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

Visual stress, its treatment with spectral filters, and its relationship to visually induced motion sickness

We review the concept of visual stress and its relation to neurological disease. Visual stress can occur from the observation of images with unnatural spatial structure and an excess of contrast energy at spatial frequencies to which the visual system is generally most sensitive. Visual stress can often be reduced using spectral filters, provided the colour is selected with precision to suit each individual. The use of such filters and their effects on reading speed are reviewed. The filters have been shown to benefit patients with a variety of neurological conditions other than reading difficulty, all associated with an increased risk of seizures. © 2009 Elsevier Ltd

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

Testing General Relativity with Atomic Clocks

We discuss perspectives for new tests of general relativity which are based on recent technological developments as well as new ideas. We focus our attention on tests performed with atomic clocks and do not repeat arguments present in the other contributions to the present volume. In particular, we present the scientific motivations of the space projects ACES and SAGAS.Comment: Contribution for "The Nature of Gravity" (eds. F. Everitt et al

Direct interaction between the catalytic subunit of Protein Phosphatase 1 and pRb

Background: The product of the retinoblastoma-susceptibility gene (pRb) is a substrate for Protein Phosphatase 1 (PP1). At mitotic exit, all three PP1 isoforms, α, γ1 and δ, bind to pRb and dephosphorylate its Ser/Thr sites in a sequential and site-specific way. The pRb-C terminal has been reported to be necessary and sufficient for PP1α binding. The present study investigated whether the three PP1 isoforms from mitotic or asynchronous HeLa cells associate differentially with wild-type and pRb mutants, as well as the holoenzyme composition of the pRb-directed PP1. Results: The requirement for the entire pRb molecule to achieve optimal PP1-binding was indicated by the fact that full-length pRb displayed the highest affinity for all three PP1 isoforms. Ser/ Thr-to-Ala substitution for up to 14 pRb sites did not affect the ability of pRb to bind the PP1 isoforms derived from mitotic or asynchronous HeLa cells, thus suggesting that the phosphate-accepting residues on pRb do not regulate the interaction with PP1. To probe for the presence of PP1 targeting subunits in the pRb-directed PP1 complex, PP1 from mitotic or asynchronous HeLa cells was isolated by affinity chromatography on GST-Rb (either full-length or its deletion mutants Rb-big pocket or Rb-C-terminal). The PP1 was always obtained as free catalytic subunit, displaying all three isoforms, thus suggesting direct interaction between pRb and PP1. The direct association was confirmed by the ability of pRb to pull-down purified PP1 catalytic subunits and by in vitro reconstitution of a complex between PP1 catalytic subunit and the pRb-C-terminal. Conclusion: The work indicated that the full length of the pRb molecule is required for optimal interaction with the PP1 isoforms and that the association between pRb and PP1 isoforms is direct

COMPARISON IN STRENGTH GAINS WITH PERIODIZED TRAINING FOR 1 SET, 1 SET-3 SET, AND 3 SET OVER 12 WEEKS

B.N. Thomas, J.W. Hardy, S.E. Kelly, S.J. Ludlow, & D.E. Lankford. Brigham Young University—Idaho, Rexburg, ID PURPOSE: The purpose of this study was to test the hypothesis that a program that begins with a single-set and switches to three-sets (1-3 set) will provide similar 1RM improvement in squat and bench press as a 3-set program and superior improvements compared to a 1-set program. METHODS: Twenty-six college-aged males and females aged 18- 25, who had not performed resistance training for at least 8 weeks, were separated into three groups which performed seven upper- and lower-body exercises 3 d/wk for 12 weeks. Subjects were divided into three groups: 1-set (n=10), 1-3 set (n=8) and 3-set (n=8). Squat and bench press 1RMs were assessed at weeks 0, 4, 8, and 12. Training loads were maintained at approximately 80% 1RM as subjects’ strength progressed. Loads were assigned so all groups would reach volitional failure after 4-6 rep/set throughout the study. When 3 sets were assigned, subjects were allowed 2-3 minutes rest between sets. All subjects participated in a familiarization session before being assessed for their first 1RM. All 1RMs were assessed in duplicate. The highest value of each assessment was used for exercise prescription and post-training analysis. Subjects were further grouped as novice (no previous experience) and previous training (those who had at least 3 months of resistance training history). Improvement comparisons within the novice group 1-set (n=9), 1-3 set (n=6) and 3-set (n=5) were analyzed using an independent ANOVA. Significance was set at \u3c 0.05. RESULTS: No differences between groups in 1RM squat improvement following 12 weeks of resistance training. 1RM bench press improvements were greater for both 3-set and 1-3 set compared to 1-set (

Inducible expression of catalytically active type 1 serine/threonine protein phosphatase in a human carcinoma cell line

Abstract Background One of the major cellular serine/threonine protein phosphatases is protein phosphatase type 1 (PP1). Studies employing many eukaryotic systems all point to a crucial role for PP1 activity in controlling cell cycle progression. One physiological substrate for PP1 appears to be the product of the retinoblastoma susceptibility gene (pRB), a demonstrated tumor suppressor. The growth suppressive activity of pRB is regulated by its phosphorylation state. Of critical importance is the question of the in vivo effect of PP1 activity on pRB and growth regulation. As a first step towards addressing this question, we developed an inducible PP1 expression system to investigate the regulation of PP1 activity. Results We have established a cell line for inducing protein expression of the type 1, alpha-isotype, serine/threonine protein phosphatase (PP1α). A plasmid encoding a fusion protein of the catalytic subunit of PP1α with a 6-histidine peptide (6His) and a peptide from hemagluttinin (HA) was transfected into the UMUC3 transitional cell carcinoma cell line, previously transfected with the reverse tetracycline transactivator plasmid pUHD172-1neo. A stable cell line designated LLWO2F was established by selection with hygromycin B. 6His-HA-PP1α protein appeared in cell lysates within two hours following addition of doxycycline to the culture medium. This protein localizes to the nucleus as does endogenous PP1α, and was shown to associate with PNUTS, a PP1-nuclear targeting subunit. Like endogenous PP1α, immunocomplexed 6His-HA-PP1α is active toward phosphorylase a and the product of the retinoblastoma susceptibility gene, pRB. When forcibly overexpressing 6His-HA-PP1α, there is a concomitant decrease in endogenous PP1α levels. Conclusions These data suggest the existence of an autoregulatory mechanism by which PP1α protein levels and activity remain relatively constant. RT-PCR analyses of isolated polysome fractions support the notion that this putative autoregulatory mechanism is exerted, at least in part, at the translational level. Implications of these findings for the study of PP1α function in vivo are discussed.</p