scholarly journals A Sub-Clone of RAW264.7-Cells Form Osteoclast-Like Cells Capable of Bone Resorption Faster than Parental RAW264.7 through Increased De Novo Expression and Nuclear Translocation of NFATc1

2020 ◽  
Vol 21 (2) ◽  
pp. 538 ◽  
Author(s):  
Laia Mira-Pascual ◽  
Anh N. Tran ◽  
Göran Andersson ◽  
Tuomas Näreoja ◽  
Pernilla Lång
Keyword(s):  
Cell Line ◽  
Nuclear Translocation ◽  
De Novo ◽  
Cathepsin K ◽  
Raw264.7 Cells ◽  
Large Numbers ◽  
Single Cell Cloning ◽  
Before And After ◽  
Low Levels ◽  
Rankl Stimulation

The murine macrophage cell line RAW264.7 is extensively used as a progenitor to study osteoclast (OC) differentiation. RAW264.7 is a heterogeneous cell line, containing sub-clones with different abilities to form OCs. The aim of this study was to identify characteristics within the heterogeneous RAW264.7 cells that define sub-clones with an augmented ability to form bone-resorbing OCs (H9), as well as sub-clones representing non-OCs (J8). RAW264.7 sub-clones were isolated by single cell cloning. Selection was based on TRAP/cathepsin K expression in sub-clone cultures without added RANKL. Sub-clones before and after differentiation with RANKL were assayed for multiple OC-characteristics. Sub-clone H9 cells presented a higher expression of OC-markers in cultures without added RANKL compared to the parental RAW264.7. After 6 days of RANKL stimulation, sub-clone H9 cells had equal expression levels of OC-markers with RAW264.7 and formed OCs able to demineralize hydroxyapatite. However, sub-clone H9 cells displayed rapid differentiation of OC already at Day 2 compared to Day 4 from parental RAW264.7, and when cultured on plastic and on bone they were more efficient in resorption. This rapid differentiation was likely due to high initial expression/nuclear translocation of OC master transcription factor, NFATc1. In contrast to H9, J8 cells expressed initially very low levels of OC-markers, and they did not respond to RANKL-stimulation by developing OC-characteristics/OC-marker expression. Hence, H9 is an additional clone suitable for experimental setup requiring rapid differentiation of large numbers of OCs.

DNA methylation in 5-aza-2'-deoxycytidine-resistant variants of C3H 10T1/2 C18 cells

1984 ◽  
Vol 4 (10) ◽  
pp. 2098-2102
Author(s):  
E Flatau ◽  
F A Gonzales ◽  
L A Michalowsky ◽  
P A Jones
Keyword(s):  
Cell Line ◽  
Cytosine Methylation ◽  
De Novo ◽  
Lethal Dose ◽  
Cytotoxic Effects ◽  
Drug Treatments ◽  
A Cell ◽  
Mouse Cells ◽  
Low Levels ◽  
Dna Cytosine Methylation

A cell line (T17) was derived from C3H 10T1/2 C18 cells after 17 treatments with increasing concentrations of 5-aza-2'-deoxycytidine. The T17 cell line was very resistant to the cytotoxic effects of 5-aza-2'-deoxycytidine, and the 50% lethal dose for 5-aza-2'-deoxycytidine was ca. 3 microM, which was 30-fold greater than that of the parental C3H 10T1/2 C18 cells. Increased drug resistance was not due to a failure of the T17 cell line to incorporate 5-aza-2'-deoxycytidine into DNA. The cells were also slightly cross-resistant to 5-azacytidine. The percentage of cytosines modified to 5-methylcytosine in T17 cells was 0.7%, a 78% decrease from the level of 3.22% in C3H 10T1/2 C18 cells. The DNA cytosine methylation levels in several clones isolated from the treated lines were on the order of 0.7%, and clones with methylation levels lower than 0.45% were not obtained even after further drug treatments. These highly decreased methylation levels appeared to be unstable, and DNA modification increased as the cells divided in the absence of further drug treatment. The results suggest that it may not be possible to derive mouse cells with vanishingly low levels of 5-methylcytosine and that considerable de novo methylation can occur in cultured lines.

X-Linked Clonality Analysis of Human Embryonic Stem Cells ( hESC ) Before and after induction of Hematopoietic Differentiation

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 4784-4784
Author(s):  
Maria Teresa Mitjavila-Garcia ◽  
Frank Yates ◽  
Marie-Laure Bonnet ◽  
Claire Rougeulle ◽  
Melanie Makhlouf ◽  
...  
Keyword(s):  
Cell Line ◽  
Cell Lines ◽  
Clonal Analysis ◽  
Hematopoietic Differentiation ◽  
Es Cell ◽  
Hematopoietic Progenitors ◽  
Liquid Cultures ◽  
Single Cell Cloning ◽  
Before And After ◽  
Clonality Analysis

Abstract Human ES cell lines represent an invaluable tool of research for the study of normal and pathological development. In this study we asked whether the human ES cell lines currently used for research purposes are derived from single outgrowths using X-linked clonality analysis. Amongst the cell lines tested, the female H9 cell line was informative at the Humara (human androgen receptor) locus by the presence of two CAG repeats allowing distinction of paternal (Xp) and maternal (Xm) X chromosomes. Clonal analysis was performed upon Hpa II digestion of DNA purified from total H9 cells (passages 27–61), single H9 cell clones generated from single cell cloning, single embryoid bodies (EB) (day 4, day 14) as well as from single hematopoietic progenitors. The same analysis was performed after induction of hematopoietic differentiation in liquid cultures in the presence of hematopoietic permissive conditions (co-culture in the presence of OP-9 cell line). Clonality was evaluated by calculating the Relative Corrected Index (RCI) which is the ratio of the intensity of peaks generated by Hpa II-digested and undigested DNA. In several experiments, all passages of H9 cell lines examined ( p27, p28, p31, p61) were found to exhibit a monoclonal pattern with disappearance of the same allele A upon Hpa II digestion (RCI > 10). 13 clonal cultures of H9 obtained by single cell cloning exhibited also the same clonal pattern with digestion of the same allele and RCI values between 6–14. Similarly, DNA purified from EB’s at different stages was found to be clonal. In DNA obtained from 75 individual hematopoietic progenitors (CFU-GM, CFU-G), clonal analysis revealed the same clonal pattern in each, with disappearance of the same allele A. However, clonal analysis performed in 10 hematopoietic colonies, revealed unexpectedly, an equal digestion of Humara alleles with RCI < 2 (polyclonal pattern). Finally, in two experiments, hematopoietic cells recovered from liquid cultures exhibited an absolute clonal pattern (Total disappearance of the same allele A). Interestingly, quantification of X-Inactivation Specific Transcript (XIST) RNA in H9 and day14 EB’s by RT-PCR did not show any evidence of XIST expression both before and after differentiation. Altogether, our data suggest that XCI has already been initiated in undifferentiated H9 cells and this inactivation is maintained, at least in the Humara locus in the absence of XIST. The monoclonal pattern of the cell line can be due to the fact that H9 ES cell line might have arisen from a limited number of stem cells having undergone XCI at a very early stage, with maintenance of the methylation status at this locus during mesodermal and hematopoietic commitment. Conversely, hematopoietic differentiation might occur from several mesodermal progenitors having inactivated the same X chromosome in H9 at the Humara locus. Despite the fact that hematopoietic progenitors and differentiated hematopoietic cells originating from H9 exhibited also a clonal pattern, a partial reactivation of the inactive X chromosome during hematopoietic differentiation could occur, and may explain the “polyclonal” pattern obtained in 10 CFU-GM analyzed. Overall, X-linked clonality testing could be an important issue before considering the potential clinical applications, in all female hES cell lines.

scholarly journals DNA methylation in 5-aza-2'-deoxycytidine-resistant variants of C3H 10T1/2 C18 cells.

1984 ◽  
Vol 4 (10) ◽  
pp. 2098-2102 ◽  
Author(s):  
E Flatau ◽  
F A Gonzales ◽  
L A Michalowsky ◽  
P A Jones
Keyword(s):  
Cell Line ◽  
Cytosine Methylation ◽  
De Novo ◽  
Lethal Dose ◽  
Cytotoxic Effects ◽  
Drug Treatments ◽  
A Cell ◽  
Mouse Cells ◽  
Low Levels ◽  
Dna Cytosine Methylation

A cell line (T17) was derived from C3H 10T1/2 C18 cells after 17 treatments with increasing concentrations of 5-aza-2'-deoxycytidine. The T17 cell line was very resistant to the cytotoxic effects of 5-aza-2'-deoxycytidine, and the 50% lethal dose for 5-aza-2'-deoxycytidine was ca. 3 microM, which was 30-fold greater than that of the parental C3H 10T1/2 C18 cells. Increased drug resistance was not due to a failure of the T17 cell line to incorporate 5-aza-2'-deoxycytidine into DNA. The cells were also slightly cross-resistant to 5-azacytidine. The percentage of cytosines modified to 5-methylcytosine in T17 cells was 0.7%, a 78% decrease from the level of 3.22% in C3H 10T1/2 C18 cells. The DNA cytosine methylation levels in several clones isolated from the treated lines were on the order of 0.7%, and clones with methylation levels lower than 0.45% were not obtained even after further drug treatments. These highly decreased methylation levels appeared to be unstable, and DNA modification increased as the cells divided in the absence of further drug treatment. The results suggest that it may not be possible to derive mouse cells with vanishingly low levels of 5-methylcytosine and that considerable de novo methylation can occur in cultured lines.

Antiparasitic Treatment of Patients with P. falciparum Malaria Reduces the Ability of Patient Serum to Induce Tissue Factor by Decreasing NF-κB Activation

1995 ◽  
Vol 73 (01) ◽  
pp. 039-048 ◽  
Author(s):  
A Bierhaus ◽  
Ch J Hemmer ◽  
N Mackman ◽  
R Kutob ◽  
R Ziegler ◽  
...  
Keyword(s):  
Tissue Factor ◽  
Falciparum Malaria ◽  
De Novo ◽  
Neutralizing Antibody ◽  
Mobility Shift ◽  
Before And After ◽  
Tf Gene ◽  
Electrophoretic Mobility Shift Assays ◽  
Antiparasitic Treatment ◽  
Stimulation Of

SummarySerum from patients with P. falciparum malaria at day 1 (pretherapy) induces tissue factor (TF) in cultured endothelial cells. TF induction depends on de novo transcription as shown in Nuclear Run On assays. Electrophoretic mobility shift assays demonstrated binding of AP-1 and NF- κB/Rel proteins to their recognition sites in the TF promotor. After therapy (day 28), stimulation of TF antigen by patient serum is reduced by 70%. When serum obtained before and after therapy was compared, a decrease of NF-κB activation was evident. Activation of NF-κB-like proteins was in part dependent on TNFα in patient serum, since a TNFα neutralizing antibody reduced induction of TF transcription and translation and induction of NF-κB-like proteins. Induction of TF activity was suppressed by pDTC, an inhibitor of NF-κB activation. When different promotor constructs of the TF gene were tested, induction was dependent upon the presence of the intact NF-κB-like binding site in the TF promotor. A mutant with deleted NF-κB, but intact AP-1 sites was not inducible. Mutation of the AP-1 sites did not prevent induction, but reduced inducibility by pretherapy serum. Therefore, NF-κB/Rel proteins are responsible for induction of TF transcription by pretherapy serum, but AP-1 is needed for highest inducibility. The effect of antiparasitic therapy on the induction of TF by serum from patients with complicated P. falciparum malaria is dependent on a therapy-mediated loss of activation of NF-κB-like proteins in post-treatment patient serum.

scholarly journals Comparative Analysis of SNP Discovery and Genotyping in Fagus sylvatica L. and Quercus robur L. Using RADseq, GBS, and ddRAD Methods

Forests ◽  
2021 ◽  
Vol 12 (2) ◽  
pp. 222
Author(s):  
Bartosz Ulaszewski ◽  
Joanna Meger ◽  
Jaroslaw Burczyk
Keyword(s):  
Population Genomics ◽  
De Novo ◽  
Genetic Studies ◽  
Genomic Libraries ◽  
Reduced Representation ◽  
Large Numbers ◽  
Broadleaved Tree Species ◽  
Fagus Sylvatica L ◽  
Reference Genomes ◽  
Future Population

Next-generation sequencing of reduced representation genomic libraries (RRL) is capable of providing large numbers of genetic markers for population genetic studies at relatively low costs. However, one major concern of these types of markers is the precision of genotyping, which is related to the common problem of missing data, which appears to be particularly important in association and genomic selection studies. We evaluated three RRL approaches (GBS, RADseq, ddRAD) and different SNP identification methods (de novo or based on a reference genome) to find the best solutions for future population genomics studies in two economically and ecologically important broadleaved tree species, namely F. sylvatica and Q. robur. We found that the use of ddRAD method coupled with SNP calling based on reference genomes provided the largest numbers of markers (28 k and 36 k for beech and oak, respectively), given standard filtering criteria. Using technical replicates of samples, we demonstrated that more than 80% of SNP loci should be considered as reliable markers in GBS and ddRAD, but not in RADseq data. According to the reference genomes’ annotations, more than 30% of the identified ddRAD loci appeared to be related to genes. Our findings provide a solid support for using ddRAD-based SNPs for future population genomics studies in beech and oak.

scholarly journals Lupeol Accumulation Correlates with Auxin in the Epidermis of Castor

Molecules ◽  
2021 ◽  
Vol 26 (10) ◽  
pp. 2978
Author(s):  
Donghai Li ◽  
Cheng Pan ◽  
Jianjun Lu ◽  
Wajid Zaman ◽  
Huayan Zhao ◽  
...  
Keyword(s):  
Auxin Transport ◽  
Biosynthesis Pathway ◽  
Comparative Transcriptome ◽  
Hormone Activity ◽  
Promoter Regions ◽  
Pentacyclic Triterpene ◽  
Large Numbers ◽  
Genes Encoding ◽  
Low Levels ◽  
Underlying Mechanisms

Lupeol, a natural lupane-type pentacyclic triterpene, possesses various pharmacological properties, and its production attracts attention. Significant quantities of lupeol are deposited on the castor aerial organ surface and are easily extractable as a predominant wax constituent. Thus, castor might be considered as a potential bioreactor for the production of lupeol. The lupeol biosynthesis pathway is well known, but how it is regulated remains largely unknown. Among large numbers of castor cultivars, we targeted one accession line (337) with high levels of lupeol on its stem surface and low levels thereof on its hypocotyl surface, implicating that lupeol synthesis is differentially regulated in the two organs. To explore the underlying mechanisms, we did comparative transcriptome analysis of the first internode of 337 stem and the upper hypocotyl. Our results show that large amounts of auxin-related genes are differentially expressed in both parts, implying some possible interactions between auxin and lupeol production. We also found that several auxin-responsive cis-elements are present in promoter regions of HMGR and LUS genes encoding two key enzymes involved in lupeol production. Furthermore, auxin treatments apparently induced the expression levels of RcHMGR and RcLUS. Furthermore, we observed that auxin treatment significantly increased lupeol contents, whereas inhibiting auxin transport led to an opposite phenotype. Our study reveals some relationships between hormone activity and lupeol synthesis and might provide a promising way for improving lupeol yields in castor.

Neurogenic urinary disorders in patients with tuberculous spondylitis before and after surgical treatment

2021 ◽  
Vol 11 (1) ◽  
pp. 27-32
Author(s):  
Aleksandr I. Gorbunov ◽  
Aleksandr N. Murav’ev ◽  
Evgenij G. Sokolovich ◽  
Petr K. Yablonsky
Keyword(s):  
Urinary Tract ◽  
Detrusor Overactivity ◽  
Lower Urinary Tract ◽  
De Novo ◽  
Spinal Tuberculosis ◽  
Neurological Status ◽  
Lower Urinary Tract Dysfunction ◽  
Detrusor Sphincter Dyssynergia ◽  
Before And After ◽  
Lower Urinary

ABSTRACT: Tuberculosis inflammation of vertebral column (spondylitis) can lead to neurogenic lower urinary tract dysfunction. There is lack of available publications for neurogenic lower urinary tract dysfunction in spinal tuberculosis. OBJECTIVE: To evaluate urodynamic disturbances in spinal tuberculosis before and after surgery for spondylitis. MATERIALS AND METHODS: We observed 19 patients with spinal tuberculosis, who had symptoms of micturitions impairment. 14 patients (73,6%) were male and 5 (26,4%) were female, average age was 43,7 7,9 years (2766). Control evaluation was performed after surgery on day 2128. RESULTS: Before surgery we found detrusor overactivity in 11 (57,9%) patients and 2 of those with detrusor overactivity had detrusor-sphincter dyssynergia. Detrusor hypo-/acontractility was diagnosed in 8 (42,1%). After surgery 5 patients (26,3%) exhibited improvement, in one case urodynamic disturbances were resolved. One patient developed detrusor overactivity and incontinence de novo and one patient had worsening neurological status, loss of sensitivity and acontractile bladder. CONCLUSION: Variable lower urinary tract dysfunction can be diagnosed in spinal tuberculosis. Only 26,3% of patients have improvement after surgery. New conditions or worsening of previous neurogenic lower urinary tract dysfunctions can be observed.

Strain-specific transgene methylation occurs early in mouse development and can be recapitulated in embryonic stem cells

Development ◽  
1995 ◽  
Vol 121 (9) ◽  
pp. 2853-2859 ◽  
Author(s):  
A. Weng ◽  
T. Magnuson ◽  
U. Storb
Keyword(s):  
De Novo ◽  
Es Cells ◽  
Embryonic Stem ◽  
Dependent Manner ◽  
Mouse Development ◽  
Partial Methylation ◽  
Distal Chromosome ◽  
Before And After ◽  
Endogenous Genes ◽  
De Novo Methylation

A murine transgene, HRD, is methylated only when carried in certain inbred strain backgrounds. A locus on distal chromosome 4, Ssm1 (strain-specific modifier), controls this phenomenon. In order to characterize the activity of Ssm1, we have investigated developmental acquisition of methylation over the transgene. Analysis of postimplantation embryos revealed that strain-specific methylation is initiated prior to embryonic day (E) 6.5. Strain-specific transgene methylation is all-or-none in pattern and occurs exclusively in the primitive ectoderm lineage. A strain-independent pattern of partial methylation occurs in the primitive endoderm and trophectoderm lineages. To examine earlier stages, embryonic stem (ES) cells were derived from E3.5 blastocysts and examined for transgene methylation before and after differentiation. Though the transgene had already acquired some methylation in undifferentiated ES cells, differentiation induced further, de novo methylation in a strain-dependent manner. Analysis of methylation in ES cultures suggests that the transgene and endogenous genes (such as immunoglobulin genes) are synchronously methylated during early development. These results are interpreted in the context of a model in which Ssm1-like modifier genes produce alterations in chromatin structure during and/or shortly after implantation, thereby marking target loci for de novo methylation with the rest of the genome during gastrulation.

Implementation of a Standardized Perioperative Pain Management Protocol to Reduce Opioid Prescriptions in Otolaryngologic Surgery

2022 ◽  
pp. 019459982110711
Author(s):  
Michael T. Chang ◽  
M. Lauren Lalakea ◽  
Kimberly Shepard ◽  
Micah Saste ◽  
Amanda Munoz ◽  
...  
Keyword(s):  
Pain Management ◽  
Postoperative Pain ◽  
Lower Amount ◽  
Opioid Use ◽  
Protocol Implementation ◽  
Perioperative Pain ◽  
Management Protocol ◽  
Before And After ◽  
Perioperative Pain Management ◽  
Low Levels

Objective To evaluate the efficacy of implementing a standardized multimodal perioperative pain management protocol in reducing opioid prescriptions following otolaryngologic surgery. Study Design Retrospective cohort study. Setting County hospital otolaryngology practice. Methods A perioperative pain management protocol was implemented in adults undergoing otolaryngologic surgery. This protocol included preoperative patient education and a postoperative multimodal pain regimen stratified by pain level: mild, intermediate, and high. Opioid prescriptions were compared between patient cohorts before and after protocol implementation. Patients in the pain protocol were surveyed regarding pain levels and opioid use. Results We analyzed 210 patients (105 preprotocol and 105 postprotocol). Mean ± SD morphine milligram equivalents (MMEs) prescribed decreased from 132.5 ± 117.8 to 53.6 ± 63.9 ( P < .05) following protocol implementation. Mean MMEs prescribed significantly decreased ( P < .05) for each procedure pain tier: mild (107.4 to 40.5), intermediate (112.8 to 48.1), and high (240.4 to 105.0). Mean MMEs prescribed significantly decreased ( P < .05) for each procedure type: endocrine (105.6 to 44.4), facial plastics (225.0 to 50.0), general (160.9 to 105.7), head and neck oncology (138.6 to 77.1), laryngology (53.8 to 12.5), otology (77.5 to 42.9), rhinology (142.2 to 44.4), and trauma (288.0 to 24.5). Protocol patients reported a mean 1-week postoperative pain score of 3.4, used opioids for a mean 3.1 days, and used only 39% of their prescribed opioids. Conclusion Preoperative counseling and standardization of a multimodal perioperative pain regimen for otolaryngology procedures can effectively lower amount of opioid prescriptions while maintaining low levels of postoperative pain.

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