technology to detect tumor-associated hotspots. However, they were designed for different sample types, of which cHOPE was specialized for cfDNA while OncoAim was for DNA from FFPE sample. The other 2 NGS pipelines, ddCAP and ddCAP-on-Tissue, used target capture as enrichment technology to scan all the exons of 10 tumor-associated genes. The input DNA of ddCAP pipeline for cfDNA could be as low as 5 ng as well, whereas 50 ng DNA was required for the ddCAP-on-Tissue pipeline for DNA from FFPE sample.In most cases that had discrepancies between CSF and plasma, CSF had more mutations detected , 8, whicOf the 19 patients who provided both plasma and CSF samples, 13 of them had mutations identified in CSF samples . Meanwhile, only 4 out of the 19 plasma samples had mutations identified, all of which were also discovered in the matched CSF samples. Previous research indicated that positive detection of EGFR was more sensitive in CSF than in the matched plasma , 10. OurIt was reported that more mutations could be detected in CSF cfDNA than in the paired CSF cells . Due to Most mutations detected in CSF samples could be found in FFPE samples except for ALK G689R in 2 individuals and KRAS Q61L in 1 individual, demonstrating that most tumors in the brain originated from the primary lung cancer. Because these 3 CSF-only mutations had low AFs, it is unlikely that they are highly instructive in the clinical treatment in the LAC patients with LM; additionally, these mutations were likely to have emerged after the metastasis of the tumor. Although the ALK G689R nutation is labeled as a variant of undetermined significance (VUS) at present, alterations in ALK gene are involved in many malignancies including lung cancer and could mediate acquired resistance to some ALK inhibitor , 12. KRANGS based detection of CSF samples of both cfDNA and cells can provide more gene information than plasma samples from LAC patients with LM. In addition, the cHOPE pipeline performed better than the other three NGS pipelines when low amount of input DNA from CSF cells was used.Additional file 1:Figure S1. Result of EGFR E746_A750del in CSF cells of Patient #12 by ddPCR system. Y-axis means the signals of each PCR amplification, X-axis means the PCR events. Evens above the cutoff threshold represent the mutated DNA in the sample tested. (TIF 1324 kb)


The surface electromyography (sEMG)-based gesture recognition with deep learning approach plays an increasingly important role in human-computer interaction. Existing deep learning architectures are mainly based on Convolutional Neural Network (CNN) architecture which captures spatial information of electromyogram signal. Motivated by the sequential nature of electromyogram signal, we propose an attention-based hybrid CNN and RNN (CNN-RNN) architecture to better capture temporal properties of electromyogram signal for gesture recognition problem. Moreover, we present a new sEMG image representation method based on a traditional feature vector which enables deep learning architectures to extract implicit correlations between different channels for sparse multi-channel electromyogram signal. Extensive experiments on five sEMG benchmark databases show that the proposed method outperforms all reported state-of-the-art methods on both sparse multi-channel and high-density sEMG databases. To compare with the existing works, we set the window length to 200ms for NinaProDB1 and NinaProDB2, and 150ms for BioPatRec sub-database, CapgMyo sub-database, and csl-hdemg databases. The recognition accuracies of the aforementioned benchmark databases are 87.0%, 82.2%, 94.1%, 99.7% and 94.5%, which are 9.2%, 3.5%, 1.2%, 0.2% and 5.2% higher than the state-of-the-art performance, respectively. Firstly, we review related works on sEMG-based gesture recognition methods, hybrid CNN and RNN architectures and the attention mechanism. Secondly, we introduce the proposed attention-based hybrid CNN-RNN architecture for sEMG-based gesture recognition, and describe the details of the new feature vector based sEMG image representation methods. Thirdly, we show the experimental results on five benchmark sEMG databases. Finally, we draw the conclusion and discuss our future work.In this section, we present related works on sEMG-based gesture recognition methods, the hybrid CNN-RNN architectures and attention mechanism in the literature.et al. [et al. [et al. [The handcrafted features and traditional machine learning classifiers have been extensively researched in early sEMG-based gesture recognition frameworks. Existing sEMG-based handcrafted features can be divided into three categories : time doet al.  utilized [et al.  applied  [et al.  used HMMet al. [et al. [et al. [et al. [The CNN architecture is the most widely used deep learning technique for sEMG-based gesture recognition, which can be divided into two categories based on different evaluation methods. The first study focuses on improving recognition accuracy of intra-session evaluation , 26. Atzet al.  construc [et al. , 27. Par [et al.  draw ada [et al.  extracteet al. [et al. [et al. [The RNN architecture has been applied for sEMG-based hand problems, such as pose estimation , 44 and  [et al.  applied  [et al.  exploiteet al. [et al. [et al. [The hybrid CNN-RNN architecture has obtained good performance in recognition of video and wearable sensors. Ebrahimi Kahou et al.  presente [et al.  designed [et al.  proposed [et al.  recommenet al. [et al. [et al. [et al. [The attention mechanism has been injected into RNN architectures for performance enhancement in many application scenarios. Dzmitry et al.  proposed [et al.  introduc [et al.  presente [et al.  providedAs mentioned above, the hybrid CNN-RNN architecture has been successfully applied to activity recognition based on video and wearable sensors. The attention mechanism is also an effective way to enhance the performance of RNN architecture.Since the electromyogram signal is noisier than other wearable sensor signals , we extretc. Therefore, we focus on how to embed the attention mechanism into the classification model and accordingly propose a novel attention-based hybrid CNN-RNN architecture for sEMG-based gesture recognition  is the number of subsegments and also time steps of RNN. Each subsegment Xt, ∀t = 1, 2, …, T has N frames with the size of 1 × C. Then, the Xt is converted into an image It with size (N × W × H), where W × H = C, W and H are width and height of the image, respectively. The detailed image representation method can be seen in section. Given the converted images {I1, I2, …, IT}, CNN is applied as feature extractor to transform them into feature vectors {F1, F2, …, FT}. The CNN model consists of seven layers. The first two layers are convolutional layers with 64 3 × 3 kernels, followed by two locally-connected layers. The locally-connected layer with 64 1 × 1 kernels is employed to extract local features of the sEMG image. Batch normalization [G-way fully-connected layer and a softmax classifier. G is the number of gestures to be recognized. The final label is decided by average-pooling of the softmax outputs.Each sample recorded from lization  is used F1, F2, …, FT} (feature vectors extracted from CNN model), the hidden states ht and outputs yt can be calculated as follows:Wih, Whh, Who are weight matrices between input, hidden and output layers. As standard RNN suffers from gradient vanishing or exploding problem, long short-term memory (LSTM) [δ is the logistic sigmoid function and i, f, o and c are input, forget, ouput gate and cell activation.The recurrent neural network contains feedback loops and encodes contextual information of a temporal sequence. Given the input sequence {y (LSTM)  has beenht is the output of the t-th hidden unit of RNN module, αt is the t-th attention weight, Wh and wT are weighted matrices and r is the output of attention module. The output r is followed by a G-way fully-connected layer and a softmax classifier.An attention layer  is emploα, β and λ are three weight parameters.X is the electromyogram signal to be recognized, y is the ground-truth label, and T is the number of time steps of RNN. G is the number of gestures to recognize, g1(X)i is the i-th dimension of g1(X) and 1i is the indicator function. fh, fa and fs stand for the hybrid CNN-RNN architecture, attention module and the last softmax layer, respectively.Xt is the t-th subsegment of X and g2(Xt)i is the i-th dimension of g2(Xt). fh and fs stand for the hybrid CNN-RNN architecture and the last softmax layer.The loss function of attention-based hybrid CNN-RNN architecture is:ion loss  and the Existing sEMG databases can be divided into two categories: sparse multi-channel sEMG database  and highL × W × H, where L is the number of frames, W is rows of the array electrode and H is columns of the array electrode. The raw signal is converted into a sEMG image with size L × W × H, where L is the number of color channels of the sEMG image, W is the width of sEMG image and H is the height of sEMG image. The sEMG image size of csl-hdemg and CapgMyo-DBa are L × 24 × 7 and L × 16 × 8, respectively.An intuitive sEMG image representation method is to use the placement of electrodes and each electrode can be regarded as a pixel of sEMG images. It is a feasible sEMG image representation method for high-density sEMG databases csl-hdemg and CapgMyo-DBa, as electromyogram signals are collected by a grid of sEMG electrodes. The detailed image representation procedure is described as follows. The input is a segment of electromyogram signal of the high-density electromyogram signal with size raw-image1, raw-image2, signal-image1, signal-image2, activity-image1 and activity-image2. The input of the sEMG image representation methods is a segment of electromyogram signal of NinaProDB1 with size L × C, where L is the number of frames, C is the number of signal channels and C = 10 for NinaProDB1. The detailed sEMG image representation methods are described as follows.However, for sparse multi-channel sEMG database, the number of electrodes is limited and the placement is sparse. Inspired by the image representation method used in  for humaraw-image1 is obtained by transforming the input into a sEMG image with size L × 1 × 10, where L is the number of color channels of the sEMG image, 1 is the width of sEMG image, and 10 is the height of sEMG image.The raw-image2 [L × 10, where 1 is the number of color channels of the sEMG image, L is the width of sEMG image, 10 is the height of sEMG image, and width × height = signal channels.The w-image2  is obtaisignal-image1 [L × 1 × 51.The l-image1  is formel-image1  with sizsignal-image2 [signal-image1 with size 1 × L × 51.The l-image2  is formeactivity-image1 [L × 1 × 51.The y-image1  is generactivity-image2 [L × 51.The y-image2  is generWe evaluate the six sEMG image representation methods using the existing CNN architecture  on NinaPfeature dimension = signal channels × feature vector dimension and conduct a sEMG image with size 1 × 1 × feature dimension which is denoted as feature-flatten-image. We have evaluated this sEMG image representation method on the NinaProDB1, and the recognition accuracy is 81.9% which is lower than that of raw signal based sEMG images . Inspired by the good performance of signal-images, we propose a new sEMG image representation method “feature-signal-image1” which makes full use of the traditional feature vector and achieves better performance among existing sEMG image representation methods.The feature extraction plays a significant role in traditional sEMG-based gesture recognition methods and many classical feature sets have achieved good performance , 13, 15.In this section, we first delineate the experimental setup, followed by the performance comparisons between proposed architecture and state-of-the-art methods on five sEMG benchmark databases. Secondly, we discuss the effectiveness of attention mechanism. Then, the results of different image representation methods are presented. Finally, we evaluate and discuss the impacts of various parameters of the architecture on recognition accuracy.In this work, we follow the experimental process which consists of data acquisition, preprocessing, segmentation and gesture recognition. The proposed architecture is implemented using MxNet , and theThe first and second sub-database of NinaPro database  are denoA subset of BioPatRec toolbox is available online  , we present the results of different numbers of subsegments on NinaProDB1. The number of subsegments is set as {2,5,10,20} and the results are shown in We compare the “feature-signal-image1” with two raw signal based methods “raw-image1” and “signal-image1” for CNN, hybrid CNN-RNN and attention-based hybrid CNN-RNN frameworks. The results in feature-signal-image1 achieves the highest accuracy in the eight evaluated sEMG image representation methods. For the input of RNN module is a vector instead of an image, we also compare the raw signal with feature vector for the RNN module and the accuracies are 79.8% and 74.5%, respectively.We also evaluate the eight image representation methods on the sparse multi-channel database NinaProDB1 and the results can be seen in In this work, we propose an attention-based hybrid CNN-RNN architecture for sEMG-based gesture recognition, which consists of feature extraction stage and attention-based sequential modeling stage. It makes full use of spatial and temporal information of electromyogram signals and the attention mechanism makes the network more intelligent to pay attention to different parts of the electromyogram signal. The evaluations are performed on five sEMG benchmark databases, namely NinaProDB1, NinaProDB2, BioPatRec26MOV, CapgMyo-DBa and csl-hdemg databases. The results show that 1) the hybrid CNN-RNN architecture outperforms both CNN and RNN modules; 2) the attention mechanism enhances the performance of the hybrid CNN-RNN architecture. Moreover, we present a new feature vector based sEMG image representation method “feature-signal-image1” for sparse multi-channel databases. Compared with the sEMG image representation method “raw-image1”, it improves the recognition accuracy from 84.8% to 87.0% on NinaProDB1, from 74.8% to 82.2% on NinaProDB2, from 92.5% to 94.1% on BioPatRec26MOV. Overall, the recognition accuracies of proposed sEMG-based gesture recognition method are 87.0% for NinaProDB1, 82.2% for NinaProDB2, 94.1% for BioPatRec26MOV, 99.7% for CapgMyo-DBa and 94.5% for csl-hdemg. The improvements are 9.2% (NinaProDB1), 3.5% (NinaProDB2), 1.2% (BioPatRec26MOV), 0.2% (CapgMyo-DBa) and 5.2% (csl-hdemg) higher than the state-of-the-art performances , 55.The electromyogram signal is a kind of biological signal which is severely affected by the difference between subjects. It makes the accuracy of Leave-One-Subject-Out cross-validation (LOSOCV) much lower than that of Within-Subject cross-validation (WSCV) in previous works , 27. Fut


Pelophylax nigromaculatus (Anura: Ranidae) attacking the bombardier beetle Pheropsophus jessoensis under laboratory conditions. In Japan, Pe. nigromaculatus is a generalist predator in grasslands where the bombardier beetle frequently occurs. Almost all the frogs (92.9%) observed rejected live bombardier beetles; 67.9% stopped their attacks once their tongues touched the beetles, and 25.0% spat out the beetles immediately after taking the beetles into their mouths. No beetle bombed a frog before being taken into a frog’s mouth. All beetles taken into mouths bombed the frogs. Only 7.1% of the frogs swallowed live bombardier beetles after being bombed in the mouth. When dead beetles were provided instead, 85.7% of the frogs rejected the dead beetles, 71.4% stopped their attacks after their tongues touched the beetles, and 14.3% spat out the beetles. Only 14.3% of the frogs swallowed the dead beetles. The results suggest that the frogs tended to stop their predatory attack before receiving a bombing response from the beetles. Therefore, bombing was not essential for the beetles to successfully defend against the frogs. Using its tongue, Pe. nigromaculatus may be able to rapidly detect a deterrent chemical or physical characteristics of its potential prey Ph. jessoensis and thus avoid injury by stopping its predatory attack before the beetle bombs it.Some animals, such as the bombardier beetles (Coleoptera: Carabidae: Brachinini), have evolved chemical defences against predators. When attacked, bombardier beetles can discharge noxious chemicals at temperatures of approximately 100 °C from the tip of their abdomens, “bombing” their attackers. Although many studies to date have investigated how bombardier beetles discharge defensive chemicals against predators, relatively little research has examined how predators modify their attacks on bombardier beetles to avoid being bombed. In this study, I observed the black-spotted pond frog  Physical and chemical defences have evolved in many organisms to protect against natural enemies . For exaAdult bombardier beetles (Coleoptera: Carabidae: Brachinini) bomb, i.e., discharge noxious chemicals from the tip of their abdomens at temperatures of approximately 100 °C, when they are disturbed or attacked . Such ejFrogs and toads are important predators of carabid beetles . HoweverPelophylax nigromaculatus  (Anura: Ranidae) to the defensive behaviour of the bombardier beetle Pheropsophus jessoensis (Morawitz). Pheropsophus jessoensis is a bombardier beetle found in East Asia  at a temperature of approximately 100 °C from their rear ends in response to predator attacks , Hyogo Prefecture, central Japan, from May to August in 2016, 2017, and 2018 (cf. Spodoptera litura (Fabricius) (Lepidoptera: Noctuidae) were provided as food , Sanda-shi , and Sayo-cho , Hyogo Prefecture, central Japan, from May to August in 2016, 2017, and 2018. The distances between these sites and the sampling site of Ph. jessoensis ranged from 15.6 to 65.4 km. Although Pe. nigromaculatus has recently been classified as near threatened (NT) in the Japanese Red Data List , this species was abundant at the collection sites. Both juveniles and adults were collected. Body weight was measured to the closest 0.01 g using an electronic balance . Small and large frogs were maintained separately in small  and large plastic cages , respectively, in the laboratory at 25 °C. Live larvae of S. litura, Tenebrio molitor Linnaeus (Coleoptera: Tenebrionidae), and Zophobas atratus Fabricius (Coleoptera: Tenebrionidae) were provided as food. Frogs were starved for 24 h before the feeding experiments to standardise their hunger level . If a frog displayed attacking behaviour  was offered to the frog several minutes after beetle rejection to determine whether the frog was hungry. If a frog swallowed the bombardier beetle, I observed whether it vomited the beetle within 330 min of swallowing it (cf. n = 28) and 213.0 ± 10.0 mg (n = 28), respectively.Feeding experiments were all conducted at 25 °C. To start, a frog was placed in a transparent plastic container . Then, a transparent glass petri dish  containing a live bombardier beetle was placed outside the plastic container where the frog could see it. Frogs that did not try to attack the beetle (34.1%) were not used for the feeding experiments. However, frogs that ignored g it cf. . VomitedPelophylax nigromaculatus usually does not attack motionless prey. However, in a pilot test, an individual of Pe. nigromaculatus attacked and ingested a dead caterpillar (S. litura) when forceps were used to move the caterpillar within the frog’s field of view. For this experiment, the bombardier beetles were killed in a freezer at −15 °C. First, a dead beetle was placed in the plastic container  within the frog’s field of view. If the frog did not initially respond to the beetle, forceps were used to move the dead beetle within the frog’s field of view again. Frogs that did not attack the dead beetles (29.2%) were not used in these experiments; frogs that ignored Ph. jessoensis did not respond to other prey . The predatory behaviours of the frogs were recorded using the same digital video camera. Frogs that did not swallow dead beetles were offered T. molitor larvae several minutes after beetle rejection to check whether they were hungry. Twenty-eight frogs and 28 dead beetles were used in this experiment. The means ± standard errors of the frog and dead beetle body weights were 8.65 ± 1.25 g (n = 28) and 214.8 ± 7.6 mg (n = 28), respectively. The mean body weight of frogs that attacked dead beetles did not differ significantly from the mean body weight of frogs that attacked live beetles . The mean body weight of dead beetles did not differ significantly from the mean body weight of live beetles A second set of frogs were presented with dead adult beetles to test whether the bombing response is essential for deterring a predatory attack. Videos of frogs responding to live and dead beetles were played back using QuickTime Player version 10.4 . Frog responses to the bombardier beetles were grouped into four categories cf. : (1) froAll experiments were performed in accordance with the Kobe University Animal Experimentation Regulations . No frogs were seriously injured or killed during the feeding experiments. My study also complies with the current laws of Japan.Generalised linear models (GLMs) featuring binomial error distributions and logit links  were used to identify factors that contributed to frogs’ successful swallowing and digestion of the bombardier beetles. The success or failure (1/0) of frogs’ swallowing and digesting beetles was used as the response variable. Frog weight, beetle weight, the frog weight × beetle weight interaction, and beetle condition (live or dead) were treated as fixed factors. When the residual deviance was larger (overdispersion) or smaller (underdispersion) than the residual degrees of freedom, a quasi-binomial error distribution was used rather than a binomial error distribution. Furthermore, fixed factors were subjected to likelihood ratio testing when marginal significance was evident. Thus, the significance of models with and without the factors of interest were compared using the GLMs. All analyses were performed using R version 3.3.2 .n = 28), 26 frogs (92.9%) rejected the beetles without swallowing them (n = 8/9) initially stopped attacking the beetles when their tongues first touched the beetles, but resumed their predatory attack soon thereafter (T. molitor larvae) soon thereafter.In the experiment using live adult bombardier beetles , 24 frogs (85.7%) rejected the dead beetles without swallowing them (n = 7/8) were initially deterred when their tongues first touched the beetles, but continued with their predatory behaviour soon afterwards. The frogs that did not swallow beetles ate other prey  soon thereafter.When dead beetles were used  was higher than that of live beetles (7.1%). However, the GLM results indicated that the frog swallowing rates of live and dead beetles did not significantly differ . WhetherThe proportion of dead beetles digested by frogs (14.3%) was higher than the proportion of live beetles swallowed (3.6%). The GLMs indicated that the difference between the digestion rates of live and dead beetles was significant . Beetle Pe. nigromaculatus frequently rejected Ph. jessoensis without attempting to swallow the beetles . Therefore, the experimental conditions used may not be appropriate for analysing foraging by certain frogs. Alternatively, the yellow-and-black pattern of Ph. jessoensis may serve as disruptive camouflage; flightless Ph. jessoensis walk on soil of forest edges, grasslands, and agricultural fields. Further work is needed to explore the significance of Ph. jessoensis colour as a defensive trait.The bombardier beetle ouration , althougouration . In factPelophylax nigromaculatus has been reported to spit out large prey that they were unable to swallow after taking the prey into their mouths   .In one study, the chemicals produced by bombardier beetles’ bombing did not stimulate the tongues of toads any less intensely than did the heat from the chemical reaction . Other tMany prey animals exhibit multiple anti-predator defences . Predati10.7717/peerj.5942/supp-1Video S1The beetle discharged toxic chemicals when its eggs were pinched with a pair of forceps.Click here for additional data file.10.7717/peerj.5942/supp-2Video S2Click here for additional data file.10.7717/peerj.5942/supp-3Video S3The frog stopped the attack immediately after its tongue touched the beetle.Click here for additional data file.10.7717/peerj.5942/supp-4Video S4The frog took the beetle into its mouth but immediately spat out the beetle.Click here for additional data file.10.7717/peerj.5942/supp-5Video S5The frog stopped its attack immediately after its tongue touched the dead beetle.Click here for additional data file.10.7717/peerj.5942/supp-6Data S1Click here for additional data file.


CSF) is used in clinical practice to mobilize cells from the bone marrow to the blood; however, it is not always effective. We show that cobalt protoporphyrin IX (CoPP) increases plasma concentrations of G‐CSF, IL‐6, and MCP‐1 in mice, triggering the mobilization of granulocytes and hematopoietic stem and progenitor cells (HSPC). Compared with recombinant G‐CSF, CoPP mobilizes higher number of HSPC and mature granulocytes. In contrast to G‐CSF, CoPP does not increase the number of circulating T cells. Transplantation of CoPP‐mobilized peripheral blood mononuclear cells (PBMC) results in higher chimerism and faster hematopoietic reconstitution than transplantation of PBMC mobilized by G‐CSF. Although CoPP is used to activate Nrf2/HO‐1 axis, the observed effects are Nrf2/HO‐1 independent. Concluding, CoPP increases expression of mobilization‐related cytokines and has superior mobilizing efficiency compared with recombinant G‐CSF. This observation could lead to the development of new strategies for the treatment of neutropenia and HSPC transplantation.Granulocyte colony‐stimulating factor (G‐ CSF is the mobilizing factor used for treating neutropenia and prior to harvesting hematopoietic stem cells for transplantation. This article describes cobalt protoporphyrin IX as a new efficient mobilizing factor upstream of G‐CSF.Recombinant G‐ Thus, CoPP is considered as a potential inducer of HO‐1 where it may have therapeutic advantages  partially blocks differentiation of myeloid progenitors toward macrophages , what is connected with increased myelocyte proliferation in the bone marrow (BM). Moreover, CoPP may inhibit the maturation of dendritic cells , iron ions, and biliverdin , whereas CoPP treatment increased the number of mature cells , but the increase after G‐CSF was more pronounced. Percentage of granulocytes with the mature phenotype (CD11b+ CD11c− Ly6Clow SSChi Ly6Ghi) was decreased after G‐CSF, but not affected by CoPP , phorbol 12‐myristate 13‐acetate (PMA) or opsonized Escherichia coli and subjected to rhodamine 123 (DHR 123) staining  and E. coli . For this purpose, G‐CSF‐ and CoPP‐mobilized or control PB was incubated with Concluding, granulocytes mobilized either by G‐CSF or by CoPP are at least as functional as the steady‐state granulocytes in tested conditions.+ Lin− Sca‐1+ (KLS) cells in the blood (−CD150+), MPP , and HPC  populations  and MPP populations  in BM; however, the decrease after CoPP was less pronounced  and checked their hematopoietic reconstitution potential.6 PBMC to the lethally irradiated GFP− recipient mice, together with 105 GFP− BM‐derived competitor cells. Two independent experiments were performed.We treated the green fluorescent protein (GFP)‐expressing mice (C57BL/6‐Tg(UBC‐GFP)30Scha/J) with CoPP, G‐CSF, or NaCl Fig . At the et al, Donor mice (C57BL/6‐Tg(UBC‐GFP)30Scha/J) are on C57BL6 genetic background, which is described as poor HSPC mobilizing strain . GFP chimerism was observed among all examined cell types  will mimic the superior mobilization effect of CoPP. We did not observe any effect of simultaneous administration of G‐CSF and rmIL‐6 compared with G‐CSF alone Fig . HoweverAs the induction of G‐CSF expression by CoPP was not reported before, we performed qPCR and Luminex assay to investigate which tissues or organs respond to CoPP by producing G‐CSF. We detected increased levels of mRNA for G‐CSF in the lysates from the spleen, liver, and gastrocnemius muscle . LuminexHmox1 gene expression, and we expected that mobilizing properties of CoPP are mediated by HO‐1 activation. To verify this supposition, we checked if CoPP induces mobilization in HO‐1−/− mice.Next, we investigated whether the mechanism of CoPP action is dependent on Nrf2/HO‐1 axis. CoPP is a known inducer of the −/− individuals have increased susceptibility to the toxic effect of many compounds, in the experiment with HO‐1+/+ and HO‐1−/− mice we administered CoPP three times, every second day (instead of daily for 5 days). We observed mobilization of total WBC and granulocytes in both CoPP‐treated groups, despite the higher basal number of granulocytes in HO‐1−/− individuals .−/− mice were initially provided by Dr. Anupam Agarwal, University of Alabama, Birmingham, USA. HO‐1−/− mice were initially created on the 129/Sv × C57BL/6 background , while retaining the original HO‐1−/− characteristics  generated by Itoh et al  conditions, with constant light/dark cycle (14/10 h) and continuous monitoring of temperature and humidity. Mice were kept in groups ≤ 5 in the individually ventilated cages with food and water ad libitum.Cobalt protoporphyrin mobilization experiment in HO‐1Cobalt protoporphyrin and SnPP (Frontier Scientific) were dissolved in DMSO (Sigma‐Aldrich) at concentration of 0.2 g/ml. The stock solutions were diluted 160× in 0.9% NaCl solution. Mice were injected intraperitoneally (i.p.) at the dose of 10 mg/kg (15 μmol/kg). Recombinant human G‐CSF  was used at the dose of 250 μg/kg. Murine recombinant IL‐6 (R&D) was used in concentration 50 μg/kg. Compounds were injected i.p. daily for 5 days or three times, every second day, depending on the experiment, as indicated in the text and Fig 1 antibodies: anti‐G‐CSF  and anti‐IL‐6 . Rat IgG1 anti‐HRP  was used as a IgG isotype control. The antibodies were administered 1 h before each CoPP dose, for five consecutive days. The experimental groups included the following: DMSO , CoPP + IgG control, CoPP + anti‐G‐CSF, and CoPP + anti‐G‐CSF + anti‐IL‐6. Each single dose was anti‐G‐CSF 12.5 μg/mice, anti‐IL‐6 130 μg/mice, and IgG control 142.5 μg/mice. Six hours after the last treatment, samples were collected for analysis  was measured using gas chromatography  were performed on −80°C frozen plasma samples according to manufacturer's instructions, and the signal was detected using FLEXMAP 3D system (Millipore).137Cs γ source at 110 cGy/min  24 h before the transplantation. Two‐ to three‐month‐old female C57BL/6‐Tg(UBC‐GFP)30Scha/J donor mice were treated for five consecutive days with CoPP, rhG‐CSF, or NaCl as described above. Six hours after the last injection, blood was collected and PBMC were isolated using Ficoll‐Paque PLUS . 5 × 106 of GFP+ PBMC with 1 × 105 GFP‐ BM‐derived competitor cells were injected into the tail vein of irradiated recipients. Fourteen weeks after the primary transplantation, 10 × 106 of BM cells were transplanted to the secondary recipients  and analyzed using BD FACSDiva and FlowJo software. The absolute numbers of cells/μl were calculated using BD Trucount Tubes (BD) and BD FACS lysing solution (BD). Antibodies for flow cytometry were used at 1:50 or 1:100 dilutions. Flow cytometry reagents and gating strategies are shown in Mice were treated with CoPP, G‐CSF, DMSO, and NaCl for 5 consecutive days as described above. Six hours after the last injection, blood samples were collected. Phagoburst assay (Glycotope Biotechnology) was performed according to manufacturer's instructions.et al, et al, et al, Heatmaps were created using heatplot function  on StepOne Plus thermocycler (Applied Biosystems) and analyzed using StepOne software. Commercially available primers M‐Csf3_2 Sigma predesigned KiCqStart™ Primers spanning exons 4–5 were used, with annealing temperature of 60°C.Quantitative real‐time PCR (qPCR) was performed using SYBRΔ), the laser flash photolysis (LKS 60 Applied Photophysics) was applied. The ΦΔ values were determined using the comparative method, which relies on the detection of 1O2 phosphorescence at λ = 1,270 nm with phenalenone (Sigma) as a standard. Investigated materials were dissolved in ethanol at the concentration necessary to produce an absorbance of 0.25 at the excitation wavelength (λexc = 355 nm). To detect the decay of singlet oxygen emission, the excitation of sample was generated using the third harmonic (λexc = 355 nm) of a Nd/YAG laser . We analyzed the intensity of singlet oxygen emission at t = 0 as a function of the relative energy of laser pulses. The ΦΔ value was calculated using the following formula:ΦΔref is a quantum yield of a reference sample, i.e., ΦΔref = 0.95 in ethanol . Exact P values for all the comparisons together with the statistical tests used are listed in In our experiments, we were not able to prespecify the expected effect size; therefore, no statistical power and sample size estimation were done. We used the maximal number of animals per experimental group that were allowed by local ethical committee. Nevertheless, we performed independent experiments to confirm majority of our conclusions. Animals in each cage were randomly assigned to all experimental groups. Injections were not done in a blinded manner, as CoPP is colored. All samples were collected and analyzed in blinded manner. Statistical analysis was done with GraphPad Prism software that estimates variance equality for analyzed datasets. Data are presented as mean with SEM or mean with individual values. For multigroup comparisons with one variable, we used one‐way ANOVA with additional post‐tests, and for multigroup comparisons with two variables, we used two‐way ANOVA with additional post‐tests. Grubbs’ test was used to determine significant outliers. Results were considered as statistically significant, when https://smart.servier.com/ with modifications.Schemes were created using images from Servier Medical Art AS, KS, and AJ designed the experiments, AS, KS, WNN, KB‐S, LM, MG, MŻ, MC, NK‐T, and MR‐B performed the experiments, AS and KS analyzed the data, WNN, KB‐S, AR, and JD assisted with experimental designs and interpretation, AS, KS, and AJ wrote the manuscript.Cobalt porphyrins for the treatment of blood‐related disorders and EP 3139917: Cobalt protoporphyrin IX for the treatment of blood‐related disorders granted to the Jagiellonian University. The remaining authors declare no conflict of interest.AS, KS, AJ, and JD are the coinventors of the patents US10010557 and US10328085: et al, Pharmacological mobilization is mainly used in two situations: (i) treatment of chemotherapy‐induced neutropenia; and (ii) mobilization of HSCs for their transplantation. Chemotherapy is often the only therapeutic option for cancer patients. Unfortunately, chemotherapy comes with significant side effects, toxicity to the hematopoietic system being one of the most dangerous. HSC transplantation is the live‐saving therapy for various hematological disorders. For many years now, mobilized blood has been the preferred source of HSCs for transplantation instead of BM biopsy. Recombinant G‐CSF (filgrastim) is the most widely used mobilizing factor. However, up to 30% healthy donors and 60% high‐risk patients fail to respond to mobilization  induces endogenous G‐CSF and other cytokines and may have superior mobilizing properties in comparison with the currently used drug, recombinant G‐CSF. First, CoPP‐mobilized PB provides faster reconstitution and higher long‐term blood chimerism upon transplantation than G‐CSF‐mobilized blood. Second, CoPP mobilizes a higher number of HSCs than G‐CSF, but in contrast to G‐CSF, CoPP does not mobilize T cells, which could be important in graft‐versus‐host disease prevention. Finally, CoPP mobilizes granulocytes with a more mature phenotype, which could find some application for the treatment of neutropenia.Our data suggest that CoPP could be used for the treatment of hematological disorders. CoPP can work more efficiently than G‐CSF. Moreover, as a simple compound it should be much cheaper in production, making the treatment available for a broader group of patients. The possibility of developing an orally absorbed CoPP formulation may further facilitate its potential clinical use.http://nicheworks.eu/ Department website: https://zbm.wbbib.uj.edu.pl/en_GB/Lab website: AppendixClick here for additional data file.Expanded View Figures PDFClick here for additional data file.Review Process FileClick here for additional data file.


CSF leak in penetrating skull base injury is relatively rare compared to close head injury involving skull base fracture. We report a 5-year-old boy presented with epistaxis and impacted pencil into the left nostril. The child was hemodynamically stable without any neurological deficit. Intraoperatively, there was a nasal septal defect posteriorly with anterior skull base fracture associated with CSF leak. The pencil was removed from the left nostril and the CSF leak was repaired using harvested abdominal fat under the same setting. Computed Tomography (CT) of the brain showed right cribriform plate fracture with small pneumocranium. Postoperatively, a prophylactic antibiotic was given for seven days and he was discharged well. Subsequent clinic visits up to one-year postoperative period showed no recurrence of the CSF leak. History taking, physical examination and CT imaging give valuable diagnostic values in managing the penetrating skull base injury. Early intervention for removal of the foreign body and repair of the CSF leak is advocated to prevent catastrophic complication.  Presence of fistulous tract in between intracranium and nasal cavity may result in cerebrospinal fluid (CSF) rhinorrhea. Craniofacial trauma contributed 80 % of the etiologies of CSF rhinorrhea. Traumatic CSF rhinorrhea is more commonly seen in skull base fractures. Majority of the skull base fractures (90 %) are caused by closed head injuries, whereas penetrating trauma constitutes the remaining 10 % of the etiology.Most of the penetrating head injuries involved oro-cranial or transorbital intracranial penetration of the sharp object with a significant external injury. Foreign bodies such as glass, air gun projectiles, metal splinters and plastic chair glides have been reported. Traumatic transnasal penetrating injury to the skull base is rare in literature. Removal of the penetrating object followed by repair of the CSF leakage depicts more challenging management.In this case report, we described an unusual case of penetrating injury of the anterior skull base by a pencil via the left nostril, without external, orbital and paranasal sinuses involvement. The importance of radiological assessment prior to surgery was emphasized in this report. We also discussed the approaches of removal of the penetrating object and endoscopic CSF leakage repair in the same setting.A 5-year-old boy presented to the emergency room with epistaxis following an impacted pencil over the left nostril. He admitted to inserting the blunted end into his left nostril and was pushed by his sibling from behind. He fell forward on his face causing impaction of the pencil into his left nostril. His mother claimed that they have successfully pulled out the pencil and the epistaxis subsequently resolved. Upon review, he was hemodynamically stable, alert and conscious. Anterior rhinoscopy showed part of the pencil shaft embedded in the left nasal cavity. The child was however uncooperative for further assessment of the extent of the injury. Otherwise, there was no external wound and deformity over the craniofacial region, bilateral eye examination and neurological examinations were unremarkable. Radiograph of the skull Figure 1 showed fHe was brought into the operating theatre for examination under anesthesia. Intraoperatively, the pencil was impacted firmly in the left nostril and nasal septum Figure 2. The penAfter removal of the foreign body, the CSF leakage was repaired with autologous graft by placing the septal cartilage and abdominal fat on to the defect until the CSF leakage ceased. The septal mucosal graft was repositioned on top of the fat graft and covered with Gelfoam and tissue glue.Post-operatively, he was ventilated for 24 hours and kept under bed rest with intravenous ceftriaxone administered for one week in view of overwhelming intraoperative findings. Computed Tomography (CT) scan of the brain, skull base and paranasal sinuses post-operatively showed right cribriform plate fracture with small pneumocranium Figure 4. No evidNasal foreign bodies are common encounter in ENT setting. It ranges from simple foreign bodies to catastrophic scenarios with skull base fractures and intracranial complications.Transnasal septal penetrating injury to the anterior skull base without an external wound and orbital involvement makes this a unique case. Furthermore, management of penetrating injury of the head and neck region in a pediatric patient can be very challenging as history may be inaccurate with limited cooperation during an examination. Additionally, clinical examination showed no external wound over the face and orbital region with intact neurological function masquerade the extent of the injury made..When the history is uncertain, the evaluation of suspected penetrating injury of the nose lies on the radiological assessment. A plain radiograph is easily available with low radiation, it offers an initial assessment of the bony framework of the facial and skull bone to detect any fracture lines. The panoramic view of plain radiograph helps in locating the foreign body. However, the visibility of the foreign body in plain radiograph depends on the types of object. Metallic foreign body is radio-opaque on radiograph whereas organic foreign body like peanut or wood would be less radio-opaque hence easily missed .CT scan gives a good osseous delineation which is important in detecting subtle fractures. This fine thin cut of the CT scan with the image in axial, coronal and sagittal plane provides an accurate assessment of the location of the foreign body. With the three-dimensional (3D) images reconstructed from the fine cut CT scan, the extent of the injury can be accurately localized and essential in planning the surgical approach. For cases suspected with vasculature or intracranial injury, a CT angiography and Magnetic Resonance Imaging (MRI) brain would be warranted .rd generation of cephalosporin with the property of crossing blood-brain barrier for prophylaxis against intracranial infection.Early removal of the penetrating foreign body is the definitive treatment in regards to avoid the unwanted sequelae . Early There are two suggested methods in the removal of the penetrating foreign body into the maxillofacial region, namely the endonasal approach and external approach . The shCSF leak was demonstrated immediately after removal of the foreign body. Most of the traumatic CSF leak will be resolved with conservative management or CSF diversion with lumbar drain. However, the incidence of ascending intracranial infection was relatively high in the group of CSF leak patient when conservative treatment was given. Thus, repair of the CSF leak was advocated whereby it significantly reduces the risk of ascending intracranial infection . Presence of fistulous tract in between intracranial and nasal cavity predisposes the patient to CSF leaks, pneumocephalus and intracranial infection such as ascending meningitis and abscess. The goals of skull base reconstruction include separation of the cranial cavity from the sinonasal tract, protection of neurovascular structures, preservation of functions and avoidance of dead spaces.As stated by Wormald and McDonough in 1997).Indications for endoscopic repair include post-traumatic CSF leak, iatrogenic and spontaneous CSF rhinorrhea associated with either malformation of the skull base, meningoencephaloceles, empty sella and increased intracranial pressure. With the improvement of skills and instrumentation, amazingly high success rates with primary closure of the defect between 88 % and 94 % were achieved in the early 1990s .The key to successful management of CSF leak is to precisely identify the site of the dural tear. Preoperative imaging such as high-resolution CT scan of the skull base and paranasal sinuses can be performed to locate the fracture site. Intrathecal application of sodium fluorescein in combination with the use of blue light and a blocking filter over the endoscope's eyepiece will precisely guide the endoscopic surgeon to the site of the lesions. Laboratory techniques such as Beta-2-transferrin and beta-trace protein testing are the standard tests that help to differentiate non-CSF-related nasal secretion from the true CSF in an unclear situation .In this case, we harvested the fat graft from the periumbilical region as the autologous graft for repair. The indentation mark over the anterior skull base was covered by the lacerated septal cartilage with septal mucosa. For defect closure, it is recommended that no mucosa should be placed under any graft or fat to avoid mucocele formation. Hence the septal cartilage with mucosa was taken out temporary, the area of skull base defect was denuded of its mucosal layer. Adequate size of the harvested fat graft was gently placed over the skull base defect with pediatric Blakesley forcep. With the use of Freers elevator, the fat graft was gently placed in between the bone and surrounding mucosa, which is described as overlay technique. Subsequently, the septal cartilage with mucosa was repositioned back on top of the fat graft. The grafted area was reinforced with Gelfoam and tissue glue. The end result of the repair was excellent without intracranial complication and further CSF leak up to date.Management of transnasal penetrating injury of the anterior skull base can be very challenging in terms of diagnosis and treatment. When the history and physical examination are in doubts, CT imaging is mandatory to rule out suspecting skull base injury. Having said that accurate diagnosis and early intervention are important in order to prevent intracranial complications, continuous follow up is advocated to detect the delayed complication.The authors declare no conflict of interest.


The blood-brain barrier (BBB) dysfunction is an initial event of various neuroinflammatory diseases. However, the absence of reliable markers and mechanisms for BBB damage greatly limits the diagnosis and treatment of neuroinflammatory diseases. Soluble CD146 (sCD146) is mainly derived from vascular endothelial cells (ECs) and highly elevated in inflammatory settings. Based on a small cohort, our previous study showed that sCD146 is elevated in the cerebrospinal fluid (CSF) of multiple sclerosis (MS), which is accompanied with BBB damage. Nevertheless, whether sCD146 monitors and regulates the BBB dysfunction remains unknown.Methods: Coupled serum and CSF samples from patients with or without neuroinflammatory diseases were collected via multicenter collaborations. sCD146 was measured by sandwich ELISA using anti-CD146 antibodies AA1 and AA98, both of which were generated in our laboratory. The correlations between sCD146 and other clinical parameters or inflammatory factors were analyzed by Spearman's correlation coefficient analysis. The role of sCD146 on BBB function was examined in an in vitro BBB model.Results: Between July 20, 2011, and February 31, 2017, we collected coupled serum and CSF samples from 823 patients, of which 562 (68.3%) had neuroinflammatory diseases, 44 (5.3%) had remitting MS, and 217 (26.4%) had non-inflammatory neurological diseases (NIND). We found that sCD146 in CSF, but not in serum, is abnormally elevated in neuroinflammatory diseases (37.3 ± 13.3 ng/mL) compared with NIND (4.7 ± 2.9 ng/mL) and remitting MS (4.6 ± 3.5 ng/mL). Abnormally elevated CSF sCD146 is significantly correlated with the hyperpermeability-related clinical parameters of BBB and neuroinflammation-related factors. Moreover, CSF sCD146 shows higher sensitivity and specificity for evaluating BBB damage. Using an in vitro BBB model, we found that sCD146 impairs BBB function by promoting BBB permeability via an association with integrin αvβ1. Blocking integrin αvβ1 significantly attenuates sCD146-induced hyperpermeability of the BBB.Conclusion: Our study provides convincing evidence that CSF sCD146 is a sensitive marker of BBB damage and neuroinflammation. Furthermore, sCD146 is actively involved in BBB dysfunction. The homeostasis of the central nervous system (CNS) microenvironment requires blood-brain barrier (BBB) integrity The adhesion molecule CD146 is primarily expressed at the intercellular junctions of ECs and is reported to be involved in a variety of physiological and pathological processes In this study, we demonstrate that CSF sCD146 is abnormally elevated in patients with various neuroinflammatory diseases and that the elevation in CSF sCD146 closely correlates with BBB damage. Moreover, we evaluate the function of sCD146 in BBB permeability and provide evidence that sCD146 contributes directly to BBB dysfunction. These findings suggest that CSF sCD146 is a sensitive marker for BBB damage, and that sCD146 is a novel driver of neuroinflammation-BBB disruption.Our previous study showed that sCD146 in the CSF could be used as a marker for disease activity in MS patients Alb), IgG synthesis and myelin basic protein (MBP), are used to evaluate BBB damage for auxiliary diagnosis. To determine the association of CSF sCD146 with BBB damage, we analyzed the correlations of CSF sCD146 with these factors. As shown in Table Alb, IgG synthesis and CSF-MBP, in patients with neuroinflammatory diseases. However, in patients with NIND, CSF sCD146 showed no or mild correlations with these factors. In both non- and neuroinflammatory diseases, CSF sCD146 had no correlations with serum sCD146 or other clinical indexes .Next, we determined whether CSF sCD146 level is correlated with BBB disruption. In clinical laboratory examinations, several parameters, such as albumin quotient , soluble adhesion molecules (sICAM and sVCAM) and inflammatory factors , have been reported in neuroinflammation and are considered to participate in BBB damage We further analyzed the correlations between CSF sCD146 and neuroinflammation-related factors. As shown in Table To evaluate whether CSF sCD146 is preferred for monitoring BBB damage and neuroinflammation, we compared the sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) of CSF sCD146 to other related molecules. Based on the cut-off value of CSF sCD146 (10.5 ng/mL), further analysis showed that the sensitivity of CSF sCD146 was much higher (100%) than those of the other clinical parameters (31.7-73.9%) and the neuroinflammation-related factors (13.3-74.4%). Moreover, we found that CSF sCD146 showed a higher specificity (93.1%), PPV (97.4%) and NPV (100%). Although sVCAM, sICAM and IFN-γ showed 100% specificity and PPV, their NPV 24.6-45.8%) were much lower than that of CSF sCD146 , including occludin, zonula occludens (ZO)-1 and junctional adhesion molecule (JAM)-1  assay showed that integrin αv and β1 were associated with sCD146 in hCMEC/D3 cells  in monitoring BBB damage. An Alb is used to evaluate BBB permeability Alb has a very low sensitivity in the diagnosis of BBB dysfunction Alb cannot be used to evaluate small local leaks or more widespread areas of a leak or smaller solutes AlbAlb is insufficient for evaluating BBB damage. The development of new biomarkers that are sensitive to BBB leakage and dysfunction is urgently needed. In this study, we provide a promising marker for evaluating BBB damage. By enrolling 823 patients with or without neuroinflammation, we found that sCD146 was markedly elevated in the CSF but not in the serum, suggesting that sCD146 reflected local inflammation, which was confirmed by examining the relationships between sCD146 and CSF inflammatory factors. Moreover, we found that the CSF sCD146 level was positively correlated with the BBB permeability tested by the routinely utilized parameters, such as the QAlb, the IgG index and CSF-MBP. Furthermore, compared to other soluble adhesion molecules that have been reported in the CSF, such as sICAM and sVCAM, CSF sCD146 showed high sensitivity and specificity, a high PPV and a high NPV for measuring BBB damage, indicating that CSF sCD146 is sensitive for monitoring early BBB damage and CNS inflammation. This study provides evidence that sCD146 is a promising marker for measuring BBB damage as well as a potential marker for monitoring inflammation development.BBB integrity maintains the homeostasis of the CNS microenvironment, and the disruption of BBB is a hallmark of many neuroinflammatory diseases. Monitoring BBB integrity facilitates predicative disease prognosis and guides treatment. Gd-enhancing MRI is the standard for evaluating BBB disruption Serum sCD146 is generally known to be a marker that reflects the severity of cardiovascular diseases and tumor development Because the BBB is an interface between the brain parenchyma and the peripheral circulation, maintaining BBB integrity is pivotal for CNS homeostasis. Malfunctions in any component of the BBB, such as brain microvascular ECs, astrocytes, pericytes and the basal membrane, could lead to critical changes in BBB function. BBB-ECs are the primary component of the BBB. The special structural features of BBB-ECs, such as their low rate of pinocytosis, higher mitochondrial content and lack of fenestrations, distinguish BBB-ECs from peripheral vascular ECs. Under physiological conditions, BBB-ECs express lower levels of adhesion molecules, which limits the entry of immune cells into the CNS. However, BBB-ECs are susceptible to damage under pathological settings, such as viral infection, trauma or systemic inflammation. The upregulation of adhesion molecules, loss tight junctions, or redistribution of cytoskeletal proteins can impair the integrity and/or permeability of the BBB. Pericytes are also important compounds for BBB development and function. Pericytes contact BBB-ECs to downregulate BBB permeability by releasing PDGF-Rβ and TGF-β. In our recent study, we found that the adhesion molecule CD146 is important for the development and maturation of BBB. The overexpression of CD146 on developing BBB-ECs triggers the recruitment of pericytes to form tight endothelial-pericyte contacts. In the mature stage, CD146 is downregulated on BBB-ECs while remaining highly expressed on pericytes. During conditions of inflammation, CD146 expression is upregulated on BBB-ECs and subsequently promotes the transmigration of inflammatory cells across the BBB Overexpressed CD146 sheds in settings in which MMPs are present, and the soluble form of CD146 then distributes into the body fluid In summary, this study provides convincing clinical evidence to show that CSF sCD146 is sensitive for monitoring BBB damage and neuroinflammation. Furthermore, we demonstrate that sCD146 directly compromises the barrier function of human brain ECs via integrin αvβ1-mediated intracellular signaling events. Based on these findings, we suggest CSF sCD146 as a promising diagnostic index for evaluating early BBB damage in multiple neurological diseases and as a potential therapeutic target for various neuroinflammatory diseases.This study enrolled 823 patients from 30 provinces of China who were diagnosed with or without neuroinflammatory diseases between 2011 and 2017. Their clinical diagnosis was confirmed at Peking University First Hospital. Written informed consent was obtained, and ethical approval was granted by the Ethics Committee of the Peking University First Hospital and Institute of Biophysics, Chinese Academy of Sciences before human sample collection. We also obtained written informed consent from guardians on behalf of the children who participated in this study. CSF samples were collected by lumbar puncture for diagnostic or surgical purposes. CSF samples were immediately centrifuged at 4 °C at 4000 rpm for 5 min. For serum collection, blood samples were taken from an antecubital vein and centrifuged at 4 °C at 4000 rpm for 5 min. The supernatants of the CSF and serum samples were separated as soon as possible, coded, frozen, stored at -80 °C and thawed just before analysis to avoid loss of biological activity.According to etiology, the patients were divided into four groups: IIDD, CNSI, PNS and NIND. IIDD included relapsing MS and neuromyelitis optical spectrum disorders (NMOSD). Relapsing MS (n=93) and remitting MS (n=44) were diagnosed according to the McDonald criteria 2SO4 was used to stop the reaction. The OD value was measured at a wavelength of 450 nm.The concentration of sCD146 in CSF or serum was measured with a sandwich ELISA protocol that was modified from that of a previous report CSF supernatants were diluted 1:1 with PBS and analyzed simultaneously for 9 different inflammation-related factors, namely, MMPs (MMP2 and MMP9), sICAM, sVCAM, IL-1β, IL-17A, IFN-γ, IL-10 and TNF-α. The multifactor detection kit  was used according to the manufacturer's instructions. All the data from the samples were analyzed using FlowCytomixPro software.3 was kindly provided by Prof. Pierre-Olivier Couraud . MycoAlert  was used to verify a lack of mycoplasma contamination every 2 months. hCMEC/D3 cells were grown on slides in 24-well plates and stimulated with the appropriate conditional medium. Treated cells were fixed with 4% paraformaldehyde (PFA) for 20 min, permeabilized with 0.1% Triton X-100 for 3 min and then washed with PBS. Cells were blocked with 5% skim milk in PBS for 30 min at 37 °C. Cells were incubated overnight at 4 °C with primary antibodies to occludin , JAM-1  and ZO-1 . Cells were incubated with appropriate fluorescence-labeled secondary antibodies for 45 min at 37 °C. Nuclei were counterstained with DAPI. Cells were mounted with cover slides in antifading agent . In addition, cytoskeletal protein F-actin was directly stained with phalloidin  for 30 min at 37 °C. Cells were analyzed using a laser scanning confocal microscope (OLYMPUS FV1000).The human BBB endothelial cell line hCMEC/Din vitro BBB permeability assay was performed using a transwell system . hCMEC/D3 cells were seeded into the upper chamber, and confluent cells were treated with appropriate conditional medium, followed by the addition of HRP in to the upper chamber. One hundred microliters of culture medium were collected from the bottom chamber, and TMB solution was used to measure the OD value (450 nm) at the appropriate time point.An EC = (TEER EC + filter - TEER filter) × S (membrane area), unit: Ω·cm2.The TEER value was measured with cell resistance meter  according to the manufacturer's instructions. The TEER 3 apoptosis, the Annexin V-FITC and 7-AAD apoptosis kit  was used for flow cytometric detection. Briefly, cells were washed with cold PBS and suspended in 1× binding buffer. A total of 1×106 cells in 100 μL were used for analysis. A total of 5 μL of Annexin V-FITC was added to the cells for 10 min at room temperature, and the mixture was protected from light. After incubation, 5 μL of 7-AAD solution was added for another 5 min at room temperature. Without washing the cells, PBS was added to the cells up to a volume of 500 μL, and the cells were analyzed by flow cytometry within 1 h.To assess hCMEC/D2, 2 mmol/L MnCl2, 1.5% Triton X-100, 2 mmol/L PMSF, and 10 μg/mL leupeptin, and the lysates were precleared with various types of nonspecific IgG antibodies and Protein G-plus agarose beads. The precleared cell lysates, each containing 1 mg of total protein, were separately incubated for 4 h at 4 °C with human Fc or Fc-sCD146, followed by the addition of 25 μL of precleared Protein G-plus agarose beads and incubation overnight at 4 °C. The proteins eluted from the Protein G-plus agarose beads were subjected to SDS-PAGE under reducing conditions and detected with an anti-integrin αv , β1  or anti-Fc antibody .For the coimmunoprecipitation assay, the cells were lysed with lysis buffer containing 20 mmol/L Tris-HCl (pH 7.4), 150 mmol/L NaCl, 1 mmol/L CaCl3 cells were harvested, preincubated in serum-free medium with various treatments for 30 min on ice, seeded into the plates, and incubated for another 30 min at 37 °C. Nonattached cells were removed by vigorous agitation and aspiration. Attached cells were counted under an optical microscope.A 96-well plate was precoated with 10 μg/mL rhsCD146. hCMEC/DCells were treated under various conditions and then lysed with a cell lysis reagent . The simple western immunoblots were performed on a Wes (ProteinSimple) using the Size Separation Master Kit with Split Buffer (12-230 KD) according to the manufacturer's standard instruction. The antibodies used included the following: p38 ; phospho-p38 ; ERK1/2 ; phospho-ERK1/2 ; JNK ; phospho-JNK ; Akt ; phospho-Akt ; p65 ; phosphor-p65 ; β-actin ; and anti-angiomotin .The results are expressed as the mean±SD. The nonparametric Mann-Whitney U test was employed for comparisons of the cytokine levels in each group. Correlations between the sCD146 level in the CSF and clinical indexes were evaluated using multiple linear regression and Spearman's Rank correlation coefficient. The cut-off value of sCD146 was calculated as the mean±2SD of the NIND group. We assessed the sensitivity, PPVs and NPVs of each biomarker to distinguish neuroinflammatory diseases from NIND. SPSS 11.0 for Windows was used to perform the analysis. The criterion for statistical significance was defined as p<0.05.Supplementary figures and tables.Click here for additional data file.