scholarly journals Epigenetics Identifier screens reveal regulators of chromatin acylation and limited specificity of acylation antibodies

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Leonie Kollenstart ◽  
Sophie C. van der Horst ◽  
Kees Vreeken ◽  
George M. C. Janssen ◽  
Fabrizio Martino ◽  
...  
Keyword(s):  
Histone Acetylation ◽  
Posttranslational Modifications ◽  
Chromatin Immunoprecipitation ◽  
Cross Reactivity ◽  
Factors Affecting ◽  
Histone Lysine ◽  
Yeast Mutants ◽  
In Cells

AbstractThe collection of known posttranslational modifications (PTMs) has expanded rapidly with the identification of various non-acetyl histone lysine acylations, such as crotonylation, succinylation and butyrylation, yet their regulation is still not fully understood. Through an unbiased chromatin immunoprecipitation (ChIP)-based approach called Epigenetics-IDentifier (Epi-ID), we aimed to identify regulators of crotonylation, succinylation and butyrylation in thousands of yeast mutants simultaneously. However, highly correlative results led us to further investigate the specificity of the pan-K-acyl antibodies used in our Epi-ID studies. This revealed cross-reactivity and lack of specificity of pan-K-acyl antibodies in various assays. Our findings suggest that the antibodies might recognize histone acetylation in vivo, in addition to histone acylation, due to the vast overabundance of acetylation compared to other acylation modifications in cells. Consequently, our Epi-ID screen mostly identified factors affecting histone acetylation, including known (e.g. GCN5, HDA1, and HDA2) and unanticipated (MET7, MTF1, CLB3, and RAD26) factors, expanding the repertoire of acetylation regulators. Antibody-independent follow-up experiments on the Gcn5-Ada2-Ada3 (ADA) complex revealed that, in addition to acetylation and crotonylation, ADA has the ability to butyrylate histones. Thus, our Epi-ID screens revealed limits of using pan-K-acyl antibodies in epigenetics research, expanded the repertoire of regulators of histone acetylation, and attributed butyrylation activity to the ADA complex.

scholarly journals lncRNA MIR22HG-Derived miR-22-5p Enhances the Radiosensitivity of Hepatocellular Carcinoma by Increasing Histone Acetylation Through the Inhibition of HDAC2 Activity

2021 ◽  
Vol 11 ◽  
Author(s):  
Qiao Jin ◽  
Hao Hu ◽  
Siqi Yan ◽  
Long Jin ◽  
Yuliang Pan ◽  
...  
Keyword(s):  
Hepatocellular Carcinoma ◽  
Histone Acetylation ◽  
Chromatin Immunoprecipitation ◽  
Promoter Region ◽  
Animal Experiments ◽  
Primary Hepatocellular Carcinoma ◽  
Acetylation Level ◽  
Expression Levels

BackgroundWith the development of radiotherapy technology, radiotherapy has been increasingly used to treat primary hepatocellular carcinoma (HCC). However, due to radioresistance and the intolerance of the adjacent organs to radiation, the effects of radiotherapy are often unsatisfactory. Therefore, it is necessary to study radiosensitization in HCC.MethodA microarray was used to analyze the genes that were significantly associated with radiosensitivity. HCC cells, HepG2 and MHCC97H, were subjected to radiation in vitro. Real-time PCR was performed to determine MIR22HG (microRNA22 host gene) and miR-22-5p expression levels. Western blotting was performed to determine histone expression levels. A histone deacetylase (HDAC) whole cell assay was used to determine the activity of HDAC2. MTT, colony formation, 5-ethynyl-2′-deoxyuridine, and wound healing assays were performed to examine the function of MIR22HG and miR-22-5p in cellular radiosensitivity. Chromatin immunoprecipitation-PCR was used to confirm that HDAC2 affects the acetylation level of the MIR22HG promoter region. Finally, animal experiments were performed to demonstrate the in vivo effect of MIR22HG on the radiosensitivity of hepatoma.ResultsIrradiation can up-regulate MIR22HG expression and down-regulate HDAC2 expression. Inhibition of HDAC2 expression promotes histone acetylation in the MIR22HG promoter region and up-regulates MIR22HG expression. MIR22HG can increase radiosensitivity via miR-22-5p in HCC.ConclusionInhibition of HDAC2 expression promotes histone acetylation in the MIR22HG promoter region, thereby up-regulating the expression of MIR22HG and promoting the production of miR-22-5p, and ultimately increasing the sensitivity of liver cancer radiotherapy.

Anti-CEA and Other Antibodies in the Study of Gastrointestinal Tumors

1992 ◽  
Vol 7 (3) ◽  
pp. 198-202 ◽  
Author(s):  
S. Lastoria ◽  
C. Caracò ◽  
E. Vergara ◽  
L. Castelli ◽  
M. Salvatore
Keyword(s):  
Monoclonal Antibodies ◽  
Distant Metastases ◽  
Gastrointestinal Tumors ◽  
Primary Tumors ◽  
Factors Affecting ◽  
Normal Tissues ◽  
Antibody Uptake ◽  
The Impact

Localization of gastrointestinal tumors by means of labeled monoclonal antibodies is a new, sensitive and suitable technique currently used in several centers. Encouraging results have been documented with several monoclonal antibodies by different authors. This article reviews our experience with radioimmunoscintigraphy in 59 patients with colorectal cancer in follow-up, using 131I and 111In labeled B72.3, and in 16 patients with primary gastrointestinal tumors using 99mTc anti-CEA monoclonal antibody (type F023C5). The sensitivity of both B72.3 and anti-CEA was greater than 70% either for primary tumors and abdominal recurrences or distant metastases except hepatic ones. A significant gradient in antibody uptake was measured on surgical biopsies between tumors and normal tissues allowing a good in vivo contrast for gamma detection. We have defined the impact of some factors affecting in vivo tumor targeting. In fact, pharmacodynamics of MAbs, percentage of injected dose bound to tissues were measured, and in particular antigenic content in tumor nodules was quantified. Furthermore, the results of RIS were compared to those obtained by CT and other imaging modalities.

scholarly journals Transcription of Genes in the Biosynthetic Pathway for Fumonisin Mycotoxins Is Epigenetically and Differentially Regulated in the Fungal Maize Pathogen Fusarium verticillioides

2011 ◽  
Vol 11 (3) ◽  
pp. 252-259 ◽  
Author(s):  
I. Visentin ◽  
V. Montis ◽  
K. Döll ◽  
C. Alabouvette ◽  
G. Tamietti ◽  
...  
Keyword(s):  
Histone Acetylation ◽  
Posttranslational Modifications ◽  
Chromatin Immunoprecipitation ◽  
Fusarium Verticillioides ◽  
Animal Health ◽  
Regulation Of Transcription ◽  
Promoter Regions ◽  
Content Type ◽  
Deacetylase Inhibitor ◽  
Class B

ABSTRACT When the fungal pathogen Gibberella moniliformis (anamorph, Fusarium verticillioides ) colonizes maize and maize-based products, it produces class B fumonisin (FB) mycotoxins, which are a significant threat to human and animal health. FB biosynthetic enzymes and accessory proteins are encoded by a set of clustered and cotranscribed genes collectively named FUM, whose molecular regulation is beginning to be unraveled by researchers. FB accumulation correlates with the amount of transcripts from the key FUM genes, FUM1 , FUM21 , and FUM8 . In fungi in general, gene expression is often partially controlled at the chromatin level in secondary metabolism; when this is the case, the deacetylation and acetylation (and other posttranslational modifications) of histones are usually crucial in the regulation of transcription. To assess whether epigenetic factors regulate the FB pathway, we monitored FB production and FUM1 , FUM21 , and FUM8 expression in the presence of a histone deacetylase inhibitor and verified by chromatin immunoprecipitation the relative degree of histone acetylation in the promoter regions of FUM1 , FUM21 , and FUM8 under FB-inducing and noninducing conditions. Moreover, we generated transgenic F. verticillioides strains expressing GFP under the control of the FUM1 promoter to determine whether its strength under FB-inducing and noninducing conditions was influenced by its location in the genome. Our results indicate a clear and differential role for chromatin remodeling in the regulation of FUM genes. This epigenetic regulation can be attained through the modulation of histone acetylation at the level of the promoter regions of the key biosynthetic genes FUM1 and FUM21 , but less so for FUM8 .

scholarly journals Small Molecule Inhibitors of the Human Histone Lysine Methyltransferase NSD2 / WHSC1 / MMSET Identified from a Quantitative High-Throughput Screen with Nucleosome Substrate

2017 ◽  
Author(s):  
Nathan P. Coussens ◽  
Stephen C. Kales ◽  
Mark J. Henderson ◽  
Olivia W. Lee ◽  
Kurumi Y. Horiuchi ◽  
...  
Keyword(s):  
High Throughput ◽  
High Throughput Screening ◽  
Point Mutations ◽  
Set Domain ◽  
Histone Lysine ◽  
Histone Lysine Methyltransferase ◽  
Attractive Therapeutic Target ◽  
Lysine Methyltransferase ◽  
In Cells

AbstractThe activity of the histone lysine methyltransferase NSD2 is thought to play a driving role in oncogenesis. Both overexpression of NSD2 and point mutations that increase its catalytic activity are associated with a variety of human cancers. While NSD2 is an attractive therapeutic target, no potent, selective and cell-active inhibitors have been reported to date, possibly due to the challenging nature of developing high-throughput assays for NSD2. To establish a platform for the discovery and development of selective NSD2 inhibitors, multiple assays were optimized and implemented. Quantitative high-throughput screening was performed with full-length wild-type NSD2 and a nucleosome substrate against a diverse collection of known bioactives comprising 16,251 compounds. Actives from the primary screen were further interrogated with orthogonal and counter assays, as well as activity assays with the clinically relevant NSD2 mutants E1099K and T1150A. Five confirmed inhibitors were selected for follow-up, which included a radiolabeled validation assay, surface plasmon resonance studies, methyltransferase profiling, and histone methylation in cells. The identification of NSD2 inhibitors that bind the catalytic SET domain and demonstrate activity in cells validates the workflow, providing a template for identifying selective NSD2 inhibitors.

scholarly journals Endothelium Response to Iron Sucrose Nanoparticles in Static Versus Dynamic Culture Model

2021 ◽  
Author(s):  
Niusha Nikravesh ◽  
Liliane Diener ◽  
Savvina Chortarea ◽  
Alexandra Rippl ◽  
Peter Wick
Keyword(s):  
Endothelial Cells ◽  
Inflammatory Responses ◽  
Dynamic Condition ◽  
Biological Fluids ◽  
Static Condition ◽  
Factors Affecting ◽  
Noticeable Increase ◽  
In Cells

Abstract Nanomedicines are promising therapeutic compounds allowing the development of new treatment approaches. However, important factors affecting the behavior of nanoparticles in vivo cannot be simulated in conventional static models. Dynamic cell cultures, where cells are cultivated in the presence of shear stress, have the potential to bridge this gap by mimicking critical features of physiological conditions. Iron-carbohydrate nanoparticles are a dominant treatment for iron deficiency unresponsive to oral iron supplements. Compared to the data available from clinical studies, little is known about the interaction of these particles with endothelial cells. Our approach implements and compares a microchannel-based dynamic human endothelium model to the static culture to explore potential differences in cells response after exposure to iron nanoparticles. Differences in cellular uptake are observable using Prussian blue assay. There is a noticeable increase on VCAM-1 and ICAM-1 gene expression on endothelial cells activated by inflammatory responses, in cells exposed to nanoparticles under static condition. Results show that cytotoxicity caused by iron particles is significantly lower under dynamic condition compared to static cultures. We demonstrate that inclusion of dynamic flow and biological fluids are positive steps towards nanoparticle evaluation in a physiologically relevant in vitro model.

scholarly journals Hsp90α Recruited by Sp1 Is Important for Transcription of 12(S)-Lipoxygenase in A431 Cells

2005 ◽  
Vol 280 (43) ◽  
pp. 36283-36292 ◽  
Author(s):  
Jan-Jong Hung ◽  
Chih-Ying Wu ◽  
Pao-Chi Liao ◽  
Wen-Chang Chang
Keyword(s):  
Chromatin Immunoprecipitation ◽  
Target Genes ◽  
Specific Inhibitor ◽  
A431 Cells ◽  
Rich Region ◽  
Immunoprecipitation Assay ◽  
Protein Levels ◽  
In Cells

Sp1 is a basic transcriptional factor that binds to the GC-rich region in the promoter of the target gene. It is involved in transcription of numerous genes by recruiting transcriptional factors to the promoters of target genes. In this study, we found in vivo and in vitro that Hsp90α was recruited to the GC-rich region of the 12(S)-lipoxygenase promoter through interaction with Sp1 in A431 cells by employing DNA affinity immunoprecipitation assay and chromatin immunoprecipitation assay. When Hsp90α was inhibited by geldanamycin (GA, a specific inhibitor of the Hsp90 family) or by siRNA of Hsp90α (to block its activity or to knockdown protein levels), respectively, luciferase activity (driven by the 12(S)-lipoxygenase promoter) and both mRNA and protein levels of 12(S)-lipoxygenase were reduced significantly in cells. In addition, the effect of GA was abolished when the Sp1 binding sites of 12(S)-lipoxygenase were mutated in A431 cells. Interestingly, binding of Sp1 to the 12(S)-lipoxygenase promoter was also decreased upon GA treatment in cells. In conclusion, our results indicate that Sp1 interacts with Hsp90α to recruit it to the promoter of 12(S)-lipoxygenase and then to regulate gene transcription by modulating the binding ability of Sp1 to promoters.

scholarly journals Phage φ29 Proteins p1 and p17 Are Required for Efficient Binding of Architectural Protein p6 to Viral DNA In Vivo

2004 ◽  
Vol 186 (24) ◽  
pp. 8401-8406 ◽  
Author(s):  
Víctor González-Huici ◽  
Martín Alcorlo ◽  
Margarita Salas ◽  
José M. Hermoso
Keyword(s):  
Dna Replication ◽  
Chromatin Immunoprecipitation ◽  
Transcription Control ◽  
Injection Process ◽  
Initiation Of Dna Replication ◽  
Cell Membrane Protein ◽  
Control Protein ◽  
Architectural Protein ◽  
In Cells

ABSTRACT Bacteriophage φ29 protein p6 is a viral architectural protein, which binds along the whole linear φ29 DNA in vivo and is involved in initiation of DNA replication and transcription control. Protein p1 is a membrane-associated viral protein, proposed to attach the viral genome to the cell membrane. Protein p17 is involved in pulling φ29 DNA into the cell during the injection process. We have used chromatin immunoprecipitation and real-time PCR to analyze in vivo p6 binding to DNA in cells infected with φ29 sus1 or sus17 mutants; in both cases p6 binding is significantly decreased all along φ29 DNA. φ29 DNA is topologically constrained in vivo, and p6 binding is highly increased in the presence of novobiocin, a gyrase inhibitor that produces a loss of DNA negative superhelicity. Here we show that, in cells infected with φ29 sus1 or sus17 mutants, the increase of p6 binding by novobiocin is even higher than in cells containing p1 and p17, alleviating the p6 binding deficiency. Therefore, proteins p1 and p17 could be required to restrain the proper topology of φ29 DNA, which would explain the impaired DNA replication observed in cells infected with sus1 or sus17 mutants.

Effect of Some Metabolic Inhibitors on Vinblastine-Induced Ribosomal-Granular Material Complexes

1971 ◽  
Vol 29 ◽  
pp. 548-549
Author(s):  
Awtar Krishan ◽  
Dora Hsu
Keyword(s):  
Granular Material ◽  
Rna Synthesis ◽  
Culture Medium ◽  
Actinomycin D ◽  
Metabolic Inhibitors ◽  
Protein And Rna Synthesis ◽  
Apparent Reduction ◽  
Plant Alkaloids ◽  
In Cells

Cells exposed to antitumor plant alkaloids, vinblastine and vincristine sulfate have large proteinacious crystals and complexes of ribosomes, helical polyribosomes and electron-dense granular material (ribosomal complexes) in their cytoplasm, Binding of H3-colchicine by the in vivo crystals shows that they contain microtubular proteins. Association of ribosomal complexes with the crystals suggests that these structures may be interrelated.In the present study cultured human leukemic lymphoblasts (CCRF-CEM), were incubated with protein and RNA-synthesis inhibitors, p. fluorophenylalanine, puromycin, cycloheximide or actinomycin-D before the addition of crystal-inducing doses of vinblastine to the culture medium. None of these compounds could completely prevent the formation of the ribosomal complexes or the crystals. However, in cells pre-incubated with puromycin, cycloheximide, or actinomycin-D, a reduction in the number and size of the ribosomal complexes was seen. Large helical polyribosomes were absent in the ribosomal complexes of cells treated with puromycin, while in cells exposed to cycloheximide, there was an apparent reduction in the number of ribosomes associated with the ribosomal complexes (Fig. 2).

Factors Affecting the Use and Consequences of Management Accounting Practices in A Transitional Economy: The Case of Vietnam

2016 ◽  
pp. 54-73 ◽  
Author(s):  
Anh Doan Ngoc Phi
Keyword(s):  
Significant Influence ◽  
Quantitative Data ◽  
Structural Equation ◽  
Qualitative Data ◽  
Management Accounting ◽  
Transitional Economy ◽  
Equation Modeling ◽  
Enterprise Performance ◽  
Factors Affecting

This study seeks to help fill an important gap in the literature by investigating factors that have facilitated the use of management accounting practices (MAPs) in Vietnam - a transitional economy. Data were collected from 220 medium-to-large enterprises. Follow-up interviews were conducted with 20 accounting heads/vice heads to obtain further information and clarification. The quantitative data collected was analyzed using both descriptive and inferential statistics (including t-tests and structural equation modeling), while the qualitative data was used to shed further light on the various relationships described by the quantitative analysis. This paper reveals that both decentralization and competition have a positive, significant influence on the use of new MAPs except for the old ones. Consequently, the use of MAPs has a positive, significant influence on enterprise performance.

STRUCTURAL AND DYNAMIC FEATURES OF EAR-LY DISABILITY DUE TO MENTAL DISEASES

2020 ◽  
pp. 62-67
Author(s):  
Shmakova O.P.
Keyword(s):  
People With Disabilities ◽  
Child And Adolescent Psychiatry ◽  
Child Psychiatrist ◽  
Childhood And Adolescence ◽  
Disabled Children ◽  
Dynamic Features ◽  
Factors Affecting ◽  
Number Of Children ◽  
Number Of Patients

Prevention of disability is one of the most significant tasks of child and adolescent psychiatry. Obtaining data on the dynamics of the number of people with disabilities and the factors affecting this indicator seems to be one of the relevant aspects. Aim: to trace the dynamics of the number of children with disabili-ties and to assess the change in the structure of early disability over the past decades. Materials and Meth-ods. A comparative analysis of two cohorts of patients was carried out: 1st - patients born in 1990-1992. (1203 patients (men - 914, 76%; women - 289, 24%)) who applied to the district neuropsychiatric dispensa-ry for outpatient care in childhood and adolescence; II - children and adolescents born in 2005 - 2018 (602 patients (male - 410, 68%; female - 192, 32%), ob-served at the time of the study by a child psychiatrist in the neuropsychiatric dispensary. Research methods: clinical and psychopathological; follow-up; statisti-cal. Results. Comparison of the number and nosologi-cal distribution of disabled children in two cohorts showed that over the 15th year there has been a shift towards an increase in the proportion of disabled children among patients observed by child and ado-lescent psychiatrists. The increase in the number of children with disabilities was due to those suffering from childhood autism and other disorders of general development. There were no statistically significant differences in the number of people with disabilities who received benefits before the age of 7, as well as differences in gender ratios among disabled people in the two cohorts. Conclusion. Early disability is a mul-tifactorial phenomenon, prevalence, dynamics, the structure of which depends not only on clinical, but also on socio-administrative realities. Children with autism require increased attention, since there has been a multiple increase in the number of patients with this diagnosis.

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