ORCID Profile
0000-0002-9605-2010
Current Organisation
University of Colorado at Boulder
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Publisher: Springer Science and Business Media LLC
Date: 08-2006
Abstract: Here we describe protocols for preparing and using fluorescent probes that respond to conformational changes by altered Foerster resonance energy transfer (FRET) efficiencies upon phosphorylation or, in principle, other posttranslational modifications (PTMs). The sensor protein, a truncated version of pleckstrin, is sandwiched between short-wavelength-excitation green fluorescent protein (GFP2) and yellow fluorescent protein (EYFP). As a result of complex conformational changes of the protein upon phosphorylation, the introduction of a second PTM consensus sequence bestows sensitivity to a second modification and yields a dual-parameter probe. The first phase of the protocol lays out the cloning strategy for single- and dual-parameter FRET sensors, including the construction of a versatile platform into which different consensus sequences may be inserted to create erse probes. Protocols for fluorescence microscopy of the probes in living cells and image processing are also described. Probe preparation takes 7 d microscopy and image processing take 2 h.
Publisher: Brazilian Society of Physical Activity and Health
Date: 05-09-2018
Abstract: High volumes of daily television viewing time increase risk of cardiovascular disease and other health problems. We report the descriptive epidemiology of high (3+ hrs/day) and very high (5+ hrs/ day) television (TV) viewing time and their correlates among Brazilian adults. Data from 60,202 participants (18 more years of age) in the National Health Survey carried in 2013 were analysed. The proportions of adults spending more than 3 hours per day and more than 5 hours/day watching TV were calculated. Descriptive and adjusted analyses examined variations in the prevalence of high and very high (of TV viewing time by age, educational attainment and physical activity level, separately for men and women. Prevalence in a normal day of high TV viewing time was 28.9% and was 8.9% for very high TV time. Overall, women and those who were less educated reported more TV time than did men and the more educated. There was a slightly inverse association between physical activity practice and TV time for men but there was not a clear association between physical activity and time spent watching TV among women. Prevalence of very high TV time was 46% lower among high-educated women than their counterparts. The prevalence of high and very high TV time suggests the need to implement actions to address high levels of TV time as a prevalent leisure- time sedentary behaviour and to promote attractive active recreational options at the population level in Brazil.
Publisher: Springer Science and Business Media LLC
Date: 11-09-2011
DOI: 10.1038/NMETH.1699
Publisher: Springer Science and Business Media LLC
Date: 28-10-2019
Publisher: Elsevier BV
Date: 12-2016
Publisher: Elsevier BV
Date: 05-2016
Publisher: Informa UK Limited
Date: 09-2008
DOI: 10.4161/EPI.3.5.7005
Abstract: Post translational modification (PTM) of histones has long been associated with epigenetic regulation. Although genomic approaches have established correlation between a handful of histone PTMs and transcriptional states, only recently have advancements in proteomics provided the tools necessary to study histone proteins and their relevant modifications in this context. Using mass spectrometry, researchers have demonstrated the ability to determine the full repertoire of histone PTMs, their residue specific location, the combinations in which they exist, and the proteins that interact with these combinations. Moving forward it will be imperative to develop novel approaches that combine proteomic and genomic technologies to determine the functional significance of these combinations of modifications. Assays with increased specificity will resolve more focused biological questions and determine to what extent, and by what mechanisms, histones influence transcription.
Publisher: Elsevier BV
Date: 11-2009
DOI: 10.1016/J.CHEMBIOL.2009.10.005
Abstract: Phosphoinositides are important signaling molecules that govern a large number of cellular processes such as proliferation, differentiation, membrane remodeling, and survival. Here we introduce a fully synthetic membrane-permeant derivative of a novel, easily accessible, and very potent phosphatidylinositol 3,4,5-trisphosphate [PtdIns(3,4,5)P(3)] mimic: phosphatidylinositol 3,4,5,6-tetrakisphosphate [PtdIns(3,4,5,6)P(4)]. The membrane-permeant PtdIns(3,4,5,6)P(4) derivative activated pathways downstream of phosphatidylinositol 3-kinase (PI3K), including protein kinase B, p70S6K, mitogen-activated protein kinase, and protein kinase C, more potently than similar membrane-permeant PtdIns(3,4,5)P(3) and PtdIns(3,4)P(2) derivatives in the absence of receptor stimulation. In addition, we demonstrate that treatment of PC12 cells with the membrane-permeant PtdIns(3,4)P(2), PtdIns(3,4,5)P(3), and PtdIns(3,4,5,6)P(4) derivatives increases the number of neurites per cell in the presence of NGF. This work establishes membrane-permeant phosphoinositides as powerful tools to study PI3K signaling and directly demonstrates that 3-phosphorylated phosphoinositides are instrumental for neurite initiation.
Publisher: Elsevier BV
Date: 02-2019
Publisher: American Chemical Society (ACS)
Date: 09-12-2005
DOI: 10.1021/JA0562200
Abstract: Cell function is regulated by complex and often interdependent networks of signaling molecules. To accurately describe these networks, it is important to monitor multiple signals in parallel. To this end, we have developed a genetically encoded, FRET-based probe that independently monitors both protein kinase A (PKA) and protein kinase C (PKC) activity in vivo. Artificial as well as physiological stimulants produced a negative or positive change in FRET efficiency following PKA or PKC activation, respectively. Mutations of the phosphate-accepting amino acids of the PKC substrate yielded a probe that was sensitive to PKA activation alone.
Publisher: The Company of Biologists
Date: 12-2019
DOI: 10.1242/DEV.182170
Abstract: Development and homeostasis rely upon concerted regulatory pathways to establish the specialized cell types needed for tissue function. Once a cell type is specified, the processes that restrict and maintain cell fate are equally important in ensuring tissue integrity. Over the past decade, several approaches to experimentally reprogram cell fate have emerged. Importantly, efforts to improve and understand these approaches have uncovered novel molecular determinants that reinforce lineage commitment and help resist cell fate changes. In this Review, we summarize recent studies that have provided insights into the various chromatin factors, post-transcriptional processes and features of genomic organization that safeguard cell identity in the context of reprogramming to pluripotency. We also highlight how these factors function in other experimental, physiological and pathological cell fate transitions, including direct lineage conversion, pluripotency-to-totipotency reversion and cancer.
Publisher: The Company of Biologists
Date: 17-10-2023
DOI: 10.1242/DEV.202169
Publisher: Elsevier BV
Date: 03-2014
Publisher: Springer Science and Business Media LLC
Date: 22-06-2015
DOI: 10.1038/NBT.3247
Publisher: Proceedings of the National Academy of Sciences
Date: 14-05-2012
Abstract: We have developed and implemented a sequence identification algorithm ( inSeq ) that processes tandem mass spectra in real-time using the mass spectrometer’s (MS) onboard processors. The inSeq algorithm relies on accurate mass tandem MS data for swift spectral matching with high accuracy. The instant spectral processing technology takes ∼16 ms to execute and provides information to enable autonomous, real-time decision making by the MS system. Using inSeq and its advanced decision tree logic, we demonstrate ( i ) real-time prediction of peptide elution windows en masse (∼3 min width, 3,000 targets), ( ii ) significant improvement of quantitative precision and accuracy (~3x boost in detected protein differences), and ( iii ) boosted rates of posttranslation modification site localization (90% agreement in real-time vs. offline localization rate and an approximate 25% gain in localized sites). The decision tree logic enabled by inSeq promises to circumvent problems with the conventional data-dependent acquisition paradigm and provides a direct route to streamlined and expedient targeted protein analysis.
Publisher: Elsevier BV
Date: 2018
Publisher: Elsevier BV
Date: 05-2011
Publisher: Springer Science and Business Media LLC
Date: 26-07-2017
DOI: 10.1038/NATURE23274
Publisher: Informa UK Limited
Date: 15-10-2011
Publisher: Springer Science and Business Media LLC
Date: 20-08-2018
Publisher: Elsevier BV
Date: 05-2017
Publisher: Elsevier BV
Date: 11-2019
Publisher: Elsevier BV
Date: 10-2012
Publisher: Proceedings of the National Academy of Sciences
Date: 18-03-2008
Abstract: Epigenetic regulation through chromatin is thought to play a critical role in the establishment and maintenance of pluripotency. Traditionally, antibody-based technologies were used to probe for specific posttranslational modifications (PTMs) present on histone tails, but these methods do not generally reveal the presence of multiple modifications on a single-histone tail (combinatorial codes). Here, we describe technology for the discovery and quantification of histone combinatorial codes that is based on chromatography and mass spectrometry. We applied this methodology to decipher 74 discrete combinatorial codes on the tail of histone H4 from human embryonic stem (ES) cells. Finally, we quantified the abundances of these codes as human ES cells undergo differentiation to reveal striking changes in methylation and acetylation patterns. For ex le, H4R3 methylation was observed only in the presence of H4K20 dimethylation such context-specific patterning exemplifies the power of this technique.
Publisher: Proceedings of the National Academy of Sciences
Date: 02-04-2012
Abstract: The transcription factor OCT4 is fundamental to maintaining pluripotency and self-renewal. To better understand protein-level regulation of OCT4, we applied liquid chromatography–MS to identify 14 localized sites of phosphorylation, 11 of which were previously unknown. Functional analysis of two sites, T234 and S235, suggested that phosphorylation within the homeobox region of OCT4 negatively regulates its activity by interrupting sequence-specific DNA binding. Mutating T234 and S235 to mimic constitutive phosphorylation at these sites reduces transcriptional activation from an OCT4-responsive reporter and decreases reprogramming efficiency. We also cataloged 144 unique phosphopeptides on known OCT4 interacting partners, including SOX2 and SALL4, that copurified during immunoprecipitation. These proteins were enriched for phosphorylation at motifs associated with ERK signaling. Likewise, OCT4 harbored several putative ERK phosphorylation sites. Kinase assays confirmed that ERK2 phosphorylated these sites in vitro, providing a direct link between ERK signaling and the transcriptional machinery that governs pluripotency.
Publisher: Elsevier BV
Date: 10-2013
DOI: 10.1016/J.STEM.2013.09.012
Abstract: Transcription factor-induced pluripotency is extremely inefficient and assumed to be stochastic. In a recent Nature publication, Rais et al. show that depletion of the methyl-binding protein 3 yields reprogramming efficiencies of up to 100% within days, suggesting that elimination of a single gene suffices to render reprogramming a deterministic process.
Publisher: Elsevier BV
Date: 2014
Publisher: Springer Science and Business Media LLC
Date: 24-09-2014
DOI: 10.1038/NMETH.3142
Location: United States of America
Start Date: 2014
End Date: 2017
Funder: National Institute of Mental Health
View Funded ActivityStart Date: 2009
End Date: 2012
Funder: National Institutes of Health
View Funded Activity