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Authors: Ke Chen , Wojciech Stach , Leila Homaeian & Lukasz Kurgan 

Introduction

Recent research resulted in the development of several 1D protein structure descriptors. They provide an important alternative for analysis/prediction of the protein structure/function. Numerous computational methods that provide accurate prediction of these descriptors from the protein sequence were proposed; they include secondary structure (1-5), secondary structure content (6-9), structural class (10-17), fold type (18-25), relative solvent accessibility (26-32), contact order and number (33-37), and residue depth (38). Recent work shows that the tertiary structure can be recovered from three 1D descriptors (39).

We developed a server that integrates predictions of several related descriptors including structural class (17), fold type (23), and secondary structure content (9). The knowledge of these three descriptors was applied in various areas including tertiary structure prediction (40), identification of domai

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Authors: Kayoko Hayashihara , Susumu Uchiyama , Shouhei Kobayashi , Masanobu Yanagisawa , Sachihiro Matsunaga & Kiichi Fukui 

Introduction

Given that DNA on which genomic information is written exists as chromosomes in a cell, handling chromosomes in vitro as experimental materials can provide varieties of information throughout life sciences. Metaphase chromosomes are highly delicate under in vitro conditions, moreover, it has been difficult to prepare massive chromosomes as experimental materials. These inconvenient points have prevented researchers to use chromosomes as the materials for in vitro experiments, although numerous microscopic observations have been so far performed. There is a standard protocol to prepare mitotic metaphase chromosomes, i.e., PA method (1, 2, 5-7). However the chromosomes prepared by the method have been found to contain lots of contaminated proteins (4). Ordinary purification processes, i.e., the sucrose density gradient centrifugation (4) often or usually result in

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Authors: Gareth Butland , Mohan Babu , Jack Greenblatt & Andrew Emili 

Introduction

Physical and functional interactions define the molecular organization of the cell. Genetic interactions, or epistasis, tend to occur between gene products involved in parallel pathways or interlinked biological processes. High-throughput experimental systems to examine genetic interactions on a genome-wide scale have been devised for Saccharomyces cerevisiae, Schizosaccharomyces pombe, Caenorhabditis elegans, and Drosophila melanogaster, but have not been reported previously for prokaryotes. Here, we describe the development of a quantitative screening procedure for monitoring bacterial genetic interactions based on conjugation of Escherichia coli deletion or hypomorphic strains to create double mutants on a genome-wide scale. The patterns of synthetic sickness and synthetic lethality (aggravating genetic interactions) we observe for certain double mutant combinations provide information about functional rela

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Authors: Angelita Simonetti , Stefano Marzi , Alexander Myasnikov , Attilio Fabbretti , Marat Yusupov , Claudio Gualerzi & Bruno Klaholz 

Introduction

Multi-component macromolecular complexes such as ribosome complexes are prone to significant heterogeneity within in vitro reconstituted samples. This can make functional and structural studies difficult, especially in the case of transiently assembling complexes. Translation initiation in prokaryotes proceeds through the formation of transient initiation complexes of the ribosome with initiation factors IF1, IF2 and IF3. The formation of the 30S initiation complex (30SIC) is accomplished in the very early phase of translation initiation, before the actual protein synthesis starts. In order to investigate the structure of the complex comprising the 30S ribosomal subunit, mRNA, fMet-tRNAfMet, IF1 and GTP-bound IF2, we have purified the components and characterized the complex formation by filter-binding assays. This was done with the aim of obtaining

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Authors: Kimi Honma & Takahiro Ochiya 

Introduction

To date, multiplexing cell-based assay is essential for high-throughput screening of molecular targets. Measuring multiple parameters of a single sample increases consistency and decrease time and cost of assay. Functional assay of living cell is useful as a first step of multiplexing assay, because live-cell assay allows following second assay using cell lysate or stained cell. However, live-cell assay of drug resistant cells that are highly activated of drug efflux mechanisms is sometimes unstable or difficult; for example, the method of measuring colored formazan products by living cells does not show correlation between the amount of formazan and the cells numbers. To this end, more reliable method to allow live-cell assay is anticipated. We described here the protocol of live-cell luciferase assay as a first step of multiplexing cell-based assay of drug-resistant cells which expresses firefly luciferase. This method has several advantages; 1)

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Authors: Makoto Nishiyama , Kazunobu Togashi & Kyonsoo Hong 

Introduction

The small size, fragility and high motility of growth cones preclude sensitive detection of growth cone electrical activity, which is essential for an elucidation of the early signaling events, such as those triggered by extracellular signaling molecules (e.g., guidance molecules) (1), that govern growth cone migration. Growth cone electrical activity was first detected using voltage-sensitive dyes (VSD) (2,3), which visualized somatic Ca2+ spikes propagating along neurites to growth cones in cultured neuroblastoma cells (N1E-115). However, the low sensitivity of VSD (ΔF/F: 1% for 100 mV, ref. 4), allowed an assessment of only relatively large growth cone membrane potential changes (> 50 mV) caused by propagating spikes. Subsequently, conventional patch clamp techniques were employed to monitor action potentials and voltage-gated macroscopic or single channel currents in large growth cones (>30 μm in diameter) in cultured *He

scientificprotocols

Authors: Nicholas Sherman & Jay Fox 

Introduction

Examination of utilized phosphorylation sites in an expressed, tagged protein requires a robust method that allows for maximum sequence coverage and identification of even low stoichiometry sites. The following protocol uses two to four enzymes to achieve high sequence coverage. Each of these digestions is analyzed un-enriched (C18) and enriched by TiO chromatography. By using this strategy and the high dynamic range and mass accuracy of the LTQFT hybrid linear ion trap – FTICR, sites modified at least down to the 0.1% level can be determined.

Reagents

1. 10 mM DTT

• 50 mM iodoacetamide

scientificprotocols

Authors: James Elsner 

Introduction

Quantile regression extends ordinary least-squares regression to quantiles of the response variable. Ordinary regression is a model for the conditional mean, where the mean is conditional on the value of the explanatory variable. Likewise, quantile regression is a model for the conditional quantiles. For trend analysis the explanatory variable is time. Quantiles are points taken at regular intervals from the cumulative distribution function of a random variable. The quantiles mark a set of ordered data into equal-sized data subsets.

The software is downloaded from the internet and installed on a computer. A data set from the internet is imported into a software session. An exploratory plot of the data is created to visualize the trends. A quantile regression model is fit to the data to quantify the trends and determine their statistical significance.

Equipment

scientificprotocols

Authors: Koji Atarashi & Kenya Honda 

Introduction

The lamina propria (LP) CD11c+ antigen-presenting cells directly sample the luminal contents and direct CD4+ T cells to differentiate into TH1, TH2 or TH17 cells (refs 1,2). Mouse CD11c+ LP cells consist of more than three subsets distinguished by different expression of cell surface markers, such as CD11b, CD70, CX3CR1, and CD103; and it has been reported that each subset has a distinct mission (refs 3-7).

This protocol details a method to isolate LP CD11c+ cells with high yield, viability and purity (see also ref. 8).

Reagents

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Authors: Bronwen Connor, Erin Firmin, Christof Maucksch, Rui Liu, Rebecca Playne, Kathryn Jones & Mirella Dottori 

Abstract

Direct reprogramming offers a unique approach by which to generate mature neural lineages for the study and treatment of neurological diseases and neurodevelopmental disorders. However, few studies have directly generated neural stem/precursor cells (iNPs) from adult human fibroblasts that are capable of producing a wide range of mature neuronal phenotypes. Further, current reprogramming protocols require the use of viral-mediated gene delivery to induce the generation of iNPs from human fibroblasts. Here we describe a robust and efficient protocol to directly reprogram adult human fibroblasts to expandable iNPs using transient non-viral gene delivery in a feeder-free cell culture system. The two transfection factors required for direct conversion to iNP cells, SOX2 and PAX6, do not generate a pluripotent cell state, reducing the potential risk of tumor formation following tran