scholarly journals CRISPR/Cas9-mediated base-editing enables a chain reaction through sequential repair of sgRNA scaffold mutations

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Tsuyoshi Fukushima ◽  
Yosuke Tanaka ◽  
Keito Adachi ◽  
Nanami Masuyama ◽  
Akiho Tsuchiya ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Regulatory Networks ◽  
Chain Reaction ◽  
Reaction Systems ◽  
Chain Reactions ◽  
Base Editing ◽  
A Chain ◽  
Loxp Sequence

AbstractCell behavior is controlled by complex gene regulatory networks. Although studies have uncovered diverse roles of individual genes, it has been challenging to record or control sequential genetic events in living cells. In this study, we designed two cellular chain reaction systems that enable sequential sgRNA activation in mammalian cells using a nickase Cas9 tethering of a cytosine nucleotide deaminase (nCas9-CDA). In these systems, thymidine (T)-to-cytosine (C) substitutions in the scaffold region of the sgRNA or the TATA box-containing loxP sequence (TATAloxP) are corrected by the nCas9-CDA, leading to activation of the next sgRNA. These reactions can occur multiple times, resulting in cellular chain reactions. As a proof of concept, we established a chain reaction by repairing sgRNA scaffold mutations in 293 T cells. Importantly, the results obtained in yeast or in vitro did not match those obtained in mammalian cells, suggesting that in vivo chain reactions need to be optimized in appropriate cellular contexts. Our system may lay the foundation for building cellular chain reaction systems that have a broad utility in the future biomedical research.

scholarly journals CRISPR/Cas9-mediated Base-editing Enables a Chain Reaction Through Sequential Repair of sgRNA Scaffold Mutations

2021 ◽  
Author(s):  
Tsuyoshi Fukushima ◽  
Yosuke Tanaka ◽  
Keito Adachi ◽  
Nanami Masuyama ◽  
Shuhei Asada ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Regulatory Networks ◽  
Chain Reaction ◽  
Cellular Behavior ◽  
Reaction Systems ◽  
Chain Reactions ◽  
Base Editing ◽  
A Chain

Abstract Cellular behavior is governed by the complex gene regulatory networks. Although studies have revealed diverse roles of individual genes, it has been a challenge to record or control the sequential genetic events in living cells. In this study, we designed two cellular chain reaction systems that enable sequential sgRNA expression in mammalian cells using a nickase Cas9 tethering of a cytosine nucleotide deaminase (nCas9-CDA). In these systems, the thymidine (T)-to-cytosine (C) substitutions in the scaffold region of sgRNA or TATA box containing loxP sequence (TATAloxP) are corrected by the nCas9-CDA, which leads to expression of next sgRNA. These reactions can proceed several times, thus generating cellular chain reactions. As a proof of the concept, we established a chain reaction through the repair of sgRNA scaffold mutations in 293T cells. Importantly, the results obtained in yeast or in vitro were not consistent with those in mammalian cells, suggesting that the in vivo chain reactions need to be optimized in appropriate cellular contexts. Our system may lay the foundation for building cellular chain reaction systems that have a broad utility in the future biomedical research.

scholarly journals Mapping transcription factor occupancy using minimal numbers of cells in vitro and in vivo

2017 ◽  
Author(s):  
Luca Tosti ◽  
James Ashmore ◽  
Boon Siang Nicholas Tan ◽  
Benedetta Carbone ◽  
Tapan K Mistri ◽  
...  
Keyword(s):  
Stem Cells ◽  
Transcription Factor ◽  
Binding Sites ◽  
Mammalian Cells ◽  
Regulatory Networks ◽  
Embryonic Stem ◽  
Specific Cell ◽  
Dna Interactions

AbstractThe identification of transcription factor (TF) binding sites in the genome is critical to understanding gene regulatory networks (GRNs). While ChIP-seq is commonly used to identify TF targets, it requires specific ChIP-grade antibodies and high cell numbers, often limiting its applicability. DNA adenine methyltransferase identification (DamID), developed and widely used in Drosophila, is a distinct technology to investigate protein-DNA interactions. Unlike ChIP-seq, it does not require antibodies, precipitation steps or chemical protein-DNA crosslinking, but to date it has been seldom used in mammalian cells due to technical impediments. Here we describe an optimised DamID method coupled with next generation sequencing (DamID-seq) in mouse cells, and demonstrate the identification of the binding sites of two TFs, OCT4 and SOX2, in as few as 1,000 embryonic stem cells (ESCs) and neural stem cells (NSCs), respectively. Furthermore, we have applied this technique in vivo for the first time in mammals. Oct4 DamID-seq in the gastrulating mouse embryo at 7.5 days post coitum (dpc) successfully identified multiple Oct4 binding sites proximal to genes involved in embryo development, neural tube formation, mesoderm-cardiac tissue development, consistent with the pivotal role of this TF in post-implantation embryo. This technology paves the way to unprecedented investigations of TF-DNA interactions and GRNs in specific cell types with limited availability in mammals including in vivo samples.

scholarly journals The proteasome cap RPT5/Rpt5p subunit prevents aggregation of unfolded ricin A chain

2013 ◽  
Vol 453 (3) ◽  
pp. 435-445 ◽  
Author(s):  
Paola Pietroni ◽  
Nishi Vasisht ◽  
Jonathan P. Cook ◽  
David M. Roberts ◽  
J. Michael Lord ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Proteasome Inhibitors ◽  
Retrograde Transport ◽  
26S Proteasome ◽  
Atpase Subunit ◽  
Aaa Atpase ◽  
A Chain ◽  
Ricin A

The plant cytotoxin ricin enters mammalian cells by receptor-mediated endocytosis, undergoing retrograde transport to the ER (endoplasmic reticulum) where its catalytic A chain (RTA) is reductively separated from the holotoxin to enter the cytosol and inactivate ribosomes. The currently accepted model is that the bulk of ER-dislocated RTA is degraded by proteasomes. We show in the present study that the proteasome has a more complex role in ricin intoxication than previously recognized, that the previously reported increase in sensitivity of mammalian cells to ricin in the presence of proteasome inhibitors simply reflects toxicity of the inhibitors themselves, and that RTA is a very poor substrate for proteasomal degradation. Denatured RTA and casein compete for a binding site on the regulatory particle of the 26S proteasome, but their fates differ. Casein is degraded, but the mammalian 26S proteasome AAA (ATPase associated with various cellular activities)-ATPase subunit RPT5 acts as a chaperone that prevents aggregation of denatured RTA and stimulates recovery of catalytic RTA activity in vitro. Furthermore, in vivo, the ATPase activity of Rpt5p is required for maximal toxicity of RTA dislocated from the Saccharomyces cerevisiae ER. The results of the present study implicate RPT5/Rpt5p in the triage of substrates in which either activation (folding) or inactivation (degradation) pathways may be initiated.

scholarly journals Leucine 232 and hydrophobic residues at the ribosomal P stalk binding site are critical for biological activity of ricin

2019 ◽  
Vol 39 (10) ◽  
Author(s):  
Yijun Zhou ◽  
Xiao-Ping Li ◽  
Jennifer N. Kahn ◽  
John E. McLaughlin ◽  
Nilgun E. Tumer
Keyword(s):  
Biological Activity ◽  
Mammalian Cells ◽  
Electrostatic Interactions ◽  
Active Site Residues ◽  
Hydrophobic Pocket ◽  
Hydrophobic Residues ◽  
A Chain ◽  
Ribosomal P

Abstract Ricin interacts with the ribosomal P stalk to cleave a conserved adenine from the α-sarcin/ricin loop (SRL) of the rRNA. Ricin toxin A chain (RTA) uses Arg235 as the most critical arginine for binding to the P stalk through electrostatic interactions to facilitate depurination. Structural analysis showed that a P2 peptide binds to a hydrophobic pocket on RTA and the last two residues form hydrogen bonds with Arg235. The importance of hydrophobic residues relative to Arg235 in the interaction with the P stalk in vivo and on the toxicity of RTA is not known. Here, we mutated residues in the hydrophobic pocket to analyze their contribution to toxicity and depurination activity in yeast and in mammalian cells. We found that Leu232, Tyr183 and Phe240 contribute cumulatively to toxicity, with Leu232 being the most significant. A quadruple mutant, Y183A/L232A/R235A/F240A, which combined mutations in critical hydrophobic residues with R235A completely abolished the activity of RTA, indicating that Arg235 and hydrophobic residues are required for full biological activity. Y183A and F240A mutants had reduced activity on RNA, but higher activity on ribosomes compared with R235A in vitro, suggesting that they could partially regain activity upon interaction with ribosomes. These results expand the region of interaction between RTA and the P stalk critical for cellular activity to include the hydrophobic pocket and provide the first evidence that interaction of P stalk with the hydrophobic pocket promotes a conformational rearrangement of RTA to correctly position the active site residues for catalytic attack on the SRL.

Vacuoles Induced in Mammalian Cells by Helicobacter Cytotoxin are Acidic Organelles

1990 ◽  
Vol 48 (3) ◽  
pp. 168-169
Author(s):  
M. H. Chestnut ◽  
C. E. Catrenich
Keyword(s):  
Acridine Orange ◽  
Mammalian Cells ◽  
Laser Scanning ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Ulcer Disease ◽  
Gastroduodenal Disease ◽  
H Pylori

Helicobacter pylori is a non-invasive, Gram-negative spiral bacterium first identified in 1983, and subsequently implicated in the pathogenesis of gastroduodenal disease including gastritis and peptic ulcer disease. Cytotoxic activity, manifested by intracytoplasmic vacuolation of mammalian cells in vitro, was identified in 55% of H. pylori strains examined. The vacuoles increase in number and size during extended incubation, resulting in vacuolar and cellular degeneration after 24 h to 48 h. Vacuolation of gastric epithelial cells is also observed in vivo during infection by H. pylori. A high molecular weight, heat labile protein is believed to be responsible for vacuolation and to significantly contribute to the development of gastroduodenal disease in humans. The mechanism by which the cytotoxin exerts its effect is unknown, as is the intracellular origin of the vacuolar membrane and contents. Acridine orange is a membrane-permeant weak base that initially accumulates in low-pH compartments. We have used acridine orange accumulation in conjunction with confocal laser scanning microscopy of toxin-treated cells to begin probing the nature and origin of these vacuoles.

Cytological changes induced by platinum-thymine in sarcoma-180 cells: Electron microscopy study

1990 ◽  
Vol 48 (3) ◽  
pp. 290-291
Author(s):  
Gustav Ofosu
Keyword(s):  
Mammalian Cells ◽  
Antitumor Agent ◽  
Microscopy Study ◽  
Electron Microscopy Study ◽  
Limiting Factor ◽  
Sarcoma 180 ◽  
Electron Microscopic ◽  
Cytological Changes

Platinum-thymine has been found to be a potent antitumor agent, which is quite soluble in water, and lack nephrotoxicity as the dose-limiting factor. The drug has been shown to interact with DNA and inhibits DNA, RNA and protein synthesis in mammalian cells in vitro. This investigation was undertaken to elucidate the cytotoxic effects of piatinum-thymine on sarcoma-180 cells in vitro ultrastructurally, Sarcoma-180 tumor bearing mice were treated with intraperitoneal injection of platinum-thymine 40mg/kg. A concentration of 60μg/ml dose of platinum-thymine was used in in vitro experiments. Treatments were at varying time intervals of 3, 7 and 21 days for in vivo experiments, and 30, 60 and 120 min., 6, 12, and 24th in vitro. Controls were not treated with platinum-thymine.Electron microscopic analyses of the treated cells in vivo and in vitro showed drastic cytotoxic effect.

Programmable in vitro Co-Encapsidation of Guest Proteins Inside Protein Nanocages

2018 ◽  
Author(s):  
Noor H. Dashti ◽  
Rufika S. Abidin ◽  
Frank Sainsbury
Keyword(s):  
Self Assembly ◽  
Mammalian Cells ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Super Resolution ◽  
Cell Engineering ◽  
Particle Analysis ◽  
Protein Cages

Bioinspired self-sorting and self-assembling systems using engineered versions of natural protein cages have been developed for biocatalysis and therapeutic delivery. The packaging and intracellular delivery of guest proteins is of particular interest for both <i>in vitro</i> and <i>in vivo</i> cell engineering. However, there is a lack of platforms in bionanotechnology that combine programmable guest protein encapsidation with efficient intracellular uptake. We report a minimal peptide anchor for <i>in vivo</i> self-sorting of cargo-linked capsomeres of the Murine polyomavirus (MPyV) major coat protein that enables controlled encapsidation of guest proteins by <i>in vitro</i> self-assembly. Using Förster resonance energy transfer (FRET) we demonstrate the flexibility in this system to support co-encapsidation of multiple proteins. Complementing these ensemble measurements with single particle analysis by super-resolution microscopy shows that the stochastic nature of co-encapsidation is an overriding principle. This has implications for the design and deployment of both native and engineered self-sorting encapsulation systems and for the assembly of infectious virions. Taking advantage of the encoded affinity for sialic acids ubiquitously displayed on the surface of mammalian cells, we demonstrate the ability of self-assembled MPyV virus-like particles to mediate efficient delivery of guest proteins to the cytosol of primary human cells. This platform for programmable co-encapsidation and efficient cytosolic delivery of complementary biomolecules therefore has enormous potential in cell engineering.

The Role of nitro (NO2-), chloro (Cl), and fluoro (F) Substitution in the Design of Antileishmanial and Antichagasic Compounds

2020 ◽  
Vol 21 ◽  
Author(s):  
Boniface Pone ◽  
Ferreira Igne Elizabeth
Keyword(s):  
Chagas Disease ◽  
Mammalian Cells ◽  
Neglected Tropical Diseases ◽  
New Drugs ◽  
Pharmacokinetic Studies ◽  
Scientific Publications ◽  
Antitrypanosomal Activity ◽  
Chemical Groups

: Neglected tropical diseases (NTDs) are responsible for over 500,000 deaths annually and are characterized by multiple disabilities. Leishmaniasis and Chagas disease are among the most severe NTDs, and are caused by the Leishmania sp, and Trypanosoma cruzi, respectively. Glucantime, pentamidine and miltefosine are commonly used to treat leishmaniasis, whereas nifurtimox, benznidazole are current treatments for Chagas disease. However, these treatments are associated with drug resistance, and severe side effects. Hence, the development of synthetic products, especially those containing N02, F, or Cl, which chemical groups are known to improve the biological activity. The present work summarizes the information on the antileishmanial and antitrypanosomal activity of nitro-, chloro-, and fluoro-synthetic derivatives. Scientific publications referring to halogenated derivatives in relation to antileishmanial and antitrypanosomal activities were hand searched in databases such as SciFinder, Wiley, Science Direct, PubMed, ACS, Springer, Scielo, and so on. According to the literature information, more than 90 compounds were predicted as lead molecules with reference to their IC50/EC50 values in in vitro studies. It is worth to mention that only active compounds with known cytotoxic effects against mammalian cells were considered in the present study. The observed activity was attributed to the presence of nitro-, fluoro- and chloro-groups in the compound backbone. All in all, nitro and h0alogenated derivatives are active antileishmanial and antitrypanosomal compounds and can serve as baseline for the development of new drugs against leishmaniasis and Chagas disease. However, efforts on in vitro and in vivo toxicity studies of the active synthetic compounds is still needed. Pharmacokinetic studies, and the mechanism of action of the promising compounds need to be explored. The use of new catalysts and chemical transformation can afford unexplored halogenated compounds with improved antileishmanial and antitrypanosomal activity.

Discovery of 3-Cinnamamido-N-Substituted Benzamides as Potential Antimalarial Agents

2020 ◽  
Vol 16 ◽  
Author(s):  
Haicheng Liu ◽  
Yushi Futamura ◽  
Honghai Wu ◽  
Aki Ishiyama ◽  
Taotao Zhang ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Antimalarial Activity ◽  
In Vitro Assays ◽  
Compound Library ◽  
Antimalarial Agents ◽  
Phenotypic Screen ◽  
Ic50 Values ◽  
Substituted Benzamides

Background: Malaria is one of the most devastating parasitic diseases, yet the discovery of antimalarial agents remains profoundly challenging. Very few new antimalarials have been developed in the past 50 years, while the emergence of drug-resistance continues to appear. Objective: This study focuses on the discovery, design, synthesis, and antimalarial evaluation of 3-cinnamamido-N-substituted benzamides. Method: In this study, a screening of our compound library was carried out against the multidrug-sensitive Plasmodium falciparum 3D7 strain. Derivatives of the hit were designed, synthesized and tested against P. falciparum 3D7 and the in vivo antimalarial activity of the most active compounds was evaluated using the method of Peters’ 4-day suppressive test. Results: The retrieved hit compound 1 containing a 3-cinnamamido-N-substituted benzamide skeleton showed moderate antimalarial activity (IC50 = 1.20 µM) for the first time. A series of derivatives were then synthesized through a simple four-step workflow, and half of them exhibited slightly better antimalarial effect than the precursor 1 during the subsequent in vitro assays. Additionally, compounds 11, 23, 30 and 31 displayed potent activity with IC50 values of approximately 0.1 µM, and weak cytotoxicity against mammalian cells. However, in vivo antimalarial activity is not effective which might be ascribed to the poor solubility of these compounds. Conclusion: In this study, phenotypic screen of our compound library resulted in the first report of 3-cinnamamide framework with antimalarial activity and 40 derivatives were then designed and synthesized. Subsequent structure-activity studies showed that compounds 11, 23, 30 and 31 exhibited the most potent and selective activity against P. falciparum 3D7 strain with IC50 values around 0.1 µM. Our work herein sets another example of phenotypic screen-based drug discovery, leading to potentially promising candidates of novel antimalarial agents once given further optimization.

scholarly journals In Silico Predicted Antifungal Peptides: In Vitro and In Vivo Anti-Candida Activity

2021 ◽  
Vol 7 (6) ◽  
pp. 439
Author(s):  
Tecla Ciociola ◽  
Walter Magliani ◽  
Tiziano De Simone ◽  
Thelma A. Pertinhez ◽  
Stefania Conti ◽  
...  
Keyword(s):  
In Silico ◽  
Mammalian Cells ◽  
Galleria Mellonella ◽  
Ex Vivo ◽  
Consensus Sequence ◽  
In Silico Analysis ◽  
Significant Activity ◽  
Synthetic Antibody

It has been previously demonstrated that synthetic antibody-derived peptides could exert a significant activity in vitro, ex vivo, and/or in vivo against microorganisms and viruses, as well as immunomodulatory effects through the activation of immune cells. Based on the sequence of previously described antibody-derived peptides with recognized antifungal activity, an in silico analysis was conducted to identify novel antifungal candidates. The present study analyzed the candidacidal and structural properties of in silico designed peptides (ISDPs) derived by amino acid substitutions of the parent peptide KKVTMTCSAS. ISDPs proved to be more active in vitro than the parent peptide and all proved to be therapeutic in Galleria mellonella candidal infection, without showing toxic effects on mammalian cells. ISDPs were studied by circular dichroism spectroscopy, demonstrating different structural organization. These results allowed to validate a consensus sequence for the parent peptide KKVTMTCSAS that may be useful in the development of novel antimicrobial molecules.

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