Identification of Total Reversible Cysteine Oxidation in an Atherosclerosis Model Using a Modified Biotin Switch Assay

Oxidative stress due to the imbalance of reactive oxygen species (ROS) and the resulting reversible cysteine oxidation (Cys_OX) are involved in the early proatherogenic aspect of atherosclerosis. Given that the corresponding redox signaling pathways are still unclear, a modified biotin switch assay was developed to quantify the reversible Cys_OX in an atherosclerosis model established by using a monocytic cell line treated with platelet releasate. The accumulation of ROS was observed in the model system and validated in human primary monocytes. Through the application of the modified biotin switch assay, we obtained the first reversible Cys_OX proteome for this model. A total of 75 peptides, corresponding to 53 proteins, were quantified with oxidative modification. The bioinformatics analysis of these Cys_OX-containing proteins highlighted biological processes including glycolysis, cytoskeleton arrangement, and redox regulation. Moreover, the reversible oxidation of three glycolysis enzymes was observed using this method, and the regulation influence was verified by an enzyme activity assay. NADPH oxidase (NOX) inhibition treatment, in conjunction with the modified biotin switch method, was used to evaluate the global Cys_OX status. In conclusion, this versatile modified biotin switch assay provides an approach for the quantification of all reversible Cys_OX and for the study of redox signaling in atherosclerosis as well as in diseases in other biological systems

Legislative Documents

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Table_2_cpubi4 Is Essential for Development and Virulence in Chestnut Blight Fungus.XLSX

<p>Ubiquitination plays key roles in eukaryotic growth, stress adaptation, and metabolic regulation. In our previous work, ubiquitin was found to be secreted in the hypovirus-infected strain of Cryphonectria parasitica, a phytopathogenic filamentous fungus responsible for the chestnut blight. Here we report the functional and molecular characterization of a polyubiquitin gene, cpubi4, in C. parasitica. The expression of cpubi4 was upregulated by the infection of a hypovirus. Deletion of cpubi4 resulted in abnormal morphology, reduced sporulation, attenuation of virulence, and significant reduction in ubiquitination. A total of 378 sites in 236 proteins were identified to be significantly decreased in ubiquitination in the absence of cpubi4. Quantitative proteome analysis revealed that 285 in 4,776 identified proteins changed in abundance (1.5-fold, P < 0.05) in the cpubi4 null mutant, as compared with the wild-type strain.</p

Table_4_cpubi4 Is Essential for Development and Virulence in Chestnut Blight Fungus.XLSX

<p>Ubiquitination plays key roles in eukaryotic growth, stress adaptation, and metabolic regulation. In our previous work, ubiquitin was found to be secreted in the hypovirus-infected strain of Cryphonectria parasitica, a phytopathogenic filamentous fungus responsible for the chestnut blight. Here we report the functional and molecular characterization of a polyubiquitin gene, cpubi4, in C. parasitica. The expression of cpubi4 was upregulated by the infection of a hypovirus. Deletion of cpubi4 resulted in abnormal morphology, reduced sporulation, attenuation of virulence, and significant reduction in ubiquitination. A total of 378 sites in 236 proteins were identified to be significantly decreased in ubiquitination in the absence of cpubi4. Quantitative proteome analysis revealed that 285 in 4,776 identified proteins changed in abundance (1.5-fold, P < 0.05) in the cpubi4 null mutant, as compared with the wild-type strain.</p

Data_Sheet_1_cpubi4 Is Essential for Development and Virulence in Chestnut Blight Fungus.DOCX

<p>Ubiquitination plays key roles in eukaryotic growth, stress adaptation, and metabolic regulation. In our previous work, ubiquitin was found to be secreted in the hypovirus-infected strain of Cryphonectria parasitica, a phytopathogenic filamentous fungus responsible for the chestnut blight. Here we report the functional and molecular characterization of a polyubiquitin gene, cpubi4, in C. parasitica. The expression of cpubi4 was upregulated by the infection of a hypovirus. Deletion of cpubi4 resulted in abnormal morphology, reduced sporulation, attenuation of virulence, and significant reduction in ubiquitination. A total of 378 sites in 236 proteins were identified to be significantly decreased in ubiquitination in the absence of cpubi4. Quantitative proteome analysis revealed that 285 in 4,776 identified proteins changed in abundance (1.5-fold, P < 0.05) in the cpubi4 null mutant, as compared with the wild-type strain.</p

Table9_Prenatal genetic diagnosis associated with fetal ventricular septal defect: an assessment based on chromosomal microarray analysis and exome sequencing.DOCX

Objective: In the study, we investigated the genetic etiology of the ventricular septal defect (VSD) and comprehensively evaluated the diagnosis rate of prenatal chromosomal microarray analysis (CMA) and exome sequencing (ES) for VSD to provide evidence for genetic counseling.Methods: We carried out chromosomal microarray analysis (CMA) on 468 fetuses with VSD and exome sequencing (ES) on 51 fetuses.Results: In our cohort, 68 (14.5%) VSD fetuses received a genetic diagnosis, including 61 (13.03%, 61/468) cases with chromosomal abnormalities and seven (13.7%, 7/51) cases with gene sequence variants. The detection rate of total pathogenic and likely pathogenic gene variations in the non-isolated VSD group (61/335, 18.2%, 55 by QF-PCR/karyotype/CMA + 6 by ES) was significantly higher than that in the isolated VSD group (7/133, 5.3%, 6 by QF-PCR/karyotype/CMA + 1 by ES, p = 0.000). The most common copy number variation (CNV) was 22q11.2 microdeletion syndrome. Additionally, we found six previously unreported variants, which expanded the variation spectrum of VSD-related genes.Conclusion: In this study, CNVs and sequence variants were found in 13.03% and 13.7% of cases, respectively. ES can be recommended for fetuses with VSD without chromosome abnormalities and pathogenic CNVs, especially those that are combined with other ultrasound abnormalities.</p

Table7_Prenatal genetic diagnosis associated with fetal ventricular septal defect: an assessment based on chromosomal microarray analysis and exome sequencing.DOCX

Objective: In the study, we investigated the genetic etiology of the ventricular septal defect (VSD) and comprehensively evaluated the diagnosis rate of prenatal chromosomal microarray analysis (CMA) and exome sequencing (ES) for VSD to provide evidence for genetic counseling.Methods: We carried out chromosomal microarray analysis (CMA) on 468 fetuses with VSD and exome sequencing (ES) on 51 fetuses.Results: In our cohort, 68 (14.5%) VSD fetuses received a genetic diagnosis, including 61 (13.03%, 61/468) cases with chromosomal abnormalities and seven (13.7%, 7/51) cases with gene sequence variants. The detection rate of total pathogenic and likely pathogenic gene variations in the non-isolated VSD group (61/335, 18.2%, 55 by QF-PCR/karyotype/CMA + 6 by ES) was significantly higher than that in the isolated VSD group (7/133, 5.3%, 6 by QF-PCR/karyotype/CMA + 1 by ES, p = 0.000). The most common copy number variation (CNV) was 22q11.2 microdeletion syndrome. Additionally, we found six previously unreported variants, which expanded the variation spectrum of VSD-related genes.Conclusion: In this study, CNVs and sequence variants were found in 13.03% and 13.7% of cases, respectively. ES can be recommended for fetuses with VSD without chromosome abnormalities and pathogenic CNVs, especially those that are combined with other ultrasound abnormalities.</p

Table8_Prenatal genetic diagnosis associated with fetal ventricular septal defect: an assessment based on chromosomal microarray analysis and exome sequencing.DOCX

Objective: In the study, we investigated the genetic etiology of the ventricular septal defect (VSD) and comprehensively evaluated the diagnosis rate of prenatal chromosomal microarray analysis (CMA) and exome sequencing (ES) for VSD to provide evidence for genetic counseling.Methods: We carried out chromosomal microarray analysis (CMA) on 468 fetuses with VSD and exome sequencing (ES) on 51 fetuses.Results: In our cohort, 68 (14.5%) VSD fetuses received a genetic diagnosis, including 61 (13.03%, 61/468) cases with chromosomal abnormalities and seven (13.7%, 7/51) cases with gene sequence variants. The detection rate of total pathogenic and likely pathogenic gene variations in the non-isolated VSD group (61/335, 18.2%, 55 by QF-PCR/karyotype/CMA + 6 by ES) was significantly higher than that in the isolated VSD group (7/133, 5.3%, 6 by QF-PCR/karyotype/CMA + 1 by ES, p = 0.000). The most common copy number variation (CNV) was 22q11.2 microdeletion syndrome. Additionally, we found six previously unreported variants, which expanded the variation spectrum of VSD-related genes.Conclusion: In this study, CNVs and sequence variants were found in 13.03% and 13.7% of cases, respectively. ES can be recommended for fetuses with VSD without chromosome abnormalities and pathogenic CNVs, especially those that are combined with other ultrasound abnormalities.</p

Image_4_Comparative Methylome Analysis Reveals Perturbation of Host Epigenome in Chestnut Blight Fungus by a Hypovirus.JPEG

<p>In eukaryotic genomes, DNA methylation is an important type of epigenetic modification that plays crucial roles in many biological processes. To investigate the impact of a hypovirus infection on the methylome of Cryphonectria parasitica, the chestnut blight fungus, whole-genome bisulfite sequencing (WGBS) was employed to generate single-base resolution methylomes of the fungus with/without hypovirus infection. The results showed that hypovirus infection alters methylation in all three contexts (CG, CHG, and CHH), especially in gene promoters. A total of 600 differentially methylated regions (DMRs) were identified, of which 144 could be annotated to functional genes. RNA-seq analysis revealed that DNA methylation in promoter is negatively correlated with gene expression. Among DMRs, four genes were shown to be involved in conidiation, orange pigment production, and virulence. Taken together, our DNA methylomes analysis provide valuable insights into the understanding of the relationship between DNA methylation and hypovirus infection, as well as phenotypic traits in C. parasitica.</p

Image_3_Comparative Methylome Analysis Reveals Perturbation of Host Epigenome in Chestnut Blight Fungus by a Hypovirus.JPEG

<p>In eukaryotic genomes, DNA methylation is an important type of epigenetic modification that plays crucial roles in many biological processes. To investigate the impact of a hypovirus infection on the methylome of Cryphonectria parasitica, the chestnut blight fungus, whole-genome bisulfite sequencing (WGBS) was employed to generate single-base resolution methylomes of the fungus with/without hypovirus infection. The results showed that hypovirus infection alters methylation in all three contexts (CG, CHG, and CHH), especially in gene promoters. A total of 600 differentially methylated regions (DMRs) were identified, of which 144 could be annotated to functional genes. RNA-seq analysis revealed that DNA methylation in promoter is negatively correlated with gene expression. Among DMRs, four genes were shown to be involved in conidiation, orange pigment production, and virulence. Taken together, our DNA methylomes analysis provide valuable insights into the understanding of the relationship between DNA methylation and hypovirus infection, as well as phenotypic traits in C. parasitica.</p