L. Ren Jenny Renaut Nicole Richoux Jan Rijstenbil Amy Ringwood F. Robledano Riccardo

Rodolfo-Metalpa G. Roesijadi Sergio Rossi G.L. Sanchez Gianluca Sarà N.S. Sarma J. Sarrazin Nicolas Savoye Felicita Scapini Doris Schiedek K.B. Schneider Michaela Schratzberger M.S. Sepulveda S. Shang Jenny Shaw Jian Shen K. Sherman Graham Sherwood P.V. Shirodkar Laura Sigg Stuart Simpson Annelie Skoog Vera Slavekova M. Smirnoff Akio Sohma Montserrat Sole Luis Soto Manu Soto M.F.L. Souza Alan Springer Annaamlai Subramanian Alex Sukhotin Teri Sutherland David Sutherland S. Taljaard Heather Tallis S. APO866 purchase Tanabe Antonio Terlizzi Jorge Terrados Johannes Teuchies Peter Thomas Richard Thompson M.F. Tognelli Moshe Tom Brant Touchette Ashley Townsend Clara Turetta Andrew Turner David Turner R.E. Turner Niklas Tysklind Wann Tzeng Richard Unsworth J.P. Valdes Herman

Van Leeuwen Jamie Vaudrey Aldo Viarengo Pierluigi Viaroli Vjercocka Vojvodic Terry Wade D. Wagner Wen Wang Wen Xiong Wang Wendy Wang Bess Ward Michel Warnau Michael Wetz Steve Widdicombe John Widdows Claudia Wiegand Steven Wilhelm Isaac Wirgin David Wright Rudolf Wu X. Xia Dawit Yemane Jennifer Yordy Jian-xin Zhao Izaskun Zorita Mikhail Zubkov Full-size table Table options View in workspace Download as CSV “
“A salient change Ivacaftor in a sound is likely to draw our attention to its location (Näätänen, 1992). Similarly, a salient change in prosody can trigger anticipatory attention to upcoming grammatical information ( Roll and Horne, 2011). An illustrative example is Central Swedish, where word stems with a high tone, e.g. lekH–‘game’ are followed by a certain class of suffixes including plural –ar, as in lekH–ar ‘games.’ Low stem tones are followed by another class of suffixes, including the singular definite morpheme –en as in lekL–en ‘the game’

( Fig. 1A). Since the choice of stem tone depends on which suffix is attached to the stem, suffixes can be referred to as ‘high tone-inducing’ (e.g. plural –ar) or ‘low tone-inducing’ (e.g. singular definite –en). The perception of Thymidylate synthase a rise to a high stem tone has previously been found to produce an increased P2 component at 200–300 ms after onset in event-related potentials (ERP) ( Roll et al., 2010). The positivity has been thought to indicate allocation of passive anticipatory attention to the tone-inducing suffixes ( Roll and Horne, 2011 and Roll et al., 2011b). High tone-inducing suffixes (e.g. plural –ar) not preceded by their required high stem tone accordingly produced a P600-like effect at 400 to 900 ms after their onset, indicating that they were unexpected.

In both models it is possible to produce a ligand distribution that is Selleckchem PI3K Inhibitor Library closer to observations than a uniform low value. Moreover, the models reproduce some observed features well, such as a decrease along the conveyor belt circulation (e.g., Thuróczy et al., 2011 and Mohamed et al., 2011) a general decrease of ligand concentrations from the mesopelagic towards the deep ocean (e.g., Ibisanmi et al., 2011), and a horizontally and temporally variable concentration of ligands near the surface, with higher ligands e.g. near the European shelf seas. Both models also make strong predictions regarding the

gradient in ligand concentrations between regions of high and low productivity (e.g. between upwelling regions and the subtropical gyres) that can hopefully be tested in future fieldwork. In the model at least, this gradient is strongly dependent on the assumed photochemical degradation rate. Ultimately, the predictions of the model are regulated by the sources and sinks associated with each specific process (Table 2). In this regard, STA-9090 datasheet process studies such as FeCycle that document

the time evolution of iron–ligand dynamics (Boyd et al., 2012) can provide important information for modeling efforts. For example, the maximum rates of ligand production from organic matter remineralization reach 0.25 and 0.05 nmol L− 1 d− 1 in PISCES and REcoM, respectively, of similar order, but towards the low end of Bay 11-7085 the two estimates of 0.3 and 1.3 nmol L− 1 d− 1 from Boyd et al. (2010). Further such experiments that normalize the rate of ligand production to carbon solubilization would prove invaluable. Equally so, experimental constraints on the bacterial, photochemical and aggregation losses of ligands

would allow tighter constraints to be placed on these parameters. Modeling the ligand distribution dynamically instead of assuming a uniform and low constant concentration brings the average vertical profile of iron closer to the observed nutrient-like profile with a maximum near the oxygen minimum in the mesopelagic. However, as the ligand concentrations are now greater than those used previously, this raises the iron concentrations in the non-iron limited regions of the ocean such as the Atlantic and Indian oceans. A useful way to evaluate this effect is by looking at the excess ligand, denoted as L⁎ (e.g. Boyd and Tagliabue, 2014-in this issue), which is defined as: ligand minus dissolved iron. Our two models clearly overestimate the prevalence of negative L⁎ regions relative to that observed ( Fig. 8). The distribution of negative L⁎ in the models reflects external inputs of dissolved iron and highlights too low scavenging rates of uncomplexed iron. In REcoM negative L⁎ regions are restricted to the dust deposition regions, while in PISCES the large sedimentary iron fluxes that are absent in REcoM are also important ( Fig. 7).

It is important to develop interventions to reduce the impact of treatment-related late effects on morbidity and mortality and to continue research regarding the etiopathogenesis of therapy-related cancers and other late effects. Malcolm A. Smith and Gregory H. Reaman Despite the enormously important and gratifying advances in cancer treatment outcomes for children with cancer, cancer remains the biggest cause of death from disease in children.

Because the etiology and biology of cancers that occur in children Pifithrin-�� mouse differ dramatically from those that occur in adults, the immediate extrapolation of efficacy and safety of new cancer drugs to childhood cancer indications is not possible. We discuss factors that will play key roles in guiding pediatric oncologists as they select lines of research to pursue in their quest for more effective treatments for children with cancer. Index 313

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“Catherine S. Manno Karen S. Fernández and Pedro A. de Alarcón This article reviews the ontogeny of hematopoiesis (embryonic/fetal/newborn phases) and its regulation and provides examples of the disorders of Hydroxychloroquine hematopoiesis that present in the newborn or infant and their pathophysiology. Many of these disorders are discussed in depth in other articles of this issue. S. Deborah Chirnomas and Gary M. Kupfer Molecular pathogenesis may be elucidated for inherited bone marrow failure syndromes (IBMFS). The study and presentation

of the details of their molecular biology and biochemistry is warranted for appropriate diagnosis and management of afflicted patients and to identify the physiology of the normal hematopoiesis and mechanisms of carcinogenesis. Molecular motor Several themes have emerged within each subsection of IBMFS, including the ribosomopathies, which include ribosome assembly and ribosomal RNA processing. The Fanconi anemia pathway has become interdigitated with the familial breast cancer syndromes. In this article, the diseases that account for most IBMFS diagnoses are analyzed. Helge D. Hartung, Timothy S. Olson, and Monica Bessler This article provides a practice-based and concise review of the etiology, diagnosis, and management of acquired aplastic anemia in children. Bone marrow transplantation, immunosuppressive therapy, and supportive care are discussed in detail. The aim is to provide the clinician with a better understanding of the disease and to offer guidelines for the management of children with this uncommon yet serious disorder. Char M. Witmer A complete blood cell count (CBC) is a frequent test sent to aid in the diagnostic evaluation of ill patients. Not uncommonly hematologic abnormalities may be the first sign of an underlying systemic disorder.

The subjects were 193 ambulatory patients with osteoporosis (189 postmenopausal women and 4 men; age range: 52–85 years, average ± SD: 70.9 ± 6.92 years), who represent a subgroup of a randomized, active comparator, double-blind study to compare the anti-fracture efficacy of ELD with that of ALF in 1054 subjects (1030 women and 24 men, BMS-354825 order aged from 46 to 92 years, mean age: 72.1 years) enrolled at 52 medical centers

in Japan [20]. In that study, subjects were randomly assigned to receive either 0.75 μg ELD or 1.0 μg ALF once daily for 144 weeks. This trial is registered with ClinicalTrials.gov, number NCT00144456. The protocol was approved by the internal human studies review board at each center, and written informed consent was obtained from each patient. The proximal femur of the 193 subjects was scanned with MDCT at 11 institutions to measure hip BMD, selleck bone geometry, and biomechanical indices. We did not intentionally select the subjects. Since not all institutes had an MDCT scanner, the 193 subjects were those examined and treated in hospitals which had MDCT scanners. All subjects in this study fulfilled the inclusion criteria of the original

study. In brief, in the original study, subjects without vertebral fractures were enrolled if their lumbar spine or total hip BMD T-score was below − 2.6 and they were over 70 years, or if their T-score was below − 3.4 and they were below 70 years. Patients with lumbar spine or total

hip BMD T-score of below − 1.7 were enrolled if they had between one and five vertebral fractures. Prevalent vertebral fractures at enrolment were assessed by lateral spine X-ray examination of the thoracic and lumbar vertebrae, and were diagnosed quantitatively according to the criteria of the Japanese Society for Bone and Mineral Research (JSBMR) [24]. Women were at least 3 years after menopause or more than 60 years of age. Patients were excluded if they had primary hyperparathyroidism, Cushing’s syndrome, premature menopause due to hypothalamic, pituitary or gonadal insufficiency, poorly controlled diabetes mellitus (HbA1c over 9%) or other causes of secondary osteoporosis, or had a history of urolithiasis. Patients Coproporphyrinogen III oxidase were also excluded if they had taken any oral bisphosphonates within 6 months before entry or for more than 2 weeks during the period 6 to 12 months before entry, or intravenous bisphosphonates at any time; had taken glucocorticoids, calcitonin, vitamin K, active vitamin D compounds, raloxifene, or hormone replacement therapy within the previous 2 months; had serum Ca levels of above 10.4 mg/dL (2.6 mmol/L) or urinary Ca excretion of over 0.4 mg/dL glomerular filtrate (GF)(0.1 mmol/L GF); had serum creatinine above 1.3 mg/dL (115 μmol/L); or had clinically significant hepatic or cardiac disorders. CT data was acquired at baseline and at completion of 144 weeks of treatment, using the following scanning and reconstruction protocol.

The first aliquot (control) was subjected to a slow freezing Selleck Trichostatin A curve previously described for collared peccaries [7]. In this protocol, the aliquot was stored in a water jacket (30 mL) at 27 °C and equilibrated for 240 min to reach 5 °C in a biological oxygen demand (BOD) incubator (Quimis, Diadema, SP, Brazil). At that point, the sample was added to the extender with 6% glycerol (also at 5 °C), which resulted in a final concentration of 3% glycerol

in the extender, and the sample was then evaluated. Finally, the semen aliquot was divided and packed into 0.25 mL or 0.50 mL plastic straws (IMV Technologies; L’Aigle, France) that were placed horizontally in an insulated box for 20 min, at 3 cm above the nitrogen (N2) vapors, and then

plunged into N2 for storage at −196 °C, following a slow http://www.selleckchem.com/products/AZD2281(Olaparib).html freezing rate at −10 °C/min. The second aliquot was cryopreserved following a fast freezing curve described by Silva et al. [35]. Semen aliquot was stored in the water jacket at 27 °C and equilibrated for 40 min to reach 15 °C in a BOD incubator (Quimis, Diadema, SP, Brazil). Further, BOD incubator was adjusted to establish at 5 °C for 30 min. Then, the glycerol addition and package was conducted as described for the first aliquot. However, the straws were placed at 5 cm above the N2 vapors for 5 min, and then finally plunged into N2 at −196 °C for storage, following a fast freezing rate at −40 °C/min. In both groups, the digital thermometer of the BOD incubator monitored the cooling rate up to 5 °C. Further, the Depsipeptide probe of an appropriate thermometer was inserted into the insulated box containing N2 vapors in order to monitor the cooling rates. After 1 week, three 0.25 mL and 0.50 mL straws derived from each of two freezing curves were thawed on a water bath, at 37 °C/1 min, and others at 70 °C/8 s, following a further 30 s at 37 °C. The semen was immediately evaluated, as per the same parameters reported for fresh semen and also for kinematic parameters of sperm motility by computer-assisted semen analysis – CASA, which will be described later. The thawed semen was

diluted in ACP-116c® on a proportion of one part semen to one part extender; then, it was evaluated by CASA, as described by Verstegen et al. [37]. Samples (10 μL) were placed in a Makler chamber, allowed to settle for 1 min and maintained at 38 °C. They were then examined in a phase contrast microcopy system with stroboscopic illumination, coupled with a video camera adapted to the Sperm Class Analyzer (SCA Version 3.2.0; Microptic s.l., Barcelona, Spain). The settings of the instrument were adjusted according to the boar semen, including temperature, 37 °C; frame rate, 25 frames/s; minimum contrast, 75; straightness threshold, 45%; low-velocity average pathway (VAP) cut-off, 10; and medium VAP cut-off, 25. Three independent and nonconsecutive microscopic fields were randomly selected and scanned.

DNA was extracted using standard methodology (QIAamp DNA kit, Qiagen, West Sussex, UK). Sequencing was performed using standard procedures with dye terminators. The fragments were separated by capillary electrophoresis (ABI Prism 3130 genetic

analyser, Hitachi Ltd., CA, USA). The candidate gene (SLC34A3) was sequenced in 12 fragments consisting of one exon plus ~ 20 base pairs on either side of the exon in the youngest affected sibling (S1*). All Apoptosis inhibitor single nucleotide polymorphisms (SNPs) were identified and the remaining siblings (n = 4) and mother were then screened for the variant SNPs. All SNPs were analysed using the NCBI (National Center for Biotechnology Information) database according to the GenBank transcript NM_080877.2. In silico mutation evaluation to predict protein structure, was conducted using two programmes: Mutation taster [8] and PolyPhen-2 [9]. Sequence alignment was performed using the Basic Local Alignment Search Tool (BLAST®) on the NCBI database. Case 1 Sibling 5* (S5*). S5* (female) was the eldest of the five siblings and first presented with knock-knee deformity and bone pain at the age of 12 y in July 2000. She was short and light for her age relative to local age matched children (Table 2). Biochemical analysis of a blood sample revealed that she had concentrations of 25OHD and

Ca within the normal range, PTH was low with elevated concentrations of 1,25(OH)2D and TALP. In addition she had low plasma 5-FU concentration P with a normal concentration of FGF23. Urine analysis confirmed a low TmP/GFR and hypercalciuria.

Radiographs confirmed S5* to have active rickets with a Thacher score of 4 and evidence of Looser zones and growth arrest lines. Table 2. Biochemical data of affected (S5*, S2* and S1*) and unaffected (S3 and S4) siblings and their mother. Z-scores were calculated from age-matched www.selleck.co.jp/products/Docetaxel(Taxotere).html data from the local community. S3 (F), S4 (F) and the mother of the children were seen in July 2006 and were aged 11, 15 and 35 y respectively (the father did not consent to examination or biochemical). They showed no clinical bone deformities, but no radiographs were taken to confirm this. S3 and S4 were both short and heavy for their ages. Their biochemical profiles were largely normal, however, S4 had a lower than average plasma P and TmP:GFR for her age, albeit not as low as her affected siblings and both the mother and S3 had a higher than average uCa excretion (uCa:uCr). No signs or symptoms of nephrocalcinosis were reported in any of the siblings or the mother. Prior to the completion of the investigations, S5* and S2* were treated with calcium and vitamin D with little or no clinical and radiological responses although biochemically 25OHD and 1,25(OH)2D concentrations did rise.

3–0.7 × 105 cells/well) and incubated to allow cell adhesion or equilibration (suspension cultures). Twenty-four hours later, extracts were added to each well (0.004–50 μg/mL). After 69 h of incubation, the supernatant was replaced with fresh medium containing 10% MTT, and the cells incubated for an additional 3 h. The plates were then centrifuged and the formazan product was dissolved in DMSO; absorbance was read at 595 nm. The selectivity

of the extracts see more was investigated in human PBMC using the Alamar Blue™ assay. PBMC were washed and resuspended (3 × 105 cells/mL) in supplemented RPMI-1640 medium plus 4% phytohemagglutinin for growth stimulation. PBMC were then plated in 96-well plates (3 × 105 cells/well selleck kinase inhibitor in 100 μL of medium). After 24 h, extracts dissolved in DMSO were added to each well (0.004–50 μg/mL) and the cells were incubated for 72 h. Twenty-four hours before the end of the incubation, 10 μL of Alamar Blue™ stock solution (0.312 mg/mL) (Resazurin; Sigma Aldrich Co., USA) were added to each well. The absorbance was read at 570 and 595 nm and the drug effect was expressed as the percentage of the control (Ferreira et al., 2011b). The extracts were assayed

for hemolytic activity according to the method of Santos et al. (2010), with some modifications. Extracts (1.56–200 μg/mL) were incubated in 96-well plates for 60 min at room temperature (25 °C) in a suspension of human erythrocytes (2%) in 0.85% NaCl containing 10 mM CaCl2. After centrifugation, hemoglobin levels in the supernatants were spectrophotometrically determined at 540 nm. The BrdU assay is a reliable in vitro non-radioactive method, which is very often used to directly quantify

cell proliferation ( Costa et al., 2008 and Ferreira et al., 2010). Accordingly, HL-60 cells were plated in 24-well tissue culture plates (1 mL/well) and treated with R. marina extracts (RMF-1, RMF-2, RMF-3, RMF-4 and RMM-5) at concentrations of 0.1 and 1 μg/mL for 24 h. Before the end of drug exposure, 10 μL of 10 mM 5-bromo-2′-deoxyuridine (BrdU) were added to each well and the cells incubated for an additional 3 h at 37 °C. To determine the amount of BrdU incorporated into DNA, cells were first Immune system harvested, transferred to cytospin slides, and allowed to dry for 2 h at room temperature ( Pera et al., 1977). Cells that incorporated BrdU were labeled by direct peroxidase immunocytochemistry, using the chromogen diaminobenzidine (DAB). Slides were counterstained with hematoxylin. Cells were scored for BrdU positivity by light microscopy (Olympus, Tokyo, Japan), where 200 cells were counted per slide to determine the percentage of BrdU-positive cells. The IC50 and EC50 values and their 95% confidence intervals were obtained by nonlinear regression using the GraphPad program (Intuitive Software for Science, San Diego, CA). Differences were evaluated by comparing data using one-way analysis of variance (ANOVA) followed by the Newman–Keuls test (p < 0.05).

The objective of this work is to provide

an assessment of the combined effect exerted by binary mixtures by measuring the spontaneous electrical activity of in vitro neural networks grown on multielectrode array (MEA) chips. In vitro neuronal networks are a simplified and accessible model of the central nervous system, exhibiting morphological and physiological properties ( Kriegstein and Dichter, 1983) and activity-dependent path-specific synaptic modification Natural Product Library high throughput similar to the in vivo tissue ( Bi and Poo, 1999 and Jimbo et al., 1999). Cortical neurons grown on MEA chips have been shown to be a valuable tool to study fundamental properties of neuronal network activity ( Gross et al., 1999, Maeda et al., 1995 and Pasquale et al., 2008), synaptic plasticity ( Jimbo et al., 1999 and Maeda et al., 1998), in vitro learning ( Eytan et al., 2003, Novellino et al., 2007 and Shahaf and Marom, 2001) and perform functional pharmacological screening ( Chiappalone et al., 2003, Gramowski et al., 2006 and Morefield et al., 2000;) and toxicological profiling ( Gross et al., 1997, Johnstone et al., 2010, Novellino et al., 2011, Shafer et al., 2008 and Streit, 1993). In a recent work published by our group (Novellino et al.,

2011) three compounds exerted inhibition of spontaneous activity at a similar magnitude compared to what previously observed in vivo and on primary cultures ( Darbin and Wichmann, 2008, Heinke et al., 2004 and Wada et al., 1995). These results support the MEAs as potential alternative toxicity testing method for neurotoxicity screening. However the Selleckchem Ivacaftor prediction of in vivo effects should rely on an integrate

approach where in vitro data are supported with other studies. There are few studies concerning the application of MEAs to study mixtures toxicity. Johnstone et al. (2009) and Losa et al. (2009) have studied the concentration–response relationships of a mixture of 5 different pyrethroid insecticides (permethrin, cypermethrin, almost cyfluthrin deltamethrin and esfenvalerate), observing a decreased spontaneous spike rate in a manner that was not effect additive. However, no detailed calculation was performed. In this work, the effects on spontaneous activity of in vitro neuronal networks coupled to MEAs have been studied using several binary mixtures. We combined inhibitory and excitatory neuroactive compounds with similar and different mode of action in binary mixtures with the aim of characterizing and assessing their joint effects. Individual and binary mixtures dose–response curves have been generated. Concentration Addition and Independent Action frameworks have been used to compare calculated and experimental results. In addition, Nuclear magnetic resonance (NMR) spectroscopy has been employed to assess that no chemical reaction or complexation took place between mixture components, as well as to monitor the presence of potential impurities.

The transition from knowledge to adaptive pain coping can be see more enhanced by using the Pain Reaction Record (Sullivan,

2003), an easily applicable measure facilitating a cognitive approach to pain coping. Pain physiology education is a continuous process initiated during the educational sessions prior to commencing active treatment (i.e. rehabilitation) and followed-up during the rehabilitation program. Indeed, pain physiology education is typically followed by various components of a biopsychosocial-oriented rehabilitation program, like stress management, graded activity and exercise therapy. It is important for clinicians to introduce these treatment components during the educational sessions, and to explain why and how the various treatment

components are likely to contribute to decreasing the hypersensitivity of the central nervous system (as explained in Nijs and Van Houdenhove, 2009 and Nijs et al., 2009). Changing illness perceptions changes the patients motivation to undertake and comply with PD 332991 the rehabilitation program. Likewise, long-term reconceptualization of pain, alterations in illness beliefs and adaptive pain cognitions are required at every stage of the rehabilitation program. This can be done easily by asking the patient to explain the treatment rationale of a specific treatment component. If during the treatment course any of the pain cognitions or illness beliefs have ‘reset’ towards maladaptive ones, then the therapist is advised to re-educate the patient. The latter can be accomplished by asking the patient to re-read the written information on pain physiology and to try to link that information with his/her current rehabilitation program. Long-term adaptive pain perceptions, and consequent adaptive pain coping strategies are required for long-term treatment compliance and

continuous Grape seed extract application of self-management strategies. Finally, frequent side-effects and symptom fluctuations can be explained using the central sensitization model (van Wilgen and Keizer, in press). The latter should shift the patient’s attention away from somatic signs towards adaptive coping strategies and reassurance. The patient’s confidence in the treatment (outcome) should be a continuous treatment goal in those with chronic musculoskeletal pain. There has been increased awareness that central sensitization provides an evidence-based explanation for many cases of ‘unexplained’ chronic musculoskeletal pain. Hence, rehabilitation of patients with chronic musculoskeletal pain should target, or at least take account of the process of central sensitization. Prior to commencing rehabilitation in such patients, it is crucial to change maladaptive illness beliefs, to alter maladaptive pain cognitions and to reconceptualise pain. This can be accomplished by patient education about central sensitization and its role in chronic pain, a strategy known as pain physiology education.

Free sulfhydryl groups were alkylated by the addition of 8.28 μL of 100 mM iodoacetamide (in 37.5 mM ammonium bicarbonate), and incubation at 37 °C for 30 min in a dry-air incubator. Any remaining iodoacetamide was quenched by the addition of 8.28 μL of 100 mM DTT (in 37.5 mM ammonium bicarbonate)and incubation at 37 °C for 30 min in a dry-air incubator. Six micro liter of sequencing-grade trypsin (0.4 μg/μL (Promega) in 37.5 mM ammonium

bicarbonate) was added to each sample. The final volume of each digest was 300 μL, and digestion was conducted at 37 °C for 16 h in a dry air incubator. Digestion was stopped by the addition of an acidified SIS peptide mixture in formic acid, to give a final formic acid concentration of 0.5% v/v

and to reduce the pH to <3, which inactivates trypsin and AZD5363 price precipitates NaDOC). Two nanogram of methionine oxidized SIS peptides were spiked into the sample per MRM run. Samples were centrifuged for 10 min 17-AAG research buy at 12,000 × g (23 °C) to remove the NaDOC precipitate. The supernatant containing the peptides was desalted and concentrated by solid phase extraction using Waters Oasis HLB 1 cc columns (10 mg). The eluted samples were frozen and lyophilized to dryness overnight. Prior to the LC/MRM-MS analysis, samples were rehydrated in a volume of Solvent A (0.1% v/v formic acid) to obtain a concentration of 0.5 μg/μL of original sample. The MS analyses were performed on an AB/MDS Sciex 4000 QTRAP equipped with an Eksigent NanoLC-1Dplus LC system. The trapping column used was a 5 × 0.3 mm C18 PepMap column, with 5 μm particles (Dionex/LC Packings). The analytical column was a 75 μm × 150 mm Reprospher 100 C18 Aqua column, packed with 3 μm particles, 100 Å pore size, packed in-house under argon. The solvent system consisted Bay 11-7085 of solvent A (100% H2O, 0.1% v/v formic acid), and solvent B (90% aqueous acetonitrile, 0.1% v/v formic acid). The on-line analyses

were 43 min in length and the gradient was constructed as follows: samples were loaded onto the trapping column at 10 μL/min (2% aqueous acetonitrile, 0.1% v/v aqueous formic acid) for 3 min, followed by a 2 min linear gradient from 3% to 13% solvent at 300 nnL/min, a 10 min linear gradient at 300 nL/min from 13% to 20% solvent B, a 9 min linear gradient at 300 nL/min from 20% to 27% solvent B, and a final 6 min linear gradient at 300 nL/min from 27% to 44% solvent B before high organic column flushing and re-equilibration. A blank solvent injection was run between all samples to prevent sample carryover on the HPLC column. An AB/MDS Sciex 4000 QTRAP with a Michrom Captive Spray source, controlled by Analyst 1.5 software (Applied Biosystems) was used for all of the LC/MRM-MS analyses. All acquisition methods used the following instrument parameters: 1300–1500 V ion spray voltage, a 110 °C interface heater temperature, an MS operating pressure of 3.5 × 10−5 Torr, and Q1 and Q3 set to unit resolution (0.6–0.8 Da FWHH).