scholarly journals An approach for accelerated isolation of genetically manipulated cell clones with reduced clonal variability

2018 ◽  
Author(s):  
Natania Casden ◽  
Oded Behar
Keyword(s):  
Conditioned Medium ◽  
Signaling Cascades ◽  
High Dilution ◽  
High Density Culture ◽  
Cell Clones ◽  
Genetic Modifications ◽  
The Common ◽  
Clonal Variability ◽  
Genomic Editing ◽  
Genetically Manipulated

Genomic editing methods, such as the CRISPR/Cas9 system, are routinely used to study gene function in somatic cells. Due to the heterogeneity of mutations, it is necessary to purify cell clones grown in high dilution until colony formation, which can be a time consuming process. Here we tested a modified approach in which we seeded cells in high dilution, together with non-edited carrier cells. As a comparison, cells were also grown in high dilution with conditioned medium from a high density culture. When using carrier cells or conditioned medium, the formation of cell colonies is accelerated. Additionally, clones grown with carrier cells are more similar to the parental lines in terms of their tumorigenic properties. Surprisingly, key signaling cascades are very divergent between clones isolated from low density cultures, even with conditioned medium, in contrast to clones isolated with carrier cells. Thus, our study uncovers a significant limitation using the common approach of isolating cell clones following genetic modifications and suggests an alternative method that mitigates the problem of heterogeneity of gene expression between clones.

Magic of signs: a non-local interpretation of homeopathy

2000 ◽  
Vol 89 (03) ◽  
pp. 127-140 ◽  
Author(s):  
H Walach
Keyword(s):  
Clinical Research ◽  
Empirical Research ◽  
Informational Content ◽  
Self Healing ◽  
High Dilution ◽  
Working Hypothesis ◽  
Non Local ◽  
The Common ◽  
Homeopathic Remedies ◽  
Repetitive Pattern

AbstractAmong homeopaths the common idea about a working hypothesis for homeopathic effects seems to be that, during the potentization process, ‘information’ or ‘energy’ is being preserved or even enhanced in homeopathic remedies. The organism is said to be able to pick up this information, which in turn will stimulate the organism into a self-healing response. According to this view the decisive element of homeopathic therapy is the remedy which locally contains and conveys this information. I question this view for empirical and theoretical reasons. Empirical research has shown a repetitive pattern, in fundamental and clinical research alike: there are many anomalies in high-dilution research and clinical homeopathic trials which will set any observing researcher thinking. But no single paradigm has proved stable enough in order to produce repeatable results independent of the researcher. I conclude that the database is too weak and contradictory to substantiate a local interpretation of homeopathy, in which the remedy is endowed with causal-informational content irrespective of the circumstances. I propose a non-local interpretation to understand the anomalies along the lines of Jung's notion of synchronicity and make some predictions following this analysis.

scholarly journals Generating new FANCA-deficient HNSCC cell lines by genomic editing recapitulate the cellular phenotypes of Fanconi anemia

2020 ◽  
Author(s):  
Ricardo Errazquin ◽  
Esther Sieiro ◽  
Pilar Moreno ◽  
María José Ramirez ◽  
Corina Lorz ◽  
...  
Keyword(s):  
Cell Lines ◽  
Fanconi Anemia ◽  
Mutant Cell ◽  
Interstrand Crosslink ◽  
Cell Clones ◽  
Highly Sensitive ◽  
Chromosome Fragility ◽  
Genomic Editing ◽  
Cellular Phenotypes ◽  
Radio Chemotherapy

AbstractFanconi anemia (FA) patients have an exacerbated risk of head and neck squamous cell carcinoma (HNSCC). Treatment is challenging as FA patients display enhanced toxicity to standard treatments, including radio/chemotherapy. Therefore better therapies as well as new disease models are urgently needed. We have used CRISPR/Cas9 editing tools in order to interrupt the human FANCA gene by the generation of insertions/deletions (indels) in exon 4 in two cancer cell lines from sporadic HNSCC having no mutation in FA-genes: CAL27 and CAL33 cells. Our approach allowed efficient editing, subsequent purification of single-cell clones, and Sanger sequencing validation at the edited locus. Clones having frameshift indels in homozygosis did not express FANCA protein and were selected for further analysis. When compared with parental CAL27 and CAL33, FANCA-mutant cell clones displayed a FA-phenotype as they i) are highly sensitive to DNA interstrand crosslink (ICL) agents such as mitomycin C (MMC) or cisplatin, ii) do not monoubiquitinate FANCD2 upon MMC treatment and therefore iii) do not form FANCD2 nuclear foci, and iv) they display increased chromosome fragility and G2 arrest after diepoxybutane (DEB) treatment. These FANCA-mutant clones display similar growth rates as their parental cells. Interestingly, mutant cells acquire phenotypes associated with more aggressive disease, such as increased migration in wound healing assays. Therefore, CAL27 and CAL33 cells with FANCA mutations are phenocopies of FA-HNSCC cells.

Allospecific proliferative human T-cell clones acquire the cytotoxic effector function after three months in culture, in IL-2 conditioned medium

1986 ◽  
Vol 17 (1) ◽  
pp. 30-36 ◽  
Author(s):  
Li-Kuang Chen ◽  
Armand Bensussan ◽  
Gordons F. Burns ◽  
Béatrice Tourvieille ◽  
Annie Soulié ◽  
...  
Keyword(s):  
T Cell ◽  
Conditioned Medium ◽  
Effector Function ◽  
T Cell Clones ◽  
Human T Cell ◽  
Cell Clones ◽  
Cytotoxic Effector

Neuronal locus and cellular signaling for stimulation of ileal giant migrating and phasic contractions

2003 ◽  
Vol 284 (5) ◽  
pp. G789-G797 ◽  
Author(s):  
Sushil K. Sarna
Keyword(s):  
Ruthenium Red ◽  
Signaling Cascades ◽  
Receptor Subtype ◽  
Calcitonin Gene ◽  
Intracellular Ca ◽  
The Common ◽  
Higher Doses ◽  
Pkc Activation ◽  
Stimulation Of

We investigated the neuronal locus, the role of PKC activation, and utilization of extracellular Ca2+ and intracellular Ca2+ release in smooth muscle cells for the generation of giant migrating contractions (GMCs) and rhythmic phasic contractions (RPCs) in intact normal and inflamed canine ileum. Calcitonin gene-related peptide (CGRP), administered close intra-arterially, stimulated GMCs at higher doses and RPCs at smaller doses. These effects were blocked by prior close intra-arterial infusions of CGRP8–37, atropine, hexamethonium, and TTX but not by tachykinin, serotonin, and histaminergic receptor subtype antagonists. Both types of contractions were blocked by verapamil in normal and inflamed ileums. Dantrolene and ruthenium red blocked only the RPCs in normal ileum but blocked both GMCs and RPCs in the inflamed ileum. PKC inhibition by chelerythrine blocked GMCs only in inflamed ileum but blocked RPCs in both normal and inflamed ileums. The inhibition of phospholipase C by neomycin blocked both RPCs and GMCs in normal and inflamed ileums. In conclusion, acetylcholine is the common neurotransmitter for the stimulation of both GMCs and RPCs, but the signaling cascades for their stimulation are partially divergent, and they differ also in the normal and inflamed states.

scholarly journals Generating New FANCA-Deficient HNSCC Cell Lines by Genomic Editing Recapitulates the Cellular Phenotypes of Fanconi Anemia

Genes ◽  
2021 ◽  
Vol 12 (4) ◽  
pp. 548
Author(s):  
Ricardo Errazquin ◽  
Esther Sieiro ◽  
Pilar Moreno ◽  
María José Ramirez ◽  
Corina Lorz ◽  
...  
Keyword(s):  
Cell Lines ◽  
Fanconi Anemia ◽  
Mutant Cell ◽  
Interstrand Crosslink ◽  
Cell Clones ◽  
Highly Sensitive ◽  
Chromosome Fragility ◽  
Genomic Editing ◽  
Cellular Phenotypes ◽  
Radio Chemotherapy

Fanconi anemia (FA) patients have an exacerbated risk of head and neck squamous cell carcinoma (HNSCC). Treatment is challenging as FA patients display enhanced toxicity to standard treatments, including radio/chemotherapy. Therefore, better therapies as well as new disease models are urgently needed. We have used CRISPR/Cas9 editing tools in order to interrupt the human FANCA gene by the generation of insertions/deletions (indels) in exon 4 in two cancer cell lines from sporadic HNSCC having no mutation in FA-genes: CAL27 and CAL33 cells. Our approach allowed efficient editing, subsequent purification of single-cell clones, and Sanger sequencing validation at the edited locus. Clones having frameshift indels in homozygosis did not express FANCA protein and were selected for further analysis. When compared with parental CAL27 and CAL33, FANCA-mutant cell clones displayed a FA-phenotype as they (i) are highly sensitive to DNA interstrand crosslink (ICL) agents such as mitomycin C (MMC) or cisplatin, (ii) do not monoubiquitinate FANCD2 upon MMC treatment and therefore (iii) do not form FANCD2 nuclear foci, and (iv) they display increased chromosome fragility and G2 arrest after diepoxybutane (DEB) treatment. These FANCA-mutant clones display similar growth rates as their parental cells. Interestingly, mutant cells acquire phenotypes associated with more aggressive disease, such as increased migration in wound healing assays. Therefore, CAL27 and CAL33 cells with FANCA mutations are phenocopies of FA-HNSCC cells.

scholarly journals Glutathione in Brain Disorders and Aging

Molecules ◽  
2022 ◽  
Vol 27 (1) ◽  
pp. 324
Author(s):  
Igor Y. Iskusnykh ◽  
Anastasia A. Zakharova ◽  
Dhruba Pathak
Keyword(s):  
Neurological Disorders ◽  
Metabolic Pathways ◽  
Neurological Diseases ◽  
Signaling Cascades ◽  
Reduced Form ◽  
Brain Disorders ◽  
Cellular Homeostasis ◽  
Physiological Conditions ◽  
The Common ◽  
The Brain

Glutathione is a remarkably functional molecule with diverse features, which include being an antioxidant, a regulator of DNA synthesis and repair, a protector of thiol groups in proteins, a stabilizer of cell membranes, and a detoxifier of xenobiotics. Glutathione exists in two states—oxidized and reduced. Under normal physiological conditions of cellular homeostasis, glutathione remains primarily in its reduced form. However, many metabolic pathways involve oxidization of glutathione, resulting in an imbalance in cellular homeostasis. Impairment of glutathione function in the brain is linked to loss of neurons during the aging process or as the result of neurological diseases such as Huntington’s disease, Parkinson’s disease, stroke, and Alzheimer’s disease. The exact mechanisms through which glutathione regulates brain metabolism are not well understood. In this review, we will highlight the common signaling cascades that regulate glutathione in neurons and glia, its functions as a neuronal regulator in homeostasis and metabolism, and finally a mechanistic recapitulation of glutathione signaling. Together, these will put glutathione’s role in normal aging and neurological disorders development into perspective.

scholarly journals Generating New FANCA-Deficient HNSCC Cell Lines by Genomic Editing Recapitulate the Cellular Phenotypes of Fanconi Anemia

2020 ◽  
Author(s):  
Ricardo Errazquin ◽  
Esther Sieiro ◽  
Pilar Moreno ◽  
María José Ramirez ◽  
Corina Lorz ◽  
...  
Keyword(s):  
Cell Lines ◽  
Fanconi Anemia ◽  
Mutant Cell ◽  
Interstrand Crosslink ◽  
Cell Clones ◽  
Highly Sensitive ◽  
Chromosome Fragility ◽  
Genomic Editing ◽  
Cellular Phenotypes ◽  
Radio Chemotherapy

Fanconi anemia (FA) patients have an exacerbated risk of head and neck squamous cell carcinoma (HNSCC). Treatment is challenging as FA patients display enhanced toxicity to standard treatments, including radio/chemotherapy. Therefore better therapies as well as new disease models are urgently needed. We have used CRISPR/Cas9 editing tools in order to interrupt the human FANCA gene by the generation of insertions/deletions (indels) in exon 4 in two cancer cell lines from sporadic HNSCC having no mutation in FA-genes: CAL27 and CAL33 cells. Our approach allowed efficient editing, subsequent purification of single-cell clones, and Sanger sequencing validation at the edited locus. Clones having frameshift indels in homozygosis did not express FANCA protein and were selected for further analysis. When compared with parental CAL27 and CAL33, FANCA-mutant cell clones displayed a FA-phenotype as they i) are highly sensitive to DNA interstrand crosslink (ICL) agents such as mitomycin C (MMC) or cisplatin, ii) do not monoubiquitinate FANCD2 upon MMC treatment and therefore iii) do not form FANCD2 nuclear foci, and iv) they display increased chromosome fragility and G2 arrest after diepoxybutane (DEB) treatment. These FANCA-mutant clones display similar growth rates as their parental cells. Interestingly, mutant cells acquire phenotypes associated with more aggressive disease, such as increased migration in wound healing assays. Therefore, CAL27 and CAL33 cells with FANCA mutations are phenocopies of FA-HNSCC cells.

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