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Recent Publications

Upon the Shoulders of Giants: Open-Source Hardware and Software in Analytical Chemistry.

Upon the Shoulders of Giants: Open-Source Hardware and Software in Analytical Chemistry.

Anal Chem. 2017 Apr 05;:

Authors: Dryden MD, Fobel R, Fobel C, Wheeler AR

Abstract
Isaac Newton famously observed that "if I have seen further it is by standing on the shoulders of giants." We propose that this sentiment is a powerful motivation for the "open-source" movement in scientific research, in which creators provide everything needed to replicate a given project online, as well as providing explicit permission for users to use, improve, and share it with others. Here, we write to introduce analytical chemists who are new to the open-source movement to best practices and concepts in this area and to survey the state of open-source research in analytical chemistry. We conclude by considering two examples of open-source projects from our own research group, with the hope that a description of the process, motivations, and results will provide a convincing argument about the benefits that this movement brings to both creators and users.

PMID: 28379683 [PubMed - as supplied by publisher]



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An RRM-ZnF RNA recognition module targets RBM10 to exonic sequences to promote exon exclusion.

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An RRM-ZnF RNA recognition module targets RBM10 to exonic sequences to promote exon exclusion.

Nucleic Acids Res. 2017 Jun 20;45(11):6761-6774

Authors: Collins KM, Kainov YA, Christodolou E, Ray D, Morris Q, Hughes T, Taylor IA, Makeyev EV, Ramos A

Abstract
RBM10 is an RNA-binding protein that plays an essential role in development and is frequently mutated in the context of human disease. RBM10 recognizes a diverse set of RNA motifs in introns and exons and regulates alternative splicing. However, the molecular mechanisms underlying this seemingly relaxed sequence specificity are not understood and functional studies have focused on 3΄ intronic sites only. Here, we dissect the RNA code recognized by RBM10 and relate it to the splicing regulatory function of this protein. We show that a two-domain RRM1-ZnF unit recognizes a GGA-centered motif enriched in RBM10 exonic sites with high affinity and specificity and test that the interaction with these exonic sequences promotes exon skipping. Importantly, a second RRM domain (RRM2) of RBM10 recognizes a C-rich sequence, which explains its known interaction with the intronic 3΄ site of NUMB exon 9 contributing to regulation of the Notch pathway in cancer. Together, these findings explain RBM10's broad RNA specificity and suggest that RBM10 functions as a splicing regulator using two RNA-binding units with different specificities to promote exon skipping.

PMID: 28379442 [PubMed - indexed for MEDLINE]



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Towards a personalized approach to aromatase inhibitor therapy: a digital microfluidic platform for rapid analysis of estradiol in core-needle-biopsies.

Towards a personalized approach to aromatase inhibitor therapy: a digital microfluidic platform for rapid analysis of estradiol in core-needle-biopsies.

Lab Chip. 2017 Apr 05;:

Authors: Abdulwahab S, Ng AH, Dean Chamberlain M, Ahmado H, Behan LA, Gomaa H, Casper RF, Wheeler AR

Abstract
Despite advances in breast cancer prevention and treatment, variability in patient-response has revealed the need for a more "personalized" approach to medicine, in which treatments are tailored to each patient's biology. Motivated by this idea, we introduce a technique that allows for quantification of small-molecule analytes directly from core needle biopsy (CNB) tissue samples on a miniaturized platform. The new technique, powered by digital microfluidics, integrates tissue-liquid extraction and magnetic bead-based competitive immunoassay for quantification of estradiol in milligram-sized CNB samples. Each measurement (from start to finish) requires ∼40 minutes, a duration consistent with a visit to a doctor's office. The performance of the new technique was validated by the gold-standard analysis method (high performance liquid chromatography coupled to tandem mass spectrometry), and was applied to evaluate human patient samples before and after a course of treatment with aromatase inhibitor therapy. We propose that the new technique has great potential for eventual use for fast, automated, and quantitative analysis of biomarkers in tissue samples, towards a personalized medicine approach.

PMID: 28379279 [PubMed - as supplied by publisher]



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A single administration of the hallucinogen, 4-acetoxy-dimethyltryptamine, prevents the shift to a drug-dependent state and the expression of withdrawal aversions in rodents.

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A single administration of the hallucinogen, 4-acetoxy-dimethyltryptamine, prevents the shift to a drug-dependent state and the expression of withdrawal aversions in rodents.

Eur J Neurosci. 2017 Apr 04;:

Authors: Vargas-Perez H, Grieder TE, Ting-A-Kee R, Maal-Bared G, Chwalek M, van der Kooy D

Abstract
Despite several studies suggesting the therapeutic use of 5-hydroxytryptamine receptors type 2A (5-HT2A ) agonists in the treatment of substance use disorders, the neurobiological basis accounting for such effects are still unknown. It has been observed that chronic exposure to drugs of abuse produces molecular and cellular adaptations in ventral tegmental area (VTA) neurons, mediated by brain-derived neurotrophic factor (BDNF). These BDNF-induced adaptations in the VTA are associated with the establishment of aversive withdrawal motivation that leads to a drug-dependent state. Growing evidence suggests that 5-HT2A receptor signaling can regulate the expression of BDNF in the brain. In this study, we observed that a single systemic or intra-VTA administration of a 5-HT2A agonist in rats and mice blocks both the aversive conditioned response to drug withdrawal and the mechanism responsible for switching from a drug-naive to a drug-dependent motivational system. Our results suggest that 5-HT2A agonists could be used as therapeutic agents to reverse a drug dependent state, as well as inhibiting the aversive effects produced by drug withdrawal. This article is protected by copyright. All rights reserved.

PMID: 28378435 [PubMed - as supplied by publisher]



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Structure-Directed and Tailored Diversity Synthetic Antibody Libraries Yield Novel Anti-EGFR Antagonists.

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Structure-Directed and Tailored Diversity Synthetic Antibody Libraries Yield Novel Anti-EGFR Antagonists.

ACS Chem Biol. 2017 May 19;12(5):1381-1389

Authors: Miersch S, Maruthachalam BV, Geyer CR, Sidhu SS

Abstract
We tested whether grafting an interaction domain into the hypervariable loop of a combinatorial antibody library could promote targeting to a specific epitope. Formation of the epidermal growth factor receptor (EGFR) signaling heterodimer involves extensive contacts mediated by a "dimerization loop." We grafted the dimerization loop into the third hypervariable loop of a synthetic antigen-binding fragment (Fab) library and diversified other loops using a tailored diversity strategy. This structure-directed Fab library and a naı̈ve synthetic Fab library were used to select Fabs against EGFR. Both libraries yielded high affinity Fabs that bound to overlapping epitopes on cell-surface EGFR, inhibited receptor activation, and targeted epitopes distinct from those of cetuximab and panitumumab. Epitope mapping experiments revealed complex sites of interaction, comprised of domains I and II but not exclusively localized to the receptor dimerization loop. These results validate the grafting approach for designing Fab libraries and also underscore the versatility of naı̈ve synthetic libraries.

PMID: 28375604 [PubMed - indexed for MEDLINE]



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Injectable and inherently vascularizing semi-interpenetrating polymer network for delivering cells to the subcutaneous space.

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Injectable and inherently vascularizing semi-interpenetrating polymer network for delivering cells to the subcutaneous space.

Biomaterials. 2017 Mar 24;131:27-35

Authors: Mahou R, Zhang DK, Vlahos AE, Sefton MV

Abstract
Injectable hydrogels are suitable for local cell delivery to the subcutaneous space, but the lack of vasculature remains a limiting factor. Previously we demonstrated that biomaterials containing methacrylic acid promoted vascularization. Here we report the preparation of a semi-interpenetrating polymer network (SIPN), and its evaluation as an injectable carrier to deliver cells and generate blood vessels in a subcutaneous implantation site. The SIPN was prepared by reacting a blend of vinyl sulfone-terminated polyethylene glycol (PEG-VS) and sodium polymethacrylate (PMAA-Na) with dithiothreitol. The swelling of SIPN was sensitive to the PMAA-Na content but only small differences in gelation time, permeability and stiffness were noted. SIPN containing 20 mol% PMAA-Na generated a vascular network in the surrounding tissues, with 2-3 times as many vessels as was obtained with 10 mol% PMAA-Na or PEG alone. Perfusion studies showed that the generated vessels were perfused and connected to the host vasculature as early as seven days after transplantation. Islets embedded in SIPN were viable and responsive to glucose stimulation in vitro. In a proof of concept study in a streptozotocin-induced diabetic mouse model, a progressive return to normoglycemia was observed and the presence of insulin positive islets was confirmed when islets were embedded in SIPN prior to delivery. Our approach proposes a biomaterial-mediated strategy to deliver cells while enhancing vascularization.

PMID: 28371625 [PubMed - as supplied by publisher]



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Systematic identification of phosphorylation-mediated protein interaction switches.

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Systematic identification of phosphorylation-mediated protein interaction switches.

PLoS Comput Biol. 2017 Mar;13(3):e1005462

Authors: Betts MJ, Wichmann O, Utz M, Andre T, Petsalaki E, Minguez P, Parca L, Roth FP, Gavin AC, Bork P, Russell RB

Abstract
Proteomics techniques can identify thousands of phosphorylation sites in a single experiment, the majority of which are new and lack precise information about function or molecular mechanism. Here we present a fast method to predict potential phosphorylation switches by mapping phosphorylation sites to protein-protein interactions of known structure and analysing the properties of the protein interface. We predict 1024 sites that could potentially enable or disable particular interactions. We tested a selection of these switches and showed that phosphomimetic mutations indeed affect interactions. We estimate that there are likely thousands of phosphorylation mediated switches yet to be uncovered, even among existing phosphorylation datasets. The results suggest that phosphorylation sites on globular, as distinct from disordered, parts of the proteome frequently function as switches, which might be one of the ancient roles for kinase phosphorylation.

PMID: 28346509 [PubMed - indexed for MEDLINE]



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Engineering Cellular Microenvironments with Photo- and Enzymatically Responsive Hydrogels: Toward Biomimetic 3D Cell Culture Models.

Engineering Cellular Microenvironments with Photo- and Enzymatically Responsive Hydrogels: Toward Biomimetic 3D Cell Culture Models.

Acc Chem Res. 2017 Mar 27;:

Authors: Tam RY, Smith LJ, Shoichet MS

Abstract
Conventional cell culture techniques using 2D polystyrene or glass have provided great insight into key biochemical mechanisms responsible for cellular events such as cell proliferation, differentiation, and cell-cell interactions. However, the physical and chemical properties of 2D culture in vitro are dramatically different than those found in the native cellular microenvironment in vivo. Cells grown on 2D substrates differ significantly from those grown in vivo, and this explains, in part, why many promising drug candidates discovered through in vitro drug screening assays fail when they are translated to in vivo animal or human models. To overcome this obstacle, 3D cell culture using biomimetic hydrogels has emerged as an alternative strategy to recapitulate native cell growth in vitro. Hydrogels, which are water-swollen polymers, can be synthetic or naturally derived. Many methods have been developed to control the physical and chemical properties of the hydrogels to match those found in specific tissues. Compared to 2D culture, cells cultured in 3D gels with the appropriate physicochemical cues can behave more like they naturally do in vivo. While conventional hydrogels involve modifications to the bulk material to mimic the static aspects of the cellular microenvironment, recent progress has focused on using more dynamic hydrogels, the chemical and physical properties of which can be altered with external stimuli to better mimic the dynamics of the native cellular microenvironment found in vivo. In this Account, we describe our progress in designing stimuli-responsive, optically transparent hydrogels that can be used as biomimetic extracellular matrices (ECMs) to study cell differentiation and migration in the context of modeling the nervous system and cancer. Specifically, we developed photosensitive agarose and hyaluronic acid hydrogels that are activated by single or two-photon irradiation for biomolecule immobilization at specific volumes within the 3D hydrogel. By controlling the spatial location of protein immobilization, we created 3D patterns and protein concentration gradients within these gels. We used the latter to study the effect of VEGF-165 concentration gradients on the interactions between endothelial cells and retinal stem cells. Hyaluronic acid (HA) is particularly compelling as it is naturally found in the ECM of many tissues and the tumor microenvironment. We used Diels-Alder click chemistry and cryogelation to alter the chemical and physical properties of these hydrogels. We also designed HA hydrogels to study the invasion of breast cancer cells. HA gels were chemically cross-linked with matrix metalloproteinase (MMP)-degradable peptides that degrade in the presence of cancer cell-secreted MMPs, thus allowing cells to remodel their local microenvironment and invade into HA/MMP-degradable gels.

PMID: 28345876 [PubMed - as supplied by publisher]



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Structure Reveals Mechanisms of Viral Suppressors that Intercept a CRISPR RNA-Guided Surveillance Complex.

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Structure Reveals Mechanisms of Viral Suppressors that Intercept a CRISPR RNA-Guided Surveillance Complex.

Cell. 2017 Mar 23;169(1):47-57.e11

Authors: Chowdhury S, Carter J, Rollins MF, Golden SM, Jackson RN, Hoffmann C, Nosaka L, Bondy-Denomy J, Maxwell KL, Davidson AR, Fischer ER, Lander GC, Wiedenheft B

Abstract
Genetic conflict between viruses and their hosts drives evolution and genetic innovation. Prokaryotes evolved CRISPR-mediated adaptive immune systems for protection from viral infection, and viruses have evolved diverse anti-CRISPR (Acr) proteins that subvert these immune systems. The adaptive immune system in Pseudomonas aeruginosa (type I-F) relies on a 350 kDa CRISPR RNA (crRNA)-guided surveillance complex (Csy complex) to bind foreign DNA and recruit a trans-acting nuclease for target degradation. Here, we report the cryo-electron microscopy (cryo-EM) structure of the Csy complex bound to two different Acr proteins, AcrF1 and AcrF2, at an average resolution of 3.4 Å. The structure explains the molecular mechanism for immune system suppression, and structure-guided mutations show that the Acr proteins bind to residues essential for crRNA-mediated detection of DNA. Collectively, these data provide a snapshot of an ongoing molecular arms race between viral suppressors and the immune system they target.

PMID: 28340349 [PubMed - indexed for MEDLINE]



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Major Roles for Pyrimidine Dimers, Nucleotide Excision Repair, and ATR in the Alternative Splicing Response to UV Irradiation.

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Major Roles for Pyrimidine Dimers, Nucleotide Excision Repair, and ATR in the Alternative Splicing Response to UV Irradiation.

Cell Rep. 2017 Mar 21;18(12):2868-2879

Authors: Muñoz MJ, Nieto Moreno N, Giono LE, Cambindo Botto AE, Dujardin G, Bastianello G, Lavore S, Torres-Méndez A, Menck CFM, Blencowe BJ, Irimia M, Foiani M, Kornblihtt AR

Abstract
We have previously found that UV irradiation promotes RNA polymerase II (RNAPII) hyperphosphorylation and subsequent changes in alternative splicing (AS). We show now that UV-induced DNA damage is not only necessary but sufficient to trigger the AS response and that photolyase-mediated removal of the most abundant class of pyrimidine dimers (PDs) abrogates the global response to UV. We demonstrate that, in keratinocytes, RNAPII is the target, but not a sensor, of the signaling cascade initiated by PDs. The UV effect is enhanced by inhibition of gap-filling DNA synthesis, the last step in the nucleotide excision repair pathway (NER), and reduced by the absence of XPE, the main NER sensor of PDs. The mechanism involves activation of the protein kinase ATR that mediates the UV-induced RNAPII hyperphosphorylation. Our results define the sequence UV-PDs-NER-ATR-RNAPII-AS as a pathway linking DNA damage repair to the control of both RNAPII phosphorylation and AS regulation.

PMID: 28329680 [PubMed - indexed for MEDLINE]



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