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Neurons communicate via chemical signals called neurotransmitters (NTs). The numerous identified NTs can have very different physiochemical properties (solubility, charge, size etc.), so quantification of the various NT classes traditionally requires several analytical platforms/methodologies. We here report that a diverse range of NTs, e.g. peptides oxytocin and vasopressin, monoamines adrenaline and serotonin, and amino acid GABA, can be simultaneously identified/measured in small samples, using an analytical platform based on liquid chromatography and high-resolution mass spectrometry (LC-MS). The automated platform is cost-efficient as manual sample preparation steps and one-time-use equipment are kept to a minimum. Zwitter-ionic HILIC stationary phases were used for both on-line solid phase extraction (SPE) and liquid chromatography (capillary format, cLC). This approach enabled compounds from all NT classes to elute in small volumes producing sharp and symmetric signals, and allowing precise quantifications of small samples, demonstrated with whole blood (100 microliters per sample). An additional robustness-enhancing feature is automatic filtration/filter back-flushing (AFFL), allowing hundreds of samples to be analyzed without any parts needing replacement. The platform can be installed by simple modification of a conventional LC-MS system.
NTs are metabolites used for communication between neurons in the brain. In humans, analysis of cerebrospinal fluid (CSF) gives the most direct measure of central NT levels. Due to their less invasive nature, neuroimaging approaches such as positron emission tomography (PET) and magnetic resonance (MR) spectroscopy1,2 are promising alternatives. However, the cost, availability and practicality aspects of CSF extraction and neuroimaging have led many studies to rely on quantification of NTs from blood, saliva or other peripheral fluids.
Measuring levels of various NTs (e.g. GABA, serotonin and its precursor tryptophan, adrenaline, oxytocin and vasopressin) in blood or other bio-fluids gives valuable information for several purposes. Some studies point to differences in peripheral NT levels as potential biomarkers for psychiatric conditions (e.g. serotonin3), or use blood measures of NTs to validate experimental neuroscience methods4,5. Peripheral NT levels have also repeatedly been shown to correlate with e.g. parent-infant attachment, e.g. neuropeptides oxytocin and vasopressin6. Central oxytocin (OT) levels are thought to mediate the link between OT levels in plasma and behaviour, although the highly limited ability of many NTs to cross the blood brain barrier render the relationship between central and peripheral levels less clear7. Therefore, reliable, valid and sensitive analytical methodologies are required to further establish the roles and predictive traits of peripheral NTs.
A further complication of employing peripheral NT levels is that reliability and validity can be questionable. Different methods of quantification do not always give comparable results. For OT, McCullough et al8 recently pointed to a discrepancy of up to 100-fold between conventional radioimmunoassay (RIA) and the newer, commercially available enzyme immunoassay (EIA) approach. The authors suggest that such EIA measures may misattribute other molecules to OT. Moreover, RIA and EIA methodologies are intended for specific compounds, and can be very time-consuming to establish, reducing method flexibility for e.g. identifying novel metabolite candidates for brain/blood level correlations9.
Another obstacle is the difficulty with obtaining convincing data from small amounts of sample. Recently, Clark et al10 analysed cerebrospinal fluid (CSF) samples from human infants and showed a correlation between neuropeptide levels and behaviour. The authors were however unable to address the correspondence between plasma and CSF peptide levels, since the amount of blood required for conventional analysis of plasma OT exceeded the amount that could safely be drawn from neonates.
An alternative to RIA and EIA is mass spectrometry (MS) based methodology. MS provides detailed information of detected compounds, including molar mass, charge, and (clues to) its molecular structure (when employing tandem MS). MS is commonly coupled with liquid chromatography (LC), which separates compounds in a sample from each other, prior to MS detection. This step enhances e.g. sensitivity, as fewer compounds enter the MS per time, reducing ion suppression11. LC-MS is common instrumentation in modern analytical laboratories, and is associated with excellent validity and high accuracy/precision measurements, especially when internal standards are used, e.g. adding known amounts of an isotopically labelled version of each analyte (e.g. the NTs) to the sample, which are used to correct for variances that can occur during sample preparation/LC-MS recording.
However, reported LC-MS methods for NT determinations are often intended for rather large amounts of sample12,13,14 and may therefore not be suitable for analysis of limited, precious samples from e.g. infants and blood banks. Also, they are typically not suited for measuring all classes of NTs at once, due to the significant variation of physiochemical properties of NTs, such as hydrophobicity and charge. Simultaneous determination of members from all NT classes would be desirable as NTs can interact extensively, as is well documented across human and non-human models. LC-MS methods may however also require a number of manual preparation steps, e.g. enriching the compounds with solid phase extraction (SPE) columns prior to LC-MS, thereby increasing analysis cost.
Our goal was to develop an LC-MS platform that would be capable of analysing samples to quantify all NT classes. Moreover, we wanted the platform to have high sensitivity, allowing analysis of minute samples. In addition, we wanted the platform to be automated, but at the same time very robust. Our proposed solution is to employ LC-MS, with LC performed in hydrophilic interaction mode (HILIC)15, coupled with on-line solid phase extraction (SPE)16 and automated filtration/filter backflushing (AFFL)17. The LC column was in capillary format (cLC). The SPE step is also performed in HILIC mode, and is compatible with protein precipitation using acetonitrile (ACN) with 0.1% formic acid (FA), allowing direct analysis of the supernatant with no additional steps. We will here describe the versatility of the method and demonstrate its sensitivity, quantification, reliability and other traits.
The main results were: neurotransmitters/precursors with a wide range of physiochemical properties (see structures and other data, Fig. 1) could be identified/measured in a single analysis, needing only 100 μL of whole blood (see cLC-MS chromatograms of endogenous NTs and isotopically labelled internal standards (IS), Fig. 2). Identifications were based on retention time matching with standards and spectra obtained with high resolution MS. A novel, automated AFFL-SPE-cLC-MS analytical platform was developed for this purpose (Fig. 3 and Supplementary Fig. S1). The automated platform could be used for reliable trace determinations, evaluated with NT monoamines and amino acids with readily available labelled internal standards; see calibration curves and validation tables (Fig. 4). Due to the platform's sensitivity and versatility, trace levels of the much larger neuropeptides oxytocin and vasopressin could also be easily detected (Fig. 2g and n). The platform provided good and similar chromatographic quality for monoamines, amino acids and peptides (Fig. 2), suggesting that AFFL-SPE-cLC-MS is a suitable platform for comprehensive NT determinations in small samples. Below is a more detailed presentation of the results and technical aspects.
Initially, two commercial zwitter-ionic HILIC columns for cLC separations were examined, (a) ZIC-HILIC18 and (b) ZIC-cHILIC19. Both have zwitter-ionic stationary phases, but while (a) has a sulphobetaine type functionality (quaternary ammonium part = positive charge, sulphonic acid part = negative charge), where the negative charge is the most accessible to interacting solutes, (b) has a phosphorylcholine type functionality (phosphate part = negative charge, quaternary ammonium part = positive charge), where the positive charge is the most accessible. Neither of the two 15 cm long columns provided a baseline separation of all the NT analytes. However, since the analytes have distinct retention times, exact masses and/or MS/MS transitions (Table 1) and the platform provides satisfactory quantitative performance (see below), baseline separation was not considered to be a necessity. The efficiency (plate number N, see Supplementary note Calculation of efficiency), and asymmetry factor (As, see Supplementary Fig. S2, for details) of the platform were somewhat better with (b) (see N values in caption of Fig. 2), hence the ZIC-cHILIC column was employed. Best resolution (Rs) and N were achieved with a mobile phase consisting of 70% ACN and 30% 100 mM ammonium formate at pH 3 (see Supplementary note Mobile phases, for brief discussion on other mobile phases examined). In addition, 300 μM ascorbic acid (anti-oxidant) was added to the mobile phase to avoid online-oxidation effects20,21 (see Supplementary note Online oxidation).
An AFFL set-up22 was used for on-line sample filtration (Fig. 3, part A), using a stainless steel filter, and subsequent back-flushing of the filter. This approach eliminated the need for single-use filters, and manual handling of these, thereby allowing for hundreds of whole blood sample injections without hardware replacement (including filter, columns etc.). On-line SPE16 (Fig. 3, part B) was employed to automatically enrich the NTs prior to cLC separation, eliminating also the need for single-use SPE cartridges (and manual handling of these). Best performance was obtained using a ZIC-HILIC SPE column, compared to porous graphitic carbon- and ion exchange SPEs (see Supplementary note SPE column materials, and discussion below), with a 100% ACN loading solvent and a two minute SPE loading time. A ZIC-cHILIC SPE column was not available to us during the study, and therefore not investigated. At least 100 μL of acetonitrilic sample volumes (resulting from the protein precipitation step, see Methods section below) could be loaded on to the SPE without an analyte breakthrough occurring (see Supplementary Fig. S3). The cLC mobile phase successfully eluted the NT analytes from the SPE to the cLC column, and the analytes were chromatographed/detected in less than 9 minutes. Including the two-minutes loading time and one-minute post-run SPE re-equilibration time, the platform analysis time cycle was 12 minutes per sample. The only sample preparation was a one-step ACN (with 0.1% FA) precipitation.
Viewing Fig. 4 in more detail, the within-day and between-day repeatabilities (n = 6 and n = 5, respectively) were satisfactory for quantification, i.e. ≤20% relative standard deviation (RSD), as according to Federal Drug Agency (FDA) validation guidelines23, from lowest concentrations (between 0.05 nM and 250 nM, depending on the analytes expected endogenous levels) and up to three orders of magnitude, with good linearity (r2 = 0.990 to 0.999) in both spiked blood and aqueous standard samples using the Orbitrap MS. Of the analytes investigated, serotonin had the lowest RSDs (within-day: 2–5%, between day: 3–7%) while adrenaline had the highest RSDs (within-day: 5–13%, between-day: 14–17%). In blood, the recoveries of the (spiked) neurotransmitters varied from 33% for dopamine up to 91% for serotonin, which were corrected for using isotope labelled internal standards added prior to sample preparation, which was one-step protein precipitation with ACN and 0.1% FA, see Methods section below). Oxytocin and vasopressin (endogenously present at low pg/mL levels8) were also easily detected at quantifiable levels (i.e. signal to noise (S/N) > 20) with the same sample preparation. The concentration limit of detection (cLOD) varied from 0.2 nM for GABA and adrenaline, up to 30 nM for tryptophan (see Supplementary note Calculation of cLOD). For all analytes, carry-over was below the LOD (S/N < 3) for all concentrations tested, except the "XH" level (see Supplementary Table S4 for details), which was approximately 50 times higher of that expected of endogenous levels. Hence, blank injections between samples were not necessary. The same columns (SPE and analytical) were used for the entire study (>1000 injections) without need for regeneration/cleaning.
The analyte stability in whole blood was demonstrated by re-analysing a number of compounds in samples after 1 week storage at 4°C and −20°C, after 24 h, 48 h and 1 week at room temperature, and after two freeze/thaw cycles. The analyte peak areas from the re-analyses were compared to the initial peak areas from day 1. At 4°C, −20°C, and after 2 freeze-thaw cycles, the stability was satisfactory for all the neurotransmitters in the sample (i.e. no decrease in peak areas). Serotonin and GABA were stable at room temperature for 48 h, but not 1 week, while the dopamine peak area was somewhat reduced after only 24 h at room temperature. The other neurotransmitters were stable at room temperature for at least 1 week. In aqueous standards (diluted with ACN/H2O, 70/30), some degree of instability was observed at room temperature and 4°C over night, hence fresh working solutions were made daily, and the stock solutions were stored at −80°C.
Neurotransmitters/precursors could be identified/measured in a single analysis, needing only 100 μL of whole blood, using a novel automated AFFL-SPE-cLC-MS analytical platform.
Several LC-MS methods12,13,14,24,25,26 have been reported for polar NT determination, but disadvantages can be the need for extensive manual sample preparation and large sample amounts, etc. For the more hydrophobic neuropeptides, standard reversed phase (RP) stationary phases have traditionally been used27,28. Disadvantages of such approaches are that they are limited to compounds of some hydrophobicity, and can therefore not be applied for small polar NT without the use of ion-pairing reagents (typically not compatible with MS) or chemical derivatization of the analytes (significantly adding to time spent on sample preparation). To our knowledge, no previous reports have demonstrated the ability to simultaneously handle both very hydrophilic and hydrophobic NTs.
Hence, key advantages of the presented AFFL-SPE-cLC-MS platform include simultaneous handling of polar NTs as well as neuropeptides with enhanced sensitivity (enabling analysis of small samples), a high degree of automation and increased speed.
The sensitivity is much attributed to the high-end mass Q-Exactive™ Orbitrap spectrometer29 employed, but also to the use of capillary columns; using cLC reduces radial dilution of the analytes during chromatography compared to conventional columns16, and employs lowered flow rates; both these features are associated with elevated sensitivity in ESI-MS16,30. The sensitivity of the platform allowed for analyte determinations with internal standards or identification of all compounds except for dopamine, which was present below LOD (S/N ≤ 3). This was somewhat expected due to its rapid metabolism. Therefore, its lack of signal served de facto as a negative control, but will likely be measured in other samples, as the cLOD (determined with spiked samples) was relatively low (below 1 nM). Conventional bore HILIC columns (e.g. 1–2.1 mm IDs) would also be able to handle monoamines, amino acids and peptides simultaneously, but one should expect a decreased sensitivity compared to using cLC. Although a ZIC-cHILIC column was chosen for this platform, the more common ZIC-HILIC column should perform nearly as well, but with some differences in selectivity31, i.e. the ability to separate various compounds.
Simultaneous determination of both polar analytes as well as neuropeptides would be far more challenging with the more common reversed phase LC approach, which separates according to hydrophobicity (typically limiting applicably to compounds with intermediate/low polarity). In contrast, HILIC columns separate compounds based on a number of factors, e.g. partitioning between an immobilized water layer in the stationary phase and the mobile phase, normal phase/adsorption interactions, electrostatic interactions, hydrogen bonding but also reversed-phase (RP) interactions18,32,33,34. The use of HILIC may also enhance sensitivity compared to RP, as relatively high amounts of ACN are used in the HILIC mobile phase, which can increase the MS signal35.
Conventional cLC injections are limited to 5–10 μL injections. Here, we used on-line SPE to perform much larger injection volumes (100 μL) to lower the detection limits without compromising chromatography. On-line SPE also greatly reduces manual steps in sample preparation, which can be a central source of error in analysis36. On the other hand, on-line SPE-LC can be prone to clogging when handling complex samples22. To avoid this, the AFFL feature was employed, ensuring robust performance. AFFL-SPE-cLC is straightforward to install17,22 and is compatible with common commercial LC-MS instrumentation, only requiring one extra pump and one switching valve. Whole blood samples are notorious for clogging LC equipment. We largely attribute the absence of problems analysing whole blood samples to the AFFL feature, which captures particulate matter with a filter that is back-flushed after each injection.
Using a zwitter-ionic HILIC phase also for SPE was superior to using ion exchange (IEX) phases or porous graphitic carbon (PGC) phases; IEX-HILIC and PGC-HILIC were associated with reduced LC performance due to poor retention, or the ZIC-cHILIC separation column being incompatible with aqueous/very salty SPE loading solvents. This does not appear to be a significant issue when employing larger bore SPE-LC systems37, but becomes a factor with small-bore systems (such as cLC), where system void volumes can play a larger role in system performance38. The "HILIC-HILIC" combination for SPE-LC has also been successfully used in proteomics and glycoproteomics38,39. To the authors' knowledge, this is the first report on "HILIC-HILIC" SPE-LC for studying endogenous NTs/metabolites.
To ensure full analyte retention on the HILIC SPE, the aqueous standards were diluted with ACN/H2O (70/30). Based on this, ACN with 0.1% FA was used to precipitate proteins from the whole blood samples, in a 1 + 7.5 ratio (blood + precipitant). This allowed the blood samples to be directly injected onto the AFFL-SPE system, without any evaporation/re-dissolving step, making sample preparation both time-efficient and easy, with a minimum of manual work. Since whole blood could be analysed without plasmafication, additional time and manual work were saved, and the blood sample could be analysed less than one hour after the blood was collected.
When performing targeted determination and quantifications, triple quadrupole (3Q) instruments are often preferred, as they are known for their excellent quantitative capabilities. Orbitrap instruments are however superior29 in resolution, and more typically used for untargeted analysis (e.g. comprehensive proteomics). This study exemplifies that precise quantifications of small metabolites can be successful with an Orbitrap instrument. In fact, linear calibration curves were obtained even without correction with internal standards (Supplementary Fig. S4), which implies relatively trustworthy quantifications for untargeted metabolomics as well. Considering that miniaturized HILIC-Orbitrap-based LC-MS systems work well for both proteomics38,39 and NTs (and likely other metabolites) as described here, it is worth considering that a single instrument set-up similar to that described here can be used for both bottom-up proteomics and metabolomics, allowing their simultaneous determination.
Few methods on quantitative determination of NTs in human whole blood have been published, and reference materials are to the authors' knowledge, not available. However, the endogenous levels quantified by our method were comparable (but somewhat higher) to levels reported in plasma samples (Supplementary Table S2). The reason is likely the high uptake of several neurotransmitters in blood platelets40, and indeed the levels of some of the analytes were lower in plasma, especially serotonin (measured with a non-validated method).
We have demonstrated that AFFL-SPE-cLC-MS can be used for quantifying NTs in small amounts of blood, with minimal sample preparation only including ACN precipitation. The platform can be used for single-run chromatography of a wide range of NTs (monoamines, amino acids and peptides). Next steps will be to continue adding analytes to the platform, towards more comprehensive "neurotransmitter-omics", and validating the quantification of e.g. oxytocin, whose labeled internal standard has become commercially available at the time of submission (Sigma Aldrich, St. Louis, MO, USA). Therapeutics affecting NT activity may also be monitored simultaneously, as HILIC is compatible with drug analysis41.
For method development/validation/measurements, compound mixtures used were: an external standard mixture consisting of commercially obtained compounds (≥99% purity) ("analyte standard mix", see Supplementary Table S3); a mixture of isotopically labelled analogs of the analytes, serving as internal standards ("internal standard mix", see Supplementary Table S3).
All subjects gave written informed consent, and the blood collection was approved by the Regional Ethics Committee (2011/1337/REK S-OE D). All methods were carried out in accordance with the approved guidelines and regulations.
Whole blood samples from veins were collected in vials containing K2 EDTA (BD, East Rutherford, NJ, USA), immediately aliquoted and stored at −80°C until the time of analysis. For validation studies, 8 blood samples were pooled prior to precipitation. In 1.5 mL polypropylene vials (Eppendorf, Hamburg, Germany), aliquots of 100 μL of thawed whole blood were mixed with 50 μL of an internal standard working solution mixture (made by diluting the internal standard mixture 50 times, see Supplementary Table S5 for details). Subsequently, 100 μL of ACN/H2O (70/30) was added (to ensure equal volumes compared to that of spiked samples in validation studies, see below) and finally 750 μL of cold ACN with 0.1% FA was rapidly added. The vials were vigorously vortexed for 20 seconds before kept at 4°C for 20 minutes, to maximize the protein precipitation. Finally, the vials were centrifuged for 10 minutes at 5000 rpm (Centrifuge 5415 R, Eppendorf). Approximately 700 μL of the supernatant were collected, and 100 μL of this were injected directly onto the AFFL-SPE-cLC-MS system.
For all chemicals and equipment used, see Supplementary Chemicals and equipment. An automated filtration/filter backflush (AFFL)-SPE-cLC-MS system22 was used in this study (see Supplementary Fig. S1 for an illustration of the entire system and Fig. 3 for details of the switching system). In position 1 (load) the auto sampler injects 100 μL of sample, and then the sample is pumped through a 1 μm stainless steel filter fitted in a union. The analytes will pass through the filter and be retained on the SPE column, while un-retained non-polar compounds and solvent will go directly to waste. The purpose of the filter is to prevent larger particles from the sample e.g. precipitates, to reach and clog the SPE column. In position 2 (inject) the 10 port valve is switched and the cLC pump elutes the analytes off the SPE column and onto the analytical column. Simultaneously, the SPE pump connected to the auto-sampler will back-flush the filter and wash off particles/precipitates. For pump 1 the loading mobile phase was 100% ACN, the flow rate was 75 μL/min and the loading time 2 minutes. For pump 2 the LC mobile phase consisted of 70% ACN and 30% 100 mM ammonium formate (pH 3, adjusted with 1% formic acid), and the flow rate was 4 μL/min. The column temperature was 30°C. After being separated on the analytical column the analytes will be transferred to the ESI where they are ionized before they enter the Orbitrap MS (See Supplementary Table S1 for all MS parameters).
The method was validated with samples made by spiking 100 μL of pooled in-house whole blood with 100 μL of the analyte standard mix at seven different concentration levels: XL, L, LM, M, HM, H and XH (Supplementary Table S4). The rest of the procedure was the same as for the sample preparation (see section above). The calibration samples were made in the same way as the validation samples, and all samples were analysed on the same day as they were prepared. The within-day repeatabilities were found by analysing 6 sample replicates of validation samples with L, M and H concentration levels on the same day, while validation samples with L, M and H concentrations were analysed on 5 different days to investigate the between-day repeatabilities. The linearity was examined with standard solutions (n = 3) and with validation samples (n = 7). The determination of the cLOD was limited by the lack of blood without NTs (blank matrix). However, a crude estimate was calculated by extrapolation (for details, see Supplementary note Calculations of cLOD).
The recovery was investigated in the following way: one pooled blood sample was spiked with NT analytes (XH level, to minimize contribution from endogenous levels) before protein precipitation, and another pooled blood sample was spiked with NT analytes after protein precipitation. To calculate the recovery, equation (1) was used. A is the peak area of the analyte and Ais is the peak area of the internal standard, respectively.
Stability was tested at room temperature, 4°C, −20°C and with thawing/freezing. Validation samples from the first validation day were re-analysed after 24 h, 48 h, 1 week and after one and two thaw/freeze cycles, and peak areas were compared with that of day 1.
To quantify the amounts of NTs in the whole blood samples, calibration curves based on calibration samples with concentrations from XL to XH (Supplementary Table S4), constructed using Excel, were used. Since the calibration solutions were made by spiking blood which already contained the neurotransmitters of interest, the endogenous levels of NTs were calculated using the regression equation from the calibration curve, corrected for the endogenous level (for details, see Supplementary note Calculation of NT concentrations, and Supplementary Fig. S5).
Supplementary information
The authors declare no competing financial interests.
Author Contributions E.F.J., S.R.W. and E.L. planned and designed the experiments. E.F.J. performed the experiments and data analysis. E.F.J., S.R.W. and S.L. wrote the manuscript. All authors reviewed and approved the paper.
PubMed Central Canada is a service of the Canadian Institutes of Health Research (CIHR) working in partnership with the National Research Council's national science library in cooperation with the National Center for Biotechnology Information at the U.S. National Library of Medicine(NCBI/NLM). It includes content provided to the PubMed Central International archive by participating publishers.


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Atlanta-based Chris Brann is a respected name with his house-music moniker Wamdue Project. His UK #1 hit "King of My Castle," went platinum in territories all over Europe. But Brann's heart lies elsewhere. "House music has never been my main interest, it's more like a bi-product, it's so easily created," says Brann. "I'd rather push the boundaries," he adds. So what makes Brann click? Try his alter egos like P'taah and the Ananda Project.Read More...
P'taah is Brann's opportunity to move out of the house-music spectrum and experiment with alternative approaches to making club and electronic music. Compressed Light was the debut album from P'taah -moving effortlessly between soundtrack-like downtempo jazz cuts to energetic fusions of percussion and club beats. Following "Compressed Light" was "Decompressed", an album of remixes including the huge broken beat anthem "The Crossing" remix by Opaque (aka Seiji). A third album of P'taah material is in the works. Tentatively titled "Staring At The Sun" it's set for release in Spring 2003, with a single featuring Atjazz remixes out in November 2002. The beauty of P'taah is in Brann's ability to combine organic instruments with cutting edge studio trickery. Percussion and future jazz beats collide to provide the back bone of deceptively minimal tracks. The sound of P'taah is unique but carries a hint of early 70s Herbie Hancock, Sun Ra's spacey soul, blaxploitation soundtracks, ECM Jazz, and new school acts like the Cinematic Orchestra and Jazzanova. Read more on Last.fm. User-contributed text is available under the Creative Commons By-SA License; additional terms may apply.
Top SongsTotal plays on Last.fm over the last 6 months
- the crossing - 9,014 plays
- Flyin'high - 8,631 plays
- Hold You Close - 30,671 plays
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- The Crossing (Evacuation of Form) (Opaque remix) - 1,395 plays
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Alternative Titles
Synonyms: Eureka 7 Novel, Eureka Seven Psalms of Planets Novel, Koukyoushi Hen: Eureka Seven Novel
Japanese: 交響詩篇エウレカセブン
Type: Novel
Volumes: 4
Chapters: Unknown
Status: Finished
Published: Oct 29, 2005 to May 31, 2006
Authors: Sugihara, Tomonori (Story), Kishiwada, Robin (Art)
Serialization: None
Score: 7.911 (scored by 283 users)
1 indicates a weighted score. Please note that 'Not yet published' titles are excluded.
Ranked: #11782
2 based on the top manga page. Please note that 'R18+' titles are excluded.
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text: Passive immunization targeting the N-terminal region of phosphorylated tau (residues 68-71) improves spatial memory in okadaic acid induced tauopathy model rats.Sarada Subramanian, Ganesh Savanur, Sowmya MadhavadasBiochemical and biophysical research…2017Alzheimer's disease (AD) is the leading cause of dementia, characterized by progressive loss of memory and other cognitive functions. The cognitive impairment in patients with AD is closely associated with loss of synapses and the formation of neurofibrillary tangles (NFT) containing hyperphosphorylated tau in the hippocampus. Effective treatment for AD is… (More)


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- Saturday, July 1, 2017, 1:30
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Probigua is an organization whose main goal is to promote education in Guatemala, and does this with different projects: A Spanish school that helps them get founds to pay for other projects, a school in Yepocapa, a project called Proyecto Biblioteca, another called Proyecto Bibliobus and scholarships.
The core of the Projects of Probigua is the school in Yepocapa. Yepocapa is an hour and a half from the beautiful Antigua and near the volcanoes Acatenango and Fuego. The school in Yepocapa is unique because it provides the entire education free of charge or offers sliding-scale fees depending on the economical situation of the family. The rest of the cost is funded by donations, the proceeds from the Probigua Spanish Academy and the Scholarship program.
The school of San Pedro in Yepocapa achieves the level of a private school in curriculum and teaching methods. The students are from Yepocapa and the villages surrounding Yepocapa. Two levels of education are offered: Primaria (6 years) and Básico (3 years). The classes align with the Guatemalan official school standard. Students learn mathematics, sciences, arts, sports and recreation. Also taught are language classes with an emphasis on English (for many students, English is their third language after their indigenous language and Spanish). Currently the library in the Yepocapa's school gives students access to substantiated literature and the computer with access to the internet, so the students can be able to expand their education and curiosity to discover new worlds.
Many kids wished they could be available to go to a school like the one in Yepocapa, that's why in 1995 the Scholarship Program started and since this moment many kids have received an opportunity to study, otherwise parents could not afford to send them to school. ach year, Probigua selects scholarship recipients based on their economic need.
With the school running, Rigoberto Zamora Chamuc noticed the need of having a library and in 1998 he developed a Project with the name Bibliobus. The idea for the Bibliobus was rooted in Rigoberto's desire for schools that were not already equipped with a full library to haveaccess to books. Together with the help of Probigua Swiss (Probigua.ch) Rigoberto financed a bus, containing approximately 2500 books. The Bibliobus is an old American school bus, similar to the buses that provide the public transportation in Guatemala. It is painted and equipped with shelves full of books, comfortable old bus seats, and a large table and benches to do homework. It is driven to two different schools each day of the week, enabling the kids to have contact to books on a fixed day every week.
The vision for the school in Yepocapa is to expand to become a boarding school. At the moment, the school in Yepocapa has 475 students. They hope to receive more students from all over the country, because they believe that by offering a more solid education, students will be better equipped to strengthen the fabric of Guatemala.
If you would like to join them in their vision, you can volunteer, make a donation and share ideas with them.
+ Info: 6a. Avenida Norte #41 B, La Antigua
Tel: 782-0860
sitio web: probigua.org
correo: firstname.lastname@example.com


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Kronos Quartet stream new album
Will check this later. Very much enjoy their work.
These guys are legit.
whoa yea reminds me of the tool/apc string quartet tribute albums
Sweet news, loved their work with Mansell.
I have nothing to contribute other than the fact that I hate that NPR has a player that pop's up in a separate window.
I actually enjoy the other window, lets me browse in Firefox while the popup is Chromed.
This is pretty great so far.
Will have to listen to this later, as Acanthus said, their work with Mansell was incredible. I get they didn't write it, so who knows what this will be like
def will listen later this group rules
"classical music band"
I can't fucking stand it when you mother fuckers say "WILL CHECK THIS LATER." No one gives a flying shit if you "will check this later." Shut the fuck up and leave your comment once you've listened or say something else. JESUS CHRIST>
I did say something else, so hopefully that was aimed at others and not me. Can anyone say how along the lines of the Soundtrack work this is?
will check this later
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by TheExcelAddict.com
How To Convert Wrongly Formatted Dates
Have you ever copied or imported data into Excel and realized that the date formats were all wrong. For example, your computer may recognize dates in dd/mm/yy format and you've just copied data, that includes a date column, from another source and the date format there is mm/dd/yy.
You'll notice that Excel recognizes some of the dates but for most it won't. You'll see the dates that Excel has recognized as right-aligned cells and those it didn't recognize, and assumed was text, as left-aligned cells. If you look closely you'll see that even the cells Excel did recognize as dates are wrong - the months and days are switched.
It can be quite a task to fix all of these dates. In the early days I tried various techniques to solve this problem, but all of them were time consuming. Then I discovered a simple little trick using Excel's 'Text to columns' option.
Here's what you can do...
Now, isn't that cool?


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HA NOI (VNS) — Viet Nam's budget private carrier, VietJetAir, intends to seek an Initial Public Offering (IPO) from overseas after the airline swung into VND120 billion (US$5.7 million) pre-tax profit for the first seven months of this year.
The airline's managing director, Luu Duc Khanh, said that the IPO might be made on the Singapore or Hong Kong stock exchanges because the Viet Nam bourses are too small.
The IPO plan was announced in the airline's second year of operation, after the carrier's profit rose beyond expectations.
VietJetAir now operated 500 flights a week with 14 domestic routes - and two international routes connecting Ha Noi and HCM City to Bangkok.
The nation's only private carrier plans to launch more domestic and international routes in the Asian region in the near future - targeting Taiwan, South Korea, Singapore, Malaysia and Japan. — VNS
FLC Group – Vietnam's 'Land Genie' to go international
Hoàng Huy Finance, Navistar Inc sign $1.8b agreement
5 Seasons – An Ideal Place for the Modern Families
Veterans' message to APEC 2017!
Domino effect in condotel investment in Hà Nội
"Live Well - Stay Healthy" at 5 Seasons
APEC 2017: Cooperation and renewal of Maternal and Child Health Programme


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I'm consciencious, clean and tidy and do my share of housework/responsibilities. More on request :)
I'm a 29 yo veterinarian and I've just recently moved back to Australia after travelling around the world for 2.5 years. I'm looking for a place to call home! I'm laid back and I am generally considered very clean and tidy. I have one very friendly 7 yo collie X called Deni who is predominantly an outdoor dog (good behaviour guaranteed by a vet!). My long list of hobbies include: photography, painting, sketching, yoga, krav maga, welding, woodworking, cooking and trying to learn Spanish. If you think we'd make amazing housemates or want to teamup, please get in touch!
Hey Guys, Its pretty important to me I can set up shop with some rad fellas/gals and hopefully around the listed areas, but open to suggestions. Migrating from Brisbane 4+ years ago I am currently working as a PM/Estimator (Mon-Fri) while looking to further myself within the electrical industry and I consider myself to be a pretty chilled out guy and get along with everyone, big pet lover, Rugby fella with the addition of occasional sporadic furniture making. I come complete list of friday afternoon drinks and my complied best beer and burger combos in Melbourne (subjectively opinionated) Scored pretty well in my house training traineeship so you can expect all the good stuff of a quality housemate and I have lived in all girl houses previously so when you need something squashed, moved or someone to anwser the door my hand will go straight up. I'm always down for a hang or a cheeky pint and will often grab my camera and go on an adventure until I get lost But eventually find myself back home with a good story! I'd love to hear about you if you think I might be a good fit otherwise good luck with the hunt! :)
Hi I'm Adelaide :) I'm under application for a couple of 2 bedroom apartments in Brunswick near Sydney Road and so I'm looking for a housemate to join me in my new home. Price range is 195-225 each per week without bills. I work full time in the city so I'm looking for someone with a similar lifestyle - basically I want somewhere calm on weeknights so I'm not sleep deprived at work. I have all the furniture we need for the communal rooms (fridge, couch, table, washing machine, dryer, tv, indoor plants which in my mind are essential) so you'd only have to worry about bedroom stuff! I'm always happy to have a chat over a wine/beer/tea but I know when to let people have their space too :) if you think we would be a good fit as housemates send me a message and I'll give you some more details on my applications :)
Hi, my name is Inken I'm a 24 year old female from Germany. I arrived in September in Australia and start traveling trough Australia with my girlfriend Kristin (28). We are both open minded girls who would love to meet new people. Kristin is looking for work in her profession as Orthopedic Technican to earn some money for our next roadtrip. I got an apprenticeship as an early childhood educator but I would rather do any other job :D! We are looking for a clean and comfortable place to stay for the next 6-8 weeks. We're not crazy party people but enjoy hanging out, having a beer with friends and listen to some good music on the weekends and some Netflix and Chill on Weekdays. If you want us to be your knew flatmates, don't hesitate to contact us! Cheers Inken and Kristin
im clean i am a bubbly person who is easy to talk to, i like going out on the weekends and easy to get along with.
Moving to Melbourne in February to start studying at the University of Melbourne. I am from Perth, enjoy cooking, live music and various other things. Hoping to find some other people in a similar situation that may want to team up, hit me up peeps!
Hey! My name is Emily, I'm a barista and graphic design student looking for somewhere short term until April. I bring home cafe food all the time! I cook tons and am a market enthusiast. I also love getting to know who I'm living with a little, chilling out and watching TV on occasion is a good time! I'm not home a heck bunch, I'm really tidy but super easygoing. Hit a sister up!
My name's Jordan and I'm a 21 year old creative from the Gold Coast. I studied Film at University and graduated in early 2016; I've since been living and working in Sydney. After over a year's production work, I've recently returned from a much needed Euro trip, where I spent a few months between Paris, Berlin and Amsterdam. I've recently made the drive down to Melbourne for a new job and am looking for a short term sublet, where I can settle in, apply for my own lease and prepare for the following working year. On my weekends I like to work on my own personal projects, taking photos, soaking up the sun in the gardens and exploring new places. I'm ultimately looking for a bunch of down to earth, open minded roommates who I can have a drink and chat with at the end of the day! Look forward to hearing from you soon.
I just finished uni in Canberra and I'm moving to Melbourne to start a job at a law firm in the CBD. Love to travel, been to around 40 countries so far. I love to play sport and try to get to the gym (but to be honest it's more of an ambition than a habit). I'm a good cook but awful at planning meals. I'm also a politics junkie and enjoy some good political satire on abc. I've previously leased an apartment in Canberra, stayed in student accommodation in the UK on uni exchange and made my way through countless hostel dorm rooms. I'm looking for housemates that are keen to hang out and chat/eat/drink but also understand that I sometimes need my own space. I'd be interested in sharing meals but am also fine to cook for myself. Location is important but I'd much rather find the right group of people who have similar values to me. If you like binging tv shows, exploring the city, going on walks and bike rides and want someone who is friendly, clean, low maintenance and easy to live with, then I could be your guy. It's a cliche but I'm looking for a home not a house. "It's the vibe" - The Castle
Hi! My name's Sam and I'm looking for a new place to call home. I'm a 29 year-old male working in tech support and studying physics at RMIT (not to be confused with psychics… I cannot tell the future, sorry in advance). I keep pretty busy during the week, but love to come home to hang out, share a meal (pumpkin pesto pasta is my specialty), have a few drinks, listen to some music or binge watch a TV series or two (currently obsessed with Stranger Things). I also apologise in advance for getting too emotionally invested in the footy. I'm really considerate and clean, especially of all the shared spaces in the house. I've lived in share houses before, so I'm pretty comfortable with all the unwritten rules of housemate life. Ideally, I'm looking at anything from $500-760 p/month. Location wise, inner city is good but mostly it's about finding great housemates who can become great friends. If you've got a spare room, or know someone who does, get in touch. Cheers Sam
I'm a 24 year old software developer who works full time in Brunswick. When I'm not working you can usually find me binging a TV show, playing some games or hitting the gym. I'm a self-professed animal lover so if your place has cats or dogs that's a big plus for me. I'm also a little too into trivia, so if you know 80s music you may be dragged along! I'm in a relationship so if the place is suitable for my partner to come and stay every once in a while that would be perfect! If not, that's also fine, it would just mean that I'm not around some nights. I look forward to meeting you!
Hey guys, I work in the renewable energy biz, teach yoga and carry out other side projects. I've lived in Melbourne for many years but it seems not long enough for my French accent to disappear! I have been sharing places for a long time too and want to be part of a house with mature peeps who are independent and yet appreciate socializing when feeling like it. My thumbs are green, I play music and like good conversations over nice dinner when the occasion is there. Melbourne is such a vibrant city so I am keen on living there with like-minded relaxed housemates (:. Cheers, Fred


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Written by ju111 on November 15, 2017
I have seen some people sharing my language/country. Thinking that it would be interesting to have a chat with them, I am planning to invite them on an external website, using private messages.
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Sheridan Healthcare Inc (Sheridan Healthcare), a subsidiary of AmSurg Corp is a healthcare service provider that offers physician services in the areas of anesthesiology, neonatology, emergency medicine and radiology. The center also provides office-based services in the areas of OBGYN, gynecology, gynecologic oncology, perinatology and pain management. It provides support, training and management in non-clinical areas. Sheridan Healthcare partners with hospitals and medical facilities. The center serves physicians, hospitals and outpatient centers. It operates through its various facilities across the US. Sheridan Healthcare is headquartered in Sunrise, Florida, the US.
Sheridan Healthcare Inc – Pharmaceuticals & Healthcare – Deals and Alliances Profile provides you comprehensive data and trend analysis of the company's Mergers and Acquisitions (M&As), partnerships and financings. The report provides detailed information on Mergers and Acquisitions, Equity/Debt Offerings, Private Equity, Venture Financing and Partnership transactions recorded by the company over a five year period. The report offers detailed comparative data on the number of deals and their value categorized into deal types, sub-sector and regions.
GlobalData derived the data presented in this report from proprietary in-house Pharma eTrack deals database, and primary and secondary research.
- Financial Deals – Analysis of the company's financial deals including Mergers and Acquisitions, Equity/Debt Offerings, Private Equity, Venture Financing and Partnerships.
- Deals by Year – Chart and table displaying information encompassing the number of deals and value reported by the company by year, for a five year period.
- Deals by Type – Chart and table depicting information including the number of deals and value reported by the company by type such as Mergers and Acquisitions, Equity/Debt Offering etc.
- Deals by Region – Chart and table presenting information on the number of deals and value reported by the company by region, which includes North America, Europe, Asia Pacific, the Middle East and Africa and South and Central America.
- Deals by Sub-sector – Chart and table showing information on the number of deals and value reported by the company, by sub-sector.
- Major Deals – Information on the company's major financial deals. Each such deal has a brief summary, deal type, deal rationale; and deal financials and target Company's (major public companies) key financial metrics and ratios.
- Business Description – A brief description of the company's operations.
- Key Employees – A list of the key executives of the company.
- Important Locations and Subsidiaries – A list and contact details of key centers of operation and subsidiaries of the company.
- Key Competitors – A list of the key competitors of the company.
- Key Recent Developments – A brief on recent news about the company.
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Reasons to Buy
Get detailed information on the company's financial deals that enable you to understand the company's expansion/divestiture and fund requirements
- The profile enables you to analyze the company's financial deals by region, by year, by business segments and by type, for a five year period.
Understand the company's business segments' expansion / divestiture strategy
- The profile presents deals from the company's core business segments' perspective to help you understand its corporate strategy.
Access elaborate information on the company's recent financial deals that enable you to understand the key deals which have shaped the company
- Detailed information on major recent deals includes a summary of each deal, deal type, deal rationale, deal financials and Target Company's key financial metrics and ratios.
Equip yourself with detailed information about the company's operations to identify potential customers and suppliers.
- The profile analyzes the company's business structure, locations and subsidiaries, key executives and key competitors.
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Stay up-to-date on the major developments affecting the company
- Recent developments concerning the company presented in the profile help you track important events.
Gain key insights into the company for academic or business research
- Key elements such as break up of deals into categories and information on detailed major deals are incorporated into the profile to assist your academic or business research needs.
Note*: Some sections may be missing if data is unavailable for the company.
View Detailed Table of Content @ https://www.htfmarketreport.com/reports/254156-sheridan-healthcare-inc-pharmaceuticals-healthcare-deals-1
Contact us
Craig Francis
+1 206 317 1218
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Then You're Going To Love This Compelling Case For Trading Jogging For Walking. By Paige Greenfield September 3, 2015. walking vs running.
Oct 15, 2013 ... Here's how many you'll burn briskly walking compared to jogging: Walking: 60- minute walk at 4 mph (15 minutes per mile): 243 calories burned
Feb 5, 2016 ... What's the difference between jogging and walking? Find out more here.
May 29, 2013 ... Gretchen Reynolds on the science of fitness. Walking and running are the most popular physical activities for American adults. But whether one ...
Take a stroll down any trail in any city and you're bound to meet at least a few people jogging or walking. While each person's reason for doing so is...
Brisk walking and jogging both fall into the category of aerobic exercise, but there are some differences between these two low-impact options. Aerobic exercise ...
2 Easy Running Plan to Lose Fat; 3 Brisk Walk vs. ... Walking and jogging are both aerobic activities that increase your heart rate, burn calories, improve bone, ...
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While walking can provide many of the same health benefits associated with running, a growing body of research suggests running may ... Running Vs. Walking.


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by Tomasz
(Calahonda Royale, Malaga, Spain)
LR Health & Beauty Natural German product for you and your family.
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Post your own submission to the Marbella Community! It's easy to do. How? Simply click here to return to INPUT Freebie.
Nov 13, 17 10:47 AM
Marbella Thanksgiving? That is right you can celebrate this American holiday right here on the Costa del Sol.
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PLEASE do not forget to enter your email address and tick the box where it says NOTIFY ME WHEN...SOMEONE COMMENTS ON MY QUESTION after you click the SUBMIT
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English Movie Listing - Original version movies showing in Marbella. Updated and published weekly. Share it with your movie buff friends.


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/v/rage 2017 remake.
an animation about the /v/ board on 4chan.4.40 / 5.00 5,565 Views
Pmation: "Unchairable"
When the lazyboy cannot be lazy…4.07 / 5.00 965 Views
Dinosaur thing
Dinosaurs are cool3.96 / 5.00 2,032 Views
The Series are so weird. I don't what kind of emotions I should have toward salad fingers.
creepy in the good way!
please make saladfingers 9!
Piece of art!
This is the greatest single flash....ever.<3
I have followed these for quite a while.
And they just keep getting better. (I like #6 the best.)
Love the salad fingers serie, great work dude !!!