Visualization of oligonucleotide probes and point mutations in interphase nuclei and DNA fibers using rolling circle DNA amplification

Rolling circle amplification (RCA) is a surface-anchored DNA replication reaction that can be exploited to visualize single molecular recognition events. Here we report the use of RCA to visualize target DNA sequences as small as 50 nts in peripheral blood lymphocytes or in stretched DNA fibers. Three unique target sequences within the cystic fibrosis transmembrane conductance regulator gene could be detected simultaneously in interphase nuclei, and could be ordered in a linear map in stretched DNA. Allele-discriminating oligonucleotide probes in conjunction with RCA also were used to discriminate wild-type and mutant alleles in the cystic fibrosis transmembrane conductance regulator, p53, BRCA-1, and Gorlin syndrome genes in the nuclei of cultured cells or in DNA fibers. These observations demonstrate that signal amplification by RCA can be coupled to nucleic acid hybridization and multicolor fluorescence imaging to detect single nucleotide changes in DNA within a cytological context or in single DNA molecules. This provides a means for direct physical haplotyping and the analysis of somatic mutations on a cell-by-cell basis.

Active galactic nuclei

Active galactic nuclei are the most powerful, long-lived objects in the Universe. Recent data confirm the theoretical idea that the power source is accretion into a massive black hole. The common occurrence of obscuration and outflows probably means that the contribution of active galactic nuclei to the power density of the Universe has been generally underestimated.

On the nucleation and growth of amyloid beta-protein fibrils: detection of nuclei and quantitation of rate constants.

We have studied the fibrillogenesis of synthetic amyloid beta-protein-(1-40) fragment (A beta) in 0.1 M HCl. At low pH, A beta formed fibrils at a rate amenable to detailed monitoring by quasi-elastic light-scattering spectroscopy. Examination of the fibrils with circular dichroism spectroscopy and electron microscopy showed them to be highly similar to those found in amyloid plaques. We determined the hydrodynamic radii of A beta aggregates during the entire process of fibril nucleation and growth. Above an A beta concentration of approximately 0.1 mM, the initial rate of elongation and the final size of fibrils were independent of A beta concentration. Below an A beta concentration of 0.1 mM, the initial elongation rate was proportional to the peptide concentration, and the resulting fibrils were significantly longer than those formed at higher concentration. We also found that the surfactant n-dodecylhexaoxyethylene glycol monoether (C12E6) slowed nucleation and elongation of fibrils in a concentration-dependent manner. Our observations are consistent with a model of A beta fibrillogenesis that includes the following key steps: (i) peptide micelles form above a certain critical A beta concentration, (ii) fibrils nucleate within these micelles or on heterogeneous nuclei (seeds), and (iii) fibrils grow by irreversible binding of monomers to fibril ends. Interpretation of our data enabled us to determine the sizes of fibril nuclei and A beta micelles and the rates of fibril nucleation (from micelles) and fibril elongation. Our approach provides a powerful means for the quantitative assay of A beta fibrillogenesis.

Very-long-baseline radio interferometry surveys of the compact structure in active galactic nuclei.

Very-long-baseline radio interferometry (VLBI) imaging surveys have been undertaken since the late 1970s. The sample sizes were initially limited to a few tens of objects but the snapshot technique has now allowed samples containing almost 200 sources to be studied. The overwhelming majority of powerful compact sources are asymmetric corejects of one form or another, most of which exhibit apparent superluminal motion. However 5-10% of powerful flat-spectrum sources are 100-parsec (pc)-scale compact symmetric objects; these appear to form a continuum with the 1-kpc-scale double-lobed compact steep-spectrum sources, which make up 15-20% of lower frequency samples. It is likely that these sub-galactic-size symmetric sources are the precursors to the large-scale classical double sources. There is a surprising peak around 90 degrees in the histogram of misalignments between the dominant source axes on parsec and kiloparsec scales; this seems to be associated with sources exhibiting a high degree of relativistic beaming. VLBI snapshot surveys have great cosmological potential via measurements of both proper motion and angular size vs. redshift as well as searches for gravitational "millilensing."

Optical evidence for the unification of active galactic nuclei and quasi-stellar objects.

There is a variety of optical evidence for some unification of different types of active galactic nuclei and quasi-stellar objects (QSOs). The case is very strong for the unification of at least some Seyfert galaxies, where polarization data show that the type assigned to the Seyfert galaxy must depend on viewing direction. It has been proposed that Fanaroff-Riley type 2 (FR2) radio galaxies are quasars seen in a direction from which the quasar is obscured, and there is some limited direct evidence for this picture. The broad absorption line QSOs may be normal QSOs seen from a special direction. Some of the sources observed to have high luminosities in the far infrared could be obscured QSOs and active nuclei. Mergers and interactions are likely to play an important role in nuclear activity, and active galaxies and QSOs could change their apparent types through these encounters followed by subsequent evolution.

Probing active galactic nuclei with H2O megamasers.

We describe the characteristics of the rapidly rotating molecular disk in the nucleus of the mildly active galaxy NGC4258. The morphology and kinematics of the disk are delineated by the point-like watervapor emission sources at 1.35-cm wavelength. High angular resolution [200 microas where as is arcsec, corresponding to 0.006 parsec (pc) at 6.4 million pc] and high spectral resolution (0.2 km.s-1 or nu/Deltanu = 1.4 x 10(6)) with the Very-Long-Baseline Array allow precise definition of the disk. The disk is very thin, but slightly warped, and is viewed nearly edge-on. The masers show that the disk is in nearly perfect Keplerian rotation within the observable range of radii of 0.13-0.26 pc. The approximately random deviations from the Keplerian rotation curve among the high-velocity masers are approximately 3.5 km.s-1 (rms). These deviations may be due to the masers lying off the midline by about +/-4 degrees or variations in the inclination of the disk by +/-4 degrees. Lack of systematic deviations indicates that the disk has a mass of <4 x 10(6) solar mass (M[symbol: see text]). The gravitational binding mass is 3.5 x 10(7) M[symbol: see text], which must lie within the inner radius of the disk and requires that the mass density be >4 x 10(9) M[symbol: see text].pc-3. If the central mass were in the form of a star cluster with a density distribution such as a Plummer model, then the central mass density would be 4 x 10(12) M[symbol: see text].pc-3. The lifetime of such a cluster would be short with respect to the age of the galaxy [Maoz, E. (1995) Astrophys. J. Lett. 447, L91-L94]. Therefore, the central mass may be a black hole. The disk as traced by the systemic velocity features is unresolved in the vertical direction, indicating that its scale height is <0.0003 pc (hence the ratio of thickness to radius, H/R, is <0.0025). For a disk in hydrostatic equilibrium the quadrature sum of the sound speed and Alfven velocity is <2.5 km.s-1, so that the temperature of the disk must be <1000 K and the toroidal magnetic field component must be <250 mG. If the molecular mass density in the disk is 10(10) cm-3, then the disk mass is approximately 10(4) M[symbol: see text], and the disk is marginally stable as defined by the Toomre stability parameter Q (Q = 6 at the inner edge and 1 at the outer edge). The inward drift velocity is predicted to be <0.007 km.s-1, for a viscosity parameter of 0.1, and the accretion rate is <7 x 10(-5) M[symbol: see text].yr-1. At this value the accretion would be sufficient to power the nuclear x-ray source of 4 x 10(40) ergs-1 (1 erg = 0.1 microJ). The volume of individual maser components may be as large as 10(46) cm3, based on the velocity gradients, which is sufficient to supply the observed luminosity. The pump power undoubtedly comes from the nucleus, perhaps in the form of x-rays. The warp may allow the pump radiation to penetrate the disk obliquely [Neufeld, D. A. & Maloney, P. R. (1995) Astrophys. J. Lett. 447, L17-L19]. A total of 15 H2O megamasers have been identified out of >250 galaxies searched. Galaxy NGC4258 may be the only case where conditions are optimal to reveal a well-defined nuclear disk. Future measurement of proper motions and accelerations for NGC4258 will yield an accurate distance and a more precise definition of the dynamics of the disk

Gravitational lensing of active galactic nuclei.

Most of the known cases of strong gravitational lensing involve multiple imaging of an active galactic nucleus. The properties of lensed active galactic nuclei make them promising systems for astrophysical applications of gravitational lensing; in particular, they show structure on scales of milliseconds of arc to tens of seconds of arc, they are variable, and they are polarized. More than 20 cases of strong gravitational lenses are now known, and about half of them are radio sources. High-resolution radio imaging is making possible the development of well-constrained lens models. Variability studies at radio and optical wavelengths are beginning to yield results of astrophysical interest, such as an independent measure of the distance scale and limits on source sizes.

The vesicular monoamine transporter 2 is present in small synaptic vesicles and preferentially localizes to large dense core vesicles in rat solitary tract nuclei.

In central neurons, monamine neurotransmitters are taken up and stored within two distinct classes of regulated secretory vesicles: small synaptic vesicles and large dense core vesicles (DCVs). Biochemical and pharmacological evidence has shown that this uptake is mediated by specific vesicular monamine transporters (VMATs). Recent molecular cloning techniques have identified the vesicular monoamine transporter (VMAT2) that is expressed in brain. This transporter determines the sites of intracellular storage of monoamines and has been implicated in both the modulation of normal monoaminergic neurotransmission and the pathogenesis of related neuropsychiatric disease. We used an antiserum against VMAT2 to examine its ultrastructural distribution in rat solitary tract nuclei, a region that contains a dense and heterogeneous population of monoaminergic neurons. We find that both immunoperoxidase and immunogold labeling for VMAT2 localize to DCVs and small synaptic vesicles in axon terminals, the trans-Golgi network of neuronal perikarya, tubulovesicles of smooth endoplasmic reticulum, and potential sites of vesicular membrane recycling. In axon terminals, immunogold labeling for VMAT2 was preferentially associated with DCVs at sites distant from typical synaptic junctions. The results provide direct evidence that a single VMAT is expressed in two morphologically distinct types of regulated secretory vesicles in central monoaminergic neurons.

A class of single-stranded telomeric DNA-binding proteins required for Rap1p localization in yeast nuclei.

We have identified a class of proteins that bind single-stranded telomeric DNA and are required for the nuclear organization of telomeres and/or telomere-associated proteins. Rlf6p was identified by its sequence similarity to Gbp1p, a single-stranded telomeric DNA-binding protein from Chlamydomonas reinhardtii. Rlf6p and Gbp1p bind yeast single-stranded G-strand telomeric DNA. Both proteins include at least two RNA recognition motifs, which are found in many proteins that interact with single-stranded nucleic acids. Disruption of RLF6 alters the distribution of repressor/activator protein 1 (Rap1p), a telomere-associated protein. In wild-type yeast cells, Rap1p localizes to a small number of perinuclear spots, while in rlf6 cells Rap1p appears diffuse and nuclear. Interestingly, telomere position effect and telomere length control, which require RAP1, are unaffected by rlf6 mutations, demonstrating that Rap1p localization can be uncoupled from other Rap1p-dependent telomere functions. In addition, expression of Chlamydomonas GBP1 restores perinuclear, punctate Rap1p localization in rlf6 mutant cells. The functional complementation of a fungal gene by an algal gene suggests that Rlf6p and Gbp1p are members of a conserved class of single-stranded telomeric DNA-binding proteins that influence nuclear organization. Furthermore, it demonstrates that, despite their unusual codon bias, C. reinhardtii genes can be efficiently translated in Saccharomyces cerevisiae cells.

Plasmid DNA entry into postmitotic nuclei of primary rat myotubes.

These studies were initiated to elucidate the mechanism of DNA nuclear transport in mammalian cells. Biotin- or gold-labeled plasmid and plasmid DNA expression vectors for Escherichia coli beta-galactosidase or firefly luciferase were microinjected into the cytoplasm of primary rat myotubes in culture. Plasmid DNA was expressed in up to 70% of the injected myotubes, which indicates that it entered intact, postmitotic nuclei. The nuclear transport of plasmid DNA occurred through the nuclear pore by a process common to other large karyophilic macromolecules. The majority of the injected plasmid DNA was sequestered by cytoplasmic elements. This understanding of plasmid DNA nuclear transport provides a basis for increasing the efficiency of gene transfer.

Disk halo size measured in individuals with monofocal versus diffractive multifocal intraocular lenses

PURPOSE: To compare disk halo size in response to a glare source in eyes with an aspheric apodized diffractive multifocal intraocular lens (IOL) or aspheric monofocal IOL. SETTING: Rementeria Ophthalmological Clinic, Madrid, Spain. DESIGN: Prospective randomized masked study. METHOD: Halo radius was measured using a vision monitor (MonCv3) with low-luminance optotypes in eyes that had cataract surgery and bilateral implantion of an Acrysof Restor SN6AD1 multifocal IOL or Acrysof IQ monofocal IOL 6 to 9 months previously. The visual angle subtended by the disk halo radius was calculated in minutes of arc (arcmin). Patient complaints of halo disturbances were recorded. Monocular uncorrected distance visual acutity (UDVA) and corrected distance visual acuity (CDVA) were measured using high-contrast (96%) and low-contrast (10%) logMAR letter charts. RESULTS: The study comprised 39 eyes of 39 subjects (aged 70 to 80 years); 21 eyes had a multifocal IOL and 18 eyes a monofocal IOL. The mean halo radius was 35 arcmin larger in the multifocal IOL group than the monofocal group (P<.05). Greater halo effects were reported in the multifocal IOL group (P<.05). The mean monocular high-contrast UDVA and low-contrast UDVA did not vary significantly between groups, whereas the mean monocular high-contrast CDVA and low-contrast CDVA were significantly worse at 0.12 logMAR and 0.13 logMAR in the multifocal than in the monofocal IOL group, respectively (P <.01). A significant positive correlation was detected by multiple linear regression between the halo radius and low-contrast UDVA in the multifocal IOL group (r = 0.72, P<.001). CONCLUSIONS: The diffractive multifocal IOL gave rise to a larger disk halo size, which was correlated with a worse low-contrast UDVA.

Large-scale clustering measurements with photometric redshifts: comparing the dark matter haloes of X-ray AGN, star-forming and passive galaxies at z ≈ 1

We combine multi-wavelength data in the AEGIS-XD and C-COSMOS surveys to measure the typical dark matter halo mass of X-ray selected active galactic nuclei (AGN) [L_X(2–10 keV) > 10^42 erg s^− 1] in comparison with far-infrared selected star-forming galaxies detected in the Herschel/PEP survey (PACS Evolutionary Probe; L_IR > 10^11 L_⊙) and quiescent systems at z ≈ 1. We develop a novel method to measure the clustering of extragalactic populations that uses photometric redshift probability distribution functions in addition to any spectroscopy. This is advantageous in that all sources in the sample are used in the clustering analysis, not just the subset with secure spectroscopy. The method works best for large samples. The loss of accuracy because of the lack of spectroscopy is balanced by increasing the number of sources used to measure the clustering. We find that X-ray AGN, far-infrared selected star-forming galaxies and passive systems in the redshift interval 0.6 < z < 1.4 are found in haloes of similar mass, log M_DMH/(M_⊙ h^−1) ≈ 13.0. We argue that this is because the galaxies in all three samples (AGN, star-forming, passive) have similar stellar mass distributions, approximated by the J-band luminosity. Therefore, all galaxies that can potentially host X-ray AGN, because they have stellar masses in the appropriate range, live in dark matter haloes of log M_DMH/(M_⊙ h^−1) ≈ 13.0 independent of their star formation rates. This suggests that the stellar mass of X-ray AGN hosts is driving the observed clustering properties of this population. We also speculate that trends between AGN properties (e.g. luminosity, level of obscuration) and large-scale environment may be related to differences in the stellar mass of the host galaxies.

Deep observation of the NGC1275 region with MAGIC: search of diffuse gamma-ray emission from cosmic rays in the Perseus cluster

Clusters of galaxies are expected to be reservoirs of cosmic rays (CRs) that should produce diffuse γ-ray emission due to their hadronic interactions with the intra-cluster medium. The nearby Perseus cool-core cluster, identified as the most promising target to search for such an emission, has been observed with the MAGIC telescopes at very-high energies (VHE, E ≥ 100 GeV) for a total of 253 hr from 2009 to 2014. The active nuclei of NGC 1275, the central dominant galaxy of the cluster, and IC 310, lying at about 0.6º from the centre, have been detected as point-like VHE γ-ray emitters during the first phase of this campaign. We report an updated measurement of the NGC 1275 spectrum, which is described well by a power law with a photon index Γ = 3.6 ± 0.2_(stat) ± 0.2_(syst) between 90 GeV and 1200 GeV. We do not detect any diffuse γ-ray emission from the cluster and so set stringent constraints on its CR population. To bracket the uncertainties over the CR spatial and spectral distributions, we adopt different spatial templates and power-law spectral indexes α. For α = 2.2, the CR-to-thermal pressure within the cluster virial radius is constrained to be ≤ 1 − 2%, except if CRs can propagate out of the cluster core, generating a flatter radial distribution and releasing the CR-to-thermal pressure constraint to ≤ 20%. Assuming that the observed radio mini-halo of Perseus is generated by secondary electrons from CR hadronic interactions, we can derive lower limits on the central magnetic field, B_(0), that depend on the CR distribution. For α = 2.2, B_(0) ≥ 5 − 8 µG, which is below the ∼25 µG inferred from Faraday rotation measurements, whereas for α ≤ 2.1, the hadronic interpretation of the diffuse radio emission contrasts with our γ-ray flux upper limits independently of the magnetic field strength.

Fluorescent Biological Aerosol Particles in Coastal Canada and the Link to Atmospheric Ice Nuclei

Bioaerosols are a subgroup of atmospheric aerosols and are often linked to the spread of human, animal and plant diseases. Bioaerosols also may play an indirect effect on environmental processes, including the formation of precipitation and alteration of the global climate through their role as nuclei for cloud droplet formation. Several types of biological organisms (e.g., fungi and bacteria) have been shown to be effective ice nuclei (IN) and cloud condensation nuclei (CCN). During 21 days in August 2013 we participated in a collaborative international campaign at a rural, coastal site near the village of Ucluelet on the west coast of Vancouver Island, British Columbia, Canada. The experiments were conducted as part of the NETCARE project (the NETwork on Climate and Aerosols: Addressing Key Uncertainties in Remote Canadian Environments), in part to examine cloud nuclei properties of marine aerosol. The study was conducted from a mobile trailer located approximately 100 m from the coast. A suite of aerosol instrumentation was operated for approximately one month. Key instruments utilized as a part of this thesis include the wideband integrated bioaerosol sensor (WIBS-4A) and the multiple orifice uniform deposition impactor (MOUDI) coupled with an off-line droplet freezing technique (DFT) for the measurement of ice nucleation activity of particles in immersion mode. The WIBS measures the concentration and properties of individual fluorescent particles suspended in the air, which can serve as a proxy for airborne biological particle content. Particles shown to be fluorescent by the WIBS instrument were divided into seven categories based on the pattern of fluorescence each particle exhibited in the three fluorescent channels. Results of the WIBS analysis show that the fluorescent particle concentration in the region correlated well with IN number. The fluorescent particle concentration correlated well with the number of particles shown to be ice active as a function of both particle size and freezing temperature. Correlations involving marine aerosols and marine biological activity indicate that the majority of IN measured at the coastal site likely are not from have marine sources.