scholarly journals Optimized Protocol for Micropropagation of Cadaman and Garnem Peach Rootstocks إستحداث بروتوکول للإکثار الدقيق لأصلي الخوخ الکادامان و الجارنم

2021 ◽  
Vol 12 (7) ◽  
pp. 731-735
Author(s):  
Doaa Abou Elyazid ◽  
M. Gawish ◽  
G. Eliwa
Keyword(s):  
Optimized Protocol ◽  
Peach Rootstocks

scholarly journals Optimized protocol for a quantitative SARS-CoV-2 duplex RT-qPCR assay with internal human sample sufficiency control

2021 ◽  
pp. 114174
Author(s):  
Aileen G. Rowan ◽  
Philippa May ◽  
Anjna Badhan ◽  
Carolina Herrera ◽  
Patricia Watber ◽  
...  
Keyword(s):  
Qpcr Assay ◽  
Optimized Protocol ◽  
Human Sample

scholarly journals An optimized protocol for isolation of S-nitrosylated proteins from C. elegans

2021 ◽  
Vol 2 (2) ◽  
pp. 100547
Author(s):  
Puneet Seth ◽  
Richard T. Premont ◽  
Jonathan S. Stamler
Keyword(s):  
C Elegans ◽  
Optimized Protocol

scholarly journals An optimized protocol for acquiring and processing cryo-EM data of human 26S proteasome with M1-Ub6

2021 ◽  
Vol 2 (1) ◽  
pp. 100278
Author(s):  
Xiang Chen ◽  
Dan Shi ◽  
Ping Zhang ◽  
Kylie J. Walters
Keyword(s):  
26S Proteasome ◽  
Optimized Protocol

scholarly journals Analysis of Polyhydroxyalkanoates Granules in Haloferax mediterranei by Double-Fluorescence Staining with Nile Red and SYBR Green by Confocal Fluorescence Microscopy

Polymers ◽  
2021 ◽  
Vol 13 (10) ◽  
pp. 1582
Author(s):  
Verónica Cánovas ◽  
Salvador Garcia-Chumillas ◽  
Fuensanta Monzó ◽  
Lorena Simó-Cabrera ◽  
Carmen Fernández-Ayuso ◽  
...  
Keyword(s):  
Fluorescence Microscopy ◽  
Culture Media ◽  
Nile Red ◽  
Sybr Green ◽  
Confocal Fluorescence Microscopy ◽  
Fluorescence Staining ◽  
Haloferax Mediterranei ◽  
Optimized Protocol ◽  
Confocal Fluorescence ◽  
Pha Production

Haloferaxmediterranei is a haloarchaeon of high interest in biotechnology because it produces and mobilizes intracellular polyhydroxyalkanoate (PHA) granules during growth under stress conditions (limitation of phosphorous in the culture media), among other interesting metabolites (enzymes, carotenoids, etc.). The capability of PHA production by microbes can be monitored with the use of staining-based methods. However, the staining of haloarchaea cells is a challenging task; firstly, due to the high ionic strength of the medium, which is inappropriate for most of dyes, and secondly, due to the low permeability of the haloarchaea S-layer to macromolecules. In this work, Haloferax mediterranei is used as a halophilic archaeon model to describe an optimized protocol for the visualization and analysis of intracellular PHA granules in living cells. The method is based on double-fluorescence staining using Nile red and SYBR Green by confocal fluorescence microscopy. Thanks to this method, the capability of PHA production by new haloarchaea isolates could be easily monitored.

scholarly journals Humans and other commonly used model organisms are resistant to cycloheximide-mediated biases in ribosome profiling experiments

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Puneet Sharma ◽  
Jie Wu ◽  
Benedikt S. Nilges ◽  
Sebastian A. Leidel
Keyword(s):  
Human Cells ◽  
Ribosome Profiling ◽  
Model Organisms ◽  
Treatment Conditions ◽  
Genome Wide ◽  
Optimized Protocol ◽  
Gene Level ◽  
Translation Inhibitor ◽  
Species Specific ◽  
Conformational Restrictions

AbstractRibosome profiling measures genome-wide translation dynamics at sub-codon resolution. Cycloheximide (CHX), a widely used translation inhibitor to arrest ribosomes in these experiments, has been shown to induce biases in yeast, questioning its use. However, whether such biases are present in datasets of other organisms including humans is unknown. Here we compare different CHX-treatment conditions in human cells and yeast in parallel experiments using an optimized protocol. We find that human ribosomes are not susceptible to conformational restrictions by CHX, nor does it distort gene-level measurements of ribosome occupancy, measured decoding speed or the translational ramp. Furthermore, CHX-induced codon-specific biases on ribosome occupancy are not detectable in human cells or other model organisms. This shows that reported biases of CHX are species-specific and that CHX does not affect the outcome of ribosome profiling experiments in most settings. Our findings provide a solid framework to conduct and analyze ribosome profiling experiments.

scholarly journals HiChIP and Hi-C Protocol Optimized for Primary Murine T Cells

2021 ◽  
Vol 4 (3) ◽  
pp. 49
Author(s):  
Tomas Zelenka ◽  
Charalampos Spilianakis
Keyword(s):  
T Cells ◽  
Long Range ◽  
Three Dimensional ◽  
Cell Types ◽  
Range Interaction ◽  
Chromatin Interactions ◽  
Optimized Protocol ◽  
Long Range Interaction ◽  
Chromatin Compactness ◽  
Detailed Protocol

The functional implications of the three-dimensional genome organization are becoming increasingly recognized. The Hi-C and HiChIP research approaches belong among the most popular choices for probing long-range chromatin interactions. A few methodical protocols have been published so far, yet their reproducibility and efficiency may vary. Most importantly, the high frequency of the dangling ends may dramatically affect the number of usable reads mapped to valid interaction pairs. Additionally, more obstacles arise from the chromatin compactness of certain investigated cell types, such as primary T cells, which due to their small and compact nuclei, impede limitations for their use in various genomic approaches. Here we systematically optimized all the major steps of the HiChIP protocol in T cells. As a result, we reduced the number of dangling ends to nearly zero and increased the proportion of long-range interaction pairs. Moreover, using three different mouse genotypes and multiple biological replicates, we demonstrated the high reproducibility of the optimized protocol. Although our primary goal was to optimize HiChIP, we also successfully applied the optimized steps to Hi-C, given their significant protocol overlap. Overall, we describe the rationale behind every optimization step, followed by a detailed protocol for both HiChIP and Hi-C experiments.

scholarly journals Optimization of a WGA-Free Molecular Tagging-Based NGS Protocol for CTCs Mutational Profiling

2020 ◽  
Vol 21 (12) ◽  
pp. 4364
Author(s):  
Giuseppa De Luca ◽  
Barbara Cardinali ◽  
Lucia Del Mastro ◽  
Sonia Lastraioli ◽  
Franca Carli ◽  
...  
Keyword(s):  
Substantial Reduction ◽  
Breast Cancer Patients ◽  
Dna Templates ◽  
Molecular Tagging ◽  
Mutational Profiling ◽  
Optimized Protocol ◽  
Set Up ◽  
Detection Of Mutations ◽  
Almost All ◽  
Next Generation Sequencing Ngs

Molecular characterization of Circulating Tumor Cells (CTCs) is still challenging, despite attempts to minimize the drawbacks of Whole Genome Amplification (WGA). In this paper, we propose a Next-Generation Sequencing (NGS) optimized protocol based on molecular tagging technology, in order to detect CTCs mutations while skipping the WGA step. MDA-MB-231 and MCF-7 cell lines, as well as leukocytes, were sorted into pools (2–5 cells) using a DEPArray™ system and were employed to set up the overall NGS procedure. A substantial reduction of reagent volume for the preparation of libraries was performed, in order to fit the limited DNA templates directly derived from cell lysates. Known variants in TP53, KRAS, and PIK3CA genes were detected in almost all the cell line pools (35/37 pools, 94.6%). No additional alterations, other than those which were expected, were found in all tested pools and no mutations were detected in leukocytes. The translational value of the optimized NGS workflow is confirmed by sequencing CTCs pools isolated from eight breast cancer patients and through the successful detection of variants. In conclusion, this study shows that the proposed NGS molecular tagging approach is technically feasible and, compared to traditional NGS approaches, has the advantage of filtering out the artifacts generated during library amplification, allowing for the reliable detection of mutations and, thus, making it highly promising for clinical use.

Optimized protocol of a frequency domain fluorescence lifetime imaging microscope for FRET measurements

2009 ◽  
Vol 72 (5) ◽  
pp. 371-379 ◽  
Author(s):  
Aymeric Leray ◽  
Franck B. Riquet ◽  
Elodie Richard ◽  
Corentin Spriet ◽  
Dave Trinel ◽  
...  
Keyword(s):  
Frequency Domain ◽  
Fluorescence Lifetime ◽  
Fluorescence Lifetime Imaging ◽  
Lifetime Imaging ◽  
Optimized Protocol

Relative Resistance of Four Peach Rootstocks to Phytophthora cactorum and Phytophthora megasperma

2001 ◽  
Vol 149 (10) ◽  
pp. 599-604 ◽  
Author(s):  
T. Thomidis ◽  
J. Cullum ◽  
K. Elena ◽  
S. N. Jeffers
Keyword(s):  
Phytophthora Cactorum ◽  
Relative Resistance ◽  
Phytophthora Megasperma ◽  
Peach Rootstocks
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