Search

Human induced pluripotent stem cell (hiPSC) to cardiomyocyte (CM) differentiation has reshaped approaches to studying cardiac development and disease. In this study, we employed a genome-wide CRISPR screen in a hiPSC to CM differentiation system and reveal here that BRD4, a member of the bromodomain and extraterminal (BET) family, regulates CM differentiation. Chemical inhibition of BET proteins in mouse embryonic stem cell (mESC)-derived or hiPSC-derived cardiac progenitor cells (CPCs) results in decreased CM differentiation and persistence of cells expressing progenitor markers. In vivo, BRD4 deletion in second heart field (SHF) CPCs results in embryonic or early postnatal lethality, with mutants demonstrating myocardial hypoplasia and an increase in CPCs. Single-cell transcriptomics identified a subpopulation of SHF CPCs that is sensitive to BRD4 loss and associated with attenuated CM lineage-specific gene programs. These results highlight a previously unrecognized role for BRD4 in CM fate determination during development and a heterogenous requirement for BRD4 among SHF CPCs., (© 2024. The Author(s), under exclusive licence to Springer Nature Limited.)

Patients with advanced hepatocellular carcinoma (HCC) have several systemic treatment options. There are many known risk factors for HCC, and although some, such as hepatitis C, are now treatable, others are not. For example, metabolic dysfunction-related chronic liver disease is increasing in incidence and has no specific treatment. Underlying liver disease, drug resistance, and an increasing number of treatment options without specific biomarkers are all challenges in selecting the best treatment for each patient. Conventional chemotherapy is almost never used for advanced-stage disease, which instead is treated with immunotherapy, tyrosine kinase inhibitors, and VEGF inhibitors. Immune checkpoint inhibitors targeting various receptors have been or are currently undergoing clinical evaluation. Ongoing trials with three-drug regimens may be the future of advanced-stage HCC treatment. Other immune-modulatory approaches of chimeric antigen receptor-modified T cells, bispecific antibodies, cytokine-induced killer cells, natural killer cells, and vaccines are in early-stage clinical trials. Targeted therapies remain limited for HCC but represent an area of potential growth. As we shift away from first-line sorafenib for advanced HCC, clinical trial control arms should comprise a standard treatment other than sorafenib, one that is a better comparator for advancing therapies.

Maternal diabetes mellitus is among the most frequent environmental contributors to congenital birth defects, including heart defects and craniofacial anomalies, yet the cell types affected and mechanisms of disruption are largely unknown. Using multi-modal single cell analyses, here we show that maternal diabetes affects the epigenomic landscape of specific subsets of cardiac and craniofacial progenitors during embryogenesis. A previously unrecognized cardiac progenitor subpopulation expressing the homeodomain-containing protein ALX3 showed prominent chromatin accessibility changes and acquired a more posterior identity. Similarly, a subpopulation of neural crest-derived cells in the second pharyngeal arch, which contributes to craniofacial structures, displayed abnormalities in the epigenetic landscape and axial patterning defects. Chromatin accessibility changes in both populations were associated with increased retinoic acid signaling, known to establish anterior-posterior identity. This work highlights how an environmental insult can have highly selective epigenomic consequences on discrete cell types leading to developmental patterning defects., Competing Interests: Competing Interests Statement: D.S. is a scientific co-founder, shareholder and director of Tenaya Therapeutics. The remaining authors declare no competing interests.

Numerous recent advances have been made in therapeutic approaches toward acute myeloid leukemia (AML). Since 2017, we have seen eleven novel Food & Drug Administration (FDA)-approved medications for AML, all of which extend beyond the classical cytarabine-based cytostatic chemotherapy. In the recent two decades, the role of immune surveillance in AML has been intensively investigated. The power of one's own innate and adaptive immunity has been harnessed pharmacologically toward the goal of clearance of AML cells. Specifically, pre-clinical studies have shown great promise for antibodies that disinhibit T cells and macrophages by blocking checkpoint receptors within the immunologic synapse, thereby resulting in the elimination of AML cells. Anti-CD33 CAR-T therapies and anti-CD3/CD123 bispecific antibodies have also exhibited encouraging results in pre-clinical and early clinical studies. However, despite these translational efforts, we currently have no immune-based therapies for AML on the market, with the exception of gemtuzumab ozogamicin. In this focused review, we discuss molecular target validation and the most relevant clinical updates for immune-based experimental therapeutics including anti-CD47 monoclonal antibodies, CAR-T therapies, and bispecific T cell engagers. We highlight barriers to the clinical translation of these therapies in AML, and we propose solutions to optimize the manufacturing and delivery of the most novel immune-based therapies in the pipeline., Competing Interests: Declaration of Competing Interest SAP served on the Acute Myeloid Leukemia Advisory Board and the COMMANDS Advisory Board for Bristol Myers Squibb. SAP served on the Multiple Myeloma Advisory Board for Pfizer. JMG serves on the Advisory Board for Novartis and holds U.S. Patent No. 9012,215, U.S. Patent No. 10,222,376, and U.S. Patent No. 11,209,435. JC serves on advisory boards for Jazz Pharmaceuticals, Pfizer, MERIT CRO, and Amgen; serves as a Data Safety Monitoring Board Member with ICON-AlloVir and ICON-Prolacta; owns stock in Actinium Pharmaceuticals, Bluebird Bio/2Seventy, Dynavax Technologies, aTyr Pharma, Gamida Cell, Miragen Therapeutics, Mustang Bio, Novavax, Ovid Therapeutics, Sorrento Therapeutics, TG Therapeutics, Vaxart, and Veru. All other authors declare no conflicts., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

The staging of intrahepatic cholangiocarcinoma (ICC) is complex, and there is no consensus among international cancer groups on how to most appropriately select candidates with nonmetastatic disease for surgical resection. Factors contributing to a higher stage of disease include larger tumor size, multiple tumors, vascular invasion (either portal venous or arterial), biliary invasion, involvement of local hepatic structures, serosal invasion, and regional lymph node metastases. For patients selected to undergo surgery, it is well-documented that R0 resection translates to a survival benefit. Estimating the risk of post-hepatectomy liver failure and post-surgical residual liver function is vital and may preclude some patients with significant tumor burden from undergoing surgery. Numerous serum and biliary biomarkers of the disease can help detect recurrence in patients undergoing surgical resection. Systemic and locoregional neoadjuvant treatments to facilitate better surgical outcomes have yielded mixed results regarding improving resectability and overall survival. Additional research is needed to identify optimal neoadjuvant treatment approaches and to evaluate which patients will benefit most from these strategies. Therapies targeting genetic mutations and protein aberrations found by tumor molecular profiling may offer additional options for future neoadjuvant treatment.

Neural Style Transfer (NST) has been a widely researched topic as of late enabling new forms of image manipulation. Here we perform an extensive study on NST algorithms and extend the existing methods with custom modifications for application to Indian art styles. In this paper, we aim to provide a comprehensive analysis of various methods ranging from the seminal work of Gatys et al which demonstrated the power of Convolutional Neural Networks (CNNs) in creating artistic imagery by separating and recombining image content and style, to the state of the art image-to-image translation models which use Generative Adversarial Networks (GANs) to learn the mapping between two domain of images. We observe and infer based on the results produced by the models on which one could be a more suitable approach for Indian art styles, especially Tanjore paintings which are unique compared to the Western art styles. We then propose the method which is more suitable for the domain of Indian Art style along with custom architecture which includes an enhancement and evaluation module. We then present evaluation methods, both qualitative and quantitative which includes our proposed metric, to evaluate the results produced by the model.

The low-density lipoprotein receptor (LDLR) controls cellular delivery of cholesterol and clears LDL from the bloodstream, protecting against atherosclerotic heart disease, the leading cause of death in the United States. We therefore sought to identify regulators of the LDLR beyond the targets of current therapies and known causes of familial hypercholesterolemia. We found that cold shock domain-containing protein E1 (CSDE1) enhanced hepatic LDLR messenger RNA (mRNA) decay via its 3' untranslated region and regulated atherogenic lipoproteins in vivo. Using parallel phenotypic genome-wide CRISPR interference screens in a tissue culture model, we identified 40 specific regulators of the LDLR that were not previously identified by observational human genetic studies. Among these, we demonstrated that, in HepG2 cells, CSDE1 regulated the LDLR at least as strongly as statins and proprotein convertase subtilisin/kexin type 9 (PCSK9) inhibitors. In addition, we showed that hepatic gene silencing of Csde1 treated diet-induced dyslipidemia in mice to a similar degree as Pcsk9 silencing. These results suggest the therapeutic potential of targeting CSDE1 to manipulate the posttranscriptional regulation of the LDLR mRNA for the prevention of cardiovascular disease. Our approach of modeling a clinically relevant phenotype in a forward genetic screen, followed by mechanistic pharmacologic dissection and in vivo validation, may serve as a generalizable template for the identification of therapeutic targets in other human disease states.

Background: GATA4 (GATA-binding protein 4), a zinc finger-containing, DNA-binding transcription factor, is essential for normal cardiac development and homeostasis in mice and humans, and mutations in this gene have been reported in human heart defects. Defects in alternative splicing are associated with many heart diseases, yet relatively little is known about how cell type- or cell state-specific alternative splicing is achieved in the heart. Here, we show that GATA4 regulates cell type-specific splicing through direct interaction with RNA and the spliceosome in human induced pluripotent stem cell-derived cardiac progenitors., Methods: We leveraged a combination of unbiased approaches including affinity purification of GATA4 and mass spectrometry, enhanced cross-linking with immunoprecipitation, electrophoretic mobility shift assays, in vitro splicing assays, and unbiased transcriptomic analysis to uncover GATA4's novel function as a splicing regulator in human induced pluripotent stem cell-derived cardiac progenitors., Results: We found that GATA4 interacts with many members of the spliceosome complex in human induced pluripotent stem cell-derived cardiac progenitors. Enhanced cross-linking with immunoprecipitation demonstrated that GATA4 also directly binds to a large number of mRNAs through defined RNA motifs in a sequence-specific manner. In vitro splicing assays indicated that GATA4 regulates alternative splicing through direct RNA binding, resulting in functionally distinct protein products. Correspondingly, knockdown of GATA4 in human induced pluripotent stem cell-derived cardiac progenitors resulted in differential alternative splicing of genes involved in cytoskeleton organization and calcium ion import, with functional consequences associated with the protein isoforms., Conclusions: This study shows that in addition to its well described transcriptional function, GATA4 interacts with members of the spliceosome complex and regulates cell type-specific alternative splicing via sequence-specific interactions with RNA. Several genes that have splicing regulated by GATA4 have functional consequences and many are associated with dilated cardiomyopathy, suggesting a novel role for GATA4 in achieving the necessary cardiac proteome in normal and stress-responsive conditions.

Central obesity with cardiometabolic syndrome (CMS) is a major global contributor to human disease, and effective therapies are needed. Here, we show that cyclic GMP-selective phosphodiesterase 9A inhibition (PDE9-I) in both male and ovariectomized female mice suppresses preestablished severe diet-induced obesity/CMS with or without superimposed mild cardiac pressure load. PDE9-I reduces total body, inguinal, hepatic, and myocardial fat; stimulates mitochondrial activity in brown and white fat; and improves CMS, without significantly altering activity or food intake. PDE9 localized at mitochondria, and its inhibition in vitro stimulated lipolysis in a PPARα-dependent manner and increased mitochondrial respiration in both adipocytes and myocytes. PPARα upregulation was required to achieve the lipolytic, antiobesity, and metabolic effects of PDE9-I. All these PDE9-I-induced changes were not observed in obese/CMS nonovariectomized females, indicating a strong sexual dimorphism. We found that PPARα chromatin binding was reoriented away from fat metabolism-regulating genes when stimulated in the presence of coactivated estrogen receptor-α, and this may underlie the dimorphism. These findings have translational relevance given that PDE9-I is already being studied in humans for indications including heart failure, and efficacy against obesity/CMS would enhance its therapeutic utility.

In diseased organs, stress-activated signalling cascades alter chromatin, thereby triggering maladaptive cell state transitions. Fibroblast activation is a common stress response in tissues that worsens lung, liver, kidney and heart disease, yet its mechanistic basis remains unclear 1,2 . Pharmacological inhibition of bromodomain and extra-terminal domain (BET) proteins alleviates cardiac dysfunction 3-7 , providing a tool to interrogate and modulate cardiac cell states as a potential therapeutic approach. Here we use single-cell epigenomic analyses of hearts dynamically exposed to BET inhibitors to reveal a reversible transcriptional switch that underlies the activation of fibroblasts. Resident cardiac fibroblasts demonstrated robust toggling between the quiescent and activated state in a manner directly correlating with BET inhibitor exposure and cardiac function. Single-cell chromatin accessibility revealed previously undescribed DNA elements, the accessibility of which dynamically correlated with cardiac performance. Among the most dynamic elements was an enhancer that regulated the transcription factor MEOX1, which was specifically expressed in activated fibroblasts, occupied putative regulatory elements of a broad fibrotic gene program and was required for TGFβ-induced fibroblast activation. Selective CRISPR inhibition of the single most dynamic cis-element within the enhancer blocked TGFβ-induced Meox1 activation. We identify MEOX1 as a central regulator of fibroblast activation associated with cardiac dysfunction and demonstrate its upregulation after activation of human lung, liver and kidney fibroblasts. The plasticity and specificity of BET-dependent regulation of MEOX1 in tissue fibroblasts provide previously unknown trans- and cis-targets for treating fibrotic disease., (© 2021. The Author(s), under exclusive licence to Springer Nature Limited.)

The number of substituted pyridine pyridinophanes found in the literature is limited due to challenges associated with 12-membered macrocycle and modified pyridine synthesis. Most notably, the electrophilic character at the 4-position of pyridine in pyridinophanes presents a unique challenge for introducing electrophilic chemical groups. Likewise, of the few reported, most substituted pyridine pyridinophanes in the literature are limited to electron-donating functionalities. Herein, new synthetic strategies for four new macrocycles bearing the electron-withdrawing groups CN, Cl, NO 2 , and CF 3 are introduced. Potentiometric titrations were used to determine the protonation constants of the new pyridinophanes. Further, the influence of such modifications on the chemical behavior is predicted by comparing the potentiometric results to previously reported systems. X-ray diffraction analysis of the 4-Cl substituted species and its Cu(II) complex are also described to demonstrate the metal binding nature of these ligands. DFT analysis is used to support the experimental findings through energy calculations and ESP maps. These new molecules serve as a foundation to access a range of new pyridinophane small molecules and applications in future work.

Alzheimer's and other neurodegenerative diseases are chronic conditions affecting millions of individuals worldwide. Oxidative stress is a consistent component described in the development of many neurodegenerative diseases. Therefore, innovative strategies to develop drug candidates that overcome oxidative stress in the brain are needed. To target these challenges, a new, water-soluble 12-membered tetraaza macrocyclic pyridinophane L4 was designed and produced using a building-block approach. Potentiometric data show that the neutral species of L4 provides interesting zwitterionic behavior at physiological pH, akin to amino acids, and a nearly ideal isoelectric point of 7.3. The copper(II) complex of L4 was evaluated by X-ray diffraction and cyclic voltammetry to show the potential modes of antioxidant activity derived, which was also demonstrated by 2,2-diphenyl-1-picrylhydrazyl and coumarin carboxylic acid antioxidant assays. L4 was shown to have dramatically enhanced antioxidant activity and increased biological compatibility compared to parent molecules reported previously. L4 attenuated hydrogen peroxide (H 2 O 2 )-induced cell viability loss more efficiently than precursor molecules in the mouse hippocampal HT-22 cell model. L4 also showed potent (fM) level protection against H 2 O 2 cell death in a BV2 microglial cell culture. Western blot studies indicated that L4 enhanced the cellular antioxidant defense capacity via Nrf2 signaling activation as well. Moreover, a low-cost analysis and high metabolic stability in phase I and II models were observed. These encouraging results show how the rational design of lead compounds is a suitable strategy for the development of treatments for neurodegenerative diseases where oxidative stress plays a substantial role.

A major theme in constraint-based modeling is unifying experimental data, such as biochemical information about the reactions that can occur in a system or the composition and localization of enzyme complexes, with high-throughput data including expression data, metabolomics, or DNA sequencing. The desired result is to increase predictive capability and improve our understanding of metabolism. The approach typically employed when only gene (or protein) intensities are available is the creation of tissue-specific models, which reduces the available reactions in an organism model, and does not provide an objective function for the estimation of fluxes. We develop a method, flux assignment with LAD (least absolute deviation) convex objectives and normalization (FALCON), that employs metabolic network reconstructions along with expression data to estimate fluxes. In order to use such a method, accurate measures of enzyme complex abundance are needed, so we first present an algorithm that addresses quantification of complex abundance. Our extensions to prior techniques include the capability to work with large models and significantly improved run-time performance even for smaller models, an improved analysis of enzyme complex formation, the ability to handle large enzyme complex rules that may incorporate multiple isoforms, and either maintained or significantly improved correlation with experimentally measured fluxes. FALCON has been implemented in MATLAB and ATS, and can be downloaded from: https://github.com/bbarker/FALCON. ATS is not required to compile the software, as intermediate C source code is available. FALCON requires use of the COBRA Toolbox, also implemented in MATLAB., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Learning a new word involves many subsystems and their interactions. The purpose of this study was to examine whether speech practice facilitates the subsequent fast mapping performance of bilingual preschool children. Participants were 18 typically developing preschoolers who learned Cantonese (L1) as a home language and English (L2) as a second language. Each participant was asked to repeat each of the four novel words after an auditory model (speech practice) before he or she was exposed to the novel word-objects in the subsequent fast mapping task. Significant speech practice and language effects on fast mapping production scores were found. These findings suggest a complex relationship between speech learning, fast mapping, and L1-L2 language skills. Our results suggest that clinical methods that facilitate bilingual children's production skills (e.g., repeat after a model) hold promise as a means of improving the initial stage of word learning in both languages., (Thieme Medical Publishers 333 Seventh Avenue, New York, NY 10001, USA.)

The goal of this research was to examine word retention in bilinguals and monolinguals. Long-term word retention is an essential part of vocabulary learning. Previous studies have documented that bilinguals outperform monolinguals in terms of retrieving newly-exposed words. Yet, little is known about whether or to what extent bilinguals are different from monolinguals in word retention. Participants were 30 English-speaking monolingual adults and 30 bilingual adults who speak Spanish as a home language and learned English as a second language during childhood. In a previous study (Kan et al., 2014), the participants were exposed to the target novel words in English, Spanish, and Cantonese. In this current study, word retention was measured a week after the fast mapping task. No exposures were given during the one-week interval. Results showed that bilinguals and monolinguals retain a similar number of words. However, participants produced more words in English than in either Spanish or Cantonese. Correlation analyses revealed that language knowledge plays a role in the relationships between fast mapping and word retention. Specifically, within- and across-language relationships between bilinguals' fast mapping and word retention were found in Spanish and English, by contrast, within-language relationships between monolinguals' fast mapping and word retention were found in English and across-language relationships between their fast mapping and word retention performance in English and Cantonese. Similarly, bilinguals differed from monolinguals in the relationships among the word retention scores in three languages. Significant correlations were found among bilinguals' retention scores. However, no such correlations were found among monolinguals' retention scores. The overall findings suggest that bilinguals' language experience and language knowledge most likely contribute to how they learn and retain new words.

Purpose: This study examines the effects of the levels of speech practice on fast mapping in monolingual and bilingual speakers., Method: Participants were 30 English-speaking monolingual and 30 Spanish-English bilingual young adults. Each participant was randomly assigned to 1 of 3 practice conditions prior to the fast-mapping task: (a) intensive speech practice, (b) moderate speech practice, or (c) no practice. In a fast-mapping experiment, each participant was briefly exposed to novel objects and their corresponding novel words. Participants' knowledge of the target novel words was assessed immediately after the exposures., Results: There were significant effects of speech practice on fast mapping for both monolingual and bilingual adults. It is important to note that participants' language experience also played a role in their fast-mapping performance., Conclusion: The findings suggest that speech practice, interacting with language experience, facilitates the processes for fast mapping.

Purpose: The study was aimed at characterizing age-related changes in speech motor performance on a nonword repetition task as a function of practice and nonword length and complexity., Method: Nonword repetition accuracy, lip aperture coordination, and nonword production durations were assessed on 2 consecutive days for 16 young and 16 elderly participants for the production of 6 novel nonwords increasing in length and complexity., Results: The effect of age on the ability to accurately and rapidly repeat long, complex nonwords was significant. However, the authors found no differences between the speech motor coordinative patterns of young and elderly adults. Further, the authors demonstrated age- and nonword-specific within- and between-session gains in speech motor performance., Conclusions: The authors speculate that cognitive, sensory, and motor factors interact in complex ways in elderly individuals to produce individual differences in nonword repetition ability at the levels of both behavioral and speech motor performance.

Selective phosphodiesterase 2 (PDE2) inhibitors are shown to have efficacy in a rat model of osteoarthritis (OA) pain. We identified potent, selective PDE2 inhibitors by optimizing residual PDE2 activity in a series of phosphodiesterase 4 (PDE4) inhibitors, while minimizing PDE4 inhibitory activity. These newly designed PDE2 inhibitors bind to the PDE2 enzyme in a cGMP-like binding mode orthogonal to the cAMP-like binding mode found in PDE4. Extensive structure activity relationship studies ultimately led to identification of pyrazolodiazepinone, 22, which was >1000-fold selective for PDE2 over recombinant, full length PDEs 1B, 3A, 3B, 4A, 4B, 4C, 7A, 7B, 8A, 8B, 9, 10 and 11. Compound 22 also retained excellent PDE2 selectivity (241-fold to 419-fold) over the remaining recombinant, full length PDEs, 1A, 4D, 5, and 6. Compound 22 exhibited good pharmacokinetic properties and excellent oral bioavailability (F=78%, rat). In an in vivo rat model of OA pain, compound 22 had significant analgesic activity 1 and 3h after a single, 10 mg/kg, subcutaneous dose., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

We identified potent, selective PDE2 inhibitors by optimizing residual PDE2 activity in a series of PDE4 inhibitors, while simultaneously minimizing PDE4 activity. These newly designed PDE2 inhibitors bind to the PDE2 enzyme in a cGMP-like mode in contrast to the cAMP-like binding mode found in PDE4. Structure activity relationship studies coupled with an inhibitor bound crystal structure in the active site of the catalytic domain of PDE2 identified structural features required to minimize PDE4 inhibition while simultaneously maximizing PDE2 inhibition., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Purpose: The aim of the present study was to examine the effect of an internal vs. external 'focus of attention' on speech motor performance in healthy young adults., Method: Twenty adults (aged 18-25) participated in a within-subjects experimental design. Nonwords, real words, and tongue twisters were produced by each participant in both attentional focus conditions (internal and external), with order of condition counterbalanced across participants. Speech motor coordinative consistency, timing, and behavioral accuracy were investigated., Results: Accuracy of nonword, real word and tongue twister productions were relatively high across both external and internal focus conditions. A robust effect of condition on timing measures was noticed such that an internal focus (on articulatory movement) resulted in longer production durations and higher durational variability for most productions. In addition, an internal focus caused an increase in movement coordination variability for the production of real words., Conclusion: Our findings offer some preliminary support to the theory that operating in an external 'focus of attention' condition, with a focus on acoustic goals, results in more efficient, automatic control of speech movements., Learning Outcomes: After reading this article, the reader will be able to: (1) describe the relevance of and challenges associated with the application of principles that govern nonspeech movements to speech performance; (2) summarize the implications of an external vs. internal focus of attention for speech motor performance; and (3) describe its role in speech motor skill learning in healthy and disordered groups., (Copyright © 2013 Elsevier Inc. All rights reserved.)

OATP1B1, OATP1B3 and OATP2B1 are important members of the organic anion transporting polypeptides (OATP) family and are implicated in the hepatic disposition of endobiotics and xenobiotics. Quantitating the expression levels of human OATP1B1, OATP1B3 and OATP2B1 in in vitro systems and tissue samples could significantly improve attempts to scale up in vitro data and result in more effective in vitro-in vivo correlation of transporter-mediated effects on drug disposition, such as hepatic clearance. In the present study, a quantification method was developed, characterized, and implemented for simultaneous determination of human OATP1B1, OATP1B3 and OATP2B1 in HEK cells transfected with OATP-expressing plasmid vectors (SLCO1B1, SLCO1B3, and SLCO2B1, respectively), human hepatocytes, human brain capillary endothelial cells, and humanized mouse liver tissue using UPLC-MRM MS. Purified membrane protein standards prepared and characterized as previously reported (Protein Expr. Purif. 2008, 57, 163-71) were first used as standards for absolute quantification of the expression levels of the three human OATP membrane proteins. The specificity of the optimized MRM transitions were characterized by analyzing the tryptic digests of the membrane protein fraction of wild type HEK cells and control mouse liver tissue using the herein reported UPLC-MRM MS method. The linearity of the calibration curve spanned from 0.2 μg mL(-1) (0.040 μg mg(-1)) to 20 μg mL(-1) (4.0 μg mg(-1)), with accuracy (% RE) within 15% at all concentrations examined for all three OATPs of interest in this study. The intra- and inter-day assay accuracy (% RE) and coefficient of variations (% CV) of triplicates are all within 15% for all levels of quality control samples prepared by mixing the membrane fraction of control mouse liver tissue with the required amount of purified human OATP1B1, OATP1B3 and OATP2B1., (Copyright © 2011 Elsevier B.V. All rights reserved.)

The endogenous cannabinoid (endocannabinoid) anandamide is principally degraded by the integral membrane enzyme fatty acid amide hydrolase (FAAH). Pharmacological blockade of FAAH has emerged as a potentially attractive strategy for augmenting endocannabinoid signaling and retaining the beneficial effects of cannabinoid receptor activation, while avoiding the undesirable side effects, such as weight gain and impairments in cognition and motor control, observed with direct cannabinoid receptor 1 agonists. Here, we report the detailed mechanistic and pharmacological characterization of N-pyridazin-3-yl-4-(3-{[5-(trifluoromethyl)pyridin-2-yl]oxy}benzylidene)piperidine-1-carboxamide (PF-04457845), a highly efficacious and selective FAAH inhibitor. Mechanistic studies confirm that PF-04457845 is a time-dependent, covalent FAAH inhibitor that carbamylates FAAH's catalytic serine nucleophile. PF-04457845 inhibits human FAAH with high potency (k(inact)/K(i) = 40,300 M(-1)s(-1); IC(50) = 7.2 nM) and is exquisitely selective in vivo as determined by activity-based protein profiling. Oral administration of PF-04457845 produced potent antinociceptive effects in both inflammatory [complete Freund's adjuvant (CFA)] and noninflammatory (monosodium iodoacetate) pain models in rats, with a minimum effective dose of 0.1 mg/kg (CFA model). PF-04457845 displayed a long duration of action as a single oral administration at 1 mg/kg showed in vivo efficacy for 24 h with a concomitant near-complete inhibition of FAAH activity and maximal sustained elevation of anandamide in brain. Significantly, PF-04457845-treated mice at 10 mg/kg elicited no effect in motility, catalepsy, and body temperature. Based on its exceptional selectivity and in vivo efficacy, combined with long duration of action and optimal pharmacokinetic properties, PF-04457845 is a clinical candidate for the treatment of pain and other nervous system disorders.

Fatty acid amide hydrolase (FAAH) is an integral membrane serine hydrolase that degrades the fatty acid amide family of signaling lipids, including the endocannabinoid anandamide. Genetic or pharmacological inactivation of FAAH leads to analgesic and anti-inflammatory phenotypes in rodents without showing the undesirable side effects observed with direct cannabinoid receptor agonists, indicating that FAAH may represent an attractive therapeutic target for the treatment of inflammatory pain and other nervous system disorders. Herein, we report the discovery and characterization of a highly efficacious and selective FAAH inhibitor PF-04457845 (23). Compound 23 inhibits FAAH by a covalent, irreversible mechanism involving carbamylation of the active-site serine nucleophile of FAAH with high in vitro potency (k(inact)/K(i) and IC(50) values of 40300 M(-1) s(-1) and 7.2 nM, respectively, for human FAAH). Compound 23 has exquisite selectivity for FAAH relative to other members of the serine hydrolase superfamily as demonstrated by competitive activity-based protein profiling. Oral administration of 23 at 0.1 mg/kg results in efficacy comparable to that of naproxen at 10 mg/kg in a rat model of inflammatory pain. Compound 23 is being evaluated in human clinical trials.

Hearing and repeating novel phonetic sequences, or novel nonwords, is a task that taps many levels of processing, including auditory decoding, phonological processing, working memory, speech motor planning and execution. Investigations of nonword repetition abilities have been framed within models of psycholinguistic processing, while the motor aspects, which also are critical for task performance, have been largely ignored. We focused our investigation on both the behavioral and speech motor performance characteristics of this task as performed in a learning paradigm by 9- and 10-year-old children and young adults. Behavioral (percent correct productions) and kinematic (movement duration, lip aperture variability - an index of the consistency of inter-articulator coordination on repeated trials) measures were obtained in order to investigate the short-term (Day 1, first five vs. next five trials) and longer-term (Day 1 vs. Day 2, first five vs. next five trials) changes associated with practice within and between sessions. Overall, as expected, young adults showed higher levels of behavioral accuracy and greater levels of coordinative consistency than the children. Both groups, however, showed a learning effect, such that in general, later Day 1 trials and Day 2 trials were shorter in duration and more consistent in coordination patterns than Day 1 early trials. Phonemic complexity of the nonwords had a profound effect on both the behavioral and speech motor aspects of performance. The children showed marked learning effects on all nonwords that they could produce accurately, while adults' performance improved only when challenged by the more complex nonword stimuli in the set. The findings point to a critical role for speech motor processes within models of nonword repetition and suggest that young adults, similar to children, show short- and longer-term improvements in coordinative consistency with repeated production of complex nonwords. There is also a clear developmental change in nonword production performance, such that less complex novel sequences elicit changes in speech motor performance in children, but not in adults.

Unlabelled: The potential role of phonological complexity in destabilizing the speech motor systems of adults who stutter was explored by assessing the performance of 17 adults who stutter and 17 matched control participants on a nonword repetition task. The nonwords varied in length and phonological complexity. Behavioral results revealed no differences between the stuttering and normally fluent groups on accuracy of nonword repetition. In contrast, dramatic differences between groups were observed in the kinematic data. Indices of the consistency of inter-articulator coordination revealed that adults who stutter were much less consistent in their coordinative patterns over repeated productions. With increasing length and complexity of the nonwords, between-group differences in coordinative consistency were more pronounced. Coordination consistency measures revealed that adults who stutter (but not normally fluent adults) showed within-session practice effects; their coordinative consistency improved in five later compared to five earlier productions. Adults who stutter produced the nonwords at a slower rate, but both groups showed increased rates of production on the later trials, indicating a practice effect for duration for both groups. We conclude that, though the adults who stutter performed behaviorally with the same accuracy as normally fluent adults, the nonword repetition task reveals remarkable differences in the speech motor dynamics underlying fluent speech production in adults who stutter compared to their normally fluent peers. These results support a multifactorial, dynamic model of stuttering in which linguistic complexity and utterance length are factors that contribute to the probability of breakdown of the speech motor system., Educational Objectives: After reading this article, the reader will be able to: (1) summarize the literature on potential language/motor interactions in stuttering, and (2) evaluate to what extent the study findings support the hypothesis that phonologically complex utterances have a destabilizing effect on the speech motor system in individuals who stutter.

Although the strategic use of enzymatic digestion combined with isotope dilution mass spectrometry has been increasingly developed and used for the absolute quantification of therapeutic and endogenous proteins in the biopharmaceutical industry over the past several years, the lack of an appropriate internal standard has become the rate-limiting step in the development of a standardized analytical approach to provide bioanalytical support for both preclinical and clinical studies. In this study, we present a universal strategy for fast development and validation (within 1-2 weeks) of a method for absolute quantification of a therapeutic monoclonal antibody in biological matrices using differential dimethyl labeling coupled with UPLC-MS/MS. Differential dimethyl labeling of tryptic peptides generated from the purified therapeutic monoclonal antibody and those derived from proteins in cynomolgus monkey serum with either d(2)- or d(0)-formaldehyde provided a fast, cost-effective, and standardized approach to generate internal standards for any surrogate peptides that are used to quantify the therapeutic monoclonal antibody in biological matrices. This labeling reaction employs inexpensive and commercially available reagents, d(0)- or d(2)-formaldehyde, to globally label the N-terminus and epsilon-amino group of Lys in a peptide via reductive amination. Moreover, the process is simple, relatively fast (<2 h reaction time), specific, and quantitative under mild reaction conditions. The chromatographic run time is 6 min per sample. The linearity of the assay for the selected monoclonal antibody was established from 1.00 to 1000 mug/mL with accuracy and precision within 15% at all concentrations. The intraday and interday assay accuracy (%RE) and coefficient of variations (CV%) are all within 15% for all QCs (2.00, 4.00, 20.0, 100, 750 mug/mL) prepared in three different serum pools from male and female cynomolgus monkeys.

Endocannabinoids are lipid signaling molecules that regulate a wide range of mammalian behaviors, including pain, inflammation, and cognitive/emotional state. The endocannabinoid anandamide is principally degraded by the integral membrane enzyme fatty acid amide hydrolase (FAAH), and there is currently much interest in developing FAAH inhibitors to augment endocannabinoid signaling in vivo. Here, we report the discovery and detailed characterization of a highly efficacious and selective FAAH inhibitor, PF-3845. Mechanistic and structural studies confirm that PF-3845 is a covalent inhibitor that carbamylates FAAH's serine nucleophile. PF-3845 selectively inhibits FAAH in vivo, as determined by activity-based protein profiling; raises brain anandamide levels for up to 24 hr; and produces significant cannabinoid receptor-dependent reductions in inflammatory pain. These data thus designate PF-3845 as a valuable pharmacological tool for in vivo characterization of the endocannabinoid system.

An LC-MS/MS method was developed and validated to quantify endogenous cyclic guanosine 3',5'-monophosphate (cGMP) in human plasma. The LC-MS/MS and competitive enzyme immunoassay (EIA) assays were compared. cGMP concentrations of 20 human plasma samples were measured by both methods. For the MS-based assay, plasma samples were subjected to a simple protein precipitation procedure by acetonitrile prior to analysis by electrospray ionization LC-MS/MS. De-protonated analytes generated in negative ionization mode were monitored through multiple reaction monitoring (MRM). A stable isotope-labeled internal standard, (13)C(10),(15)N(5)-cGMP, which was biosynthesized in-house, was used in the LC-MS/MS method. The competitive EIA was validated using a commercially available cGMP fluorescence assay kit. The intra-assay accuracy and precision for MS-based assay for cGMP were 6-10.1% CV and -3.6% to 7.3% relative error (RE), respectively, while inter-assay precision and accuracy were 5.6-8.1% CV and -2.1% to 6.3% RE, respectively. The intra-assay accuracy and precision for EIA were 17.9-27.1% CV and -4.9% to 24.5% RE, respectively, while inter-assay precision and accuracy were 15.1-39.5% CV and -30.8% to 4.37% RE, respectively. Near the lower limits of detection, there was little correlation between the cGMP concentration values in human plasma generated by these two methods (R(2)=0.197, P=0.05). Overall, the MS-based assay offered better selectivity, recovery, precision and accuracy over a linear range of 0.5-20ng/mL. The LC-MS/MS method provides an effective tool for the quantitation of cGMP to support clinical mechanistic studies of curative pharmaceuticals.

Purpose: The authors examined the effects of utterance length and linguistic complexity on speech movement consistency for 210 participants between the ages of 5 and 22 years. Variability and durational analyses were conducted to (a) determine a more complete picture of the developmental course of earlier observations of the effects of linguistic constructs on speech motor variability and (b) describe trends for duration of the same sequence of words in different sentential contexts across development., Method: Lower-lip movement was recorded during the production of "buy Bobby a puppy" spoken in isolation as well as embedded as a phrase in 2 longer, more complex sentences., Results: Compared with young adults, children demonstrated higher variability in producing repeated movement sequences for the target word sequence across all conditions. Also, for all age groups except young adults, increased processing demands resulted in significantly increased movement trajectory variability. Duration analyses suggest that around age 9 years, children begin to use adult-like pre-speech processes to plan the timing of sentence internal phrases, and maturation of these planning processes continues through late adolescence., Conclusion: These results provide further evidence for language-motor interactions and for a protracted course of speech motor development that continues well into adolescence.

Background: Recent evidence suggests that succinate, long known as an intermediate in the citric acid cycle, may also have a role as a signaling molecule through GPR91 and that activation of this receptor results in blood pressure (BP) elevation via the renin-angiotensin system. We sought to test the hypothesis that GPR91 contributes to BP elevation in hypertension. In addition we investigated whether elevated succinate in diabetes could contribute to the increased rate of gluconeogenesis in that condition., Methods: Circulating succinate concentration was measured using liquid chromatography tandem mass spectrometry in rodent models of hypertension and metabolic disease as well as in human hypertensives and type 2 diabetics in comparison to control subjects., Results: Elevated succinate was detected in spontaneously hypertensive rats (SHR), ob/ob mice, db/db mice, and fa/fa rats in comparison to their non-diseased controls. The changes in concentration are consistent with activation of GPR91. In contrast, neither human hypertensives nor diabetic patients had elevated succinate in comparison to controls., Conclusions: These findings are consistent with a role of GPR91 signaling in rodent hypertension and diabetes models but not in the analogous human diseases.

The use of a cassette incubation of probe substrates with human liver microsomes (HLM) - also known as the 'cocktail' approach - is becoming a widely accepted approach to determine the interaction of new chemical entities (NCEs) with cytochrome P450 enzymes (CYP450) in early drug discovery. This article describes two LC-MS/MS-based analytical methods used at the high-throughput (HT) stage and late discovery (LD) stage for analysis of 'cocktail' incubates to analyze the probe metabolites 1'-hydroxymidazolam (CYP3A4), 4'-hydroxydiclofenac (CYP2C9), dextrorphan (CYP2D6), 1'-hydroxytacrine (CYP1A2) and 4'-hydroxymephenytoin (CYP2C19). The analytical methods are advantageous over currently reported methods due to their sensitivity, shorter analyses times (<2 min/sample for the HT method and 4 min/sample for the LD method) and their ability to monitor a unique set of clinically relevant probe metabolites from a biological incubate containing low microsomal protein (0.1mg/mL). The analytical methods employ the same mobile phase, acetonitrile and 0.1% formic acid, under similar LC-MS/MS conditions. In the HT method, the chromatographic method consists of a short robust step-gradient where the probe metabolites are simultaneously and quickly eluted to enhance throughput. The probe metabolites are chromatographically resolved in the LD stage by utilizing a true linear gradient to obtain optimal peak separation. The IC50 data generated by both analytical methods using single incubations versus cocktail incubations for various test compounds are in good agreement (correlation coefficient (r2)>or=0.98). The scientist conducting the analysis is provided with a choice of method selection depending on the stage of the test compound and on whether throughput or minimizing interference from other co-eluting metabolites is the most important criterion.

Individuals with Parkinson's disease (PD) demonstrate low vocal intensity (hypophonia) which results in reduced speech intelligibility. We examined the effects of three cues to increase loudness on respiratory support in individuals with PD. Kinematic data from the rib cage and abdomen were collected using respiratory plethysmography while participants read a short passage. Individuals with PD and normal age- and sex-matched controls (OC) increased sound pressure level (SPL) to a similar extent. As compared to OC, individuals with PD used larger rib cage volume excursions in all conditions. Further, they did not slow their rate of speech in noise as OC speakers did. Respiratory strategies used to support increased loudness varied with the cue, but the two groups did not differ in the strategies used. When asked to target a specific loudness, both groups used more abdominal effort than at comfortable loudness. Speaking in background noise resulted in the largest increase in SPL with the most efficient respiratory patterns, suggesting that natural or implicit cues may be best when treating hypophonia in individuals with PD. Data demonstrate the possibility that both vocal loudness and speech rate are impacted by cognitive mechanisms (attention or self-perception) in individuals with PD.

Present study aims to improve efficiency and capacity of in vivo rat pharmacokinetic studies for rapid assessment of systemic exposure (AUC and C(max)) of new chemical entities. Plasma concentration-time profiles in rats from structurally diverse compounds were extracted from the Pfizer database. AUC(0-8) was calculated with 7 data points or a reduced subset of 3 data points. AUC values determined with 7 data points were compared to subset AUC values. A < or = 30% difference in values for 90% of cases was acceptance criteria. In parallel, samples were analyzed individually and pooled at each time point across compounds. For 96% of cases, AUC values estimated using 1, 4, and 8 h were comparable to AUC values obtained from 7 data points suggesting 1, 4, and 8 h sampling should be sufficient to estimate AUC. For C(max), the difference between 1, 4, and 8 h data-point analysis versus 7 data-point analysis is less than 30% for 72% of cases. Concentrations from individual versus pooled sample analysis were found to be equivalent. A rapid rat PK screening paradigm was created by the combination of 1, 4, and 8 h sampling and pooled sample analysis, which improves throughput and cycle time of in vivo PK studies., (Copyright 2006 Wiley-Liss, Inc.)

An online extraction/mass spectrometry technique was evaluated for direct analysis of plasma samples. A simple user-friendly online extraction system that consists of two pumps, an autosampler, a six-port switching valve and a mass spectrometer is described. The system was controlled by the LC-MS software (Masslynx 3.5, Waters Corporation, Beverly, MA). Various analytical conditions such as extraction column, mobile phases, run time and wash solvent were optimized to establish an analytical method that was simple, easy to set up and generic. Sample preparation effort was minimal, which included dilution of plasma with water and centrifugation conducted in 96-well plate format. The system was used to analyze in vivo plasma samples from rat n-in-one cassette dosing studies. Concentration and pharmacokinetic (PK) data obtained from the online extraction method were comparable with data obtained from the protein precipitation extraction method. Overall, the simple, robust online extraction system provides cost savings by minimizing sample preparation and method development time. The system was used to analyze compounds from different structural classes. These studies suggest that calculated lipophilicity of a compound can be used as a tool for pre-selection of extraction column, which would save method development time for early discovery studies.

Drug-metabolizing enzymes and drug transporters are key regulators of drug disposition and pharmacodynamics, which are closely linked to drug efficacy and safety. In this article, current challenges and future solutions to predicting their influence on pharmacokinetics and inter-organ distribution in humans, from data generated during the drug discovery decision-making process, are presented. In vitro phenotyping strategies for drug metabolizing enzymes (eg, CYP3A4, UGT1A1) and transporters (eg, OATP1B1) are offered, including perspectives on a selection of in vitro systems, novel in vitro phenotyping reagents and remaining technology gaps, challenges in extrapolating in vitro data to the in vivo situation, in silico models for the prediction of whether compounds are enzyme or transporter substrates, and the impact of pharmacogenomics.

Avasimibe, an acyl-CoA:cholesterol acyltransferase inhibitor, has been previously shown to be a potent inducer of CYP3A4 and multiple drug resistance protein 1. We have further characterized the drug interaction potential of avasimibe by studying the inductive and inhibitory effect of this compound on major drug-metabolizing enzymes. Enzymes known to be involved in the metabolism of drugs likely to be coadministered with avasimibe, such as CYP1A1/2, CYP2C, and CYP2B6, were evaluated further by microarray analysis, Western immunoblotting, and activity assays, using rifampicin and beta-naphthoflavone as positive controls. No change was observed in CYP1A1/2 mRNA or activity levels after avasimibe treatment. Differential induction of CYP2C9- and CYP2B6-immunoreactive protein and activity was observed depending on drug concentration and donor. Microarray analysis showed a similar increase in CYP2C and CYP2B6 mRNA levels. The inhibition potential of avasimibe on the major drug-metabolizing enzymes was assessed using pooled human liver microsomes. Avasimibe inhibited CYP2C9 (IC50 2.9 microM), CYP1A2 (IC50 13.9 microM), and CYP2C19 (IC50 26.5 microM). A clinical drug interaction study was conducted to determine whether avasimibe might interact with the CYP2C9 substrate warfarin. Volunteers received 750 mg of avasimibe and showed a 54.2% reduction in trough concentrations of S-warfarin and decreased prothrombin times by 12, 15, 19, and 21% on days 6 through 9, respectively. These results demonstrate that avasimibe's inductive spectrum resembles that of rifampin.

Induction of drug-metabolizing enzymes and transporters can cause drug-drug interactions and loss of efficacy. In vitro induction studies traditionally use primary hepatocyte cultures and enzyme activity with selected marker compounds. We investigated the use of a novel human hepatocyte clone, the Fa2N-4 cell line, as an alternative reagent, which is readily available and provides a consistent, reproducible system. We used the Invader assay to monitor gene expression in these cells. This assay is a robust, yet simple, high-throughput system for quantification of mRNA transcripts. CYP1A2, CYP3A4, CYP2C9, UGT1A, and MDR1 transcripts were quantified from total RNA extracts from Fa2N-4 cells treated with a panel of known inducers and compared with vehicle controls. In addition, we used enzyme activity assays to monitor the induction of CYP1A2, CYP2C9, and CYP3A4. The Fa2N-4 cells responded in a similar manner as primary human hepatocytes. Treatment with 10 microM rifampin resulted in increases in CYP3A4 mRNA (17-fold) and activity (6-beta-hydroxytestoterone formation, 9-fold); and in CYP2C9 mRNA (4-fold) and activity (4'-hydroxydiclofenac formation, 2-fold). Treatment with 50 microM beta-naphthoflavone resulted in increases in CYP1A2 mRNA (15-fold) and activity (7-ethoxyresorufin O-dealkylation, 27-fold). UGT1A mRNA was induced by beta-naphthoflavone (2-fold), and MDR1 (P-glycoprotein) mRNA was induced by rifampin (3-fold). These preliminary data using a few prototypical inducers show that Fa2N-4 cells can be a reliable surrogate for primary human hepatocytes, and, when used in conjunction with the Invader technology, could provide a reliable assay for assessment of induction of drug-metabolizing enzymes and transporters.

This longitudinal case study examined the emergence of a wide range of oral language skills in a deaf child whose cochlear implant was activated at 20 months. The main purposes of this study were to determine "Hannah's" rate of spoken language development during her second to fourth year of implant experience and to estimate the efficiency of her progress by comparing her performance to that of typically developing children. Mother-child interactions were also examined to determine changes in Hannah's communication competence. Normal or above-normal rates of development were observed in the following areas: (a) decreased production of nonwords, (b) increased receptive vocabulary, (c) type-token ratio, (d) regular use of word combinations, and (e) comprehension of phrases. Below-normal rates of development were observed in the following areas: (a) speech intelligibility, (b) number of word types and tokens, and (c) mean length of utterance in morphemes. Analysis of parent-child interactions showed a large increase in responses to questions during the third year of implant use. Data from Hannah's first post-implantation year (D. J. Ertmer & J. A. Mellon, 2001) indicated that some early language milestones were attained quite rapidly (e.g., canonical vocalizations and emergence of first word combinations). In contrast, the current study revealed that progress had slowed for related, but more advanced skills (e.g., production of intelligible speech and consistent use of word combinations). These changes in rate of development suggest that any advantages for language learning due to Hannah's advanced maturity (or other unknown factors) decreased with time and increasing linguistic complexity.

Cyclophosphamide (CP) and its metabolite, hydroxycyclophosphamide (OH-CP) have been quantitated in mouse plasma and tissue by derivatization combined with liquid chromatography-tandem mass spectrometry (LC-MS-MS). The derivatization was conducted immediately upon sample collection, to trap the OH-CP metabolite intermediate prior to further conversion to phosphoramide mustard or other reaction products. This simple and straightforward derivatization procedure, combined with sample extraction via protein precipitation, allowed quantitation of CP and the oxime derivative of OH-CP in plasma for concentrations ranging from approximately 12.5-3333 ng/ml, and in spleen tissue for concentrations of 1,250-50,000 ng/g. The short cycle time (2.5 min) of the LC-MS-MS method allowed high throughput analysis with minimal matrix interference. Mouse plasma levels were quantitated for doses of 40, 65 and 120 mg/kg; spleen concentrations were determined for mice dosed at 120 mg/kg. The CP and oxime plasma levels correlated well with dose amounts. The CP levels in the spleen and plasma were similar while the oxime levels in the spleen were significantly lower than the plasma.

Mass spectrometry of charged derivatives of peptides has been a growing area of interest in the past decade. Fragmentation of charged derivatives of peptides is believed to be different from than that of protonated peptides when analyzed by collisionally activated dissociation-tandem mass spectrometry (CAD-MS/MS). The charged derivatives fragment by charge-remote fragmentation mechanisms, which are usually classified as high-energy (HE)-CAD processes. Our objective in the present study is to investigate the mechanism of fragmentation of charged derivatives of peptides when analyzed by matrix-assisted laser desorption/ionization-postsource decay-mass spectrometry (MALDI-PSD-MS) and electrospray ionization (ESI)-CAD-MS/MS (ion trap), which involve low-energy processes. Three major types of hydrogens (alpha, beta, and amide) are available for migration during the formation of the *a(n) ions (the predominant ion series produced from these charged derivatives). To pinpoint which of the three hydrogens is involved in the formation of the *a(n) ions, deuterium-labeled peptide derivatives with labels at specific sites were synthesized and analyzed by MALDI-PSD-MS and ESI-CAD-MS/MS. Our results suggest that the amide hydrogen of the residue at which the cleavage occurs shifts during the formation of *a(n); this observation serves as evidence for the mechanism proposed earlier by Liao et al. for fragmentation of such charged derivatives. The results also help elucidate the structure of the *a(n) ions, *b(n) ions, and others formed during cleavage at the proline residue, as well as the ions formed during loss of the C-terminal residue from these charged derivatives.

Charged derivatives of peptides are useful in obtaining simpler collision-activated dissociation (CAD) mass spectra. An N-terminal charge-derivatizing reagent capable of reacting with picomole levels of peptide has been recently reported (Huang et al. Anal. Chem. 1997, 69, 137-144) in the contexts of analyses by fast atom bombardment (FAB) and matrix-assisted laser desorption/ionization (MALDI) mass spectrometry. Electrospray ionization (ESI) mass spectrometric investigation of these tris(trimethoxyphenylphosphonium) acetyl derivatives are described in this article, including studies by in-source fragmentation (ISF) and tandem mass spectrometry (MS/MS). Results from ISF are compared with those from MS/MS. Similarities and differences between ESI-ISF, MALDI-post-source decay (PSD), and FAB-CAD data are presented. Differences in fragmentation of these charged derivatives in the triple quadrupole and ion trap mass spectrometers also are discussed. Application of this derivatizing procedure to tryptic digests and subsequent analysis by liquid chromatography-mass spectrometry is also shown.

The analysis of peptide derivatives by fast atom bombardment, liquid secondary-ionization mass spectrometry, plasma desorption, electrospray ionization, and matrix-assisted laser desorption/ionization is reviewed. The fragmentation patterns of peptides and of charge-derivatized peptides are compared, and the proposed fragment ion structures are summarized. A variety of derivatization approaches and the distinguishing features of mass spectra produced from these derivatives are described. The most promising derivatization approaches are evaluated, and the strengths and limitations of these approaches are discussed.