The effect of type of atrial fibrillation (AF) on adverse outcomes in Chinese patients without oral anticoagulants (OAC) was controversial.The type of AF associated with adverse outcomes in Chinese patients without OAC.A total of 1358 AF patients without OAC from a multicenter, prospective, observational study was included for analysis. Univariable and multivariable Cox regression models were utilized. Net reclassification improvement analysis was performed for the assessment of risk prediction models.p < .001), thromboembolism . After multivariable adjustment, NPAF was a strong predictor of thromboembolism , all‐cause death . Net reclassification improvement analysis indicated that the addition of NPAF to the CHA2DS2‐VASc score allowed an improvement of 0.37 in risk prediction for thromboembolic events .There were 896(66%) patients enrolled with non‐paroxysmal AF (NPAF) and 462(34%) with paroxysmal AF (PAF). The median age was 70.9 ± 12.6 years, and 682 patients (50.2%) were female. During 1 year of follow‐up, 215(16.4%) patients died, and 107 (8.1%) patients experienced thromboembolic events. Compared with the PAF group, NPAF group had a notably higher incidence of all‐cause mortality (20.2% vs. 9.4%, 2DS2‐VASc score allowed an improvement in the accuracy of the prediction of thromboembolic events.In Chinese AF patients who were not on OAC, NPAF was an independent predictor of thromboembolism and mortality. The addition of NPAF to the CHA The type of AF was defined according to the 2006 ACC/AHA/ESC guidelines for the management of patients with AF.The registry was designed to have a 1‐year follow‐up. The follow‐up was completed in November 2012 by trained research personnel via clinic visit, telephone or delivery of medical records. The status of OAC during the follow‐up period was collected again at the visit. In this study, the primary outcome was thromboembolic events , and secondary outcomes were defined as all‐cause death, cardiovascular death and stroke. Cardiovascular death included sudden cardiac death and death caused by heart failure, stroke, myocardial infarction, pulmonary embolus, peripheral embolus, aortic dissection.SDs or medians with quartiles; categorical variables were expressed as frequencies and percentages. Differences in continuous variables between groups according to the type of AF were analyzed using unpaired t‐test or the Mann–Whitney U test; comparison of categorical variables was performed using χ2 test or Fisher's exact test. Kaplan–Meier curves and log‐rank tests were performed to illustrate the discrepancies among the AF patterns. Univariable and multivariable Cox regression analysis was utilized to evaluate the effects of AF type on the TE events, stroke, all‐cause death and cardiovascular death. The following covariables were adjusted in the multivariable model: sex, age ≥75‐years‐old, body mass index, admission systolic blood pressure, admission diastolic blood pressure, admission heart rate, tobacco use, previous stroke or transient ischemic attack (TIA), coronary artery disease (CAD), prior myocardial infarction, hypertension, HF, significant valvular heart disease, diabetes mellitus, emphysema/chronic obstructive pulmonary disease, hyperthyroidism, sleep apnea, previous major bleeding, dementia or cognitive defects, antiplatelet drug, β‐blocker, ACEI/ARB, calcium channel blocker, diuretics, digoxin, statin, antiarrhythmic drug. Further, we conducted subgroup analyses to assess whether the difference between types on the risk of TE events and all‐cause death existed among the following specific subsets of patients, including sex, age, presence of CAD, hypertension, HF, CHA2DS2‐VASc score and antiplatelet drugs at discharge. Hazard ratio was estimated using Cox proportional hazards models fitted separately in subgroups of patients. The association between the rhythm at discharge (AF/flutter group vs other rhythm group) and adverse outcomes were also explored as the sensitivity analysis. The incremental contribution of AF type in predicting the risk of TE events based on CHA2DS2‐VASc score was presented as net reclassification improvement and integrated discrimination improvement using the PredicABEL, an R package for the assessment of risk prediction models. The prognostic utility of CHA2DS2‐VASc score and after adding NPAF as a risk factor (1 point) into CHA2DS2‐VASc score was assessed by C‐statistic estimates. The comparison between the two scores was also made.Continuous variables were expressed as means with p‐value <.05 were considered significant.Hazard ratio and 95% confidence intervals (CI) were calculated. The software package SPSS version 25.0 , the R software version 4.0.2  and the MedCalc version 19.0.7 were used for statistical analysis. GraphPad Prism version 6.01 was utilized for figures. All statistical tests were two‐tailed, and a 32DS2‐VASc score, but lower body mass index, diastolic blood pressure, and heart rate. They suffered from comorbidities more frequently. Patients with NPAF were more likely to take antiplatelet drugs, diuretics and digoxin, while they had less proportion of antiarrhythmic agents. Baseline characteristics of patients classified by the rhythm at discharge were given in Table Baseline characteristics were given in Table 2DS2‐VASc score and other confounders, NPAF was still a strong independent predictor of thromboembolism, stroke, and all‐cause death and the tendency remained for cardiovascular death. The prognostic value of AF patterns in the subgroups was also assessed . As for the TE events, the results were consistent in all subgroups. Regarding the all‐cause death, the trend existed in all subgroups except for the patients discharged without antiplatelet agents (p for interaction = 0.006). Furthermore, sensitivity analysis suggested that the main findings were consistent . The integrated discrimination improvement was 0.34% . After adding NPAF into CHA2DS2‐VASc score, the c‐statistic (CHA2DS2‐VASc score as continuous variable) increased from 0.622 to 0.638 .Besides, net reclassification improvement analysis indicated that the addition of NPAF to the CHA42DS2‐VASc score allowed an improvement in the accuracy of the prediction of TE events.Our data, from a multicenter, prospective, observational study, showed an increased risk of TE events in non‐anticoagulated patients with NPAF compared with those with PAF. After adjusting for variables, NPAF was still an independent predictor of thromboembolism and all‐cause death. The trend preserved when the AF pattern was classified into AF/flutter group or other rhythm group according to the electrocardiogram at discharge. The main findings were consistent in subgroups. Further, the addition of NPAF to the CHA2DS2‐VASc score and more comorbidities.p = .012). Recently data from 5 AF registries in Japan also indicated that NPAF increased the risk of ischemic stroke after adjusting for OAC administration at enrollment .Patients with NPAF had a higher arrhythmic burden than those with PAF. The AF pattern  is still the easiest manner to evaluate the burden in daily practice. The effect of the AF pattern on outcomes has been controversial for nearly two decades. Furthermore, there were few data in the Chinese population. In our study, the risk of all‐cause death increased from paroxysmal to persistent to permanent, which was in line with the results reported in The EORP‐AF General Pilot Registry.The role of AF types in the subgroups was also explored. Regarding thromboembolism, the results were consistent in all subgroups. As for the all‐cause death, the trend preserved in all subgroups except for the patients discharged without antiplatelet agents. The limited sample size might contribute to the difference. Further, considering the admission classification defined by physician's interpretation might be subjective, we performed a sensitivity analysis using a relatively objective classification according to the electrocardiogram rhythm at discharge. The main findings kept consistent. All results proved that NPAF was still an independent predictor of adverse outcomes, even after adjusting for the previously reported risk factors.p = .005). The literature suggested that reducing the burden of AF can improve prognosis. The statistical difference on the risk of TE events was reached in the post hoc analysis of the large clinical trials,2DS2‐VASc score of 0, one might wish to consider additional risk factors (including NPAF or AF burden) beyond those in scores to decide whether there was a need for thromboembolic protection that outweighs the bleeding risk. The present study assessed the role of NPAF in patients without OAC and evaluated its value by adding it to the CHA2DS2‐VASc score, and an improvement was seen. The arbitrary classification of AF type might limit the assistance for improving the predictive utility of the CHA2DS2‐VASc score on TE events. While last year investigators reported2DS2‐VASc score. NPAF was assigned a weight of 4 or 10 points in that paper, yet it was only 1 point in our study. In that case, the AF pattern might be used to identify patients who needed anticoagulation. Meanwhile, the weight of the AF pattern in the risk prediction model needs more investigated.We also evaluated the potential of the AF pattern in decision‐making for stroke prevention. Though increasing evidence showed that patterns or duration of AF associated with adverse events,2DS2‐VASc score. Our results only propose the hypothesis that adding NPAF in the CHA2DS2‐VASc score could improve the prediction accuracy for thromboembolism in AF patients. Further studies with larger sample size were needed to validate the results and investigate how much weight should be given to NPAF when integrating it into the existing risk prediction model. In addition, the present study could not accurately assess the AF burden. Further studies based on remote monitoring devices are needed.There were several limitations in the present study. First, the database did not consist of all known risk factors related to TE events, such as left atrial diameter, high‐sensitivity troponin, N‐terminal pro‐B‐type natriuretic peptide. We cannot rule out the possibility of unmeasured or residual confounding. Second, the relatively small sample size might lead to our exaggerative estimation of the association between NPAF and thromboembolic risk. Nevertheless, a similar trend was previously reported. Third, the diagnosis of the type of AF was physician‐defined and arbitrary, and the distinction between paroxysmal and persistent AF is often not made correctly without access to long‐term monitoring. Considering that, we performed a sensitivity analysis by classifying the patients according to the electrocardiogram at discharge to explore the association. And the results were consistent. Fourth, the AF pattern might progress from paroxysmal to persistent or permanent AF,52DS2‐VASc score allowed an improvement in the accuracy of the prediction of thromboembolic events. Further investigations are needed to confirm the results and assess its utility in therapy strategy.In Chinese non‐anticoagulated AF patients, NPAF was an independent predictor of thromboembolism and mortality. The addition of NPAF to the CHAThe authors declare no potential conflict of interest.Figure S1 Flowchart of the study patients.Click here for additional data file.Table S1 Baseline characteristics of patients divided by electrocardiogram at discharge.Click here for additional data file.Table S2 The risk of outcomes in patients discharged with atrial fibrillation/flutter comparing to sinus rhythm.Click here for additional data file.Table S3 The baseline risk factors of thromboembolic events.Click here for additional data file.


Leucine-rich repeat kinase 2 (LRRK2) mutations are among the most significant genetic risk factors for developing late onset Parkinson’s disease (PD). To understand whether a therapeutic can modulate LRRK2 levels as a potential disease modifying strategy, it is important to have methods in place to measure the protein with high sensitivity and specificity. To date, LRRK2 measurements in cerebrospinal fluid (CSF) have used extracellular vesicle enrichment via differential ultracentrifugation and western blot detection. Our goal was to develop a methodology which could be deployed in a clinical trial, therefore throughput, robustness and sensitivity were critical. To this end, we developed a Stable Isotope Standard Capture by Anti-peptide Antibody (SISCAPA) assay which is capable of detecting LRRK2 from 1 ml of human CSF. The assay uses a commercially available LRRK2 monoclonal antibody (N241A/34) and does not require extracellular vesicle enrichment steps. The assay includes stable isotope peptide addition which allows for absolute quantitation of LRRK2 protein. We determined that the assay performed adequately for CSF measurements and that blood contamination from traumatic lumbar puncture does not pose a serious analytical challenge. We then applied this technique to 106 CSF samples from the MJFF LRRK2 Cohort Consortium which includes healthy controls, sporadic PD patients and LRRK2 mutation carriers with and without PD. Of the 105 samples that had detectable LRRK2 signal, we found that the PD group with the G2019S LRRK2 mutation had significantly higher CSF LRRK2 levels compared to all other groups. We also found that CSF LRRK2 increased with the age of the participant. Taken together, this work represents a step forward in our ability to measure LRRK2 in a challenging matrix like CSF which has implications for current and future LRRK2 therapeutic clinical trials. To advance LRRK2 therapeutic development, measuring LRRK2 in cerebrospinal fluid (CSF) as an indirect central nervous system (CNS) target engagement and/or patient stratification biomarker would be advantageous. For instance, CSF LRRK2 concentrations could serve as a patient enrichment tool and/or a pharmacodynamic endpoint if a therapy aims to modulate aberrant CNS levels, e.g., with an antisense oligonucleotide (ASO) or gene therapy approach. Importantly, an outstanding question in the field is whether PD patients with LRRK2 mutations (such as G2019S) have altered expression of total or phosphorylated LRRK2 in brain and CSF. To date, CSF LRRK2 detection has proven technically challenging. This has led the Michael J Fox Foundation (MJFF) to sponsor the LRRK2 Detection Consortium which is an industry/academia initiative aimed at promoting the development of technologies enabling LRRK2 detection in different matrices including PBMCs, urine and CSF.Leucine-rich Repeat Kinase 2 (LRRK2) is a large (280 kDa) GTPase/kinase involved in intracellular vesicle dynamics, autophagy and inflammation processes . Given tLRRK2 in human CSF has been successfully measured using extracellular vesicle enrichment strategies . DiffereAn alternative to WB-based protein detection is enzyme-linked immunosorbent assay (ELISA) which is suited toward measuring proteins with far higher throughput. A number of high quality total LRRK2 mAbs and commercially available ELISA kits are available, however, to date there have been no reports demonstrating reliable CSF LRRK2 detection with these. Internal efforts from Biogen and other industry groups with support from the MJFF have developed ultrasensitive immunoassays  to enable CSF LRRK2 detection . DespiteBecause of the limited applicability of ultracentrifugation/WB analyses and issues developing a high sensitivity ELISA based platform, our group sought to develop a SISCAPA  including 8G10 (DEDGHFP), SIG-39840 (FPNEF) and N241A/34 (EGDLLVNPDQ). Of these three antibodies, preliminary experiments led us to select N241A/34 as a candidate anti-peptide antibody to isolate and measure the tryptic peptide AEEGDLLVNPDQPR (AA 1834–1847). This peptide was shown to be unique to LRRK2 protein .According to epitope mapping data provided by MJFF , several20 + 0.1% formic acid and 5% acetonitrile (I). A Thermo Q Exactive Plus  orbitrap mass spectrometer was operating in data dependent acquisition (DDa) mode to search for the most abundant peptides eluted from beads. Full MS settings were 60,000 resolution, AGC target 1e6 and max IT time was 100 ms with a scan range of 200–2000 m/z. dd MS2 settings were 15,0000 resolution, AGC target 1e6, max IT time was 100 ms and scan range was 200–2000 m/z.To test the hypothesis that N241A/34 could isolate the unique LRRK2 peptide that contains its epitope , we conjugated Neuromab N241A/34  onto M-280 Tosylactivated Dynabeads  at a concentration of 1 ug N241A/34 per 1 μl of beads. We then digested 10 μg wild type recombinant LRRK2  using 5 μg TPCK-treated trypsin  at 40°C for 3 h shaking at 1400 RPM on an Eppendorf Thermomixer. Digestion was stopped by adding 5 μg of protease inhibitor AEBSF . Ten μl of N241A/34 bead solution  was added to the protein digest and immunoprecipitation was performed on an end over end Hula Mixer  for 1.5 h at 4°C. Sample was then placed on a DynaMag-2 magnetic tube holder  and beads were isolated from digest mixture. Beads were then washed using 1 ml of PBS + 0.05% Tween (PBST) on and end over end mixer for 1 min. PBST was removed and then washed twice using 1 ml PBS. Peptides were eluted off beads with 50 μl of HOne hundred ug Neuromab N241A/34 monoclonal antibody  was desalted with a Zeba Spin desalting column, 7 K MWCO, 0.5 ml  and then biotinylated using a One-Shot biotinylation kit . Degree of biotinylation was measured according to One-Shot protocol using a Thermo Fisher NanoDrop spectrophotometer  where 2 μL of biotinylated antibody is loaded to read the absorbance at 280 and 354 nm. Absorbances at both wavelengths are input into ChromaLink Biotin Molar Substitution Ratio (MSR) calculator to determine precise degree of biotinylation. An average of six biotin molecules was calculated per one N241A/34 antibody. Biotinylated N241A/34 was combined with streptavidin-coated magnetic beads  at a ratio of 100 μg antibody to 1 mg of beads according to the manufacturers protocol. Final conjugated antibody mixture was stored in 0.1% BSA PBS solution at 4C until use.13C1215N6 KAEEGDLLVNPDQPR  labeled at both C (K) and (R) termini (m/z 566.9641+++) was added to each sample. Then, 10 μl of N241A/34 on beads was added to each sample and incubated at 4°C for 1.5 h on an end over end Hula Mixer . Beads were then washed using 1 ml of PBS + 0.05% Tween (PBST) on and end over end mixer for 1 min. PBST was removed and then beads were washed twice using 1 ml PBS at 1 min each time. Peptides were eluted off beads with 50 μl of H20 + 0.1% formic acid and 5% acetonitrile  buffer  was added. Twenty micrograms of TPCK-treated trypsin were added to each sample and digested at 40°C for 1.5 h at 1400 RPM shaking on a Thermomixer . Following digestion, samples were placed on ice for 5 min then 50 μl of 2 pg/ml (100 fg) heavy synthetic peptide ile ACN; .m/z. Inclusion list contained both light (m/z 560.9566+++) and heavy (m/z 566.9641+++). Samples were analyzed using Skyline 64-bit  software and signal was considered detectable if cumulative peak area was >5000 units and contained a minimum of four fragment ions. Most intense fragment ions typically observed were y5, y6, y7, and b6, b7, b8.A RSLC  nanoflow autosampler and HPLC system was used for sample separation. Peptide eluent was injected onto a Thermo C18 Pepmap nano trap column  at 20 μl/min for 4.5 min. Peptides were then eluted onto an E800A EasySpray nanoLC column  nanoLC column at 0.3 μl/min. For all other experiments Q Exactive HFX was operating in parallel reaction monitoring (PRM) mode at 120,000 resolution, AGC target set to 1e^6, maximum injection time (IT) set to 240 ms and isolation window set to 1.0 To determine the concentration of each sample (in pg/ml), the light:heavy ratio was taken and then multiplied by two since the internal standard was 2 pg/ml. Finally, we considered the fraction of the entire protein that is being detected, i.e., the peptide is 1/170.4 of the total mass of total LRRK2  protein. Therefore, we multiplied the ratio by 170.4 to give us an accurate concentration of the total LRRK2 protein in the sample. This is expressed by:T-test.Since previous work showing LRRK2 measurements in CSF used a vesicle enrichment step followed by vesicle lysis with detergents, we aimed to determine whether detergent addition  was necessary for the detection of LRRK2 in our CSF samples. Four 2 ml pools of CSF were made and then aliquoted into separated 1 ml tubes. Half the samples (four) had 100 μl 10X RIPA  while the other half (four) did not. Signals were compared using a paired two-tailed An 8 ml pool of CSF  was split into 8–1 ml aliquots. Each aliquot had a different volume of fresh whole blood spiked in, ranging from 10 ηl to 10 μl, i.e., 0.001 to 1% v/v whole blood in 1 ml CSF. To be consistent with sample processing at a clinical trial site, samples were frozen at −80°C following whole blood addition then thawed. After thawing the samples, a small volume of the blood-spiked CSF (5 μl) was removed from each aliquot to assess hemoglobin (HgB) levels . The rest of the sample was processed using the SISCAPA workflow described here for total LRRK2 detection. To measure Hgb, we used a commercial ELISA kit from Abcam(ab157707). CSF was diluted 1:100 in assay diluent to a final volume of 500 μL. HgB present in the test samples was captured by anti-HgB antibody pre-adsorbed on the surface of microtiter wells after a 20 min incubation under room temperature no shaking. After sample binding, unbound proteins and molecules were washed off, and an enzyme-antibody conjugate was added to the wells and allowed to bind to captured HgB. After 20 min incubation, unbound proteins and molecules were washed and Chromogen substrate solution was then added to catalyze the reaction. After 10 min incubation, stop solution was added. Light intensity, which was proportional to the amount of HgB present, was measured at 435 nm on a SpectraMax plate reader . HgB concentrations were determined on a standard curve by plotting OD vs concentration using a five-parameter logistical curve-fit. The calibration curve range of this method is 6.25 ηg/ml-200 ηg/ml.106 CSF samples were collected as part of the MJFF LRRK2 Cohort Consortium and were shipped to Biogen, blinded, in 200 μl aliquots. Five aliquots were pooled (1 ml) in 1.5 ml Eppendorf LoBind tubes and then processed by two separate operators according to the protocol described here including detergent, trypsin and internal standard addition to all samples. Unblinding only took place until after all sample analyses were conducted and raw data was submitted back to MJFF. Age of cohort ranged from 26 to 83 years old. LRRK2+PD+ group included G2019S mutation carriers while the LRRK2-PD+ group had no known mutations. Of the 106 samples analyzed, 105 of samples had evaluable levels of LRRK2.We confirmed that N241A/34 could isolate AEEGDLLVNPDQPR, but also unexpectedly found that it isolated a second high confidence peptide containing a missed cleavage, i.e., KAEEGDLLVNPDQPR (AA 1833–1847). Proteome Discoverer  processed the DDa data against the human proteome and showed that KAEEGDLLVNPDQPR gave an 8X higher peak (2.68e8) area than AEEGDLLVNPDQPR 3.27e7; . In addiR2 = 0.999) at these concentrations. Bias was calculated for each of the concentration points. Furthermore, endogenous QC (EQC) was included in 4 of the runs  solution . We alsothe runs . Based othe runs .Since the SICAPA workflow appeared to provide consistent data using rLRRK2 , we moveT-test, p = 0.0165 (To determine whether or not detergent addition (RIPA buffer) had an effect on LRRK2 detection, presumably through vesicle disruption during proteolysis, we compared CSF with and without 100 μl RIPA in each sample . Data sh= 0.0165 .CSF blood contamination caused by traumatic lumbar puncture can have a negative impact on CSF measures particularly when protein analyte is highly expressed in blood such as alpha synuclein (aSYN). Since LRRK2 is expressed in circulating white blood cells including monocytes, macrophages and leukocytes , we wantn = 28) was 31.7 ± 22.7 pg/ml (range = 5–104 pg/ml). For sporadic (non LRRK2 carriers) PD (n = 34) it was 31.1 ± 24.8 pg/ml (range = 7–120 pg/ml). For LRRK2+PD- non manifesting carriers (n = 29) it was 40.8 ± 32.1 pg/ml (range = 9–122 pg/ml). For LRRK2+PD+ patients (n = 14) mean levels were 67.8 ± 39.6 pg/ml (range = 19–139 pg/ml). The effect of age was measured by plotting total LRRK2 levels against age for each of the 4 groups separately  but not in the PD-LRRK2+ (One hundred and six 106) human CSF samples were received from the MJFF LRRK2 Cohort Consortium in a blinded fashion. Of the 106 samples, 105 had evaluable levels of LRRK2 , 6. Mean human CSparately . Data shD-LRRK2+  or PD+LRD-LRRK2+ .p = 0.0007; p = 0.0013), idiopathic PD (p = 0.0007), and compared to non-manifesting LRRK2 carriers (p = 0.029).We then analyzed group differences in total LRRK2 levels. A one-way ANOVA showed a significant effect between the four groups  in the model, for each cohort . A BonfeThe current work describes a novel quantitative methodology for reliably detecting total LRRK2 levels in 1 ml of human CSF. The method does not require an exosome isolation step and uses a commercially available antibody (N241A/34) within the SISCAPA workflow which is scalable and amenable to higher throughput analyses. We demonstrated that the assay meets our basic fit for purpose qualification criteria such as dilutional linearity across a physiological dynamic range and acceptable precision (12.6%) and accuracy (10.8%). Furthermore, we show that blood contamination in CSF does not pose a serious analytical challenge compared to other analytes that are more highly expressed in blood such as aSYN. Finally, using this methodology we showed that PD patients harboring the G2019S LRRK2 mutation have significantly higher CSF LRRK2 levels compared to healthy individuals, sporadic PD patients, and non-manifesting LRRK2 carriers.The search for a sensitive and high throughput assay to detect LRRK2 and/or pLRRK2 levels in CSF has been a challenge for both academic investigators and industry teams seeking to advance clinical stage LRRK2 therapeutic programs. Our internal work suggested that a number of high quality antibody reagents could be used to develop ultrasensitive immunoassays  that could measure low pg/ml concentrations . The use of a missed cleavage peptide as a surrogate for LRRK2 protein detection was initially a concern because it was not clear how well we could control its generation compared to the fully cleaved peptide . Tryptic miscleavage occurs for many reasons such as adjacent cleavage sites, nearby glutamic or asparatic acid residues or phosphorylation . In the Using this assay we showed that blood contamination did not cause a significant pre-analytical issue for LRRK2 detection as it does for aSYN , consistThe main finding of the current study is that LRRK2+ PD patients have elevated CSF LRRK2 levels compared to healthy controls, sporadic PD patients or LRRK2+ non-manifesting carriers. Even after adjusting for age and gender , LRRK2 lIn summary, the assay described here provides a reliable means to measure total LRRK2 levels in human CSF which could be used to support interventional clinical trials where LRRK2 is targeted. Future iterations of the assay should include techniques to capture kinase activity  and should be optimized to reduce sample volume requirements. Additional steps can also be taken to automate this assay to improve throughput. Importantly we showed that in a set of 105 CSF samples that the LRRK2+PD+ group had roughly 2× higher CSF LRRK2 levels compared to other groups. Although the precise reasoning for this increase has yet to be elucidated, future studies can use this quantitative methodology to probe the relationship between disease progression and longitudinal CSF LRRK2 levels.The datasets generated for this study can be found in the LRRK2 Cohort Consortium database.Ethical review and approval was not required for the de-identified sample analysis in accordance with the local legislation and institutional requirements. The patients/participants provided their written informed consent to participate in this study.OM developed the assay described here and executed the experiments. SC and AE performed the experiments. MY performed statistical analyses on the LRRK2 Cohort Consortium CSF the data. WH and DG conceptualized experiments and gave guidance throughout the assay development process.All authors are employed by Biogen, Cambridge, MA, United States. Biogen has obtained a non-exclusive patent license for SISCAPA assay research use from SISCAPA ASSAY Technologies, Inc. for the assay.


Staphylococcus aureus virulence and their increased production in community-associated methicillin-resistant S. aureus (CA-MRSA) likely contributes to the enhanced virulence of MRSA strains. Here, we analyzed the differences in bacterial cell aggregation according to PSM presence in the specific human cerebrospinal fluid (CSF) environment. CSF samples from the intraventricular or lumbar intrathecal area of each patient and tryptic soy broth media were mixed at a 1:1 ratio, inoculated with WT and PSM-deleted mutants (Δpsm) of the CA-MRSA strain, USA300 LAC, and incubated overnight. Cell aggregation images were acquired after culture and image analysis was performed. The cell aggregation ratio in WT samples differed significantly between the two sampling sites . The cell aggregation ratio in Δpsm samples also differed significantly between the two sampling sites . Division of the study cases into two groups according to the aggregated area ratio  showed that the median aggregation ratio value differed significantly between groups A and B . The differences in CSF distribution and PSM presence within the specific CSF environment are significant factors affecting bacterial cell aggregation.Phenol-soluble modulins (PSMs) are major determinants of  Staphylococcus aureus (MRSA) is one of the most infamous antibiotic-resistant pathogens [S. aureus toxins [Methicillin-resistant athogens . While Mathogens . These iathogens . With reathogens . Nonetheathogens ,5. This s toxins ,6. By fas toxins . Howevers toxins . Furthers toxins . HoweverCA-MRSA virulence is highly dependent upon its characteristic toxins. Phenol-soluble modulin (PSM) peptides, alpha-toxin, and Panton-Valentine leucocidin are key factors highly linked to CA-MRSA virulence and distinguish CA-MRSA from other MRSAs . Of thesTo investigate the role of PSMs in human CSF and prepare for the potential increases in CNS CA-MRSA infection, it is necessary to study the MRSA growth characteristics that are exclusively present in the CNS environment. CSF is the best representative for pathological CNS changes owing to its close anatomical relationship with critical CNS structures . Thus, tThis ex vivo study was conducted with institutional review board approval from Eulji University . The study was initiated in September 2018 and aimed to investigate the factors affecting CA-MRSA bacterial cell aggregation in CSF (used to represent the CNS environment). Patients were recruited from the neurosurgery or neurology departments of Eulji University Hospital between July 4, 2018 and June 28, 2019. Neurosurgery patients were diagnosed with normal pressure hydrocephalus (NPH) or obstructive hydrocephalus due to intraventricular hemorrhage and underwent ventricular puncture during their operations. Intraventricular CSF, which is inevitably excreted during these operations, was collected. In case of the neurology patients involved in this study, a CSF sample via lumbar puncture was collected to enable a differential diagnosis for the various nervous system disorders and the remaining sample was collected for research purposes. Collected CSF samples were stored at 4 °C until use and analyzed as soon as possible. All samples were obtained by the clinician during the collection processes with written, informed patient consent and patients’ personal data were protected at all times.The wild-type (WT) and PSM-deleted mutant (Δpsm) strains of CA-MRSA LAC (USA300), as well as MW2 (USA400) used in the current study were provided by the Duksung Women’s University Laboratory, which had stored these strains that were obtained during previous research . Each stVarying concentrations  of human CSF in TSB media were tested to determine the best culture conditions for distinguishing cell aggregation patterns between WT and Δpsm strains (data not shown). Test tubes (5 mL) and 96-well microtiter plates  were used to investigate cell aggregation. After testing, 50% CSF in TSB was selected to cultivate cells in 96-well microtiter plates for cell aggregation observation. In each well, CSF and TSB media (50 µL each) were mixed in each well 50 µL of CSF from each patient and TSB media were mixed at a 1:1 ratio. Precultured WT or Δpsm LAC strains (5 μL) were inoculated into the wells and incubated at 37 °C with shaking (200 rpm) overnight. Plate outer wells were filled with 200 μL of sterile distilled water to maintain humidity and avoid water evaporation from cell cultures in the inner wells. Cell aggregation images were acquired from the bottom of each well of the plates after overnight culture. An attachable magnifying lens  was equipped on a smartphone  camera and used to magnify the images 30´. For convenient manipulation of the smartphone camera under the plate, the phone was connected to a laptop computer with the LonelyScreen mirroring program . Image J software  are was used to measure the aggregation area . The folAfter the overnight culture was centrifuged at 4000 rpm for 10 min, 100 μL of each culture supernatant was transferred from the microtiter plate to sample vials for liquid chromatography-mass spectrometry (LC/MS) analysis. PSMs in the culture supernatants were quantified as described previously ,19 with A biofilm assay was conducted in the form of a preliminary study after selecting 10 intraventricular and 10 lumbar intrathecal CSF samples from the total number of cases. After overnight culture at 37 °C, the culture broth in the 96-well plate was discarded and residual liquid was further removed by tapping the plate upside down on several layers of paper towel. Biofilms attached inside each well were washed three times with 200 µL of phosphate buffered saline  and at every washing the residual liquid was removed by tapping on paper towels. After washing, biofilms in the plate were stained with 100 μL of 0.1% (w/v) crystal violet solution for 10 min at room temperature. After discarding the crystal violet solution, microtiter plate wells were washed three times with 200 μL of double distilled water and air-dried for 1 h. Finally, 200 μL of 95% ethanol was added into each well and crystal violet was eluted from the biofilms by pipetting followed by incubation at room temperature for 15 min. After another pipetting to mix the solutions well, the optical density of the eluted dye solution was determined using a SpectraMax ABS Plus Microplate Reader  at 550 nm.p value less than 0.05.Data are shown as the number (%) for categorical variables and medians (25th–75th percentile) for continuous variables. To compare the characteristics of the study groups, the chi-squared test was used for categorical variables, and the Student’s t test for continuous variables where appropriate. A multivariate logistic regression was then used to evaluate the independent associations of each potential explanatory variable. All variables that had been previously identified in the literature were considered eligible for inclusion in the model. Statistical significance was defined as a n = 19) followed by migraine or tension-type headache (n = 18), infection (n = 12), and cerebrovascular disease (n = 11). Of the 68 cases, normal CSF, which is a clear body fluid free of red blood cells (RBCs) and containing only a few (<5) white blood cells (WBCs), was found in 40 cases. Increased RBCs, increased WBCs, and an increase in both RBCs and WBCs were observed in 17, 5, and 6 cases, respectively. The clinical and laboratory data of the 68 CSF samples obtained from 64 enrolled patients are summarized in The growth characteristics observed in this study were divided roughly into three types as follows: (1) Active cell aggregation with three different patterns ; (2) robust bacterial culture with minimal aggregation; or (3) neither aggregation nor bacterial culture observed . Of the p < 0.001) and in Δpsm samples . CSF protein content differed significantly between the two groups .We divided study cases into two groups according to the CSF sampling site  and compared the clinical and laboratory data. In four patients diagnosed with NPH, samples were collected from both the intraventricular and the lumbar intrathecal area at a certain time interval . A compap < 0.001) (p = 0.002). After multivariate analysis for confounding factors, the aggregation ratio was significantly lower in the ventricular CSF than in the lumbar intrathecal CSF  (p < 0.001). Differences were also significant for showing increased WBCs (p = 0.049) and protein in CSF (p = 0.002). < 0.001) . In the = 0.002) . Given t= 0.002) . In thisp < 0.0001)  , which cIn this study, we investigated the interaction of CA-MRSA with human CSF dependent on the presence of PSMs. Various CA-MRSA aggregation patterns were observed from 68 CSF samples. The cell aggregation patterns are illustrated in CSF plays an essential role in maintaining CNS homeostasis. The functions of CSF include maintaining the buoyancy of the brain, spinal cord, and nerves, volume adjustment in the cranial cavity, nutrient transport, protein or peptide transport, brain volume regulation, buffering against external forces, signal transduction, drug transport, immune system control, elimination of metabolites, and cooling of heat generated by neural activity . The proS. aureus aggregation factor (Saaf), in human CSF. As deduced from the cell aggregation results, the Saaf concentration seems very low in intraventricular CSF and high in lumbar intrathecal CSF. We have only preliminary characterization data of Saaf including the reduced cell aggregation both in the heat-treated CSF or in the filtrate of CSF after ultrafiltration (data not shown). Based on these observations, we might cautiously expect that Saaf is a protein. However, the identification of Saaf and its biochemical characterization including cell aggregation mechanism in human CSF is currently under investigation.Intraventricular CSF rarely induced aggregation, whereas lumbar intrathecal CSF induced cell aggregation with various patterns. This is demonstrated in p < 0.001). Even in lumbar intrathecal CSF, the cell aggregation ratio was significantly affected by the presence of PSM. This result indicates that PSM is the main effector that controls the ratio of aggregation, and thus, plays an important role in defining the different forms of MRSA infection. Of the numerous identified CA-MRSA virulence factors, PSMs are a key virulence factor ,25. The We hypothesized that the use of antibiotics passing through the blood brain barrier may be related to the suppression of bacterial culture as well as cell aggregation. Although five of the eight cases in which antibiotics were administered before CSF sampling showed neither bacterial culture nor cell aggregation, LC/MS analysis failed to detect residual antibiotics in all eight CSF samples. The definitive role of antibiotics in this study remains inconclusive.S. aureus biofilm development. Dastgheyb et al. [Previous studies showed diverse mechanisms through which PSMs impact b et al.  demonstrb et al.  found thb et al.  althoughLimitations of this study included the following: First, whole CSF was used directly after sampling without any separation or purification. Although minimal relationships between various human cells that may exist in the CSF and bacterial cell aggregation were observed (data not shown), the removal of human cells prior to cell aggregation assay is recommended to standardize the experiment. Second, the biofilm assay was only performed on 20 samples  to match the sample sizes. Thus, the results of the present study might be influenced by selection bias. Third, PSM quantification by LC/MS was only performed to double-check the presence and absence of PSMs in the WT and Δpsm precultures, respectively, prior to their inoculation for the cell aggregation assay. PSM production from the WT strain cultured in intraventricular and lumbar intrathecal CSFs was investigated and the PSM amount confirmed constant after culture (data not shown). Finally, to determine the differences between strains, we conducted a preliminary experiment with USA400 CA-MRSA strain MW2 in the same manner and the same result  was observed (data not shown). Further study is needed to elucidate the effect of different types of PSMs on cell aggregation and biofilm formation among other staphylococcal species.This study is the first report examining staphylococcal growth characteristics in human CSF. A distinct difference in cell aggregation was observed between intraventricular and lumbar intrathecal CSF. This finding suggests that the distribution (source compartment) and composition  of CSF are important factors affecting CA-MRSA cell aggregation. In addition, Saaf related to cell aggregation may exist in human CSF. Future research focusing on discovery of the biomolecules that participate in cell aggregation and revealing the aggregation mechanism is necessary to understand and treat CA-MRSA CNS infection.


The brain ventricles are interconnected, elaborate cavities that traverse the brain. They are filled with cerebrospinal fluid (CSF) that is, to a large part, produced by the choroid plexus, a secretory epithelium that reaches into the ventricles. CSF is rich in cytokines, growth factors and extracellular vesicles that glide along the walls of ventricles, powered by bundles of motile cilia that coat the ventricular wall. We review the cellular and biochemical properties of the ventral part of the third ventricle that is surrounded by the hypothalamus. In particular, we consider the recently discovered intricate network of cilia-driven flows that characterize this ventricle and discuss the potential physiological significance of this flow for the directional transport of CSF signals to cellular targets located either within the third ventricle or in the adjacent hypothalamic brain parenchyma. Cilia-driven streams of signalling molecules offer an exciting perspective on how fluid-borne signals are dynamically transmitted in the brain.This article is part of the Theo Murphy meeting issue ‘Unity and diversity of cilia in locomotion and transport’. The physician philosophers thought that the ‘Common’ and the ‘Imaginative’ senses reside in the two large lateral ventricles located mirror-symmetrically in the front of the brain. The bipartite middle ventricle would encompass the ‘Estimative’ and ‘Phantastic’ senses. The last ventricle in the back of the brain would be devoted to the ‘Memorative’ sense . Modern et al. [Despite their ‘downgrading’, brain ventricles continue to attract attention, perhaps more now than in the past when physiologists viewed them primarily as toxic waste dumps for metabolites that the brain eliminated. Contemporary neurobiologists view the cerebrospinal fluid (CSF)-filled ventricles as dynamic reservoirs of signalling substances and nutrients that can reach neurons, glia cells and stem cells . This waet al.  have ideet al. ,4 but noThe aim of this brief review is to provide background knowledge for selected components of the ventricular system , and inf2.The central nervous system of vertebrate animals develops from the neural tube. The wall of the tube consists of neuronal progenitor cells and encloses the CSF-filled ventricle. Through a combination of proliferation, migration and differentiation of the progenitor cells, fore-, mid- and hindbrain form at the front end of the neural tube, while the back end of the neural tube gives rise to the spinal cord. Brain ventricles co-develop with the nervous system so that around the time of birth, four distinct brain ventricles have formed. In the postnatal brain, ventricles are enclosed by the ependyma that consists mostly of E1 cells  inside the lateral ventricles of the human brain is shown in the detailed anatomical drawings by Vesalius [The intensely vascularized structure of the Vesalius . This paVesalius . The conVesalius ,14–17. IIn the brain of lampreys, a primitive vertebrate animal, the CP distributes throughout the ventricles . In mammThe CSF also contains extracellular vesicles (EVs) ,29. EVs Adult neurogenic stem cell niches in which neurogenesis persists after birth are found in the subventricular zone of the lateral ventricle, in the subgranular zone of the dentate gyrus and along and beneath the v3v wall ,24,41,42In summary, the CP is a rich source of signalling compounds, some of which are dissolved single molecules, while others are packaged into EVs or other types of lipid particles . Because4.Knowing the biology and biochemistry of the ependymal cells is crucial since these cells form the boundary between CSF and brain parenchyma ,6. Throuvice versa. Tanycytes of the E3 type also populate the organum vasculosum of the laminae terminalis (OVLT) located at the anterior wall of the v3v. These tanycytes are in direct contact with v3v through the apical area of the tanycytes that sprouts sensory cilia [The current literature discusses four types of tanycytes ,52, but ry cilia ,57.Taken together, the wall of the third ventricle is histologically complex and is made of several cell types. The dominant cells are the ependymocytes that sustain a lifelong complex CSF flow. Tanycytes provide a conduit between ventricular CSF and the brain parenchyma. Tanycytes also form bona fide stem cell niches in the adult brain, and these niches respond to CSF-infused growth factors by proliferation and differentiation.5.a). To generate this flow map, 1 µm diameter fluorescent beads are added in bulk to the medium in which the v3v explants are kept. The movement of beads evoked by beating cilia bundles is captured and a particle tracking software is used to compute individual bead tracks [b) or fluorescent liposomes (c) to the ependyma of the explant. Rates of transport of beads and liposomes across the ependyma are in the range of several hundred micrometres per second. Since in the mouse, the v3v is about 2 mm long, moving of CSF from the inflow to the outflow occurs within a few seconds. Either of these methods reveals a complex flow pattern in the v3v that consists of straight flows, bent flows and whirls capable of directional transport and of subdividing the ventricular volume [Does CSF flow through the ventricles like water would flow in a system of interconnected rivers and lakes? This is definitely not the case in the v3v. Explants of the ventricular walls show complex flow patterns that consist of eight distinct streams that are interconnected like roads in a map a. To gend tracks . These tiposomes c to the r volume ,58–60.F6.in vivo owing to heart beating [in vivo [CSF flow in the ventricular system of mammalian brains is driven by CSF secretion into the ventricles and by motile cilia beating, and plays an important role in the delivery of CSF components and washout of waste . CSF is  beating ,17,61, r beating  and even beating . Differe beating  and the [in vivo ,61,62 buet al. [a) [et al. simulations [Alternatively, detailed CSF flow in the 3D ventricles can be investigated with computational fluid dynamics. Previous numerical studies focused on the human brain and investigated the CSF flow in idealized geometry or subject-specific geometry of ventricles reconstructed from medical imaging –67. In met al.  took int al. [a) . Interesulations  show thaCompared with the human brain ventricles, the two parts  of the 3V of the mouse brain are much more separated. The ventral part is also much narrower than its dorsal counterpart. As a result, most oscillatory CSF net flow will take the dorsal route owing to its smaller resistance and, therefore, the flow in the ventral part will be dominated by cilia beating.7.in vivo? There are two lines of evidence that suggest that flow directions in explants and in the live animal are very similar. First, it was observed that in the lateral ventricle, cilia effective stroke and flow directions, as detected by particle tracking (a), are correlated [a). The corresponding movies of cilia beating in the ependyma underneath flows 3 and 4 show that cilia beat in the opposite direction as well . One co8.Why is there a ventricle in the hypothalamus and what would be the purpose of a complex directional flow pattern in the v3v? The answer could be that CSF delivers factors to specific hypothalamic nuclei, especially those that are located at or near the basal side of ependyma. Ependymal flow 8 passes above the suprachiasmatic nucleus, which regulates the circadian clock. Flow 6 passes by the arcuate nucleus, which controls various metabolic functions. Tanycytes provide a direct exchange between CSF and neuronal or vascular targets. Flow 6 streams to the tanycyte region, while flow 7 leads away from the tanycytes. In the explant, there is no flow above the tanycytes, but in the closed ventricle in the brain, flows 6 and 7 would provide coordinate transport of CSF solutes to and then away from the tanycyte region.This view of the ventricular system as a site of targeted flow provides a number of challenges. What are the substances that are being delivered? There are many factors in CSF, and they not only need to be identified but assays for a function in the hypothalamus need to be developed. These functions could relate to metabolism, circadian timing or the control of stem cell growth and differentiation. How can CSF constituents transit from CSF to their subependymal targets? For solutes such as growth factors, there may be specific receptors on E1, E2 or E3 cells. The uptake of EVs may involve endocytosis . Where dProtein targeting mechanisms in cells may serve as a useful paradigm for thinking about the ventricular transport. A key insight for understanding protein targeting within cells was the realization that proper sorting and delivery relies on information encoded in the protein itself. In analogy, it is possible that EVs, potential carriers of CSF signals in the ventricular system, expose on their surface protein sequences that are used as a postal code for sorting into specific streams and for proper delivery to the apical face of specific E1, E2 and E3 cells. Addressing all these points experimentally is a daunting task. The reward of such an effort could be the discovery of a cilia motility-based signalling system in an important and ancient part of the brain.


Streptococcus pneumoniae has been reported as the most common cause of bacterial meningitis; however, there is limited data on pneumococcal meningitis in Indonesia. This cross-sectional study aimed to isolate and identity S. pneumoniae strains from cerebrospinal fluid (CSF) specimens collected as part of routine testing from patients with clinically diagnosed central nervous system infection at a national referral hospital in Jakarta, Indonesia in 2017. S. pneumoniae isolation and identification were performed using conventional culture and molecular tools. Antibiotic susceptibility patterns were monitored through minimum inhibitory concentration testing. From 147 CSF specimens, one S. pneumoniae strain was identified from a patient with bacterial meningitis symptoms. The isolate was serotype 6B (ST5661) and susceptible to 18 antimicrobial agents tested, including penicillin, tetracycline, and the macrolide group. Our data provide insights into the epidemiology of invasive pneumococcal disease in Indonesia.CNS infection is a life-threatening condition in developing countries and  S. pneumoniae has reported as the most common cause of bacterial meningitis [et al. reported that S. pneumoniae serotypes causing bacterial meningitis in hospitalized children under five years of age in India were serotypes 6B, 14, 6A, and 19F which is included in pneumococcal vaccine (PCV) [et al. reported that tuberculosis, toxoplasmosis, and cryptococcosis are common causes of CNS infections in adult patients in a referral hospital in Jakarta, Indonesia [S. pneumoniae strains in cerebrospinal fluid (CSF) from patients clinically diagnosed with CNS infection at a national referral hospital, Jakarta, Indonesia.Bacteria, amoebae, fungi, and viruses are capable of central nervous system (CNS) invasion causing significant morbidity and mortality . The incningitis . Jayaramne (PCV) . It was ne (PCV) . Currentg and the supernatant was removed. This step was performed sequentially for each 1000 µl of CSF specimen [2 for 18–20 h. The plate was re-examined after 48 h of incubation [S. pneumoniae ATCC 49619 as positive control for the testing. The remaining CSF specimens were stored at −80 °C until further processing. Matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) was used to confirm the bacterial identification [This cross-sectional study was approved by the Institutional Review Board of Faculty of Medicine, Universitas Indonesia, Jakarta, Indonesia. A total of 147 CSF specimens from patients who were diagnosed with CNS infection were collected as a part of a routine testing at a national referral hospital, Jakarta from July to December 2017 . These Cspecimen . Direct cubation . The alpcubation , using fication .lytA gene, was performed by using the Invitrogen Platinum Master Mix  [http://www.pubmlst.org). Antibiotic susceptibility testing was performed by using the microdilution MIC Plate for S. Pneumoniae (Sensititre Thermoscientific) containing 20 antibiotics in a 96-well round bottom plate. This test was performed using an overnight culture to prepare the suspension in Mueller-Hinton broth equal to 0.5 McFarland. The suspension (100 µl) was transferred into 11 ml of lysed-horse blood and vortexed; next 100 µl of the mixture was transferred into each well. The MIC plate was incubated at 37 °C incubator for 18–20 h and examined using a mirror to observe the first clear well defined as the MIC value, which was interpreted using Clinical and Laboratory Standard Institute (CLSI 28th Edition) breakpoints for defining sensitivity.Bacterial DNA was extracted as described previously . The qPCen, USA) . Serotypen, USA)  and confen, USA) . Allele The patient characteristics are described in S. pneumoniae. Furthermore, this isolate was determined as serotype 6B and the sequence type (ST) of this strain was identified as ST5661. This isolate was susceptible to the following 18 antibiotics: moxifloxacin, levofloxacin, tetracycline, cefuroxime, ceftriaxone, chloramphenicol, penicillin, meropenem, ertapenem, amoxicillin/clavulanic acid (2 : 1 ratio), linezolid, clindamycin, cefepime, azithromycin, erythromycin, trimethoprim, sulfametoxazole, and vancomycin. Using qPCR targeting the lytA gene, we detected three specimens  to be positive for S. pneumoniae with Ct values of 19, 26, and 34, respectively. Specimen IDs 670710542 and 371017224 were found to be negative through direct microscopy and culture (One specimen (Specimen ID: 371016106) displayed Gram-positive lanceolate-shaped diplococci following direct Gram-staining , and alp culture .Staphylococcus epidermidis (n=8), Staphylococcus saprophyticus (n=3), Streptococcus dysgalactiae (n=1), Bacillus sp. (n=5), Enterobacter cloacae (n=1), Corynebacterium sp. (n=1), and Acinetobacter baumannii (n=2) were isolated. Colonies on the blood agar plate from two other specimens were identified as Cryptococcus sp., which were confirmed by India ink examination . We also observed from routine laboratory test results that 65 of the 147 CSF specimens (44.2%) were directly examined for acid-fast bacilli (Mycobacterium tuberculosis) either through Ziehl-Neelsen staining, culture, or real time PCR. Tuberculous meningitis was confirmed for 11 specimens (16.9%), i.e. one positive culture and 10 positive real time PCR results (data not shown)In addition, other microbial colonies in the culture were identified using MALDI-TOF mass spectrometry and conventional assays following routine laboratory analyses in the hospitals. From these CSF specimens, S. pneumoniae following direct microscopic examination and culture belonged to a patient who was admitted to the emergency department with a major complaint of asphyxia in the last 6 h prior to admission (S. pneumoniae (data not shown). CSF was cloudy and yellow in appearance with a cell count of 582 µl–1 (60 % polymorphonuclear), glucose level of 11 mg dl–1 (serum glucose level was 98 mg dl–1), and protein content of 215 mg dl–1, which was typical for bacterial meningitis. Accordingly, the patient was administered 300 mg ceftriaxone every 12 h intravenously for 14 days. The first administration as an empirical therapy initiated approximatly 3 h before the CSF specimen was collected.One specimen (Specimen ID 371016106) in this study that was positive for dmission . In addiS. pneumoniae following qPCR analyses , but negative for direct microscopic and culture. Specimen ID 670710542 was collected from a patient, who had lost consciousness and was clinically diagnosed with tuberculous meningitis. The CSF of this patient was clear and colourless with a cell count of 76 µl–1 (90 % mononuclear), glucose level of 57 mg dl–1 (serum glucose level was 93 mg dl–1), and protein content of 60 mg dl–1. M. tuberculosis culture and PCR analysis showed negative results (–1 (65 % mononuclear), glucose level of 43 mg dl–1 (serum glucose level was not examined), and protein content of 250 mg dl–1. M. tuberculosis culture and Ziehl-Neelsen staining showed negative results (The other two specimens (Specimen IDs 670710542 and 371017224) were positive for  results . Specime results . The CSF results . Both paS. pneumoniae in CSF using conventional  and molecular methods from clinically diagnosed CNS infection patients in Jakarta, Indonesia. Overall, three cases of pneumococcal meningitis were identified; one was detected through culture, Gram-staining, and qPCR analyses, while the remaining cases were only detected by qPCR. A study from Bangladesh reported a higher proportion of pneumococcal meningitis, 25 of 401 (6.2 %) [S. pneumoniae was identified from blood specimen as an etiological agent of the disease in only one child [S. pneumoniae is still circulating and causing disease in Indonesia.To the best of our knowledge, this is the first study to detect  (6.2 %) . Howeverne child . This seS. pneumoniae. Bacterial blood counts >103 ml–1 results in higher incidence of meningitis than those <103 ml–1 [et al. reported that high pneumococcal DNA loads in blood and CSF are associated with fatal outcomes in children with IPD [The sensitivity of Gram-staining and culture from CSF specimens depends on the bacterial load, meningeal pathogen type, and initiation of antibiotic therapy before lumbar puncture . The use103 ml–1 . Howeverwith IPD .S. pneumoniae serotype 6B detected in this study is one of the predominant serotypes in Indonesia [The ndonesia . The Worndonesia . Howeverndonesia . Furtherndonesia .S. pneumoniae using qPCR, these limitations may have slightly affected the success rate of bacterial isolation using blood agar media.The limitation of this study is that we used remaining CSF specimens obtained from a hospital because the CSF sample were required for other routine hospital tests. Additionally, some CSF samples were stored temporarily at −20 °C before being processed in the laboratory. Although we detected S. pneumoniae of serotype 6B (ST5661) was isolated in one patient with CNS infection in Jakarta, Indonesia. This finding showed invasive serotypes of S. pneumoniae are still circulating in Indonesia and causing diseases. This study suggests that CSF should be collected prior to antibiotic treatment to increase the rate of isolation. A further study about the effect of antibiotic treatment should be done to have better understanding of antibiotic treatment affecting the success rate of S. pneumoniae isolation.In conclusion,


A bioinformatics analysis was employed to investigate the function mechanism of the circRNAs.  circRNA expression was analysed in six cerebrospinal fluid (CSF) samples from three patients of the infectious and noninfectious phases using an Arraystar Human circRNA Array. Differentially altered circRNAs were validated by quantitative real-time polymerase chain reaction (qRT-PCR) in the 66 CSF samples of 33 patients of the infectious and noninfectious phases.  Firstly, 142 circRNAs were found significantly different in 6 CSF samples of the infection and noninfection phases of 3 patients. Fourteen circRNAs with the top largest fold changes were chosen from the 142 circRNAs for PCR validation in the same 6 CSF samples of 3 patients. Three circRNAs were selected to be validated in 60 CSF samples of 30 patients using the PCR test. In infection CSF, an upregulated hsa_circRNA_402632 and downregulated hsa_circRNA_008636 and hsa_circRNA_405481 were confirmed by PCR test. A bioinformatics analysis was used to investigate the function mechanism of the 3 circRNAs. hsa_circRNA_402632 is enriched in the insulin resistance pathway, the FoxO and AMPK signaling pathways are the most important pathways for hsa_circRNA_008636 gene expression, and hsa_circRNA_405481 is enriched in the endometrial cancer signaling pathway, Fc epsilon RI signaling pathway, and TGF-beta signaling pathway.  hsa_circRNA_402632, hsa_circRNA_008636, and hsa_circRNA_405481 may be potential diagnostic markers for central nervous system infection after neurological surgery. Postoperative central nervous system infection (PCNSI) is a common and serious complication –5. The iSelf-control samples were used for analysis from 33 selected patients at infected and noninfected phases from our center between February 2018 and November 2019. First, six CSF samples of three patients in infectious and noninfectious phases were obtained, and differentially expressed circRNAs were detected in the infection and noninfection groups by the Arraystar Human circRNA Array. Then, a qRT-PCR test was performed to validate the differentially expressed circRNAs with the same six CSF samples of the three patients used in the Arraystar Human circRNA Array. Following this, 60 CSF samples from 30 patients were prepared for the qRT-PCR test to validate the candidate circRNAs. The candidate circRNAs underwent a bioinformatics analysis to predict their molecular function and gene expression .The study protocol was approved by the Ethics Committee of Anyang People's Hospital based on the ethical standards of the 1964 Declaration of Helsinki and its later amendments. The patients or their families were informed about the study, and their consent to participate in it was obtained.The patients who enrolled in this study had central nervous system infection after neurological surgery therapy. The PCNSI was diagnosed according to the diagnostic criteria of the Centers for Disease Control and Prevention (CDC) . PatientThe study included 33 patients, 9 males (27.3%) and 24 females (72.7%). The average age was 58.88 ± 12.12 years (26 to 85 years). The initial diagnoses were cerebral hemorrhage in 20 patients (60.6%), subarachnoid hemorrhage (SAH) caused by intracranial aneurysm or AVM in 7 patients (21.3%), trigeminal neuralgia or facial spasm in 3 patients (9.1%), brain trauma in 1 case (3.0%), hydrocephalus in 1 case (3.0%), and cerebellar tonsillar hernia malformation with syringomyelia in 1 case (3.0%) .The neurological surgery therapy was ventricular drainage in 10 patients (30.3%), craniotomy combined with ventricular drainage in nine cases (27.2%), craniotomy in five cases (15.2%), ventricular drainage combined with lumbar drainage in three cases (9.1%), craniotomy combined with decompressive craniectomy in two cases (6.1%), lumbar drainage in two cases (6.1%), decompressive craniectomy in one case (3.0%), and a ventriculoperitoneal shunt in one case (3.0%).Among them, 26 cases (78.8%) underwent an emergency operation, and seven cases (21.2%) underwent selective operation.Sixty-six CSF samples of thirty-three patients in infectious and noninfectious phases were obtained. Among these, 17 noninfected CSF samples were obtained on the first day after neurological surgery when the patients were in uninfected phases; the infected CSF samples from the same 17 patients were obtained at the time of diagnosis of infection. In the other 18 patients, the infected CSF samples were obtained first at the time of diagnosis of infection, and the noninfected CSF samples from the same 18 patients were obtained after the PCNSL was cured.Sixty-six CSF samples from thirty-three patients were collected, centrifuged at 3000 rpm for three minutes and frozen at -80°C in the refrigerator. Each sample was 5 ml in size. Among these, 6 CSF samples from 3 patients were used for circRNA profiling using the Arraystar Human circRNA Array. The criteria for selecting patients for circRNA array were random; any patient enrolled in our study might be selected. For the convenience of the experiment, we chose the first three patients enrolled in our study for circRNA array. First, the total RNA extracted and purified was quantified using the NanoDrop ND-1000.Microarray hybridization was performed based on the Arraystar's standard protocols. The RNAs extracted were digested with RNase R  to remove linear RNAs and enrich circular RNAs. Then, the enriched circular RNAs were amplified and transcribed into fluorescent circRNAs utilizing a random priming method . The labeled circRNAs were hybridized onto the Arraystar Human circRNA Array V2 . After having washed the slides, the arrays were scanned by the Agilent Scanner G2505C. Agilent Feature Extraction software (version 11.0.1.1) was used to analyze the acquired array images. Quantile normalization and subsequent data processing were performed using the R software limma package. Differentially expressed circRNAs with statistical significance between the noninfection and infection groups were identified through volcano plot filtering. Differentially expressed circRNAs between the noninfection and infection groups were identified through fold-change filtering. Hierarchical clustering was performed to show the distinguishable circRNA expression pattern among samples.Primers were designed and synthetized from the six samples which were used for circRNA profiling, and these were also used for the PCR experiment to validate the preliminary and candidate circRNAs. Sixty samples from thirty patients were prepared for PCR of the expanded sample size to validate the candidate circRNAs.CircInteractome through the NIH web was used to analyze RNA-binding protein sites that matched with circRNAs and analyze circRNAs for predictions through a microRNA (miRNA) sponge. According to the endogenous RNA (ceRNA) mechanism, the TargetScan, miRTarBase, and miRDB databases were used to analyze the target proteins for miRNA, and R software was used to analyze all the target proteins that bind to miRNA. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEEG) pathway analyses were used to identify the ceRNA target proteins' function and signal pathways.t-test. circRNAs having fold changes ≥ 1.5 and p values ≤ 0.05 are selected as the significantly differentially expressed. We filtered the analysis outputs and ranked the differentially expressed circRNAs according to fold change and p value, using Microsoft Excel's Data/Sort & Filter functionalities.When comparing differences of circRNA expression profiling between the infected and noninfected groups, the fold change between the groups for each circRNA was computed. The statistical significance of the difference was analyzed by p values ≤ 0.05 were selected as the significantly differentially expressed circRNAs. A volcano plot was used to visualize the differential expression of circRNAs between noninfection CSF and infection CSF groups , altering RBP activity, miRNA sponge activity, and rolling circle translation. It also demonstrates conservation, abundance, cell type-specific expression, and tissue-specific expression and participates in the development of various diseases. Therefore, it will gradually be used as a new clinical diagnostic marker or a treatment target for many diseases such as intracranial infection.In this study, the circRNA array detected a large number of differentially expressed circRNAs between noninfected and infected CSF (6141 upregulated and 6446 downregulated). A qRT-PCR test was used to validate the differentially expressed circRNAs. Finally, three circRNAs were identified, including hsa_circRNA_402632, hsa_circRNA_405481, and hsa_circRNA_008636. A bioinformatics analysis was conducted to explore the potential functions of these circRNAs, which revealed the following results: hsa_circRNA_008636 can enrich the FoxO signaling pathway and the AMPK signaling pathway and hsa_circRNA_402632 can enrich the insulin resistance pathway, while hsa_circRNA_405481 can enrich the endometrial cancer signaling pathway, Fc epsilon RI signaling pathway, and TGF-beta signaling pathway, all of which may be related to oxidative stress and inflammatory response. Therefore, they have a strong underlying application prospect as biomarkers in the diagnosis of intracranial infection.


Chronic osteoarthritic pain is not well understood in terms of its pathophysiological mechanism. Activated glial cells are thought to play a role in the maintenance of chronic pain. T98G glioblastoma cell line was previously observed to release higher amounts of interleukin-6 (IL-6) when treated with cerebrospinal fluid (CSF) from patients with another chronic pain condition, post-herpetic neuralgia. In this study, we investigated the ability of CSF from patients diagnosed with knee osteoarthritis suffering from chronic pain, to trigger the release of pro-inflammatory cytokines, IL-6, IL-1beta and tumour necrosis factor alpha (TNF-α) from T98G. Characterization of upstream signalling was also explored.Fifteen osteoarthritis patients undergoing total knee replacement due to chronic knee pain and 15 patients without pain undergoing other surgeries with spinal anaesthesia were prospectively recruited. CSF was collected during anaesthesia. CSF were added to cultured T98G cells in the presence of lipopolysaccharide. IL-6, IL-1β and TNF-α release from T98G cells were measured using enzyme immunoassay. Antibody array and western blotting were performed using CSF-triggered T98G cell lysates to identify possible signalling targets. Age, gender and pain scores were recorded. Mann-Whitney U test was used to compare IL-6 release and protein expression between groups. Association between IL-6 and pain score was analysed using linear regression.p = 0.001). Antibody array conducted with 6 pooled T98G cell lysate induced with osteoarthritis pain patient CSF identified greater than 2-fold proteins including STE20-related kinase adaptor protein and spleen tyrosine kinase. Further validation done using western blotting of individual CSF-triggered T98G cell lysate showed non-significant increase.Significant higher levels of IL-6 were released by T98G cells when induced by osteoarthritis patients’ CSF in the presence of LPS. The IL-6 levels showed positive association with pain score ; Higher IL-6 release from T98G when triggered by OA-CSF, in the presence of LPS, suggest the presence of “unknown molecule” in CSF that may be crucial in the maintenance phase of chronic pain in our osteoarthritis population. Further studies on the signalling pathways involved in pain and relevance of IL-6 release from T98G cells in other pain models are needed. Osteoarthritis (OA) is a common joint degenerative disease affecting many patients worldwide. It is characterised by the progressive loss of articular cartilage, joint space narrowing, secondary bone hyperplasia and atrophy of joint muscles . This diThere is a lack of understanding about the signalling pathways involved in pain. Mechanisms underlying chronic pain may involve activation of a series of signalling molecules leading to increased peripheral and/or central sensitisation. Some studies evaluated inflammatory cytokines such as tumour necrosis factor alpha (TNFα), interleukin-1 beta (IL-1β), IL-6 and IL-8 and IL-10 in patients with chronic pain-related diseases. For instance, IL-6 is elevated in cerebrospinal fluid (CSF) of patients with complex regional pain syndrome (CRPS)  and elevOther studies showed chronic pain in OA being mediated by changes in neurotrophic factors, chemokines and interleukins as a response by chondrocytes or synovial cells at the joint in the synovial fluid . Some shOur group previously showed that CSF of post-herpetic neuralgia (PHN) chronic pain patients induced elevated IL-6 release from human glioblastoma T98G cells [The procedure for CSF collection was approved by our Institutional Ethics Board and written informed consent was obtained from each patient (DSRB Ref No. 2011/01710). Patients (both the pain group and the group without pain) were prospectively recruited by the attending anaesthesiologists prior to surgery. All patients recruited in the pain group satisfied the following inclusion criteria: (a) Confirmed OA of knees from history and x-ray of the knees by orthopaedic surgeons, (b) Numeric pain rating scale of more than 3 , (c) Knee pain of more than 6 months duration, (d) Scheduled for total knee replacement surgery under spinal anaesthesia, (e) Age of 21 years and above, (f) No other concurrent chronic pain conditions. Patients without pain (NP) and undergoing surgery with spinal anaesthesia served as the control group. The inclusion criteria for no pain (NP) group include: (a) Patients who do not have knee osteoarthritis, (b) No history of any type of chronic pain conditions over the past 1 year; (c) Patient consented for surgery below the umbilicus under a spinal anaesthesia, (d) Age of 21 years and above. Pregnant patients or patients with severe end-organ function impairment  were excluded.CSF was obtained during the conduct of spinal anaesthesia, where two-ml of CSF was withdrawn after the dura was punctured, before injecting local anaesthetic into the subarachnoid space to effect the spinal anaesthesia. A total of 15 chronic knee OA patients and 15 NP patients were recruited. CSF samples were then stored at − 80 °C. Patients’ demographics such as gender and age were also recorded.Human glioblastoma cell line T98G  was cultured as previously described [Escherichia coli 055:B5  to the T98G cells. Cells were induced for 48 h at 37 °C and 5% CO2 environment. LPS was used to enhance cytokine release from the cells [T98G cells were grown to approximately 90% confluency in 12-well plates. CSF from patients were filtered using a 0.22 μm filter to remove any existing cells in the CSF prior to all subsequent experiments. Filtered CSF was added into cultured T98G cells. The culture medium was replaced with FBS-free EMEM medium before addition of 50 μl CSF along with 32 μg/ml lipopolysaccharide (LPS) from he cells . Each weUsing supernatant from the above 48 h CSF-triggered T98G protocol, TNFα and IL-1β levels were also determined with ELISA assays- Human TNFα Duoset ELISA (DY210) and Human IL-1β/IL-1F2 Duoset (DY201) respectively . Detection range of standards for TNFα was 15.6 pg/ml to 1000 pg/ml; and IL-1β was 3.91 pg/ml to 250 pg/ml.n = 6) were pooled in equal quantity and added to the antibody array slide. Similarly, the same amount of T98G cell lysates from NP samples (n = 6) were pooled and added to another antibody array slide. The assay was then performed according to manufacturer’s instructions. Array image was captured using array scanner . Analysis of array data was done using Genescan software. Comparison of signals between pooled OA pain sample and pooled NP sample was done after normalization with glyceraldehyde-3-phosphate dehydrogenase (GAPDH).Nuclear factor kappa light chain enhancer of activated B cells (NF-κB) phospho antibody array  was used to screen for changes in protein expression and phosphorylation profile in our samples. This array applied an ELISA-based technique where samples were biotinylated before adding to the array slide containing the affixed antibodies. Biotin on samples upon interaction with dye-labelled streptavidin on array would generate the fluorescence signal. Patient CSF-triggered T98G cells, in the presence of LPS, were harvested and lysed with CelLytic MT Cell Lysis Reagent . Total protein from these T98G cell lysate were quantified using bicinchoninic acid (BCA) assay . Due to limited amount of CSF drawn from each patient, and to obtain a minimum amount of protein for each antibody array, the triggered T98G cell lysates from OA pain samples (n = 15 each) were checked for protein expression level of targets with western blotting. Ten micrograms of each CSF-triggered T98G cell lysates were loaded per lane of 10% Tris-glycine gels and run using Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). Proteins on gel were transferred onto Polyvinylidene difluoride (PVDF) membrane. 0.1% Tween-20 added into Tris-buffered saline (TBS-T) was used to wash membranes and prepare antibodies. Non-specific binding sites on the membranes were blocked in 5% w/v Bovine serum albumin (BSA)-TBS-T or milk-TBS-T. BSA was used when checking for spleen tyrosine kinase (Syk); while milk was used for STE20-related kinase adaptor protein (STRAD) and GAPDH. Primary antibodies used in this study are: STRAD (G-8) ; Syk (D3Z1E) ; and GAPDH (MAB374) . GAPDH, with a molecular weight of 37 kDa, was used as the loading control for normalization. Membranes were imaged with ChemiDoc XRS+ system. Densitometry quantification was done with ImageLab software.To validate the fold increase protein expression and phosphorylation changes as observed in the antibody array for OA triggered T98G cells, all individual OA and NP-CSF triggered T98G cell lysates  and SPSS v24.0 . Cytokine levels was checked for normality and deemed not normally distributed, thus non-parametric Mann Whitney-U test was used. Mann-Whitney U test was also used to analyse STRAD and Syk protein levels from western blotting between groups. Simple and multivariate linear regression model analyses were used to study the relationship of pain score and cytokine level. A n = 7), hernia repair (n = 2) and ligation of piles (n = 6).The average age of patients in our cohort is 61 ± 13 years. Demographics of OA pain and NP patients are described in Table The IL-6 levels in the CSF samples from OA and NP patients were below the lowest detection limit of the ELISA kit 9.38 pg/ml) used. In order to detect IL-6 levels using the ELISA method, the additional induction methodology used in [.38 pg/mln = 15) whereas 23.7 ± 3.4 pg/ml of IL-6 was detected for NP-CSF group (n = 15). There was a statistically significant higher amount of IL-6 released from T98G cells triggered with OA-CSF as compared to NP-CSF .In the absence of CSF, addition of LPS to T98G cells did not trigger detectable levels of IL-6 release [TNFα and IL-1β (lowest detection range of 15.6 pg/ml and 3.91 pg/ml respectively), were evaluated and found to be undetectable in T98G cell culture media when induced with CSF (OA/NP) and LPS, with their respective ELISA kits.p = 0.007). After adjusting for age and gender in the multivariate model, the significant association persists (adjusted B estimate = 10.1 (95% CI = 4.3–15.9); p = 0.001).There was a significant positive relationship between pain score and IL-6 cytokine levels  = 2.1–12.1); The antibody array used consisted of 6 replicates of 215 antibodies for targets related to the NF-κB pathway. Forty microgram of T98G cell lysates, harvested from each of the 6 individual CSF samples trigger, in the presence of LPS, were pooled together to give a total 240 μg protein for the array testing. The 240 μg pooled OA-CSF triggered T98G cell lysate was added to one array slide; while pooled NP-CSF triggered T98G cell lysate was added to another slide. The fluorescence signal of all 6 antibody replicates within each array were recorded and the average was calculated. GAPDH was used to normalise the signal for both OA and NP samples. Those with a greater than 2-fold increase in OA-CSF triggered T98G cell lysate as compared to that of NP-CSF were noted. The more notable ones having greater than 3-fold increase were STRAD (4.25 fold), Syk (3.40 fold), lymphocyte-specific protein tyrosine kinase (3.30 fold) and IkappaB-alpha (3.07 fold)  as compared to NP patients . However, the difference was not statistically significant (p = 0.298)  Fig. .Fig. 3Np = 0.183)  as compared to NP patients 1.96 ± 1.4; n = 15) for Syk protein. This higher protein expression in OA patients was not statistically significant  such as those influencing immune responses, inflammation and wounds . IL-6 haWe explored the signalling pathway upstream of IL-6 in our T98G model. Specifically, NF-κB explorer array was chosen as it is one of the common upstream molecular players of IL-6. Several protein targets were found to be elevated with greater than two-fold in OA-CSF triggered T98G cells as compared to NP-CSF triggered ones Fig. , these tTwo protein targets which showed an increase of 4.2 and 3.4 fold in OA-CSF in the array were STRAD and Syk respectively. STRAD, predominantly found in CNS, is known to complex with serine/threonine kinase LKB1 and scaffold protein MO25α to activate AMP-activated protein kinase (AMPK) signalling pathway which includes release of pro-inflammatory cytokines . Syk plaThe strength of our study is the use of human CSF to provide an objective evaluation of chronic pain. CSF, as compared to peripheral blood, has a more direct contact with the CNS environment. It bathes the CNS and therefore the contents in CSF may be crucial in understanding changes of the underlying disease associated with chronic knee pain . Our finAlthough we found that a 48-h treatment time point for measuring IL-6 release provides sufficient signal strength in the ELISA, it may not be the peak protein expression or phosphorylation changes for STRAD and Syk, thus leading to overall lack of significance in western blotting results. Another limitation was that only a limited number of antibodies were tested.In summary, CSF from chronic knee pain patients suffering from OA knees was able to induce higher levels of IL-6 release from T98G cells than NP-CSF in the presence of LPS. This finding suggests the presence of an as-yet-unknown molecule(s) in the CSF of patients with chronic pain which could potentially serve as clinically informative biomarker(s). Further studies are needed to investigate the relevance of IL-6 release by T98G glial cells in other chronic pain models and to evaluate any potential upstream players.


Accumulating data suggest cerebrospinal fluid (CSF) neurogranin (Ng) as a potential biomarker for cognitive decline and neurodegeneration in Alzheimer disease (AD). To investigate whether the CSF Ng can be used for diagnosis, prognosis, and monitoring of AD, we examined 111 cognitively normal (CN) controls, 193 mild cognitive impairment (MCI) patients and 95 AD patients in the Alzheimer’s Disease Neuroimaging Initiative (ADNI) cohort. Correlations were tested between baseline CSF Ng levels and baseline core AD biomarkers and longitudinal glucose metabolism, brain atrophy and cognitive decline. We detected that CSF Ng levels increased with disease severity, and correlated with phosphorylated tau and total tau levels within each diagnostic group. High baseline CSF Ng levels correlated with longitudinal reductions in cortical glucose metabolism within each diagnostic group and hippocampal volume within MCI group during follow-up. In addition, high baseline CSF Ng levels correlated with cognitive decline as reflected by decreased cognitive scale scores. The CSF Ng levels predicted future cognitive impairment  in CN controls. These data demonstrate that CSF Ng offers diagnostic utility for AD and predicts future cognitive impairment in CN individuals and, therefore, may be a useful addition to the current AD biomarkers. Alzheimer’s disease (AD) is a multifactorial age-related neurodegenerative disease whose pathology starts decades before the clinical symptoms appear . PatholoNg is a calmodulin-binding postsynaptic protein and plays a critical role in plasticity, synapse repair, and long-term potentiation , 5. It iIn the present study, we present results on CSF Ng in the Alzheimer’s Disease Neuroimaging Initiative (ANDI) cohort of cognitively normal (CN) controls, patients with mild cognitive impairment (MCI) and patients with AD. We tested the specific hypotheses that the CSF Ng levels were altered in patients with AD and had diagnostic utility for AD, that the CSF Ng correlated with AD core biomarkers  and total tau (t-tau)), cognitive decline and imaging evidence of neurodegeneration and that the CSF Ng levels predicted future cognitive impairment at the early asymptomatic stage.The baseline characteristics of participants are shown in Mean CSF Ng levels were higher in AD subjects compared with sMCI subjects (P = 0.011) or CN controls (P < 0.001). Mean CSF Ng levels were higher in pMCI subjects compared with sMCI subjects (P = 0.028) or CN controls (P < 0.001). Mean CSF Ng levels were higher in sMCI subjects compared with CN controls (P = 0.042) . When co18F-Fluorodeoxyglucose positron emission tomography (FDG-PET) was identified in subjects with CN , MCI  and AD . The CSF Ng levels were negatively correlated with hippocampal volume in subjects with MCI . The CSF Ng levels also related to the decline of ADNI_MEM  and ADNI_EF  scores in subjects with MCI and AD  and t-tau  levels in subjects with CN, MCI and AD, respectively (P < 0.001). No correlations were observed between CSF Ng levels and CSF Aβ levels in patients with AD  or CN controls . The CSF Ng levels negatively correlated with CSF Aβ levels in subjects with MCI . Significant association of baseline CSF Ng levels and I and AD .We assessed the ability of CSF biomarkers to predict future cognitive impairment in cognitively normal controls over time. The CSF Ng levels provided higher predictive accuracy than the core CSF biomarkers . The meaIn this study, we found that (1) CSF Ng levels were elevated in AD, pMCI and sMCI groups compared with CN group and the diagnostic utility of CSF Ng levels differentiating patients with AD from CN controls was comparable to that of core CSF biomarkers; (2) CSF Ng levels associated with CSF p-tau and t-tau levels within each diagnostic group and with CSF Aβ levels within MCI group; (3) high baseline CSF Ng levels correlated with longitudinal reduction of cognitive scale scores within MCI and AD groups, decreased cortical glucose metabolism within each diagnostic group, and hippocampal volume atrophy within MCI group; (4) CSF Ng levels predicted conversion from CN to MCI. Taken together, these findings suggest that CSF Ng is a very early and potentially presymptomatic biomarker for AD. This biomarker may be helpful in AD diagnosis, predicting disease progression and staging severity of AD even in its preclinical stage. Our study also provided clues to how Ng participated in the pathophysiological process in AD, to monitor drug effects on synaptic degeneration in clinical treatment trials, and provided evidence for drug development.We found that mean CSF Ng levels were higher in female individuals compared with those in male individuals in the Aβ+ group. A potential explanation of this difference maybe that sex is a crucial variable in disease heterogeneity of AD. The cumulative evidence indicates that women exhibit steeper cognitive decline and higher rates of brain atrophy after diagnosis of MCI or AD . Women mWe observed positive correlations between CSF Ng levels and CSF p-tau and t-tau levels in each diagnostic group and negative correlations between CSF Ng levels and CSF Aβ levels in MCI group. This relationship gives support to the utility of CSF Ng as a biomarker, for it is sensitive to AD-related biological changes. There is evidence that tau pathology is involved in synapse degeneration and contributes to cognitive decline , 29. TheLogistic regression analysis was used to assess the impact of CSF analytes on risk for disease progression. The AUC (reflect predictive probabilities of the logistic regression models) of the CSF Ng model was great in predicting progression from CN to MCI. High CSF Ng levels are strongly associated with risk of MCI in cognitive normal participants, suggesting that CSF Ng is a marker of clinical progression in asymptomatic population. The CSF Ng levels reflect synaptic loss in a very early stage but not in later stages of the AD spectrum. Our data showed that synaptic dysfunction was, to some extent, involved in AD pathophysiology. Low CSF Aβ is considered a biomarker of an individual in the Alzheimer’s continuum . Thus, wThere were several limitations of our study. Firstly, CSF biomarkers included in the cox regression models as dichotomous variables might conceal an underlying continuum. Secondly, the newly published NIA-AA criteria conducts an ATN classification system and the ATN system is flexible in that new biomarkers can be added to the three existing ATN groups . WhetherTaken together, our findings suggest that the CSF Ng can be used as a biomarker for synaptic pathology in AD, and CSF Ng is a valuable biomarker of early neurodegeneration. In addition to the “core CSF biomarkers” Aβ, p-tau, and t-tau, the CSF Ng might have added value.http://www.adni-info.org.Data used in this article were obtained from the ADNI database (adni.loni.usc.edu) . ADNI waWe included all CN controls, MCI patients and AD patients with available baseline CSF Ng samples. CN controls had Mini-Mental State Examination (MMSE) score between 25 and 30; clinical dementia rating (CDR) score of 0. CN controls were grouped into those that remained cognitively stable for at least 1 follow-up and those who progressed to MCI during follow-up. Subjects with MCI had MMSE score between 24 and 30; CDR score of 0.5; objective memory loss as shown on scores on delayed recall of the Wechsler memory scale logical memory II [> 1 standard deviations (SD) below the normal mean]; preserved activities of daily living, and the absence of dementia. In our study, patients with MCI were grouped into those that remained cognitively stable for at least 1 follow-up (sMCI) and those who progressed to AD dementia during follow-up (pMCI). Patients with AD fulfilled the National Institute of Neurological Communicative Disorders and Stroke-Alzheimer Disease and Related Disorders Association criteria for probable AD, had MMSE scores between 20-26 and a Clinical Dementia Rating scale of 0.5 or 1.0. As to “ATN” categories: amyloid positive and negative (A+ versus A-) were separated by a cutoff value of 192 pg/mL for CSF Aβ level; tau pathology positive and negative (T+ versus T-) were separated by a cutoff value of 23 pg/mL for CSF p-tau level –40.CSF Ng was analyzed by electrochemiluminescence technology using Ng7, which is a monoclonal antibody specific for Ng, as coating antibody and polyclonal Ng anti-rabbit  as detector antibody . Values Magnetic resonance (MR) images were collected from a variety of 1.5/3.0 Tesla MR system, using protocols optimized for each MR scanner. The FreeSurfer pipeline was used to generate hippocampus estimates . We usedMean FDG uptake was obtained per subject within a set of predefined and previously validated regions of interest  based on a literature as described elsewhere in detail . Each suSummary cognitive scores were chosen over individual cognitive tests to use more comprehensive and robust measures of domain-specific cognitive performance. Summary metric for the memory cognitive domain was ADNI-MEM , AD Assessment Schedule-Cognition , Mini-Mental State Examination (MMSE), and Logical Memory data)  and for Tests of inter-group differences were performed using Chi-square analysis for frequencies or Mann-Whitney U test/Kruskal-Wallis test for continuous measures. Linear regression models were constructed to examine the cross-sectional associations between CSF Ng levels and core CSF biomarkers  at baseline. Longitudinal associations between CSF Ng levels and cognitive, metabolic and structural data were assessed using linear mixed-effects model. Each CSF variable, hippocampal volume, FDG-PET and cognitive scale scores were z-scale transformed to ensure normality. Model was adjusted for age, sex, educational and APOE genotype . Logistic regression analysis was used to assess the impact of different CSF analytes on the risk of disease progression. The receiver-operator curves and the area under the curves were derived from the predictive probabilities of the logistic regression models. Cox proportional hazard regression models access whether the CSF biomarkers  predict cognitive impairment. The cut-off value of CSF Ng was obtained from receiver operating characteristic curve. Participants were followed up until a diagnosis of MCI/AD, death, or last follow-up visit. Time to event was defined as time from baseline CN to first visit defined as MCI/ baseline MCI to first visit defined as AD.All tests were two-sided, statistical significance was set at P < 0.05. All statistics were performed using R 3.6.2 and IBM SPSS Statistics 25.Supplementary Figure 1Supplementary Tables