Nrf2)-mediated pathway in HaCaT cells via the phosphorylation of p38 and extracellular signal-regulated kinase (ERK). FBTA also downregulated the expression of matrix metalloproteinase-1 (MMP-1) while upregulating type I procollagen by modulating Nrf2 signaling in UVB-irradiated HaCaT cells. Collectively, our results show that FBTA might be useful as a functional food while being a good candidate in the development of cosmetic products and medicines for the remedy of UVB-induced skin photo-aging.Ultraviolet B (UVB) irradiation is viewed as the principal inducer of skin photo-aging, associated with acceleration of collagen degradation and upregulation of matrix metalloproteinases (MMPs). The ethnic groups of southern/western China use Fuzhuan brick-tea (FBT) as a beverage and as a nutritional supplement. In this study, we scrutinized the antagonistic effects of aqueous extract of Fuzhuan-brick tea (FBTA) on skin photo-aging in UVB-exposed human keratinocyte (HaCaT) cells. FBTA exhibited strong antioxidant activity and quenched UVB-induced generation of cellular reactive oxygen species (ROS) without showing any toxicity. FBTA was capable of combating oxidative stress by augmenting messenger RNA (mRNA) and protein levels of both phase I and phase II detoxifying enzymes, especially heme oxygenase 1 (HO-1), by upregulating the nuclear factor erythroid 2-related factor 2 ( Ultraviolet (UV) irradiation is viewed as one of the main factors causing structural and functional alterations in the skin, triggering skin aging . AccumulNrf2) is crucial for the upregulation of HO-1. Under quiescence, Nrf2 is dormant in the cytoplasm due to the Kelch-like ECH-associated protein 1 (Keap1). However, responses to oxidative stress or conformational changes of Keap1 by inducers facilitate the nuclear translocation of Nrf2 and binding to antioxidant response elements (ARE), modulating the expression of various antioxidant enzymes and mitigating ROS generation . M. MCamell the tea  comparedThe Fuzhuan brick tea (FBT) was purchased from the Hunan Yiyang Tea Factory . The voucher specimens of the plant and extracts have been deposited in the Laboratory of Enzyme Biotechnology, Kyungpook National University, Daegu, Republic of Korea. After air dying, 100 g tea powder were mixed with 15-folds of distilled water (DW) and placed in a shaking incubator at 60 °C for 24 h. Then, the supernatant was collected with filter paper , and dried using a rotary vacuum evaporator . Finally, the aqueous extracts of FBT (FBTA) were subjected to lyophilization and dissolved in deionized water at a concentration of 30 mg/mL as a stock solution.2,2-diphenyl-1-picrylhydrazyl (DPPH-) and 2,20-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid (ABTS-) radical scavenging assays, a ferric reducing antioxidant power (FRAP) assay, a cupric-reducing antioxidant capacity (CUPRAC) assay, and an oxygen radical absorbance capacity (ORAC) assay were carried out to evaluate the hydrogen and electron-donating capacity of FBTA, by which we confirmed the cell-free antioxidant potentiality of FBTA using previously described methods .5 cells/mL) were cultured in DMEM medium supplemented with fetal bovine serum (FBS) and penicillin/streptomycin at 37 °C in 5% CO2 incubator. Then, sub-confluent cells were treated with indicated concentration  3, 10, 30, or 100 μg/mL) of FBTA for 24 h. Subsequently, the cells was exposed to UVB at a dose of 60 mJ/cm2 using a UVB source  set at a spectral peak of 312-nm for 20 s. After UVB irradiation, the cells were cultured in serum-free medium for 24 h. Cell viability was determined using the 3--2,5-diphenyltetrazolium bromide (MTT) colorimetric assay as described previously [HaCaT cells (1 × 10eviously .5 cells/mL) were cultured with indicated concentration of FBTA  in 96-well black plates for 24 h and then exposed to UVB-irradiation (60 mJ/cm2), followed by a change in the media and further incubation for 24 h. After that, the cells were washed with PBS twice and treated with 25 μM 2’,7’-dichlorofluorescin diacetate (DCF-DA) for 30 min at 37 °C in a CO2 incubator. Finally, fluorescence intensity was measured at excitation and emission wavelengths of 485 and 528 nm, respectively, by a fluorescence microplate reader .HaCaT cells (1 × 105 cells/mL) were cultured with indicated concentration of FBTA  in 6-well plates for 24 h. TRIzol reagent  was used for the extraction of total RNA and complementary DNA (cDNA) was prepared using RT & GO Mastermix  and served as the PCR template. A PCR Thermal Cycler Dice TP600  was used to carry out RT-PCR using the various primer sequences  buffer with a phosphatase and protease inhibitor cocktail  and the bicinchoninic acid (BCA) method was applied to quantify the protein content. A nuclear/cytosolic fractionation kit  was used for the extraction of nuclear proteins. Aliquots of 50 μg of total proteins were used to carry out the Western blot analysis using various antibody  accordinn = 3) and analyzed using the GraphPad Prism Software . Statistical analysis was performed using one-way analysis of variance (ANOVA), followed by Dennett’s test. A value of p < 0.05 was considered as significant.The data were expressed as the mean ± standard deviation  and liubao tea (74.56 ± 3.08%) at a dose of 300 μg/mL . In ABTSTo examine the cytotoxic effects of UVB and FBTA on HaCaT cells, an MTT assay was performed. Since gallic acid at its putative concentration (9~10 μM) in FBTA has shown the highest antioxidant effects in cell free in vitro antioxidant assays, we used gallic acid as a positive control for cell-based assays. Gallic acid (f.c. 10 μM) and FBTA (f.c. 3 to 300 μg/mL) treatment did not show any significant cytotoxicity for 24 h A. Thus wSpectrofluorometric analysis disclosed that UVB exposure significantly increased the intracellular ROS production in HaCaT cells  through Results of immunoblotting analysis revealed that UVB-irradiation dramatically lessened the protein expression of phase I antioxidant enzymes such as superoxide dismutase 1 (SOD1), catalase (CAT), and glutathione peroxidase 1 (GPx-1). Interestingly, FBTA and gallic acid treatment expressively upregulated the protein levels in a dose-dependent manner A. LikewiNrf2 inevitably harmonizes the cellular antioxidant function by the triggering of a series of antioxidant genes, thereby acting against photo-aging in the skin [Nrf2 in FBTA-treated HaCaT cells. As shown in Nrf2 was steadily raised in FBTA- and gallic acid-treated HaCaTs. Likewise, immunoblotting analysis revealed that FBTA and gallic acid enhanced the nuclear translocation of Nrf2, while simultaneously lessening the cytosolic Nrf2 level , a specific inhibitor of Nrf2, before FBTA and gallic acid treatment. As expected, brusatol significantly suppressed Nrf2 expression, and reserved the FBTA as well as gallic acid effects . Interestingly, FBTA treatment substantially augmented the phosphorylation of p38 and ERK1/2 after 30 min  components such as MMPs and collagen, thereby hastening skin photo-aging . Among te stress . Our resIt is well known that elderly people have lower endogenous antioxidants activity resulting in more vulnerable to UV-irradiated skin damage, thus the improved strategies for skin photoprotection are needed. Botanicals with antioxidant properties are viewed as potential therapeutic agents to treat skin disorders such as photo-aging . FBTA shThe UVB irradiation of the epidermis causes ROS generation, resulting in attenuation of SOD, CAT, and GPx1 activity and hastening of oxidative damage . Here, wNrf2, the key regulator of HO-1 activation, was studied. Furthermore, Hirota et al. [Nrf2 gene in mice exhibited the acceleration of UVB-induced photoaging process. Thus, Nrf2, a well-known redox-sensitive transcription factor, plays a critical role to protect cells against UVB-stimulated photoaging. The pharmacological approach for the activation of Nrf2 has drawn substantial attention as a tactic for skin photoprotection [Nrf2 enters into the nucleus and triggers phase II-detoxifying enzymes such as HO-1, thereby indirectly ameliorating the cellular antioxidant defense system, and can protect skin against oxidative damage [Nrf2 translocation of the nucleus, while inhibition of Nrf2 strongly alleviated the upregulation of HO-1 , a phase II-detoxifying enzymes, can convert heme into bilirubin, which acts as a strong antioxidant capable of protecting against cell death from oxidative insult . Besidesa et al.  discoverotection . Upon ele damage . In our  of HO-1 A, confirr injury . The apoadiation . FBTA ca and JNK . Our resaT cells A. Pharmafication B. A curr-3 cells . Gallic ll death . Based oFoeniculum vulgare Mill extract attenuated UVB-induced skin photo-aging by suppression of ROS production and expression of MMP-1, while increasing the type I procollagen level in hairless mice [Youngia denticulatum, taken as a wild vegetables, has been reported to act as an antioxidant, abolishing UVB-induced upregulation of MMP-1 and degradation of procollagen I in HaCaT cells [Nrf2 , semifermented (oolong tea), and fermented  teas are some of the most widely consumed beverages in the world. The major chemical constituents of teas are polyphenols. Among them, flavan-3-ol such as EGCG and ECG are dominant. Flavonoids, gallic acid, caffeine, and amino acid are also present. Interestingly, during the production of black tea, catechins such as EC, ECG, EGC, and EGCG are oxidized by polyphenol oxidase (PPO) and peroxidase (POD) and consequently dimerized to theaflavins and polymers (thearubigins). There are some studies reporting on the pharmacokinetics profile of tea polyphenols, while the pharmacokinetics profiles of black tea polyphenols, theaflavins, and thearubigins have not been studied extensively . Howeversumption . Mountinsumption . Thus, isumption . This isFurther studies should be focused on (1) finding active ingredients contributing anti-skin aging potential by investigating synergistic effects of polyphenolics, small molecules, and/or undetected compounds in FBTA, and (2) the pharmacokinetic parameters of tea polyphenols after oral administration of FBTA in a mice model. These are rewarding in that the future data will be beneficial to consumers for better skin and inner beauty care.Nrf2 via induction of p38 and ERK1/2 phosphorylation and subsequently induced HO-1, thereby successively quenching UVB-induced ROS production. Most prominently, significant stimulation of MMP-1 and downregulation of type I procollagen by UVB was restored by FBTA in HaCaT cells, probably through the activation of Nrf2. Collectively, our results demonstrated molecular evidence that FBTA could inhibit UVB-induced photoaging via quenching of ROS and triggering of Nrf2 signaling cascades.Our findings revealed for the first time that FBTA pretreatment mitigated UVB-induced photoaging in human keratinocyte HaCaT cells . FBTA st


Early diagnosis and treatment of neurosyphilis is of great significance for regression. There is no gold standard for the diagnosis of neurosyphilis. We did this study to explore the factors associated with the clinical diagnosis of neurosyphilis and assess their accuracy for the diagnosis of neurosyphilis.We retrospectively reviewed 100 cases of syphilis patients who underwent lumbar puncture at a major dermatology hospital in Guangzhou, China between April 2013 and November 2016. Fifty patients who were clinically diagnosed with neurosyphilis were selected as case group. Control group consisted of 50 general syphilis patients who were matched with age and gender. The records of patients were reviewed to collect data of socio-demographic information, clinical symptom, and laboratory indicators. Multivariable logistic regression was used to explore diagnostic indictors, and ROC analysis was used to assess diagnostic accuracy.P = 0.001), cerebrospinal fluid (CSF) Treponema pallidum particle agglutination (TPPA) titer , CSF protein , and CSF white blood cell (WBC)  were found to be statistically associated with neurosyphilis. In ROC analysis, CSF TPPA titer had a sensitivity of 90%, a specificity of 84%, and an area under curve (AUC) of 0.941.Neurological symptoms (odds ratio (OR) = 59.281, 95% CI:5.215–662.910, CSF TPPA can potentially be considered as an alternative test for diagnosis of neurosyphilis. Combining with neurological symptoms, CSF protein, CSF WBC, the diagnosis would have a higher sensitivity. Treponema pallidum  is the causative agent of syphilis, which can invade the central nervous system (CNS) at any stage after exposure [treponema pallidum affects the central nervous system and can lead to asymptomatic meningitis, which can further progress to severe and irreversible symptomatic neurosyphilis if left untreated [exposure , 2. In antreated . Therefontreated . Becausentreated –7.There is no gold standard for the diagnosis of neurosyphilis. One commonly used diagnostic criteria developed by the Centers for Disease Control and Prevention (CDC) of the United States mentioned that neurosyphilis can be divided into two categories. One is “confirmed” neurosyphilis which can be diagnosed by the criterion that a reactive Venereal Disease Research Laboratory test (VDRL) in cerebrospinal fluid (CSF). The other one is “presumptive” neurosyphilis which can be diagnosed by the following criteria: (1) a nonreactive VDRL in CSF, (2) elevated CSF protein or leukocyte count, and (3) clinical symptoms or signs consistent with neurosyphilis without alternate known causes accounting for these . AccordiAlthough VDRL is considered as a definitive diagnosis test of neurosyphilis, there are some limitations. First, while VDRL has a high specificity, its sensitivity ranges from as low as 27 to 70% –12. A noThis study was conducted at a major dermatology hospital in Guangzhou, China. We retrieved data on syphilis patients who underwent the lumbar puncture between April 2013 and November 2016. In this study, we included 50 patients who were clinically diagnosed with neurosyphilis (NS), and then 50 general syphilis patients with the matched age ranges and gender ratio were randomly selected as control group. The exclusion criteria were as follows: Human Immunodeficiency Virus (HIV) infection, return visit, without lumbar puncture. The records of patients were reviewed to collect data such as age, gender, native place, occupation, marital status, HIV infection status, serum toludine red unheated serum test (TRUST) titer, serum TPPA titer, serum treponema pallidum immunoglobulin M (TP-IgM), CSF VDRL, CSF TRUST titer, CSF TPPA titer, CSF protein, CSF WBC, CSF glucose, CSF chlorides, and neurological symptoms . We only collected the results of laboratory tests within 90 days before or after the lumbar puncture. If a patient underwent multiple laboratory tests, we selected the results of the first test conducted before treatment into analysis. This study was approved by the Ethics Committee of the Dermatology Hospital of Southern Medical University. The ethics committee approval number is GDDHLS-20171004.r is ratio of controls to cases, σ is the standard deviation of the variable which were compared, difference is the difference between the means of the case group and the control group, α is significance level, β is desired power. We used CSF WBC and CSF protein to calculate the sample size separately, and chose the larger one which was calculated with parameters of CSF protein as the sample size of our study. In the calculation process, α = 0.05, Zα/2 = 1.96, β = 0.8, Zβ = 0.84, r = 1, Difference = 150 mg/L, σ = 350 mg/L. The values of Difference and σ were derived from previous research [Before we started our study, we have calculated the sample size. According to previous studies, we assumed that CSF WBC and CSF protein could be used as diagnostic indictors of neurosyphilis . We seleference2 . In thisThe diagnosis of neurosyphilis were based on the guidelines of the Centers for Disease Control in Europe and America , 9. The Reciprocal and logarithmic transformation were performed to titer data before data analysis.P value< 0.1 were retained. The multivariable models were created through stepwise elimination of variables from univariable analysis. In the multivariable analysis, odds ratios were adjusted for age and gender. Then the receiver operating characteristics (ROC) analysis was used to assess the indicators which were significant in logistic regression. Accuracy for the diagnosis of neurosyphilis. We used SPSS 20.0 and MedCalc 15.10 to perform statistical analyses. P values less than 0.05 were considered to be statistically significant.Median and interquartile range (IQR) were used to describe continuous variables, while frequencies and percentages were used to describe categorical variables. The Mann-whtiney U test was used to compare continuous variables and the chi-square test was used to compare categorical variables. Logistic regression was used to calculate the odds ratio (OR) of laboratory indicators and clinical symptoms. We conducted a univariable analysis firstly. The variables which p = 0.953). Overall, 75% (75/100) of the study population were males, accounting for 68% in the Non-NS group and 82% the NS group (p = 0.106). In all patients, 84% were from Guangdong Province, with 82% in the Non-NS group and 86% in the NS group (p = 0.585). The Non-NS group had more patients who were single (18%) than the NS group (4%) (p = 0.025). Sixty two percent in the Non-NS group and 54% in the NS group (p = 0.418) had a permanent job. More patients (46%) in the NS group had neurological symptoms than patients (10.2%) in the Non-NS group (p < 0.001). Patients in the NS group had significantly higher serum TRUST titer , Serum TPPA titer , Serum TP-IgM positive rate , CSF TRUST titer , and CSF TPPA titer  than patients in the Non-NS group. CSF protein and CSF WBC count were higher and CSF glucose and chlorides levels lower in the NS group than in the Non-NS group .The characteristics of the 100 patients were shown in Table Univariable logistic regression indicated that the following variables were significantly associated with NS: Neurological symptoms, Serum TPPA titer, Serum TP-IgM, CSF TPPA titer, CSF protein, CSF WBC, CSF chlorides Table .Table 2p = 0.001), CSF TPPA titer , CSF protein , and CSF WBC  :5.215–662.910, P < 0.001). However, there were no significant differences in AUC among neurological symptoms, CSF protein, and CSF WBC (P > 0.05). When we combined neurological symptoms, CSF protein, and CSF WBC  exceeded the cutoff values, the patient was considered to be neurosyphilis), the sensitivity and specificity were 92.00 and 33.30%, respectively. When we combined neurological symptoms, CSF protein, CSF WBC, and CSF TPPA using the same method mentioned above, the sensitivity and specificity were 98.00 and 40.80%, respectively. We transformed the CSF TPPA titer, CSF protein, and CSF WBC into dichotomous variables by cutoff values and then performed logistic regression analysis. The results of multivariable logistic regression also showed neurological symptoms , CSF TPPA titer , CSF protein , and CSF WBC  were included in the model.We conducted ROC analyses to assess the diagnosis accuracy of neurological symptoms, CSF TPPA titer, CSF protein, and CSF WBC. For different cutoff points, the ROC analyses showed different sensitivity, specificity and area under curve (AUC). The AUC was the best, when CSF TPPA titer was at 1:160, CSF protein was at 497 mg/L, and CSF WBC was at 3 cells/μL, respectively. The AUC of neurological symptoms was 0.679 (Fig. Neurosyphilis is still difficult to diagnose because there is no definitive test for diagnosis. Nowadays, CSF-VDRL is considered as widely used diagnostic criteria for neurosyphilis. However, VDRL still has some shortcomings, such as high specificity and low sensitivity –12, specIn our study, CSF TPPA titer had a sensitivity of 90%, a specificity of 84%, and an AUC of 0.941. The diagnostic accuracy was much higher than other three predictors. The results of our study were similar with some previous studies . Castro Sensitivity of neurological symptoms, CSF protein, CSF WBC changed from 46 to 54%. These predictors were insensitive and nonspecific. A literature review indicated that part of early neurosyphilis patients presented neurological symptoms of typical aseptic meningitis, including headache, stiff neck, nausea, vomiting. The most common symptoms included papilledema, convulsions, confusion, and focal and cranial nerve abnormalities. In the advanced stage of neurosyphilis, the neurological symptoms of patients were usually dementia and tabes dorsalis. Meanwhile, there were still some asymptomatic neurosyphilis patients. So the indicator of neurological symptoms showed a high specificity and low sensitivity .When we took neurological symptoms, CSF protein, CSF WBC into account at the same time, the sensitivity rose to 92.00%, and when we combined neurological symptoms, CSF protein, CSF WBC, and CSF TPPA, the sensitivity rose to 98.00%. There were some things to be noticed when we used these predictors to diagnose neurosyphilis. Previous studies mentioned that elevated CSF protein and WBC can occur in HIV-infected patients without neurosyphilis , 19, 20.Our study was limited by the design of retrospective study. The data for this study were collected from patients who underwent lumbar puncture examination, which may lead to potential bias in patient selection. Potential selection bias may exist since patients of control group were included base on matched age and gender. There was a possibility that some cases were misclassified because of the lack of gold standard for the diagnosis of neurosyphilis.CSF-VDRL is highly specific but insensitive as a widely used diagnostic method. When CSF-VDRL is nonreactive, some other indicators should be considered. In our study, neurological symptoms, CSF TPPA titer, CSF protein, CSF WBC were identified as independent predictors of neurosyphilis. Especially, CSF TPPA had high sensitivity, specificity and AUC. Therefore, we suggested that CSF TPPA should be considered as an alternative test for the diagnosis of neurosyphilis. Combining with neurological symptoms, CSF protein, CSF WBC, the diagnosis would have a higher sensitivity.


ABCD1 gene. Proteomic analysis of cerebral spinal fluid (CSF) from young males with active cALD revealed markers of inflammation including APOE4. APOE4 genotype has been associated with an inferior prognosis following acute and chronic neurologic injury. We assessed APOE4 inheritance among 83 consecutive young males with cALD prior to hematopoietic cell transplant and its association with markers of cerebral disease. The allele frequency of APOE4 was not significantly different from that of the general population at 17%. Young males with cALD that were APOE4 carriers had similar CSF protein and chitotriosidase activity to that of non-carriers. In contrast, APOE4 carriers had an increased burden of cerebral disease involvement as determined by MRI severity score , higher gadolinium intensity score , inferior neurologic function , and elevated CSF MMP2 levels compared to that of non-carriers . These are the first data showing that APOE4 is associated with increased severity of cerebral disease in cALD and suggest it may be a modifier of disease.Cerebral adrenoleukodystrophy (cALD) is an inflammatory neurodegenerative disease associated with mutation of the  ABCD1 gene, a member of the ATP-binding cassette (ABC) transporter superfamily1. Loss of function of ABCD1 leads to VLCFA accumulation, and adrenal and neurologic dysfunction. Up to 40% of affected young males develop idiopathic, progressive, fatal cerebral inflammation and demyelination  characterized by gadolinium-enhancing T2 lesions on magnetic resonance imaging (MRI); suggesting blood-brain-barrier (BBB) disruption2. While hematopoietic cell transplant (HCT) can arrest cALD progression, many young males are diagnosed too late in their disease for transplant to salvage favorable neurologic function4.Adrenoleukodystrophy (ALD) is a rare X-linked peroxisomal disease caused by mutations in the very long chain fatty acid (VLCFA) transporter encoded by the 5. The genetic polymorphisms of APOE are APOE2, APOE3 , and APOE46. Previous work reported by Bell et al. has shown that astrocyte-derived ApoE3 protein maintains the BBB integrity through down regulation of the pro-inflammatory protein, cyclophilin A (CypA)7. However, when the APOE4 allele is present, CypA is unregulated (i.e. increased) leading to activation of MMP9 and loss of BBB integrity. This BBB-disruptive mechanism is thought to contribute to the increased risk of Alzheimer’s and Parkinson’s disease in APOE4 carriers10.Composed of astrocytes, endothelial cells, and perivascular cells, the BBB is selectively impenetrable unless disrupted through malignancy, injury, or inflammation. Apolipoprotein E (ApoE) is a major lipoprotein found in the plasma via liver secretion and is also found in the CSF secreted by astrocytesAPOE genotype (carriers of APOE4 versus non-carriers) correlated with our previously described cALD disease biomarkers.To explore new biomarkers in the cerebral spinal fluid (CSF), we conducted proteomic analysis of cALD CSF compared to that non-ALD CSF followed by gene ontogeny analysis. In the present study, we identified ApoE family member proteins in the CSF and investigated whether the specific 14. In this study, we sought to undertake a comprehensive analysis of the CSF from young males with cALD using a proteomics-based approach. To investigate the proteomic composition of CSF in young males with cALD, we pooled the CSF from 18 young males with cALD prior to bone marrow transplant. A reference sample derived from non-ALD CSF was used for comparison as we have previously published15. The pooled samples were subjected to trypsin digestion followed by fragment labeling using iTRAQ isobaric tags, separation by liquid chromatography, and peptide identification by tandem mass spectrometry.We have previously published several individual CSF biomarkers that are associated with disease burden in cALDWith a 1-peptide threshold and 90% confidence setting to identify proteins, we found 450 proteins in the CSF (5316 spectra); the false discovery rate (FDR) was 1%. The median amino acid length for the CSF proteome was 463 (range 45 to 4753) Fig. . The medTo understand the ontogeny of the proteins expressed at a higher level in cALD CSF, we performed Ingenuity Pathway Analyses (IPA) from the top quartile of proteins expressed in cALD Fig. . Top qua18. Therefore, we sought to determine if ApoE4 played a role in the manifestation of cerebral disease in cALD.The protein sequence coverage from MSMS-identified proteins was at an average of 13.7% with a range of 0.2% to 85% Fig. . We note19. We evaluated the total ApoE concentration in the CSF of young males with cALD and found a mean of 17 µg/mL, consistent within the range of previously published results22  to ApoE4 for 72 hours and measured ROS production using H2DCFDA, a dye with fluorescent activity in an increased ROS state  of human endothelial cells and found that the baseline oxygen consumption rate (OCR) was significantly reduced with the addition of ApoE4 to the cell culture media . Measurement of peak OCR and mitochondrial reserve were also significantly reduced in the presence of ApoE4   Fig. . These dAPOE4 allele to compare the extent of cerebral disease versus non-carriers of APOE4. We assessed 83 young males with cALD during pre-treatment evaluations. We found the distribution of APOE2, APOE3, and APOE4 alleles to be 0.060, 0.765, 0.175, respectively, which is very similar to that of the general population , which was not statistically different from non-APOE4 carriers at 8.4 years .Given that cALD is manifest by blood brain barrier breakdown and cerebral inflammation, we next evaluated young males with cALD who carried the 26. Exposure of fibroblasts from patients with ALD to excess levels of fatty acid gives rise to mitochondrial ROS production and reduced mitochondrial respiration as demonstrated by Lopez-Erauskin et al.26. Given that young males with cALD present with neuro-inflammation and BBB  disruption, we sought to further investigate any modifying effect that inheriting APOE4 could have on patients with cALD. We evaluated CSF total protein concentration, which we previously have described as a biomarker of disease burden12, and found that carriers of APOE4 had a mean CSF total protein concentration of 40.5 mg/dL. This was not different from APOE4 non-carriers    Fig. . Monocyt56) Fig. .28. We determined Loes scores of the young males in this cohort and found those carrying APOE4 had a significantly more MRI white matter involvement with a mean Loes score of 10.5 compared to APOE4 non-carriers with a mean Loes score of 7 (p = 0.01) suggesting that more demyelination was present in young males carrying APOE4 11. In the current study, young males with APOE4 had a significantly higher mean GIS (mean score 2.0) compared to APOE4 non-carriers  07) Fig. .29  as compared to non-carriers  indicating more neurologic dysfunction at the time of evaluation.Neurologic function is an important indicator of quality of life and ability to participate in everyday activities. We have used a 25-point scoring system developed by Moser and Raymond to assess the neurologic function where points are assessed in several domains when dysfunction is present and a neurologic function score (NFS) is derivedAPOE4 allows for uninhibited matrix metalloproteinase (MMP) activation in mouse models, which ultimately leads to a permissive BBB7. We evaluated the MMP1, 2, 9, 10 concentrations of the CSF and found the APOE4 carriers had significantly higher levels of MMP2 in their CSF (mean 13168 pg/mL) compared to that of non-carriers  suggesting that a similar APOE4 – MMP association may exist . A receiver operating characteristic curve (ROC) from nominal logistic fit analysis indicated reasonable association between the above biomarkers and APOE4 status   Fig. .APOE4 allele. Frequency of APOE4 inheritance among patients with cALD (17.5%) was slightly higher than the 12% rate reported in the general population6, but this difference was not significantly different on Chi-square analysis. Our data indicate that young males with cALD carrying APOE4 had a higher cerebral disease burden at the time of evaluation for HCT. Our data show a 50% higher average Loes score in the APOE4 carrier group . Elevated Loes score in cALD has been shown to be associated with inferior outcomes after HCT30. Similarly, GIS was also increased in the presence of APOE4 among the young males with cALD. GIS has also been associated with a higher risk of poor neurologic function after HCT11. More intense gadolinium enhancement suggests a more permissive BBB in APOE4 carriers. Bell et al. have shown that a mechanism through which ApoE3  may maintain the BBB is via the inhibition of cyclophilin A, an activator of MMP9. It was further demonstrated that carriage of ApoE4 allowed cyclophilin A to activate MMP9 and led to increased BBB leakiness. Unlike the prior work that showed MMP9 was the key mediator of BBB disruption, we found MMP2 is higher in the CSF of APOE4 carriers suggesting there may be a difference between murine and human biologic mechanisms. Furthermore, work recently reported by Paik et al. demonstrated attenuation of endothelial cell permeability with reduction in Aß induction of MMP-2 expression, suggesting that MMP-2 could be involved in BBB permeability, but it may vary depending on the pathophysiological process (in this case ß-amyloid toxicity). Interestingly, while CSF total protein may be a sign of increased BBB disruption, we did not find an association between APOE4 and CSF protein. In contrast, we did find an increase in CSF chitotriosidase activity, suggesting that inflammation and BBB disruption is higher in APOE4 carriers.In this large cALD cohort, we report a potential genetic modifier of cALD disease related to the inheritance of the 34. Interestingly, one of the common immunosuppressive drugs given to cALD patients post-HCT to suppress graft versus host disease is cyclosporine, which binds intracellular cyclophilins in T-cells and inhibits their activity35. Cyclosporine can also bind and inhibit extracellular cyclophilins such as CypA36. Furthermore, Bell et al. has shown the APOE4 transgenic mice treated with cyclosporine had restored BBB integrity7. It is interesting to speculate that perhaps one of the mechanisms by which HCT stabilizes cerebral disease in young males with cALD is through the action of cyclosporine on the BBB apart from the allogeneic donor cell engraftment.It is well described that HCT is the only established treatment modality to stabilize cALD once neuroinflammation has startedAPOE4 status truly modulates the onset of cerebral disease as well as learn if there is any change in the velocity of cerebral disease progression observed by serial MRIs and neurologic function.Our study was limited in that it was a cross-sectional analysis of young males with a new diagnosis of ALD, and all evaluated patients already manifested cerebral disease. Ideally, future studies should focus on longitudinal evaluation of young males with ALD who do not yet manifest cerebral disease to learn if APOE4 is linked to poorer neurologic function following onset of acute or chronic neurologic injury39. In Alzheimer’s disease, studies have shown a link between APOE4 inheritance and increased pericyte destruction leading to increased BBB permeability as contributors to the disease process8. Persons with neuropsychiatric disease have an increased prevalence of both anti-N-methyl-D-aspartate receptor (NMDA) antibodies and APOE4, demonstrating the a between BBB permeability allowing the penetrance of antibody producing immune cells with a neurologic illness41. Finally, APOE genotype can somewhat modify disease onset in metachromatic leukodystrophy, another leukodystrophy often beginning in childhood42.There are numerous examples in the literature in which APOE4 have more advanced than those who do not carry APOE4. We speculate that they may have an accelerated course of disease, but without a true longitudinal study, this is unknown. As newborn screening for ALD becomes more prevalent, longitudinal studies looking at cALD disease modifiers will become increasingly important. Such studies will help inform us which patients may be at greatest risk of developing the cerebral form of the disease, perhaps allowing for increased surveillance with MRI and other biomarker testing.Our data suggest that young males who inherit Patients with cALD  had CSF sampling done 2 to 6 months prior to hematopoietic stem cell transplant at the University of Minnesota. This study and the use of all bodily fluids were approved by the Committee on the Use of Human Subjects in Research at the University of Minnesota. Informed consent was obtained for all patient samples from the parents or legal guardians on behalf of the child participants. Written assent was also obtained if patients were greater than 8 years of age. the methods were carried out in accordance with the relevant guidelines and regulations.CSF samples were concentrated with Amicon Ultra 3 K molecular weight spin filters(Millipore) and buffer exchanged by passing two rounds of 500 µL 0.5 M triethylammonium bicarbonate (Sigma) through the spin columns. The retentates is that a word? from the spin filters was collected and a Bradford assay was done to determine protein concentrations of all samples. Thirty micrograms of each sample was suspended in 160 µl of protein denaturing buffer  on ice, and the sample was transferred to a PCT tube  with a 150 µl cap. A Barocycler NEP2320  was cycled between 35 kpsi (30 sec) and 0 kpsi (15 sec) for 40 cycles at 37 °C. A volume of 5.83 µl of 200 mM methyl methanethiosulfonate (MMTS) were added to each sample . The samples were mixed by a brief vortex and incubated (15 min) at room temperature. The sample was transferred to a new 1.5 mL microfuge Protein LoBind tube .in vacuo. Each sample was then cleaned with a 4 mL Extract Clean™ C18 SPE cartridge . Eluents were vacuum-dried and suspended in dissolution buffer (0.5 M triethylammonium bicarbonate) from the iTRAQ kit to a final 2 µg/µl concentration.The 20-µg aliquot of each sample was diluted four fold with ultra-pure water. A pooled control sample of 125 µg was made from equivalent amounts of all the samples in the experiment (6.9 µg per sample) and diluted four-fold with ultra-pure water. Trypsin digestion was performed by adding 1:35 ratio of trypsin  to total protein. Samples were incubated (16 hrs) at 37 C, frozen at −80 C (30 minutes), and dehydrated st dimension fractionation was done as previously described followed by second dimension separation using liquid chromatography43.20 µg of a research sample or pooled reference was labeled for each iTRAQ channel in the set. A single iTRAQ assay consisted of iTRAQ isobaric tags 113–118 that were used to label CSF proteins from ALD patients 1–6. iTRAQ isobaric tags 119 and 121 were used to label a reference CSF protein extraction from children without adrenoleukodystrophy which we have previously published. The CSF protein from a total of 18 young males with cALD were analyzed in three iTRAQ experiments. The iTRAQ labeling and offline 144. We made minor modifications to the LC and MS parameters: HCD activation time was 20 msec; lock mass was not employed; dynamic exclusion settings were: repeat count = 1, exclusion list size = 500, exclusion duration = 30 seconds, exclusion mass width (high and low) was 15 ppm and early expiration was disabled.We analyzed the first dimension LC fractions by capillary LC-MS on an Orbitrap Velos MS system as previously describedHomo sapiens (taxonomy ID 9606) protein FASTA sequences downloaded from UniProt on Dec 13, 2016 after concatenation with the common lab contaminants database from http://www.thegpm.org/crap/ with a total of 92,719 protein sequences. Sequest was searched with a fragment ion mass tolerance of 0.100 Da and a precursor ion tolerance of 50 ppm with trypsin as the enzyme. Methylthio modification of cysteine was specified in Sequest as a fixed modification. Pyro-glutamic acid, deamidation of asparagine, oxidation of methionine, dioxidation of methionine and iTRAQ 8-plex of lysine and peptide N-terminus were specified in Sequest as variable modifications. Quantification results will be reported in a separate manuscript.Tandem mass spectra were extracted by roteome Discoverer version 2.1.0.81. Charge state deconvolution and deisotoping were not performed. All MS/MS samples were analyzed using Sequest  set up to search the 46.Scaffold  was used to validate MS/MS based peptide and protein identifications. Peptide identifications were accepted if they could be established at greater than 97.0% probability to achieve an FDR less than 1.0% by the Scaffold Local FDR algorithm. Protein identifications were accepted if they could be established at greater than 99.0% probability to achieve an FDR less than 1.0% and contained at least 2 identified peptides. Protein probabilities were assigned by the Protein Prophet algorithmAPOE genotyping was performed on prepared DNA samples by assessing APOE single nucleotide polymorphism (SNP) variants rs7412 and rs429358 using the SNP Genotyping Assay 44.HCT candidates’ CSF Human Apolipoprotein E was quantified using the enzyme-linked immunosorbent assay: Apolipoprotein E /ApoE Quantikine Kit . The CSF was not diluted.2. After incubation, wells were trypsinized with 200 µL of 1x trypsin and washed three times with PBS/0.2% BSA. Cells were next stained with 100 µL CM-H2DCFDA solution according to the manufacturer’s protocol (Thermo Fisher) at room temperature for 5 minutes. Samples were washed three times with PBS/0.2% BSA. 5 µL of 7-AAD (Invitrogen) was added tube for live-dead discrimination. CM-H2DCFDA was read in the FL1 channel; 7AAD was read in the FL3 channel on a BD Accuri C6 analyzer.Human dermal microvascular endothelial cells (HDMECs) were plated in 24-well plate at 10,000 cells/well/mL media +/− 500 nM ApoE4, and incubated for 24 hours at 37C, 5% CO2, 37 C for 72 hours prior to analysis. The plate was gently washed and each well received 500 µL Assay Medium  that was pre-warmed to 37 C. The plate was incubated at 37 C for a minimum of 1 hour without CO2 as is standard for Seahorse biochemical analysis experiments.HDMECs were plated at 15,000 cells/well +/− 500 nM ApoE4. Plate was incubated at 5% COOxidative phosphorylation determination was performed on a Seahorse XL-24 biochemical analyzer using the following injections: 2 µM oligomycin (PortA), 1 µM FCCP (Port B), 0.5 µM Antimycin A/Rotenone (Port C) and the following parameters: 3 cycles  for each pre-port injection and each post-port injection.Samples were analyzed as a multiplex for MMP-1, 2, 9, and 10 using the Luminex platform performed as a multiplex. The magnetic bead set  was purchased from EMD Millipore Corporation . Samples were assayed according to manufacturer’s instructions. Fluorescent color-coded beads coated with a specific capture antibody were added to each sample. After incubation, and washing, biotinylated detection antibody was added followed by phycoerythrin-conjugated streptavidin. The beads were read on a Luminex dual-laser fluidics based instrument (Bioplex 200). Samples were run in duplicate and values were interpolated from 5-parameter fitted standard curves.et al.28. Qualitative enhancement was assessed using the gadolinium intensity score (GIS) developed by Miller et al.11 Loes and GIS scores were determined by a single neuroradiologist (D.R.N.).Young males with cALD are defined by finding T2-weighted white matter signal abnormalities as well as gadolinium enhancement on MRI. The extent of cerebral involvement  can be quantified using a 35-point scoring system develop by Loes 47.Clinical cerebral disease severity was scored at the time of initial patient assessment in clinic by T.C.L., W.M., or G.V.R. using the ALD neurologic function score (NFS)et al.48. CSF samples were diluted in buffer , and 20 μl aliquots of these dilutions were incubated with 20 µl of 22 µM 4-methylumbelliferyl-beta-D-N,N′,N′-triacetyl-chitotriose  in 0.5 M citrate-phosphate buffer, pH 5.2, in 0.1% albumin  pre-coated 96-well plates for 1 hour at 37 C. The reaction was stopped after 1 hour with 250 µl 0.5 M Na2CO3-NaHCO3, pH 10.7. Enzymatic cleavage of MUTAC produces a fluorescent product, 4-methylumbelliferone (4-MU), which was read on a Molecular Devices, SpectraMAX Gemini fluorometer with 365 nm excitation and 450 nm emission settings. The comparison of relative fluorescent units (RFU) with standards  ranging from 0.4–12.5 ng/well allowed calculation of activity, which was expressed as nmoles 4-MU generated/mL of CSF per hour14.Chitotriosidase activity was measured using a modification of the technique described by Sotgui https://www.qiagenbio-informatics.com/products/ingenuity-pathway-analysis)49. Two-way comparisons were analyzed using a Student’s unpaired t-test. Least squares multivariate fit modeling, regression, and nominal logistic fit analyses was performed using JMP (version 13.0). P-values are given in each figure.The functional analyses were generated through the use of IPA (QIAGEN Inc.,


Hand hygiene, including handwashing by children, has been reported to contribute to the prevention of various infectious conditions. This study aims to explore the correlates of handwashing behavior among 1690 fourth to sixth grade primary school students in 19 Tibetan primary schools . The theory of reasoned action (TRA) was applied. Data was collected by questionnaire. Structural equation modeling (SEM) analysis showed that students’ attitude  and subjective norms in terms of compliance to teachers’, parents’ and peers’ suggestions to wash hands  were directly associated with students’ handwashing behavior. Students’ knowledge  had an indirect association with handwashing behavior, mediated by students’ attitudes and subjective norms. Subjective norms  were also indirectly correlated with handwashing through students’ attitudes. Therefore, our study supported the theory of reasoned action through our findings that students’ attitude and knowledge, and also attitudes from teachers, parents and peers were correlated with student handwashing behavior. Students reported higher level of compliance to teachers than to their parents and classmates. Based on this information, we recommend teacher-involved participatory hygiene education to promote students’ handwashing behaviors in areas at high risk for infectious diseases that can be prevented by handwashing. Hand hygiene has been recommended as an important way of keeping individuals from diseases such as foodborne parasitic infections ,2,3. SysAlthough handwashing is a simple and efficient method for reducing the risk of infectious diseases, studies have shown the relatively low adherence to the recommended handwashing; the reported percentages of adherence vary between 1.8% and 78%, depending on the population, and context ,7. A larStudies on the behavioral determinants of adequate hand hygiene remain scarce and lack conclusive results . A few sRural Tibetan areas in China are characterized by a high incidence of foodborne parasitic diseases, especially echinococcosis . StudiesThe specific study questions are (1) what are the handwashing habits of primary school children in rural Tibetan primary schools, and (2) what are the correlates of school children’s handwashing. The theory of reasoned action was applied in this study to explore the social and behavioral determinants that are associated with handwashing among children, as recommended by Gellman . The theBased on the theory of reasoned action, our hypotheses are: (1) The knowledge and attitudes of students are associated with adequate hand hygiene; (2) the subjective norms, including the normative belief concerning important referent individuals and the motivation to comply with them, are associated with adequate hand hygiene; (3) families and peers, as well as teachers, play a role regarding the intention of students to wash their hands, and regarding adequate hand hygiene of the students. Therefore, the following model in The study was reviewed and approved by the Institutional Review Board of the Western China School of Public Health, Sichuan University (project identification code: 0040405502146). Formal permission was obtained from the local Education Bureau of the Tibetan autonomous prefecture of Golog, and initial written consent was obtained from school principals, who were the students’ statutory guardians when they were boarding at school. Most students’ home was far away from the schools in Tibetan areas, and they could not go home often. Therefore, informed consent was obtained from school principals instead of parents for students’ participation in this study.The study design was a cross-sectional survey. The survey consisted of a one-time paper and pencil questionnaire to be completed by the students and was conducted in the Tibetan autonomous prefecture of Golog, Qinghai Province, one of the poorest areas in China . Living Golog has a high prevalence (1.91%–11.93%) of human echinococcosis. Echinococcosis is a foodborne parasitic disease that can largely be prevented by adequate handwashing ,25. It iIn 2016, a multistage random cluster sampling method was applied to select the sample. Firstly, we randomly selected two of the six counties in Golog. Secondly, all nineteen townships from the sample counties were involved. Thirdly, we decided to include all the nineteen central primary schools in the nineteen townships, since there was only one central primary school in each township in the study area. Lastly, we purposively invited all students from the fourth to sixth grades to participate in our study; therefore, the students’ literacy level and language ability were adequate to understand and answer the questions.Informed by the theory of reasoned action, and given the characteristics of the most important condition to be prevented in the study area, the questionnaire was designed based on a review of the literature and expert consultation. The questionnaire included the knowledge about preventing echinococcosis through handwashing, attitudes towards handwashing, and subjective norms including the normative belief concerning important referent individuals and the motivation to comply with them  to 5  was utilized to measure participants’ responses to such knowledge based questions.Attitude towards handwashing was measured by three items such as “do you agree you should wash your hands before eating and after using the toilet?” A five-point Likert scale ranging from 1  to 5  was utilized to measure these items. The higher the score, the more positive the attitude towards handwashing.The subjective norms were measured by the degree to which individuals were likely to comply with advice from three important referent individuals: family, teacher, and peer. Six pairs of items were included, such as “did your family ever tell you to wash hands before eating and after using the toilet?” followed by “to what extent are you likely to follow your family when they tell you to wash hands before eating and after using the toilet?” The first item on normative belief was measured by 0 (never told) and 1 (told). The second item on motivation to comply was measured by a five-point Likert scale ranging from 1  to 5 . The higher the total score, the more positive the subjective norm one perceived.The outcome variable is actual behavior since it is still unclear whether behavior intention could lead to actual behavior in the theory of reasoned action . Four quWe first examined sample demographic characteristics of participants and the rate of handwashing using descriptive statistics. We conducted confirmatory factor analysis to assess whether observed variables were adequate indicators of latent variables. 2/degrees of freedom (χ2/df) less than 5.00, and the root mean square error of approximation (RMSEA) less than 0.05 [Several indicators were used to evaluate the fit of the model, goodness-of-fit index (GFI), comparative fit index (CFI), normed fit index (NFI) of 0.90 or above, χhan 0.05 . The ana2/df = 4.432), goodness-of-fit index (GFI = 0.965), comparative fit index (CFI = 0.952), normed fit index (NFI = 0.961) as well as root mean square error of approximation (RMSEA = 0.045). The SEM was established to reflect the relationships pertaining to the variables in the research model. We fitted the data and the theoretical model through the maximum likelihood and bias-corrected bootstrap with 2000 replications, and modified the theoretical model until the AMOS reached a local minimum according to model fit indices. The model fitness was within the acceptable ranges, namely, the normed chi–square (χp < 0.001), as well as washing hands after touching dogs  compared to families and peers. This result supports our hypothesis that teachers have a relatively stronger social influence on primary school students’ handwashing compliance than families and peers. Primary school students may be more compliant to their teachers than to their families and peers regarding handwashing.The total effects, direct effects and indirect effects among the four latent variables were shown in In the current study, we constructed a structural equation model based on the theory of reasoned action to explore the correlates of primary school students’ handwashing behavior. All hypotheses were supported by our results. The results supported the first hypothesis that subjective norms and attitude could directly affect handwashing. Furthermore, knowledge and subjective norms indirectly affect handwashing via attitude . Lastly,The results showed a small percentage of students frequently wash hands before meals and after toilet at home or school, and this percentage is lower than the findings of other studies in adjacent regions ,30. SeveThe results of the structural equation model showed that attitudes to handwashing had a strong direct association with primary school students’ handwashing behavior. Students who had favorable attitudes to handwashing were more likely to wash their hands, which is consistent with findings from a previous study based on the RANAS  model . In thisThe present study also found that subjective norms not only had the direct positive effects on students’ handwashing behavior, but also had important indirect effects via attitude. This finding indicated that subjective norms played an important role in students’ handwashing behavior, which is consistent with previous research findings ,37. ThusAn important new finding of this study is that teachers had a stronger influence on students’ handwashing behavior, compared with families and peers. In remote rural areas in China, most primary students board in their schools and spend more time with their teachers than parents except during vacation time ,32. TeacFindings of this study provide important information for health care practitioners, families, and educators to design educational and intervention programs to improve hand hygiene among students. Given the setting of the study, the results may be especially relevant for the development of interventions in economically disadvantaged areas.Several limitations of this study need to be taken into consideration. First, because of the cross-sectional design, we cannot infer causal relationships between the factors that we identified and the handwashing behaviors. Second, social desirability may have affected the answers given by the participants. This study was conducted in remote and poor areas. Therefore, we recommend replicating this study in other populations and regions. We also suggest future studies to apply a longitudinal design and to validate the measures of handwashing behavior by observations.The findings of this study may be used to develop interventions to promote adequate handwashing among school children. When evaluating such interventions, it is important not only to evaluate the handwashing behaviors, but also to evaluate whether the intervention and the handwashing indeed lower the risk for the infectious conditions that are targeted by the intervention in a specific context or region . So, in In this study, regarding the measurement of the attitude towards ‘handwashing before meals and after toilet’, it should be considered that the attitude towards these two elements  can vary within one person. Therefore, we propose future studies to measure these two attitudes separately.Finally, the theory of reasoned action model was applied, which does not include perceived threat as a determinant of behavior. Perceived threat contains ‘severity’ and ‘perceived susceptibility’ . Yang foThis study found that the students in this study had poor handwashing behaviors before meals and after toilet both at home and in school in echinococcosis endemic areas. Students’ handwashing behaviors were directly associated with attitudes, and subjective norms affected handwashing behaviors both directly and indirectly. Teachers had a stronger influence on students’ subjective norms compared to families and peers. Findings from this study suggest that teacher-involved participatory hygiene education could be an effective way to promote students’ handwashing behaviors in echinococcosis endemic areas.


High-efficiency thermally activated delayed fluorescence (TADF) is leading the third-generation technology of organic light-emitting diodes (OLEDs). TADF emitters are designed and synthesized using inexpensive organic donor and acceptor derivatives. TADF emitters are a potential candidate for next-generation display technology when compared with metal-complex-based phosphorescent dopants. Many studies are being conducted to enhance the external quantum efficiencies (EQEs) and photoluminescent quantum yield of green TADF devices. Blue TADF reached an EQE of over 35% with the support of suitable donor and acceptor moieties based on a suitable molecular design. The efficiencies of green TADF emitters can be improved when an appropriate molecular design is applied with an efficient device structure. The triazine acceptor has been identified as a worthy building block for green TADF emitters. Hence, we present here a review of triazine with various donor molecules and their device performances. This will help to design more suitable and efficient green TADF emitters for OLEDs. Organic light-emitting diodes (OLEDs) and the application of organic materials in emitting technology have attracted much attention from industrial and research communities since 1987. The advantages of OLED technology, such as its light weight, image quality, high contrast, fast response time, thin film, and wide-view angle, have made it a potential candidate in commercial applications instead of liquid crystal displays (LCDs) ,3,4,5. MThe spin rule explains the singlet and triplet emission possibilities of OLED dopants, where 25% are only responsible for singlet emission while 75% are from the triplet state. First-generation fluorescent emitters harvest only singlet emission with an internal quantum efficiency (IQE) of 25%. The remaining 75% of the IQE does not hold any emission responsibility for colors ,23,24,25Current studies are exploring new technologies using metal-free dopants and a type of emission referred to as thermally activated delayed fluorescence (TADF). This TADF mechanism can also achieve 100% IQE by activating an efficient up-conversion process of reverse intersystem crossing (RISC) from an excited triplet state to an excited singlet state ,49,50,51Many donor–acceptor-based structures have been reported for TADF emitters. The most commonly reported donor moieties are carbazole, diphenylamine, acridine, phenoxazine, phenothiazine, and their derivatives. The electron-withdrawing groups of cyano, boron, pyridine, pyrimidine, triazine, sulfone, dicyanofluorene, pyrazine, and ketone have been employed as acceptor moieties ,64,65,663B and 2DAC-Mes3B revealed EQEs of 22.8% and 21.6%, respectively, which were higher than those of thioxanthone-based green emitters -2-yl)-9-phenyl-9H-carbazole (PhCzTAZ) molecule -9'-phenyl-9H,9'H-3,3'-bicarbazole (CzT)  moleculemolecule  displayeTmCzTrz) and 9,9'--5--1,3-phenylene)bis(9H-carbazole) (DCzmCzTrz) revealed PLQYs of 100% and 98%, respectively, and the energy difference between the singlet and triplet states were 0.07 and 0.20 eV, respectively. The higher PLQY of the TmCzTrz molecule showed greater efficiencies  than the DCzmCzTrz molecule. The number of carbazole donors with even HOMO distribution increased, and the device efficiencies were enhanced furan-3-yl)-9,9’’-diphenyl-9H,9’H,9’’H-3,3’:6’,3’’-tercarbazole (TCzTrzDBF), and 12-dibenzofuran-3-yl)-5-phenyl-5,12-dihydroindolocarbazole (IDCzTrzDBF) were synthesized and used for green TADF emitters  and IDCzTrzDBF (12.2%). Moreover, the three-carbazole-unit-based TCzTrzDBF enhanced the current and power efficiencies  compared with the two-carbazole-unit-based BCzTrzDBF . Further studies were carried out by Jung et al. by changing the donor and acceptor positions with a dibenzofuran linker unit (2Cz3Trz) and 9-dibenzofuran-3-yl)-9'-phenyl-9H,9'H-3,3'-bicarbazole (3Cz2Trz) showed low PLQYs and device efficiencies compared with TCzTrzDBF. Changing the acceptor attached position and reducing the amount of carbazole did not reveal any interesting efficiency enhancements. However, a dibenzofuran linker can suppress the nonradiative mechanism when compared with the presence of a phenyl linker moiety furan-3-yl)-9'-phenyl-9H,9'H-3,3'-bicarbazole  than the monosubstituted CzT molecule (6%). Symmetrical molecules of DPA-TRZ and DACT-II with a phenyl linker unit exhibited better device properties. Carbazole with diphenyl amine at the third and sixth positions (DACT-II) enhanced the device quantum efficiency up to 29.6%, while diphenyl amine, at the third and sixth positions of diphenylamine (DPA-TRZ), showed a low efficiency of 13.8%. So, carbazole with a diphenylamine donor at the third and sixth positions resulted in a more interesting effect with the triazine acceptor than a similar molecular design with diphenylamine donor derivatives. A IDCzTrzDBF molecule was constructed with a furan linker between an indolocarbazole donor and a triazine acceptor, but this linker moiety and substituted position did not have a successful effect on the external quantum efficiency. A furan linker moiety attached to a symmetrical donor of carbazole with third- and sixth-position-substituted phenyl carbazole (TCzTrzDBF) showed better performance. When the number of carbazole donor moieties was increased and attached to the phenyl group at meta and para positions (TmCzTrz), the result was an EQE over 25%. Overall, a carbazole donor containing a symmetrical structure, along with free rotating substitutions at the third and sixth positions, and a number of carbazole donors attached through meta and para substitution further helped to achieve high EQEs for green TADF emitters compared with carbazole-based rigid donor derivatives of indolocarbazole. Carbazole and its derivatives with a triazine acceptor were the subject of an interesting study on device performances. The monosubstituted indolocarbazole donor moiety TrzIAc) molecule . The indeno–acridine donor moiety not only enhanced the thermal stability but also improved device efficiencies with green color emission (511 nm) acridine (molecule  was repo(511 nm) . BFAcTRZ) and benzothiophene–acridine phenyl)-5,5-dimethyl-5,13-dihydrobenzothienoacridine, BTAcTRZ) with a triazine acceptor  and BTAcTRZ (21.8%) were higher than 20%, and BTAcTRZ exhibited better efficiencies than that of the indeno–acridine-based TrzIAc molecule. The BTAcTRZ molecule showed a red-shifted electroluminescent emission of 526 nm due to its donor moiety acridine, acceptor . The PLQr moiety ,92.TrzIAc, BFAcTrz, and BTAcTrz revealed better EQEs, which were higher than those of indolocarbazole-based green TADF emitters.Rigid donor moieties of indeno–acridine-, dibenzofuro–acridine-, and benzothieno–acridine-based PXZ-TRZ) material with a phenoxazine donor (Tri-PXZ-TRZ) revealed an EQE of 13.3%, which was higher than that of the single-substituted donor molecule. Moreover, the trisubstituted material showed red-shifted emission, and the PLQY was 58% [The 10-phenyl)-10H-phenoxazine  TADF emitter phenyl)-10H-phenothiazine   emission was notices at 548 nm, which showed little red shifting due to its long conjugation donor molecule [Shizu et al. reported another molecule phenyl)-N1-(4-(diphenylamino)phenyl)-N4,N4-diphenylbenzene-1,4-diamine, DPA-TRZ)  with a 1molecule ,95. DMAC-TRZ) phenyl)-9,9-dimethyl-9,10-dihydroacridine (MAC-TRZ)  with a d of RISC ,93.3ACR-TRZ) TADF emitters -yl)phenyl)-1,3,5-triazine  and diphenyl acridine -1,3-phenylene)bis, TRZ-DDPAc) were constructed -1,3-phenylene)bis, structed  with a Dectively .DACT-II-, TRZ-DDPAc-, DMAC-TRZ-, and TmCzTrz-based devices exhibited good EQEs of 29.6%, 27.3%, 26.5%, and 25.5%, respectively. The DACT-II molecular design had a carbazole donor with symmetrically attached diphenylamine units, while the TmCzTrz molecule was constructed with three carbazole units at the meta and para positions of the phenyl linker. TRZ-DDPAc had two diphenyl acridine moieties at the meta positions of the phenyl linker group. The molecular design containing only donors of phenoxazine and phenothiazine did not show any impact on efficiency enhancement. Acridine derivatives had prominent effects on quantum efficiencies. The number of donor moieties with symmetrical attachments with the linker phenyl group enhanced the device properties.Regarding the device performances of triazine-based green TADF emitters, PXZ-TRZ, PTZ-TRZ, Bis-PXZ-TRZ, Tris-PXZ-TRZ, and DPA-TRZ showed red-shifted emissions of 529, 532, 552, 553, and 548 nm, respectively. The carbazole-donor-based CC2TA, PIC-TRZ, PIC-TRZ2, DCzmCzTrz, and TmCzTrz showed blue-shifted emissions of 490, 500, 505, 496, and 500 nm, respectively. So, future works should consider elucidating which type of donor moiety is suitable for designing green TADF emitters with color purity emission. Many donor derivatives were incorporated with triazine acceptors to design various green TADF emitters. The strong donor moieties of acridine, phenothiazine, diphenylamine, and phenoxazine and the weak donor moieties of carbazole, benzofurocarbazole, and benzothioenocarbazole were employed, showing suitable attachment to acceptor and linker units. We noticed that strong-donor-based TrzIAc and DCzmCzTrz showed EQEs of 20.9% and 21.3%, respectively, and an emission layer thickness of 25 nm. The 30 wt % doped BFAcTrz, TmCzTrz, and TRZ-DDPAc exhibited quantum efficiencies of 20.4%, 25.5%, and 27.3%, respectively, and had the same emission layer thickness of 25 nm. A DACT-II-based device showed better device properties at a low doping concentration of 6%, but the thickness of the emission layer was reported to be 40 nm, and we believe that the greater thickness of the host material (CBP) supported an effective energy flow to achieve an EQE of 29.6%. DMAC-TRZ showed good device characteristics (EQE of 26.5%) and employed an 8% doping (20 nm) concentration and hole-blocking layer (DPSS). The acridine-based 3ACR-TRZ was 16 wt % doped with CBP as the host material, and the emission layer thickness was as high as 55 nm, but the device could not reach an EQE over 19%. So, for device optimization, using various doping concentrations and host materials is crucial to obtain an effective device. Host materials play a major role in device performance as they are responsible for supplying energy to the emission layer. At the same time, host materials control the charge recombination of collected electrons and holes from the cathode and anode, respectively. The choice of host material depends on the triplet energy of the dopant material, and high triplet energy host materials dope with dopant to establish an effective device. Moreover, the selection of host materials, adjacent layers, and doping concentrations is important to ensure the effectiveness of the device. Among the above-reported triazine-based green TADF emitters, 20 wt % doped emitters of Triazine-acceptor-based green TADF emitters with suitable donor derivatives and host materials have shown great performance in terms of device efficiency. The EQEs were over 29%, which were higher than those of any red TADF emitters. Still, many improvements are needed in the molecular design to achieve a high efficiency. Host materials play a major role in device efficiency by supporting effective energy transfer to the dopant. Moreover, a proper doping concentration also enhances device performance. Triazine has exhibited good withdrawing characteristics and a suitable donor moiety connecting the appropriate position, which should result in a highly efficient and stable molecular design for green TADF emitters.


Background: Neurocysticercosis (NCC) is the most common helminthic infection of the central nervous system (CNS). The diagnosis of NCC is sometimes challenging due to its heterogenous clinical manifestations and the variable sensitivity and specificity of neuroimaging and serological tests.Methods: Next-generation sequencing (NGS) of cerebrospinal fluid (CSF) was used to detect pathogens in patients with clinically suspected CNS infections. A series of patients diagnosed with NCC is reviewed here.Results: Using NGS of CSF, four patients were diagnosed with NCC. The reads corresponding to Taenia solium ranged from 478 to 117,362, with genomic coverage of 0.0564–11.15%. Reads corresponding to T. solium were not found in non-template controls and far exceeded those of the background microorganisms in patients with NCC, facilitating the interpretation of the NGS results.Conclusions: This case series demonstrates that NGS of CSF is promising in the diagnosis of NCC in difficult to diagnose cases. Larger studies are needed in the future. Taenia solium (Neurocysticercosis (NCC) is an infection of the central nervous system (CNS) and its meningeal coverings caused by the larval stage of the tapeworm a solium . It is ea solium –3. The ca solium . The diaa solium –7. Compaa solium . Second,a solium .The use of DNA-based techniques for NCC diagnosis has been reported , 10, 11.A multi-center prospective research project that includes 26 hospitals has been conducted in China since 2016, with the goal of detecting pathogens in patients with clinically suspected CNS infections using NGS of CSF. CSF from patients with encephalitis or meningitis was sent for NGS. The inclusion and exclusion criteria for encephalitis and meningitis in the multicenter study are provided in Supplementary Table This study was approved by the Institutional Review Board of Peking Union Medical College Hospital (PUMCH) and the Beijing Genomics Institute, Shenzhen (IRB no. JS-890). The use of the patients' clinical data and CSF samples was approved by the Ethics Committee of PUMCH. Written informed consent was obtained from each patient or their legal surrogate in accordance with the Declaration of Helsinki.DNA was extracted from 300 μL of CSF (per patient and negative “no-template” controls) using the TIANamp Micro DNA Kit . Proteinase K (10 mL) and buffer GB  were added. Then the sample was incubated for 10 min at 56°C. After adding 300 μL of cold absolute ethyl alcohol, the tube was incubated for 5 min at room temperature. The DNA-containing liquid was transferred to a new adsorption column and washed with buffer GD and buffer PW. Finally, the DNA was dissolved with 40 mL of Tris-ethylenediaminetetraacetic acid buffer.The extracted DNA was fragmented into 200–300 bp fragments using a Bioruptor Pico, according to the manufacturer's instructions. DNA libraries were constructed using through end-repair, poly(A)-tailing, adapter ligation, and PCR amplification. After quality control using an Agilent 2100 Bioanalyzer , the libraries were sequenced on a BGISEQ-100 platform.ftp://ftp.ncbi.nlm.nih.gov/genomes/), which includes the genome sequences of 1494 bacteria, 2700 viruses, 73 fungi, and 48 parasites. The mapped data were used for further analysis. The depth and coverage were calculated for each species with Soap.coverage (SOAP).High-quality sequencing data were generated after removing short (<35 bp), low-quality, and low-complexity reads. The reads were then mapped to the human reference genome (hg19 and YH sequences) using the Burrows-Wheeler Aligner. The remaining data were aligned to the NCBI microbial genome database . The sequences were then mapped to the NT database with the online software NCBI blast.Species-specific PCR identification of Cysticercus cellulosae IgG in serum or the CSF sample was detected using a commercial enzyme linked immunosorbent assay (ELISA) kit using semi-purified antigen extracted from the scolices of Taenia solium cysticerci .The presence of Of the 381 patients with clinically suspected CNS infections, four patients with NCC were identified by NGS of CSF. Their clinical features are summarized in Table Case 1: A 53-year-old woman presented with a recurrent headache, blurred vision, and progressive memory loss. The headache first appeared 5 years earlier and worsened gradually. One and a half years ago, she developed blurred vision. Brain magnetic resonance imaging (MRI) at that time showed hydrocephalus. Repeated lumbar punctures revealed increased opening pressure, elevated protein and pleocytosis without identifing the etiology. Two months ago, she developed progressive memory loss. She also had recurrent grand mal seizures about 20 years ago. On admission, head computed tomography (CT) showed scattered parenchymal calcified lesions in the right frontal lobe, right parietal lobe, right thalamus, left temporal lobe, left occipital lobe, and bilateral basal ganglia area  and was treated with albendazole and dexamethasone. She also underwent an endoscopic third ventriculostomy (ETV) because of the severe hydrocephalus. The patient's symptoms, neuroimaging and CSF findings improved markedly after treatment.a Figure . Brain Ma Figure . CSF cyt Figures . TherefoCase 2: A 57-year-old man presented with paroxysmal blurred vision for 2 months. When he was admitted 1 month ago, lumbar puncture revealed increased opening pressure, pleocytosis, elevated protein level, and reduced glucose level. CSF cytology showed lymphocytic inflammation. Cryptococcus antigen test and Mycobacterium PCR of the CSF were negative. He was diagnosed with possible tuberculous meningitis and started on empirical anti-tuberculous treatment. However, he was readmitted after 1 month when his symptoms were not relieved. Lumbar puncture was repeated and NGS of CSF was negative. Serum and CSF samples were both positive for C. cellulosae IgG. Head CT revealed a single calcified lesion in the left frontal lobe , and progressive memory loss. Eight years before admission, his symptoms began with recurrent headache and transient LOC. Lumbar puncture revealed increased opening pressure, pleocytosis, elevated protein level, and reduced glucose level. He was diagnosed with possible tuberculous meningitis and given empirical anti-tuberculous treatment for more than 1 year. Six years ago, he was admitted with the same symptoms and diagnosed with possible cryptococcal meningitis, for which he received fluconazole for more than 6 months and amphotericin B for 1 month. Three months before admission, he developed progressive memory loss. On admission, brain MRI showed an enhanced lesion posterior to the medulla . Another 377 other patients with clinically suspected CNS infections, including patients with tuberculous meningitis, cryptococcal meningitis, and neurobrucellosis (T. solium (unpublished data).cellosis  were alsT. solium ranged from 478 to 117,362, with genomic coverage ranging from 0.0564 to 11.15%, and the reads per million (RPM) ranging from 25.24 to 4654.18. A few reads aligned with other members of the genus Taenia, including T. saginata and T. asiatica  are not sufficiently accurate but, rather, because clinicians may not think to perform these tests. Note that, subarachnoid NCC presents as chronic meningitis and has the same endemic area in northern China as tuberculous meningitis. Therefore, some patients might be misdiagnosed with tuberculous meningitis and not undergo serological tests for NCC. As shown in our cases, the diagnosis of NCC was difficult in these patients with extraparenchymal NCC. The correct diagnosis was made 5 and 8 years after the onset of the initial symptoms in Cases 1 and 3, respectively. Fortunately, these patients were diagnosed with NCC “unexpectedly” with NGS of CSF, which can identify pathogens without prior information. In June 2017, a similar case demonstrating the usefulness of NGS in the diagnosis of NCC was published in This case series is part of a multi-center research project involving some rural hospitals in China. Case 3 was referred from one of these rural hospitals. With advances in sample handling and transportation, CSF samples from less-developed areas can now reach research centers faster. NGS testing was performed in a timely manner and the result was returned to the rural hospital promptly. With the result, the hospital organized further investigations and initiated targeted treatment with good effect. To some extent, NGS of CSF may help close the gap between developed and less-developed areas in the diagnosis of difficult cases of CNS infections.This study demonstrates that NGS of CSF is promising in the diagnosis of NCC in difficult to diagnose cases. Larger studies are needed in the future.HG and SF contributed conception and design of the study. SF, XQ, LL, YH, CM, JY, QL, YG, HR, JW, LC, and WZ collected the clinical data. HW, JZ, RS, QC, and YL performed next-generation sequencing of cerebrospinal fluid and bioinformatics analysis. SF wrote the first draft of the manuscript after discussions with XQ, LL, WZ, and HG. HW, JZ, QC, and RS wrote some of the methods part. All authors contributed to manuscript revision, read and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


In DLB patients, low VGF levels were related to a more advanced cognitive decline at time of first presentation, whereas high levels of VGF were associated with steeper subsequent longitudinal cognitive decline. Hence, CSF VGF levels were lower in DLB compared to both AD and controls across different analytical methods. The strong associations with cognitive decline further points out VGF as a possible disease stage or prognostic marker for DLB.In a previous proteomic study, we identified the neurosecretory protein VGF (VGF) as a potential biomarker for dementia with Lewy bodies (DLB). Here, we extended the study of VGF by comparing levels in cerebrospinal fluid (CSF) from 44 DLB patients, 20 Alzheimer’s disease (AD) patients, and 22 cognitively normal controls selected from the Amsterdam Dementia Cohort. CSF was analyzed using two orthogonal analytical methods: (1) In-house-developed quantitative ELISA and (2) selected reaction monitoring (SRM). We further addressed associations of VGF with other CSF biomarkers and cognition. VGF levels were lower in CSF from patients with DLB compared to either AD patients or controls. VGF was positively correlated with CSF tau and α-synuclein (0.55 <  Dementia with Lewy bodies (DLB) is a common cause of dementia in the elderly, accounting for up to 20% of dementia cases . Next toIn a recent study done by our group, the secretory granule protein VGF (non-acronymic) was identified as a novel potential biomarker candidate for DLB in cerebrospinal fluid (CSF) using an in-depth proteomics workflow. In fact, VGF was decreased in CSF from patients with DLB compared to cognitively normal (age-matched) controls. VGF is a secreted neuronal and endocrine precursor protein whose expression is induced by brain-derived neurotrophic factor (BDNF) ,10. VGF The main aim of the present study was to extend the clinical validation of CSF VGF in a relatively large cohort consisting of patients with DLB, compared to either AD and cognitively normal controls. We used two different analytical methods: (1) In-house-developed quantitative competitive enzyme linked immunosorbent assays (ELISA), and (2) selected reaction monitoring (SRM). In addition, we aimed to explore possible correlations between VGF and other CSF biomarkers, as well as to investigate whether VGF might have a role in predicting cognitive decline and survival in DLB patients.p < 0.001; DLB vs. controls: p < 0.05). Similarly, patients with dementia had lower cognitive domain scores compared to controls (p < 0.05). CSF Aβ1-42 was lower, and tau and p-tau levels were higher, in patients with AD (p < 0.001), whereas their levels in patients with DLB were in-between those of patients with AD and controls (p < 0.05). There were no significant differences in CSF α-synuclein levels between DLB patients and controls (p > 0.05).Demographic and clinical characteristics of the diagnostic groups are presented in p < 0.05 and p < 0.001, respectively; p < 0.01 and p < 0.001, respectively; p > 0.05).Levels of VGF373-417, as measured by ELISA, were lower in DLB patients than in either AD patient, or in controls . In DLB and controls, but not in AD, VGF373-417 and VGF SRM correlated positively with CSF tau, p-tau, and α-synuclein . No associations between VGF and Aβ1-42 were found among patients with DLB and AD. In controls, however, VGF levels correlated positively with CSF Aβ1-42 .We performed Spearman correlation analysis to assess associations between the CSF VGF levels and other CSF biomarker levels throughout all investigated groups . VGF373-Subsequently, we used age-, sex-, and education-adjusted linear mixed models to assess the associations between CSF VGF and cognitive decline. We first investigated associations between CSF VGF levels and MMSE scores. VGF373-417 and VGF SRM showed a distinct association with MMSE, both at patients’ first assessment at the memory clinic and over follow-up . Next, wp = 0.26; VGF SRM: HR = 0.79 [0.5–1.2], p = 0.27).Finally, to assess the association between CSF VGF and survival, we performed Cox proportional hazards models adjusted for sex and age. During follow-up, 25 DLB patients (56%) died and 19 DLB patients were alive ). Cox proportional hazard models revealed that CSF VGF levels peptides were not associated with mortality  due to AD compared to controls subjects and patients with AD dementia . In addiAmong the strengths of our study are the relatively large number of DLB patients for this type of experiment and the use of two different analytical methods. The fact that both analytical methods showed essentially similar results confirms the robustness of our findings. Furthermore, etiological diagnosis was either supported by biomarkers or confirmed by autopsy. In addition, control subjects included in the present study had normal CSF AD biomarker levels at first presentation and persevered normal cognitive function for at least two years. Using these inclusion criteria, we could almost certainly rule out AD in these individuals with subjective cognitive decline (SCD). We therefore consider it unlikely that our results are significantly biased by clinical misdiagnosis. Some limitations of the present study also need to be mentioned. First, we focused here on two peptides derived from the VGF protein only, rather than including a broader comparison with other VGF peptides. In view of pathophysiological implication of different VGF peptides which are endowed with differing bioactivity and roles ,10, the We included 44 patients with DLB, 20 patients with AD, and 22 individuals with subjective cognitive decline (SCD) who served as controls. The above-mentioned patients and controls were selected from the Amsterdam Dementia Cohort , consistAll selected patients and controls underwent an extensive standardized and multidisciplinary work-up, including medical history, physical and neurological examination, neuropsychological testing, electroencephalography (EEG), brain magnetic resonance imaging (MRI), and laboratory testing, including lumbar puncture. Diagnoses were made in a multidisciplinary consensus meeting.n = 41, 93%) or was confirmed at autopsy . Clinical diagnosis of AD was established according to National Institute on Aging and Alzheimer’s Association (NIA-AA) criteria. All AD patients had positive AD CSF biomarkers . Individuals were labeled as SCD when they presented at the memory clinic with subjective cognitive complaints, but (i) ancillary investigations were normal, and (ii) criteria for Mild Cognitive Impairment (MCI), dementia, or other neurological or psychiatric disorder(s) known to cause cognitive complaints were not met . In addition, the individuals with SCD we selected showed negative AD CSF biomarkers, as well as their cognitive function on neuropsychological testing remained normal for at least two years at follow-up after first presentation at the memory clinic.DLB was diagnosed according to the international diagnostic consensus criteria for probable DLB . The diaThe study was approved by the local medical ethics committee and all subjects gave their written informed consent for the use of their clinical data and CSF for research purposes.n = 533, age = 59.7 ± 9.8 years, 46% male, MMSE = 28.9 ± 1.0). Next, we created composite cognitive domain scores by averaging z-scores for the corresponding tests per domain. Composite scores were only calculated if there were data available on ≥2 tests within that specific domain, otherwise that domain score was classified as missing. In addition, a global cognitive composite score that combined all 16 neuropsychological test scores across five different cognitive domains was calculated.A standardized neuropsychological test battery was used to measure performance on five cognitive domains. The memory domain included the total immediate and delayed recall of the Dutch version of Rey Auditory Verbal Learning Test and the total recall on condition A of the Visual Association Test. The attention domain consisted of Digit Span Forward, Trail Making Test (TMT) part A and the Stroop word and color tasks. To measure executive functioning, we used TMT part B, color-word task of the Stroop test, Digits Span Backward, and Letter Fluency . The language domain included Category Fluency test  and VAT naming condition. The visuospatial domain was assessed using Dot Counting and Number Location of the Visual Object and Space Perception battery (VOSP). In addition, we used Mini-Mental State Examination (MMSE) as an indication of global cognitive performance. Raw test scores for TMT and Troop were inverted as higher scores indicate worse performance. To obtain cognitive domain scores, all raw test scores were converted to z-scores using the mean and standard deviation of equivalent neuropsychological test scores from an independent reference group of healthy controls  DLB patients, with a mean follow-up time of 2.7 ± 2.2 years. Follow-up extended up to nine years for individual patients.For each DLB patient, all-cause mortality information  was updated using the Dutch municipal population register (date searched: 30 December 2018). Causes of death were not indicated in this register. We defined survival time as the time between (i) the date of the patient’s first visit at the memory clinic, and (ii) either the date of death or the above date (30 December 2018) for alive patients.g at 4 °C for 10 min, and aliquoted in polypropylene tubes of 0.5 mL. Part of the CSF was used for routine analyses, including leukocyte and erythrocyte count, glucose concentration, total protein concentration and levels of Aβ1-42, total tau and p-tau were measured with commercially ELISA assays . Previously validated reference values for AD based on the biobank data were used: <813 pg/mL for Aβ1-42, >375 pg/mL for tau and >52 pg/mL for p-tau [In line with international biobanking consensus guidelines , CSF wasor p-tau ,44. The or p-tau . The remCSF samples assessed in the present study were taken at the patient’s first presentation at the memory clinic.CSF levels of α-synuclein were assessed with an in-house validated immunoassay from ADx Neurosciences  .4, pH 7.2–7.4, 0.15 mol/L NaCl, 0.5 g/L Tween 20) containing normal donkey serum (90 mL/L), aprotinin (20 nmol/L) and EDTA (1 g/L), and incubated  with a mixture of primary antibody (in the same medium) and serial dilutions of either standard peptide, or sample/s. After primary incubation, plates were washed, hence treated with biotinylated secondary antibody , streptavidin-peroxidase conjugate , and tetramethylbenzidine . The reaction was stopped with HCl (1 mol/L), and optical density was measured at 450 nm using a multilabel plate reader . Synthetic peptides corresponding to the full natural peptides  were used as standards. Assay characterization showed: 50% inhibition of signal with 1 nmol/L and 10 pmol/L standard peptide; intra- and inter-assay variation (CV%) of 3% and 11%, 4% and 10%; deviation from parallelism in serial sample dilutions <15% and <10% for the VGF485-503 and VGF373-417 assay, respectively. Recovery of standard peptide added to human CSF was >80% for both assays. To gain some insight as to assay specificity for N-terminally cleaved peptides, versus the same sequence within N-terminally extended forms, or the VGF precursor, synthetic peptides were used containing an additional N-terminal amino acid residue . Cross-reactivity with such N-terminally extended peptides was 0.1% and 0.5% in the VGF485-503 and VGF373-417 assay, respectively, hence indicating a high specificity of either assay for the relevant cleaved peptide.The natural peptides (a) VGF485-503 or NAPP-19  and (b) VGF373-417 or GGEE-45 (as above) were identified in human CSF, and/or in the TT human thyroid cell line, respectively ,47. SyntThe results reported mostly focus on the VGF373-417 peptide. Analysis of the VGF485-503 peptide yielded comparable findings, depicted in For SRM analysis, 200 µL of CSF was spiked with Triethylammonium bicarbonate (TEAB) buffer and a quantitative protein epitope signature tag (QPrEST), kindly provided by Atlas Antibodies AB, #QPrEST20926) of VGF as internal standard. Samples were reduced and alkylated with 1 mM (Tris(2-carboxyethyl)phosphine hydrochloride) (TCEP) and 1 mM 2-chloroacetamide (CAA) at 95 °C for 10min. Proteins were digested for 16 h at 37 °C by adding 1.2 µg trypsin/LysC (Promega). Digestion was stopped by addition of 800 µL 1.25% trifluoroacetic acid (TFA) and peptides were transferred to strong cation exchange STAGE-Tips  by centr2 test, or Kruskal Wallis H test variance with post hoc FDR correction when appropriate. Differences in CSF biomarkers were assessed using general linear model (GLM) corrected for age and sex. CSF tau, p-tau, and VGF were logarithmically transformed before the analyses because of skewed values. We assessed associations of VGF with the established CSF AD biomarkers, Aβ1-42, tau and p-tau, and α-synuclein using Spearman’s correlations. FDR corrections were used to adjust p-values for multiple comparisons. Associations between VGF and cognition at baseline and longitudinal changes over time were assessed with linear mixed models adjusted for age, sex, and education. The models included terms for time (years), VGF levels and an interaction term of VGF level*time as independent variables and MMSE scores, cognitive domain z-scores and global cognition composite score as dependent variables. For all models, a random intercept was assumed. VGF levels were transformed to z-scores. A Beta-coefficient of one (β = 1) therefore implies that one standard deviation increase in VGF levels was associated with one standard deviation increase in cognition score. For illustrative purposes, we dichotomized VGF levels . Finally, we tested the effect of CSF VGF levels on time to death. Cox proportional hazards models with age and sex as covariates were used to calculate hazards ratios for determining the effect of CSF VGF levels on time to death. Statistical significance was set at p < 0.05 for main effects and p < 0.10 for interaction effects.Statistical analyses were performed in R v.3.5.1 ‘Feather Spray’. We assessed differences in patient characteristics using univariate analysis of variance (ANOVA) with post-hoc False Discovery Rate (FDR) correction, χIn conclusion, the present study represents a first step in validating novel synaptic biomarkers for DLB, by showing that VGF levels are lower in patients with DLB compared to both AD patients and cognitively normal controls across different analytical methods. Our results highlight the importance of synaptic dysfunction in DLB. The strong association with cognitive decline might indicate that VGF is useful as a disease stage or prognostic marker in an early symptomatic stage of the disease. Further investigation of synaptic pathophysiology in the clinical continuum of DLB is likely to provide new insight.