The cerebrospinal fluid sample was collected by standard lumbar puncture technique using a single-use traumatic needle. The samples were analysed on an Agilent 1260 Series liquid chromatograph comprising a degasser, a quaternary pump, a light-tight autosampler unit set, a thermostated column compartment, and a UV/VIS detector. Agilent ChemStation software, the statistical software Prism4, version 5.0 , and IBM® SPSS® Statistics were used for the analysis of the results. The difference in plasma concentration of donepezil after 12 h  and after 24 h (29.38 ± 1.71 ng/ml) was nonsignificant. In contrast, the donepezil concentration in the cerebrospinal fluid was significantly higher in the 24-h interval (7.54 ± 0.55 ng/ml) compared with the 12-h interval . Based on these data, it is plausible to predict that donepezil might produce a stronger AChE inhibition in the brain at 24 h compared with 12 h following the administration. This information may help physicians individually adjust the time of drug administration in the patients according to time course of the disease symptoms.Although some studies have described the pharmacokinetics and pharmacodynamics of donepezil in the peripheral compartment, studies focused on drug transport across the blood–brain barrier are still very rare. To our knowledge, the fluctuation in the cerebrospinal fluid concentration of donepezil after administration of the drug has not been described in the literature so far. We recruited 16 patients regularly taking a standard therapeutic dose of donepezil (10 mg per day). All patients (Caucasian race) were treated for at least three months with a stable dose of 10 mg per day prior to sample collection. Patients were divided into two groups depending on the time of plasma and cerebrospinal fluid sampling: 12 h ( Alzheimer’s disease (AD) is a progressive neurodegenerative disorder affecting a significant proportion of the elderly population. With respect to continual increase of life expectancy, AD is expected to affect more than 100 million individuals worldwide in the near future  and 24 h  following donepezil administration.Sixteen patients (Caucasian race) who regularly use a standard therapeutic dose of donepezil (10 mg per day) were recruited into the study. All patients were treated for at least 3 months with a stable dose of 10 mg per day prior to sample collection. The patients were randomly divided into two groups depending on the time of plasma and CSF sampling: 12 h  measurement were purchased from Sigma-Aldrich  and were of the best available quality: acetonitrile, gradient grade; LiChrosolv in analytical grade; n-hexane and 1-butanol in gradient grade. All other reagents used in analytical methods and sample preparation were of analytical grade. The water used in the study was double-distilled and was of deionised HPLC grade.The samples were analysed on an Agilent 1260 Series liquid chromatograph  composed of a degasser, a quaternary pump, a light-tight autosampler unit set, a thermostated column compartment, and a UV/VIS detector. Agilent ChemStation software  and the statistical software Prism4  were used for data analysis.An analytical column  was used for the analysis. The mobile phase contained acetonitrile and 50 mM sodium dihydrogen phosphate . The pH was adjusted to 3.1 with phosphoric acid  to 1 ml plasma or CSF; subsequently, the samples were intensely shaken for 5 min and centrifuged at 11,000×Calibration standards were established using blank human plasma or human CSF supplemented with donepezil in a range of concentrations ; see Fig. r2 > 0.9967 in plasma; r2 > 0.9983 in CSF) between the peak areas and calibration standards in the range of 3.125–100 ng/ml in the plasma and CSF.Regression analysis of the relationship between peak areas and theoretical concentrations of calibration standards was performed with the least-squares method using Prism4 . Endogenous compounds in the plasma and CSF were efficiently separated from donepezil by sample preparation and elution for 23 min. The donepezil concentration exhibited a linear relationship (n = 4) on the same day. Inter-day precision was determined by calculating the means of four replicates of samples spiked with donepezil  on 3 subsequent days.Intra-day precision was determined by repeating the measurement of spiked samples ten times with five different concentrations of plasma and CSF  of donepezil are summarised in Table Donepezil in plasma samples  was quantified once a day on 10 different days. The accuracy was 98.4 % (6.25 ng/ml), 98.7 % (12.5 ng/ml), 98.9 % (25 ng/ml), 99.3 % (50 ng/ml), and 99.7 % (100 ng/ml), respectively.Donepezil in CSF samples at the same concentrations was quantified in the same way. The accuracy was 98.1 % (6.25 ng/ml), 98.3 % (12.5 ng/ml), 99.0 % (25 ng/ml), 99.0 % (50 ng/ml), and 99.1 % (100 ng/ml), respectively.LOQ was calculated as the lowest concentration of samples which were measured with a precision of 20 % and a relative error of ±20 %. The LOD was derived from LOQ according to the equation LOD = (3.3 × LOQ)/10.The donepezil LOQ in plasma samples was 1.56 ng/ml and the LOD was 0.52 ng/ml. The donepezil LOQ in CSF samples was 3.13 ng/ml and the LOD was 1.03 ng/ml.The concentrations of donepezil in human plasma and human CSF were calculated from the areas under the peaks using the calibration curve. The calibration curve was calculated by Prism4 statistical software .® SPSS® Statistics. The mean and SEM (Standard Error of the Mean) were calculated for plasma and CSF concentrations of donepezil. Difference between sampling intervals  were evaluated via nonparametric Mann–Whitney U test, and the differences were considered significant at a significance level 2α = 0.05.Statistical analysis was performed using software IBMSixteen patients regularly taking a standard therapeutic dose of donepezil (10 mg per day) were recruited into this clinical study. The patients were randomly divided into two groups depending on the time of plasma and CSF sampling: 12 or 24 h after donepezil administration. Differences in plasma and CSF donepezil concentrations were found between the 12- and 24-h groups, and the concentration changes are summarised in Table U = 22.0, Z = −1.006, p = 0.35). In contrast, elevated CSF concentration of donepezil was found after 24 h (7.54 ± 0.55 ng/ml) compared with 12-h interval , and this effect was statistically significant . No correlation between plasma and CSF concentrations of donepezil was found.The mean plasma concentration of donepezil was subjectively elevated after 12 h (39.99 ± 5.90 ng/ml) compared with 24-h interval (29.38 ± 1.71 ng/ml), although this difference was not statistically significant , respectively.The profile of donepezil with respect to its absorption, metabolic pathways, drug–drug interaction, and clearance is well established Seltzer . Hence, Modern imaging methods showed that the dose of donepezil 5 and 10 mg inhibited cortical AChE activity by only 20–40 % in vivo . According to previously published studies, P-gp (P-glycoprotein) is an important determinant for the disposition of donepezil, influencing its efflux from the central compartment to the blood stream . On the other hand, donepezil concentration measured by the selected method was well detected also in low concentrations. Despite these limitations, our study is a original work focused on donepezil concentrations in the CSF between doses. Further studies with larger numbers of patients are necessary to verify our results.The limitation of this study is relatively small sample size due to the difficulty of recruiting patients consenting to undergo the invasive lumbar puncture procedure. In addition, the compliance of patients could not be reliably assessed. Also, we expected to find higher concentrations of donepezil in plasma and CSF . Based on CSF data, it is plausible to predict that donepezil might produce a stronger AChE inhibition in the brain at 24 h compared with 12 h following the administration. This information may help physicians individually adjust the time of drug administration in the patients according to time course of the disease symptoms.This study supported the use of higher doses of donepezil in the treatment of AD, since its concentration in the CSF is relatively low and unstable throughout the whole day. The CSF concentration of donepezil achieved 25.97 ± 2.09 % of plasma concentration in 24 h and only 11.25 ± 1.18 % in 12 h compared with actual plasma concentrations (


Red blood cells are the most abundant cells in the blood that deliver oxygen to the whole body. Erythropoietin (EPO), a positive regulator of erythropoiesis, is currently the major treatment for chronic anemia. Granulocyte colony-stimulating factor (G-CSF) is a multifunctional cytokine and a well-known regulator of hematopoietic stem cell proliferation, differentiation, and mobilization. The use of EPO in combination with G-CSF has been reported to synergistically improve erythroid responses in a group of patients with myelodysplastic syndromes who did not respond to EPO treatment alone; however, the mechanism remains unclear.C57BL/6 J mice injected with G-CSF or EPO were used to compare the erythropoiesis status and the efficiency of erythroid mobilization by flow cytometry.In this study, we found that G-CSF induced more orthochromatophilic erythroblast production than did EPO in the bone marrow and spleen. In addition, in contrast to EPO treatments, G-CSF treatments enhanced the efficiency of the mobilization of newly synthesized reticulocytes into peripheral blood. Our results demonstrated that the effects of G-CSF on erythropoiesis and erythrocytic mobilization were independent of EPO secretion and, in contrast to EPO, G-CSF promoted progression of erythropoiesis through transition of early stage R2 (basophilic erythroblasts) to late stage R4 (orthochromatophilic erythroblasts).We demonstrate for the first time that G-CSF treatments induce a faster erythropoiesis-enhancing response than that of EPO. These findings suggest an alternative approach to treating acute anemia, especially when patients are experiencing a clinical emergency in remote areas without proper blood bank supplies. Red blood cells (RBCs) are the most abundant cells in the blood and are essential for oxygen transport around the body. After birth, the site of erythropoiesis switches from the fetal liver to the bone marrow (BM) and spleen. In humans, the BM is the major site for steady-state erythropoiesis. In contrast, in mice, in addition to the BM, the spleen plays a minor role 10%) in steady-state erythropoiesis 0% in ste. Under sAnemia can develop from loss of RBCs, a reduction in RBC production, increased destruction of RBCs, or a shorter RBC lifespan. The World Health Organization defines anemia as a hemoglobin level lower than 12 g/dl in women and lower than 13 g/dl in men , 15. OthOur previous study found that granulocyte colony-stimulating factor (G-CSF) could mobilize newly synthesized erythrocytes to the peripheral blood (PB) and promote erythrocytic differentiation and proliferation in vitro and ex vivo . The preB. anthracis lethal toxin (LT) was provided by the Institute of Preventive Medicine, National Defense Medical Center , and purified as previously described [escribed . EGFP miescribed  and C57BC57BL/6 J mice  were retro-orbitally injected with 2 IU/g rhEPO  or 55 μg/kg recombinant human G-CSF  once daily on 3 consecutive days. Animals treated with an identical volume of saline served as negative controls. BM cells were isolated as previously described . SpleensEnhanced green fluorescent protein (EGFP) mice  were injected with 55 μg/kg G-CSF or 2 IU/g EPO retro-orbitally once daily on 3 consecutive days. PB was collected retro-orbitally at 20, 40, and 60 h after initial injection. Cells were incubated with rat anti-mouse APC-conjugated TER-119 antibody at 37 °C for 1 h and subsequently with 5 μM of RNA-selective dye F22 [C57BL/6 J mice  were treated with 55 μg/kg G-CSF administered by retro-orbital injection once daily on 2 consecutive days for the EPO immunoassay or 5 consecutive days for the soluble P-selectin immunoassay. Animals treated with an identical volume of saline served as negative controls. C57BL/6 J mice that were retro-orbitally injected with 1.5 mg/kg LT and untreated served as positive controls for the EPO immunoassay and the soluble P-selectin immunoassay, respectively. PB (serum for the EPO immunoassay and plasma for the soluble P-selectin immunoassay) was collected retro-orbitally at 22, 44, and 66 h after initial injection for the EPO immunoassay, and day 0 before initial injection and days 1–5 after initial injection for the soluble P-selectin immunoassay. The EPO and soluble P-selectin immunoassays were performed using an enzyme-linked immunosorbent assay (ELISA) in accordance with the manufacturers’ instructions .C57BL/6 J mice  were treated with 55 μg/kg G-CSF or 1 mg/kg recombinant mouse P-selectin  once daily on 2 consecutive days through retro-orbital injection. The hematopoietic parameters were measured on day 2 after initial injection using an automated hematology analyzer .t test using the SPSS software, version 17.0 . P values lower than 0.05 indicated significant differences.All quantifiable data are presented as mean ± standard deviation (SD). Statistical analysis was conducted by one-way analysis of variance (ANOVA) followed by the post-hoc Bonferroni-corrected high/TER-119med), basophilic erythroblasts , late basophilic and polychromatophilic erythroblasts , and orthochromatophilic erythroblasts   [To compare the erythropoiesis status after G-CSF and EPO treatments, C57BL/6 J mice were retro-orbitally injected with G-CSF or EPO once daily for 3 consecutive days. Erythropoiesis progression was analyzed in the BM Fig.  and spleh) Figs.  and 2b [Figs. −)  than EPOThe spleen is a secondary organ for erythropoiesis but not for granulopoiesis –4, 25. Ihigh/TER-119high) were higher at 20 h under G-CSF treatment than under EPO, and were similar between G-CSF and EPO treatments at 40 and 60 h after initial injection  to a greater extent than EPO did  . The samThis study demonstrated that stimulation of erythropoiesis in mice with G-CSF is different to that with EPO. Treatment with G-CSF elicited greater R4 erythroblast production than did treatment with EPO in the BM and spleen. Additionally, G-CSF induced higher mobilization levels of newly synthesized reticulocytes from the BM and spleen to the PB than EPO did.G-CSF has been a US Food and Drug Administration (FDA)-approved drug for treating various hematopoietic defects for many years. In contrast to EPO, elicitation of neutralizing antibodies after G-CSF treatments has not been reported. Myelodysplastic syndromes (MDS) are a diverse group of diseases characterized by ineffective hematopoiesis and peripheral cytopenia with unknown mechanisms , 35. As In conclusion, although G-CSF combined with EPO has been administered to treat anemia in patients with MDS, aplastic anemia, and HIV for many years , 46, 47,


Obesity has been linked to several types of cancers including ovarian cancer. Retinol binding protein 4 (RBP4) is an adipokine that drives the development of hyperinsulinemia and type II diabetes in obesity patients and animals. Previously, we have identified RBP4 as a serum marker for ovarian cancer. Here we further explored the consequence of RBP4 upregulation in ovarian cancer cells and its molecular mechanism.Our results show that RBP4 is overexpressed in ovarian cancer cells to the same extent as in adipose tissues. The overexpression of RBP4 in ovarian cancer cells promotes cancer cell migration and proliferation. At molecular level, cancer progression factors MMP2 and MMP9 are induced in response to RBP4 overexpression. We further investigated which signaling pathways are utilized by RBP4 to activate ovarian cancer cell migration. We found RhoA/Rock1 pathway is turned on and CyclinD1 is upregulated in RBP4 overexpressed cells. Inhibition of RhoA/Rock1 pathway reduces the RBP4-induced MMP2 and MMP9 expression. The RBP4 action is depend on its associated ligand vitamin A/retinol acid (RA) and possibly involves similar pathways as for conferring insulin resistance. Moreover, we show that knockdown of RBP4 significantly reduce cancer cell migration and proliferation as well as expressions of oncogenic factors.Our results indicated that RBP4 can drive ovarian cancer cell migration and proliferation via RhoA/Rock1 and ERK pathway. It suggests that RBP4 act as a oncogene in ovarian cancer cells. Thus, RBP4 could be a molecular bridge between obesity and cancers and a potential target for treating obese cancer patients. Obesity is a well-established cancer risk factor and its occurrence is strongly associated with several types of cancers, including breast, colon, endometrial, ovarian, gastric, pancreatic and liver cancers , 2. HoweRetinol binding protein 4 (RBP4) is secreted by liver and adipose tissues , 4. RBP4Besides its transportation function, RBP4 has recently been recognized as an adipokine –15. SeveOvarian cancer is the most lethal type of gynecological cancer in the world . OvarianWe here investigated whether RBP4 is a tumorgenic factor that connects obesity and ovarian cancer. Our data showed that RBP4 was up-regulated in ovarian cancer cells and overexpression of RBP4 promoted cancer cell migration. The MMP-2 and MMP-9, key factors in cancer metastasis, were induced by RBP4 overexpression. We further identified RhoA/Rock1 pathway as mediators for RBP4 action. RhoA and Rock1 were overexpressed in response to RBP4 and inhibition of RhoA/Rock1 reduced MMP-2 and MMP-9 expression. The RBP4 action was dependent on its associated ligand RA. Moreover, knockdown of RBP4 greatly reduced cancer migration. Our data not only established RBP4 as a direct linkage between obesity and ovarian cancer, but also suggested RBP4 was a possible target for cancer treatment, especially in those associated with obesities.This study was approved by the Medical Ethical Committee of Beijing Chaoyang Hospital . The written informed consents were obtained from all the participants enrolled in the study. Specimens were sampled from patients undergoing surgery for ovarian carcinoma or benign ovarian tissues at the Beijing Chaoyang Hospital.All procedures were approved by the Animal Care and Use Committee of Beijing Chaoyang Hospital . All experiment methods were performed in accordance with the relevant guidelines and regulations. In brief, healthy specific-pathogen-free (SPF) male SD rats were purchased from the Vital River. All rats were preserved under standard housing laboratory conditions. After one week of adaptation to the diet and the new environment, female SD rats were divided into two diet groups: the normal control (NC) group fed ad libitum a standard rodent chow, the high-fat (HF) group fed ad libitum a high-fat chow. After six weeks to induce obesity, the ovarian tissues were obtained after euthanasia.Antibodies against RBP4 (#ab109193), actin (#ab8226), RhoA (#ab187027), p-RhoA (#ab41435), ROCK1 (#ab45171), Erk (#ab54230), p-Erk (#ab51100), Cyclin D1 (#ab134175), MMP2 (#ab37150), MMP9 (#ab38898) were obtained from Abcam, USA. ROCK1 inhibitor Y27632 was purchased from Sigma, USA. All primers for qPCR were ordered from Invitrogen .2 at 37 °C.Human ovarian cancer cell line A2780 was obtained from Prof. Haiteng Deng laboratory , 29. SKOTo upregulate the expression of RBP4, the human RBP4 full length cDNA was amplified and inserted into the pCMV-Flag vector, and a scramble sequence was inserted into the pCMV-flag vector as the control vector. To knock down the expression of RBP4, a RBP4 siRNA was designed and obtained from Jima Inc. . For transfection, the cells were seeded into 6-well plates. When cell confluency reached 50%, RBP4 siRNA or RBP4-pCMV-Flag was transfected into the cells using lipo2000 according to the manufacturer’s instructions.Cells were lysed in RIPA buffer with protease and phosphatase inhibitor cocktail. Equal amount of protein samples was loaded onto 12% SDS-PAGE and was then transferred to PVDF membranes. After blocking with 5% BSA for 1 h at room temperature, the membranes were incubated with primary antibodies at 4 °C overnight. Then, the membranes were incubated with horseradish peroxidase-conjugated secondary antibody  for 1 h at room temperature. The protein bands were visualized by ChemiDoc XRS+ . Data analysis was done using Quantity one.−ΔΔCt method. Each experiment was performed in triplicate. The primers used in real-time PCR were as follow: RBP4 F: AGGAGAACTTCGACAAGGCTC; RBP4 R: GAGAACTCCGCGACGATGTT; GAPDH F: GGAGCGAGATCCCTCCAAAAT; GAPDH R: GGCTGTTGTCATACTTCTCATGG; RHOA F: AGCCTGTGGAAAGACATGCTT; RHOA R: TCAAACACTGTGGGCACATAC; Rock1 F: AACATGCTGCTGGATAAATCTGG; Rock1 R: TGTATCACATCGTACCATGCCT; cyclinD1(CCND1) F: GCTGCGAAGTGGAAACCATC; cyclinD1(CCND1) R: CCTCCTTCTGCACACATTTGAA; ERK1 F: CTACACGCAGTTGCAGTACAT; ERK1 R: CAGCAGGATCTGGATCTCCC; MMP2 F: TACAGGATCATTGGCTACACACC; MMP2 R: GGTCACATCGCTCCAGACT; MMP9 F: TGTACCGCTATGGTTACACTCG; MMP9 R: GGCAGGGACAGTTGCTTCT.Total RNA was isolated from the cells using trizol method. RNA was reverse transcribed using the PrimeScript RT Master Mix  according to the manufacturer’s instructions. The PCR amplifications were performed using SYBR Premix Ex Taq II . The expression level of each sample was internally normalized against that of the glyceraldehyde 3-phosphate dehydrogenase (GAPDH). The relative quantitative value was calculated using 2μm thick. RBP4 antibody was applied on the sections for 30 min and incubated overnight at 4 °C then shaking at room temperature for 30 min. Antibody binding was amplified using biotin and streptavidin HRP for 10 min each and the complex was visualized using DAB. ALL sections were assessed microscopically for positive DAB staining. The immunostained sections were examined under microscopy and the expression level of RBP4 was scored on the basis of the intensity of staining.The expression of RBP4 was assessed using immunohistological staining as described previously . BrieflyA 24-well Transwell chamber  was used to examine the invasive ability of the ovarian cancer cells. Cells were suspended in DMEM medium and were added into the upper Transwell chamber. The lower Transwell chamber was filled with DMEM medium supplemented with 10% FBS. After incubation of 16 h at 37 °C, the non-migrated cells were removed with a sterile cotton swab, and the migrated cells were stained with 0.1% crystal violet for 20 min at room temperature. The numbers of cells were calculated under a light microscope in five random fields.Cell proliferation was determined by MTT assay. Cells were seeded at 1000 cells/well in a 96-well plate. After incubation for indicated time, MTT was added into the plate incubated for 4 h. The optical density (OD) was measured 490 nm at designated time.5 cells / well were seeded in 6-well plates. The cells were then harvested, fixed with 70% ice cold ethanol, and stored at 4 °C until analysis. After fixation, the cells were washed twice with cold phosphate-buffered saline (PBS) and centrifuged, following which the supernatants were removed. The pellet was resuspended and stained with PBS containing 50 μg/ml PI and 100 μg/ml RNaseA for 20 min in the dark. The cell cycle data were analyzed using Modifit software.Cell cycle distribution was analyzed by PI staining and flow cytometry. The 1 × 10P value of more than 0.01 was considered as statistical significance. Graphpad 5.0 software was used for all the statistical analyses.All the continuous variables were expressed as average ± standard deviation (SD). Student’s t-test was used for the difference analysis. A We first detected the RBP4 expression levels in ovarian cancer tissues. Western blot results showed that the RBP4 protein was upregulated by nearly 4-fold in ovarian cancer tissues comparing to the benign ovarian tissues Fig. . The higWe used ovarian cancer cell line A2780 and SKOV3 to test the effects of RBP4 expression on ovarian cancer. Firstly, we confirmed the effect of RBP4 overexpression and knocked down in A2780 and SKOV3 cells Fig. . Then thWe have shown that RBP4 overexpression can greatly stimulate ovarian cancer cell migration. Then, we tested the expression level of MMP2 and MMP9, which are essential for cancer metastasis . Both prTo explore how RBP4 exerts its effect on cancer cells, we tested the expression level of several key players in tumorigenesis related signaling. RBP4 overexpression stimulated the expression of both RhoA and Rock1. In RBP4 overexpression groups, the RhoA and Rock1 levels were elevated at both mRNA and protein level Fig. . MoreoveRock1 inhibitor Y-27632 had been shown to effectively inhibit Rock1 and its associated pathways . To testRBP4 is a major RA transport protein . We testObese patients are associated with high cancer risk, poor prognosis and reduced response to anti-cancer therapies , 2. ObesRBP4 and its associated RA have been shown to trigger several downstream pathways to confer insulin resistance –19. The Adding to the existing knowledge, we showed that RhoA/Rock1 pathway was turned on in response to RBP4 overexpression. RhoA/Rock1 pathway played pivot roles in cell morphogenesis, adhesion, and motility and was often activated in malignant cancers . PreviouWe observed that knockdown of RBP4 can greatly suppress ovarian cancer cell migration and proliferation. Considering RBP4 as a circulating protein, targeting RBP4 could be a relative easy option for ovarian cancer treatment, especially those associated with obesities.In conclusion, this study described the function of RBP4 in driving ovarian cancer cell migration and proliferation. Moreover, the underlying molecular mechanism of RBP4 was activation of RhoA/Rock1 pathway and CyclinD1 expression. Therefore, RBP4 could be a molecular bridge between obesity and cancers and a potential target for treating obese cancer patients.


Clinical outcome studies of schizoaffective disorder patients have yielded conflicting results. One reason is the heterogeneity of samples drawn from the schizoaffective disorder population. Here, we studied schizoaffective disorder patients who showed marked functional impairment and continuous signs of illness for at least 6 months .We assessed 176 chronic psychosis patients with a structured interview (SCID-IV-TR) and the Diagnostic Interview for Genetic Studies schizoaffective disorder module. We diagnosed 114 patients with schizophrenia and 62 with schizoaffective disorder. The two groups were similar with regard to age, gender, and race. We tested for group differences in antecedent risk factors, clinical features, and functional outcome.p < 0.01; more hospitalizations to prevent suicide, p < 0.01) and higher anxiety disorder comorbidity (p < 0.01).The schizoaffective disorder group differed from the schizophrenia group on two measures only: they showed higher rates of suicidality (more suicide attempts, When schizoaffective disorder patients meet DSM criteria B and C for schizophrenia, they resemble schizophrenia patients on several measures used to assess validity. The increased rate of anxiety disorders and suicidality warrants clinical attention. Our data suggest that a more explicit definition of schizoaffective disorder reduces heterogeneity and may increase validity. A recent meta-analysis of 50 studies comparing illness course and clinical outcomes in schizoaffective disorder, schizophrenia, and affective disorders reports the use of 10 different sets of diagnostic criteria for schizoaffective disorder .Schizophrenia and schizoaffective disorder are defined by three domains of psychopathology: psychosis, mood symptoms, and functional impairment. Figure By contrast, the schizoaffective disorder population occupies a considerably larger space, including patients with no functional impairment and with lower levels of psychosis. Within these differently sized spaces, we diagnose schizophrenia three times more often than schizoaffective disorder . The resIn an attempt to reduce diagnostic heterogeneity, Williams and McGlashan employed a stringent definition of schizoaffective disorder: patients met DSM-III diagnostic criteria for both schizophrenia and an affective disorder . Their sHere, we build on the study by Williams and McGlashan by comparing patients who met DSM-IV-TR criteria of schizoaffective disorder and schizophrenia, respectively. While the DSM-IV-TR diagnoses schizophrenia and schizoaffective disorder share criterion A , the schizophrenia criterion B  and criterion C (continuous signs for at least 6 months) are permissible, but not required for a diagnosis of schizoaffective disorder Table . This reIn this study, we compared a group of schizoaffective disorder patients, who met DSM-IV-TR diagnostic criteria for schizoaffective disorder and also met DSM criteria B and C for schizophrenia, with a group of schizophrenia patients. The two groups were comparable in terms of sex, age, race, and duration of illness. We hypothesized that the groups would not differ with regard to antecedent risk factors, clinical features, and functional outcome.The sample for this study was taken from an ongoing data repository at Vanderbilt University Medical Center, the Psychiatric Genotype/Phenotype Project. Subjects were recruited from inpatient units  and the outpatient clinic  of an academic medical center, and a local community mental health clinic with many Medicaid/Medicare patients . Subjects with a schizophrenia or schizoaffective disorder diagnosis were identified by medical record review and approached for participation in the repository. All patients enrolled were free of significant head injury, major medical or neurological illness, and active substance abuse or dependence. All study procedures were approved by the Vanderbilt University Institutional Review Board , and written consent forms accompanied by comprehensive explanations of all study procedures were provided. Subjects were given monetary compensation for their participation.N = 114) or schizoaffective disorder (N = 62) in the repository. Details concerning diagnostic criteria and review are outlined below.The sample for the present study included all subjects who met diagnostic criteria for schizophrenia .Diagnostic and Statistical Manual of Mental Disorders, Fourth Edition, Text Revision (SCID-IV-TR)  .The reliability of the DSM-IV-TR schizoaffective disorder diagnosis is low, largely due to criterion C  , which hAll of our schizophrenia and schizoaffective disorder patients met DSM-IV-TR criteria A–C for schizophrenia. All patients met not only the DSM-IV-TR criterion A but also the DSM-5 criterion A for schizophrenia, i.e., they presented with at least two criteria A, at least one being delusions, hallucinations, or disorganized speech. Criterion B  was defined as having functioning “markedly below the level achieved prior to the onset” in at least one of the following areas: (a) work, (b) personal relations, or (c) self-care. We considered an additional fourth area, (d) receiving social security disability payments for psychiatric illness, when making these judgments .Distress or impairment is also a diagnostic criterion for a major depressive episode (DSM-IV-TR and DSM-5 criterion B) or manic episode (DSM-IV-TR and DSM-5 criterion C). However, the functional impairment in schizophrenia is more severe and of longer duration, leading to a level of functioning below the level achieved prior to the onset. All schizoaffective disorder patients in this study had significant impairment in at least one of the four impairment areas, and over half had impairment across all areas Table , making For further characterization of the patient sample and to assess dimensions of current psychopathology, we complemented the SCID interview with the Global Assessment of Functioning (GAF) scale, the 17-item Hamilton Depression (HAM-D) rating scale, the Young Mania Rating Scale (YMRS), and the Positive and Negative Syndrome Scale (PANSS). We used the Wechsler Test of Adult Reading (WTAR) to assess premorbid IQ as an antecedent factor and the Screen for Cognitive Impairment in Psychiatry (SCIP) for the assessment of current cognitive function. We assessed functional outcome by collecting information about marital status, employment history, disability status, number of hospitalizations, and number of arrests during the SCID interview.U tests to assess all continuous variables . Pearson chi-square analyses were used to assess categorical variables .We tested for group differences between the schizophrenia and schizoaffective disorder patients. Kolmogorov–Smirnov, skewness, and Kurtosis tests for normality revealed that a number of our continuous variables were not normally distributed. We used non-parametric Mann–Whitney U = 2,403.50, p < 0.01) , and years of education  . Patients with schizoaffective disorder also reported more suicide attempts  and more hospitalizations to prevent suicide .Both groups displayed moderate degrees of psychosis (PANSS scores) and few signs of mania (YMRS scores). The schizoaffective disorder patients were more depressed than the schizophrenia patients  = 0.54, p > 0.05], but the schizoaffective disorder group showed a higher rate of anxiety disorders , particularly PTSD .Both groups showed similar rates of substance use disorders comorbidity , number of arrests , percentage of patients employed , percentage qualifying for social security disability , or number of hospitalizations .There was no difference between the two groups on a variety of measures of functional or clinical outcome, including history of marital status [χPrevious comparisons of schizoaffective disorder and schizophrenia have been hampered by significant heterogeneity of the schizoaffective disorder samples. Here, we present results from a comparison of patients who met DSM criteria A–C for schizophrenia but differed with respect to meeting DSM criteria for schizoaffective disorder. When defined with this stringency, schizoaffective disorder and schizophrenia show similarities in antecedent risk factors, clinical features, and functional outcome. However, the schizoaffective disorder patients were more depressed, reported greater suicidality, and showed increased rates of anxiety disorders, especially PTSD. Trauma shapes the phenotype of psychosis , but wheThese results differ from the conventional view that schizoaffective disorder is a more benign clinical condition than schizophrenia . But theSchizoaffective disorder has been defined in the context of a categorical diagnostic system, but there is compelling reason to redefine the disorder in the context of a dimensional system . The twoSchizoaffective disorder is a common clinical diagnosis , despiteWe were able to examine only one subgroup of schizoaffective disorder patients, which is a significant limitation of this study. When reviewing our cohort of chronic psychosis patients eligible for inclusion in the study, only 1 of 177 met schizoaffective disorder criteria but not schizophrenia criteria B and C. This might indicate a greater prevalence of the schizoaffective disorder subpopulation examined here; however, we need to consider an ascertainment bias since our patient sample was primarily recruited from psychiatric inpatient units. It is an essential next step to compare schizoaffective disorder patients, who do not meet schizophrenia B and C criteria, with the two groups considered here and with a group of psychotic bipolar disorder patients. Another limitation of our study is the narrow set of clinical and functional outcome data. Future studies should explore the antecedent, concurrent, and predictive validators of schizoaffective disorder more thoroughly.Schizoaffective disorder has been considered a better outcome diagnosis when compared with schizophrenia, but the literature has been inconclusive. The present study supports the need for a more detailed clinical characterization of schizoaffective disorder patients for treatment and endophenotype studies, thus reducing heterogeneity and increasing reliability and validity.This study was carried out in accordance with the recommendations of the Vanderbilt University Institutional Review Board with written informed consent from all subjects. All subjects gave written informed consent in accordance with the Declaration of Helsinki. The protocol was approved by the Vanderbilt University Institutional Review Board.KS, KA, and SH conceptualized the study. KS setup and performed all data analyses; wrote the original draft of and prepared the manuscript. SH, MS, and KA edited the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The reviewer R-DS and handling Editor declared their shared affiliation, and the handling Editor states that the process nevertheless met the standards of a fair and objective review.


Brucellosis is a zoonotic bacterial infection which is transmitted to humans from infected animals and is endemic in many parts of the world including Saudi Arabia. In this article, we report a case of occupational neurobrucellosis that presented with a space-occupying lesion mimicking a brain tumor. We stress on the importance of obtaining detailed social history including occupation to reach the diagnosis in several conditions including brucellosis. We also stress on taking universal precautions when handling any specimens. It may be advisable that manipulation of all unknown specimens arriving at the laboratory should occur in biological safety cabinet until a highly infectious organism is ruled out. Neurobrucellosis should be included in the differential diagnosis in patients presenting with solitary mass lesion mimicking brain tumor especially in endemic areas or high occupational risk group. Brucella was first identified from autopsy material of a patient who died on the island of Malta in 1887 by David Bruce. Nine years later, neurobrucellosis was first reported by Matthew Hughes [Brucellosis is a zoonotic bacterial infection which is transmitted to humans from infected animals and is endemic in many parts of the world including Saudi Arabia. It is a multisystem disease that may present with a broad spectrum of clinical manifestations including undulant fever and musculoskeletal symptoms and signs . Brucellw Hughes . The het Brucella melitensis immunoglobulins were both high measuring IgM 12.2 U/ml and IgG 127.4 U/ml . Using enzyme-linked immunosorbent assay (ELISA) for cerebrospinal fluid (CSF), Brucella titer was high at 1 : 40 . CSF analysis showed lymphocytic pleocytosis at 63 cells and 88% lymphocytes. Protein was slightly increased, but glucose was normal. Her bacterial culture was negative for both aerobic and anaerobic organisms. Polymerase chain reaction (PCR) for tuberculosis, herpes simplex virus, Epstein–Barr virus, and cytomegalovirus was negative. Both CSF Brucella total antibodies and oligoclonal bands were positive with five well defined gamma restriction bands which were not present in the corresponding serum sample. Magnetic resonance imaging (MRI) of the brain revealed peritrigonal  deep white matter mass measuring 2.6 × 3.5 cm with surrounding vasogenic edema. There was no restricted diffusion with slightly increased flow at the affected area and normal cerebral blood volume on MR perfusion. Axial and sagittal T1 postgadolinium studies showed minimal enhancement  followed by abortus and suis. Laboratory infection could be due to accidents, direct contact, inoculation through needle-stick injuries, and contamination of skin and mucous membranes through spills or splashes into eyes, mouth, or nose. Many of the exposures were caused by handling specimens where brucellosis was not suspected clinically [inically .Communication between clinicians and laboratory workers is important to help the laboratory staff take extra precautions during the identification of a specimen. It may be advisable that manipulation of all unknown specimens arriving at the laboratory should occur in biological safety cabinet until a highly infectious organism is ruled out . In our Brucellosis is a systemic disease that may involve almost every organ system. The exact mechanism by which the organism reaches the nervous system is still unclear. Involvement of the central nervous system (CNS) has only been detected in 3–5% of patients. Once bacteremia occurs, the organism travels to the meninges producing polyradiculoneuropathy, meningitis, or meningoencephalitis. One of the rare consequences of the direct deleterious effect of the organism invading the CNS is the occurrence of a mass lesion or brain abscess which can be documented radiologically and pathologically. Another possible mechanism is an immune-mediated damage of nervous tissue due to the release of circulating endotoxins or to the immunological and inflammatory reaction of the host to the presence of these organisms within the nervous system or other tissues of the body [Clinical features of neurobrucellosis include back pain, areflexia, paraparesis, cranial nerve involvement, myelitis, and meningovascular involvement including strokes, neuropathy, or depression. The vestibulocochlear nerve has been reported to be the most commonly affected cranial nerve where the patients present with vestibuloacoustic neuritis or hearing loss . Brucella isolation from the CSF (<20%) and the long time needed for the results had made it suboptimal. Therefore, the diagnosis usually relies on the detection of antibodies to Brucella lipopolysaccharide (LPS) in CSF by agglutination tests (positive Wright's agglutination or Coombs' test at C1:160 titers) or ELISA. Although brain biopsy is the gold standard investigation to diagnose several conditions, clinical examination and paraclinical tests including serology might score the diagnosis and avoid performing this invasive procedure. Imaging findings can range from normal imaging to inflammatory changes , white matter, or vascular changes [Laboratory tests conducted for the diagnosis of neurobrucellosis include blood and CSF cultures, serum and CSF agglutination tests, and ELISA. Examination of the CSF typically reveals an elevated protein concentration, a normal or slightly depressed glucose concentration, and a moderate lymphocytic pleocytosis. Although positive blood and CSF cultures are the gold standard for diagnosis, the low rate of changes .To date, neurobrucellosis presenting with a space-occupying mass mimicking a cerebral tumor has been documented in only three patients –10. BactThe treatment of neurobrucellosis is still controversial, and no consensus regarding the best treatment has been established. The primary drugs of choice due to their enhanced CNS penetrance, tolerability, and high gastrointestinal absorption are doxycycline, rifampicin, sulfamethoxazole-trimethoprim, ciprofloxacin, and ceftriaxone. In addition to neurotoxicity, streptomycin has low CSF penetrance and has been accused of being inappropriate. Treatment duration in neurobrucellosis is suggested to be several months (a minimum of 6–8 weeks) depending on the patient's response and should generally be continued until the CSF analysis has returned to normal and the MRI abnormalities disappear .We stress on the importance of obtaining detailed social history including occupation to reach the diagnosis in several conditions including brucellosis. We also stress on taking universal precautions when handling any specimens. It may be advisable that manipulation of all unknown specimens arriving at the laboratory should occur in biological safety cabinet until a highly infectious organism is ruled out. Neurobrucellosis should be included in the differential diagnosis in patients presenting with solitary mass lesion mimicking brain tumor especially in endemic areas or high occupational risk group.


In the present study, CFLs harvested from cadavers were categorized according to the differences in the angle of the CFL with respect to the long axis of the fibula and their shape, and then three-dimensional reconstructions of the CFLs were used to simulate and examine the differences in the angles of the CFLs with respect to the long axis of the fibula and how they affect CFL function.The study sample included 81 ft from 43 Japanese cadavers. CFLs were categorized according to their angle with respect to the long axis of the fibula and the number of fiber bundles. Five categories were subsequently established: CFL20° (angle of the CFL with respect to the long axis of the fibula from 20° to 29°); CFL30° (range 30–39°); CFL40° (range 40–49°); CFL50° (range 50–59°); and CFL2 (CLFs with two crossing fiber bundles). Three-dimensional reconstructions of a single specimen from each category were then created. These were used to simulate and calculate CFL strain during dorsiflexion (20°) and plantarflexion (30°) on the talocrural joint axis and inversion (20°) and eversion (20°) on the subtalar joint axis.In terms of proportions for each category, CFL20° was observed in 14 ft (17.3%), with CFL30° in 22 ft (27.2%), CFL40° in 29 ft (35.8%), CFL50° in 15 ft (18.5%), and CFL2 in one foot (1.2%). Specimens in the CFL20° and CFL30° groups contracted with plantarflexion and stretched with dorsiflexion. In comparison, specimens in the CFL40°, CFL50°, and CFL2 groups stretched with plantarflexion and contracted with dorsiflexion. Specimens in the CFL20° and CFL2 groups stretched with inversion and contracted with eversion.CFL function changed according to the difference in the angles of the CFLs with respect to the long axis of the fibula. Many studies have investigated the functional role of the CFL in addition to the ATFL because the ATFL and CFL are largely involved in ankle inversion restriction , 2. HoweFor example, Ozeki et al. examined cadavers to explore the functional role of the CFL and observed that the CFL becomes taut at dorsiflexion angles ≥18°, and it is nearly relaxed at other angles . FurtherAnalyzing data from autopsies and operations, Ruth identified four categories of CFLs based on angles relative to the long axis of the fibula and morphology: 0° 14 ft, 18.7%), 10–45° , 80–90° , and fan-shaped CFLs  .7%, 10–4, but theTherefore, In the present study, CFLs harvested from cadavers were categorized according to the differences in the angle of the CFL with respect to the long axis of the fibula and the shape, and then three-dimensional reconstructions of the CFLs were used to simulate and examine the differences in the angles of the CFLs with respect to the long axis of the fibula and how they affect CFL function.A total of 81 legs from 43 Japanese cadavers  that had been switched to alcohol after placement in 10% formalin were examined. None showed signs of previous major surgery around the ankle. This study was approved by the Ethics Committee at our institution., Taiwan). Five categories were established: CFL20° (angle of the CFL with respect to the long axis of the fibula from 20° to 29°); CFL30° (range 30–39°); CFL40° (range 40–49°); CFL50° (range 50–59°); and CFL2 (CFLs with two crossing fiber bundles). All measurements were carefully done in an intermediate position of ankle plantarflexion/dorsiflexion of 0° and foot inversion/eversion of 0°. A single specimen from each category was then selected, and three-dimensional reconstructions were created from five feet, using the MicroScribe system  to digitize two points, the origin and terminus, of the CFL  dissected the CFL ligaments alone. The lower limbs were cut 10 cm above the knee to produce isolated specimens. The CFLs were carefully dissected after removal of skin, subcutaneous tissue, muscle-tendon tissue, and crural fascia. With reference to a previous study , the CFLThe MicroScribe system is an instrument with high precision . However, measurements must be performed manually. In addition, although the study cadavers were thoroughly fixed to the examination table such that they did not move, it was necessary to test whether they had moved, since the measurements entail dissection of the ligament tissue. A previous study by the authors found the intraclass correlation coefficient  to be 0.97–0.99 , which iA chi-squared test with a significance level of 5% was used to compare CFL running angle differences between men and women and between left and right feet.Five categories were identified: CFL20° in 14 ft (17.3%); CFL30° in 22 ft (27.2%); CFL40° in 29 ft (35.8%); CFL50° in 15 ft (18.5%); and CFL2 in one foot (1.2%).p = 0.101).A comparison between males and females showed that, among men, 7 legs (8.6%) were CFL20°, 13 ft (16.1%) were CFL30°, 18 ft (22.2%) were CFL40°, 13 ft (16.1%) were CFL50°, and 0 ft (0%) were CFL2, while, among women, 7 ft (8.6%) were CFL20°, 9 ft (11.1%) were CFL30°, 11 ft (13.6%) were CFL40°, 2 ft (2.5%) were CFL50°, and 1 ft (1.2%) was CFL2. No significant differences were seen (p = 0.665).With respect to left-right differences, both legs could be measured in 37 cadavers . Among the right feet, 5 ft (6.7%) were CFL20°, 9 ft (12.2%) were CFL30°, 15 ft (20.3%) were CFL40°, 8 ft (10.8%) were CFL50°, and 0 ft (0%) were CFL2. Among the left feet, 7 ft (9.4%) were CFL20°, 11 ft (14.9%) were CFL30°, 11 ft (14.9%) were CFL40°, 7 ft (9.4%) were CFL50°, and 1 ft (1.4%) was CFL2. No significant differences were seen  with an angle respect to the long axis of the fibula between 10° and 50°, which was similar to the finding of 56 ft 74.7%) reported in the previous study [4.7% repoThe mean change in strain for all categories during ankle flexion and extension ranged from −1.3% to 0.3%, indicating that the CFL was not stretched for the most part. Previous studies using cadavers found that, during dorsiflexion and plantarflexion, the CFL was nearly relaxed , and it In regards to changes in strain for each category when 20° inversion and 20° eversion were applied during ankle flexion and extension, the means for all categories showed that the sample as a whole stretched with inversion in both dorsiflexion and plantarflexion. Past studies with frozen cadavers reported that the CFL is highly involved in limiting ankle inversion , 2. The In the present study, at 0° ankle flexion and extension, specimens displayed elongation with inversion and contraction with eversion, especially in the CFL20° and CFL2 categories. Sarrafian and Kelikian observed that, because the CFL terminus plane of motion is perpendicular to the subtalar joint axis of motion when the ankle is in the neutral position, the CFL will contract with subtalar inversion and stretch with subtalar eversion when the running angle is perpendicular to the fibula and show the exact opposite tendency when the angle of the CFL with respect to the long axis of the fibula is parallel to the fibula . AccordiThis study did have a number of limitations. First, it involved simulations with cadavers. Therefore, gravity, weight bearing, muscle activity, and the posture of the foot were not considered. In the future, we believe that it will be necessary to perform biomechanical research using our basic data with in vivo samples. Second, all cadavers used in this study were Japanese. It is not certain whether the present findings apply to cadavers from other ethnicities. Many studies have raised the possibility of skeletal muscle and tendon variations across ethnicities –23, and The findings of the present study suggest that CFL function may change in relation to the angle of the CFL with respect to the long axis of the fibula and morphologies. In the future, we believe that it will be necessary to perform biomechanical research using our basic data with in vivo samples which may lead to the elucidation of the functional role of CFL. Furthermore, we believe that it would be valuable to confirm whether individual anatomical differences may represent risk factors for CFL injury.


Luminescent gold nanocrystals (AuNCs) are a recently-developed material with potential optic, electronic and biological applications. They also demonstrate energy transfer (ET) acceptor/sensitization properties which have been ascribed to Förster resonance energy transfer (FRET) and, to a lesser extent, nanosurface energy transfer (NSET). Here, we investigate AuNC acceptor interactions with three structurally/functionally-distinct donor classes including organic dyes, metal chelates and semiconductor quantum dots (QDs). Donor quenching was observed for every donor-acceptor pair although AuNC sensitization was only observed from metal-chelates and QDs. FRET theory dramatically underestimated the observed energy transfer while NSET-based damping models provided better fits but could not reproduce the experimental data. We consider additional factors including AuNC magnetic dipoles, density of excited-states, dephasing time, and enhanced intersystem crossing that can also influence ET. Cumulatively, data suggests that AuNC sensitization is not by classical FRET or NSET and we provide a simplified distance-independent ET model to fit such experimental data. Cheng et al. estimated a tryptophan-AuNC donor-acceptor separation distance using FRET formalism for human serum albumin- (HSA) stabilized AuNCset al. extracted distances from tryptophan to the AuNC emission center based on FRET for both BSA and HSA-stabilized AuNCs25 clusters which could be recovered after dissociating the clusters25 clusters by graphene oxide and use of Au10,15,18,25 glutathione-stabilized clusters as light harvesting antennae for subsequent interactions with the electroactive dye methyl viologen41R4 donor-acceptor distance dependence due to the acceptor’s 2-dimensional nature4344Beyond just characterizing the ET interactions and observing sensitization in the experimental donor-AuNC systems, publications are also starting to accumulate where FRET theory is directly applied to the data to extract further physical information about a given configuration. For example, Russell via traditional FRET or NSET as we currently understand it. We consider the role of additional factors that take into account the unique electronic properties of ultra-small luminescent AuNCs including their exceptionally high density of excited states, rapid dephasing time and strong electron confinement as well as the role of the cluster’s paramagnetic properties. Lastly, we provide a simplified distance independent ET model to fit such experimental data.Clearly, AuNC sensitization by ET is a repeatable phenomenon, however, the difference in suggested governing ET mechanisms likely does not arise from a wide diversity of underlying ET process(es), but rather reflects the general lack of understanding that currently exists regarding ET to- and especially sensitization of AuNCs. Here, we investigate the ET characteristics of luminescent AuNCs as acceptors with a series of structurally and functionally distinct luminescent donor systems, including Cy3 and Rhodamine-Red (R-Red) molecular dyes, Ru- and Tb-metal ion complexes and two different QDs using clearly defined-stoichiometric conjugates, see Φ) did not change over a period of ~2 years and were not affected by the presence of different buffers  are presented in Φ values were calculated by comparing the photoluminescence (PL) intensities and absorbance to those of Rhodamine-6G and Indocyanine Green dye standards. The absorption and PL spectra of AuNCs and the donor sample set utilized are shown in 182248Φ was relatively high for water-soluble AuNCs at ~6% with an average PL lifetime of ~0.75 μs when excited at 355 nm and monitored at 800 nm. Lifetime data were fit to bi-exponential decay functions  (PEG)-dithiolane ligands terminated with either a carboxyl, amine, azide, or methoxy group buffers  similar unctions , 4 and 5N, were achieved by controlling the reaction stoichiometry and conjugation was verified in representative samples by UV-vis absorption following purification of the conjugate with size exclusion chromatography (N/QD625 configuration and estimated the distance between the different NPs using TEM and dynamic light scattering (DLS) measurements; these were then compared to the theoretical predictions of distance (details in vs. size). N/QD625 (N = 40) conjugates assembled by electrostatic attraction between the negatively charged CL1/MUA-capped QD625 and positively charged AuNC-TA-PEG-NH2. TEM images confirmed that more than 30 AuNCs surrounded each QD625 when assembled with a reaction stoichiometry of N ~ 40. Importantly, the TEM analysis did not show any QDs stacking on top of the other QDs which would arise from 3-D aggregation or crosslinking. Rather, they reflect well-separated conjugates in a 2-D packing arrangement which would arise from the dehydration of homogeneous samples as expected. The separation distance (R) between AuNC and QD625 was measured to be ~7.7 nm in the dehydrated TEM sample; note this is not the value utilized for extrapolating the experimental QD-AuNC separation. The averaged hydrodynamic distance between the AuNC and QD625 in water was determined to be ~11.7 nm by considering the hydrodynamic size of the QD  for the predicted ET efficiencies obtained from calculations.Photophysical characteristics of the molecular dyes, metal chelates and the QD donors are summarized in tography : a full e QD see . The PEGN/donor N < 1) and in Configuration 2, the QD donor is surrounded by varying numbers of AuNC acceptors (AuNCN/donor N > 1). In comparison to the use of a single donor-to-acceptor ratio, the use of different ratios improves the accuracy of the data as it surveys a wide cross-section of interactions and thus provides more insight into the underlying trends. The following provides a brief overview of results for each configuration. Where applicable, values for both donor steady state and lifetime quenching along with any apparent acceptor PL sensitization within each configuration/ratio are presented in We selected three classes of donors commonly used for ET studies with significantly different physicochemical properties, electronic structures and lifetimes. In Configuration 1, the AuNC acceptors are surrounded by varying numbers of dyes or metal chelate donors  with emission originating from their lowest excited singlet states and with lower energy, non-emissive triplet states. N = 0.6 . To extrN = 0.6  and 5. FN = 0.6, -inset whN = 0.6 (vs. 0.73 μs).Similar phenomena were observed for the R-Red/AuNC conjugates . Steady N = 0.6 . No corrN = 0.6 -inset. REQ, N = 0.6 (N = 0.6) 60% sensitization efficiency,  while changes in their relative magnitudes inversely track each other. Changes in the PL lifetime of Ru(bpy), as a function of the conjugation ratio are shown in N = 0.6 while that of the AuNC acceptor increased ~8%.We next examined ET dynamics for metal ion complexes conjugated to AuNCs.The Ru(bpy) and Tb(chelate) complexes are characterized by emission from symmetry forbidden, lowest energy excited states with relatively long luminescence lifetimes of 0.39 μs and 910 μs, respectively N = 0.6 . The insy, ESen, . Indeed,N is varied between 0.08 and 0.6 (−56% to −94%). An unexpectedly large increase in the PL from the AuNCs was observed as the conjugation ratio was increased. In fact, the AuNC signal enhancement was much higher than that expected from the donor quenching measurements, suggesting additional processes could be occurring in these conjugates. In addition to the expected Tb PL lines in the visible, a bright blue emission was observed from the isolated Tb(chelate) . This suggested a direct ET or quantum efficiency augmentation from cs-124 to the AuNCs in addition to any ET from the Tb. After correcting and accounting for this, the recalculated sensitization efficiency from the AuNCs reached a maximum of 94% for N = 0.6, consistently tracking with the donor quenching measurements (N varied from 0.08 to 0.6 (N = 0.08 to 1.07 μs for N = 0.6 (Donor quenching and acceptor sensitization characteristics of the AuNC/Tb(chelate) system are summarized in chelate) , indicaturements . Quenchiurements . The res8 to 0.6 . AuNC li N = 0.6 .τD0) of 7.3 ns and Φ of 0.16 and red emitting QD625 with a τD0 of 61 ns and Φ of 0.50. For these experiments, Configuration 2 was used where a single QD was surrounded by varying numbers of AuNCs  FRET; (2) NSET; and (3) Nanovolume Energy Transfer (NVET) which is modified from Persson’s volume damping theory; pertinent details of each mechanism are discussed below or in the kD0 is the decay rate of the donor in the absence of acceptor and is a summation of the radiative (kr0) and nonradiative decay (knr0) rates (kD0 = kr0 + knr0 = 1/τD0). kET is the ET rate from donor to acceptor and N is the ratio of acceptors to donors. In the case of FRET, the interaction between donor and acceptor is characterized by a dipole-dipole interaction resulting in an ET rate that follows a 1/R6 dependency14R is the donor-to-acceptor center-to-center separation distance. The ET rate can be expressed in the following generalized form:where R0 is the critical separation distance for which the ET efficiency is 50%. Here, the exponent, X, depends on the interaction strength between donors and acceptors as well as their physical nature, their relative size and the separation distance between them. In FRET theory, X = 6, since donors and acceptors are considered point dipoles142566R4 dependence (X = 4) where R is the distance from the AuNC surface to the donor.where leff = 1.64 × 0.75 nm (AuNC radius) ≈1.23 nm) and the corrected electron mean free path (lCor ~ 2.34 nm)6970X = 3. The corresponding expressions for each ET model along with their derivation can be found in the R0, the spectral overlap integral, J, the estimated donor-acceptor separation, R, . We also back-calculated an experimental R0 (R-Exp.0) from the experimental donor quenching data and donor native lifetime using R-Exp.0 = (kET/kD0)1/X × R for comparison to the theoretically estimated R0 value postulated by each model. R values were calculated based on the approximated length of the ligands or chemical linkers  is provided in R, the donor-acceptor separation for NSET and NVET, are also very favorable for efficient quenching (3.4~9.3 nm) as the calculated NSET R0 values range from 4.6~7.6 nm while the NVET R0 values span 7.8~15.2 nm. Given their weaker distance dependency, these are much larger than the FRET R0 values which ranged from 3.9~5.4 nm.Our experimental data, however, indicates a weaker dependence of the ET rate on separation distance between the donors and AuNCs, which led us to also consider a further modified version of Persson’s volume damping theory661cm−1nm4  and the N increases with no significant changes to its lifetime. R-Red PL undergoes even stronger quenching along with a slight decrease in lifetime versus N. The AuNC acceptor in these assemblies exhibits a net decrease in PL intensity, effectively a negative sensitization. Experimental ET efficiency (EET) from donor steady state fluorescence quenching efficiency (EQ = 1 − F/F0) and acceptor sensitization efficiency, ESen 77For these organic dyes, the lack of significant changes in lifetime when assembled with AuNCs, in combination with a lack of any AuNC sensitization lead us to rule out the traditional ET mechanisms that have previously been used to describe similar systems. We hypothesize that the dramatic quenching observed in the steady state luminescence of the molecular dyes is the result of efficient intersystem crossing (ISC) from the dye’s first excited singlet state to the triplet state followed by nonradiative relaxation to the ground state. Moreover, the resulting spectra will only reflect dye molecules that are not affected by ISC and thus the PL intensity will change while that of lifetime should not. The ISC rate can be enhanced by strong spin-orbit coupling between the dye and magnetic dipoles on the AuNC surface that are associated with unpaired spins on dangling Au-S bonds, or magnetic properties of the Au core i.e., net sensitization per acceptor) for this system along with the QD systems that follow below. The luminescence lifetime of the Ru(bpy) complex decreases modestly with increasing N. ET efficiencies, based on steady state luminescence quenching, donor lifetime quenching and AuNC sensitization are shown in N = 0.6 and this matched with the concomitant acceptor sensitization which reached the same value of 94% for the same ratio. Again, the observed ET efficiency was much higher than the maximum efficiency predicted by FRET but similar to NVET (96%). Additionally, non-conjugate solution phase only control experiments implemented with the metal chelates and the AuNCs at the same concentrations showed almost no ET (data not shown), confirming the critical need for a close proximity between donors and AuNC acceptors for significant ET to occur.Data from the AuNC/Ru(bpy) assemblies shows the Ru donor undergoing significant PL quenching while the AuNC acceptor sensitization component concomitantly increases as a function of donor number. See N and were followed by a concomitant AuNC acceptor sensitization. A similar pattern was observed for the QD625 assemblies, with the exception that donor PL did not continue to decrease beyond N = 5. Experimental quenching efficiencies for the AuNC/QD assemblies, along with efficiencies predicted by FRET, NSET and NVET are summarized in N = 15 (see N = 5 (64%) and then remained approximately the same with increasing N. Maximum AuNC acceptor sensitization by QD625 in this complex was roughly comparable within the error at 72%. The initial quenching efficiency is consistent with the NSET and NVET models and is significantly greater than expected from FRET. Despite the clear and overt AuNC sensitization, the reason for the apparent saturation in the QD625 donor quenching efficiency at N = 5 is not clear at this time. ET between QDs and AuNPs has been previously ascribed to an NSET type process24265960616263Structurally, this configuration represents a true NP-NP system. Within the AuNC/QD545 assemblies, donor PL intensity and lifetime decreased with increasing = 15 see . The PL ee N = 5 % and theτavg) composed of multiple exponential decays which have both short and long-lived components79Φ’s than those associated with the short-lived componentsEnsemble measurements of QD lifetimes are typically described by an ensemble average value  and Tb(chelate) donor assemblies, they still do not fully reproduce the observed behavior. To provide further insight into the ET mechanism(s), in kET) for the three models from donor quenching and acceptor sensitization efficiency using R0) and our experimentally-derived data for the distance R-Exp0 (1/τD0), we estimated the donor decay rate (kD0) and extracted the ratio of experimental ET rate to donor decay rate, kET/kD0, shown in kET/kD0 directly affects the ET efficiency, EET , and an associated fast dephasing time, τcA ~ 10 fs, based on the ~410 meV spectral bandwidth of the AuNC PLet al.The electronic structure of the luminescent AuNCs is significantly different from that of traditional molecular dye acceptors. AuNCs have a very high density of excited states, kET = (2π/ℏ)(VDA)2ρ(E), where the matrix element VDA describes the coupling between the donor and acceptor excited states. The density of excited states in these types of small-sized AuNCs can be estimated as ρ(E) ≈ MτcA/ℏ, where M is the number of acceptor excited states, ℏ is the reduced Planck constant and ℏ/τcA is the line-width of the acceptor resonance. The donor-AuNC-acceptor ET rate can then be estimated as:Here, we define the resonant ET rate from a donor to a AuNC acceptor as M ≫ 1. This explains the efficient ET to the AuNC acceptors that was observed from the metal ion complexes and QDs. As M becomes larger, kET will also become larger in proportion to M. Thus, compared to an acceptor having a smaller M value, the AuNC is expected to have a more efficient ET rate from the same donor.where via an ET process. The time dependence of the excited donor and acceptor populations, nD and nA, respectively, under steady state (continuous wave) excitation conditions can be described by the kinetic equations:We next look to the excitation dynamics in an ensemble of optically active donors and AuNC acceptors which are coupled GD and GA are the probabilities of donor and acceptor excitation, kD and kA are the decay rates of excited donor and acceptor, N is the ratio of acceptor to donor in the ensemble, and kET is the ET rate. The steady state solutions of where kET = 0), the donor and acceptor populations are determined by the two standard independent expressions: nD = GD/kD and nA = GA/kA. Assuming the M ≫ 1 condition is satisfied and transitions from the acceptors to donors are suppressed, transparent expressions for the donor and acceptor populations become:From nA, and reduces the population of donor states, nD, consequently increasing the PL intensity of the acceptors while suppressing donor PL. The PL intensities of donor and acceptor, nDkD and nAkAN, respectively, depend on the product of the acceptor-to-donor ratio, N, and the ET rate kET. Sensitization of acceptors as introduced by Dexter is described as N(nAkA − GA)N(nAkA − GA) = EETGD with the ET efficiency given by VDA, based on experimental data.Data from the Cy3 and R-Red organic dye-AuNC assemblies show very effective quenching of both donors and a slight quenching of the acceptor PL. Suppression of PL from Cy3 and R-Red molecules in the presence of heavy atoms has been well documented2858687kD, is increased significantly and this relaxation becomes mainly nonradiative. The populations of the emissive states in the donor and acceptor are still governed by kD = kr + knr. Although the solutions for nD and nA can be written in the same form as in knr ≫ kET. The PL intensity of the donor, which is proportional to ID = krnD and can be rewritten using ID = [kr/(N kET + kD)]GD ≈ (kr/knr) GD ≪ GD. Resonant energy transfer from the AuNCs to the dyes, for example, is described by i) sufficient spectral overlap exists for this condition, in reality the actual values are negligible and circa 2 orders of magnitude less (ii) kD ≫ kET in The quenching of both donor molecules and the AuNC acceptor can be described using the system of ude less  than thoude less ; and .Here, one can see that ET depletes the acceptor population and consequently it decreases the PL intensity which is proportional to vs. temperature (T) were analyzed using a modified Curie-Weiss equation to derive the magnetic susceptibility, see SIθ) value indicates minimal electron-electron exchange interactions typical of isolated paramagnetic centers. The quality of fit to the nearly 1/T behavior of susceptibility also indicates the presence of localized moments in the compound. The total number of spins (~2 × 1017) measured along with the total weight of AuNC (~6 mg) allows us to roughly estimate the average number of surface dangling bond spins at ~2 spins per AuNC  magnetic measurements using a Quantum Design Magnetic Properties Measurement SystemEET = 1 − FD/FD0) always appear larger than those based on time decay experiments . The PL intensity of donor, FD, is proportional to (1/τDr)/(1/τD), where τDr andτD are the radiative and total decay time of a donor, respectively. The total decay rate, 1/τD, in the presence of the AuNCs includes the ET rate. As a result, the ratio of donor PL intensity in the presence of AuNCs, FD, and in their absence, FD0, can be written as FD/FD0 = (τD/τD0)(τDr0/τDr) where τDr0 and τD0 are the radiative and total decay times of the donor in the absence of the AuNCs. One can see that steady state measurements of the ET efficiency, 1 − FD/FD0, contains the additional factor τDr0/τDr, which is usually “1” for traditional ET calculations; assuming the radiative decay rate of donor does not change with/without acceptor. This can explain the difference if the radiative decay time, τDr, becomes longer in the presence of AuNCs (τDr0/τDr < 1), and 1 − FD/FD0 = 1 − (τD/τD0)(τDr0/τDr) is larger than the traditional lifetime based EET calculation, 1 − τD/τD0. Supporting the notion of variation in radiative/nonradiative decay rates by gold nanoparticulates, an increase in Lissamine and Cy5 donor dye decay times in the presence of AuNCs was observed in refs Additional evidence for AuNC acceptors influencing organic dye donors may be found in the difference in quenching efficiency estimates when using either steady state or lifetime measurements. The quenching efficiencies based on steady state experiments  and QDs may indeed represent the first reports of this phenomena with these particular donor materials. We also note that many of the observed photophysical changes in donor to AuNC acceptor properties during ET are quite similar to those expected as “Hallmarks” or defining characteristics of classical FRET and this can serve to confuse the interpretation. For example, steady state donor quenching was matched with a concomitant increase in AuNC acceptor sensitization and, similarly, donor lifetime decreases with increases in that of the paired AuNC acceptor. The magnitude of these photophysical changes track to some extent directly with the number of acceptors or donors in the experimental configuration towards a plateau. Moreover, the observed ET efficiency is also dependent upon the requisite spectral overlap being present along with close donor-acceptor proximity; this strongly suggests the need for resonance between donor-acceptor dipoles. The experimentally determined ET efficiencies were rigorously compared to predicted efficiencies based on the FRET, NSET and NVET models all of which have a strong distance-dependent coupling. In all cases, the FRET model dramatically underestimated the efficiencies while closer approximations to the experimental data were provided by modifying Persson’s surface and volume damping theory; the latter is not based on a traditional FRET coupling.Here, we confirm that luminescent AuNCs do indeed function as efficient ET acceptors when paired with appropriate donors displaying the requisite electronic characteristics, In contrast to the above, the Cy3 and R-Red organic dyes do not appear to manifest any notable role as ET donor to the AuNCs. The slight AuNC quenching in the presence of dyes may be due to ET from directly-excited AuNCs to the dyes. However, the small amount of underlying spectral overlap , suggest8410851110i.e., incoherent) and has been referred to as coherent ET via strong coupling between the donor and acceptoralbeit of much smaller magnitude. Indeed, such data is quite analogous to the dynamic process originally observed for sensitization of near-IR emitting 1.8 nm diameter tiopronin-protected AuNCs with (Ru(bpy)3)2+ donorsCumulative contributions from their electronic structure, exceptionally high density of excited states, fast dephasing time, and even magnetism all contribute to making AuNCs a “non-classical” material when it comes to ET as these factors must now all be considered and even accounted for. Additionally, in the role of an acceptor the net result of several AuNC properties  mean that, in addition to ET from donors, there will also be a direct acceptor excitation component which can significantly complicate experimental formats. Using a theoretical analysis extended from our results, we suggest that AuNC properties, including especially the rapid dephasing time (~10 fs), play an important role in efficient ET by effectively preventing resonant back-transfer to the donor which has a relatively long lifetime compared to this. This situation is different from a classical Förster mechanism . As the electronic properties of luminescent AuNCs are elucidated, there will also be a need for more physical evidence supporting coupling How to cite this article: Oh, E. et al. Energy Transfer Sensitization of Luminescent Gold Nanoclusters: More than Just the Classical Förster Mechanism. Sci. Rep.6, 35538; doi: 10.1038/srep35538 (2016).


To clarify the pathogenesis of two different types of adult-onset normal-pressure hydrocephalus (NPH), we investigated cerebrospinal fluid distribution on the high-field three-dimensional MRI. The subarachnoid spaces in secondary NPH were smaller than those in the controls, whereas those in idiopathic NPH were of similar size to the controls. In idiopathic NPH, however, the basal cistern and Sylvian fissure were enlarged in concurrence with ventricular enlargement towards the z-direction, but the convexity subarachnoid space was severely diminished. In this article, we provide evidence that the key cause of the disproportionate cerebrospinal fluid distribution in idiopathic NPH is the compensatory direct CSF communication between the inferior horn of the lateral ventricles and the ambient cistern at the choroidal fissure. In contrast, all parts of the subarachnoid spaces were equally and severely decreased in secondary NPH. Blockage of CSF drainage from the subarachnoid spaces could cause the omnidirectional ventricular enlargement in secondary NPH. Since 1965, adult-onset normal pressure hydrocephalus (NPH) has been largely classified into idiopathic NPH (iNPH) or secondary NPH (sNPH), which develops after subarachnoid haemorrhage, trauma or infection2345789109101213151617Fifty-two patients were diagnosed with iNPH, 15 patients were diagnosed with sNPH, and 31 participants were allocated to the control group. The mean ages were not significantly different among the three groups . The mea23457821Further evidence indicating a direct pathway of CSF flow at the inferior choroidal point of the choroidal fissure is presented in head CT images taken before and immediately after shuntography performed on a patient with iNPH with a suspected shunt malfunction . On the In the video clip showing an amygdalohippocampectomy , it is eThis study shows that CSF distribution markedly differs between the two categories of NPH. et al. reported that the direction of the interstitial fluid flow towards the ventricles or subarachnoid spaces varies in an unpredictable manner2331373839252641232428444546474849444546474849Recently, classical understanding of CSF bulk flow and the concept of the third circulation have changed2324252627282930323325262729303135et al. found that the interface between the third ventricle and the quadrigeminal cistern also serves as a compensatory direct CSF pathway in addition to the choroidal fissureOur observation of CSF distribution reduced subarachnoid spaces in the high parietal convexity in iNPH supports the current view of CSF absorption. The main pathogenic mechanism associated with iNPH is thought to be the disruption of the most downstream site of CSF drainage systems, because patients with iNPH have large volumes of intracranial CSF. The subarachnoid spaces might be harder to expand than the ventricles because there are many trabeculas and partitions in the subarachnoid spaces, as shown in 151617On the other hand, the main pathogenic factor for the omnidirectional expansion of ventricles in sNPH is thought to be the disruption of CSF drainage from subarachnoid spaces, because the subarachnoid spaces in sNPH extensively diminished and the total intracranial CSF volume was smaller than that in the age-matched controls. Some clinical studies have reported that significantly increased inflammatory fibrogenic cytokines or ferritin levels in CSF are associated with sNPH after subarachnoid haemorrhage5152545555In conclusion, the present study revealed the characteristics of CSF distribution in patients with iNPH, which strongly differed from those in patients with sNPH. Patients with iNPH had a substantially larger total CSF volume than control subjects, whereas patients with sNPH had a smaller total CSF volume. Under normal conditions, the ventricles directly communicate with the subarachnoid spaces via the foramina of Luschka and Magendie. However, in iNPH, the ventricles and basal cistern are enlarged, along with the opening of the inferior choroidal point of the choroidal fissure. This provides a direct compensatory CSF communication route between the ventricles and subarachnoid spaces. Thus, this is a key cause of the disproportionate CSF distribution in iNPH, named “Disproportionately Enlarged Subarachnoid-space Hydrocephalus (DESH)”The study protocol was designed in accordance with guidelines outlined in the Declaration of Helsinki and was approved by the ethics committee for human research at Rakuwakai Otowa Hospital, Kyoto, Japan . The methods were performed in accordance with the approved guidelines. Written informed consent was obtained from all patients or their family members.Details regarding clinical data collection, image acquisition, and segmentation and quantification of the ventricles and subarachnoid spaces were described in our prior publications9All MRI examinations were performed with a 64-channel 3-Tesla MRI system . The sagittal source images of the T2-weighted 3D-SPACE were automatically processed to create 3D volume-rendering reconstruction and multiplanar reconstruction images by using an independent 3D volume analyser workstation . The intracranial volumes were segmented by the utilization of the combined techniques of the edge-guided nonlinear interpolation and user-steered live-wire segmentation582345in situ during the amygdalohippocampectomy of one patient diagnosed with hippocampal glioma.We subjectively assessed whether there was an opening at the inferior choroidal point of the choroidal fissure using 3D-CISS sequences to evaluate the fine structures in the CSF spaces. Additionally, one patient who was diagnosed with iNPH and who had received a ventriculo-peritoneal shunt four years ago at another hospital needed to undergo shuntography to test the patency of his shunt system because his gait and cognitive function had worsened gradually. A CT scan was performed before and immediately after shuntography, in which a small amount of OMNIPAQUE-240  was injected into the left inferior horn of the lateral ventricles via reservoir and ventricular catheter of the shunt system. Furthermore, the actual CSF movement through the thin arachnoid membrane between the inferior horn of the lateral ventricles and the ambient cistern was observed p) of less than 0.05. All missing data were treated as deficit data that did not affect other variables. Statistical analysis was performed using R software .Mean values and standard deviations for age, sex, and several MRI parameters were calculated and compared among patients with iNPH, sNPH, and control groups by a Mann-Whitney-Wilcoxon test. The Chi-square test was used to compare the two proportions. Statistical significance was assumed at a probability value .Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


We also show that an extracellular chaperone clusterin; a nanobody specific to the amyloid-β peptide (Aβ); and bapineuzumab, a humanized monoclonal antibody raised against Aβ, could all reduce the Ca2+ influx caused by synthetic Aβ oligomers but are less effective in CSF. These assays could be used to characterize potential therapeutic agents in CSF before clinical trials.One potential therapeutic strategy for Alzheimer’s disease (AD) is to use antibodies that bind to small soluble protein aggregates to reduce their toxic effects. However, these therapies are rarely tested in human CSF before clinical trials because of the lack of sensitive methods that enable the measurement of aggregate-induced toxicity at low concentrations. We have developed highly sensitive single vesicle and single-cell-based assays that detect the Ca  •2+ influxHuman cerebrospinal fluid (CSF) can permeabilize membranes and induce Ca•2+ influxCSF of control individuals and those with Alzheimer’s disease show similar Ca•2+ influxAn extracellular chaperone clusterin and a nanobody Nb3 can inhibit Ca•2+ influx but is less effective in CSFBapineuzumab reduces Aβ-aggregate-induced Ca in vitro assays to quantitatively measure the Ca2+ influx caused by human cerebrospinal fluid. If a given chaperone, antibody, or nanobody is effective in reducing Ca2+ influx, the authors determine what concentration is needed to prevent Ca2+ influx.Drews et al. develop and implement sensitive  Monomeric amyloid-β peptide (Aβ) is released from the amyloid precursor protein (APP) by proteolysis, aggregates into a range of soluble oligomers, and, ultimately, aggregates into insoluble amyloid fibrils deposited in the brain . A body al death . Picomolpoptosis . The solpoptosis . Moreoveely 1 pM . This isPrevious work to test potential therapeutic strategies, for example antibodies, has used aggregates derived from human sources , such as2+ influx into hundreds of surface immobilized single lipid vesicles -based imaging method to count the total number of ThT-active species in CSF using sensitive single-molecule fluorescence measurements  (2+ influx in the cellular assay at either antibody concentration (2+ influx caused by CSF samples compared to the concentration needed to counteract the effect of synthetic Aβ42 oligomers in buffer solution. This reduced effectiveness of bapineuzumab in the inhibition of CSF-induced Ca2+ influx can have a number of causes including (1) molecular crowding and non-specific binding to a multitude of components present in CSF, which might reduce the effective interaction between the species causing Ca2+ influx and the antibody; (2) the morphology of the species present that are responsible for Ca2+ influx in CSF might be different from the species in the experiments with purified Aβ; (3) there might be post-translationally modified species of Aβ present in CSF in which the binding epitope of bapineuzumab is absent or inaccessible to the antibody. A combination of these effects could reduce the binding of the antibody to the aggregates, and as a consequence bapineuzumab is less effective at reducing the Ca2+ influx induced by CSF.Next, we probed the efficacy of bapineuzumab, which is a bivalent antibody that binds soluble Aβ monomer and oligomers at the N terminus . Using rle assay A and S3B+ influx A, and thmpletely C. Bapinell assay B and 3D.ll assay , indicatll assay . In the  of ∼12% B and S3Dase 18%) C and S3Entration E and 3F.The mechanisms by which amyloid aggregates cause toxic effects and neuronal cell death remain a topic that is under investigation. One commonly proposed mechanism is that aggregates permeabilize membranes, leading to altered calcium homeostasis and, ultimately, to cell death. However, aggregates can also act by a range of other mechanisms such as by causing oxidative stress, neuroinflammation, or synaptic dysfunction. It is therefore extremely important to develop and apply techniques that explore the significance of particular molecular processes that are likely to contribute to the development of AD in humans.2+ influx in our assays using both single vesicles and astrocytes. In addition, our results show that Nb3 and bapineuzumab, both of which were designed to bind to the Aβ peptide, can both inhibit this Ca2+ influx. Interestingly, we detected no significant difference in the number of ThT-active species (an indication of β-sheet containing aggregates) or between Ca2+ influx induced by aliquots of AD CSF and HC CSF. These results are in agreement with the results of ELISA measurements, using the same antibody to capture and detect aggregates  was aggregated as described previously .Nb3 is an Aβ-specific nanobody isolated from a llama and was prepared as described previously . ClusterThe bapineuzumab equivalent antibody was prepared as described in its U.S. patent (US 7179892 B2) in 25 mM histidine, 7% sucrose, and 0.02% polysorbate 80 (pH 6.0) at 48 mg/mL. Endotoxin levels were < 0.005 (EU/mg).All CSF samples were imaged with the single aggregate visualization through enhancement (SAVE) method as previously described . In shor1-42, T-tau, and P-tau181 were quantified with sandwich ELISAs . Intra-assay coefficients of variation were below 10%. All controls had no cognitive symptoms and a normal CSF T-Tau/Aβ1-42 ratio < 0.52. Patients with AD had CSF Aβ1-42 < 600 ng/L and T-tau > 350 ng/L. Study protocols were approved by the Queen Square ethics committee (references 12_LO_1504 & 12_LO_005), and all individuals gave written informed consent.Control CSF samples were collected by lumbar puncture from 6 cognitively normal individuals (aged 49–68 years) and 6 individuals with an AD diagnosis (aged 51–68 years). A standardized protocol for the collection and storage of CSF was followed. In short, lumbar puncture in the L3/L4 interspace was performed between 9 a.m. and 12 a.m. to collect 15 mL of CSF in sterile polypropylene tubes. The samples were de-identified, spun at 3,000 rpm for 10 min, and divided into aliquots each containing 1 mL that were frozen on dry ice and stored at −80°C in 1.5 mL capacity LoBind micro-centrifuge tubes . Sample collection, centrifugation, and freezing was completed within 1 hr. CSF Aβg and stored at −80°C pending experimental use. CSF Aβ1-42, T-tau and P-tau181 were quantified with sandwich ELISAs . All measurements were performed in one round of analyses using one batch of reagents by board-certified laboratory technicians. Intra-assay coefficients of variation were below 10%. All AD positive samples had protein levels of Aβ1-42 < 600 ng/L, T-tau > 350 ng/L and P-tau181 > 80 ng/L according to The AD CSF used for the cell assays was collected by lumbar puncture from patients who sought medical advice because of memory problems. The samples were de-identified and aliquoted into 0.5 mL aliquots in polypropylene cryo tubes following centrifugation at 2,200 × Astrocyte dosing experiments were performed as described previously  using raVesicles with a mean diameter of 200 nm were prepared by freeze-and-thaw cycles combined with extrusion as previously described . BrieflyGlass coverslips were cleaned by sonicating in 2% (v/v) Hellmanex III in water for 10 min followed by sonicating twice in water and twice in methanol for 10 min each . Then th2+ influx into individual vesicles was performed using a home-built total internal reflection fluorescence microscope (TIRFM) based on an inverted Olympus IX-71 microscope. A 488-nm laser  beam was expanded and focused on the back focal plane of the 60×, 1.49 NA oil-immersion objective lens  to excite the Cal-520 dye incorporated into the vesicle. The emerging fluorescence was collected by the same objective. The emission beam was separated from the excitation beam by a dichroic , passed through a set of filters , and imaged onto an air-cooled EMCCD camera . Excitation power density was fixed to ∼10 W/cm2 for 50 frames with a scan speed of 20 Hz and bit depth of 16 bits.The measurements of Ca2+ containing buffer solution Leibovitz’s L-15 (L15). 16 different fields of view were recorded under three different conditions . The distance between each field of view was set to 100 μm and was automated  to avoid any user bias. First, images were acquired in the presence of only L15 buffer, which we denote as blank (Fblank). For each field of view, 50 images were taken with an exposure time of 50 ms. Thereafter, a solution of a sample was added and incubated for 10 min before images of the exact same fields of view were recorded (Fsample). Next, 10 μL of a solution of ionomycin was added to the coverslip and incubated for 5 min, and, subsequently, images of the vesicles were acquired in the same fields of view.Hundreds of single vesicles tethered to coverslips via biotin-neutrAvidin linkage were incubated with 50 μL of HEPES buffer (50 mM [pH 6.5]) and imaged as previously described . Just beblank), in the presence of the sample (Fsample), and after the addition of ionomycin (FIonomycin). The relative influx of Ca2+ into an individual vesicle due to aggregates of the Aβ42 peptide was then determined using the following equation:The recorded images were analyzed using Fiji to determine the fluorescence intensity of each spot under the three different conditions, namely background  peptide (MDAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVVIA), here called Aβ42, was used for the vesicle assay experiments and prepared as describe previously . Briefly2+ influx were taken at a time point corresponding to the end of the lag phase (approximately 70 min) as previously described (Aliquots of monomeric Aβ42 were diluted with buffer to a concentration of 2 μM in low-binding Eppendorf tubes on ice before individual samples were then pipetted into multiple wells of a 96-well half-area plate. Then the plate was incubated under quiescent conditions at 37°C, and aliquots for measurements of Caescribed .Aliquots were taken from an aggregation reaction of recombinant Aβ42 corresponding to the end of the lag phase. To measure the change of the fluorescence intensity in the single vesicle assay, we diluted the sample to 5 nM with L15 buffer as described previously.Indicated concentrations of nanobodies and antibodies were added to an aliquot taken from an aggregation reaction of recombinant Aβ42 and incubated for 15 min. The change in the fluorescence intensity was measured as described above.