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Pooled Lentiviral CRISPR-Cas9 Screens for Functional Genomics in Mammalian Cells.

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Pooled Lentiviral CRISPR-Cas9 Screens for Functional Genomics in Mammalian Cells.

Methods Mol Biol. 2019;1869:169-188

Authors: Aregger M, Chandrashekhar M, Tong AHY, Chan K, Moffat J

Abstract
CRISPR-Cas9 technology provides a simple way to introduce targeted mutations into mammalian cells to induce loss-of-function phenotypes. The CRISPR-Cas9 system has now successfully been applied for genetic screens in many cell types, providing a powerful tool for functional genomics with manifold applications. Genome-wide guide-RNA (gRNA) libraries allow facile generation of a pool of cells, each harboring a gene knockout mutation that can be used for the study of gene function, pathway analysis or the identification of genes required for cellular fitness. Furthermore, CRISPR genetic screens can be applied for the discovery of genes whose knockout sensitizes cells to drug treatments or mediates drug resistance. Here, we provide a detailed protocol discussing the necessary steps for the successful performance of pooled CRISPR-Cas9 screens.

PMID: 30324523 [PubMed - in process]



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EPH Profiling of BTIC Populations in Glioblastoma Multiforme Using CyTOF.

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EPH Profiling of BTIC Populations in Glioblastoma Multiforme Using CyTOF.

Methods Mol Biol. 2019;1869:155-168

Authors: Hu AX, Adams JJ, Vora P, Qazi M, Singh SK, Moffat J, Sidhu SS

Abstract
The ability to elucidate the phenotype of brain tumor initiating cell (BTIC) in the context of bulk tumor in glioblastoma multiforme (GBM) provides significant therapeutic benefits for therapeutic evaluation. For the identification of such an elusive and rare subpopulation of cells, a single cell analysis technology with deep profiling capabilities known as Mass Cytometry (CyTOF) can prove to be highly useful. CyTOF circumvents the spectral overlap limitations of traditional flow cytometry by replacing fluorophores with metal isotope tags, allowing the accurate detection of significantly more parameters at the same time. In this chapter, we demonstrate that synthetic antibodies can be conjugated with metal isotope tags for CyTOF analysis, resulting in the development of a highly tailored, custom multi-parameter panel. This toolset was used to stain patient-derived GBM cells, which was analyzed via CyTOF. Analysis software viSNE and SPADE were applied to study the co-expression patterns of the Eph Receptor (EphR) family and several putative BTIC markers in GBM, resulting in the identification of a distinct group of cells consistent with a BTIC subpopulation. This approach can be readily adapted to the detection of cancer stem-like cells in other cancer types.

PMID: 30324522 [PubMed - in process]



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Patterned Optoelectronic Tweezers: A New Scheme for Selecting, Moving, and Storing Dielectric Particles and Cells.

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Patterned Optoelectronic Tweezers: A New Scheme for Selecting, Moving, and Storing Dielectric Particles and Cells.

Small. 2018 Oct 11;:e1803342

Authors: Zhang S, Shakiba N, Chen Y, Zhang Y, Tian P, Singh J, Chamberlain MD, Satkauskas M, Flood AG, Kherani NP, Yu S, Zandstra PW, Wheeler AR

Abstract
Optical micromanipulation has become popular for a wide range of applications. In this work, a new type of optical micromanipulation platform, patterned optoelectronic tweezers (p-OET), is introduced. In p-OET devices, the photoconductive layer (that is continuous in a conventional OET device) is patterned, forming regions in which the electrode layer is locally exposed. It is demonstrated that micropatterns in the photoconductive layer are useful for repelling unwanted particles/cells, and also for keeping selected particles/cells in place after turning off the light source, minimizing light-induced heating. To clarify the physical mechanism behind these effects, systematic simulations are carried out, which indicate the existence of strong nonuniform electric fields at the boundary of micropatterns. The simulations are consistent with experimental observations, which are explored for a wide variety of geometries and conditions. It is proposed that the new technique may be useful for myriad applications in the rapidly growing area of optical micromanipulation.

PMID: 30307718 [PubMed - as supplied by publisher]



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Predicting the Effect of Mutations on Protein Folding and Protein-Protein Interactions.

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Predicting the Effect of Mutations on Protein Folding and Protein-Protein Interactions.

Methods Mol Biol. 2019;1851:1-17

Authors: Strokach A, Corbi-Verge C, Teyra J, Kim PM

Abstract
The function of a protein is largely determined by its three-dimensional structure and its interactions with other proteins. Changes to a protein's amino acid sequence can alter its function by perturbing the energy landscapes of protein folding and binding. Many tools have been developed to predict the energetic effect of amino acid changes, utilizing features describing the sequence of a protein, the structure of a protein, or both. Those tools can have many applications, such as distinguishing between deleterious and benign mutations and designing proteins and peptides with attractive properties. In this chapter, we describe how to use one of such tools, ELASPIC, to predict the effect of mutations on the stability of proteins and the affinity between proteins, in the context of a human protein-protein interaction network. ELASPIC uses a wide range of sequential and structural features to predict the change in the Gibbs free energy for protein folding and protein-protein interactions. It can be used both through a web server and as a stand-alone application. Since ELASPIC was trained using homology models and not crystal structures, it can be applied to a much broader range of proteins than traditional methods. It can leverage precalculated sequence alignments, homology models, and other features, in order to drastically lower the amount of time required to evaluate individual mutations and make tractable the analysis of millions of mutations affecting the majority of proteins in a genome.

PMID: 30298389 [PubMed - in process]



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Two-Dimensional Biochemical Purification for Global Proteomic Analysis of Macromolecular Protein Complexes.

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Two-Dimensional Biochemical Purification for Global Proteomic Analysis of Macromolecular Protein Complexes.

Methods Mol Biol. 2019;1871:445-454

Authors: Pourhaghighi R, Emili A

Abstract
A high-resolution two-dimensional (2-D) proteomic fractionation technique for the systematic purification and subsequent mass spectrometry-based identification of endogenous protein macromolecular complexes is described. The method hyphenates preparative isoelectric focusing (IEF) with mixed-bed ion exchange chromatography (IEX) to efficiently separate cell- or tissue- derived soluble protein mixtures, allowing for more effective and less biased physiochemical characterization of stable multiprotein assemblies. After comprehensive 2D fractionation of cell-free lysates, each fraction is subjected to quantitative tandem mass spectrometry (MS/MS) and subsequent computational analysis to map high-confidence protein-protein interactions (PPIs). Herein, the experimental component (workflow protocols) for this global "interactome" network mapping platform is described.

PMID: 30276754 [PubMed - in process]



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A Neurosphere Assay to Evaluate Endogenous Neural Stem Cell Activation in a Mouse Model of Minimal Spinal Cord Injury.

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A Neurosphere Assay to Evaluate Endogenous Neural Stem Cell Activation in a Mouse Model of Minimal Spinal Cord Injury.

J Vis Exp. 2018 Sep 13;(139):

Authors: Lakshman N, Xu W, Morshead CM

Abstract
Neural stem cells (NSCs) in the adult mammalian spinal cord are a relatively mitotically quiescent population of periventricular cells that can be studied in vitro using the neurosphere assay. This colony-forming assay is a powerful tool to study the response of NSCs to exogenous factors in a dish; however, this can also be used to study the effect of in vivo manipulations with the proper understanding of the strengths and limitations of the assay. One manipulation of the clinical interest is the effect of injury on endogenous NSC activation. Current models of spinal cord injury provide a challenge to study this as the severity of common contusion, compression, and transection models cause the destruction of the NSC niche at the site of the injury where the stem cells reside. Here, we describe a minimal injury model that causes localized damage at the superficial dorsolateral surface of the lower thoracic level (T7/8) of the adult mouse spinal cord. This injury model spares the central canal at the level of injury and permits analysis of the NSCs that reside at the level of the lesion at various time points following injury. Here, we show how the neurosphere assay can be utilized to study the activation of the two distinct, lineally-related, populations of NSCs that reside in the spinal cord periventricular region - primitive and definitive NSCs (pNSCs and dNSCs, respectively). We demonstrate how to isolate and culture these NSCs from the periventricular region at the level of injury and the white matter injury site. Our post-surgical spinal cord dissections show increased numbers of pNSC and dNSC-derived neurospheres from the periventricular region of injured cords compared to controls, speaking to their activation via injury. Furthermore, following injury, dNSC-derived neurospheres can be isolated from the injury site - demonstrating the ability of NSCs to migrate from their periventricular niche to sites of injury.

PMID: 30272658 [PubMed - in process]



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Molecular classification of primary mediastinal large B-cell lymphoma using routinely available tissue specimens.

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Molecular classification of primary mediastinal large B-cell lymphoma using routinely available tissue specimens.

Blood. 2018 Sep 26;:

Authors: Mottok A, Wright G, Rosenwald A, Ott G, Ramsower C, Campo E, Braziel RM, Delabie J, Weisenburger DD, Song JY, Chan WC, Cook JR, Fu K, Greiner T, Smeland E, Holte H, Savage KJ, Glinsmann-Gibson BJ, Gascoyne RD, Staudt LM, Jaffe ES, Connors JM, Scott DW, Steidl C, Rimsza LM

Abstract
Primary mediastinal large B-cell lymphoma (PMBCL) is recognized as a distinct entity in the World Health Organization classification. Currently, diagnosis relies on consensus of histopathology, clinical variables and presentation, giving rise to diagnostic inaccuracy in routine practice. Previous studies have demonstrated that PMBCL can be distinguished from subtypes of diffuse large B-cell lymphoma (DLBCL) based on gene expression signatures. However, requirement of fresh-frozen biopsy material has precluded the transfer of gene expression-based assays to the clinic. Here, we developed a robust and accurate molecular classification assay (Lymph3Cx) for the distinction of PMBCL from DLBCL subtypes based on gene expression measurements in formalin-fixed, paraffin-embedded tissue. A probabilistic model accounting for classification error, comprising 58 gene features, was trained on 68 cases of PMBCL and DLBCL. Performance of the model was subsequently evaluated in an independent validation cohort of 158 cases and showed high agreement of the Lymph3Cx molecular classification with the clinico-pathological diagnosis of an expert panel (frank misclassification rate 3.8 %). Furthermore, we demonstrate reproducibility of the assay with 100 % concordance of subtype assignments at two independent laboratories. Future studies will determine Lymph3Cx's utility for routine diagnostic purposes and therapeutic decision making.

PMID: 30257882 [PubMed - as supplied by publisher]



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How bacterial xenogeneic silencer rok distinguishes foreign from self DNA in its resident genome.

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How bacterial xenogeneic silencer rok distinguishes foreign from self DNA in its resident genome.

Nucleic Acids Res. 2018 Sep 25;:

Authors: Duan B, Ding P, Hughes TR, Navarre WW, Liu J, Xia B

Abstract
Bacterial xenogeneic silencers play important roles in bacterial evolution by recognizing and inhibiting expression from foreign genes acquired through horizontal gene transfer, thereby buffering against potential fitness consequences of their misregulated expression. Here, the detailed DNA binding properties of Rok, a xenogeneic silencer in Bacillus subtilis, was studied using protein binding microarray, and the solution structure of its C-terminal DNA binding domain was determined in complex with DNA. The C-terminal domain of Rok adopts a typical winged helix fold, with a novel DNA recognition mechanism different from other winged helix proteins or xenogeneic silencers. Rok binds the DNA minor groove by forming hydrogen bonds to bases through N154, T156 at the N-terminal of α3 helix and R174 of wing W1, assisted by four lysine residues interacting electrostatically with DNA backbone phosphate groups. These structural features endow Rok with preference towards DNA sequences harboring AACTA, TACTA, and flexible multiple TpA steps, while rigid A-tracts are disfavored. Correspondingly, the Bacillus genomes containing Rok are rich in A-tracts and show a dramatic underrepresentation of AACTA and TACTA, which are significantly enriched in Rok binding regions. These observations suggest that the xenogeneic silencing protein and its resident genome may have evolved cooperatively.

PMID: 30252102 [PubMed - as supplied by publisher]



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Colloidal aggregation: from screening nuisance to formulation nuance.

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Colloidal aggregation: from screening nuisance to formulation nuance.

Nano Today. 2018 Apr;19:188-200

Authors: Ganesh AN, Donders EN, Shoichet BK, Shoichet MS

Abstract
It is well known that small molecule colloidal aggregation is a leading cause of false positives in early drug discovery. Colloid-formers are diverse and well represented among corporate and academic screening decks, and even among approved drugs. Less appreciated is how colloid formation by drug-like compounds fits into the wider understanding of colloid physical chemistry. Here we introduce the impact that colloidal aggregation has had on early drug discovery, and then turn to the physical and thermodynamic driving forces for small molecule colloidal aggregation, including the particulate nature of the colloids, their critical aggregation concentration-governed formation, their mechanism of protein adsorption and subsequent inhibition, and their sensitivity to detergent. We describe methods that have been used extensively to both identify aggregate-formers and to study and control their physical chemistry. While colloidal aggregation is widely recognized as a problem in early drug discovery, we highlight the opportunities for exploiting this phenomenon in biological milieus and for drug formulation.

PMID: 30250495 [PubMed]



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Generating Intracellular Modulators of E3 Ligases and Deubiquitinases from Phage-Displayed Ubiquitin Variant Libraries.

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Generating Intracellular Modulators of E3 Ligases and Deubiquitinases from Phage-Displayed Ubiquitin Variant Libraries.

Methods Mol Biol. 2018;1844:101-119

Authors: Zhang W, Sidhu SS

Abstract
Ubiquitination is a posttranslational protein modification pathway regulating diverse cellular processes that are implicated in numerous human diseases. However, targeting the enzymes in the ubiquitination cascade potently and selectively remains a major challenge. Recently we devised a methodology to generate ubiquitin-based modulators for E3 ligases and deubiquitinases, enzymes that control the specificity of protein ubiquitination and deubiquitination, respectively. Here, we describe methods to generate libraries of ubiquitin variants and perform phage display selections to isolate high-affinity binders for target proteins. Importantly, the strategy introduced here can be applied to other small protein domains mediating protein-protein interactions to engineer tools for target validation and potential therapeutic development.

PMID: 30242706 [PubMed - in process]



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