Unidirectional alteration of methylation and hydroxymethylation at the promoters and differential gene expression in oral squamous cell carcinoma.

Background: Oral squamous cell carcinoma (OSCC) is one of the most common types of cancer worldwide. Although overall losses of 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) have been previously observed, a genome-wide, single-base-resolution, and simultaneous mapping of 5mC and 5hmC in OSCC is still unaccomplished. Similarly, the mechanism of how 5mC and 5hmC collectively lead to abnormal gene expression in OSCC is largely unexplored. Using parallel whole-genome bisulfite sequencing (WGBS) and whole-genome oxidative bisulfite sequencing (oxWGBS), we characterized 5mC- and 5hmC-profiles at single-nucleotide resolution in paired primary OSCC samples and their normal adjacent tissues (NATs). We also analyzed the effect of 5mC- and 5hmC-modifications on differential gene expression in OSCC using multi-omics analysis. Results: An overall reduction of both 5mC and 5hmC in various genomic regions have been observed in OSCC samples. At promoter regions, a total of 6,921 differentially methylated regions and 1,024 differentially hydroxymethylated regions were identified in OSCC. Interestingly, compared to bidirectional modification with 5mC and 5hmC, unidirectional modification with 5mC and 5hmC at the promoters is associated with bigger change in the gene expression. Additionally, genes bearing unidirectional modification with 5mC and 5hmC at the promoters are enriched in signaling pathways like cell proliferation, cell differentiation, and receptor tyrosine kinase pathway that are essential for the tumorigenesis. Finally, the grouped expression signature of top 20 genes bearing promoter-unidirectional-modification with 5mC and 5hmC tends to correlate with the clinical outcome of certain subtypes of head and neck squamous cell carcinoma. Conclusion: Using parallel WGBS and oxWGBS analyses, we observed an overall reduction of 5mC- and 5hmC-modifications at various genomic regions in OSCC. Unidirectional modification with 5mC and 5hmC at the promoters is associated with enhanced changes in gene expression in OSCC tissues. Furthermore, such differentially expressed genes bearing unidirectional modifications with 5mC and 5hmC at the promoters might have clinical relevance to the outcome of OSCC.

Characterization of Mitochondrial DNA Methylation of Alzheimer's Disease in Plasma Cell-Free DNA.

Noninvasive diagnosis of Alzheimer's disease (AD) is important for patients. Significant differences in the methylation of mitochondrial DNA (mtDNA) were found in AD brain tissue. Cell-free DNA (cfDNA) is a noninvasive and economical diagnostic tool. We aimed to characterize mtDNA methylation alterations in the plasma cfDNA of 31 AD patients and 26 age- and sex-matched cognitively normal control subjects. We found that the mtDNA methylation patterns differed between AD patients and control subjects. The mtDNA was predominantly hypomethylated in the plasma cfDNA of AD patients. The hypomethylation sites or regions were mainly located in mt-rRNA, mt-tRNA, and D-Loop regions. The hypomethylation of the D-Loop region in plasma cfDNA of AD patients was consistent with that in previous studies. This study presents evidence that hypomethylation in the non-protein coding region of mtDNA may contribute to the pathogenesis of AD and potential application for the diagnosis of AD.

Integrative Multi-Omics Analysis Reveals Candidate Biomarkers for Oral Squamous Cell Carcinoma.

Oral squamous cell carcinoma (OSCC) is one of the most common types of cancer worldwide. Due to the lack of early detection and treatment, the survival rate of OSCC remains poor and the incidence of OSCC has not decreased during the past decades. To explore potential biomarkers and therapeutic targets for OSCC, we analyzed differentially expressed genes (DEGs) associated with OSCC using RNA sequencing technology. Methylation-regulated and differentially expressed genes (MeDEGs) of OSCC were further identified via an integrative approach by examining publicly available methylomic datasets together with our transcriptomic data. Protein-protein interaction (PPI) networks of MeDEGs were constructed and highly connected hub MeDEGs were identified from these PPI networks. Subsequently, expression and survival analyses of hub genes were performed using The Cancer Genome Atlas (TCGA) database and the Gene Expression Profiling Interactive Analysis (GEPIA) online tool. A total of 56 upregulated MeDEGs and 170 downregulated MeDEGs were identified in OSCC. Eleven hub genes with high degree of connectivity were picked out from the PPI networks constructed by those MeDEGs. Among them, the expression level of four hub genes (CTLA4, CDSN, ACTN2, and MYH11) were found to be significantly changed in the head and neck squamous carcinoma (HNSC) patients. Three hypomethylated hub genes (CTLA4, GPR29, and TNFSF11) and one hypermethylated hub gene (ISL1) were found to be significantly associated with overall survival (OS) of HNSC patients. Therefore, these hub genes may serve as potential DNA methylation biomarkers and therapeutic targets of OSCC.

Targeted Sequencing of Genomic Repeat Regions Detects Circulating Cell-free Echinococcus DNA.

BACKGROUND: Echinococcosis is a chronic zoonosis caused by tapeworms of the genus Echinococcus. Treatment of the disease is often expensive and complicated, sometimes requiring extensive surgery. Ultrasonographic imaging is currently the main technique for diagnosis, while immunological analysis provides additional information. Confirmation still needs pathological analysis. However, these diagnostic techniques generally detect infection in late stages of the disease. An accurate, early and non-invasive molecular diagnostic method is still unavailable. METHODOLOGY/PRINCIPAL FINDINGS: We sequenced the cell-free DNA (cfDNA) from plasma of echinococcosis patients and confirmed the presence of Echinococcus DNA. To improve detection sensitivity, we developed a method based on targeted next-generation sequencing of repeat regions. Simulation experiments demonstrate that the targeted sequencing is sensitive enough to detect as little as 0.1% of an Echinococcus genome in 1 mL of plasma. Results obtained using patient plasma shows that the Area Under the Curve (AUC) of the method is 0.862, with a detection sensitivity of 62.50% and specificity of 100%, corresponding to a Youden-index of 0.625. CONCLUSIONS/SIGNIFICANCE: This study provides evidence that hydatid cysts release cfDNA fragments into patient plasma. Using the repeat region targeted sequencing method, highly specific detection of Echinococcus infection was achieved. This study paves a new avenue for potential non-invasive screening and diagnosis of echinococcosis.

Mitochondrial DNA heteroplasmy rises in substantial nigra of aged PINK1 KO mice.

Mutations in PINK1 and Parkin result in early-onset autosomal recessive Parkinson's disease (PD). PINK1/Parkin pathway maintain mitochondrial function by mediating the clearance of damaged mitochondria. However, the role of PINK1/Parkin in maintaining the balance of mtDNA heteroplasmy is still unknown. Here, we isolated mitochondrial DNA (mtDNA) from cortex, striatum and substantia nigra of wildtype (WT), PINK1 knockout (PINK1 KO) and Parkin knockout (Parkin KO) mice to analyze mtDNA heteroplasmy induced by PINK1/Parkin deficiency or aging. Our results showed that the Single Nucleotide Variants (SNVs) of late-onset somatic variants mainly increased with aging. Conversely, the early-onset somatic variants exhibited significant increase in the cortex and substantia nigra of PINK1 KO mice than WT mice of the same age. Increased average variant allele frequency was observed in aged PINK1 KO mice and in substantial nigra of aged Parkin KO mice than in WT mice. Cumulative variant allele frequency in the substantia nigra of PINK1 KO mice was significantly higher than that in WT mice, further supporting the pivotal role of PINK1 in mtDNA maintenance. This study presented a new evidence for PINK1 and Parkin in participating in mitochondrial quality control and provided clues for further revealing the role of PINK1 and Parkin in the pathogenesis of PD.

Charge-Dependent Regulation in DNA Adsorption on 2D Clay Minerals.

DNA purification is essential for the detection of human clinical specimens. A non-destructive, controllable, and low reagent consuming DNA extraction method is described. Negatively charged DNA is absorbed onto a negatively charged montmorillonite to achieve non-destructive DNA extraction based on cation bridge construction and electric double layer formation. Different valence cation modified montmorillonite forms were used to validate the charge-dependent nature of DNA adsorption on montmorillonite. Electric double layer thickness thinning/thickening with the high/lower valence cations exists, and the minerals tended to be sedimentation-stable due to the Van der Waals attraction/electrostatic repulsion. Li-modified montmorillonite with the lowest charge states showed the best DNA adsorption efficiency of 8-10 ng/µg. Charge-dependent regulating research provides a new perspective for controllable DNA extraction and a deep analysis of interface engineering mechanisms.

ELMOD3, a novel causative gene, associated with human autosomal dominant nonsyndromic and progressive hearing loss.

Autosomal dominant nonsyndromic hearing loss (ADNSHL) is a highly genetically heterogeneous disorder. Up to date only approximately 37 ADNSHL-causing genes have been identified. The goal of this study was to determine the causative gene in a five-generation Chinese family with ADNSHL. A Chinese family was ascertained. Simultaneously, two affected individuals and one normal hearing control from the family were analyzed by whole exome capture sequencing. To assess the functional effect of the identified variant, in-vitro studies were performed. novel missense variant, c.512A>G (p.His171Arg) in exon 8 of the ELMO domain-containing 3 (ELMOD3) gene, was identified as a causative variant in this family affected by late-onset and progressive ADNSHL. The variant was validated by Sanger sequencing and found to co-segregate with the phenotype within the pedigree and was absent in 500 ethnically matched unrelated normal hearing control subjects. To our knowledge, this is the first report of a family with ADNSHL caused by ELMOD3 mutation. Western blots and immunofluorescence staining demonstrated that p.His171Arg resulted in abnormal expression levels of ELMOD3 and abnormal subcellular localization. Furthermore, the analysis of the stability of the wild-type (WT) and mutant ELMOD3 protein shows that the decay of p.His171Arg is faster than that of the WT, suggesting a shorter halflife of the c.512A > G variant. A novel variant in the ELMOD3 gene, encoding a member of the engulfment and cell motility (ELMO) family of GTPase-activating proteins, was identified for the first time as responsible for ADNSHL.

Effects of Commonly Used Pesticides in China on the Mitochondria and Ubiquitin-Proteasome System in Parkinson's Disease.

Evidence continues to accumulate that pesticides are the leading candidates of environmental toxins that may contribute to the pathogenesis of Parkinson's disease. The mechanisms, however, remain largely unclear. According to epidemiological studies, we selected nine representative pesticides (paraquat, rotenone, chlorpyrifos, pendimethalin, endosulfan, fenpyroximate, tebufenpyrad, trichlorphon and carbaryl) which are commonly used in China and detected the effects of the pesticides on mitochondria and ubiquitin-proteasome system (UPS) function. Our results reveal that all the nine studied pesticides induce morphological changes of mitochondria at low concentrations. Paraquat, rotenone, chlorpyrifos, pendimethalin, endosulfan, fenpyroximate and tebufenpyrad induced mitochondria fragmentation. Furthermore, some of them (paraquat, rotenone, chlorpyrifos, fenpyroximate and tebufenpyrad) caused a significant dose-dependent decrease of intracellular ATP. Interestingly, these pesticides which induce mitochondria dysfunction also inhibit 26S and 20S proteasome activity. However, two out of the nine pesticides, namely trichlorphon and carbaryl, were found not to cause mitochondrial fragmentation or functional damage, nor inhibit the activity of the proteasome, which provides significant guidance for selection of pesticides in China. Moreover, our results demonstrate a potential link between inhibition of mitochondria and the UPS, and pesticide-induced Parkinsonism.

BNIP3 Protein Suppresses PINK1 Kinase Proteolytic Cleavage to Promote Mitophagy.

Mutations in PINK1 (PTEN-induced putative kinase 1) cause early onset familial Parkinson's disease (PD). PINK1 accumulates on the outer membrane of damaged mitochondria followed by recruiting parkin to promote mitophagy. Here, we demonstrate that BCL2/adenovirus E1B 19-kDa interacting protein 3 (BNIP3), a mitochondrial BH3-only protein, interacts with PINK1 to promote the accumulation of full-length PINK1 on the outer membrane of mitochondria, which facilitates parkin recruitment and PINK1/parkin-mediated mitophagy. Inactivation of BNIP3 in mammalian cells promotes PINK1 proteolytic processing and suppresses PINK1/parkin-mediated mitophagy. Hypoxia-induced BNIP3 expression results in increased expression of full-length PINK1 and mitophagy. Consistently, expression of BNIP3 in Drosophila suppresses muscle degeneration and the mitochondrial abnormality caused by PINK1 inactivation. Together, the results suggest that BNIP3 plays a vital role in regulating PINK1 mitochondrial outer membrane localization, the proteolytic process of PINK1 and PINK1/parkin-mediated mitophagy under physiological conditions. Functional up-regulation of BNIP3 may represent a novel therapeutic strategy to suppress the progression of PD.