Non-Random mtDNA Segregation Patterns Indicate a Metastable Heteroplasmic Segregation Unit in m.3243A>G Cybrid Cells

Many pathogenic mitochondrial DNA mutations are heteroplasmic, with a mixture of mutated and wild-type mtDNA present within individual cells. The severity and extent of the clinical phenotype is largely due to the distribution of mutated molecules between cells in different tissues, but mechanisms underpinning segregation are not fully understood. To facilitate mtDNA segregation studies we developed assays that measure m.3243A>G point mutation loads directly in hundreds of individual cells to determine the mechanisms of segregation over time. In the first study of this size, we observed a number of discrete shifts in cellular heteroplasmy between periods of stable heteroplasmy. The observed patterns could not be parsimoniously explained by random mitotic drift of individual mtDNAs. Instead, a genetically metastable, heteroplasmic mtDNA segregation unit provides the likely explanation, where stable heteroplasmy is maintained through the faithful replication of segregating units with a fixed wild-type/m.3243A>G mutant ratio, and shifts occur through the temporary disruption and re-organization of the segregation units. While the nature of the physical equivalent of the segregation unit remains uncertain, the factors regulating its organization are of major importance for the pathogenesis of mtDNA diseases

A Research Agenda for Hybrid Intelligence:Augmenting Human Intellect With Collaborative, Adaptive, Responsible, and Explainable Artificial Intelligence

We define hybrid intelligence (HI) as the combination of human and machine intelligence, augmenting human intellect and capabilities instead of replacing them and achieving goals that were unreachable by either humans or machines. HI is an important new research focus for artificial intelligence, and we set a research agenda for HI by formulating four challenges

A frameshift polymorphism in P2X5 elicits an allogeneic cytotoxic T lymphocyte response associated with remission of chronic myeloid leukemia

Minor histocompatibility antigens (mHAgs) constitute the targets of the graft-versus-leukemia response after HLA-identical allogeneic stem cell transplantation. Here, we have used genetic linkage analysis to identify a novel mHAg, designated lymphoid-restricted histocompatibility antigen–1 (LRH-1), which is encoded by the P2X5 gene and elicited an allogeneic CTL response in a patient with chronic myeloid leukemia after donor lymphocyte infusion. We demonstrate that immunogenicity for LRH-1 is due to differential protein expression in recipient and donor cells as a consequence of a homozygous frameshift polymorphism in the donor. Tetramer analysis showed that emergence of LRH-1–specific CD8(+) cytotoxic T cells in peripheral blood and bone marrow correlated with complete remission of chronic myeloid leukemia. Furthermore, the restricted expression of LRH-1 in hematopoietic cells including leukemic CD34(+) progenitor cells provides evidence of a role for LRH-1–specific CD8(+) cytotoxic T cells in selective graft-versus-leukemia reactivity in the absence of severe graft-versus-host disease. These findings illustrate that the P2X5-encoded mHAg LRH-1 could be an attractive target for specific immunotherapy to treat hematological malignancies recurring after allogeneic stem cell transplantation

A Research Agenda for Hybrid Intelligence: Augmenting Human Intellect With Collaborative, Adaptive, Responsible, and Explainable Artificial Intelligence

We define hybrid intelligence (HI) as the combination of human and machine intelligence, augmenting human intellect and capabilities instead of replacing them and achieving goals that were unreachable by either humans or machines. HI is an important new research focus for artificial intelligence, and we set a research agenda for HI by formulating four challenges

Heteroplasmy evolution of cybrid clone V_50.

<p>(A) Average cellular mutation load decreases with increasing passage number of clone V_50. Conventional gel-based PCR-RFLP was used for quantitation of the m.3243A>G heteroplasmy. (B) Relative mutation load PCR-RFMT histograms of flow sorted single cells of selected passages of clone V_50. n refers to the number of single cells evaluated in the histogram. (C) Frequency histograms of m.3243A>G Padlock/RCA mutation loads of cells in V_50 P52 at increasing stringency of the dots/cell number criterion, cells with >0 (all), ≥20 and ≥40 dots/cell. See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0052080#pone.0052080.s001" target="_blank">Figure S1</a> for choice of stringency and bin size. (D) Microscopic image of V_50 P52 after m.3243A>G Padlock/RCA <i>in situ</i> genotyping. Note in this microscopic field the presence of homoplasmic wild type cells (arrow heads) amidst the heteroplasmic cells. See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0052080#pone.0052080.s002" target="_blank">Figure S2</a> for discussion on the number and nature of the signals.</p

Cellular heteroplasmy evolution of V_50 P48 sub-clone V_3.2.

<p>(A) Relative m.3243A>G Padlock/RCA mutation load frequency histograms of cells in passages 1, 12 and 81 of sub-clone V_3.2. Histograms from cells with ≥60 dots/cell had similar shapes. Numbers in parentheses represent the total number of cells analyzed. (B) Relative mutation load frequency histograms of cells in passages 1, 12 and 81 of V_50 P42 sub-clone V_3.2 generated by computer simulation of random segregation (mtDNA copy number input 1,800/cell). Additional computer simulations showed that an mtDNA copy number input of 12,000 is required to explain the experimental distribution at P81 by random mtDNA segregation.</p