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Although oral hygiene is known to impact self-confidence and self-esteem, little is known about how it influences our interpersonal behavior. Using a wearable, multi-sensor device, we examined differences in consumers’ individual and interpersonal confidence after they had or had not brushed their teeth. Students (N = 140) completed nine one-to-one, 3-minute “speed dating” interactions while wearing a device that records verbal, nonverbal, and mimicry behavior. Half of the participants brushed their teeth using Close-Up toothpaste (Unilever) prior to the interactions, whilst the other half abstained from brushing that morning. Compared to those who had not brushed their teeth, participants who had brushed were more verbally confident (i.e., spoke louder, over-talked more), showed less nonverbal nervousness (i.e., fidgeted less), and were more often perceived as being “someone similar to me.” These effects were moderated by attractiveness but not by self-esteem or self-monitoring.

The amino acid sequence of the M-protein for multiple myeloma is unique compared to the polyclonal antibodies in patients' blood. This uniqueness is exploited to develop an ultrasensitive M-protein detection method utilizing mass spectrometry (MS). The method involves the de novo amino acid sequencing of the full-length M-protein, and a targeted MS/MS assay to detect and quantify the unique M-protein sequence in serum samples. Healthy control serum spiked with NISTmAb and serial samples from an MM patient were used to demonstrate the ability of the platform to sequence and monitor a target M-protein. The de novo NISTmAb protein sequence obtained matched the published sequence, confirming the ability of the platform to accurately sequence a target M-protein in serum. NISTmAb was quantified down to 0.0002 g/dL in serum, a level hundreds of times more sensitive than conventional blood-based tests such as SPEP and IFE. The M-protein in the patient sample could be quantified throughout complete remission, demonstrating the utility of the assay to track M-protein considerably beyond the sensitivities of current blood-based tests. Notably, the assay detected a 2-fold rise in M-protein levels 10 months before any changes were detected by conventional IFE. The MS-based assay is highly sensitive, noninvasive, and requires only a small amount of serum, less than 100 μL. Sequencing data is deposited into PRIDE with identifier PXD022784, and quantification data can be found in Panorama Public with identifier PXD022980.

Reports about repeated experiences tend to include more schematic information than information about specific instances. However, investigators in both forensic and intelligence settings typically seek specific over general information. We tested a multi-method interviewing format (MMIF) to facilitate recall and particularisation of repeated events through the use of the self-generated cues mnemonic, the timeline technique, and follow-up questions. Over separate sessions, 150 adult participants watched four scripted films depicting a series of meetings in which a terrorist group planned attacks and planted explosive devices. For half of our sample, the third witnessed event included two deviations (one new detail and one changed detail). A week later, participants provided their account using the MMIF, the timeline technique with self-generated cues, or a free recall format followed by open-ended questions. As expected, more information was reported overall in the MMIF condition compared to the other format conditions, for two types of details, correct details, and correct gist details. The reporting of internal intrusions was comparable across format conditions. Contrary to hypotheses, the presence of deviations did not benefit recall or source monitoring. Our findings have implications for information elicitation in applied settings and for future research on adults’ retrieval of repeated events.

Mentalizing is the powerful cognitive ability to understand others. By attributing mental states to others, we become able to explain and predict their behavior. The right temporoparietal junction (rTPJ) plays a key role in processing models of mental states. Yet, a different line of research suggests that the rTPJ is crucially involved in attentional control, prompting debates on its cognitive function. In this pre-registered neuro-navigated event-related TMS study, we tested for the rTPJ's specificity in mentalizing and attentional control. We interfered with its activity in a recently developed spatial cueing paradigm in which another's mental states were apparently task-relevant, allowing direct comparison of TMS effects on attention and mentalizing. We contrasted effects with a nearby control TMS site. Our confirmatory analysis showed no evidence for an involvement of the rTPJ in mentalizing or attentional control, presumably due to an observed large inter-individual variability of TMS effects on context and validity. To follow up this finding, we conducted exploratory analyses which revealed that rTPJ TMS had an influence on both attentional control and mentalizing. TMS effects on attention and mentalizing co-varied across participants: participants responding most to rTPJ TMS on mentalizing were also those for whom rTPJ TMS increased the attentional effect the most. This provides further evidence against total absolute segregation between mentalizing and attention within the rTPJ. Rather, our results suggest a common cognitive mechanism in both domains for which the rTPJ is necessary, paving the way for future research to cross-validate and extend these findings.

International audience; Serine hydroxymethyltransferase 2 (SHMT2) converts serine plus tetrahydrofolate (THF) into glycine plus methylene-THF and is upregulated at the protein level in lung and other cancers. In order to better understand the role of SHMT2 in cancer a model system of HeLa cells engineered for inducible over-expression or knock-down of SHMT2 was characterized for cell proliferation and changes in metabolites and proteome as a function of SHMT2. Ectopic over-expression of SHMT2 increased cell proliferation in vitro and tumor growth in vivo. Knockdown of SHMT2 expression in vitro caused a state of glycine auxotrophy and accumulation of phosphoribosylaminoimidazolecarboxamide (AICAR), an intermediate of folate/1-carbon-pathway-dependent de novo purine nucleotide synthesis. Decreased glycine in the HeLa cell-based xenograft tumors with knocked down SHMT2 was potentiated by administration of the anti-hyperglycinemia agent benzoate. However, tumor growth was not affected by SHMT2 knockdown with or without benzoate treatment. Benzoate inhibited cell proliferation in vitro, but this was independent of SHMT2 modulation. The abundance of proteins of mitochondrial respiration complexes 1 and 3 was inversely correlated with SHMT2 levels. Proximity biotinylation in vivo (BioID) identified 48 mostly mitochondrial proteins associated with SHMT2 including the mitochondrial enzymes Acyl-CoA thioesterase (ACOT2) and glutamate dehydrogenase (GLUD1) along with more than 20 proteins from mitochondrial respiration complexes 1 and 3. These data provide insights into possible mechanisms through which elevated SHMT2 in cancers may be linked to changes in metabolism and mitochondrial function.

This research examines how the cultural dimension of uncertainty avoidance-a person's (in)tolerance for uncertain or unknown situations-impacts communication alignment in crisis negotiations. We hypothesized that perpetrators high on uncertainty avoidance would respond better to negotiators who use formal language and legitimize their position with reference to law, procedures, and moral codes. Data were transcriptions of 53 negotiations from a Dutch-German police training initiative, where police negotiators interacted with a high (German) and low (Dutch) uncertainty-avoidant mock perpetrator. Consistent with accounts of cross-cultural interaction, negotiators tended to achieve more alignment in within-culture interactions compared to cross-cultural interactions. Moreover, German negotiators, who scored higher on uncertainty avoidance than the Dutch negotiators, were found to use more legitimizing messages and more formal language than their Dutch counterparts. Critically, irrespective of the negotiators cultural background, the use of these behaviors was a significant moderator of the degree to which negotiator and perpetrator aligned their communicative frames: Using legitimizing and formal language helped with German perpetrators but had no effect on Dutch perpetrators. Our findings show the effects of cultural background on communication alignment and demonstrate the benefits of using more formal language and messages that emphasize law and regulations when interacting with perpetrators high on uncertainty avoidance. (PsycINFO Database Record

Renal cell tumors with mixed morphology resembling multiple renal cell carcinoma (RCC) subtypes are generally regarded as unclassified RCC. However, occasionally, papillary adenoma or RCC appears admixed with a larger, different tumor histology. We retrieved 17 renal tumors containing a papillary adenoma or papillary RCC component admixed with another tumor histology and studied them with immunohistochemistry and fluorescence in situ hybridization (FISH). Larger tumors were oncocytomas (n = 10), chromophobe RCCs (n = 5), borderline oncocytic tumor (n = 1), and clear cell RCC (n = 1). The size of papillary component ranged from 1 to 34 mm. One tumor was an oncocytoma encircled by a cyst (2.0 cm) with papillary hyperplasia of the lining. The papillary lesions were diffusely cytokeratin 7 positive (17/17), in contrast to "host" tumors. Alpha-methylacyl-coA-racemase labeling was usually stronger in the papillary lesions (13/15). KIT was negative in all papillary lesions and the clear cell RCC and positive in 16/16 oncocytic or chromophobe tumors. Eight of 15 (53%) collision tumors had differing FISH results in the two components. A papillary renal cell proliferation within another tumor is an uncommon phenomenon with predilection for oncocytoma and chromophobe RCC, possibly related to their common entrapment of benign tubules. When supported by distinct morphology and immunohistochemistry in these two components, this phenomenon should be diagnosed as a collision of two processes. A diagnosis of unclassified RCC should be avoided, due to potential misrepresentation as an aggressive renal cancer.

Drawing on theories of mimicry as a schema-driven process, we tested whether the degree of verbal mimicry is dependent on the congruence between interactants' power dynamic (symmetric versus asymmetric), task type (cooperative versus competitive), and interaction context (negotiation versus social). Experiment 1 found higher verbal mimicry among dyads who successfully completed a cooperative problem-solving task compared with those who did not, but only under conditions of symmetric, not asymmetric, power. Experiment 2 had dyads complete either a cooperative or a competitive negotiation task, under conditions of symmetric versus asymmetric power. Verbal mimicry was associated with improved negotiation outcomes under conditions of cooperation and symmetry, and competition and asymmetry. Experiment 3 completes this picture by separating cooperative-competitive orientation from the interaction context. Consistent with Experiment 2, verbal mimicry was associated with task success during a negotiation context with asymmetric power, and during a social interaction context with symmetric power. Our results point to the contextual link between verbal mimicry and task outcome. (PsycINFO Database Record (c) 2019 APA, all rights reserved).

We present a new signal for detecting deception: full body motion. Previous work on detecting deception from body movement has relied either on human judges or on specific gestures (such as fidgeting or gaze aversion) that are coded by humans. While this research has helped to build the foundation of the field, results are often characterized by inconsistent and contradictory findings, with small-stakes lies under lab conditions detected at rates little better than guessing. We examine whether a full body motion capture suit, which records the position, velocity, and orientation of 23 points in the subject’s body, could yield a better signal of deception. Interviewees of South Asian (n = 60) or White British culture (n = 30) were required to either tell the truth or lie about two experienced tasks while being interviewed by somebody from their own (n = 60) or different culture (n = 30). We discovered that full body motion–the sum of joint displacements–was indicative of lying 74.4% of the time. Further analyses indicated that including individual limb data in our full body motion measurements can increase its discriminatory power to 82.2%. Furthermore, movement was guilt- and penitential-related, and occurred independently of anxiety, cognitive load, and cultural background. It appears that full body motion can be an objective nonverbal indicator of deceit, showing that lying does not cause people to freeze.

Hereditary leiomyomatosis and renal cell carcinoma (HLRCC) is an inherited cancer syndrome associated with a highly aggressive form of type 2 papillary renal cell carcinoma (PRCC). Germline inactivating alterations in fumarate hydratase (FH) cause HLRCC and result in elevated levels of reactive oxygen species (ROS). Recent work indicates that FH−/− PRCC cells have increased activation of ABL1, which promotes tumor growth, but how ABL1 is activated remains unclear. Given that oxidation can regulate protein-tyrosine phosphatase (PTP) catalytic activity, inactivation of an ABL-directed PTP by ROS might account for ABL1 activation in this malignancy. Our group previously developed “q-oxPTPome,” a method that globally monitors the oxidation of classical PTPs. In this study, we present a refined q-oxPTPome, increasing its sensitivity by >10×. Applying q-oxPTPome to FH-deficient cell models showed that multiple PTPs were either highly oxidized (including PTPN12) or overexpressed. Highly oxidized PTPs were those with relatively high sensitivity to exogenous H2O2. Most PTP oxidation in FH-deficient cells was reversible, although nearly 40% of PTPN13 was irreversibly oxidized to the sulfonic acid state. Using substrate-trapping mutants, we mapped PTPs to their putative substrates and found that only PTPN12 could target ABL1. Furthermore, knockdown experiments identified PTPN12 as the major ABL1 phosphatase, and overexpression of PTPN12 inhibited ABL1 phosphorylation and HLRCC cell growth. These results show that ROS-induced oxidation of PTPN12 accounts for ABL1 phosphorylation in HLRCC-associated PRCC, revealing a novel mechanism for inactivating a tumor suppressor gene product and establishing a direct link between pathologic PTP oxidation and neoplastic disease. Significance: This work identifies a novel mechanism of activation of the oncogenic kinase ABL1 via ROS-induced, oxidation-mediated inactivation of cognate protein tyrosine phosphatases.

Three-dimensional cultures of primary epithelial cells including organoids, enteroids and epithelial spheroids have become increasingly popular for studies of gastrointestinal development, mucosal immunology and epithelial infection. However, little is known about the behavior of these complex cultures in their three-dimensional culture matrix. Therefore, we performed extended time-lapse imaging analysis (up to 4 days) of human gastric epithelial spheroids generated from adult tissue samples in order to visualize the dynamics of the spheroids in detail. Human gastric epithelial spheroids cultured in our laboratory grew to an average diameter of 443.9 ± 34.6 µm after 12 days, with the largest spheroids reaching diameters of > 1,000 µm. Live imaging analysis revealed that spheroid growth was associated with cyclic rupture of the epithelial shell at a frequency of 0.32 ± 0.1/day, which led to the release of luminal contents. Spheroid rupture usually resulted in an initial collapse, followed by spontaneous re-formation of the spheres. Moreover, spheroids frequently rotated around their axes within the Matrigel matrix, possibly propelled by basolateral pseudopodia-like formations of the epithelial cells. Interestingly, adjacent spheroids occasionally underwent luminal fusion, as visualized by injection of individual spheroids with FITC-dextran (40 kDa). In summary, our analysis revealed unexpected dynamics in human gastric spheroids that challenge our current view of cultured epithelia as static entities and that may need to be considered when performing spheroid infection experiments.

Src homology 2 (SH2) domains are modular protein structures that bind phosphotyrosine (pY)-containing polypeptides and regulate cellular functions through protein-protein interactions. Proteomics analysis showed that the SH2 domains of Src family kinases are themselves tyrosine phosphorylated in blood system cancers, including acute myeloid leukemia, chronic lymphocytic leukemia, and multiple myeloma. Using the Src family kinase Lyn SH2 domain as a model, we found that phosphorylation at the conserved SH2 domain residue Y(194) impacts the affinity and specificity of SH2 domain binding to pY-containing peptides and proteins. Analysis of the Lyn SH2 domain crystal structure supports a model wherein phosphorylation of Y(194) on the EF loop modulates the binding pocket that engages amino acid side chains at the pY+2/+3 position. These data indicate another level of regulation wherein SH2-mediated protein-protein interactions are modulated by SH2 kinases and phosphatases.

Numb is an adaptor protein that functions in the endocytosis and intracellular trafficking of membrane receptors and adhesion molecules. Previous studies have indicated that Numb localization and function are regulated through phosphorylation by atypical protein kinase C at several key sites. Here, using LC-MS/MS, we report the identification of 25 serine/threonine Numb phosphorylation sites, and a single tyrosine phosphorylation site. Amino acid sequences flanking several of the sites identified conform to consensus motifs for cyclin-dependent kinase 5 (CDK5). In vitro kinase assays and immunoblotting confirmed that CDK5 phosphorylates Numb. LC-MS/MS analysis identified specific CDK5-directed phosphorylation of Numb at position S288 and at two additional regions. Therefore, Numb is likely to exist in multiple phospho-isoforms, and may be subject to phosphorylation-mediated regulation downstream of CDK5. These findings provide a basis for further investigations into the complex role of Numb phosphorylation in regulating its subcellular localization, protein interactions, and function. All MS data have been deposited in the ProteomeXchange with identifier PXD000997 (http://proteomecentral.proteomexchange.org/dataset/PXD000997).

Aberrant expression and activation of FGFR3 is associated with disease states including bone dysplasia and malignancies of bladder, cervix, and bone marrow. MS analysis of protein-phosphotyrosine in multiple myeloma cells revealed a prevalent phosphorylated motif, D/EYYR/K, derived from the kinase domain activation loops of tyrosine kinases including FGFR3 corresponding to a recognition sequence of protein-tyrosine phosphatase PTPN1. Knockdown of PTPN1 or the related enzyme PTPN2 by RNAi resulted in ligand-independent activation of FGFR3. Modulation of FGFR3 activation loop phosphorylation by both PTPN1 and PTPN2 was a function of receptor trafficking and phosphotyrosine phosphatase (PTP) compartmentalization. The FGFR3 activation loop motif DYYKK(650) is altered to DYYKE(650) in the oncogenic variant FGFR3(K650E) , and consequently it is constitutively fully activated and unaffected by activation loop phosphorylation. FGFR3(K650E) was nevertheless remarkably sensitive to negative regulation by PTPN1 and PTPN2. This suggests that in addition to modulating FGFR3 phosphorylation, PTPN1 and PTPN2 constrain the kinase domain by fostering an inactive-state. Loss of this constraint in response to ligand or impaired PTPN1/N2 may initiate FGFR3 activation. These results suggest a model wherein PTP expression levels may define conditions that select for ectopic FGFR3 expression and activation during tumorigenesis.

This research examined the coordination of interrogator and suspects' verbal behavior in interrogations. Sixty-four police interrogations were examined at the aggregate and utterance level using a measure of verbal mimicry known as Language Style Matching. Analyses revealed an interaction between confession and the direction of language matching. Interrogations containing a confession were characterized by higher rates of the suspect matching the interrogators' language style than interrogations without a confession. A sequence analysis of utterance-level Language Style Matching revealed a divergence in the type of matching that occurred across outcome. There was a linear increase in interrogator-led matching for interrogations containing a confession and an increase in suspect-led matching for nonconfession interrogations. These findings suggest that police interrogations play out, in part, at the basic level of language coordination.

Background & Aims: Gastric dendritic cells (DCs) control the adaptive response to infection with Helicobacter pylori, a major risk factor for peptic ulcer disease and gastric cancer. We hypothesize that DC interactions with the gastric epithelium position gastric DCs for uptake of luminal H pylori and promote DC responses to epithelial-derived mediators. The aim of this study was to determine whether the gastric epithelium actively recruits DCs using a novel co-culture model of human gastric epithelial spheroids and monocyte-derived DCs. Methods: Spheroid cultures of primary gastric epithelial cells were infected with H pylori by microinjection. Co-cultures were established by adding human monocyte-derived DCs to the spheroid cultures and were analyzed for DC recruitment and antigen uptake by confocal microscopy. Protein array, gene expression polymerase chain reaction array, and chemotaxis assays were used to identify epithelial-derived chemotactic factors that attract DCs. Data from the co-culture model were confirmed using human gastric tissue samples. Results: Human monocyte-derived DCs co-cultured with gastric spheroids spontaneously migrated to the gastric epithelium, established tight interactions with the epithelial cells, and phagocytosed luminally applied H pylori. DC recruitment was increased upon H pylori infection of the spheroids and involved the activity of multiple chemokines including CXCL1, CXCL16, CXCL17, and CCL20. Enhanced chemokine expression and DC recruitment to the gastric epithelium also was observed in H pylori–infected human gastric tissue samples. Conclusions: Our results indicate that the gastric epithelium actively recruits DCs for immunosurveillance and pathogen sampling through chemokine-dependent mechanisms, with increased recruitment upon active H pylori infection. Keywords: Stomach, Organoid, Mononuclear Phagocyte, In Vitro Model

Background: Melatonin is synthesized in the pineal gland and is an important circadian phase marker, especially in the determination of sleep patterns. Both temporary and permanent abnormal sleep patterns occur in children; therefore, it is desirable to have methods for monitoring melatonin in biological fluids in the diagnosis and treatment of such disorders. Objective: The objective of the study is to develop a liquid chromatography-tandem mass spectrometry method for the determination of melatonin in saliva and to apply it to monitoring salivary concentrations in children with sleep disorders. Methods: A deuterated internal standard (d7-melatonin) was added to a diluted saliva sample (20 mL) in an autosampler vial insert, and 50 mL were injected. Plasticware was strictly avoided, and all glassware was scrupulously cleaned and then baked at 120°C for at least 48 hours to obtain satisfactory performance. Reverse-phase chromatography was performed on a C8 column using a linear gradient elution profile comprising mobile phases A (0.1% aqueous formic acid) and B (15% methanol in acetonitrile containing 0.1% formic acid), pumped at a total flow rate of 0.8 mL/min. The run time was 8 minutes. After atmospheric pressure chemical ionization, mass spectrometric detection was in positive ion mode. Mass detection was by selected reaction monitoring mode with the following mass transitions used for quantification: melatonin, m/z 233.0 / 173.8 and d7-melatonin, m/z 240.0 / 178.3. Results: Linearity (r< 0.999) was established from 3.9 to 1000 pg/mL. Imprecision (coefficient of variation percent) was less than 11%, and accuracy was 100-105% (7.0-900 pg/mL). The method was selective, and the mean (range) ratio of the slopes of calibrations in water to those in daytime saliva samples collected from 10 healthy adult subjects was 0.989 (0.982-0.997), indicating negligible matrix effects. The application of the assay was demonstrated in healthy adults and in children being clinically investigated for sleep disturbances. Conclusions: A validated liquid chromatography-tandem mass spectrometry method suitable for monitoring salivary melatonin in children with circadian rhythm sleep disorders is reported. The method also has potential application to pediatric population pharmacokinetic studies using sparse sampling of saliva as the biological sample matrix.

Numb is an endocytic adaptor protein that regulates the endocytosis and trafficking of transmembrane receptors including Notch, E-cadherin, and integrins. Vertebrate Numb is alternatively spliced at exons 3 and 9 to give rise to four protein isoforms. Expression of these isoforms varies at different developmental stages, and although the function of Numb isoforms containing exon 3 has been studied, the role of exon 9 inclusion has not been shown. Here we use affinity purification and tandem mass spectrometry to identify Numb associated proteins, including novel interactions with REPS1, BMP2K, and BCR. In vitro binding measurements indicated exon 9-independent Numb interaction with REPS1 and Eps15 EH domains. Selected reaction monitoring mass spectrometry was used to quantitatively compare the proteins associated with the p72 and p66 Numb isoforms, which differ by the exon 9 region. This showed that significantly more EPS15 and three AP-2 subunit proteins bound Numb isoforms containing exon 9. The EPS15 preference for exon 9-containing Numb was confirmed in intact cells by using a proximity ligation assay. Finally, we used multiplexed selected reaction monitoring mass spectrometry to assess the dynamic regulation of Numb association with endocytic proteins. Numb hyper-phosphorylation resulted in disassociation of Numb endocytic complexes, while inhibition of endocytosis did not alter Numb association with the AP-2 complex but altered recruitment of EPS15, REPS1, and BMP2K. Hence, quantitative mass spectrometric analysis of Numb protein-protein interactions has provided new insights into the assembly and regulation of protein complexes important in development and cancer.

Therapeutic drug monitoring (TDM) and more recently target concentration intervention (TCI) have been widely used in clinical practice for the optimization of drug treatment. TDM and TCI have been applied most frequently in the cardiovascular, respiratory, neurology, and infectious disease areas because the medications used here have both narrow therapeutic indices and a clear relationship between concentration and effect. However, apart from drugs such as methotrexate and 5-fluorouracil, the clinical application of TDM/TCI in oncology is minimal. An important reason for this is that a therapeutic index for most anticancer agents has not been established. However, in the last 20 years, relationships between plasma drug concentrations and clinical outcome have been defined for various chemotherapeutic agents. Defining concentration-effect relationships is also complicated by the fact that cancer is almost always treated with multiple drugs given in combination making the precise definition of the pharmacodynamics of individual agents difficult. The increase in patients with obesity and also those underweight adds to the complexity of effective oncology treatment. This review describes some of the evidence that supports the use of TDM/TCI in oncology. It is proposed that as more patients previously ineligible for chemotherapy become eligible, TDM/TCI may play a critical role in optimizing chemotherapy outcomes. However, pharmacokinetic-pharmacodynamic research to investigate both therapeutic benefit and feasibility in daily clinical practice is required.

Therapeutic drug monitoring (TDM)6 of certain drugs with a narrow therapeutic index significantly improves patient outcome. For example, the quantification of immunosuppressive drugs in samples from patients after organ transplantation is an essential prerequisite for the prevention of both adverse drug reactions and rejection events. The need for accurate, precise, and standardized measurement of drugs presents a major challenge for clinical laboratories and the diagnostics industry. A plethora of different techniques have been developed in the past to meet these requirements. Currently, liquid chromatography–tandem mass spectrometry (LC-MS/MS)-based methods and immunoassays appear to be the most prevalent approaches in clinical laboratories. Since these techniques differ in many aspects, in making the choice the laboratory must consider the technical, clinical, and economic criteria, as well as staff qualifications. In this Q&A, 4 leading experts in the field of immunosuppressive drug monitoring present their opinions on the advantages and disadvantages of the analytical methods and provide a general guide for the optimization of analytical strategies in the field of TDM. From your perspective, what are the advantages and disadvantages of mass spectrometry–based assays and immunoassays in the context of TDM? Gregory Maine: One of the main advantages of mass spectrometry is that users can develop their own assays. Mass spectrometry–based assays can be analytically sensitive, specific, and capable of measuring several compounds in a single run. The disadvantages of this technique include that the instrument is not a “turnkey” analyzer, it requires large capital investment and service costs, it is subject to matrix effects and ion suppression, and the results from different laboratories are not necessarily comparable. The advantages of drug immunoassays include their easy integration into the core laboratory and the ability to measure them along with other immunoassays, such as those for serologic factors and hormones. In addition, TDM immunoassays …

Therapeutic drug monitoring of tacrolimus by high-performance liquid chromatography-tandem mass spectrometry has become standard practice. We report on the long-term (4.5 years) use of one such method. Whole blood samples (25 mu L) were treated with zinc sulphate (100 mu L) and acetonitrile containing ascomycin (internal standard, 2504). A high-performance liquid chromatography-tandem mass spectrometer operating in positive ion mode with an electrospray interface was used. Chromatography was performed on a TDM C-18 cartridge column (10 mm x 2.1 mm, 10 mu m, Waters) using a switch gradient. A total of 4029 batches were analyzed for tacrolimus: this comprised of 81950 analyses of which 61027 were patient samples. Calibration curves (1.0-50 mu g/L) were run on 1765 occasions (mean r(2) = 0.999; range r(2) = 0.988-0.999). Inter-batch accuracy and imprecision of the method (2.5, 12.5 and 30.0 mu g/L), when in routine use, was 97.6-98.5% and

The most popular screening test for primary aldosteronism is plasma aldosterone/renin ratio (ARR). Because both estrogen and progesterone affect aldosterone and renin levels, we studied effects of two contraceptives commonly used in our population, one oral and one subdermal, on ARR, measuring renin as both direct renin concentration (DRC) and plasma renin activity (PRA).Normotensive, healthy women underwent measurement (seated, midmorning) of plasma aldosterone, DRC, PRA, electrolytes, and creatinine and urinary aldosterone, cortisol, electrolytes, and creatinine at baseline (menses) and after either 1) 3 wk treatment with oral ethinylestradiol plus drospirenone (n = 17) or 2) 1 wk and 6 wk treatment with subdermal etonogestrel (n = 15), a third-generation progestin.Treatment with oral ethinylestradiol plus drospirenone was associated with significant increases in aldosterone [median (range) at baseline = 131 (85-590) pmol/liter; at 1 wk, 200 (130-784) pmol/liter; and at 3 wk, 412 (199-1010) pmol/liter (P0.001, Friedman test)] and PRA [2.1 (1.2-4.7), 3.6 (1.5-7.1), and 4.9 (1.5-10.8) ng/ml · h, P0.001] but decreases in DRC [22 (11-36), 21 (8.7-41), and 14 (8.5-39) mU/liter, P0.01] leading to increases in ARR calculated by DRC [6.6 (3.3-31.3), 10.9 (5.2-58.9), and 29.8 (5.1-88.5), P0.001]. There were no significant changes in ARR calculated by PRA, plasma electrolytes and creatinine, and all urinary measurements. In contrast, treatment with subdermal etonogestrel was associated with no significant changes in PRA, DRC, aldosterone, or ARR at either 1 or 6 wk.The combined oral contraceptive ethinylestradiol plus drospirenone is capable of significantly increasing ARR with risk of false-positive results during screening for primary aldosteronism, but only if DRC is used to calculate the ratio. Subdermal etonogestrel had no effect on ARR after 6 wk.

Plasma aldosterone to renin ratio (ARR) is the most popular screening test for primary aldosteronism (PAL). Certain medications are known to cause false-negative or -positive ARRs by affecting renin and aldosterone levels. There are no previously published data on the effects of antidepressants on ARR.Normotensive, depressed male patients (n = 26) underwent measurement (seated, midmorning) of plasma aldosterone, direct renin concentration (DRC), renin activity (PRA), electrolytes and creatinine and urinary aldosterone, cortisol, electrolytes, and creatinine at baseline and after 2 and 6 wk treatment with sertraline (n = 14) or escitalopram (n = 12).For both antidepressants, treatment was associated with rises in aldosterone [sertraline: baseline, mean ± sd, 243 ± 34; 2 wk, 256 ± 33; 6 wk, 267 ± 34 pmol/liter (P0.01 by ANOVA); escitalopram, 261 ± 36, 269 ± 38, 282 ± 40 pmol/liter (P0.05)], DRC [19.5 ± 2.2, 33.5 ± 2.0, 39.0 ± 2.4 mU/liter (P0.001); 24.5 ± 2.4, 34.0 ± 2.7, 42.8 ± 2.4 mU/liter (P0.001)], and PRA [2.24 ± 0.21, 2.58 ± 0.26, 4.68 ± 0.42 ng/ml · h (P0.001); 4.31 ± 0.22, 5.57 ± 0.36, 6.42 ± 0.53 ng/ml · h (P0.001)]. ARR fell significantly whether calculated using DRC [sertraline, 13.7 ± 2.2, 7.5 ± 0.7, 6.8 ± 0.7 (P0.001); escitalopram, 11.5 ± 1.9, 8.0 ± 1.1, 6.6 ± 1.0 (P0.001)], or PRA [116.6 ± 15.8, 108.4 ± 15.6, 60.4 ± 6.2 (P0.001); 61.2 ± 8.1, 50.0 ± 7.7, 45.6 ± 6.0 (P0.01)].Selective serotonin reuptake inhibitor antidepressants can significantly reduce ARR and therefore potentially increase the risk of false-negative results when screening for PAL. Further studies in hypertensive patients, including patients with confirmed PAL, are required.

Because primary aldosteronism is not uncommon, specifically treatable and in some cases curable, and carries higher risks for cardiovascular morbidity and mortality than essential hypertension, screening hypertensive patients for its presence by measuring aldosterone to renin ratio (ARR) is increasingly common. A significantly higher false-positive ARR rate for women than men, resulting in unnecessary suppression tests has previously been reported.Using a new, highly accurate aldosterone assay and both of the currently widely used renin assays, ARR was measured in 19 normal, ovulating women at three time points in the menstrual cycle and compared with single measurements in 21 normal males of similar age.ARRs in males were possibly too well down in the current normal range. Although normotensive and normokalemic, two women had raised ARRs in the luteal phase but only when direct renin concentration (DRC) was used. Their DRC levels were low at all sampling times [despite midrange plasma renin activity levels], whereas their progesterone and aldosterone levels were highest for the group. Saline suppression testing, performed in one of them, showed normal aldosterone suppressibility.False-positive ARRs in normal women during the luteal phase only when DRC is used may explain the higher incidence of false-positive ARRs in hypertensive women than men and suggest the following: 1) plasma renin activity is preferable to DRC in determination of ARR and 2) new reference ranges for ARR that take into account gender and sex hormone levels are required.

Nonsmall cell lung carcinoma (NSCLC) accounts for 80% of lung cancers. The most prevalent subtypes of NSCLC are adenocarcinoma (ADC) and squamous cell carcinoma (SCC), which combined account for approximately 90%. Ten resected NSCLC patient tumors (5 ADC and 5 SCC) were directly introduced into severely immune deficient (NOD-SCID) mice, and the resulting xenograft tumors were analyzed by standard histology and immunohistochemistry (IHC) and by proteomics profiling. Mass spectrometry (MS) methods involving 1- and 2-dimensional LC-MS/MS, and multiplexed selective reaction monitoring (SRM, or MRM), were applied to identify and quantify the xenograft proteomes. Hierarchical clustering of protein profiles distinguished between the ADC and SCC subtypes. The differential expression of 178 proteins, including a comprehensive panel of intermediate filament keratin proteins, was found to constitute a distinctive proteomic signature associated with the NSCLC subtypes. Epidermal growth factor receptor (EGFR) was expressed in ADC and SCC xenografts, and EGFR network activation was assessed by phosphotyrosine profiling by Western blot analysis and SRM measurement of EGFR levels, and mutation analysis. A multiplexed SRM/MRM method provided relative quantification of several keratin proteins, EGFR and plakophilin-1 in single LC-MS/MS runs. The protein quantifications by SRM and MS/MS spectral counting were associated with superior dynamic range and reproducibility but were otherwise consistent with orthogonal methods including IHC and Western immuno blotting. These findings illustrate the potential to develop a comprehensive MS-based platform in oncologic pathology for better classification and potentially treatment of NSCLC patients.

Multiple limited sampling strategies (LSSs) have been proposed for estimation of mycophenolic acid (MPA) area under the concentration-time curve from 0 to 12 hours postdose (AUC 0-12) after mycophenolate mofetil intake. The aim of this study was to provide summary information on all published LSSs for MPA and to evaluate their predictive performance in an independent population of kidney transplant recipients. Seventy-eight LSSs for MPA were identified. Sixty-nine full AUC profiles were collected from 45 subjects (25 cotreated with cyclosporine and 20 with tacrolimus). Predicted MPA AUC 0-12, calculated by applying the relevant concentration measurements within the LSS equations, was compared with full AUC calculated by using all concentration measurements in the linear trapezoidal rule. Four error indices (median prediction error, median percentage prediction error [MPPE], root median squared prediction error, and median absolute percentage prediction error [MAPE]) were used to evaluate bias and imprecision. Twelve of the 25 LSSs for cyclosporine-cotreated recipients and one of the 53 LSSs for tacrolimus-cotreated recipients displayed acceptable (less than 15%) bias and imprecision. In the cyclosporine group, two equations demonstrated the highest predictive power, one that used four time points in the first 6 hours postdose (r2 = 0.84, MPPE 1.6%, MAPE 7.8%) and one that used four time points in the first 4 hours postdose (r2 = 0.76, MPPE -0.8%, MAPE 10.2%). In the tacrolimus group, an equation that used two time points in the first 4 hours postdose was superior (r2 = 0.80, MPPE -3.0%, MAPE 13.6%). Application of the LSSs most appropriate for cyclosporine-cotreated patients to the tacrolimus-cotreated group resulted in clinically unacceptable bias and imprecision and vice versa. High variability in performance of LSSs highlights the importance of validating any LSS before applying it to an alternative population. Attention to comedication use is of particular relevance when choosing a LSS.

Purpose: Neuroblastoma (NB) is an aggressive tumor of the developing peripheral nervous system that remains difficult to cure in the advanced stages. The poor prognosis for high-risk NB patients is associated with common disease recurrences that fail to respond to available therapies. NB tumor-initiating cells (TICs), isolated from metastases and primary tumors, may escape treatment and contribute to tumor relapse. New therapies that target the TICs may therefore prevent or treat tumor recurrences. Experimental Design: We undertook a system-level characterization of NB TICs to identify potential drug targets against recurrent NB. We used next-generation RNA sequencing and/or human exon arrays to profile the transcriptomes of 11 NB TIC lines from six NB patients, revealing genes that are highly expressed in the TICs compared with normal neural crest-like cells and unrelated cancer tissues. We used gel-free two-dimensional liquid chromatography coupled to shotgun tandem mass spectrometry to confirm the presence of proteins corresponding to the most abundant TIC-enriched transcripts, thereby providing validation to the gene expression result. Results: Our study revealed that genes in the BRCA1 signaling pathway are frequently misexpressed in NB TICs and implicated Aurora B kinase as a potential drug target for NB therapy. Treatment with a selective AURKB inhibitor was cytotoxic to NB TICs but not to the normal neural crest-like cells. Conclusion: This work provides the first high-resolution system-level analysis of the transcriptomes of 11 primary human NB TICs and identifies a set of candidate NB TIC-enriched transcripts for further development as therapeutic targets. Clin Cancer Res; 16(18); 4572–82. ©2010 AACR.

Oxypurinol is the active metabolite of allopurinol which is used to treat hyperuricaemia associated with gout. Both oxypurinol and allopurinol inhibit xanthine oxidase which forms uric acid from xanthine and hypoxanthine. Plasma oxypurinol concentrations vary substantially between individuals and the source of this variability remains unclear. The aim of this study was to develop an HPLC-tandem mass spectrometry method to measure oxypurinol in urine to facilitate the study of the renal elimination of oxypurinol in patients with gout. Urine samples (50 microL) were prepared by dilution with a solution of acetonitrile/methanol/water (95/2/3, v/v; 2 mL) that contained the internal standard (8-methylxanthine; 1.5 mg/L), followed by centrifugation. An aliquot (2 microL) was injected. Chromatography was performed on an Atlantis HILIC Silica column (3 microm, 100 mm x 2.1mm, Waters) at 30 degrees C, using a mobile phase comprised of acetonitrile/methanol/50 mM ammonium acetate in 0.2% formic acid (95/2/3, v/v). Using a flow rate of 0.35 mL/min, the analysis time was 6.0 min. Mass spectrometric detection was by selected reactant monitoring (oxypurinol: m/z 150.8-->108.0; internal standard: m/z 164.9-->121.8) in negative electrospray ionization mode. Calibration curves were prepared in drug-free urine across the range 10-200 mg/L and fitted using quadratic regression with a weighting factor of 1/x (r(2) > 0.997, n=7). Quality control samples (20, 80, 150 and 300 mg/L) were used to determine intra-day (n=5) and inter-day (n=7) accuracy and imprecision. The inter-day accuracy and imprecision was 96.1-104% and

A method for the simultaneous quantification of eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA) and arachidonic acid (AA) in human plasma by HPLC-tandem mass spectrometry (HPLC-MS/MS) was developed and validated. Free and esterified forms of fatty acids were hydrolysed from plasma samples in the presence of an internal standard and subjected to liquid-liquid extraction. The chromatographic run time was 3.5 min per sample. The assay was linear from 0.5 to 300 mg/L (r(2) > 0.997, n = 18). Based on matrix addition, accuracy deviation was

Mycophenolic acid is now the second most widely used immunosuppressant in solid organ transplantation. Overestimation of mycophenolic acid concentration is a recognized problem of immunoassay, and high-performance liquid chromatography with ultraviolet detection methods have long analysis times and a risk of analyte coelution which may compromise high sample throughput in a clinically meaningful time frame. A novel liquid chromatography-tandem mass spectrometry assay for mycophenolic acid was developed using very small (10 microL) sample volumes and evaluated in comparison with an established immunological assay. The enzyme mediated immunoassay showed a median positive bias compared with liquid chromatography-tandem mass spectrometry of 14.6%. Linear regression analysis showed a significant positive impact of bilirubin (r2 = 0.230) on bias with further increases of r2 to 0.261, 0.286, and 0.294 with the stepwise addition of creatinine, hematocrit, and gamma-glutamyl transpeptidase, respectively. The impact of comedication and transplant type depended on the patient population: analysis of all samples showed opposing effects to analysis of those samples lacking data with biochemical variables above. The liquid chromatography-tandem mass spectrometry method described in this report is capable of measuring mycophenolic acid concentrations in very small sample volumes and in a timely fashion without the significant overestimates characterizing enzyme mediated immunoassay measurements in patients with serologic features characterizing liver or renal graft rejection.

A multi-center evaluation (3 sites) of the LC/MS/MS MassTrak tacrolimus Immunosuppressants Kit (Kit) was undertaken.Ten aspects of the analytical performance of the Kit were investigated based on FDA and CLSI guidelines.The linear analytical range of the procedure was between 0.68 and 31.7ng/mL. Within-run and total imprecision were6% and8% (n=240), respectively. Recoveries of tacrolimus added to clinical samples that contained between 5 and 10ng/mL of tacrolimus before addition were 99, 102 and 105% at 5.0, 10 and 20ng/mL, respectively. Comparison of in-house and Kit procedures in samples from liver (n=50-58) or kidney (n=50 or 51) transplant recipients yielded method mean biases between -2.0 and +10.7% at 5 and 15ng/mL.This evaluation indicates that the Kit is suitable for the monitoring of tacrolimus in kidney and liver transplant recipients.

The stoichiometry of protein phosphorylation at specific amino acid sites may be used to infer on the significance of the modification, and its biological function in the cell. However, detection and quantification of phosphorylation stoichiometry in tissue remain a significant challenge. Here we describe a strategy for highly sensitive, label-free quantification of protein phosphorylation stoichiometry. Method development included the analysis of synthetic peptides in order to determine constants to relate the mass spectrometry signals of cognate peptide/phosphopeptide pairs, and the detection of the cognate peptides by using high resolution Fourier Transform mass spectrometry (FTMS) and selected reaction monitoring mass spectrometry (SRM). By analyzing extracted ion currents by FTMS, the phosphorylation stoichiometries of two tyrosine residues (tyrosine-194 and tyrosine-397) in the protein tyrosine kinase Lyn were determined in transfected human HEK293T cells and two cultured human multiple myeloma strains. To achieve high sensitivity to measure phosphorylation stoichiometry in tissue, SRM methods were developed and applied for the analysis of phosphorylation stoichiometries of Lyn phospho-sites in multiple myeloma xenograft tumors. Western immuno-blotting was used to verify mass spectrometry findings. The SRM method has potential applications in analyzing clinical samples wherein protein phosphorylation stoichiometries may represent important pharmacodynamic biomarkers.

Background: Reliable measurement of aldosterone with less interlaboratory variation than RIA would help standardize testing for primary aldosteronism. We set out to validate a high-performance liquid chromatography–tandem mass spectrometry (HPLC-MS/MS) method for aldosterone in human plasma. Methods: We prepared samples (EDTA plasma, lithium heparin plasma, and serum from separator and plain clot tubes) and measured aldosterone using online HPLC-MS/MS with d7-aldosterone as internal standard. We also analyzed EDTA plasma samples by immunoassay. We established a reference range for HPLC-MS/MS aldosterone by analyzing blood collected midmorning from 97 normotensive seated subjects. Results: The linear range was 69.4–5548.0 pmol/L (2.5–200 ng/dL) (r2 > 0.994, n = 14). Inter- and intraday analytical recovery and imprecision for quality control samples of 166.4, 1109.6, and 4161.0 pmol/L (6.0, 40.0, and 150.0 ng/dL) were 92.2%–102.0% and Conclusions: This first reported aldosterone method using online HPLC-MS/MS is precise across the clinically relevant range, not influenced by collection tube type, and offers semiautomated sample preparation and high throughput.

One hundred seven management training evaluations were meta-analyzed to compare effect sizes for the transfer of managerial training derived from different rating sources (self, superior, peer, and subordinate) and broken down by both study- and training-related variables. For studies as a whole, and interpersonal management skills training studies in particular, transfer effects based on trainees' self-ratings, and to a lesser extent ratings from their superiors, were largest and most varied across studies. In contrast, transfer effects based on peer ratings, and particularly subordinate ratings, were substantially smaller and more homogeneous. This pattern was consistent across different sources of studies, features of evaluation design, and within a subset of 14 studies that each included all 4 rating sources. Across most rating sources, transfer of training was greatest for studies conducted in nonmilitary settings, when raters were likely to have known whether the manager being rated had attended training, when criteria were targeted to training content, when training content was derived from an analysis of tasks and skill requirements, and when training included opportunities for practice. (PsycINFO Database Record (c) 2009 APA, all rights reserved).

The immunosuppressant drug tacrolimus has a narrow therapeutic index and is subject to a large variation in individual bioavailability and clearance. With its narrow therapeutic index, therapeutic drug monitoring is standard clinical practice in the management of transplant recipients. In this study, we report the evaluation of the cloned enzyme donor immunoassay (CEDIA) for the determination of whole-blood tacrolimus concentrations compared with high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) and microparticle enzyme immunoassay (MEIA) using samples obtained from liver (n = 100) and renal (n = 88) transplant recipients. Linear regression analysis showed a relationship of CEDIA = 1.24 HPLC-MS/MS -0.18 (r = 0.81). The mean bias (+/-SEM) for all patients when compared with HPLC-MS/MS was 22.2% (+/-2.1%). The precision of the CEDIA method for all samples showed a root mean square error of 3.1 microg/L. Liver transplant recipient samples showed a mean (+/-SEM) bias compared with HPLC-MS/MS of 12.5% (+/-1.6%). The precision of the CEDIA method for these samples showed a root mean square error of 1.5 microg/L. The data suggest that in the renal transplant group, the CEDIA and MEIA methods have a bias of 33.3% and 20.1%, respectively, compared with HPLC-MS/MS. The CEDIA tacrolimus immunoassay has been shown to be a rapid method for the determination of whole-blood tacrolimus concentrations and may be considered when HPLC-MS/MS is not available. When used in the clinical setting with other parameters, it would be a useful adjunct in the management of liver transplant recipients, but a significant bias in renal transplant patients needs to be further investigated.

We report here the validation of an HPLC-electrospray-tandem mass spectrometry method for the quantification of everolimus, an immunosuppressant drug. Whole blood samples (100 microl) were extracted by protein precipitation which involved sample pre-treatment with zinc sulphate followed by acetonitrile (containing internal standard, 40-O-(3'-hydroxy)propyl-rapamycin). HPLC was performed using a step-gradient at a flow rate of 0.6 ml/min on a Waters TDM C18 column (10 mm x 2.1mm I.D.) with a resultant chromatographic analysis time of 2 min. Mass spectrometric detection by selected reaction monitoring (everolimus m/z 975.5-->908.3; internal standard m/z 989.5-->922.3). The assay was linear from 0.5 to 40 microg/l (r2>0.994, n=11). The inter- and intra-day analytical recovery and imprecision for quality control samples (1.25, 12.5 and 30 microg/l) were 93.4-98.2% and

Economy is considered to be a key factor for the determination of performance in endurance events such as cycling. There have been no investigations which have related cycling economy to simultaneous measurements of 3D kinematics and muscular activation. This study examined selected biomechanical and neuromuscular parameters which have the strongest association with cycling economy.Twenty-five trained cyclists (31.27 ± 3.19 years) completed steady state cycling time trials at a workload of 180 W. Simultaneous measurements of 3D kinematics and electromyographical parameters were obtained. Continuous measurements of expired gases were used to provide a measure of cycling economy.A multiple regression analysis showed that key parameters of peak knee extension velocity and mean activity of the rectus femoris muscles were significant predictors of VO2 during steady state cycling (P0.01).This study has documented the key biomechanical parameters pertinent to cycling economy. As economy has been shown to influence aerobic performance, future work should focus on optimising these parameters to improve cycling economy.

We report here a validated method for the quantification of a new immunosuppressant drug FTY720, using HPLC-tandem mass spectrometry. Whole blood samples (500 microl) were subjected to liquid-liquid extraction, in the presence of an internal standard (Y-32919). Mass spectrometric detection was by selected reaction monitoring with an atmospheric pressure chemical ionization source in positive ionization mode (FTY720: m/z 308.3-->255.3). The assay was linear from 0.2 to 25 microg/l (r(2)>0.997, n=5). The inter- and intra-day analytical recovery and imprecision for quality control samples (0.5, 7 and 15 microg/l) were 95.8-103.2 and

Therapeutic monitoring with dosage individualization of sirolimus drug therapy is standard clinical practice for organ transplant recipients. For several years sirolimus monitoring has been restricted as a result of lack of an immunoassay. The recent reintroduction of the microparticle enzyme immunoassay (MEIA (R)) for sirolimus on the IMx (R) analyser has the potential to address this situation. This Study, using patient samples, has compared the MEIA (R) sirolimus method with an established HPLC-tandem mass spectrometry method (HPLC-MS/MS). An established HPLC-UV assay was used for independent cross-validation. For quality control materials (5, 11, 22 mu g/L), the MEIA (R) showed acceptable validation criteria based on intra-and inter-run precision (CV) and accuracy (bias) of < 8% and < 13%, respectively. The lower limit of quantitation was found to be approximately 3 mu g/L. The performance of the immunoassay was compared with HPLC-MS/MS using EDTA whole-blood samples obtained from various types of organ transplant recipients (n = 116). The resultant Deming regression line was: MEIA = 1.3 x HPLC-MS/MS+ 1.3 (r = 0.967, s(y/x) = 1) with a mean bias of 49.2% +/- 23.1 % (range, -2.4% to 128%; P < 0.001). The reason for the large and variable bias was not explored in this study, but the sirolimus-metabolite cross-reactivity with the MEIA (R) antibody could be a substantive contributing factor. Whereas the MEIA (R) sirolimus method may be an adjunct to sirolimus dosage individualization in transplant recipients, users must consider the implications of the substantial and variable bias when interpreting results. In selected patients where difficult clinical issues arise, reference to a specific chromatographic method may be required.

Clinical tissue samples provide valuable information for understanding human diseases. One major type of clinical tissue sample that is amenable to various kinds of analysis is fresh frozen and optimal cutting temperature (OCT)-embedded primary patient tissue. Recent advances in mass spectrometry (MS) technologies have been widely applied to study human proteomes by using clinical specimens. However, polymeric compounds such as OCT can interfere with MS analyses. Here we present methods that enable the preparation and analysis of fresh frozen and OCT embedded primary tissue samples by LC-MS/MS. A scraping method was first introduced to reduce the heterogeneity of OCT-embedded non-small cell lung carcinoma tumor sections. OCT compound was reproducibly removed by a series of washing steps involving ethanol and water prior to trypsin digestion. In data-dependent acquisition mode, optimized dynamic exclusion duration settings were established to maximize peptide identifications. These sample preparation conditions and MS parameter settings should be utilized or carefully adjusted in order to achieve optimal comprehensive proteome characterization starting from fresh frozen and OCT embedded clinical tissue specimens.

Aims The pharmacokinetics of mycophenolic acid and its glucuronide are complex. This study investigated the pharmacokinetics, pharmacodynamics and protein binding of mycophenolic acid and its glucuronide metabolite, early post-transplant in renal allograft recipients. Methods Forty-two de novo renal transplant recipients receiving mycophenolate mofetil and concomitant cyclosporin (n = 32) or tacrolimus (n = 10) participated in the study. Blood samples were taken on day 5 post-transplant for measurement of free and total concentrations of mycophenolic acid, mycophenolic acid glucuronide and relevant biochemistry. Associations between free fraction and biochemistry were investigated. Free and total 6-h area under the concentration–time curve (AUC0−6) of mycophenolic acid was assessed relative to clinical outcomes in the first month post-transplant. Results Kinetic variability of free and total mycophenolic acid and its glucuronide was greater in patients on cyclosporin (12- to 18-fold variation) than on tacrolimus (four- to fivefold) cotherapy. Cyclosporin-treated patients also had significantly lower predose total mycophenolic acid concentrations than tacrolimus-treated patients (median 0.8 mg l−1 and 1.6 mg l−1, respectively, P = 0.002). Mycophenolic acid glucuronide predose concentration correlated positively with mycophenolic acid glucuronide AUC0−6 (r > 0.95). Mycophenolic acid free fraction varied 11-fold, from 1.6% to 18.3%, whilst the glucuronide free fraction varied threefold, from 17.4% to 54.1%. Urea and creatinine concentrations correlated positively (r > 0.46), whilst albumin correlated negatively (r = −0.54) with free fraction of mycophenolic acid. Similar relationships were found for the free fraction of mycophenolic acid glucuronide. Mycophenolic acid free fraction was on average 70% higher in patients with albumin concentrations below a specified albumin cut-off concentration of 31 g l−1[free fraction = 7 ± 4% for lower albumin and 4 ± 3% for higher albumin, respectively; P = 0.001; 95% confidence interval (CI) for the difference 1.9, 4.2]. Neither free nor total mycophenolic acid AUC0−6 was related to rejection (P > 0.07). Free AUC0−6 was significantly higher in those patients with thrombocytopenic, leukopenic and/or infectious outcomes than in those without (mean ± SD 1.9 ± 0.3 mg h−1 l−1 and 1.1 ± 0.1 mg h−1 l−1, P = 0.0043; 95% CI for the difference 0.3, 1.4). Conclusions The marked variability in mycophenolic acid/glucuronide pharmacokinetics occurring early post-transplant during the current study was greater in cyclosporin (12–18-fold) than in tacrolimus (four- to fivefold) treated patients. Concomitant cyclosporin was associated with total mycophenolic acid concentrations approximately half that of tacrolimus. Patients with marked renal impairment had the highest free fractions reported to date. The exposure to unbound mycophenolic acid was significantly related to infections and haematological toxicity.

A meta-analysis of 117 studies evaluated the effects of behavior modeling training (BMT) on 6 training outcomes, across characteristics of training design. BMT effects were largest for learning outcomes, smaller for job behavior, and smaller still for results outcomes. Although BMT effects on declarative knowledge decayed over time, training effects on skills and job behavior remained stable or even increased. Skill development was greatest when learning points were used and presented as rule codes and when training time was longest. Transfer was greatest when mixed (negative and positive) models were presented, when practice included trainee-generated scenarios, when trainees were instructed to set goals, when trainees' superiors were also trained, and when rewards and sanctions were instituted in trainees' work environments.

The aim of this study was to determine the most informative sampling time(s) providing a precise prediction of tacrolimus area under the concentration-time curve (AUC). Fifty-four concentration-time profiles of tacrolimus from 31 adult liver transplant recipients were analyzed. Each profile contained 5 tacrolimus whole-blood concentrations (predose and 1, 2, 4, and 6 or 8 hours postdose), measured using liquid chromatography-tandem mass spectrometry. The concentration at 6 hours was interpolated for each profile, and 54 values of AUC(0-6) were calculated using the trapezoidal rule. The best sampling times were then determined using limited sampling strategies and sensitivity analysis. Linear mixed-effects modeling was performed to estimate regression coefficients of equations incorporating each concentration-time point (C0, C1, C2, C4, interpolated C5, and interpolated C6) as a predictor of AUC(0-6). Predictive performance was evaluated by assessment of the mean error (ME) and root mean square error (RMSE). Limited sampling strategy (LSS) equations with C2, C4, and C5 provided similar results for prediction of AUC(0-6) (R-2 = 0.869, 0.844, and 0.832, respectively). These 3 time points were superior to C0 in the prediction of AUC. The ME was similar for all time points; the RMSE was smallest for C2, C4, and C5. The highest sensitivity index was determined to be 4.9 hours postdose at steady state, suggesting that this time point provides the most information about the AUC(0-12). The results from limited sampling strategies and sensitivity analysis supported the use of a single blood sample at 5 hours postdose as a predictor of both AUC(0-6) and AUC(0-12). A jackknife procedure was used to evaluate the predictive performance of the model, and this demonstrated that collecting a sample at 5 hours after dosing could be considered as the optimal sampling time for predicting AUC(0-6).

The authors describe a reverse-phase high-performance liquid chromatography-electrospray-tandem mass spectrometry method for the measurement of nicotine in human plasma. Samples (500 microL) with added deuterium-labeled d3-nicotine as an internal standard (IS) were treated with a 2-step process of ether extraction (6 mL) followed by back-extraction into 0.1% formic acid (50 microL). Chromatography was performed on a phenyl Novapak column with a mobile phase consisting of 50% 10 mM ammonium formate (pH 3.3) and acetonitrile (50:50, vol/vol). A flow rate of 0.2 mL/min resulted in a total analysis time of 5 minutes per sample. Mass spectrometric detection was by selected reactant monitoring (nicotine m/z 163.2 --130.2; IS m/z 166.2 --87.2). The assay was linear from 0.5 to 100 microg/L (r0.993, n = 9). The accuracy and imprecision of the method for quality control samples were 87.5% to 113% and10.2%, respectively. Interday accuracy and imprecision at the limit of quantification (0.5 microg/L) was 113% and 7.2% (n = 4). The process efficiency for nicotine in plasma was75%. The method described has good process efficiency, stabilized nicotine, avoided concentration steps, and most importantly minimized potential contamination. Further, we have established that water-based standards and controls are interchangeable with plasma-based samples. This method was used successfully to measure the pharmacokinetic profiles of subjects involved in the development of an aerosol inhalation drug delivery system.

BACKGROUND The development of hyperlipidemia after liver transplant is frequently treated with hydroxymethylglutaryl coenzyme A reductase inhibitors (statins) such as atorvastatin. As atorvastatin and the primary immunosuppressant drug, cyclosporine, are metabolized by the same pathway, there is the potential for an interaction. OBJECTIVE To determine the effect of atorvastatin on cyclosporine pharmacokinetics in liver transplant recipients. METHODS Six stable, long-term adult liver transplant recipients from a single center who developed posttransplant dyslipidemia were recruited to participate in a 14-day, open-label study of atorvastatin 10 mg/d coadministered with standard posttransplant immunosuppression using constant oral doses of cyclosporine and corticosteroids. A 10-point pharmacokinetic profile was performed prior to and on day 14 after commencement of atorvastatin therapy. Cyclosporine concentrations were measured by HPLC-electrospray-tandem mass spectrometry. The AUC was calculated by the linear trapezoidal rule, with other parameters determined by visual inspection. RESULTS Atorvastatin coadministration increased the cyclosporine AUC by 9% (range 0–20.6%; 3018 vs 3290 ng•h/mL; p = 0.04). No significant change was evident for other cyclosporine pharmacokinetic parameters. Total cholesterol and low-density lipoprotein cholesterol levels were significantly lower on day 14 than at baseline (p < 0.02). One patient developed a twofold increase in transaminases after 2 weeks of atorvastatin therapy, but no other clinical or biochemical adverse events were recorded. CONCLUSIONS Atorvastatin coadministration increases the cyclosporine AUC by approximately 10% in stable liver transplant recipients. This change in systemic exposure to cyclosporine is of questionable clinical significance. Atorvastatin is effective in reducing cholesterol levels in liver transplant recipients.

The immunosuppressant drug mycophenolic acid (MPA) and its major metabolite, mycophenolic acid glucuronide (MPAG), are highly bound to albumin. An HPLC-tandem-MS (HPLC/MS/MS) and an HPLC-UV assay were developed to measure free (unbound) concentrations of MPA and MPAG, respectively. Ultrafiltrate was prepared from plasma (500 microl) by ultrafiltration at 3000 x g for 20 min (20 degrees C). Both MPA and MPAG were isolated from ultrafiltrate (100 microl) by acidification and C18 solid-phase extraction. Free MPA was measured by electrospray tandem mass spectrometry using selected reactant monitoring (MPA: m/z 338.2--206.9) in positive ionisation mode. Chromatography was performed on a PFPP column (50 mm x 2 mm, 5 microm). Total analysis time was 7 min. The assay was linear over the range 1-200 microg/l with a limit of quantification of 1 microg/l. The inter-day accuracy and imprecision of quality controls (7.5, 40, 150 microg/l) were 94-99% and7%, respectively. Free MPAG was chromatographed on a C18 Nova-Pak column (150 mm x 3.9 mm, 5 microm) using a binary gradient over 20 min. The eluent was monitored at 254 nm. The assay was linear over the range 1-50 mg/l with the limit of quantification at 2.5 mg/l. The inter-day accuracy and imprecision of quality controls (5, 20, 45 mg/l) was 101-107% and8% (n = 4), respectively. For both methods no interfering substances were found in ultrafiltrate from patients not receiving MPA. The methods described have a suitable dynamic linear range to facilitate the investigation of free MPA and MPAG pharmacokinetics in transplant patients. Further, this is the first reported HPLC-UV method to determine free MPAG concentrations.

Although monitoring of tacrolimus blood concentrations is standard clinical practice following liver transplantation, a greater understanding of the relationship between trough concentrations and clinical outcome is required. The aim of this study was to perform a pharmacodynamic investigation of tacrolimus in pediatric liver transplant recipients. A retrospective analysis was performed on 35 pediatric liver recipients who received oral tacrolimus as the primary immunosuppressant. Outcome data were recorded corresponding to the times that tacrolimus trough concentrations had been determined (using a validated high-performance liquid chromatography-tandem mass spectrometry assay). A Mann-Whitney rank sum test was used to investigate differences between median concentrations at which drug toxicity, infection, and organ rejection did and did not occur. Outcome data and trough concentrations were recorded from the immediate post-transplant time to over 3 years (656 concentration-effect measures). Seventy one percent of data was obtained after the first 90 days post-transplant. Patients were identified as having experienced hypomagnesemia (20), hypertension (14), hyperkalemia (12), infection (11), nephrotoxicity (8), diarrhea (6), hyperglycemia (3), neurotoxicity(3), and rejection (3) during retrospective follow-up. Median trough concentrations were significantly higher (P

Patient outcomes in transplantation would improve if dosing of immunosuppressive agents was individualized. The aim of this study is to develop a population pharmacokinetic model of tacrolimus in adult liver transplant recipients and test this model in individualizing therapy. Population analysis was performed on data from 68 patients. Estimates were sought for apparent clearance (CL/F) and apparent volume of distribution (V/F) using the nonlinear mixed effects model program (NONMEM). Factors screened for influence on these parameters were weight, age, sex, transplant type, biliary reconstructive procedure, postoperative day, days of therapy, liver function test results, creatinine clearance, hematocrit, corticosteroid dose, and interacting drugs. The predictive performance of the developed model was evaluated through Bayesian forecasting in an independent cohort of 36 patients. No linear correlation existed between tacrolimus dosage and trough concentration (r(2) = 0.005). Mean individual Bayesian estimates for CL/F and V/F were 26.5 +/- 8.2 (SD) L/hr and 399 +/- 185 L, respectively. CL/F was greater in patients with normal liver function. V/F increased with patient weight. CL/F decreased with increasing hematocrit. Based on the derived model, a 70-kg patient with an aspartate aminotransferase (AST) level less than 70 U/L would require a tacrolimus dose of 4.7 mg twice daily to achieve a steady-state trough concentration of 10 ng/mL. A 50-kg patient with an AST level greater than 70 U/L would require a dose of 2.6 mg. Marked interindividual variability (43% to 93%) and residual random error (3.3 ng/mL) were observed. Predictions made using the final model were reasonably nonbiased (0.56 ng/mL), but imprecise (4.8 ng/mL). Pharmacokinetic information obtained will assist in tacrolimus dosing; however, further investigation into reasons for the pharmacokinetic variability of tacrolimus is required.

The Response to Offender Personality Disorder Consultation was released in October 2011. For some this is a welcome step in the right direction due to its therapeutic optimism, however for practitioners operating in the secure estate there are significant challenges ahead. This aim of this article is to discuss the increasing convergence of health and criminal justice and their inherent ideological and practical difficulties. It does so with reference to the consultation on offender personality disorder pathways and in particular the implications regarding multi-disciplinary and cross agency approaches to risk, public protection and personality disorder respectfully. It concludes that before embarking on a new wave of determining and responding to those with personality disorder, offender or otherwise, a more in-depth and empirically informed critical reflection is warranted.

The aims of this study were to investigate the population pharmacokinetics of tacrolimus in adult kidney transplant recipients and to identify factors that explain variability.Population analysis was performed on retrospective data from 70 patients who received oral tacrolimus twice daily. Morning blood trough concentrations were measured by liquid chromatography-tandem mass spectrometry. Maximum likelihood estimates were sought for apparent clearance (CL/F) and apparent volume of distribution (V/F), with the use of NONMEM (GloboMax LLC, Hanover, Md). Factors screened for influence on these parameters were weight, age, gender, postoperative day, days of tacrolimus therapy, liver function tests, creatinine clearance, hematocrit fraction, corticosteroid dose, and potential interacting drugs.CL/F was greater in patients with abnormally low hematocrit fraction (data from 21 patients only), and it decreased with increasing days of therapy and AST concentrations (P.01). Average parameter estimates were as follows: CL/F = 31.8 L/h (hematocrit0.33), CL/F = 24.2 L/h (hematocrit0.33), and V/F = 2080 L. Marked interindividual variability (42% to 111%) and residual random error (3.7 ng/mL) were observed. On the basis of the derived model, a patient with normal AST (20 U/L) or high AST (200 U/L) concentrations 7 days after commencement of therapy would require a tacrolimus dose of 4.6 mg or 4.0 mg, respectively, to achieve a steady-state trough concentration of 10 ng/mL.The population pharmacokinetics of tacrolimus in adult kidney transplant recipients showed wide variability. Thus it is not possible to use a standard tacrolimus dose as an empiric predictor of concentration in this population. An understanding of factors that influence the pharmacokinetics of tacrolimus may assist in drug dosage decisions.