Project description:Background: Cell free DNA (cfDNA) in plasma has received increasing attention and has been studied in a broad range of clinical conditions implicating inflammation, cancer, and aging. However, few studies have focused on mitochondrial DNA (mtDNA) in the cell free form. This study characterized the size distribution and sequence characteristics of plasma cell free mtDNA (cf mtDNA) in humans.Methods and Results: We optimized DNA isolation and next-generation sequencing library preparation protocols to better retain short DNA fragments from plasma, and applied these optimized methods to plasma samples from patients with sepsis. After massive parallel sequencing, we verified that our methods can retain substantially shorter DNA fragments than the standard isolation method, resulting in an average of 11.5 fold increase in short DNA fragments yield (DNA < 100bp). We report that cf mtDNA in plasma is highly enriched in short-size cfDNA (30 ~ 60 bp), which is much shorter than the value previously reported (~140 bp). Motivated by this unique size distribution, we size-selected short cfDNA fragments from the sequencing library, which further increased the mtDNA recovery rate by an average of 10.4 fold. Using this approach we detected mixtures of different mtDNA sequences, termed heteroplasmy, in plasma from 3 patients. In one patient who previously received bone marrow transplantation, different minor allele frequencies were observed between plasma and white blood cells (WBC) at heteroplasmic mtDNA sites, consistent with mixed-tissue origin for plasma DNA.Conclusion: mtDNA in plasma exists as very short fragments that exhibit mtDNA heteroplasmy distribution differences from that found in a single organ/tissue. This study is the first report of genome wide identification of mtDNA heteroplasmy in human plasma. Our optimized method can be used to investigate the potential utility of cf mtDNA fragments and heteroplasmy as biomarkers in various diseases.
Project description:The physical properties of cell-free DNA fragments in plasma, such as fragment sizes and ends, have attracted much recent interest, leading to the emerging field of fragmentomics. This study attempted to further characterize the end structure of plasma DNA.
Project description:We discovered that the cell-free chromatin assembly system senses free DNA ends and mounts a DNA double-strand break response. DNA ends are first recognized by the Ku complex and later resected. The phosphorylation of H2A.V (homologous to gH2A.X) initiates at DNA breaks and spreads over ten thousands of base pairs of DNA within a few minutes. The phosphorylation of gH2A.V remains tightly associated with the damaged DNA in cis and does not transfer to intact DNA circles in the same reaction. Our descriptions of the damage-related proteome and phospho-proteome provide a rich resource for in-depth mechanistic analyses of the DNA chromosome break response in this model system.
Project description:Proteomic analysis of injured human peripheral nerves, particularly focusing on events occurring in the proximal and distal nerve ends, remains relatively underexplored. This study aimed to investigate the molecular patterns underlying a digital nerve injury, concentrating on differences in protein expression between the proximal and distal nerve ends. A total of 26 human injured digital nerve samples (24 men; 2 women; median age 47 [30-66] years), harvested during primary nerve repair within 48 hours post-injury from proximal and distal nerve ends, were analyzed using mass spectrometry. A total of 3914 proteins were identified, with 127 proteins showing significant differences in abundance between the proximal and the distal nerve ends. The downregulation of proteins in the distal nerve end was associated with synaptic transmission, autophagy, neurotransmitter regulation, cell adhesion and migration. Conversely, proteins upregulated in the distal nerve end were implicated in cellular stress response, neuromuscular junction stability and muscle contraction, neuronal excitability and neurotransmitter release, synaptic vesicle recycling and axon guidance and angiogenesis. Investigation of proteins, with functional annotations analysis, in proximal and the distal ends of human injured digital nerves, revealed dynamic cellular responses aimed at promoting tissue degeneration and restoration, while suppressing non-essential processes.
Project description:In the bacterium Escherichia coli, RecBCD coordinates repair of two ends at a DNA double-strand break, preventing aberrant chromosome amplification