Volume: 10 Issue: 10 —BIO-PROTOCOL

Biochemistry

Permethylation and Microfractionation of Sulfated Glycans for MS Analysis Authors: Shin-Yi Yu, Sergei Snovida and Kay-Hooi Khoo, date: 05/20/2020, view: 1984, Q&A: 0

Sulfated glycans are barely detectable in routine mass spectrometry (MS)-based glycomic analysis due to ion suppression by the significantly more abundant neutral glycans in the positive ion mode, and sialylated non-sulfated glycans in the negative ion mode, respectively. Nevertheless, the negative charge imparted by sulfate can be advantageous ...

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Negative Ion Mode nanoLC-ESI-MS/MS Analyses of Permethylated Sulfated Glycans Authors: Shin-Yi Yu, Chu-Wen Cheng and Kay-Hooi Khoo, date: 05/20/2020, view: 1931, Q&A: 0

We have developed enabling techniques for sulfoglycomics based on MALDI-MS mapping and MS/MS sequencing of permethylated sulfated glycans. We then extended further the analytical workflow to C18 reverse phase (RP)-nanoLC-nanoESI-MS/MS analyses of permethylated sulfated glycans in the negative ion mode. The advantages are that extra sulfates on ...

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Evaluation of the Efficiency of Genome Editing Tools by a Frameshift Fluorescence Protein Reporter Authors: Balaji T. Moorthy, Akhilesh Kumar, Lauren X. Lotenfoe and Fangliang Zhang, date: 05/20/2020, view: 2298, Q&A: 0

In the last decade, genome editing has been the center of attention as a novel tool for mechanistic investigations and for potential clinical applications. Various genome editing tools like meganucleases, zinc finger nucleases (ZFNs), transcription activator-like effector-based nucleases (TALEN), and the clustered regularly interspaced short ...

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In vitro Assay for Bacterial Membrane Protein Integration into Proteoliposomes Authors: Hanako Nishikawa, Masaru Sasaki and Ken-ichi Nishiyama, date: 05/20/2020, view: 2469, Q&A: 0

It is important to experimentally determine how membrane proteins are integrated into biomembranes to unveil the roles of the integration factors, and to understand the functions and structures of membrane proteins. We have developed a reconstitution system for membrane protein integration in E. coli using purified factors, in which the ...

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Cancer Biology

Modeling NOTCH1 driven T-cell Acute Lymphoblastic Leukemia in Mice Authors: Agnieszka A. Wendorff and Adolfo A. Ferrando, date: 05/20/2020, view: 2526, Q&A: 0

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematological malignancy that arises from transformation of T-cell primed hematopoietic progenitors. Although T-ALL is a heterogenous and molecularly complex disease, more than 65% of T-ALL patients carry activating mutations in the NOTCH1 gene. The majority of T-ALL–associated ...

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Isolation of Tumor Cells Based on Their Distance from Blood Vessels Authors: Saran Kumar, Husni Sharife, Tirzah Kreisel, Libat Bar-Lev, Myriam Grunewald and Eli Keshet, date: 05/20/2020, view: 2472, Q&A: 0

Differential exposure of tumor cells to microenvironmental cues greatly impacts cell phenotypes, raising a need for position based sorting of tumor cells amenable to multiple OMICs and functional analyses. One such key determinant of tumor heterogeneity in solid tumors is its vasculature. Proximity to blood vessels (BVs) profoundly affects tumor ...

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Cell Biology

Spatial Image Correlation Spectroscopy (ICS): A Technique for Average Size Determination of Subcellular Accumulated Structures from Fluorescence Microscopic Images Authors: Akira Kitamura and Masakata Kinjo, date: 05/20/2020, view: 2037, Q&A: 0

Size determination of subcellular structures such as inclusion bodies (IBs) and granules from fluorescent images is important for identification and structural characterization. However, it is often time-consuming just for the comparison of the average size of the structures. Here, we introduce a high-throughput procedure to represent the average ...

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Pancreatic Acinar Cell Preparation for Oxygen Consumption and Lactate Production Analysis Authors: Jane A. Armstrong, Robert Sutton and David N. Criddle, date: 05/20/2020, view: 1716, Q&A: 0

Mitochondrial dysfunction is a principal feature of acute pancreatitis (AP) although the underlying mechanisms are still unclear. AP precipitants induce Ca²⁺-dependent formation of the mitochondrial permeability transition pore (MPTP) in pancreatic acinar cells (PACs), leading to ATP depletion and necrosis. Evaluations of mitochondrial ...

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Analysis of Generalized Fibrosis in Mouse Tissue Sections with Masson’s Trichrome Staining Authors: Diantha van de Vlekkert, Eda Machado and Alessandra d’Azzo, date: 05/20/2020, view: 2989, Q&A: 0

Expansion of fibrous connective tissue and abnormal deposition of extracellular matrix (ECM) are at the basis of many fibrotic diseases. Fibrosis can occur in response to both physiological and pathological cues, including wound healing, tissue remodeling/repair and inflammation. Chronic fibrosis can lead to severe tissue damage, organ failure and ...

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Developmental Biology

Direct Reprogramming of Mouse Embryonic Fibroblasts to Conventional Type 1 Dendritic Cells by Enforced Expression of Transcription Factors Authors: Fábio F. Rosa, Cristiana F. Pires, Olga Zimmermannova and Carlos-Filipe Pereira, date: 05/20/2020, view: 2953, Q&A: 0

Ectopic expression of transcription factor combinations has been recently demonstrated to reprogram differentiated somatic cells towards the dendritic cell (DC) lineage without reversion to a multipotent state. DCs have the ability to induce potent and long-lasting adaptive immune responses. In particular, conventional type 1 DCs (cDC1s) excel on ...

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Immunology

Flow Cytometry Analysis and Fluorescence-activated Cell Sorting of Myeloid Cells from Lung and Bronchoalveolar Lavage Samples from Mycobacterium tuberculosis-infected Mice Authors: Alissa C. Rothchild, Dat Mai, Alan Aderem and Alan H. Diercks, date: 05/20/2020, view: 2865, Q&A: 0

Mycobacterium tuberculosis (Mtb) is transmitted by aerosol and can cause serious bacterial infection in the lung that can be fatal if left untreated. Mtb is now the leading cause of death worldwide by an infectious agent. Characterizing the early events of in vivo infection following aerosol challenge is critical for ...

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Microbiology

Bacterial Lawn Avoidance and Bacterial Two Choice Preference Assays in Caenorhabditis elegans Authors: Jogender Singh and Alejandro Aballay, date: 05/20/2020, view: 2314, Q&A: 0

Physical avoidance of pathogens is a crucial defense strategy used by the host to reduce pathogen infection. Hosts display the use of multiple strategies to sense and avoid pathogens, ranging from olfaction to sensing of damage caused by pathogen infection. Understanding various mechanisms of pathogen avoidance has the potential to uncover ...

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Molecular Biology

Molecular Size Analysis of Recombinant Importin-histone Complexes Using Analytical Ultracentrifugation Authors: Abhilash Padavannil, Chad A. Brautigam and Yuh Min Chook, date: 05/20/2020, view: 1971, Q&A: 0

Histones constitute the protein components of nucleosomes. Despite their small sizes, histones do not diffuse through the nuclear pore complex. Instead, they are transported to the nucleus by importins, either alone or in complex with histone chaperones. Determining the molecular size of the importin-histone complexes is key to understanding the ...

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Neuroscience

Generation, Analyzing and in-vivo Drug Treatment of Drosophila Models with IBMPFD Authors: Ting Zhang, Bruce A. Hay and Ming Guo, date: 05/20/2020, view: 2015, Q&A: 0

Missense mutations of p97/cdc48/Valosin-containing protein (VCP) cause inclusion body myopathy, Paget disease with frontotemporal dementia (IBMPFD) and other neurodegenerative diseases. The pathological mechanism of IBMPFD is not clear and there is no treatment. We generated Drosophila models of IBMPFD in adult flight muscle in ...

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Plant Science

Purification of Protein-complexes from the Cyanobacterium Synechocystis sp. PCC 6803 Using FLAG-affinity Chromatography Authors: Minna M. Koskela, Petra Skotnicová, Éva Kiss and Roman Sobotka, date: 05/20/2020, view: 2498, Q&A: 0

Exploring the structure and function of protein complexes requires their isolation in the native state–a task that is made challenging when studying labile and/or low abundant complexes. The difficulties in preparing membrane-protein complexes are especially notorious. The cyanobacterium Synechocystis sp. PCC 6803 is a widely used model ...

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