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Systematic protein-protein interaction mapping for clinically relevant human GPCRs.

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Systematic protein-protein interaction mapping for clinically relevant human GPCRs.

Mol Syst Biol. 2017 Mar 15;13(3):918

Authors: Sokolina K, Kittanakom S, Snider J, Kotlyar M, Maurice P, Gandía J, Benleulmi-Chaachoua A, Tadagaki K, Oishi A, Wong V, Malty RH, Deineko V, Aoki H, Amin S, Yao Z, Morató X, Otasek D, Kobayashi H, Menendez J, Auerbach D, Angers S, Pržulj N, Bouvier M, Babu M, Ciruela F, Jockers R, Jurisica I, Stagljar I

Abstract
G-protein-coupled receptors (GPCRs) are the largest family of integral membrane receptors with key roles in regulating signaling pathways targeted by therapeutics, but are difficult to study using existing proteomics technologies due to their complex biochemical features. To obtain a global view of GPCR-mediated signaling and to identify novel components of their pathways, we used a modified membrane yeast two-hybrid (MYTH) approach and identified interacting partners for 48 selected full-length human ligand-unoccupied GPCRs in their native membrane environment. The resulting GPCR interactome connects 686 proteins by 987 unique interactions, including 299 membrane proteins involved in a diverse range of cellular functions. To demonstrate the biological relevance of the GPCR interactome, we validated novel interactions of the GPR37, serotonin 5-HT4d, and adenosine ADORA2A receptors. Our data represent the first large-scale interactome mapping for human GPCRs and provide a valuable resource for the analysis of signaling pathways involving this druggable family of integral membrane proteins.

PMID: 28298427 [PubMed - in process]



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New Molecular Assay for the Proliferation Signature in Mantle Cell Lymphoma Applicable to Formalin-Fixed Paraffin-Embedded Biopsies.

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New Molecular Assay for the Proliferation Signature in Mantle Cell Lymphoma Applicable to Formalin-Fixed Paraffin-Embedded Biopsies.

J Clin Oncol. 2017 Mar 14;:JCO2016707901

Authors: Scott DW, Abrisqueta P, Wright GW, Slack GW, Mottok A, Villa D, Jares P, Rauert-Wunderlich H, Royo C, Clot G, Pinyol M, Boyle M, Chan FC, Braziel RM, Chan WC, Weisenburger DD, Cook JR, Greiner TC, Fu K, Ott G, Delabie J, Smeland EB, Holte H, Jaffe ES, Steidl C, Connors JM, Gascoyne RD, Rosenwald A, Staudt LM, Campo E, Rimsza LM, Lymphoma/Leukemia Molecular Profiling Project

Abstract
Purpose Mantle cell lymphoma is an aggressive B-cell neoplasm that displays heterogeneous outcomes after treatment. In 2003, the Lymphoma/Leukemia Molecular Profiling Project described a powerful biomarker-the proliferation signature-using gene expression in fresh frozen material. Herein, we describe the training and validation of a new assay that measures the proliferation signature in RNA derived from routinely available formalin-fixed paraffin-embedded (FFPE) biopsies. Methods Forty-seven FFPE biopsies were used to train an assay on the NanoString platform, using microarray gene expression data of matched fresh frozen biopsies as a gold standard. The locked assay was applied to pretreatment FFPE lymph node biopsies from an independent cohort of 110 patients uniformly treated with rituximab plus cyclophosphamide, doxorubicin, vincristine, and prednisone. Seventeen biopsies were tested across three laboratories to assess assay reproducibility. Results The MCL35 assay, which contained a 17-gene proliferation signature, yielded gene expression of sufficient quality to assign an assay score and risk group in 108 (98%) of 110 archival FFPE biopsies. The MCL35 assay assigned patients to high-risk (26%), standard-risk (29%), and low-risk (45%) groups, with different lengths of overall survival (OS): a median of 1.1, 2.6, and 8.6 years, respectively (log-rank for trend, P < .001). In multivariable analysis, these risk groups and the Mantle Cell Lymphoma International Prognostic Index were independently associated with OS ( P < .001 for both variables). Concordance of risk assignment across the three independent laboratories was 100%. Conclusion The newly developed and validated MCL35 assay for FFPE biopsies uses the proliferation signature to define groups of patients with significantly different OS independent of the Mantle Cell Lymphoma International Prognostic Index. Importantly, the analytic and clinical validity of this assay defines it as a reliable biomarker to support risk-adapted clinical trials.

PMID: 28291392 [PubMed - as supplied by publisher]



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Diverse Applications of Nanomedicine.

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Diverse Applications of Nanomedicine.

ACS Nano. 2017 Mar 14;:

Authors: Pelaz B, Alexiou C, Alvarez-Puebla RA, Alves F, Andrews AM, Ashraf S, Balogh LP, Ballerini L, Bestetti A, Brendel C, Bosi S, Carril M, Chan WC, Chen C, Chen X, Chen X, Cheng Z, Cui D, Du J, Dullin C, Escudero A, Feliu N, Gao M, George M, Gogotsi Y, Grünweller A, Gu Z, Halas NJ, Hampp N, Hartmann RK, Hersam MC, Hunziker P, Jian J, Jiang X, Jungebluth P, Kadhiresan P, Kataoka K, Khademhosseini A, Kopeček J, Kotov NA, Krug HF, Lee DS, Lehr CM, Leong KW, Liang XJ, Ling Lim M, Liz-Marzán LM, Ma X, Macchiarini P, Meng H, Möhwald H, Mulvaney P, Nel AE, Nie S, Nordlander P, Okano T, Oliveira J, Park TH, Penner RM, Prato M, Puntes V, Rotello VM, Samarakoon A, Schaak RE, Shen Y, Sjöqvist S, Skirtach AG, Soliman MG, Stevens MM, Sung HW, Tang BZ, Tietze R, Udugama BN, VanEpps JS, Weil T, Weiss PS, Willner I, Wu Y, Yang L, Yue Z, Zhang Q, Zhang Q, Zhang XE, Zhao Y, Zhou X, Parak WJ

Abstract
The design and use of materials in the nanoscale size range for addressing medical and health-related issues continues to receive increasing interest. Research in nanomedicine spans a multitude of areas, including drug delivery, vaccine development, antibacterial, diagnosis and imaging tools, wearable devices, implants, high-throughput screening platforms, etc. using biological, nonbiological, biomimetic, or hybrid materials. Many of these developments are starting to be translated into viable clinical products. Here, we provide an overview of recent developments in nanomedicine and highlight the current challenges and upcoming opportunities for the field and translation to the clinic.

PMID: 28290206 [PubMed - as supplied by publisher]



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Leveraging increased cytoplasmic nucleoside kinase activity to target mtDNA and oxidative phosphorylation in AML.

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Leveraging increased cytoplasmic nucleoside kinase activity to target mtDNA and oxidative phosphorylation in AML.

Blood. 2017 Mar 10;:

Authors: Liyanage SU, Hurren R, Voisin V, Bridon G, Wang X, Xu C, MacLean N, Siriwardena TP, Gronda M, Yehudai D, Sriskanthadevan S, Avizonis D, Shamas-Din A, Minden MD, Bader GD, Laposa R, Schimmer AD

Abstract
Mitochondrial DNA (mtDNA) biosynthesis requires replication factors and adequate nucleotide pools from the mitochondria and cytoplasm. We performed gene expression profiling analysis of 542 human AML samples and identified 55% with upregulated mtDNA biosynthesis pathway expression compared to normal hematopoietic cells. Genes that support mitochondrial nucleotide pools, including mitochondrial nucleotide transporters and a subset of cytoplasmic nucleoside kinases, were also increased in AML compared to normal hematopoietic samples. Knockdown of cytoplasmic nucleoside kinases reduced mtDNA levels in AML cells, demonstrating their contribution in maintaining mtDNA. To assess cytoplasmic nucleoside kinase pathway activity, we employed a nucleoside analog 2'3'-dideoxycytidine (ddC), which is phosphorylated to the activated anti-metabolite, 2'3'-dideoxycytidine triphosphate (ddCTP) by cytoplasmic nucleoside kinases. ddC is a selective inhibitor of the mitochondrial DNA polymerase, POLG. ddC was preferentially activated in AML cells compared to normal hematopoietic progenitor cells. ddC treatment inhibited mtDNA replication, oxidative phosphorylation, and induced cytotoxicity in a panel of AML cell lines. Furthermore, ddC preferentially inhibited mtDNA replication in a subset of primary human leukemia cells and selectively targeted leukemia cells while sparing normal progenitors cells. In animal models of human AML, treatment with ddC decreased mtDNA, electron transport chain proteins, and induced tumor regression without toxicity. ddC also targeted leukemic stem cells in secondary AML xenotransplantation assays. Thus, AML cells have increased cytidine nucleoside kinase activity that regulates mtDNA biogenesis and can be leveraged to selectively target oxidative phosphorylation in AML.

PMID: 28283480 [PubMed - as supplied by publisher]



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Structural and functional characterization of a ubiquitin variant engineered for tight and specific binding to an alpha-helical ubiquitin interacting motif.

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Structural and functional characterization of a ubiquitin variant engineered for tight and specific binding to an alpha-helical ubiquitin interacting motif.

Protein Sci. 2017 Mar 09;:

Authors: Manczyk N, Yates BP, Veggiani G, Ernst A, Sicheri F, Sidhu SS

Abstract
Ubiquitin interacting motifs (UIMs) are short α-helices found in a number of eukaryotic proteins. UIMs interact weakly but specifically with ubiquitin conjugated to other proteins, and in so doing, mediate specific cellular signals. Here we used phage display to generate ubiquitin variants (UbVs) targeting the N-terminal UIM of the yeast Vps27 protein. Selections yielded UbV.v27.1, which recognized the cognate UIM with high specificity relative to other yeast UIMs and bound with an affinity more than two orders of magnitude higher than that of ubiquitin. Structural and mutational studies of the UbV.v27.1-UIM complex revealed the molecular details for the enhanced affinity and specificity of UbV.v27.1, and underscored the importance of changes at the binding interface as well as at positions that do not contact the UIM. Our study highlights the power of the phage display approach for selecting UbVs with unprecedented affinity and high selectivity for particular α-helical UIM domains within proteomes, and it establishes a general approach for the development of inhibitors targeting interactions of this type. This article is protected by copyright. All rights reserved.

PMID: 28276594 [PubMed - as supplied by publisher]



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Detecting Membrane Protein-protein Interactions Using the Mammalian Membrane Two-hybrid (MaMTH) Assay.

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Detecting Membrane Protein-protein Interactions Using the Mammalian Membrane Two-hybrid (MaMTH) Assay.

Curr Protoc Chem Biol. 2017 Mar 02;9(1):38-54

Authors: Saraon P, Grozavu I, Lim SH, Snider J, Yao Z, Stagljar I

Abstract
Protein-protein interactions (PPIs) play an integral role in numerous cellular processes. Membrane protein interactions, in particular, are critical in cellular responses to stresses and stimuli, with dysfunction of these PPIs (e.g., due to aberrant expression and/or mutation of interaction partners) leading to a diverse array of pathological states. Exploration of the interaction space and dynamics of membrane proteins is difficult due to the limitations of current techniques used to study proteins in the biochemically complex environment of biological membranes. In the protocols below, we describe a newly developed membrane protein interaction assay called the Mammalian-Membrane Two-Hybrid (MaMTH), designed specifically for the detection of integral membrane PPIs in the context of living mammalian cells. Prior to using MaMTH, cell lines of interest are genetically modified to encode a reporter of choice. MaMTH "bait" and "prey" constructs of interest are also generated using Gateway cloning technology. The assay is then performed by co-transfection of baits and preys, with bait-prey interaction quantifiably assessed by way of a reporter signal (e.g., light (luciferase), fluorescence (GFP). © 2017 by John Wiley & Sons, Inc.

PMID: 28253435 [PubMed - indexed for MEDLINE]



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Engineering a humanized bone organ model in mice to study bone metastases.

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Engineering a humanized bone organ model in mice to study bone metastases.

Nat Protoc. 2017 Apr;12(4):639-663

Authors: Martine LC, Holzapfel BM, McGovern JA, Wagner F, Quent VM, Hesami P, Wunner FM, Vaquette C, De-Juan-Pardo EM, Brown TD, Nowlan B, Wu DJ, Hutmacher CO, Moi D, Oussenko T, Piccinini E, Zandstra PW, Mazzieri R, Lévesque JP, Dalton PD, Taubenberger AV, Hutmacher DW

Abstract
Current in vivo models for investigating human primary bone tumors and cancer metastasis to the bone rely on the injection of human cancer cells into the mouse skeleton. This approach does not mimic species-specific mechanisms occurring in human diseases and may preclude successful clinical translation. We have developed a protocol to engineer humanized bone within immunodeficient hosts, which can be adapted to study the interactions between human cancer cells and a humanized bone microenvironment in vivo. A researcher trained in the principles of tissue engineering will be able to execute the protocol and yield study results within 4-6 months. Additive biomanufactured scaffolds seeded and cultured with human bone-forming cells are implanted ectopically in combination with osteogenic factors into mice to generate a physiological bone 'organ', which is partially humanized. The model comprises human bone cells and secreted extracellular matrix (ECM); however, other components of the engineered tissue, such as the vasculature, are of murine origin. The model can be further humanized through the engraftment of human hematopoietic stem cells (HSCs) that can lead to human hematopoiesis within the murine host. The humanized organ bone model has been well characterized and validated and allows dissection of some of the mechanisms of the bone metastatic processes in prostate and breast cancer.

PMID: 28253234 [PubMed - indexed for MEDLINE]



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Combinatorial Therapies After Spinal Cord Injury: How Can Biomaterials Help?

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Combinatorial Therapies After Spinal Cord Injury: How Can Biomaterials Help?

Adv Healthc Mater. 2017 Mar 01;:

Authors: Führmann T, Anandakumaran PN, Shoichet MS

Abstract
Traumatic spinal cord injury (SCI) results in an immediate loss of motor and sensory function below the injury site and is associated with a poor prognosis. The inhibitory environment that develops in response to the injury is mainly due to local expression of inhibitory factors, scarring and the formation of cystic cavitations, all of which limit the regenerative capacity of endogenous or transplanted cells. Strategies that demonstrate promising results induce a change in the microenvironment at- and around the lesion site to promote endogenous cell repair, including axonal regeneration or the integration of transplanted cells. To date, many of these strategies target only a single aspect of SCI; however, the multifaceted nature of SCI suggests that combinatorial strategies will likely be more effective. Biomaterials are a key component of combinatorial strategies, as they have the potential to deliver drugs locally over a prolonged period of time and aid in cell survival, integration and differentiation. Here we summarize the advantages and limitations of widely used strategies to promote recovery after injury and highlight recent research where biomaterials aided combinatorial strategies to overcome some of the barriers of spinal cord regeneration.

PMID: 28247563 [PubMed - as supplied by publisher]



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Optimization of Satellite Cell Culture Through Biomaterials.

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Optimization of Satellite Cell Culture Through Biomaterials.

Methods Mol Biol. 2017;1556:329-341

Authors: Davoudi S, Gilbert PM

Abstract
Hydrogels, a type of biomaterial, are an invaluable part of biomedical research as they are highly hydrated and properties such as elasticity, porosity, and ligand density can be tuned to desired values. Recently, culture substrate stiffness was found to be an important regulator of muscle stem cell self-renewal. Polyethylene glycol (PEG), a synthetic polymer, can be fabricated into hydrogels that match the softness of skeletal muscle tissue, thereby providing a culture surface that is optimal for maintaining muscle stem cell self-renewal potential ex vivo. In this Chapter, we describe a method to produce flat PEG hydrogels across a range of stiffnesses, including a formulation that matches the bulk stiffness of healthy skeletal muscle (12 kPa), while maintaining a constant ligand density. Since PEG is inert to protein adsorption, the steps required to surface functionalize the hydrogel with an adhesive interface (e.g., laminin) are also described.

PMID: 28247359 [PubMed - in process]



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Neisseria gonorrhoeae Sequence Typing for Antimicrobial Resistance (NG-STAR): a novel antimicrobial resistance multilocus typing scheme for tracking the global dissemination of N. gonorrhoeae strains.

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Neisseria gonorrhoeae Sequence Typing for Antimicrobial Resistance (NG-STAR): a novel antimicrobial resistance multilocus typing scheme for tracking the global dissemination of N. gonorrhoeae strains.

J Clin Microbiol. 2017 Feb 22;:

Authors: Demczuk W, Sidhu S, Unemo M, Whiley DM, Allen VG, Dillon JR, Cole M, Seah C, Trembizki E, Trees DL, Kersh EN, Abrams AJ, de Vries HJ, van Dam AP, Medina I, Bharat A, Mulvey MR, Van Domselaar G, Martin I

Abstract
A curated web-based user-friendly sequence typing tool based on antimicrobial resistance determinants in Neisseria gonorrhoeae was developed and is publicly accessible at https://ngstar.canada.ca The N. gonorrhoeae Sequence Typing for Antimicrobial Resistance (NG-STAR) molecular typing scheme uses the DNA sequences of 7 genes (penA, mtrR, porB, ponA, gyrA, parC, 23S rRNA) associated with resistance to β-lactam antimicrobials, macrolides, or fluoroquinolones. NG-STAR uses the entire penA sequence combining the historical nomenclature for penA types I-XXXVIII with novel nucleotide sequence designations; the full mtrR sequence and a portion of its promoter region; portions of ponA, porB, gyrA and parC; and 23S rRNA sequences. NG-STAR grouped 768 isolates into 139 sequence types (STs) (n=660) consisting of 29 CCs having a maximum of a single locus variation; and 76 NG-STAR STs (n=109) were identified as unrelated singletons. NG-STAR had a high Simpson's diversity index of 96.5% (CI 95%=0.959-0.969). The most common STs were NG-STAR: ST-90 (n=100, 13.0%), ST-42 and ST-91 (n=45, 5.9%), ST-64 (n=44, 5.72%), and ST-139 (n=42, 5.5%). Decreased susceptibility to azithromycin was associated with NG-STAR ST-58, ST-61, ST-64, ST-79, ST-91 and ST-139(n=156, 92.3%); decreased susceptibility to cephalosporins with NG-STAR ST-90, ST-91 and ST-97 (n=162, 94.2%); and ciprofloxacin resistance with NG-STAR ST-26, ST-90, ST-91, ST-97, ST-150 and ST-158 (n=196, 98.0%). All isolates of NG-STAR ST-42, ST-43, ST-63, ST-81, and ST-160 (n=106) were susceptible to all four antimicrobials. The standardization of nomenclature associated with antimicrobial resistance determinants through an internationally available database will facilitate the monitoring of the global dissemination of antimicrobial resistant N. gonorrhoeae strains.

PMID: 28228492 [PubMed - as supplied by publisher]



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