Generation of human induced pluripotent stem cells by simple transient transfection of plasmid DNA encoding reprogramming factors

<p>Abstract</p> <p>Background</p> <p>The use of lentiviruses to reprogram human somatic cells into induced pluripotent stem (iPS) cells could limit their therapeutic usefulness due to the integration of viral DNA sequences into the genome of the recipient cell. Recent work has demonstrated that human iPS cells can be generated using episomal plasmids, excisable transposons, adeno or sendai viruses, mRNA, or recombinant proteins. While these approaches offer an advance, the protocols have some drawbacks. Commonly the procedures require either subcloning to identify human iPS cells that are free of exogenous DNA, a knowledge of virology and safe handling procedures, or a detailed understanding of protein biochemistry.</p> <p>Results</p> <p>Here we report a simple approach that facilitates the reprogramming of human somatic cells using standard techniques to transfect expression plasmids that encode OCT4, NANOG, SOX2, and LIN28 without the need for episomal stability or selection. The resulting human iPS cells are free of DNA integration, express pluripotent markers, and form teratomas in immunodeficient animals. These iPS cells were also able to undergo directed differentiation into hepatocyte-like and cardiac myocyte-like cells in culture.</p> <p>Conclusions</p> <p>Simple transient transfection of plasmid DNA encoding reprogramming factors is sufficient to generate human iPS cells from primary fibroblasts that are free of exogenous DNA integrations. This approach is highly accessible and could expand the use of iPS cells in the study of human disease and development.</p

Assessing amino acid racemization variability in coral intra-crystalline protein for geochronological applications.

Over 500 Free Amino Acid (FAA) and corresponding Total Hydrolysed Amino Acid (THAA) analyses were completed from eight independently-dated, multi-century coral cores of massive Porites sp. colonies. This dataset allows us to re-evaluate the application of amino acid racemization (AAR) for dating late Holocene coral material, 20 years after Goodfriend et al. (GCA56 (1992), 3847) first showed AAR had promise for developing chronologies in coral cores. This re-assessment incorporates recent method improvements, including measurement by RP-HPLC, new quality control approaches (e.g. sampling and sub-sampling protocols, statistically-based data screening criteria), and cleaning steps to isolate the intra-crystalline skeletal protein. We show that the removal of the extra-crystalline contaminants and matrix protein is the most critical step for reproducible results and recommend a protocol of bleaching samples in NaOCl for 48 h to maximise removal of open system proteins while minimising the induced racemization. We demonstrate that AAR follows closed system behaviour in the intra-crystalline fraction of the coral skeletal proteins. Our study is the first to assess the natural variability in intra-crystalline AAR between colonies, and we use coral cores taken from the Great Barrier Reef, Australia, and Jarvis Island in the equatorial Pacific to explore variability associated with different environmental conditions and thermal histories. Chronologies were developed from THAA Asx D/L, Ala D/L, Glx D/L and FAA Asx D/L for each core and least squares Monte Carlo modelling applied in order to quantify uncertainty of AAR age determinations and assess the level of dating resolution possible over the last 5 centuries. AAR within colonies follow consistent stratigraphic aging. However, there are systematic differences in rates between the colonies, which would preclude direct comparison from one colony to another for accurate age estimation. When AAR age models are developed from a combined dataset to include this natural inter-colony variability THAA Asx D/L, Glx D/L and Ala D/L give a 2σ age uncertainty of ±19, ±38 and ±29 year, for the 20th C respectively; in comparison 2σ age uncertainties from a single colony are ±12, ±12 and ±14 year. This is the first demonstration of FAA D/L for dating coral and following strict protocols 2σ precisions of ±24 years can be achieved across different colonies in samples from the last 150 years, and can be ±10 years within a core from a single colony. Despite these relatively large error estimates, AAR would be a valuable tool in situations where a large number of samples need to be screened rapidly and cheaply (e.g. identifying material from mixed populations in beach or uplift deposits), prior to and complementing the more time-consuming geochronological tools of U/Th or seasonal isotopic timeseries

Dynamic Interactions between TIP60 and p300 Regulate FOXP3 Function through a Structural Switch Defined by a Single Lysine on TIP60

SummaryThe human FOXP3 molecule is an oligomeric transcriptional factor able to mediate activities that characterize T regulatory cells, a class of lymphocytes central to the regulation of immune responses. The activity of FOXP3 is regulated at the posttranslational level, in part by two histone acetyltransferases (HATs): TIP60 and p300. TIP60 and p300 work cooperatively to regulate FOXP3 activity. Initially, p300 and TIP60 interactions lead to the activation of TIP60 and facilitate acetylation of K327 of TIP60, which functions as a molecular switch to allow TIP60 to change binding partners. Subsequently, p300 is released from this complex, and TIP60 interacts with and acetylates FOXP3. Maximal induction of FOXP3 activities is observed when both p300 and TIP60 are able to undergo cooperative interactions. Conditional knockout of TIP60 in Treg cells significantly decreases the Treg population in the peripheral immune organs, leading to a scurfy-like fatal autoimmune disease

2004 Wild Blueberry Project Progress Reports

The 2004 edition of the Wild Blueberry Project Progress Reports was prepared for the Wild Blueberry Commission of Maine and the Wild Blueberry Advisory Committee by researchers at the University of Maine, Orono. Projects in this report include: 1. Determination of Pesticide Residue Levels in Freshly Harvested and Processed Lowbush Blueberries 2. Effect of Wild Blueberry Products on Physical, Chemical, Microbiological and Sensory Quality of Soy-Based and Ground Beef Patties 3. Evaluation of Emerging Disinfection Technologies for Wild Blueberry Processing 4. Detection of Infested Blueberries using Near-Infrared Spectroscopy-Spectra Collection 5. Health Claims for Wild Blueberries 6. Wild blueberries and Arterial Functional Properties 7. Irrigation Water Use in Wild Blueberry Production 8. Insect Control Tactics for Blueberry Pest Insects & Program Base 9. Integrated Pest Management (IPM) Strategies 10. Biology and Ecology of Blueberry Insect Pests 11. Stem Blight/Dieback and Leaf Spot Diseases in Lowbush Blueberry Fields 12. . Evaluation of fungicide control of mummy berry blight in wild blueberries: a) ground application and b) aerial application 13. Effect of Foliar Copper Application on Growth and Yield of Wild Blueberries 14. Effect of Soil pH on Nutrient Uptake 15. Effect of Fertilizer Timing (prune year vs. crop year) on Wild Blueberry Growth and Productivity 16. Raising Foliar Nitrogen by Application of CoRoN 17. Effect of Manganese on Growth and Yield of Wild Blueberry 18. Assessment of Hexazinone Alternatives for Weed Control in Wild Blueberries and Field Cover Program Base 19. Evaluation of Fall Applications of Sulfonylurea Herbicides for Bunchberry Control in Wild Blueberries 20. Evaluation and Demonstration of Techniques for Filling in Bare Spots in Wild Blueberry Fields 21. Assessment of Evitol for Sedge Control in Wild Blueberries 22. Cultural Weed Management Using pH 23. 2004 Pesticide Groundwater Survey 24. Wild Blueberry Extension Education Program in 200

2003 Wild Blueberry Project Reports

The 2003 edition of the Wild Blueberry Project Reports was prepared for the Wild Blueberry Commission of Maine and the Wild Blueberry Advisory Committee by researchers at the University of Maine, Orono. Projects in this report include: 1. Factors Affecting the Microbial and Pesticide Residues Levels on Lowbush Blueberries 2. Effect of Blueberry Products on Oxidation in Ground Beef Patties 3. Infestation Detection using Near-Infrared Spectroscopy 4. Whole Wild Blueberries and Arterial Functional Properties 5. Irrigation Water use in Wild Blueberry Production 7. Control Tactics for Blueberry Pest Insects 8. IPM Strategies 9. Biology and Ecology of Blueberry Pest Insects, 2003 10. Wild Blueberry Pollination Research 11. Stem Blight/Dieback and Leaf Spot Diseases in Wild Blueberry Fields 12. Effect of Foliar N spray on Leaf N Concentration, Growth and Yield of Wild Blueberries 13. Effect of Foliar Spray (4-13-15) on Leaf Nutrient Concentration, Growth and Yield of Wild Blueberries 14. Effect of Foliar Copper Application on Growth and Yield of Wild Blueberries 15. Effect of Foliar Copper and/or Iron Application on Growth and Yield of Wild Blueberries 16. Effect of Soil pH on Nutrient Uptake. 17. Effect of Gibberellic Acid (GA3) and CPPU on Fruit Set and Yield of Wild Blueberry after low temperature flower stress 18. Effect of Fertilizer Timing (prune year vs. crop year) on Wild Blueberry Growth and Productivity. 19. Assessment of Hexazinone Alternatives for Weed Control in Wild Blueberries and Weed Control and Field Cover Program Base 20. Evaluation of Fall Applications of Sulfonylurea Herbicides for Bunchberry Control in Wild Blueberries 21. Assessment of clean-cut adapter on hand clippers for weed control in wild blueberries 22. Evaluation and Demonstration of Techniques for Filling in Bare Spots in Wild Blueberry Fields 23. Blueberry Extension Education Program in 2003 24. 2003 Pesticide Groundwater Survey 25. Cultural Weed Management using Sulfur to lower the p

Lysine acetyltransferase Tip60 is required for hematopoietic stem cell maintenance.

Hematopoietic stem cells (HSCs) have the potential to replenish the blood system for the lifetime of the organism. Their 2 defining properties, self-renewal and differentiation, are tightly regulated by the epigenetic machineries. Using conditional gene-knockout models, we demonstrated a critical requirement of lysine acetyltransferase 5 (Kat5, also known as Tip60) for murine HSC maintenance in both the embryonic and adult stages, which depends on its acetyltransferase activity. Genome-wide chromatin and transcriptome profiling in murine hematopoietic stem and progenitor cells revealed that Tip60 colocalizes with c-Myc and that Tip60 deletion suppress the expression of Myc target genes, which are associated with critical biological processes for HSC maintenance, cell cycling, and DNA repair. Notably, acetylated H2A.Z (acH2A.Z) was enriched at the Tip60-bound active chromatin, and Tip60 deletion induced a robust reduction in the acH2A.Z/H2A.Z ratio. These results uncover a critical epigenetic regulatory layer for HSC maintenance, at least in part through Tip60-dependent H2A.Z acetylation to activate Myc target genes.Cancer Research UK, Wellcome Trust, National Institutes of Health, Singapore state fundin

The population of merging compact binaries inferred using gravitational waves through GWTC-3

We report on the population properties of 76 compact binary mergers detected with gravitational waves below a false alarm rate of 1 per year through GWTC-3. The catalog contains three classes of binary mergers: BBH, BNS, and NSBH mergers. We infer the BNS merger rate to be between 10 $\rm{Gpc^{-3} yr^{-1}}$ and 1700 $\rm{Gpc^{-3} yr^{-1}}$ and the NSBH merger rate to be between 7.8 $\rm{Gpc^{-3}\, yr^{-1}}$ and 140 $\rm{Gpc^{-3} yr^{-1}}$ , assuming a constant rate density versus comoving volume and taking the union of 90% credible intervals for methods used in this work. Accounting for the BBH merger rate to evolve with redshift, we find the BBH merger rate to be between 17.9 $\rm{Gpc^{-3}\, yr^{-1}}$ and 44 $\rm{Gpc^{-3}\, yr^{-1}}$ at a fiducial redshift (z=0.2). We obtain a broad neutron star mass distribution extending from $1.2^{+0.1}_{-0.2} M_\odot$ to $2.0^{+0.3}_{-0.3} M_\odot$. We can confidently identify a rapid decrease in merger rate versus component mass between neutron star-like masses and black-hole-like masses, but there is no evidence that the merger rate increases again before 10 $M_\odot$. We also find the BBH mass distribution has localized over- and under-densities relative to a power law distribution. While we continue to find the mass distribution of a binary's more massive component strongly decreases as a function of primary mass, we observe no evidence of a strongly suppressed merger rate above $\sim 60 M_\odot$. The rate of BBH mergers is observed to increase with redshift at a rate proportional to $(1+z)^{\kappa}$ with $\kappa = 2.9^{+1.7}_{-1.8}$ for $z\lesssim 1$. Observed black hole spins are small, with half of spin magnitudes below $\chi_i \simeq 0.25$. We observe evidence of negative aligned spins in the population, and an increase in spin magnitude for systems with more unequal mass ratio

Search of the Orion spur for continuous gravitational waves using a loosely coherent algorithm on data from LIGO interferometers

We report results of a wideband search for periodic gravitational waves from isolated neutron stars within the Orion spur towards both the inner and outer regions of our Galaxy. As gravitational waves interact very weakly with matter, the search is unimpeded by dust and concentrations of stars. One search disk (A) is 6.87° in diameter and centered on 20h10m54.71s+33°33′25.29′′, and the other (B) is 7.45° in diameter and centered on 8h35m20.61s-46°49′25.151′′. We explored the frequency range of 50-1500 Hz and frequency derivative from 0 to -5×10-9 Hz/s. A multistage, loosely coherent search program allowed probing more deeply than before in these two regions, while increasing coherence length with every stage. Rigorous follow-up parameters have winnowed the initial coincidence set to only 70 candidates, to be examined manually. None of those 70 candidates proved to be consistent with an isolated gravitational-wave emitter, and 95% confidence level upper limits were placed on continuous-wave strain amplitudes. Near 169 Hz we achieve our lowest 95% C.L. upper limit on the worst-case linearly polarized strain amplitude h0 of 6.3×10-25, while at the high end of our frequency range we achieve a worst-case upper limit of 3.4×10-24 for all polarizations and sky locations. © 2016 American Physical Society

First low-frequency Einstein@Home all-sky search for continuous gravitational waves in Advanced LIGO data

We report results of a deep all-sky search for periodic gravitational waves from isolated neutron stars in data from the first Advanced LIGO observing run. This search investigates the low frequency range of Advanced LIGO data, between 20 and 100 Hz, much of which was not explored in initial LIGO. The search was made possible by the computing power provided by the volunteers of the Einstein@Home project. We find no significant signal candidate and set the most stringent upper limits to date on the amplitude of gravitational wave signals from the target population, corresponding to a sensitivity depth of 48.7 [1/root Hz]. At the frequency of best strain sensitivity, near 100 Hz, we set 90% confidence upper limits of 1.8 x 10(-25). At the low end of our frequency range, 20 Hz, we achieve upper limits of 3.9 x 10(-24). At 55 Hz we can exclude sources with ellipticities greater than 10(-5) within 100 pc of Earth with fiducial value of the principal moment of inertia of 10(38) kg m(2)