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Protein footprinting

Stefan Wanning, Richard Süverkrüp, Alf Lamprecht
In spray freeze-srying (SFD), a solution is sprayed into a refrigerant medium, frozen, and subsequently sublimation dried, which allows the production of flowable lyophilized powders. SFD allows commonly freeze-dried active pharmaceutical ingredients (e.g., proteins and peptides) to be delivered using new applications such as needle-free injection and nasal or pulmonary drug delivery. In this study, a droplet stream was injected into a vortex of cold gas in order to reduce the risk of droplet collisions and therefore droplet growth before congelation, which adversely affects the particle size distribution...
October 19, 2016: AAPS PharmSciTech
Juliana Blin, Emiliano P Ricci
Next Generation Sequencing (NGS) techniques have revolutionized most biomedical research fields over the past decade by allowing a broader vision on biological processes that occur at the molecular level. Among these, ribosome profiling or footprinting is a powerful tool to study mRNA translation in a transcriptome-wide manner. Ribosome profiling has been used to study the impact of translational control of gene expression under many different cellular conditions including viral infections. Indeed, translation is a critical step during the viral replication cycle in which the infected cell is embezzled to produce viral proteins...
October 2016: Médecine Sciences: M/S
Buyong Ma, Ruth Nussinov
No abstract text is available yet for this article.
October 18, 2016: Nature Chemical Biology
Luke Pett, Konstantinos Kiakos, Vijay Satam, Pravin Patil, Sarah Laughlin-Toth, Matthew Gregory, Michael Bowerman, Kevin Olson, Mia Savagian, Megan Lee, Moses Lee, W David Wilson, Daniel Hochhauser, John A Hartley
BACKGROUND: Sequence specific polyamide HxIP 1, targeted to the inverted CCAAT Box 2 (ICB2) on the topoisomerase IIα (topo IIα) promoter can inhibit NF-Y binding, re-induce gene expression and increase sensitivity to etoposide. To enhance biological activity, diamino-containing derivatives (HxI*P 2 and HxIP* 3) were synthesised incorporating an alkyl amino group at the N1-heterocyclic position of the imidazole/pyrrole. METHODS: DNase I footprinting was used to evaluate DNA binding of the diamino Hx-polyamides, and their ability to disrupt the NF-Y:ICB2 interaction assessed using EMSAs...
October 14, 2016: Biochimica et Biophysica Acta
Sang C Lee, Jack Zhang, Josh Strom, Danzhou Yang, Thai Nho Dinh, Kyle Kappeler, Qin M Chen
Inhibition of protein synthesis serves as a general measure of cellular consequence to chemical stress. A few proteins are translated selectively and influence cell fate. How these proteins can bypass the general control of translation remains unknown. We found that low to mild doses of oxidants induce de novo translation of NRF2 protein. Here we demonstrate the presence of a G-quadruplex structure in 5' Untranslated Region (5' UTR) of NRF2 mRNA, as measured by Circular Dichroism, Nuclear Magnetic Resonance, and Dimethylsulfate Footprinting...
October 10, 2016: Molecular and Cellular Biology
Shawn W Foley, Brian D Gregory
Every eukaryotic RNA transcript undergoes extensive post-transcriptional processing from the moment of transcription up through degradation. This regulation is performed by a distinct cohort of RNA-binding proteins which recognize their target transcript by both its primary sequence and secondary structure. Here, we describe protein interaction profile sequencing (PIP-seq), a technique that uses ribonuclease-based footprinting followed by high-throughput sequencing to globally assess both protein-bound RNA sequences and RNA secondary structure...
October 10, 2016: Current Protocols in Molecular Biology
Gerben A Bekker, Hilde Arnout R H Fischer, Hilde Tobi, Hans C M van Trijp
Cultured meat is an unfamiliar emerging food technology that could provide a near endless supply of high quality protein with a relatively small ecological footprint. To understand consumer acceptance of cultured meat, this study investigated the influence of information provision on the explicit and implicit attitude toward cultured meat. Three experiments were conducted using a Solomon four-group design to rule out pretest sensitization effects. The first experiment (N = 190) showed that positive or negative information about cultured meat changed the explicit attitude in the direction of the information...
October 4, 2016: Appetite
Kathryn M Hart, Chris M W Ho, Supratik Dutta, Michael L Gross, Gregory R Bowman
TEM β-lactamase confers bacteria with resistance to many antibiotics and rapidly evolves activity against new drugs. However, functional changes are not easily explained by differences in crystal structures. We employ Markov state models to identify hidden conformations and explore their role in determining TEM's specificity. We integrate these models with existing drug-design tools to create a new technique, called Boltzmann docking, which better predicts TEM specificity by accounting for conformational heterogeneity...
October 6, 2016: Nature Communications
Katherine N Clayton, Janelle W Salameh, Steven T Wereley, Tamara L Kinzer-Ursem
As the field of colloidal science continues to expand, tools for rapid and accurate physiochemical characterization of colloidal particles will become increasingly important. Here, we present Particle Scattering Diffusometry (PSD), a method that utilizes dark field microscopy and the principles of particle image velocimetry to measure the diffusivity of particles undergoing Brownian motion. PSD measures the diffusion coefficient of particles as small as 30 nm in diameter and is used to characterize changes in particle size and distribution as a function of small, label-free, surface modifications of particles...
September 2016: Biomicrofluidics
Ngoc-Phuong-Thao Nguyen, Sonja Linder, Stefanie K Flitsch, Bettina Schiel-Bengelsdorf, Peter Dürre, Philippe Soucaille
: An operon comprising two genes, CA_P0037 and CA_P0036, that encode proteins of unknown function that were previously shown to be highly expressed in acidogenic cells and repressed in solventogenic and alcohologenic cells is located on the pSOL1 megaplasmid of Clostridium acetobutylicum upstream of adhE2 A CA_P0037::int (189/190s) mutant in which an intron was inserted at position 189/190 in the sense strand of CA_P0037 was successfully generated by the Targetron technique. The resultant mutant showed significantly different metabolic flux patterns in acidogenic (producing mainly lactate, butyrate, and butanol) and alcohologenic (producing mainly butyrate, acetate, and lactate) chemostat cultures but not in solventogenic or batch cultures...
October 4, 2016: MBio
Aimee Rinas, Vishaal S Mali, Jessica A Espino, Lisa M Jones
Fast photochemical oxidation of proteins (FPOP) has become a valuable tool for protein structural characterization. The method has recently been demonstrated to oxidatively modify solvent-accessible sites of proteins inside live cells (IC-FPOP). However, the flow system used for in vitro analysis is not well-suited for IC-FPOP as a number of factors can lead to cell aggregation, causing inconsistent labeling and clogging. Here, we present an IC-FPOP flow system that centrally focuses the cells, ensuring consistent radiation exposure...
October 7, 2016: Analytical Chemistry
Marianne Ngure, Moheshwarnath Issur, Nikoloz Shkriabai, Hsiao-Wei Liu, Gonzalo Cosa, Mamuka Kvaratskhelia, Matthias Götte
Domain II of the nonstructural protein 5 (NS5A) of the hepatitis C virus (HCV) is involved in intermolecular interactions with the viral RNA genome, the RNA-dependent RNA polymerase NS5B, and the host factor cyclophilin A (CypA). However, domain II of NS5A (NS5A(DII)) is largely disordered, which makes it difficult to characterize the protein-protein or protein-nucleic acid interfaces. Here we utilized a mass spectrometry-based protein footprinting approach in attempts to characterize regions forming contacts between NS5A(DII) and its binding partners...
October 5, 2016: ACS Infectious Diseases
Mary F Barbe, Jessica J Krueger, Regina Loomis, Jessica Otte, Jennifer Gordon
Pur-alpha is a highly conserved sequence-specific DNA and RNA binding protein with established roles in DNA replication, RNA translation, cell cycle regulation, and maintenance of neuronal differentiation. Prior studies have shown that mice lacking Pur-alpha (-/-) display decreased neurogenesis and impaired neuronal differentiation. We sought to examine for the first time, the behavioral phenotype and brain histopathology of mice that are heterozygous (+/-) for Pur-alpha. Standardized behavioral phenotyping revealed a decreased escape response to touch, limb and abdominal hypotonia, and gait abnormalities in heterozygous Pur-alpha (+/-) mice, compared to wild-type (+/+) littermates...
September 17, 2016: Neuroscience
Kishore Reddy Katikireddy, Thore Schmedt, Marianne O Price, Francis W Price, Ula V Jurkunas
Human corneal endothelial cells are derived from neural crest and because of postmitotic arrest lack competence to repair cell loss from trauma, aging, and degenerative disorders such as Fuchs endothelial corneal dystrophy (FECD). Herein, we identified a rapidly proliferating subpopulation of cells from the corneal endothelium of adult normal and FECD donors that exhibited features of neural crest-derived progenitor (NCDP) cells by showing absence of senescence with passaging, propensity to form spheres, and increased colony forming efficacy compared with the primary cells...
October 2016: American Journal of Pathology
Yi Zhong, Theofanis Karaletsos, Philipp Drewe, Vipin Sreedharan, David Kuo, Kamini Singh, Hans-Guido Wendel, Gunnar Rätsch
MOTIVATION: Deep sequencing based ribosome footprint profiling can provide novel insights into the regulatory mechanisms of protein translation. However, the observed ribosome profile is fundamentally confounded by transcriptional activity. In order to decipher principles of translation regulation, tools that can reliably detect changes in translation efficiency in case-control studies are needed. RESULTS: We present a statistical framework and an analysis tool, RiboDiff, to detect genes with changes in translation efficiency across experimental treatments...
September 14, 2016: Bioinformatics
Paloma Bonato, Lysangela R Alves, Juliana H Osaki, Liu U Rigo, Fabio O Pedrosa, Emanuel M Souza, Nan Zhang, Jörg Schumacher, Martin Buck, Roseli Wassem, Leda S Chubatsu
Herbaspirillum seropedicae is a diazotrophic β-Proteobacterium found endophytically associated with gramineae (Poaceae or graminaceous plants) such as rice, sorghum and sugar cane. In this work we show that nitrate-dependent growth in this organism is regulated by the master nitrogen regulatory two-component system NtrB-NtrC, and by NtrY-NtrX, which functions to specifically regulate nitrate metabolism. NtrY is a histidine kinase sensor protein predicted to be associated with the membrane and NtrX is the response regulator partner...
September 16, 2016: FEBS Journal
Erin E Swinstead, Ville Paakinaho, Diego M Presman, Gordon L Hager
Transcription factor (TF) signaling regulates gene transcription and requires a complex network of proteins. This network includes co-activators, co-repressors, multiple TFs, histone-modifying complexes, and the basal transcription machinery. It has been widely appreciated that pioneer factors, such as FoxA1 and GATA1, play an important role in opening closed chromatin regions, thereby allowing binding of a secondary factor. In this review we will focus on a newly proposed model wherein multiple TFs, such as steroid receptors (SRs), can function in a pioneering role...
September 16, 2016: BioEssays: News and Reviews in Molecular, Cellular and Developmental Biology
Christopher Connelly, Thomas Cicuto, Jason Leavitt, Alexander Petty, Amy Litman, Henry C Margolis, Aren E Gerdon
Amelogenin, the predominant extracellular matrix protein secreted by ameloblasts, has been shown to be essential for proper tooth enamel formation. In this study, amelogenin adsorption to hydroxyapatite (HAP) surfaces, a prototype for enamel mineral, has been studied using a quartz crystal microbalance (QCM) to interrogate effects of protein phosphorylation and solution pH. Dynamic flow-based experiments were conducted at pH 7.4 and 8.0 using native phosphorylated porcine amelogenin (P173) and recombinant non-phosphorylated porcine amelogenin (rP172)...
September 8, 2016: Colloids and Surfaces. B, Biointerfaces
Beth A Bachert, Soo J Choi, Paul R LaSala, Tiffany I Harper, Dudley H McNitt, Dylan T Boehm, Clayton C Caswell, Pawel Ciborowski, Douglas R Keene, Anthony R Flores, James M Musser, Flavia Squeglia, Daniela Marasco, Rita Berisio, Slawomir Lukomski
The streptococcal collagen-like proteins 1 and 2 (Scl1 and Scl2) are major surface adhesins that are ubiquitous among group A Streptococcus (GAS). Invasive M3-type strains, however, have evolved two unique conserved features in the scl1 locus: (i) an IS1548 element insertion in the scl1 promoter region and (ii) a nonsense mutation within the scl1 coding sequence. The scl1 transcript is drastically reduced in M3-type GAS, contrasting with a high transcription level of scl1 allele in invasive M1-type GAS. This leads to a lack of Scl1 expression in M3 strains...
2016: Frontiers in Cellular and Infection Microbiology
Atsushi Yoshino, Natalia Polouliakh, Akira Meguro, Masaki Takeuchi, Tatsukata Kawagoe, Nobuhisa Mizuki
Components of fish roe possess antioxidant and antiaging activities, making them potentially very beneficial natural resources. Here, we investigated chum salmon eggs (CSEs) as a source of active ingredients, including vitamins, unsaturated fatty acids, and proteins. We incubated human dermal fibroblast cultures for 48 hours with high and low concentrations of CSE extracts and analyzed changes in gene expression. Cells treated with CSE extract showed concentration-dependent upregulation of collagen type I genes and of multiple antioxidative genes, including OXR1, TXNRD1, and PRDX family genes...
2016: Clinical Interventions in Aging
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