Spatial Transcriptomics: Constructing a Single-Cell Resolution Transcriptome-Wide Expression Atlas.
Abstract The method described here aims at the construction of a single-cell resolution gene expression atlas for an animal or tissue, combining in situ hybridization (ISH) and single-cell mRNA-sequencing (scRNAseq).A high resolution and medium-coverage gene expression atlas of an animal or tissue of interest can be obtained by performing a series of ISH experiments, followed by a process of image registration and gene expression averaging. Using the overlapping fraction of the genes, concomitantly obtained scRNAseq data can be fitted into the spatial context of the gene expression atlas, complementing the coverag...
Source: Mol Biol Cell - November 14, 2017 Category: Molecular Biology Authors: Achim K, Vergara HM, Pettit JB Tags: Methods Mol Biol Source Type: research

Single mRNA Molecule Detection in Drosophila.
Abstract Single molecule fluorescent in situ hybridization (smFISH) enables quantitative measurements of gene expression and mRNA localization. The technique is increasingly popular for analysis of cultured cells but is not widely applied to intact organisms. Here, we describe a method for labeling and detection of single mRNA molecules in whole embryos of the fruit fly Drosophila melanogaster. This method permits measurements of gene expression in absolute units, enabling new studies of transcriptional mechanisms underlying precision and reproducibility in cell specification. PMID: 29130194 [PubMed - in proc...
Source: Mol Biol Cell - November 14, 2017 Category: Molecular Biology Authors: Little SC, Gregor T Tags: Methods Mol Biol Source Type: research

Detection and Automated Analysis of Single Transcripts at Subcellular Resolution in Zebrafish Embryos.
Abstract Single molecule fluorescence in situ hybridization (smFISH) is a method to visualize single mRNA molecules. When combined with cellular and nuclear segmentation, transcripts can be assigned to different cellular compartments resulting in quantitative information on transcript levels at subcellular resolution. The use of smFISH in zebrafish has been limited by the lack of protocols and an automated image analysis pipeline for samples of multicellular organisms. Here we present a protocol for smFISH on zebrafish cryosections. The protocol includes a method to obtain high-quality sections of zebrafish embryo...
Source: Mol Biol Cell - November 14, 2017 Category: Molecular Biology Authors: Stapel LC, Broaddus C, Vastenhouw NL Tags: Methods Mol Biol Source Type: research

Detection of mRNA and Associated Molecules by ISH-IEM on Frozen Sections.
Abstract The use of tagged RNA probes to directly hybridize frozen sections of chemically fixed tissues, followed by the tag detection with specific antibodies and gold conjugates form the core of the in situ hybridization (ISH)-immunoelectron microscopy (IEM) method that we have developed and successfully used to detect endogenous gurken and bicoid mRNAs in Drosophila oocytes. PMID: 29130197 [PubMed - in process] (Source: Mol Biol Cell)
Source: Mol Biol Cell - November 14, 2017 Category: Molecular Biology Authors: Rabouille C Tags: Methods Mol Biol Source Type: research

Padlock Probes to Detect Single Nucleotide Polymorphisms.
Abstract Rapid development of high-throughput DNA analyzation methods has enabled global characterization of genetic landscapes and aberrations in study subjects in a time and cost effective fashion. In most methods, however, spatial tissue context is lost since sample preparation requires isolation of nucleic acids out of their native environment. We hereby present the most recent protocol for multiplexed, in situ detection of mRNAs and single nucleotide polymorphisms using padlock probes and rolling circle amplification. We take advantage of a single nucleotide variant within conserved ACTB mRNA to successfully ...
Source: Mol Biol Cell - November 14, 2017 Category: Molecular Biology Authors: Krzywkowski T, Nilsson M Tags: Methods Mol Biol Source Type: research

Quantifying Gene Expression in Living Cells with Ratiometric Bimolecular Beacons.
Abstract Molecular beacons (MBs), a class of oligonucleotide-based probes, have enabled researchers to study various RNA molecules in their native live-cell contexts. However, it is also increasingly recognized that, when delivered into cells, MBs have the tendency to be sequestered into the nucleus where they may generate false positive signals. In an attempt to overcome this issue, MBs have been synthesized with chemically modified oligonucleotide backbones to confer greater biostability. Alternatively, strategies have been developed to minimize nuclear entry. In the latter approach, we have combined functional ...
Source: Mol Biol Cell - November 14, 2017 Category: Molecular Biology Authors: Yang Y, Chen M, Krueger CJ, Tsourkas A, Chen AK Tags: Methods Mol Biol Source Type: research

Optimizing Molecular Beacons for Intracellular Analysis of RNA.
Abstract Conventional molecular beacons (MBs) have been used extensively for imaging specific endogenous RNAs in living cells, but their tendency to generate false-positive signals as a result of nuclease degradation and/or nonspecific binding limits sensitive and accurate imaging of intracellular RNAs. In an attempt to overcome this limitation, MBs have been synthesized with various chemically modified oligonucleotide backbones to confer greater biostability. We have recently developed a new MB architecture composed of 2'-O-methyl RNA (2Me), a fully phosphorothioate (PS) modified loop domain and a phosphodiester ...
Source: Mol Biol Cell - November 14, 2017 Category: Molecular Biology Authors: Chen M, Yang Y, Krueger CJ, Chen AK Tags: Methods Mol Biol Source Type: research

Live Imaging of Nuclear RNPs in Mammalian Complex Tissue with ECHO-liveFISH.
Abstract Multiplex RNA detection with fluorescence microscopy offers high spatial and temporal resolution required for addressing complex behaviors of RNA in living cells. Using chemically engineered linear oligonucleotide probes that emit fluorescence upon hybridization to target RNA, we have devised an imaging method suitable for studies of the dynamic regulation of nuclear RNPs, an important and yet poorly understood cellular pathway of gene expression. This new method labels specific sequences of RNA components in RNPs and thus avoids overexpression of fluorescent marker proteins that may result in entangled e...
Source: Mol Biol Cell - November 14, 2017 Category: Molecular Biology Authors: Wang DO Tags: Methods Mol Biol Source Type: research

In Vivo Visualization and Function Probing of Transport mRNPs Using Injected FIT Probes.
Abstract Fluorogenic hybridization methods, such as the use of FIT probes, enable the in vivo detection of specific mRNAs transcribed from their endogenous, genetically nonmodified loci. Here, we describe the design, synthesis and injection of nuclease resistant FIT probes into developing Drosophila oocytes to detect endogenous localizing mRNAs as wells as to probe function of structural RNA elements. PMID: 29130204 [PubMed - in process] (Source: Mol Biol Cell)
Source: Mol Biol Cell - November 14, 2017 Category: Molecular Biology Authors: Chamiolo J, Gaspar I, Ephrussi A, Seitz O Tags: Methods Mol Biol Source Type: research

Visualizing RNA in Live Bacterial Cells Using Fluorophore- and Quencher-Binding Aptamers.
e A Abstract To elucidate the roles, dynamics, and regulation of RNAs, it is vital to be able to visualize the RNA of interest (ROI) in living cells noninvasively. Here, we describe a novel live-cell RNA imaging method using fluorophore- and quencher-binding aptamers, which can be genetically fused to the ROI. In this method, new membrane permeable and nonfluorescent fluorophore-quencher conjugates were utilized, and we showed that their fluorescence increases dramatically upon binding to fluorophore- or quencher-binding aptamers. This phenomenon allowed for labeling the ROI with many different colored fluorophore...
Source: Mol Biol Cell - November 14, 2017 Category: Molecular Biology Authors: Sunbul M, Arora A, Jäschke A Tags: Methods Mol Biol Source Type: research

Method for Imaging Live-Cell RNA Using an RNA Aptamer and a Fluorescent Probe.
Abstract Live-cell imaging of mRNA dynamics is increasingly important to understanding spatially restricted gene expression. We recently developed a convenient and versatile method that uses a gene-specific RNA aptamer and a fluorescent probe to enable spatiotemporal imaging of endogenous mRNAs in living cells. The method was validated by live-cell imaging of the endogenous mRNA of β-actin. The new RNA-imaging technology might be useful for live-cell imaging of any RNA molecules. PMID: 29130206 [PubMed - in process] (Source: Mol Biol Cell)
Source: Mol Biol Cell - November 14, 2017 Category: Molecular Biology Authors: Sato SI, Yatsuzuka K, Katsuda Y, Uesugi M Tags: Methods Mol Biol Source Type: research

Live Imaging of mRNA Synthesis in Drosophila.
Abstract mRNA synthesis is one of the earliest readouts of the activity of a transcribed gene, which is of particular interest in the context of metazoan cell fate specification. These processes are intrinsically dynamic and stochastic, which makes in vivo single-cell measurements inevitable. Here, we present the application of a technology that has been widely used in single celled organisms to measure transcriptional activity in developing embryos of the fruit fly Drosophila melanogaster. The method allows for quantification of instantaneous polymerase occupancy of active gene loci and thereby enables the develo...
Source: Mol Biol Cell - November 14, 2017 Category: Molecular Biology Authors: Garcia HG, Gregor T Tags: Methods Mol Biol Source Type: research

Imaging Translation Dynamics of Single mRNA Molecules in Live Cells.
Abstract mRNA translation is a key step in decoding the genetic information stored in DNA. Regulation of translation efficiency contributes to gene expression control and is therefore important for cell fate and function. Here, we describe a recently developed microscopy-based method that allows for visualization of translation of single mRNAs in live cells. The ability to measure translation dynamics of single mRNAs will enable a better understanding of spatiotemporal control of translation, and will provide unique insights into translational heterogeneity of different mRNA molecules in single cells. PMID: 2...
Source: Mol Biol Cell - November 14, 2017 Category: Molecular Biology Authors: Ruijtenberg S, Hoek TA, Yan X, Tanenbaum ME Tags: Methods Mol Biol Source Type: research

Isolation and Characterization of Endogenous RNPs from Brain Tissues.
Abstract Identification of physiological target RNAs and protein interactors bound to RNA-binding proteins is a key prerequisite to understand the underlying mechanisms of posttranscriptional expression control and RNA granule assembly. Here, we describe a multistep biochemical approach to isolate endogenous ribonucleoprotein particles from brain tissues by exploiting differential centrifugation and gradient fractionation followed by immunoprecipitation with monospecific, affinity-purified antibodies directed against selected RNA-binding proteins. This protocol results in highly enriched endogenous ribonucleoprote...
Source: Mol Biol Cell - November 14, 2017 Category: Molecular Biology Authors: Schieweck R, Ang FY, Fritzsche R, Kiebler MA Tags: Methods Mol Biol Source Type: research

Construction of Rabbit Immune Antibody Libraries.
Abstract Rabbits have distinct advantages over mice as a source of target-specific antibodies. They produce higher affinity antibodies than mice, and may elicit strong immune response against antigens or epitopes that are poorly immunogenic or tolerated in mice. However, a great majority of currently available monoclonal antibodies are of murine origin because of the wider availability of murine fusion partner cell lines and well-established tools and protocols for fusion and cloning of mouse hybridoma. Phage-display selection of antibody libraries is an alternative method to hybridoma technology for the generatio...
Source: Mol Biol Cell - November 10, 2017 Category: Molecular Biology Authors: Nguyen TTH, Lee JS, Shim H Tags: Methods Mol Biol Source Type: research

Targeting Intracellular Antigens with pMHC-Binding Antibodies: A Phage Display Approach.
Abstract Antibodies that bind peptide-MHC (pMHC) complex in a manner akin to T-cell receptor (TCR) have not only helped in understanding the mechanism of TCR-pMHC interactions in the context of T-cell biology, but also spurred considerable interest in recent years as potential cancer therapeutics. Traditional methods to generate such antibodies using hybridoma and B-cell sorting technologies are sometimes inadequate, possibly due to the small contribution of peptide to the overall B-cell epitope space on the surface of the pMHC complex (typical peptide MW = 1 kDa versus MHC MW = 45 kD...
Source: Mol Biol Cell - November 10, 2017 Category: Molecular Biology Authors: Wu Z, Santich BH, Liu H, Liu C, Cheung NV Tags: Methods Mol Biol Source Type: research

Phage Display and Selections on Cells.
Abstract Antibody identification by phage display on protein or peptide targets is well established and many protocols are available. But there are many targets that cannot be expressed recombinantly or, like peptides, do not reflect correct folding of the protein. Most of these targets are cell surface receptors. Here, we describe a protocol for a panning strategy on cells to obtain specific binders to cell surface receptors. A depletion step is included to prevent enrichment of antibodies that bind to unwanted targets. Each step of the protocol is explained and variations of this protocol are given. PMID: 2...
Source: Mol Biol Cell - November 10, 2017 Category: Molecular Biology Authors: Fahr W, Frenzel A Tags: Methods Mol Biol Source Type: research

Combine Phage Antibody Display Library Selection on Patient Tissue Specimens with Laser Capture Microdissection to Identify Novel Human Antibodies Targeting Clinically Relevant Tumor Antigens.
Abstract A functional approach to generate tumor-targeting human monoclonal antibodies is through selection of phage antibody display libraries directly on tumor cells. Although technically convenient, the use of cancer cell lines for the selection has limitations as those cell lines often undergo genetic and epigenetic changes during prolonged in vitro culture and alter their cell surface antigen expression profile. The key is to develop a technology that allows selection of phage antibody display libraries on tumor cells in situ residing in their natural tissue microenvironment. Laser capture microdissection (LC...
Source: Mol Biol Cell - November 10, 2017 Category: Molecular Biology Authors: Su Y, Bidlingmaier S, Lee NK, Liu B Tags: Methods Mol Biol Source Type: research

Antibody Selection on FFPE Tissue Slides.
MK Abstract Standard antibody phage-display panning uses purified proteins, antigen-transfected cells, or tumor cell lines as target structure to generate specific antibodies. Here, we describe a method for the selection of specific antibodies by phage panning against routine formalin-fixed paraffin-embedded (FFPE) tissue biopsies immobilized on glass slides. Selected antibody fragments recognize disease-associated antigens in its native conformation, suitable for the development of targeted diagnostic and therapeutic agents. PMID: 29116517 [PubMed - in process] (Source: Mol Biol Cell)
Source: Mol Biol Cell - November 10, 2017 Category: Molecular Biology Authors: Ten Haaf A, Gattenlöhner S, Tur MK Tags: Methods Mol Biol Source Type: research

High-Throughput IgG Reformatting and Expression.
Abstract We have recently described a one-step zero-background IgG reformatting method that enables the rapid reformatting of phage-displayed antibody fragments into a single-mammalian cell expression vector for full IgG expression (Chen et al. Nucleic Acids Res 42:e26, 2014). The strategy utilizes our unique positive selection method, referred to as insert-tagged (InTag) positive selection, where a positive selection marker (e.g. chloramphenicol-resistance gene) is cloned together with the antibody inserts into the expression vector. The recombinant clones containing the InTag adaptor are then positively selected...
Source: Mol Biol Cell - November 10, 2017 Category: Molecular Biology Authors: Chen CG, Sansome G, Wilson MJ, Panousis C Tags: Methods Mol Biol Source Type: research

Monitoring Phage Biopanning by Next-Generation Sequencing.
Abstract Phage display has enabled the rapid isolation of antigen-specific antibodies from combinatorial libraries of the variable heavy chain gene (VH) and variable light chain gene (VL). The method is based on genetic engineering of bacteriophages and repeated rounds of antigen-guided selection by phage biopanning.Next-Generation Sequencing (NGS) coupled with bioinformatics are powerful tools for analyzing the large number of DNA sequences present in an immune library.Here, we describe a method that demonstrates how NGS analysis enhances phage biopanning of complex antibody libraries as well as facilitates the a...
Source: Mol Biol Cell - November 10, 2017 Category: Molecular Biology Authors: Vaisman-Mentesh A, Wine Y Tags: Methods Mol Biol Source Type: research

Metasecretome Phage Display.
Abstract Metasecretome is a collection of cell-surface and secreted proteins that mediate interactions between microbial communities and their environment. These include adhesins, enzymes, surface structures such as pili or flagella, vaccine targets or proteins responsible for immune evasion. Traditional approaches to exploring matasecretome of complex microbial communities via cultivation of microorganisms and screening of individual strains fail to sample extraordinary diversity in these communities, since only a limited fraction of microorganisms are represented by cultures. Advances in culture-independent sequ...
Source: Mol Biol Cell - November 10, 2017 Category: Molecular Biology Authors: Ciric M, Ng F, Rakonjac J, Gagic D Tags: Methods Mol Biol Source Type: research

Phagekines: Screening Binding Properties and Biological Activity of Functional Cytokines Displayed on Phages.
Abstract The current chapter focuses on the use of filamentous phages to display, modify, and characterize cytokines, which are proteins belonging to a versatile group of essential mediators involved in cell-cell communication. Cytokines exhibit a considerable diversity, both in functions and in structural features underlying their biological effects. A broad variety of cytokines have been successfully displayed on phages, allowing the high-throughput study of their binding properties and biological activities and the discovery of novel therapeutics through directed evolution. The technical singularities and some ...
Source: Mol Biol Cell - November 10, 2017 Category: Molecular Biology Authors: Rojas G, Carmenate T Tags: Methods Mol Biol Source Type: research

Recovery and Characterization of Bacteria Resisting Infection by Lytic Bacteriophage.
Abstract Bacteria and bacteriophages coexist and coevolve, bacteriophages being obligatory predators exerting an evolutionary pressure on their prey. Mechanisms in action vary depending on the bacterial genomic content and on the regulation of the bacteriophage cycle. To assess the multiplicity of bacterial genes involved in resistance as well as the changes in the bacteriophage interactions with the bacteria, it is necessary to isolate and investigate large numbers of independent resistant variants. Here we describe protocols that have been applied to the study of Pseudomonas aeruginosa and four of its virulent b...
Source: Mol Biol Cell - November 10, 2017 Category: Molecular Biology Authors: Latino L, Pourcel C Tags: Methods Mol Biol Source Type: research

Methods for Bacteriophage Preservation.
Abstract In a view of growing interest in bacteriophages as the most abundant members of microbial communities and as antibacterial agents, reliable methods for bacteriophage long-term preservation, that warrant the access to original or mutant stocks of unchanged properties, have become of crucial importance. A storage method that retains the infectivity of any kind of bacteriophage virions, either in a cell lysate or in a purified suspension, does not exist, due to the enormous diversity of bacteriophages and hence the differentiation of their sensitivity to various storage conditions. Here, we describe a method...
Source: Mol Biol Cell - November 10, 2017 Category: Molecular Biology Authors: Łobocka MB, Głowacka A, Golec P Tags: Methods Mol Biol Source Type: research

Conceptual Challenges of the Systemic Approach in Understanding Cell Differentiation.
Abstract The cells of a multicellular organism are derived from a single zygote and genetically identical. Yet, they are phenotypically very different. This difference is the result of a process commonly called cell differentiation. How the phenotypic diversity emerges during ontogenesis or regeneration is a central and intensely studied but still unresolved issue in biology. Cell biology is facing conceptual challenges that are frequently confused with methodological difficulties. How to define a cell type? What stability or change means in the context of cell differentiation and how to deal with the ubiquitous m...
Source: Mol Biol Cell - November 10, 2017 Category: Molecular Biology Authors: Paldi A Tags: Methods Mol Biol Source Type: research

A Primer on Mathematical Modeling in the Study of Organisms and Their Parts.
vil M Abstract Mathematical modeling is a very powerful tool for understanding natural phenomena. Such a tool carries its own assumptions and should always be used critically. In this chapter, we highlight the key ingredients and steps of modeling and focus on their biological interpretation. In particular, we discuss the role of theoretical principles in writing models. We also highlight the meaning and interpretation of equations. The main aim of this chapter is to facilitate the interaction between biologists and mathematical modelers. We focus on the case of cell proliferation and motility in the context of mu...
Source: Mol Biol Cell - November 10, 2017 Category: Molecular Biology Authors: Montévil M Tags: Methods Mol Biol Source Type: research

Parameters Estimation in Phase-Space Landscape Reconstruction of Cell Fate: A Systems Biology Approach.
Abstract The thermodynamical formalism of irreversible processes offers a theoretical framework appropriate to explain the complexity observed at the macroscopic level of dynamic systems. In this context, together with the theory of complex systems and systems biology, the thermodynamical formalism establishes an appropriate conceptual framework to address the study of biological systems, in particular cancer.The Chapter is organized as follows: In Subheading 1, an integrative view of these disciplines is offered, for the characterization of the emergence and evolution of cancer, seen as a self-organized dynamic s...
Source: Mol Biol Cell - November 10, 2017 Category: Molecular Biology Authors: Montero S, Martin R, Mansilla R, Cocho G, Nieto-Villar JM Tags: Methods Mol Biol Source Type: research

Complexity of Biochemical and Genetic Responses Reduced Using Simple Theoretical Models.
Abstract Living systems are known to behave in a complex and sometimes unpredictable manner. Humans, for a very long time, have been intrigued by nature, and have attempted to understand biological processes and mechanisms using numerous experimental and mathematical techniques. In this chapter, we will look at simple theoretical models, using both linear and nonlinear differential equations, that realistically capture complex biochemical and genetic responses of living cells. Even for cases where cellular behaviors are stochastic, as for single-cell responses, randomness added to well-defined deterministic models...
Source: Mol Biol Cell - November 10, 2017 Category: Molecular Biology Authors: Selvarajoo K Tags: Methods Mol Biol Source Type: research

Endogenous Molecular-Cellular Network Cancer Theory: A Systems Biology Approach.
Abstract In light of ever apparent limitation of the current dominant cancer mutation theory, a quantitative hypothesis for cancer genesis and progression, endogenous molecular-cellular network hypothesis has been proposed from the systems biology perspective, now for more than 10 years. It was intended to include both the genetic and epigenetic causes to understand cancer. Its development enters the stage of meaningful interaction with experimental and clinical data and the limitation of the traditional cancer mutation theory becomes more evident. Under this endogenous network hypothesis, we established a co...
Source: Mol Biol Cell - November 10, 2017 Category: Molecular Biology Authors: Wang G, Yuan R, Zhu X, Ao P Tags: Methods Mol Biol Source Type: research

Spatiotemporal Fluctuation Analysis of Molecular Diffusion Laws in Live-Cell Membranes.
Abstract A present challenge of membrane biophysics is deciphering the dynamic behavior of molecules, such as lipids and proteins, within the natural environment of a living-cell membrane. Here, a fluorescence fluctuation-based approach will be described, which makes it possible to probe the "diffusion law" of molecules directly from imaging, in the form of a mean square displacement vs time-delay plot (iMSD), with no need for interpretative models. Of note, the presented approach does not require extraction of the molecular trajectories nor the use of bright fluorophores. Conversely, it can be used at h...
Source: Mol Biol Cell - November 10, 2017 Category: Molecular Biology Authors: Cardarelli F Tags: Methods Mol Biol Source Type: research

Normalized Screening of Protein Engineering Libraries by Split-GFP Crude Cell Extract Quantification.
UT Abstract The different expression level and solubility showed by each protein variant represents an important challenge during screening campaigns: Usually, the total activity measurement constitutes the only criterion for identifying improved variants. This hampers the chances of finding interesting mutants, especially if the aim is to improve activity: On the one hand, interesting but poorly soluble variants will remain undetectable. On the other hand, a mutation might not increase activity, but improve expression level or solubility. The split-GFP technology offers an affordable and technically simple manne...
Source: Mol Biol Cell - November 2, 2017 Category: Molecular Biology Authors: Santos-Aberturas J, Dörr M, Bornscheuer UT Tags: Methods Mol Biol Source Type: research

Functional Analysis of Membrane Proteins Produced by Cell-Free Translation.
k S Abstract Cell-free production is a valuable and alternative method for the synthesis of membrane proteins. This system offers openness allowing the researchers to modify the reaction conditions without any boundaries. Additionally, the cell-free reactions are scalable from 20 μL up to several mL, faster and suitable for the high-throughput protein production. Here, we present two cell-free systems derived from Escherichia coli (E. coli) and Spodoptera frugiperda (Sf21) lysates. In the case of the E. coli cell-free system, nanodiscs are used for the solubilization and purification of membrane proteins. ...
Source: Mol Biol Cell - November 2, 2017 Category: Molecular Biology Authors: Dondapati SK, Wüstenhagen DA, Kubick S Tags: Methods Mol Biol Source Type: research

Solid-Phase Agar Plate Assay for Screening Amine Transaminases.
ml;hne M Abstract Agar plate assays represent a useful method for high-throughput prescreening of larger enzyme libraries derived from for example error-prone PCR or multiple site-saturation mutagenesis to decrease screening effort by separating promising variants from less active, inactive, or neutral variants. In order to do so, colonies are directly applied for enzyme expression and screening on adsorbent and microporous membranes instead of elaborately preparing cell lysates in 96-well plates. This way, 400-800 enzyme variants can be prescreened on a single membrane, 10,000-20,000 variants per week and per sin...
Source: Mol Biol Cell - November 2, 2017 Category: Molecular Biology Authors: Weiß MS, Bornscheuer UT, Höhne M Tags: Methods Mol Biol Source Type: research

Ultrahigh-Throughput Screening of Single-Cell Lysates for Directed Evolution and Functional Metagenomics.
Abstract The success of ultrahigh-throughput screening experiments in directed evolution or functional metagenomics strongly depends on the availability of efficient technologies for the quantitative testing of a large number of variants. With advanced robotics, libraries of up to 10(5) clones can be screened per day as colonies on agar plates or cell lysates in microwell plates, albeit at high cost of capital, manpower and consumables. These cost considerations and the general need for high-throughput make miniaturization of assay volumes attractive. To provide a general solution to maintain genotype-phenotype li...
Source: Mol Biol Cell - November 2, 2017 Category: Molecular Biology Authors: Gielen F, Colin PY, Mair P, Hollfelder F Tags: Methods Mol Biol Source Type: research

Isolation of pH-Sensitive Antibody Fragments by Fluorescence-Activated Cell Sorting and Yeast Surface Display.
olmar H Abstract Fluorescence-activated cell sorting (FACS) in combination with yeast surface display (YSD) has proven to be a valuable tool for the engineering of antibodies. It enables the fast and robust identification and isolation of candidates with prescribed characteristics from combinatorial libraries. A novel application for FACS and YSD that has recently evolved addresses the engineering of antibodies toward pH-switchable antigen binding, aiming at reduced binding at acidic pH, compared to neutral pH. Therefore, we give guidance for the incorporation of such pH switches into antibody variable domains usi...
Source: Mol Biol Cell - November 2, 2017 Category: Molecular Biology Authors: Schröter C, Krah S, Beck J, Könning D, Grzeschik J, Valldorf B, Zielonka S, Kolmar H Tags: Methods Mol Biol Source Type: research

Noncoding RNAs in DNA Damage Response: Opportunities for Cancer Therapeutics.
Abstract DNA repair machinery preserves genomic integrity, which is frequently challenged through endogenous and exogenous toxic insults, and any sort of repair machinery malfunctioning ultimately manifests in the form of several types of terrible human diseases such as cancers (Hoeijmakers, Nature 411(6835): 366-374, 2001). Noncoding RNAs (ncRNAs) are crucial players of DNA repair machinery in a cell and play a vital role in maintaining genomic stability, which is essential for its survival and normal functioning thus preventing tumorigenesis. To preserve the integrity of the genome, cells initiate a specific cel...
Source: Mol Biol Cell - November 2, 2017 Category: Molecular Biology Authors: Arjumand W, Asiaf A, Ahmad ST Tags: Methods Mol Biol Source Type: research

MicroRNAs in Breast Cancer: Diagnostic and Therapeutic Potential.
Abstract MicroRNAs (miRNAs) are a large family of small, approximately 20-22 nucleotide, noncoding RNAs that regulate the expression of target genes, at the post-transcriptional level. miRNAs are involved in virtually diverse biological processes and play crucial roles in cellular processes, such as cell differentiation, proliferation, and apoptosis. Accumulating lines of evidence have indicated that miRNAs play important roles in the maintenance of biological homeostasis and that aberrant expression levels of miRNAs are associated with the onset of many diseases, including cancer. It is possible that the diverse ...
Source: Mol Biol Cell - November 2, 2017 Category: Molecular Biology Authors: Asiaf A, Ahmad ST, Arjumand W, Zargar MA Tags: Methods Mol Biol Source Type: research

Combination of Anti-miRNAs Oligonucleotides with Low Amounts of Chemotherapeutic Agents for Pancreatic Cancer Therapy.
Abstract Pancreatic ductal adenocarcinoma (PDAC) is the most predominant type of pancreatic cancer and presents one of the highest mortality rates when compared with other carcinomas. The absence of efficient treatment options for PDAC prompted us to investigate whether microRNA inhibition, combined or not with chemotherapeutic agents, would constitute a promising therapeutic approach for this disease. In this chapter, we describe several methods and procedures that can be used to evaluate the potential of new therapeutic strategies involving oligonucleotides against overexpressed microRNAs, in PDAC, either alone ...
Source: Mol Biol Cell - November 2, 2017 Category: Molecular Biology Authors: Passadouro M, Faneca H Tags: Methods Mol Biol Source Type: research

Angiogenesis Analysis by In Vitro Coculture Assays in Transwell Chambers in Ovarian Cancer.
e;nchez E, López-Camarillo C Abstract Angiogenesis is an important biological process in tumor growth and metastasis of tumor cells, and it has been associated with poor clinical outcomes in ovarian cancer. In vitro assays are useful tools for understanding the complex mechanisms of angiogenesis under a variety of conditions. Capillary-like formation and transwell migration assays are two of the most common techniques used in angiogenesis research. Here, we show an easy coculture model to study the role of microRNAs on angiogenesis that combines tube formation and cell migration assays. Recently, we reporte...
Source: Mol Biol Cell - November 2, 2017 Category: Molecular Biology Authors: Flores-Pérez A, Rincón DG, Ruiz-García E, Echavarria R, Marchat LA, Álvarez-Sánchez E, López-Camarillo C Tags: Methods Mol Biol Source Type: research

Construction of Multi-Potent MicroRNA Sponge and Its Functional Evaluation.
Abstract MicroRNA is a small, endogenous RNA that inhibits specific gene expression by interacting mostly on the UTR region of target mRNA. Among various methods to inhibit the microRNA, microRNA sponge is a construct designed to inhibit specific microRNA by providing excess amount of target mRNA. Here, we describe a method to generate multi-potent miRNA sponge which can inhibit multiple microRNAs simultaneously. In addition, two methods to examine the functionality of miRNA sponge will be introduced. This tool can be used to stably inhibit multiple microRNAs either in cell or in vivo, expanding the scope of funct...
Source: Mol Biol Cell - November 2, 2017 Category: Molecular Biology Authors: Chang S Tags: Methods Mol Biol Source Type: research

MicroRNA Sequencing Data Analysis Toolkits.
Abstract MicroRNA (miRNA) is a non-protein-coding small RNA molecule that negatively regulates gene expression by degradation of mRNA or suppression of mRNA translation. MiRNAs play important roles in biological processes such as cellular development, differentiation, proliferation, apoptosis and stem cell self-renewal and cancer development. The expression profile of microRNAs is tissue-, cell-type specific. PCR- and microarray-based arrays are the commonly used for differential expression of microRNAs between different diseased conditions. With the next-generation sequencing or massively parallel DNA sequencing ...
Source: Mol Biol Cell - November 2, 2017 Category: Molecular Biology Authors: Wu W Tags: Methods Mol Biol Source Type: research

Identification of Plant Nuclear Proteins Based on a Combination of Flow Sorting, SDS-PAGE, and LC-MS/MS Analysis.
ána J, Doležel J, Šebela M Abstract In the plant nucleus, the majority of cellular DNA content is stored and maintained. This makes this highly specialized organelle the major coordinator of almost all essential processes in plant cells such as transcription, DNA replication, and repair. None of these biological pathways can be fully understood without a comprehensive characterization of nuclear proteins. Nevertheless, the interest of the proteomic community in the plant nuclear proteome has been very limited so far. This is probably due to the high integrity of plant cell, presence of many interfer...
Source: Mol Biol Cell - November 2, 2017 Category: Molecular Biology Authors: Chamrád I, Uřinovská J, Petrovská B, Jeřábková H, Lenobel R, Vrána J, Doležel J, Šebela M Tags: Methods Mol Biol Source Type: research

Proteomic Analysis of Rice Golgi Membranes Isolated by Floating Through Discontinuous Sucrose Density Gradient.
Abstract The Golgi apparatus is an endomembrane system organelle and has roles in glycosylation, sorting, and secretion of proteins in the secretory pathway. It has a central function in living organism and is also essential for plant growth. Proteomic approaches to identify the Golgi membrane proteins have been performed in cell suspension cultures and many Golgi membrane-associated proteins were found, whereas it has well established in rice seedling yet. In this chapter, our recent improving published methods for isolated rice Golgi membranes by floating through a discontinuous sucrose density gradient are prov...
Source: Mol Biol Cell - November 2, 2017 Category: Molecular Biology Authors: Oikawa K, Inomata T, Hirao Y, Yamamoto T, Baslam M, Kaneko K, Mitsui T Tags: Methods Mol Biol Source Type: research

Analyzing the Vacuolar Membrane (Tonoplast) Proteome.
Abstract A large number of proteins in the vacuolar membrane (VM; tonoplast), including transporters and receptors, support the various functions of the vacuole. Molecular analysis of membrane proteins is an essential step in understanding how the vacuole operates but so far only a small number of tonoplast proteins have been identified at the molecular level. Accordingly, mutant lines with altered level of tonoplast proteins for characterizing their physiological roles have been developed sparsely. Also, detecting activities of tonoplast proteins remains difficult as it requires a certain degree of enrichment of ...
Source: Mol Biol Cell - November 2, 2017 Category: Molecular Biology Authors: Ohnishi M, Yoshida K, Mimura T Tags: Methods Mol Biol Source Type: research

Isolation of Intact Thylakoid Membranes from Heterocysts of Filamentous, Nitrogen-Fixing Cyanobacteria.
Abstract The isolation of thylakoid membranes, including intact membrane protein complexes, from heterocysts of filamentous cyanobacteria such as Nostoc punctiforme, is described. Protocols for BN-PAGE/SDS-PAGE 2-D electrophoresis are not included. However, the chapter ends with advisory notes on sample preparation for blue-native PAGE of thylakoid membrane proteins, which can then be used together with any standard protocol. PMID: 29086401 [PubMed - in process] (Source: Mol Biol Cell)
Source: Mol Biol Cell - November 2, 2017 Category: Molecular Biology Authors: Magnuson A, Cardona T Tags: Methods Mol Biol Source Type: research

Sample Preparation for Analysis of the Plant Mitochondrial Membrane Proteome.
Abstract Containing plastids and vacuoles in addition to those organelles also found in other (heterotrophic) cells, the plant cell displays an extraordinary level of compartmentalization, largely obtained by the utilization of membranes. These membranes not only confine reaction spaces but must also facilitate cross-talk between organelles and other cell compartments. They also host important components of the plant energy metabolism, i.e., the electron transport chains of mitochondria and chloroplasts. Characterization of the proteomes of these membranes requires isolation of pure and intact organelles from plan...
Source: Mol Biol Cell - November 2, 2017 Category: Molecular Biology Authors: Schikowsky C, Thal B, Braun HP, Eubel H Tags: Methods Mol Biol Source Type: research

Assay of Plasma Membrane H(+)-ATPase in Plant Tissues under Abiotic Stresses.
Abstract Plasma membrane (PM) H(+)-ATPase, which generates the proton gradient across the outer membrane of plant cells, plays a fundamental role in the regulation of many physiological processes fundamental for growth and development of plants. It is involved in the uptake of nutrients from external solutions, their loading into phloem and long-distance transport, stomata aperture and gas exchange, pH homeostasis in cytosol, cell wall loosening, and cell expansion. The crucial role of the enzyme in resistance of plants to abiotic and biotic stress factors has also been well documented. Such great diversity of phy...
Source: Mol Biol Cell - November 2, 2017 Category: Molecular Biology Authors: Janicka M, Wdowikowska A, Kłobus G Tags: Methods Mol Biol Source Type: research

Applications and Caveats on the Utilization of DNA-Specific Probes in Cell-Based Assays.
Abstract To perform cell-based assays using fluorescence as the readout there is a fundamental need to identify individual cellular objects. In the majority of cases this requires the addition of a DNA dye or so-called nuclear counterstain and these have become integral to assay design. End-point assays can use live or fixed cells and thus it is beneficial if such reagents are cell membrane-permeant.Further, membrane-permeant DNA dyes can open new opportunities in dynamic real time assays with caveats according to the impact of their interaction with the chromatin in live cells. As cell-based assays offer informat...
Source: Mol Biol Cell - October 31, 2017 Category: Molecular Biology Authors: Edward R Tags: Methods Mol Biol Source Type: research

General Staining and Segmentation Procedures for High Content Imaging and Analysis.
Abstract Automated quantitative fluorescence microscopy, also known as high content imaging (HCI), is a rapidly growing analytical approach in cell biology. Because automated image analysis relies heavily on robust demarcation of cells and subcellular regions, reliable methods for labeling cells is a critical component of the HCI workflow. Labeling of cells for image segmentation is typically performed with fluorescent probes that bind DNA for nuclear-based cell demarcation or with those which react with proteins for image analysis based on whole cell staining. These reagents, along with instrument and software se...
Source: Mol Biol Cell - October 31, 2017 Category: Molecular Biology Authors: Chambers KM, Mandavilli BS, Dolman NJ, Janes MS Tags: Methods Mol Biol Source Type: research