Four patients  with meningeal relapse after alloSCT and subsequent treatment with nilotinib were identified. A total of 17 cerebrospinal fluid (csf) and serum samples were assessed for nilotinib concentration and patient outcome was recorded. Nilotinib concentrations showed a low median csf/plasma ratio of 0.53% (range 0.23–1.5%), yet pronounced clinical efficacy was observed with long-lasting responses (>1 year) in three patients. Comparison with historical data showed a trend towards superior efficacy of nilotinib versus imatinib. Despite poor csf penetration, nilotinib showed significant clinical activity in CNS relapse of BCR-ABL+ leukemias. As nilotinib has a high protein-binding affinity, the low-protein concentration in csf could translate into a relatively higher amount of free and therefore active nilotinib in csf as compared to blood, possibly explaining the observed efficacy. Thus, treatment with a 2nd generation TKI warrants further investigation and should be considered in cases of CNS relapse of BCR-ABL-positive leukemia after alloSCT.Central nervous system (CNS) involvement is a severe complication of BCR-ABL-positive leukemia after allogenic stem cell transplantation  associated with fatal outcome. Although second-generation tyrosine-kinase inhibitors (TKI) such as nilotinib have shown activity in systemic BCR-ABL Nonetheless, 15–20% of patients (pts) with BCR-ABL+ ALL or CML-BC develop CNS relapse during ongoing imatinib therapy [In BCR-ABL-positive acute lymphoblastic leukemia (ALL) and advanced stages of chronic myeloid leukemia ) central nervous system (CNS) involvement is a lethal complication, typically occurring late in the course of the disease, particularly after allogeneic stem cell transplantation . Imatinib was the first specific BCR-ABL tyrosine kinase inhibitor (TKI) to be approved for the treatment of BCR-ABL therapy  despite  In vitro tests of these new TKIs show considerably higher activity in comparison with imatinib, with a 40-fold increased potency for nilotinib and a 325-fold for dasatinib [Second-generation TKIs such as dasatinib and nilotinib have been approved for the treatment of CML patients who are refractory or intolerant to imatinib.+ CNS disease however is not clear and warrants further investigation. Efficacy of dasatinib in BCR-ABL+ CNS leukemia has been demonstrated in an animal model and was furthermore observed in 11 patients. This study also analysed the csf/plasma ratios of dasatinib in 3 patients, showing low levels of dasatinib in csf. For nilotinib, no data on csf penetration and clinical efficacy against CNS relapse of BCR-ABL+ leukemia is available so far. Specifically, in pediatric patients very little data has been published on treatment with second generation TKIs at all.Moreover, nilotinib and dasatinib were able to overcome imatinib resistance caused by several BCR-ABL tyrosine kinase domain mutations. The potential of these second-generation TKIs in targeting BCR-ABL+ aggressive leukemia after alloSCT.We therefore investigated the clinical activity and csf penetration of nilotinib in four patients with CNS relapse of BCR-ABL+ ALL, 1 CML-BC; 3 male, 1 female, age 15–49 years) in two centers  treated with nilotinib for CNS relapse after alloSCT were identified. Previous therapeutic regimens include imatinib and dasatinib TKI-therapy for systemic relapse in patients 1–3 while patient 4 had received dasatinib in combination with intrathecal therapy and radiotherapy for meningeal relapse. Investigation of nilotinib csf concentration was performed within a noninterventional diagnostic study, approved by the local ethics committee of the Charite Berlin, Germany (EA4/023/08). Informed consent was obtained from all patients or their legal representatives according to Good Clinical Practice guidelines and in concordance with the Declaration of Helsinki.Four patients  scan, csf analysis by lumbar puncture with analysis consisting of cytology including csf differential cell analysis, csf clinical chemistry , csf immunophenotyping, chimerism analysis of the csf, and molecular testing for BCR-ABL using PCR for detection of minimal residual disease (MRD).Further lumbar puncture for assessment of leukemia and nilotinib concentration in csf was performed as clinically indicated. Blood samples were drawn concurrently to calculate csf/plasma ratios for nilotinib. Response assessment was based on analysis of cell count, cytomorphology, immunophenotyping, BCR-ABL quantitative RT-PCR (qPCR) , and chiA csf complete remission (CR) was defined as negative csf cytology, normal csf cell count, immunophenotyping, and cytogenetic and chimerism analysis; a csf partial remission (PR) was defined as negative cytology and csf cell count, but detection of residual disease by either csf immunophenotyping, cytogenetics, or chimerism analysis. Csf minimal residual disease (MRD) negativity was defined as a CR without detection of BCR-ABL amplicons in csf PCR.All four patients experienced CNS relapse after alloSCT. Clinical signs and symptoms that lead to the suspicion of CNS relapse consisted of headache (patients 1–4), facial palsy (patients 1–3), vomiting (patients 1 and 4), and seizures (patient 4). In addition, MRI scans and csf analysis showed the following results for these patients, confirming the diagnosis.Initial diagnosis of CNS relapse was established by positive cytology, detection of BCR-ABL transcripts, and decrease in donor chimerism in csf with concurrent radiological findings  in patient 1. In patient 2, a positive csf cytology and immunophenotyping showing lymphatic blasts, a decrease in donor-chimerism, and positivity for BCR-ABL qPCR in csf could be found serologically while MRI scans showed a slight enhancement and an increase in contrast agent uptake. Patient 3 initially had an unremarkable MRI; however in the csf BCR-ABL transcripts could be repeatedly and unequivocally detected, while csf cytology, cell count, chimerism analysis, and immunophenotyping were negative. Patient 4 showed positive radiomorphological results , leukemic blasts in csf cytology, positive immunophenotyping, and BCR-ABL qPCR in csf.Median time from alloSCT to first CNS relapse was 54 months (range 17–132 months). Median duration of nilotinib treatment was 541 days (range 99–1786 days). Nilotinib treatment was discontinued in patient 1 on day 431 because of progressive disease , in patient 2 on day 99 because of detection of T315I mutation and systemic disease progression. Both patients died of progressive leukemia. Patient 3, while on nilotinib monotherapy for 847 days, experienced meningeal progression and periodically received additional intrathecal chemotherapy and rituximab from thereon until systemic progression with detection of T315I and F317L mutation. Subsequently TKI treatment was switched after 1786 days of nilotinib treatment to ponatinib. The patient is alive at the time of the paper writing and continuing ponatinib treatment. Patient 4 is currently in remission and continuing nilotinib treatment.Patient 1 achieved a complete remission (CR) with resolution of neurological symptoms, normalization of cytology/immunophenotyping, and restoration of csf donor chimerism; however CNS minimal residual disease (MRD) defined by csf BCR-ABL qPCR positivity persisted. Patient 2 had stable csf cell count and stable csf BCR-ABL transcript levels with minor clinical improvement. Patient 3 showed considerable improvement in symptoms and achieved csf MRD negativity on days 191 and 224 after start of nilotinib treatment. However, minimal amounts of csf BCR-ABL transcripts intermittently reappeared, while csf cytology and chimerism remained negative. When meningeal progression with positive cytology was detected 847 days after start of nilotinib monotherapy intrathecal chemotherapy was added to nilotinib, resulting in intermittent csf MRD negativity until systemic detection of T315I and F317L mutation on day 1786 of nilotinib treatment. Patient 4 achieved a CR in bone marrow and CSF and MRD negativity after three months of treatment with nilotinib and concomitant intrathecal chemotherapy. This patient is continuing nilotinib treatment in complete remission.In summary, long-lasting responses of more than one year were observed. Patients 1, 3, and 4 had sustained clinical improvement and csf responses while on nilotinib for 431 days (patient 1), 1786 days (patient 3), and >650 days (patient 4), respectively. Both patient 3 and patient 4 are alive; patient 4 is continuing nilotinib treatment.P > 0.17, Median time to progression (TTP) leading to discontinuation in the nilotinib-treated patients was 541 days with 1 of 4 patients still in remission. Compared with historical data published for imatinib [n = 5, median csf/plasma ratio 0.26%; range 0.23–0.47%), 13 ng/mL in patient 2 , 7 ng/mL in patient 3 , and 2.7 ng/mL  in patient 4. Additionally, a ratio of csf free (unbound) nilotinib and free plasma nilotinib was calculated based on the published nilotinib protein binding of 98%  with the pediatric patient (patient 4) representing a single case. However, CNS relapse of BCR-ABL+ leukemia after allogeneic stem cell transplantation is a rare event with lethal consequences, impeding observation of larger patient cohorts. The number of patients with available pharmacological data (n = 4) is similar to that of patients for dasatinib by Porkka et al. (n = 3) [n = 7) [To our knowledge, this is the first report on nilotinib csf pharmacology and efficacy in patients with CNS manifestations of BCR-ABL (n = 3)  and the  [n = 7) .+ leukemia the authors found limited csf penetration with low csf/plasma ratios for all 3 patients evaluated. Complete responses were observed in 7/11 patients; the majority received concomitant antileukemic treatment  but csf MRD analysis for response evaluation was not reported. Additional animal experiments showed superior survival of mice inoculated with CNS leukemia and treated with dasatinib compared to imatinib in the control group. In summary, these combined findings suggest improved efficacy of dasatinib over imatinib in targeting CNS BCR-ABL+ leukemia despite poor csf penetration.Csf nilotinib concentrations were within the range of those reported for imatinib . For das+-CNS leukemia patients suggests similar clinical activity with sustained responses for more than 1 year  despite low csf drug levels.Our experience of nilotinib treatment in BCR-ABL in vitro data suggest considerably higher activity compared to imatinib and there might not be a strict dose-response relationship. As csf is a low protein compartment TKI and is more likely to exist as protein-unbound and therefore as active drug. The ratio of free (not-protein-bound) nilotinib in csf relative to free drug in plasma could therefore be increased 5–30 folds compared to the csf/plasma ratio of total nilotinib  underlining the necessity of effective BCR-ABL suppression. The pronounced influence of mutation status is also highlighted by the clinical course of the other patients. Patient 1 experienced meningeal progression during dasatinib and INNO406 treatment, both drugs supposedly penetrating into the CNS [+ CNS disease should be strongly considered if the patient fails to respond, a strategy that has been proven effective in systemic treatment [Evaluating the exact influence of TKI-therapy in patients with BCR-ABL the CNS , 11. Des the CNS . The outreatment .+ CNS relapse after alloSCT despite limited csf concentrations which may be attributed to the low-protein environment of csf. As treatment options are limited for these heavily pretreated patients with poor prognosis, clinical trials should further elucidate the role of different TKIs in this clinical setting. In light of our data, the addition of a 2nd generation TKI should be considered in BCR-ABL+ meningeal relapse after allogeneic stem cell transplantation.In summary, our findings suggest relevant activity of nilotinib in BCR-ABL


The identification of reliable biomarkers for Alzheimer’s disease (AD) remains challenging. Recently, abnormal levels of microRNAs (miRNAs) miR-27a, miR-29a, miR-29b, and miR-125b in cerebrospinal fluid (CSF) of AD patients were reported. We aimed to confirm the biomarker potential of these miRNAs for AD diagnosis. Additionally, we examined the influence of blood contamination on CSF miRNA levels as potential confounding factor. We studied expression levels of the four miRNAs by quantitative PCR in CSF samples of AD patients and non-demented controls, and in blood-spiked CSF. Levels of miR-29a, but not of the other three miRNAs, were increased by a factor of 2.2 in CSF of AD patients. Spiking of small amounts of blood into CSF revealed that miR-27a and miR-29a, but not miR-125b levels were strongly influenced by the number of blood cells in the sample. In conclusion, miR-29a may be a candidate biomarker for AD, but only when used in cell-free CSF. Alzheimer’s disease (AD) is the most common form of dementia worldwide. Diagnosis of AD is currently supported by measurement of total tau (t-tau), phosphorylated tau (p-tau), and amyloid-β42 (Aβ42) in cerebrospinal fluid (CSF). However, diagnostic accuracy of this panel of biomarkers has its limitations . MicroRNAltered expression of several miRNAs in CSF of AD patients has been previously reported. In two recent publications, it was reported that the levels of miR-27a and miR-125b are lower in CSF of AD patients than in controls , 9, wherCSF of 18 AD patients and 20 healthy individuals was obtained at the Radboud University Medical Center, Nijmegen. The samples had been collected by lumbar puncture in polypropylene tubes, centrifuged, aliquoted, and stored at -80 °C. AD patients had been diagnosed as probable AD according to the criteria of the National Institute of Neurological and Communicative Disorders and Stroke as well as Alzheimer’s Disease and Related Disorders Association (NINCDS/ADRDA), based on clinical evaluation, magnetic resonance imaging, and neuropsychological testing . More reFresh whole blood from a healthy individual was spiked into 500 μl aliquots of cell-free CSF to final concentrations of 10 to 20,000 erythrocytes/μl CSF. Cell count was confirmed using flow cytometry. Samples were incubated at room temperature for 3 h, followed by centrifugation to remove cells to imitate CSF processing in clinical circumstances. Likewise, the effect of incubation time was examined by applying incubation periods of 0 to 180 min between spiking of 20,000 cells/μl and centrifugation.http://appliedbiosystems.com.RNA isolation, reverse transcription, pre-amplification, and qPCR were performed as previously described by us . Primer -ΔCt, with ΔCt = CtmiRNA − GM, and t test, and the Mann Whitney U test was used for non-parametric data. Correlations were determined using Pearson r. Statistical outliers were identified using the Dixons Q test and the Grubbs test for outliers. P values were considered significant when p < 0.05. Receiver operator characteristics (ROCs) analysis was used to determine the diagnostic value of miR-29a and miR-125b. Area under the curve (AUC) was calculated with 95 % confidence intervals. The optimal cut-off value was determined using the Youden index (sensitivity + specificity − 1.0) and likelihood ratio (sensitivity/(1 − specificity)).To normalize expression levels of miRNAs, the geometric mean (GM) of three reference RNAs was used. Those had similar cycle threshold (Ct) values in the CSF of AD and control groups. The following formulas were used to calculate relative expression levels (RELs) and GM: REL = 2p = 0.0001, Fig. p = 0.025, Fig. p = 0.058).We measured miR-27a, miR-29a, miR-29b, and miR-125b levels in (nearly) cell-free CSF samples of AD patients and controls. Numbers of leukocytes and erythrocytes and total protein levels did not differ between groups Table , but mear = –0.89, p = 0.001, Fig. r = –0.88, p = 0.002, Fig. We studied the effect of blood contamination, which often occurs during lumbar puncture of CSF, on miRNA levels. We simulated this effect by spiking whole blood into cell-free CSF. Mean Ct values of miR-27a and miR-29a were negatively correlated with the numbers of erythrocytes  have been described that are differentially expressed in the brain and regulate proteins involved in AD pathogenesis (see for an overview  and [14]We found similar levels of miR-27a in CSF samples of AD patients and controls, which contradicts the previously reported decrease of miR-27a levels in AD CSF . A possiMiRNAs are found in plasma and in serum , 18, andThis study demonstrated that reproducing previously reported CSF miRNA expression data can be challenging and that blood contamination may be a major factor that influences miRNA levels. Hereby, blood-derived miRNAs may alter the levels in CSF, but possibly also other factors such as miRNA degrading enzymes present in the blood may play a role in influencing miRNA CSF levels. For our study, we also cannot exclude that the mean difference in age, although not statistically significant, between the AD and the control group may have biased our results. This, however, requires further study. Other factors that may account for contradictory miRNA expression data are normalization methods  and coho


Expression of soluble CD163 (sCD163), a macrophage/microglia biomarker, is increased in inflammatory conditions, and sCD163 levels in the cerebrospinal fluid (CSF) have recently been shown to be elevated in patients with multiple sclerosis (MS): the sCD163 CSF/serum ratio was elevated in patients with relapsing-remitting MS (RRMS), primary progressive MS (PPMS), and clinically isolated syndrome (CIS) compared with symptomatic controls.To investigate the contributions of the sCD163 CSF/serum ratio to a biomarker panel focusing on inflammation and axonal degeneration in newly diagnosed MS; thus optimising a diagnostic biomarker panel for MS.After a full MS diagnostic work-up, including collection of paired samples of CSF and serum, 125 patients were included in this study. Patients were divided into groups based on their diagnosis, and patients with normal clinical and paraclinical findings were defined as symptomatic controls. Serum and CSF levels, ratios, and indices of sCD163, CXCL13, osteopontin, neopterin, and CSF levels of neurofilament light polypeptide were determined by enzyme-linked immunosorbent assays (ELISAs). For sCD163 the results constitute a post-hoc analysis of already published data.All tested biomarkers, notably the sCD163 ratio, the CXCL13 ratio, the NEO ratio, the CSF level of NfL, the IgG index, and the serum level of OPN, were significantly correlated to RRMS, PPMS, and/or CIS. The individual biomarkers in single tests had a lower performance than the IgG index, however, their combined receiver operating characteristic (ROC) curve demonstrated excellent diagnostic discriminatory power.The biomarker panel showed distinct profiles for each patient group and could be a valuable tool for clinical differentiation of MS subgroups. The combined ROC analysis showed that sCD163 contributes positively as a diagnostic marker to a panel of established MS biomarkers. Patients with PPMS were demonstrated to have significantly elevated levels of both inflammatory and degenerative markers. Multiple sclerosis (MS) is the most common demyelinating disease of the central nervous system (CNS) in young adults. The cause of the disease is unknown, yet it is generally assumed to be complex interactions between environmental factors and genetic susceptibility –3. At thA characteristic feature of MS lesions (plaques) is the abundant presence of macrophages in all types (I-IV) of MS lesions . MacrophWe have recently demonstrated a significant association between levels of the sCD163 CSF/serum ratio (sCD163 ratio) and MS/CIS . Here, iThe objectives of the present study were to investigate the contributions of the sCD163 ratio to a biomarker panel focusing on inflammation and axonal degeneration in patients with newly diagnosed MS. The study was performed on a large cohort of patients diagnosed with remitting relapsing MS (RRMS), primary progressive MS (PPMS), or clinically isolated syndrome (CIS). All cases were compared with a group of symptomatic controls (SC) . The appThe study was conducted in accordance with the Ethical Declaration of Helsinki and all patients gave written, informed consent. The study and the material for informed consent were approved by The Central Denmark Region Committee on Biomedical Research Ethics .Patients admitted for further diagnostic investigations to the MS clinic, Department of Neurology, Aarhus University Hospital were consecutively included in the study during a period from September 2009 to December 2011. A full diagnostic work-up included medical history, clinical examination, MRI of the entire neural axis, CSF analyses  index), and evoked potentials as recommended . CSF andIn total 183 patients agreed to participate in this study and in accordance with consensus guidelines  serum anThis patient cohort was also the cohort in the recent study of sCD163 levels and macrophage activity in MS . In the Levels of sCD163 were analysed by ELISA as previously described . In brieLevels of CXCL13, NEO, and OPN were analysed in both serum and CSF and levels of NfL were only analysed in CSF by enzyme-linked immunosorbent assay (ELISA) following the instructions given by the manufacturers. The available kits for NfL at the time of analysis were restricted to be used only with CSF. Samples were run in duplicates and a coefficient of variation (CV) was calculated, accepting only values ≤15%. Intra assay variations were calculated from six individual measurements of a known standard on each plate and values ≤15% were accepted (ranges: CXCL13 (2.02–8.57), NEO (3.54–13.67), NF-Light (0.58–5.94%), and OPN (2.00–9.80%)). Samples with values exceeding the highest point of the standard curve were diluted and reanalysed. The diagnoses were established before the results of the sCD163 analyses were received. For the other four biomarkers each plate contained 36 randomly selected samples, and each sample were labelled with a study ID and assayed in a manner blinded to the clinical status of the patients.2SO4) were added and the absorbance was measured on an ELISA plate reader  at 450 and 540 nm. Concentrations were calculated by linear regression in Excel.CXCL13 levels  were measured in a competitive ELISA assay by adding 100 μL assay diluent and 50 μL of sample to each well, followed by a two hour incubation period. After 4 × wash, 200 μL of conjugate (mAb-HPR) were added, followed by two hours incubation. After 4 × wash, 200 μL substrate solution (TMB) were added and the plate was incubated for 30 min protected from light. Finally, 50 μL of stop solution (H2SO4) were added and the absorbance was measured on an ELISA plate reader  at 450 and 620 nm. Concentrations were calculated by 4-parametric statistical regression in GraphPad Prism.NEO levels  were measured by adding 100 μL enzyme conjugate (NEO), 20 μL of sample and 50 μL of NEO antiserum (pAb-HPR) to each well, followed by a 90 min incubation period. After 4 × wash, 150 μL of substrate solution (TMB) were added and the plate was incubated for 10 min protected from light. Finally, 150 μL of stop solution (H2SO4) were added and the absorbance was measured on an ELISA plate reader  at 450 and 540 nm. Concentrations were calculated by 4-parametric statistical regression in GraphPad Prism.NfL (NF-light)  levels were measured by adding 50 μL sample dilution buffer and 50 μL of sample to each well, followed by one hour incubation. After 3 × wash, 100 μL of tracer (mAb) were added, followed by 45 min incubation. After 3 × wash, 100 μL conjugate (HPR) were added and the plate was incubated for 30 min. After 3 × wash, 100 μL substrate solution (TMB) were added and the plate was incubated for 15 min protected from light. Finally, 50 μL of stop solution  were measured by adding 100 μL assay diluent and 50 μL of sample (diluted 1:25) to each well, followed by a two hour incubation period.2SO4) were added and the absorbance was measured on an ELISA plate reader  at 450 and 540 nm. Concentrations were calculated by 4-parametric statistical regression in GraphPad Prism.After 4 × wash, 200 μL of conjugate (pAb-HPR) were added, followed by two hours incubation. After 4 × wash, 200 μL substrate solution (TMB) were added and the plate was incubated for 30 min protected from light. Finally, 50 μL of stop solution . Descriptions of MRI data was conducted by a neuro-radiologist and confirmed by a senior neurologist who viewed all scans in the IMPAX system at the Department of Neurology, AUH.For data collection we used Microsoft Excel and all statistical analysis was performed using STATA12. Please see Figure A in For calculations of ratios (i.e. sCD163 ratio) the CSF concentration was divided by the serum concentration, and indices were calculated by dividing the biomarker CSF/serum ratio by the albumin ratio.The receiver operating characteristic (ROC) analyses were performed for the variables that were most significantly different from the SC group in the regression analysis. The parameter results for patients with RRMS, PPMS, and CIS are combined as true positives and plotted against SC as true negatives. A logistic regression analysis was performed for the combined diagnostic properties for each biomarker and the IgG index, and the entire biomarker panel including the IgG index . For a vSoluble CD163 was detectable in CSF and serum from all individuals, as reported in . The sCDCXCL13 was detectable in serum from all individuals, but CXCL13 in CSF was undetectable in samples from one patient each from the RRMS and CIS patient groups, respectively, and in samples from five patients from the SC group. For calculating statistics the lower detection limit of the assay (0.0016 μg/L) was used where concentrations of proteins were below the lower detection limit. The CXCL13 levels, ratio and index values are presented in NEO was detectable in CSF and serum from all individuals. The NEO levels, ratio and index values are presented in NfL was detectable in CSF from all individuals. The NfL CSF levels are shown in OPN was detectable in CSF and serum from all individuals. The OPN levels, ratio and index values are presented in Spearman and Pearson correlation analyses for the biomarkers are presented without and with the Bonferroni correction in Patients in the PPMS group were significantly older than patients in the RRMS, CIS and SC groups; and levels of sCD163 in both serum and CSF were found to correlate with age, however this was not the case for the sCD163 ratio.The levels of sCD163 in serum and CSF were not significantly correlated to gender or any of the clinical measures: time since last attack, EDSS, mean no. of relapses, mean disease duration, enhancing lesions or the total number of MRI white matter lesions (TNL). There were moderate to strong correlations between most biomarker variables and CSF protein, CSF cells and IgG index.Pearson pairwise correlation analysis of biomarkers is presented without and with the Bonferroni correction in Results from the linear regression analysis are presented in The geometric mean (GM) values of the sCD163 ratio was significantly increased for the patients with RRMS: 1.36 fold , CIS: 1.33 fold , and especially PPMS: 1.57 fold .Of the other variables, most notably the CXCL13 ratio was elevated 13 fold  in patients with RRMS, but also significantly elevated in patients with PPMS: 5.52 fold  and CIS: 6.86 fold (95%CI: p = 0.003). The NEO ratio was significantly elevated in all groups, especially RRMS: 1.44 . The NfL CSF was notably increased for patients with PPMS: 2.49 fold .It is noteworthy, that OPN was a variable in which serum levels were elevated compared with the SC group, significantly for the CIS group: 1.62 fold .The biomarkers with the optimal differentiating properties were established from the regression analysis: the sCD163 ratio, the CXCL13 ratio, the NEO ratio, the NfL CSF, and the OPN serum. These parameters were further analyzed in a biomarker profile , a ROC aThe biomarker profile was determined by categorizing the GM for each patient group in the following manner: Very high ; High . To examine the sensitivity and specificity of each biomarker a ROC analysis was performed for each biomarker . The sCDThe ROC analyses were followed by a logistic regression model see  in whichet al [The biomarkers selected for this study have all documented discriminating potency providing differentiation between diseased and healthy subjects. As pointed out by Xia et al  this soret al , 63. As et al . In our et al , we demoet al [For sCD163 the results constitute a post-hoc analysis of already published data . In the et al  none of et al . HoweverOverall, we find it noteworthy that (except for OPN) the biomarker ratios and indexes (i.e. levels in CSF in relation to levels in serum) were optimal in differentiating between patient groups rather than levels in either CSF or serum. The well-established MS diagnostic marker, the IgG index, also measures a relation between levels in the CNS and in the systemic circulation. This underscores that measuring biomarker levels concomitantly in both compartments is advantageous.In the present study patients with PPMS had significantly elevated sCD163 CSF median levels compared with SC. This is also the case for the NEO CSF levels. Interestingly, the levels of sCD163 and NEO in serum and CSF are decreased / increased in similar ways Fig  and 2j. For the other biomarkers analysed, the results were as follows: the median levels of the CXCL13 ratio were significantly increased for all patient groups compared with SC. This increase attained the highest level of significance for patients with RRMS, followed by CIS and PPMS. This is in agreement with the general hypothesis that the pathological mechanism in these two groups is mainly driven by inflammation of which CXCL13 is a marker .no. of attacks (r = 0.32) but there was no correlation to time since last attack  were significantly elevated in PPMS.S1 Dataset(DOCX)Click here for additional data file.S1 Previous Publication(PDF)Click here for additional data file.


MetAMOS represents an important step towards fully automated metagenomic analysis, starting with next-generation sequencing reads and producing genomic scaffolds, open-reading frames and taxonomic or functional annotations. MetAMOS can aid in reducing assembly errors, commonly encountered when assembling metagenomic samples, and improves taxonomic assignment accuracy while also reducing computational cost. MetAMOS can be downloaded from:  Meta[*MH0002  (run acc[*MH0002 ,91; *MH0[*MH0024  (run acc[*MH0024 ,94; *MH0[*MH0082  (run acc[*MH0082 ,97.MetAMOS is available from . MetAMOSbp: base pair; GB: Gigabyte; HMP: Human Microbiome Project; MetaHIT: Metagenomics of the Human Intestinal Tract; HTML: Hyper-Text Markup Language; kbp: thousand base pairs; Mbp: million base pairs; ORF: open reading frame; SNP single-nucleotide polymorphism; SRA: Sequence Read Archive.The authors declare that they have no competing interests.MP, AP, SK, and TT conceived the project and designed the experiments and software. BO and AD contributed to the design of experiments and software. TT, SK, DS, BO, and AD implemented and tested the software. TT, SK, IA, BL, and AD performed the experiments. SK and TT collected and analyzed the results. MP, TT, SK and AP wrote the manuscript. All authors read and approved the final manuscript. SK and TT contributed equally to this work.Figure S1 and Table S1.Click here for file


The 7th annual Global Handwashing Day will be observed October 15, 2014. This observance increases awareness and understanding of handwashing with soap as an effective and affordable way to prevent disease around the world.Handwashing with soap has an important role to play in child survival and health. Approximately 2.2 million children aged <5 years die each year from diarrheal diseases and pneumonia, the top two causes of death among young children globally . HandwasAlthough persons around the world clean their hands with water, few use soap to wash their hands. Washing hands with soap removes bacteria much more effectively .http://www.cdc.gov/features/globalhandwashing. General handwashing information is available from at http://www.cdc.gov/handwashing. Information on water-related hygiene is available at http://www.cdc.gov/healthywater/hygiene/index.html.Additional information on Global Handwashing Day is available from CDC at


Clostridium difficile or more complex disease states, such as inflammatory bowel disease (IBD). The “gut microbial organ” from the donor that is used in these transplants may serve to transfer genetic material between donor and recipient via virus-like particles, specifically bacteriophages, that infect the bacterial component of the microbiota. The recently published study by Chehoud et al. provides evidence for not only the transfer of bacteriophages during FMT but also the transfer of multiple populations of bacteriophages to recipients from the donor microbiota used . While the clinical significance of these findings remains unclear, nothing short of a diligent and persistent effort is needed to define the intended and unintended consequences of FMT.Fecal microbiota transplantation (FMT) has received increased attention as a therapy for correcting intestinal dysbiosis and restoring a state of health in patients suffering from either recalcitrant infection by  Clostridium difficile have been shown, capturing the attention and imagination of the press, public, and medical profession , which was first alluded to in the 4th century in China, is the transfer of a donor’s gut microbiota to a recipient with the intent of correcting intestinal dysbiosis and restoring states of health , 2. Its ofession –7. Not sfession –. Adding fession – and, in C. difficile colitis, where a patient’s microbial organ is essentially absent, replacement with a donor microbial organ has a strong rationale and proven efficacy. However, in most other cases, the donor microbiota would have to restore or supersede functional problems of the existing microbiota, a task that is both formidable and unpredictable. Like “traditional organ” transplants, potential problems with pathogens, the functionality of donor microbiota, and “rejection” arising from microbe-microbe and microbe-host incompatibilities need to be taken into consideration. The work presented by Chehoud et al. highlights a unique feature of the “gut microbial organ” that should be monitored as well, and it provides a framework for how to do this . Furthermore, by constructing longer contigs (>3 kb), they were able to track the transfer of phage between donor and recipient during FMT. They additionally supported these proposed transfers by using quantitative PCR targeting four specific contigs detected in at least two of the three recipient patients. More interesting was the detection of conserved protein domain families that correlated with the frequency of transmission. These included initiator replication proteins and lambda integrases, which are indicative of a temperate replication strategy. Likewise, based on the assembled contigs, the viral family that was preferentially transferred between donor and recipient was the Siphoviridae , known to be rich in temperate phages. The fact that numerous temperate phages were transferred suggests a common strategy used by phages to optimize their transmission and dispersal between hosts. By observing and tracking the transfer of bacteriophage populations between individuals, the study authors proposed that the transfer of bacteriophage populations is a general characteristic of FMT. However, the longer-term clinical significance of these findings remains uncertain. Though the recipient patients were monitored throughout the course of the study and were found to be symptom-free for at least 4 weeks, all three eventually relapsed and reverted to therapy with immunomodulators.This study looked at a single healthy adult donor and three pediatric ulcerative colitis recipient patients who received 22 to 30 FMT treatments over the course of 6 to 12 weeks. Samples collected from the donor and recipients  were enriched for virus-like particles (VLPs) and analyzed using metagenomic-enabled approaches. This strategy for enriching VLPs from samples up front allowed for a more detailed analysis, since the recovered sequences and functional gene “payload” could be directly ascribed to the enriched fraction of metagenomic data. The initial comparisons of VLP-derived sequences with reference databases yielded a low fraction of aligned reads (average of 17.9%), highlighting their generally underdescribed nature. The investigators also assembled the VLP-enriched fraction of sequence data to provide genomic context (synteny) for functional genes that were then annotated using several protein databases . Despite these efforts, they were only able to annotate an average of 7.3% of the detected proteins (based on predicted open reading frames), which also highlights the difficulties associated with ascribing functions to VLPs. Prominent conserved protein domains found were phage-associated structural components , but domains associated with effector proteins were also detected, including bacteriocins, beta-lactamases, and reverse transcriptases. Despite the low success for annotating functional proteins, the investigators were able to produce circular contigs by assembling the reads, indicating sequencing of full viral genomes. As a result, many contigs could be ascribed to probable viral families . As highlighted in their manuscript, examples included incomplete annotation resources and, worse yet, incomplete functional characterization. DNA sequencing-enabled approaches to studying microbial communities and their array of populations have been augmented by bioinformatics tools for annotating and tracking specific sequences . However, there is a current lag in the study of viral/phage elements, owing to a much less structured set of reference data and databases. Recognizing that bacteriophages are a characteristic of FMT and have the potential to alter the composition, features, and functions of the microbiota underscores how ill-defined the “gut microbial organ” currently is. The report by Chehoud et al. serves as a reminder that systematic efforts are sorely needed for understanding baseline bacteriophage dynamics in the gut in order to identify and circumvent problems associated with use of the gut microbiota itself as a therapy for treating disease.While many have touted FMT as generally safe as a therapeutic approach, this notion is based on rather limited clinical observations—mostly in subjects with


Introduction: Gases, such as nitric oxide (NO), carbon monoxide (CO), or hydrogen sulfide (H2S), termed gasotransmitters, play an increasingly important role in understanding of how electrical signaling of cells is modulated. H2S is well-known to act on various ion channels and receptors. In a previous study we reported that H2S increased calcium-activated potassium (BK) channel activity.Aims: The goal of the present study is to investigate the modulatory effect of BK channel phosphorylation on the action of H2S on the channel as well as to recalculate and determine the H2S concentrations in aqueous sodium hydrogen sulfide (NaHS) solutions.Methods: Single channel recordings of GH3, GH4, and GH4 STREX cells were used to analyze channel open probability, amplitude, and open dwell times. H2S was measured with an anion selective electrode.Results: The concentration of H2S produced from NaHS was recalculated taking pH, temperature salinity of the perfusate, and evaporation of H2S into account. The results indicate that from a concentration of 300 μM NaHS, only 11–13%, i.e., 34–41 μM is effective as H2S in solution. GH3, GH4, and GH4 STREX cells respond differently to phosphorylation. BK channel open probability (Po) of all cells lines used was increased by H2S in ATP-containing solutions. PKA prevented the action of H2S on channel Po in GH4 and GH4 STREX, but not in GH3 cells. H2S, high significantly increased Po of all PKG pretreated cells. In the presence of PKC, which lowers channel activity, H2S increased channel Po of GH4 and GH4 STREX, but not those of GH3 cells. H2S increased open dwell times of GH3 cells in the absence of ATP significantly. A significant increase of dwell times with H2S was also observed in the presence of okadaic acid.Conclusions: Our results suggest that phosphorylation by PKG primes the channels for H2S activation and indicate that channel phosphorylation plays an important role in the response to H2S. Nevertheless, action appeared required to carefully estimate the H2S concentrations derived from the H2S donor  used in our experiments.The physiological concentrations of H2+ as well as by membrane voltage and are modulated by a wide variety of intra- and extracellular factors, including protein kinases, phosphatases causing phosphorylation/dephosphorylation or changes in their redox environment in particular at the C-terminal region . We used these three different but related cell lines, because they all express BK channels who differ in their sequences and respond differently to phosphorylation or in the responsiveness to arachidonic acid. The sequences of BK α-subunits in GH3 and GH4 splice variants differ most prominently by the presence or absence of 27 amino acids in the COOH terminus of the channel  and prolactin and express various receptors. Stressors applied to GH cells lead to the expression of a 59 amino acid cysteine rich insert at the pore forming α-subunit C-terminus of BK channels—termed GH STREX cells  and hence destabilization of the reference electrode. Recordings from excised outside-out patches were made with an Axopatch-200B amplifier connected to a Digidata 1322A interface using pClamp10 software for data acquisition and analysis. Data were filtered at 1 kHz offline and analyzed with Clampfit software . Raw recordings were idealized with the built in feature of pClamp's Clampfit module using the half-amplitude threshold method with automatic baseline and level tracking. Dwell time analysis was done by fitting open dwell time distributions to standard exponentials with the appropriate number of terms to get an optimal fit. Since BK channels are localized in clusters in cell membranes, all of our patches contained more than one channel. For this reason we analyzed only open dwell times but not closed channel kinetics. Single channel current amplitudes were calculated by fitting amplitude histograms to a Gaussian distribution. Channel open probability was expressed as Popen = NPo/n, where NPo = [(to)/(to + tc)], Po = open probability for one channel, to = sum of open times, tc = sum of closed times, N = actual number of channels in the patch, and n = maximum number of individual channels observed in the patch. All equations used were standard built in equations from Clampfit. Experiments were repeated at least three times and the mean, as well as the s.e.m. (standard error of the mean) were calculated.In brief: pipettes for single channel patch clamp recordings were drawn from borosilicate glass (Science Products/FRG) with resistances of 3–6 MegaOhm . For rapid solution exchange (about 300–500 ms) membrane patches were held in a stream of the experimental solution from a second pipette.2, 1 CaCl2, 10 HEPES, pH 7.2. The regular pipette solution contained in mM: 145 KCl, 1 MgCl2, 10 HEPES, pH 7.2, and 5 EGTA, 3.63 CaCl2 – resulting in 0.5 μM free Ca2+ as calculated using the Webmaxc extended calculator (http://www.stanford.edu/~cpatton/webmaxcE.htm). Experiments were carried out at room temperature between 20 and 22°C. Sodium hydrosulfide  was used as a source of H2S. NaHS at concentrations usually used in the present study did not change the pH of the buffered solution. NaHS solutions prepared shortly before experiments were clear and were usually used 3–5 min but no longer than 20 min. For details of making and working with H2S, see Hughes et al.  0.1 units/ml; PKC inhibitor fragment 19–31 (PKCin) – 500 nM; PKG – 400 units/ml plus 50 μM cGMP; Staurosporine (STS) – 1 μM; Okadaic Acid (OA) – 100 nM.+ and HS−, and HS− associates with H+ to produce H2S. Previously as a rule of thumb we calculated for neutral solutions that one-third of NaHS exists as H2S and the remaining two-thirds are present as HS−  together with an Apollo 1000 free radical Analyser . The response time of the sensor is less than 5 s, with a sensitivity of 2 pA/nM and a detection range from 5 nM to 100 μM H2S. For calibration of the sensor instructions according to the WPI manual were used. After we had established a calibration curve we determined the rate of loss of H2S versus time for the NaHS donor. As depicted from Figure 2S obtained after 20 min is 42%. A loss of about 33% after 15 min and 90% after 30 min was reported previously by Kimura et al. . For NaHS = 300 μM and pK1 = 6.89, the H2S concentration calculates to 97 μM, which is quite similar to the rule of thumb of 1/3 H2S in solution (see above). Calculated from pK1 = 7.04 for standard conditions  K1 = 10−7.04  + (−0.157 × (S0.5)) + 0.0135 × S  = is the natural logarithm of T, and S = salinity). Taken salinity as zero for water and temperature 20°C equal to 293.15 Kelvin we calculate pK1 = 7.056, which is very close to literature value . The relationship is linear up to about 50 μM H2S, respectively 226 μM NaHS. Table 2S concentrations. During dilution from the stock to the final solution which took about 30–60 s we may have lost about 5–10% H2S by evaporation  containing the experimental fluid. The syringe opening was covered with parafilm with a small whole inserted to allow for pressure exchange. The tip of the perfusion pipette was located in the recording chamber at about 1 mm distance to the measured cell or patch. The constant perfusion rate of 1 ml/80 s ensured that the channels recorded were always submerged by the desired HThere are some problematic features of proportional data, like open probabilities (Po), which often hinder the use of parametric statistical procedures, such as ANOVAs and regression analysis. Basic assumptions of these methods are often violated, because all values coming from single channel recordings are set within the interval 0 < Po < 1. To be more precise, proportional data tend to be non-normally distributed and heteroscedastic in nature, which actually means that the size of sample variances is not evenly distributed along the interval of 0 < Po < 1.p = ln(p/(1 − p)), which “normalized” this kind of data and substantially reduced heteroscedasticity. After this transformation, “usual” parametric methods could be used. Due to similar reasons, dwell-time measurements were also log-transformed prior to statistical analysis.Besides using less powerful non-parametric methods, there are other strategies to overcome these limitations, e.g., data transformation , high significant , and most significant . Data described in the text as percentage were calculated by setting control values to 100% and expressing experimental values as percentual change of controls.Evaluation of statistic analysis is given as: significant  (Table ***) different to ATP0. PKC  in the pipette solution decreased BK Po non-significantly compared to ATP1, but Po was significantly different from ATP0 (*). PKC is known to cause inhibition of BK channel activity from GH cells  if compared to ATP1. The experiments support previous results of an inhibitory role of PKC at BK channels from GH3 cells  for GH3, GH4, and GH4 STREX cells are listed in Table *) Table . Similar*) Table , and rev*) Table . All fur*) Table , however***) (Table **) with high significance compared to ATP1, as previously reported by Zhou et al. (All drug concentrations used for these cells were similar to those used with GH3 cells. With ATP1 Po increased high significantly compared to ATP0 (*) Table . Using Pu et al. . Use of u et al. . With th+ conductance (RCK1 and RCK2). This insert bears various cysteine residues which can be phosphorylated. The STREX insert speeds activation and slows deactivation kinetics and their half-activation voltage is shifted to the left . The increase caused by ATP was, however, less compared to BK channels from GH3 or GH4 cells (Table ***) when compared to all other conditions, implying that inhibition of the BK channel attached phosphatase allows for unrestricted phosphorylation of the channel protein.These cells contain a 59 amino-acid insert, so called STREX (stress axis regulated) exon at the BK channel α-subunit C terminus, located between the two regulatory domains of Kls Table . PKA is ls Table . PKA inc2S effects are shown in Figure We again present data separately for all three cell types used in our study. Original recordings of H2S concentration 34–41 μM, see Materials and Methods), applied to the extracellular bath solution most significantly increased Po of BK channels from 0.01522 ± 0.00302 to 0.0295 ± 0.00722  , V = +30 mV), equal to 190.58 ± 13.81% of control (=100%) during the first minute of H2S perfusion. Po decayed after 1 min H2S application during the following 3 min. A similar transient response was observed previously using ethanol  with NaHS, i.e., to 191.65 ± 23.52% of control (=100%) , i.e., to 169.42 ± 19.99% of control. PKG (50 units/ml) also increased Po from 0.0724 ± 0.0118 (control) to 0.1395 ± 0.0236(n = 8) in NaHS by 205.56 ± 19.08% (***). PKC catalytic subunit  decreased BK channel activity to 0.04434 ± 0.00752 and NaHS had no significant effect . After addition of PKC inhibitor pseudo-substrate , Po was 0.07408 ± 0.01808 (control), but NaHS again had no significant effect . During application of the kinase inhibitor staurosporine  Po was decreased to 0.04683 ± 0.01493, and in this case NaHS significantly increased Po to 0.07483 ± 0.01892 , i.e., to 168.56 ± 11.87% of control. The most significant increase of Po compared to either ATP0 or ATP1 was obtained with the phosphatase inhibitor okadaic acid (100 nM) which increased Po to 0.3075 ± 0.0581, however, Po was not further significantly altered by NaHS .As reported previously  Figure . All furn = 8 (***), i.e., to 183.48 ± 22.12% (***) after application of NaHS. With ATP1 solution, Po increased significant from 0.11647 ± 0.01969 (control) to 0.20591 ± 0.02146, in NaHS, n = 11 (***), i.e., to 201.36 ± 19.12%. After application of PKA, Po was 0.12569 ± 0.02224 (control), but with NaHS no significant alteration of Po 0.12693 ± 0.02414, n = 11, i.e., to 107.33 ± 11.86% was observed. With PKG Po increased highly significant from 0.05229 ± 0.01732 (control) to 0.1430 ± 0.04916 (n = 7) in NaHS, i.e., by 258.95 ± 33.78% (***). Application of PKC subunit  decreased Po to 0.02771 ± 0.00647 and NaHS increased Po significantly to 0.0465 ± 0.01669, i.e., to 161 ± 28%, n = 7 (*). A similar Po of 0.02089 ± 0.00553 was obtained with PKC inhibitor , and Po was high significantly increased with NaHS to 0.04093 ± 0.0108, n = 7 (***), i.e., to 186.78 ± 19.85%. With staurosporine  Po was 0.0505 ± 0.01474 and increased with NaHS highly significant to 0.087 ± 0.02195, n = 5 (***), i.e., to 181.66 ± 15.35%. Okadaic acid (100 nM) substantially increased Po to 0.44223 ± 0.0403 compared to either ATP0 or ATP1 controls, whereas NaHS decreased Po under this condition to 0.31953 ± 0.03142, n = 7 (***), i.e., to 74.841 ± 7.8%.All basic conditions and concentrations of drugs were similar to the experiments using GH3 cells. In ATP0, Po increased highly significant from 0.00968 ± 0.00285 (control) to 0.01632 ± 0.00452, n = 7, i.e., to 148 ± 11.99%. With ATP1 Po increased to 0.03428 ± 0.00931, n = 13 compared to ATP0, but this increase was much less compared to Po-values obtained in GH3 and GH4 cells , i.e., to 177.74 ± 15.91%. PKA subunit (50 units/ml) increased Po to 0.08221 ± 0.03214, n = 8 under control conditions, however, NaHS application had no significant effect on Po, 0.05739 ± 0.02129, n = 8, i.e., 78.65 ± 9.38% of control. PKG high-significantly increased Po from 0.05043 ± 0.01330 (control) to 0.08829 ± 0.01635 in NaHS, by 201.94 ± 40.37%, n = 8 (***).In ATP0 BK channel Po of 0.00652 ± 0.00224 (control) was low compared to Po of GH3 and GH4 cells. NaHS non-significantly increased Po to 0.00921 ± 0.00286, n = 6 (control), however, NaHS increased Po to 0.06833 ± 0.01267, i.e., to 322.44 ± 74.48%, although with low significance (*). With PKC inhibitor  Po of 0.0400 ± 0.00879, n = 5 was similar to ATP1, and increased with NaHS to 0.10552 ± 0.02854, n = 5, i.e., to 253.07 ± 28.82%, however not significantly. Also staurosporine  non-significantly increased Po from 0.0375 ± 0.00975 (control) to 0.10114 ± 0.02397 in NaHS, n = 8, i.e., to 306.14 ± 49.76%. In okadaic acid (100 nM) which significantly increased Po to 0.39927 ± 0.09621 compared to ATP1, NaHS had no additional significant effect on Po, 0.42796 ± 0.08584, n = 7, i.e., to 114.93 ± 9.22%.As with previous results from GH3 and GH4 cells, using PKC subunit  BK channel Po was low, 0.02533 ± 0.00604, 2S, whereas BK channels from GH4-STREX cell did not respond to H2S. Po of BK channels of all cell types phosphorylated by ATP was highly significantly increased and H2S further increased Po at high significance. PKA and PKG were not able to increase Po beyond the ATP1 effect. H2S was able to increase Po of BK channels from GH3, but not those of GH4 and GH4 STREX cells. H2S high significantly increased Po of all cell types pretreated with PKG. PKC-subunit (PKCsu) either had no effect (GH4-STREX) or decreased Po  but H2S increased Po of BK channels pretreated with PKCsu in GH4 and GH4 STREX but not those of GH3 cells. Staurosporin had no effect on BK channel Po activity but did not prevent the activating effect of H2S. Okadaic acid most significantly increased BK channel Po of all cell types and H2S had no further effect on channel Po, it even decreased BK channel Po of GH4 cells.In summary, our result show Table  that Po ***) increased mean open dwell times from 0.74 ± 0.087 ms (ATP0) to 2.022 ± 0.18 ms (ATP1). Open dwell times were also significantly increased with PKA to 1.78 ± 0.22 ms (***), with PKG to 1.56 ± 0.15 ms (**) and with PKC subunit to 1.78 ± 0.6 ms (*), compared to an ATP0 solution. A significant increase of mean dwell times was also observed with BK channels from GH4 cells from 0.77 ± 0.10 ms (ATP0) to 1.49 ± 0.11 ms (**) after application of ATP1. With PKA dwell times increased to 3.00 ± 0.44 ms (***) and with PKG to 1.54 ± 0.12 ms (**) if compared to ATP1 , PKCin vs. PKA (***), staurosporine vs. PKA (***) and okadaic acid vs. PKA (***) and PKG vs. PKA (*). NaHS had a significant increasing effect on open dwell times only in okadaic acid (***). In GH4 STREX cells open dwell times were only significantly increased when PKG is compared with ATP0 (*).Values of BK channel Po, amplitude and dwell time are shown in Figures 1 Figure . Signifi**), with GH4 cells in okadaic acid from 0.10 ± 26 ms to 1.73 ± 0.46 ms (***), and in ATP0 in GH4 STREX cells from 0.69 ± 0.10 ms to 0.99 ± 0.16 ms (*). NaHS had no effect at dwell times with all other experimental settings  to (1) obtain reliable, well visible effects at the channels, and (2) being able to compare our results with previous investigations. Previous studies  increased Po was about 30 μM NaHS, which effectively amounts to 3.4–4.1 μM free H2S. Hence, H2S appears to induce BK channel activation at a physiological relevant concentration range. The effective H2S level at its target site(s) is unknown, however. H2S may be stored as bound sulfane sulfur and/or neuronal excitation can trigger its release from surrounding astrocytes  applied for a few minutes were completely reversible  at BK channel Po  was only about 6–8%. As indicated from a previous dose-response plot , HS− (~80%), and traces of S2−, are always simultaneously present in the solution or in the cytosol. This mixture is usually referred to in the literature as H2S for simplicity  which in turn may prime the channels for H2S activation.With pipette solutions containing no ATP H2S only had a high significant effect on BK channel Po from GH3 cells but had no effect on GH4 and GH4 STREX cells. Phosphorylation of BK channels by PKA therefore appears to prevent further H2S activation in these cells. PKG had no significant effect on Po compared to ATP1 of all cell types , whereas NaHS significantly increased Po of BK channels phosphorylated by PKG of all cell types. In all of our experiments the initial phosphorylation state of BK channels at the start of experiments was not determined and remains unknown. Also the amount of phosphorylation by ATP or the addition of kinases was not measured. This should be kept in mind when considering the interpretation of the results.In all three cell lines PKA effect on Po is not significant different compared to ATP1. The result indicates that the channels are already sufficiently phosphorylated in the presence of ATP. PKA has been reported to activate, inhibit or have no effect on BK channels as reviewed by Schubert and Nelson . These d2S in this context did not alter BK channel Po from GH3 cells, but increased Po of GH4 and even more of GH4 STREX cells. With PKC inhibitor, H2S had no effect at GH3 cells but high significantly increased Po of BK channels from GH4 and GH4 STREX cells. Although PKC phosphorylation suppresses Po of BK channels H2S appears able to overcome this effect and to stimulate the channels. The kinase inhibitor, staurosporine, had no significant effect on BK channel Po of all three cell lines. H2S under these conditions was able, however, to significantly increase Po. Hence, in all three cell types Po of non-phosphorylated (ATP0) and phosphorylated channels by ATP1 and by PKG can be elevated by H2S. PKC phosphorylation prevented the Po increasing H2S effect in GH3 cells but increased Po in GH4 and GH4 STREX cells. PKA also differently affected cell types with increasing Po of GH3 cells but not of GH4 and GH4 STREX cells (which appear even decreased by H2S).PKC subunit and PKC inhibitor decrease channel Po but this effect was only significant with GH4 cells. Our data are in congruence with a decrease of channel activity by PKC reported previously . This peculiar action of okadaic acid may be due to the fact that a very active protein phosphatase is associated with the BK channels  channel Po is increased. PKA prevented the action of H2S on channel Po in GH4 and GH4 STREX but increased BK Po of GH3 cells at high significance. This suggests that PKA phosphorylates the channels of GH4 and GH4 STREX cells at some other site which rearranges the protein in a way that H2S is no longer able to act at the channels. Zhou et al.  or if phosphorylated by ATP can be activated by Hu et al.  showed t2S on BK channels Po which may be dependent on the channels conformation which exposes or impedes phosphorylation sites and this way may govern the access of H2S to reach its effective location.Our experimental results indicate that phosphorylation may prime or prevent the action of HThe 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.


Neonates are at high risk of meningitis and of resulting neurologic complications. Early recognition of neonates at risk of poor prognosis would be helpful in providing timely management. From January 2008 to June 2014, we enrolled 232 term neonates with bacterial meningitis admitted to 3 neonatology departments in Shanghai, China. The clinical status on the day of discharge from these hospitals or at a postnatal age of 2.5 to 3 months was evaluated using the Glasgow Outcome Scale (GOS). Patients were classified into two outcome groups: good  or poor . Neonates with good outcome had less frequent apnea, drowsiness, poor feeding, bulging fontanelle, irritability and more severe jaundice compared to neonates with poor outcome. The good outcome group also had less pneumonia than the poor outcome group. Besides, there were statistically significant differences in hemoglobin, mean platelet volume, platelet distribution width, C-reaction protein, procalcitonin, cerebrospinal fluid (CSF) glucose and CSF protein. Multivariate logistic regression analyses suggested that poor feeding, pneumonia and CSF protein were the predictors of poor outcome. CSF protein content was significantly higher in patients with poor outcome. The best cut-offs for predicting poor outcome were 1,880 mg/L in CSF protein concentration . After 2 weeks of treatment, CSF protein remained higher in the poor outcome group. High CSF protein concentration may prognosticate poor outcome in neonates with bacterial meningitis. Neonates are at high risk of meningitis and of resulting neurologic complications . DespiteEarly recognition of neonates at risk of poor prognosis would be helpful in providing timely management and treatment to improve outcome , and to The aim of the present study was to identify possible risk factors that may predict prognosis in neonates with bacterial meningitis. Moreover, we wanted to clarify whether the CSF parameters are independently associated with poor prognosis.6/L in conjunction with a positive blood culture [From January 2008 to June 2014, we enrolled 232 term neonates with bacterial meningitis admitted to 3 neonatology departments in Shanghai, China, including Xinhua Hospital, Ninth People’s Hospital and Children's Hospital of Shanghai. Neonatal bacterial meningitis was defined as clinical symptoms and signs of meningitis plus either a positive culture in the cerebrospinal fluid (CSF), or CSF WBC of >20×10 culture ,12. InclOutcome data were determined on the day of discharge from hospital or at a postnatal age of 2.5 to 3 months. The clinical status was evaluated with the Glasgow Outcome Scale (GOS) which ranges from 1 to 5: 1 = death; 2 = persistent vegetative state; 3 = severe disability; 4 = moderate disability; 5 = good recovery . ModeratP values <0.05 were considered statistically significant.All statistical analyses were performed using SPSS 16.0 . Chi square test or Fisher’s exact test was used for categorical variable comparisons. Student's t test or Mann-Whitney test was used to compare continuous variables. Multivariate logistic regression was used to assess the predictors of poor outcome (prognosis) in neonatal meningitis. The cut-off point for CSF protein to predict prognosis in neonatal bacterial meningitis was calculated according to receiver operating characteristic (ROC). Area under the curve (AUC) and sensitive-specific values with 95% confidence intervals were calculated. Results are presented as mean ± standard deviation or n and percentage. Among the 232 patients with meningitis, 88 patients were male and 144 were female, all were born at term. The mean age at onset of meningitis was 13.0 ± 7.7 days. 92 (39.7%) neonates were born by cesarean section (CS), and 140 (60.3%) neonates were vaginal deliveries. The mean birth weight was 3072 ± 422 g. There were 10 neonates with low birth weight (< 2500 g) and 2 neonates with high birth weight (> 4000 g). The infection was observed in 29 (12.5%) infants within the 3 days of life (early onset infection), including 9 (3.9%) on the first day. The average duration of hospital stay was 28.6 ± 14.3 days (range: 1 to 93 days). The overall mortality was 3.0% (7 of 232 patients).The predisposing factors for neonatal bacterial meningitis included maternal fever greater than 38°C in 38 (16.4%) cases and prolonged rupture of membranes over 12 h in 45 (19.4%) cases. In one case, the cord was cut using a pair of contaminated scissors. 30 (12.9%) cases of vaginal colonization with group B streptococcus were noted. Furthermore, 17 cases had other purulent infections .Bacteriological confirmation of meningitis was obtained in 232 cases. Positive cultures of blood and cerebrospinal fluid were obtained in 95 (40.9%) cases and 169 (72.8%) cases, respectively, including 32 (13.7%) cases with a positive culture in both blood and cerebrospinal fluid. Bacteria identified as the causal agent for meningitis were: E. coli, 73 cases; Group B streptococcus, 65 cases; Coagulase negative Staphylococci, 24 cases; Streptococcus pneumoniae, 18 cases; Pseudomonas Aeruginosa, 16 cases; Corynebacterium, 12 cases; Listeria Monocytogenes, 10 cases; Haemophilus influenzae, 8 cases; Meningococcus, 2 cases; Proteusbacillus vulgaris, 2 cases; and Enterococcus feces, 2 case.According to the definition of GOS, patients were classified into two outcome groups: good  or poor . The poor outcome group included mortality in 7 patients, severe disability in 23 patients and moderate disability in 35 patients.Neonatal meningitis has been associated with a variety of neurologic complications . In our The clinical features of neonates with bacterial meningitis are summarized in 9/L with a mean of 16.28×109/L, including 67 cases with a leukocytosis more than 20.0×109/L and 2 cases with a leucopenia less than 4.0×109/L. Anemia with a hemoglobin less than 145 g/L in 151 cases. 72.0% of cases had a C-reaction protein (CRP) level over 8 mg/dl. Procalcitonin (PCT) was 0.05 μg / L or over in 77.2% of cases.Peripheral blood and CSF cyto-biochemical findings are also summarized in 6/L with a median of 80×106/L, including 105 cases with the number of cells more than 100×106/L. CSF polykaryocyte (%) > 50% was observed in 65.9% of cases. Protein concentration varied between 438 and 4750 mg/L, with a median of 1400 mg/L. CSF glucose <1.5 mmol/L in 25.0% of cases, and CSF glucose / blood glucose ratio <0.5 in 62.1% of cases.CSF cell count varied between 11 and 17870×10p<0.05). The good outcome group also had less pneumonia and more diarrhea than the poor outcome. Besides, there were statistically significant differences in hemoglobin, mean platelet volume, platelet distribution width, C-reaction protein, procalcitonin, CSF glucose and CSF protein. , pneumonia  and CSF protein  were the predictors of poor outcome. . p<0.001). . Newborns are especially vulnerable to infection. The cellular and humoral immune systems are immature, including the phagocytic function. The incidence of meningitis ranges from 0.2 to 6.1 cases per 1,000 live newborns ,16. The The incidence of meningitis confirmed by culture in the CSF was not high. But there is a strong association of meningitis and sepsis with positive blood cultures . TherefoEarly recognition of meningitis infants at risk of poor prognosis would be helpful in providing prompt management and identifying those who warrant long-term follow-up and early intervention –25. PrevBesides, we found that the poor feeding, pneumonia and CSF protein were predictive of poor outcome in our study. Weber et al found that reduced feeding ability may be an independent predictor of severe disease . HoweverIt should be emphasized that CSF protein in the poor outcome group was extremely high in our study. The level of cerebrospinal fluid protein content significantly influenced patient prognosis. Though previous study showed that high CSF protein was associated with poor prognosis in childhood bacterial meningitis , no studIt has been recognized that WBCs, immunoglobulins, and complements are normally sparse or absent in CSF. At the initial stage of neonatal meningitis, micro-organisms can cross the blood—brain barrier via microbial interactions with host receptors, and replicate within the subarachnoid space concomitantly. Later bacteria release endotoxins, teichoic acid, and other substances that trigger an inflammatory response with mediators such as WBCs and TNF resulting in increasing protein levels in CSF. Besides, the production of cytokines leads to the attraction and activation of polymorphonuclear leukocytes and the production of high amounts of reactive oxygen species. These free radicals are highly reactive and may cause impairment of lipids, proteins, carbohydrates or nucleic acids, thus increasing the risk of poor sequelae. Because of the high lipid content in the brain and low cerebral antioxidant defense, the central nervous system is particularly susceptible to the deleterious properties of oxidative stress ,31. So wIn summary, CSF protein may be a biomarker of severe CNS infection with poor prognosis for identifying patients for timely and intensive treatment.


Rana japonica, which usually possesses an ochre or brown back; this coloration enables the organs, blood vessels, and eggs to be observed through the skin without performing dissection. We crossed two kinds of recessive color mutant (black-eyed and gray-eyed) frogs through artificial insemination, and F2 offspring produced frogs whose skin is translucent throughout the life cycle. Three kinds of dermal chromatophores—xanthophores, iridophores, and melanophores—are observed in a layered arrangement in the skin of wild-type frogs, but few chromatophores were present in the skin of the see-through frogs. The translucent skin enables observation of organ growth and cancer formation and progression in the animal, which can be monitored over its entire life without the need for dissection. See-through frogs thus provide a useful animal model for environmental, medical, and biological research.We have succeeded in creating see-through frogs from natural color mutants of the Japanese brown frog  Dissection, particularly when performed in schools, has become increasingly controversial in much of the world. However, if the internal organs of an animal were clearly visible through the skin over the animal’s entire life history, then there would be a greatly reduced need for dissection. In frogs, the skin is generally covered with dermal chromatophore units consisting of xanthophores, iridophores, and melanophores24Rana japonica121415Nature)The black-eyed and gray-eyed recessive color mutants seen in several frog species lack the normal iridophores and melanophores, respectively, resulting in pale or albino frogs6789101112https://www.youtube.com/watch?v=3TumwZ40LQM; tadpole https://www.youtube.com/watch?v=tYRjZtaxOgg).Upon crossing two color mutant (gray-eyed and black-eyed) frogs with recessive genes through artificial insemination, all of the offspring appeared normal (wild-type) due to the presence of dominant genes , Table 12 = 2.7445, P = 0.433; 3:3:1:1, χ2 = 4.8660, P = 0.182). The most effective crossing combinations were between Bbgg female and BbGg male parents. See-through frogs produced by the F2 generation had low viability, probably due to the presence of two recessive genes.As shown in Three kinds of dermal chromatophores—xanthophores, iridophores, and melanophores—were observed in a layered arrangement in the skin of wild-type frogs . PterinoThe see-through frogs produced in this study allowed us to observe changes in the internal tissues and organs in detail, externally, in both early development and senescence, to evaluate the effects of chemicals on the viscera and bones in a simple and inexpensive manner, to view the development and progression of cancer, and to assess the effects of toxins over time. See-through frogs do not need to be dissected to perform such viewing and therefore enable the repeated ongoing observation of the viscera over the entire life course in a single frog. Furthermore, the dramatic changes in organs that occur during the metamorphosis of tadpoles into adult frogs can be easily seen.sukeru-pyon”, combining the Japanese words for “see-through” and “frog”.Genetic engineering of see-through frogs is an approach that could be used to produce see-through, fluorescent frogs. In the near future, we plan to produce frogs that are both see-through and fluorescent by injecting fluorescent GFP-tagged genes into see-through frogs, causing the frogs to fluoresce to indicate expression of the tagged geneRana japonica. We hope to create completely see-through frogs in the near future. It is worth noting that it is unrealistic to apply the present method to mammals such as mice, given the differences in skin structure, although the development of whole-body imaging at single-cell resolution has recently enabled system-level approaches to studying cellular circuits in mice by using tissue decolorizationThe see-through frogs produced in the present study appear pale when viewed dorsally, as xanthophores remain in the dorsal skin. However, these frogs are almost transparent when viewed ventrally, because there are little to no xanthophores in the ventral skin. If we could obtain color mutants that lack xanthophores, we would be able to produce completely see-through frogs by using these mutants to produce triple mutants lacking genes for all three chromatophores. In fact, variant blue ranid frogs have been found in several frog species192021in vitro and in vivo has been reported242526in vivo. Evidence that melanosomes and pterinosomes are homologous organelles has been reported in several studies282930in vitro. In the cloned xanthophore, pre-existing pterinosomes disappeared and melanosomes were formed instead during proliferation. Ide considered these melanosomes to originate de novo from melanosomes via premelanosomes, because of the presence of premelanosomes in the melanized xanthophores. The results of the present study also suggest that xanthophores may be converted into melanophores, demonstrating the de novo formation of melanosomes via premelanosomes.Immature xanthophore-like cells containing melanosome-like structures were observed in the skin of the see-through frogs. Some evidence suggesting the conversion of xanthophores into melanophores Another possible explanation may be that xanthophores contain melanosome-like organelles, but not transdifferentiated chromatophores, as melanosome-like structures were also observed in xanthophores in the skin of gray-eyed frogs . Accordi133334353513In light of these findings, further studies will be necessary to clarify the processes involved in chromatophore formation in more detail, and to examine the fine structure of pigment organelles by observing pigment cell development in see-through tadpoles and frogs using electron microscopy.This study has received a patent under “Creation and Use of See-through Frogs”, application number (2006-203987), open number (2008-029223) on application date (2006.7.26), open date (2008.2.14), applicant (Hiroshima University), creator (Masayuki Sumida).All animal experiments were performed in accordance with the approved guidelines of Hiroshima University regarding protection and management of animals and standards for the breeding, safekeeping, and minimization of suffering of experimental animals. All experimental protocols were approved by the Hiroshima University Animal Experiment Committee, and were in keeping with the basic guidelines of Hiroshima University regarding animal experiments .We used two color mutants (black-eyed and gray-eyed) that were originally caught in the field, and then bred and maintained in the Institute for Amphibian Biology, Hiroshima University1238We observed the dermal chromatophores in the dorsal skin of wild-type, black-eyed, gray-eyed, and see-through adult frogs with an electron microscope to examine the microstructure of the skin of see-through frogs. Pieces of the dorsal skins removed from each of these frogs were cut into minute pieces in cold 0.1 M phosphate buffer (pH 7.4) containing 3% glutaraldehyde and kept in the same solution for 2 h after renewal of the fluid. The minute pieces were then postfixed in 0.1 M phosphate buffer (pH 7.4) containing 2% osmic acid for 2 h. These fixation procedures were performed at 2–4 °C. The fixed samples were dehydrated in an ethanol series and embedded in Epon 812. Sections were cut on a Porter-Blum MT-1 ultramicrotome with a glass knife and double stained with saturated uranyl acetate and alkaline lead citrate. Observations were made using a Hitachi Hs-8 electron microscope.How to cite this article: Sumida, M. et al. The first see-through frog created by breeding: description, inheritance patterns, and dermal chromatophore structure. Sci. Rep.6, 24431; doi: 10.1038/srep24431 (2016).


There is an increasing burden of responsibility for intensivists to optimize donation potential after the declaration of brain death in patients with catastrophic brain injury. Best practice for donor management, if present, has been formed on low quality and mainly observational studies or consensus. In particular, research into the use of corticosteroids has shown varied benefit. The specific and limited results of the CORTICOME study are less important than the systematic methodology and the development of rigour in the study of deceased organ donation. Donor management would benefit from continued systematic analysis of current literature, understanding of the physiologic basis for therapy, and further prospective controlled trials. Worldwide collaboration partnerships and funding are needed to optimize the management of deceased organ donation. Organ donation continues to save lives. There is an increasing burden of responsibility for intensivists to optimize donation potential after the declaration of brain death in patients with catastrophic brain injury. Until recently, organ donor management has been considered a low priority. As such, rigorous research to guide improved organ procurement and graft survival has been limited. Pinsard and colleagues  have comUntil now, research into the use of corticosteroids has shown varied benefit. The overall quality of studies has been poor with few randomized controlled studies and mostly observational reports with limited patients and high potential for confounding factors. Most of these studies included other hormonal therapies rather than corticosteroids alone . A recenPinsard and colleagues should be commended for accomplishing the challenging task of conducting a prospective multi-centre study involving 259 subjects in order to identify the benefits of low-dose corticosteroids after brain death. The authors found that the need for norepinephrine was reduced . AlthougThough hypothesized, the mechanism for reduced dose and duration of vasopressors with low dose hydrocortisone is still unclear. Similar results have been found with corticosteroid use in other disease states such as septic shock , perhapsin situ of another patient, the transplant recipient. Numerous uncontrollable variables independent of donor management impact on transplant outcomes . While improving transplant outcomes is clearly the most important goal, it may not be the most scientifically valid outcome. The scientific question should first assess whether a specific strategy improves organ function in the brain-dead donor themselves, and then second, assess whether improved donor organ function leads to increased organ utilization and transplant graft survival. Outcomes analysis should change from traditional graft survival to specific physiologic goal-directed targets relevant at the bedside of the donor. We would propose that physiologic outcomes and end organ function, such as haemodynamics, cardiac ejection fraction, oxygenation status, hepatic markers, and renal function, need to be prospectively measured to identify specific benefits of specific donor management therapies.Current donor management studies often suffer from paradigm challenges. Traditional ICU interventional studies aim to improve organ function and meaningful outcomes in the patient receiving those interventions. Meanwhile, donor management studies seek to improve organ function The systematic approach to research in organ donation management is evolving slowly and is long overdue. The current work on the use of corticosteroids is an example. The specific and limited results of the CORTICOME study are less important than the systematic methodology and the development of rigour in the study of deceased organ donation. The same must be accomplished for other therapies, individually and combined, including fluid management and haemodynamic supports. In the future, more aggressive, novel, and multimodal interventions need to be studied. Collaboration with transplant research, to identify immunomodulating targets in potential donors, should be pursued. Donor management would benefit from continued systematic analysis of current literature, understanding of the physiologic basis for therapy, and further prospective controlled trials.For deceased organ donation research to flourish, national and worldwide collaborative partnerships need to be formed as they were developed for other disease states such as sepsis and traumatic brain injury. Appropriate funding must also be committed to prospective research and initiatives. The basis of this collaboration and support would effectively eliminate the 'art' and create a new scientific rigour to the management of deceased organ donation. With increased collaboration and funding, the potential to save lives through optimized organ donation, given the multi-patient recipient impact of each donor, may be greater than that seen with other recent initiatives.


The aim of this study was to characterize Ng in plasma and CSF using mass spectrometry and to investigate the performance of plasma Ng as an AD biomarker.Synaptic dysfunction and degeneration are central events in Alzheimer’s disease (AD) pathophysiology that are thought to occur early in disease progression. Synaptic pathology may be studied by examining protein biomarkers specific for different synaptic elements. We recently showed that the dendritic protein neurogranin (Ng), including the endogenous Ng peptide 48 to 76 (NgPaired plasma and CSF samples from patients with AD (n = 25) and healthy controls (n = 20) were analyzed in parallel using an immunoassay developed in-house on the Meso Scale Discovery platform and hybrid immunoaffinity-mass spectrometry (HI-MS). A second plasma material from patients with AD (n = 13) and healthy controls (n = 17) was also analyzed with HI-MS. High-resolution mass spectrometry was used for identification of endogenous plasma Ng peptides.48–76 was not present in plasma. CSF Ng was significantly increased in AD compared with controls (P < 0.0001), whereas the plasma Ng levels were similar between the groups in both studies. Plasma and CSF Ng levels showed no correlation. CSF Ng was stable during storage at −20°C for up to 2 days, and no de novo generation of peptides were detected.Ng in human plasma is present as several endogenous peptides. Of the 16 endogenous Ng peptides identified, seven were unique for plasma and not detectable in CSF. However, Ng48–76, might reflect the neurodegenerative processes within the brain, indicating a role for Ng as a potential novel clinical biomarker for synaptic function in AD.For the first time, to our knowledge, we have identified several endogenous Ng peptides in human plasma. In agreement with previous studies, we show that CSF Ng is significantly increased in AD as compared with healthy controls. The origin of Ng in plasma and its possible use as a biomarker need to be further investigated. The results suggest that CSF Ng, in particular NgThe online version of this article (doi:10.1186/s13195-015-0124-3) contains supplementary material, which is available to authorized users. Lowers for AD .A central event in AD pathology is synaptic dysfunction and degeneration . Synaptiin situ hybridization studies on the human brain have shown that the Ng mRNA is selectively translocated to dendrites and that such translocation is impaired in the cortex of patients with AD [Neurogranin (Ng) is a postsynaptic protein with a critical role in long-term potentiation, where it regulates the levels of calmodulin in response to intracellular calcium levels after neuronal excitation -12. In t with AD .48–76) was significantly increased in AD CSF as compared with controls [Recently, we generated three monoclonal anti-Ng antibodies with epitopes near the C-terminal part of the protein . By immucontrols .If a synaptic marker reflecting the ongoing synaptic degeneration in the central nervous system (CNS) could be identified in blood or plasma, it would be a very valuable clinical tool because CSF sampling is considered to be a somewhat more invasive procedure. In addition, simple and repeated sampling would also be made possible, allowing for dynamic changes to be measured over time at a regular clinic. Therefore, in this study of paired plasma and CSF samples, we aimed to first characterize Ng in plasma using MS and then investigate whether plasma Ng is a marker of synaptic degeneration in AD.de novo. The endogenous peptide Ng48–76, which in our previous study was found to be significantly increased in both patients with AD and individuals with prodromal AD, was not found in plasma. The findings presented in the present study imply that CSF Ng might reflect the ongoing neurodegenerative processes within the CNS, indicating a role for Ng, and in particular Ng48–76, as a potential novel clinical biomarker for synaptic function in AD.Using MS, we show—for the first time, to our knowledge—that Ng is expressed as a variety of endogenous Ng peptides in human plasma. Although plasma Ng was not found to differ between AD patients and healthy controls, we replicated the previous finding of increased CSF Ng in the AD group using an in-house assay developed on the Meso Scale Discovery (MSD) platform. Finally, we also show that CSF Ng is stable during storage and that no peptides are generated Anonymized plasma samples collected in the routine workflow at the Neurochemistry Laboratory at Sahlgrenska University Hospital, Mölndal, Sweden, were used for method development, as approved by the ethics committee at the University of Gothenburg.AD. The patients were recruited in Erlangen, Germany, and each underwent a thorough clinical investigation, including a medical history as well as a physical, neurological and psychiatric examinations. The diagnosis of AD was made according to the National Institute of Neurological and Communicative Disorders and Stroke-Alzheimer’s Disease and Related Disorders association (NINCDS-ADRDA) criteria [1–42, Aβ1–40 , t-tau and p-tau181. The subjects were included in the study only if the clinical, neuroimaging and neuropsychological diagnoses were in accordance with the neurochemical findings as described elsewhere [1–40 and Aβ1–42 concentrations were determined using established ELISA methods as described elsewhere [The paired CSF and plasma study included 25 AD patients (mean age: 76 ± 8 years) and 20 control subjects without dementia (mean age: 54 ± 14 years)  and 17 individuals without dementia  at the Memory Clinic, Skåne University Hospital, Sweden  was used as the calibration curve, and the blank was included as zero concentration of Ng. All samples were analyzed without knowledge of clinical diagnosis.The monoclonal mouse antibody Ng7, with an epitope including amino acids 52 to 65 , was usen-octyl-β-d-glucopyranoside  had been added. The beads and sample were transferred to a KingFisher magnetic particle processor  for automatic washing and elution of the Ng peptides and protein. Eluted Ng was collected and dried in a vacuum centrifuge and redissolved in 5 μl of 0.1% formic acid (FA) in 20% acetonitrile and subsequently analyzed using an ultrafleXtreme matrix-assisted laser desorption/ionization (MALDI) time-of-flight/time-of-flight (TOF/TOF) MS . All solvents used were of high-performance liquid chromatography (HPLC) quality.To minimize nonspecific binding of other proteins during the immunoprecipitation, all plasma samples were pretreated twice with 20 μl of Novex Dynabeads Protein G  per milliliter of plasma and incubated for 1 hour at RT before the beads were magnetically removed as described previously, with some modifications . In brie43–75), with glycine labeled with double 13C , was added to the plasma prior to sample preparation and was used as an internal standard. Samples were analyzed blinded .In both the paired cohort and validation study where MALDI-TOF/TOF MS was used, the custom Ng peptide RKKIKSGERGRKGPGPGGPGGAGVARGGAGGGP (corresponding to Ngm/z) ratio range at a resolution setting of 70,000. Subsequent tandem mass spectra (MS/MS) were produced using the higher-energy collisional dissociation fragmentation cell and acquired at the same resolution setting. An inclusion list was employed to ensure that we obtained MS/MS data of the compounds of interest. Mass spectral raw files were then charge-deconvoluted by using Mascot Distiller software  and subsequently submitted to a Mascot database search (Matrix Science).The identities of the Ng peptides were confirmed using a top-down approach as previously described , but witPlasma and CSF samples were depleted of native Ng by three consecutive immunoprecipitations before full-length recombinant Ng  was added to the samples, followed by 24-hour incubation at RT. After the incubation, samples were analyzed by HI-MS to screen for degradation products from the full-length Ng protein in the form of newly formed Ng peptides.Pooled CSF samples with or without a stabilizing agent , were kept at RT, 4°C or −20°C for 1 to 7 days before they were placed in long-term storage at −80°C pending analysis. As a reference sample, a freshly prepared aliquot, with or without stabilizer, was stored at −80°C on the day of the preparation . All other samples were normalized against the Ng content of the respective reference sample for either group. Ng content was analyzed by MSD.U test, and significance was determined at P < 0.05. Correlations were determined using Spearman’s correlation.For statistical analysis, Prism 6 for Windows software  was used. Because biomarker values were skewed, nonparametric tests were used. Differences between groups were assessed using the Mann–Whitney m/z ratio corresponding roughly to full-length Ng  was detected   or in the validation study . No correlation between CSF and plasma Ng was found in the paired cohort . Owing to the large CSF volume needed for HI-MS analysis (1 ml), the CSF samples were analyzed only by MSD.In contrast, MSD analysis of the CSF samples showed a marked increase of Ng in patients with AD  Figure D. As theWith the purpose of investigating the stability of CSF Ng during storage, CSF was kept at RT, 4°C or −20°C with or without the addition of a stabilizer for between 1 and 7 days before it was stored at −80°C pending analysis. The samples were analyzed using the MSD Ng assay, and all values were normalized against a reference sample, which was stored at −80°C on the day of preparation. The CSF Ng levels were stable at RT for 2 days, after which the concentration dropped to around 70% compared with the day of preparation. Samples kept at either 4°C or −20°C were both stable for all time points investigated Figure A. Similade novo could be detected in the spiked samples after the incubation (data not shown). In contrast, 14 Ng peptides were detected in the spiked plasma samples, indicating that Ng is degraded in plasma  and incubated at RT for 24 hours. The samples were then analyzed for newly generated Ng peptides using HI-MS. In CSF, no Ng peptides generated In this study, we characterized the Ng content in plasma and investigated the potential of plasma Ng as a biomarker for AD. We also addressed the stability of CSF Ng during storage and investigated any potential degradation of Ng in both CSF and plasma.In total, we identified 16 endogenous Ng peptides in plasma. We also show that full-length Ng most likely is present, but mostly in posttranslationally modified forms. Of the endogenous plasma peptides, seven had not been detected previously in either brain tissue or CSF [48–76 showed the most pronounced increase in AD CSF compared with controls. Importantly, Ng48–76 was also found to be the dominant peptide in AD brain tissue [In a previous study, we showed increased CSF Ng levels in AD using ELISA . In the n tissue , but thiin vivo with regard to activity and/or specificity. These differences could be due to a number of different factors, such as pH or storage stability. However, a characterization of the active enzymes in plasma after −80°C storage that are capable of cleaving full-length Ng to generate the peptides observed in such samples is beyond the scope of this article.In a series of experiments designed to test the stability of Ng in plasma, we show that recombinant full-length protein incubated in plasma, depleted of native Ng, is metabolized into a variety of fragments  (Table Limitations of both studies are their small sample size and that healthy control subjects were significantly younger than the AD patient groups P < 0.00 (Table 1de novo formation of peptides in CSF and that CSF Ng is stable for storage at – 20°C, as well as that there are no native plasma Ng peptides ending with amino acid 76. This suggests that CSF Ng, especially Ng48–76, might reflect ongoing neurodegenerative processes within the brain, indicating a role for Ng as a potential novel clinical biomarker for synaptic function in AD.The present study shows that Ng is present in plasma at high concentrations, both as posttranslationally modified full-length protein and as a number of endogenous peptides. The previously reported difference in Ng levels in CSF between controls and AD patients is a consistent finding that is not reflected in the plasma. The most important discoveries are that there is no degradation of full-length Ng or