Quantitative mitochondrial DNA copy number determination using droplet digital PCR with single cell resolution

Mitochondria are involved in a number of diverse cellular functions, including energy production, metabolic regulation, apoptosis, calcium homeostasis, cell proliferation, and motility, as well as free radical generation. Mitochondrial DNA (mtDNA) is present at hundreds to thousands of copies per cell in a tissue-specific manner. mtDNA copy number also varies during aging and disease progression and therefore might be considered as a biomarker that mirrors alterations within the human body. Here, we present a new quantitative, highly sensitive droplet digital PCR (ddPCR) method, droplet digital mitochondrial DNA measurement (ddMDM), to measure mtDNA copy number not only from cell populations but also from single cells. Our developed assay can generate data in as little as 3 h, is optimized for 96-well plates, and also allows the direct use of cell lysates without the need for DNA purification or nuclear reference genes. We show that ddMDM is able to detect differences between samples whose mtDNA copy number was close enough as to be indistinguishable by other commonly used mtDNA quantitation methods. By utilizing ddMDM, we show quantitative changes in mtDNA content per cell across a wide variety of physiological contexts including cancer progression, cell cycle progression, human T cell activation, and human aging

Telomere length and telomerase activity in T cells are biomarkers of high-performing centenarians

It is generally recognized that the function of the immune system declines with increased age and one of the major immune changes is impaired T-cell responses upon antigen presentation/stimulation. Some "high-performing" centenarians (100+ years old) are remarkably successful in escaping, or largely postponing, major age-related diseases. However, the majority of centenarians ("low-performing") have experienced these pathologies and are forced to reside in long-term nursing facilities. Previous studies have pooled all centenarians examining heterogeneous populations of resting/unstimulated peripheral blood mononuclear cells (PBMCs). T cells represent around 60% of PBMC and are in a quiescence state when unstimulated. However, upon stimulation, T cells rapidly divide and exhibit dramatic changes in gene expression. We have compared stimulated T-cell responses and identified a set of transcripts expressed in vitro that are dramatically different in high- vs. low-performing centenarians. We have also identified several other measurements that are different between high- and low-performing centenarians: (a) The amount of proliferation following in vitro stimulation is dramatically greater in high-performing centenarians compared to 67- to 83-year-old controls and low-performing centenarians; (b) telomere length is greater in the high-performing centenarians; and (c) telomerase activity following stimulation is greater in the high-performing centenarians. In addition, we have validated a number of genes whose expression is directly related to telomere length and these are potential fundamental biomarkers of aging that may influence the risk and progression of multiple aging conditions

Recent Advances in Understanding Particle Acceleration Processes in Solar Flares

We review basic theoretical concepts in particle acceleration, with particular emphasis on processes likely to occur in regions of magnetic reconnection. Several new developments are discussed, including detailed studies of reconnection in three-dimensional magnetic field configurations (e.g., current sheets, collapsing traps, separatrix regions) and stochastic acceleration in a turbulent environment. Fluid, test-particle, and particle-in-cell approaches are used and results compared. While these studies show considerable promise in accounting for the various observational manifestations of solar flares, they are limited by a number of factors, mostly relating to available computational power. Not the least of these issues is the need to explicitly incorporate the electrodynamic feedback of the accelerated particles themselves on the environment in which they are accelerated. A brief prognosis for future advancement is offered.Comment: This is a chapter in a monograph on the physics of solar flares, inspired by RHESSI observations. The individual articles are to appear in Space Science Reviews (2011

Theory and Applications of Non-Relativistic and Relativistic Turbulent Reconnection

Realistic astrophysical environments are turbulent due to the extremely high Reynolds numbers. Therefore, the theories of reconnection intended for describing astrophysical reconnection should not ignore the effects of turbulence on magnetic reconnection. Turbulence is known to change the nature of many physical processes dramatically and in this review we claim that magnetic reconnection is not an exception. We stress that not only astrophysical turbulence is ubiquitous, but also magnetic reconnection itself induces turbulence. Thus turbulence must be accounted for in any realistic astrophysical reconnection setup. We argue that due to the similarities of MHD turbulence in relativistic and non-relativistic cases the theory of magnetic reconnection developed for the non-relativistic case can be extended to the relativistic case and we provide numerical simulations that support this conjecture. We also provide quantitative comparisons of the theoretical predictions and results of numerical experiments, including the situations when turbulent reconnection is self-driven, i.e. the turbulence in the system is generated by the reconnection process itself. We show how turbulent reconnection entails the violation of magnetic flux freezing, the conclusion that has really far reaching consequences for many realistically turbulent astrophysical environments. In addition, we consider observational testing of turbulent reconnection as well as numerous implications of the theory. The former includes the Sun and solar wind reconnection, while the latter include the process of reconnection diffusion induced by turbulent reconnection, the acceleration of energetic particles, bursts of turbulent reconnection related to black hole sources as well as gamma ray bursts. Finally, we explain why turbulent reconnection cannot be explained by turbulent resistivity or derived through the mean field approach.Comment: 66 pages, 24 figures, a chapter of the book "Magnetic Reconnection - Concepts and Applications", editors W. Gonzalez, E. N. Parke

Development of a Peripherally Restricted 5-HT2B Partial Agonist for Treatment of Pulmonary Arterial Hypertension

Ligands for the serotonin 2B receptor (5-HT2B) have shown potential to treat pulmonary arterial hypertension in preclinical models but cannot be used in humans because of predicted off-target neurological effects. The aim of this study was to develop novel systemically restricted compounds targeting 5-HT2B. Here, we show that mice treated with VU6047534 had decreased RVSP compared with control treatment in both the prevention and intervention studies using Sugen-hypoxia. VU6047534 is a novel 5-HT2B partial agonist that is peripherally restricted and able to both prevent and treat Sugen-hypoxia–induced pulmonary arterial hypertension. We have synthesized and characterized a structurally novel series of 5-HT2B ligands with high potency and selectivity for the 5-HT2B receptor subtype. Next-generation 5-HT2B ligands with similar characteristics, and predicted to be systemically restricted in humans, are currently advancing to investigational new drug–enabling studies