scholarly journals Characterization of the tracrARN-DRARN genetic complex associated with the CRISPR-Cas9 system of the phytosymbiont Acholeplasma palmae: biotechnological interest

2021 ◽  
Vol 38 (4) ◽  
pp. 970-992
Author(s):  
Luis Moncayo ◽  
Alex Castro ◽  
Diego Arcos ◽  
Paulo Centanaro ◽  
Diego Vaca ◽  
...  
Keyword(s):  
Gene Editing ◽  
Genetic Manipulation ◽  
Plant Biotechnology ◽  
Data Bank ◽  
Molecular Tools ◽  
Biophysical Characterization ◽  
Guide Rna ◽  
Alignment Algorithms ◽  
Docking Power

The CRISPR-Cas9 technology used in plant biotechnology is based on the use of Cas9 endonucleases to generate precise cuts in the genome, and a duplex consisting of a trans-activating CRISPR RNA (tracrRNA) and a CRISPR RNA (DRRNA) which are precursors of guide RNA (sgRNA) commercially redesigned (sgRNA-Cas9) to guide gene cleavage. Most of these tools come from clinical bacteria. However, there are several CRISPR-Cas9 systems in environmental microorganisms such as phytoendosymbionts of plants of the genus Acholeplasma. But the exploitation of these systems more compatible with plants requires using bioinformatics tools for prediction and study. We identified and characterized the elements associated with the duplex in the genome of A. palmae. For this, the protein information was obtained from the Protein Data Bank and the genomics from GenBank/NCBI. The CRISPR system was studied with the CRISPRfinder software. Alignment algorithms and NUPACK software were used to identify the tracrRNA and DRRNA modules, together with various computational software for genetic, structural and biophysical characterization. A CRISPR-Cas system was found in A. palmae with type II-C characteristics, as well as a thermodynamically very stable duplex, with flexible regions, exhibiting a docking power with Cas9 thermodynamically favored. These results are desirable in programmable gene editing systems and show the possibility of exploring native molecular tools in environmental microorganisms applicable to the genetic manipulation of plants, as more research is carried out. This study represents the first report on the thermodynamic stability and molecular docking of elements associated with the tracrRNA-DRRNA duplex in the phytosymbiont A. palmae.

scholarly journals Rapid, efficient and activation-neutral gene editing of polyclonal primary human resting CD4+ T cells allows complex functional analyses

2021 ◽  
Author(s):  
Manuel Albanese ◽  
Adrian Ruhle ◽  
Jennifer Mittermaier ◽  
Ernesto Mejías-Pérez ◽  
Madeleine Gapp ◽  
...  
Keyword(s):  
T Cells ◽  
Gene Editing ◽  
Genetic Manipulation ◽  
Immune Functions ◽  
Hiv Replication ◽  
Guide Rna ◽  
Ribonucleoprotein Complexes ◽  
Neutral Gene ◽  
Endogenous Loci

AbstractCD4+ T cells are central mediators of adaptive and innate immune responses and constitute a major reservoir for human immunodeficiency virus (HIV) in vivo. Detailed investigations of resting human CD4+ T cells have been precluded by the absence of efficient approaches for genetic manipulation limiting our understanding of HIV replication and restricting efforts to find a cure. Here we report a method for rapid, efficient, activation-neutral gene editing of resting, polyclonal human CD4+ T cells using optimized cell cultivation and nucleofection conditions of Cas9–guide RNA ribonucleoprotein complexes. Up to six genes, including HIV dependency and restriction factors, were knocked out individually or simultaneously and functionally characterized. Moreover, we demonstrate the knock in of double-stranded DNA donor templates into different endogenous loci, enabling the study of the physiological interplay of cellular and viral components at single-cell resolution. Together, this technique allows improved molecular and functional characterizations of HIV biology and general immune functions in resting CD4+ T cells.

Purification and Biophysical Characterization of an 11S Globulin from Wrightia tinctoria Exhibiting Hemagglutinating Activity

2013 ◽  
Vol 20 (5) ◽  
pp. 499-509 ◽  
Author(s):  
Pramod Kumar ◽  
Dipak N. Patil ◽  
Anshul Chaudhary ◽  
Shailly Tomar ◽  
Dinesh Yernool ◽  
...  
Keyword(s):  
Hemagglutinating Activity ◽  
Biophysical Characterization ◽  
11S Globulin ◽  
Wrightia Tinctoria

Biophysical Characterization of Glycodendrimers As Nano-carriers for HIV Peptides

2013 ◽  
Vol 20 (31) ◽  
pp. 3935-3943 ◽  
Author(s):  
M. Ionov ◽  
K. Ciepluch ◽  
B. Moreno ◽  
D. Appelhans ◽  
J. Sanchez-Nieves ◽  
...  
Keyword(s):  
Biophysical Characterization

scholarly journals Remote Sensing Methods for the Biophysical Characterization of Protected Areas Globally: Challenges and Opportunities

2021 ◽  
Vol 10 (6) ◽  
pp. 384
Author(s):  
Javier Martínez-López ◽  
Bastian Bertzky ◽  
Simon Willcock ◽  
Marine Robuchon ◽  
María Almagro ◽  
...  
Keyword(s):  
Remote Sensing ◽  
Protected Areas ◽  
Large Scale ◽  
Anthropogenic Activities ◽  
Global Scale ◽  
Area Network ◽  
Biophysical Characterization ◽  
Challenges And Opportunities ◽  
Scale Characterization

Protected areas (PAs) are a key strategy to reverse global biodiversity declines, but they are under increasing pressure from anthropogenic activities and concomitant effects. Thus, the heterogeneous landscapes within PAs, containing a number of different habitats and ecosystem types, are in various degrees of disturbance. Characterizing habitats and ecosystems within the global protected area network requires large-scale monitoring over long time scales. This study reviews methods for the biophysical characterization of terrestrial PAs at a global scale by means of remote sensing (RS) and provides further recommendations. To this end, we first discuss the importance of taking into account the structural and functional attributes, as well as integrating a broad spectrum of variables, to account for the different ecosystem and habitat types within PAs, considering examples at local and regional scales. We then discuss potential variables, challenges and limitations of existing global environmental stratifications, as well as the biophysical characterization of PAs, and finally offer some recommendations. Computational and interoperability issues are also discussed, as well as the potential of cloud-based platforms linked to earth observations to support large-scale characterization of PAs. Using RS to characterize PAs globally is a crucial approach to help ensure sustainable development, but it requires further work before such studies are able to inform large-scale conservation actions. This study proposes 14 recommendations in order to improve existing initiatives to biophysically characterize PAs at a global scale.

scholarly journals Fluorescence Fluctuation Spectroscopy enables quantification of potassium channel subunit dynamics and stoichiometry

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Giulia Tedeschi ◽  
Lorenzo Scipioni ◽  
Maria Papanikolaou ◽  
Geoffrey W. Abbott ◽  
Michelle A. Digman
Keyword(s):  
Plasma Membrane ◽  
Protein Diffusion ◽  
Ion Diffusion ◽  
Fluorescence Fluctuation Spectroscopy ◽  
Biophysical Characterization ◽  
Kv Channels ◽  
Subunit Stoichiometry ◽  
Spatio Temporal ◽  
Voltage Sensing Domain

AbstractVoltage-gated potassium (Kv) channels are a family of membrane proteins that facilitate K+ ion diffusion across the plasma membrane, regulating both resting and action potentials. Kv channels comprise four pore-forming α subunits, each with a voltage sensing domain, and they are regulated by interaction with β subunits such as those belonging to the KCNE family. Here we conducted a comprehensive biophysical characterization of stoichiometry and protein diffusion across the plasma membrane of the epithelial KCNQ1-KCNE2 complex, combining total internal reflection fluorescence (TIRF) microscopy and a series of complementary Fluorescence Fluctuation Spectroscopy (FFS) techniques. Using this approach, we found that KCNQ1-KCNE2 has a predominant 4:4 stoichiometry, while non-bound KCNE2 subunits are mostly present as dimers in the plasma membrane. At the same time, we identified unique spatio-temporal diffusion modalities and nano-environment organization for each channel subunit. These findings improve our understanding of KCNQ1-KCNE2 channel function and suggest strategies for elucidating the subunit stoichiometry and forces directing localization and diffusion of ion channel complexes in general.

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