Clonal analysis of a human antibody response. Quantitation of precursors of antibody-producing cells and generation and characterization of monoclonal IgM, IgG, and IgA to rabies virus.

We quantitated and characterized the changes in the human B cell repertoire, at the clonal level, before and after immunization with rabies virus. Moreover, we generated 10 monoclonal cell lines producing IgM, IgG, and IgA antibodies to the virus. We found that in healthy subjects, not previously exposed to the virus, nearly 2% of the circulating B lymphocytes were committed to the production of antibodies that bound the virus. These B cells expressed the surface CD5 molecule. The antibodies they produced were polyreactive IgM that displayed a relatively low affinity for the virus components (Kd, 1.0-2.4 x 10(-6) g/microliters). After immunization, different anti-virus (IgG and IgA) antibody-producing cells consistently appeared in the circulation and increased from less than 0.005% to greater than 10% of the total B cells committed to the production of IgG and IgA, respectively. Most of such B cells do not express CD5 and produce monoreactive antibodies of high affinity for rabies virus (Kd, 6.5 x 10(-9) to 1.2 x 10(-10) g/microliters). One of these IgG mAbs efficiently neutralized rabies virus in vitro and in vivo, as detailed elsewhere (Dietzschold, B., P. Casali, Y. Ueki, M. Gore, C. E. Rupprecht, A. L. Notkins, and H. Koprowski, manuscript submitted for publication). Hybridization experiments using probes specific for the different human V gene segment families revealed that cell precursors producing low affinity IgM binding to rabies virus utilized a restricted number of VH gene segments (i.e., only members of the VHIIIb subfamily), whereas cell precursors producing high affinity IgG and IgA to rabies virus utilized an assortment of different VH gene segments (i.e., members of the VHI, VHIII, VHIV, and VHVI families and VHIIIb subfamily). In conclusion, our studies show that EBV transformation in conjunction with limiting dilution technology and somatic cell hybridization techniques are useful methods for quantitating, at the B cell clonal level, the human antibody response to foreign Ags and for generating human mAbs of predetermined specificity and high affinity

PG 1700+518 Revisited: Adaptive Optics Imaging and a Revised Starburst Age for the Companion

We present the results of adaptive-optics imaging of the z=0.2923 QSO PG 1700+518 in the J and H bands. The extension to the north of the QSO is clearly seen to be a discrete companion with a well-defined tidal tail, rather than a feature associated with the host galaxy of PG 1700+518 itself. On the other hand, an extension to the southwest of the QSO (seen best in deeper, but lower-resolution, optical images) does likely comprise tidal material from the host galaxy. The SED derived from images in J, H, and two non-standard optical bands indicates the presence of dust intermixed with the stellar component. We use our previously reported Keck spectrum of the companion, the SED found from the imaging data, and updated spectral-synthesis models to constrain the stellar populations in the companion and to redetermine the age of the starburst. While our best-fit age of 0.085 Gyr is nearly the same as our earlier determination, the fit of the new models is considerably better. This age is found to be remarkably robust with respect to different assumptions about the nature of the older stellar component and the effects of dust.Comment: 11 pages; includes two eps figures. Latex (AASTEX). Two additional figures in gif format. Postscript version including all figs. (424 kb) can be obtained from http://www.ifa.hawaii.edu/~canaguby/preprints.html To appear in ApJ. Letter

Measurements of the Cerenkov light emitted by a TeO2 crystal

Bolometers have proven to be good instruments to search for rare processes because of their excellent energy resolution and their extremely low intrinsic background. In this kind of detectors, the capability of discriminating alpha particles from electrons represents an important aspect for the background reduction. One possibility for obtaining such a discrimination is provided by the detection of the Cerenkov light which, at the low energies of the natural radioactivity, is only emitted by electrons. In this paper, the results of the analysis of the light emitted by a TeO2 crystal at room temperature when transversed by a cosmic ray are reported. Light is promptly emitted after the particle crossing and a clear evidence of its directionality is also found. These results represent a strong indication that Cerenkov light is the main, if not even the only, component of the light signal in a TeO2 crystal. They open the possibility to make large improvements in the performance of experiments based on this kind of material

Constraints on the Space Density of Methane Dwarfs and the Substellar Mass Function from a Deep Near-Infrared Survey

We report preliminary results of a deep near-infrared search for methane-absorbing brown dwarfs; almost five years after the discovery of Gl 229b, there are only a few confirmed examples of this type of object. New J band, wide-field images, combined with pre-existing R band observations, allow efficient identification of candidates by their extreme (R-J) colours. Follow-up measurements with custom filters can then confirm objects with methane absorption. To date, we have surveyed a total of 11.4 square degrees to J~20.5 and R~25. Follow-up CH_4 filter observations of promising candidates in 1/4 of these fields have turned up no methane absorbing brown dwarfs. With 90% confidence, this implies that the space density of objects similar to Gl 229b is less than 0.012 per cubic parsec. These calculations account for the vertical structure of the Galaxy, which can be important for sensitive measurements. Combining published theoretical atmospheric models with our observations sets an upper limit of alpha <= 0.8 for the exponent of the initial mass function power law in this domain.Comment: 11 pages + 2 figures To be published in Astrophysical Journal Letter

Measurements and optimization of the light yield of a TeO2_2 crystal

Bolometers have proven to be good instruments to search for rare processes because of their excellent energy resolution and their extremely low intrinsic background. In this kind of detectors, the capability of discriminating alpha particles from electrons represents an important aspect for the background reduction. One possibility for obtaining such a discrimination is provided by the detection of the Cherenkov light which, at the low energies of the natural radioactivity, is only emitted by electrons. This paper describes the method developed to evaluate the amount of light produced by a crystal of TeO$_2$ when hit by a 511 keV photon. The experimental measurements and the results of a detailed simulation of the crystal and the readout system are shown and compared. A light yield of about 52 Cherenkov photons per deposited MeV was measured. The effect of wrapping the crystal with a PTFE layer, with the aim of maximizing the light collection, is also presented

Whole Genome Sequence Analysis of a Large Isoniazid-Resistant Tuberculosis Outbreak in London: A Retrospective Observational Study

BACKGROUND: A large isoniazid-resistant tuberculosis outbreak centred on London, United Kingdom, has been ongoing since 1995. The aim of this study was to investigate the power and value of whole genome sequencing (WGS) to resolve the transmission network compared to current molecular strain typing approaches, including analysis of intra-host diversity within a specimen, across body sites, and over time, with identification of genetic factors underlying the epidemiological success of this cluster. METHODS AND FINDINGS: We sequenced 344 outbreak isolates from individual patients collected over 14 y (2 February 1998-22 June 2012). This demonstrated that 96 (27.9%) were indistinguishable, and only one differed from this major clone by more than five single nucleotide polymorphisms (SNPs). The maximum number of SNPs between any pair of isolates was nine SNPs, and the modal distance between isolates was two SNPs. WGS was able to reveal the direction of transmission of tuberculosis in 16 cases within the outbreak (4.7%), including within a multidrug-resistant cluster that carried a rare rpoB mutation associated with rifampicin resistance. Eleven longitudinal pairs of patient pulmonary isolates collected up to 48 mo apart differed from each other by between zero and four SNPs. Extrapulmonary dissemination resulted in acquisition of a SNP in two of five cases. WGS analysis of 27 individual colonies cultured from a single patient specimen revealed ten loci differed amongst them, with a maximum distance between any pair of six SNPs. A limitation of this study, as in previous studies, is that indels and SNPs in repetitive regions were not assessed due to the difficulty in reliably determining this variation. CONCLUSIONS: Our study suggests that (1) certain paradigms need to be revised, such as the 12 SNP distance as the gold standard upper threshold to identify plausible transmissions; (2) WGS technology is helpful to rule out the possibility of direct transmission when isolates are separated by a substantial number of SNPs; (3) the concept of a transmission chain or network may not be useful in institutional or household settings; (4) the practice of isolating single colonies prior to sequencing is likely to lead to an overestimation of the number of SNPs between cases resulting from direct transmission; and (5) despite appreciable genomic diversity within a host, transmission of tuberculosis rarely results in minority variants becoming dominant. Thus, whilst WGS provided some increased resolution over variable number tandem repeat (VNTR)-based clustering, it was insufficient for inferring transmission in the majority of cases

Moloney murine leukemia virus decay mediated by retroviral reverse transcriptase degradation of genomic RNA

AbstractRetroviral vectors are powerful tools for the introduction of transgenes into mammalian cells and for long-term gene expression. However, their application is often limited by a rapid loss of bioactivity: retroviruses spontaneously loose activity at 37 °C, with a half-life of 4 to 9 h depending on the retrovirus type. We sought to determine which components of the retrovirus are responsible for this loss in bioactivity and to obtain a quantitative characterization of their stability. To this end, we focused on RNA and viral proteins, two major components that we hypothesized may undergo degradation and negatively influence viral infectivity. Reverse transcription PCR (RT-PCR) targeting RNA encoding portions of the viral genome clearly demonstrated time-dependent degradation of RNA which correlated with the loss in viral bioactivity. Circular dichroism spectroscopy, SDS-PAGE and two-dimensional SDS-PAGE analyses of viral proteins did not show any change in secondary structure or evidence of proteolysis. The mechanism underlying the degradation of viral RNA was investigated by site-directed mutagenesis of proteins encoded by the viral genome. Reverse transcriptase and protease mutants exhibited enhanced RNA stability in comparison to wild type recombinant virus, suggesting that the degradation of RNA, and the corresponding virus loss of activity, is mediated by the reverse transcriptase enzyme

New application of superconductors: high sensitivity cryogenic light detectors

In this paper we describe the current status of the CALDER project, which is developing ultra-sensitive light detectors based on superconductors for cryogenic applications. When we apply an AC current to a superconductor, the Cooper pairs oscillate and acquire kinetic inductance, that can be measured by inserting the superconductor in a LC circuit with high merit factor. Interactions in the superconductor can break the Cooper pairs, causing sizable variations in the kinetic inductance and, thus, in the response of the LC circuit. The continuous monitoring of the amplitude and frequency modulation allows to reconstruct the incident energy with excellent sensitivity. This concept is at the basis of Kinetic Inductance Detectors (KIDs), that are characterized by natural aptitude to multiplexed read-out (several sensors can be tuned to different resonant frequencies and coupled to the same line), resolution of few eV, stable behavior over a wide temperature range, and ease in fabrication. We present the results obtained by the CALDER collaboration with 2x2 cm2 substrates sampled by 1 or 4 Aluminum KIDs. We show that the performances of the first prototypes are already competitive with those of other commonly used light detectors, and we discuss the strategies for a further improvement

Characterization of the KID-Based Light Detectors of CALDER

The aim of the Cryogenic wide-Area Light Detectors with Excellent Resolution (CALDER) project is the development of light detectors with active area of $5\times5$ cm$^2$ and noise energy resolution smaller than 20 eV RMS, implementing phonon-mediated kinetic inductance detectors. The detectors are developed to improve the background suppression in large-mass bolometric experiments such as CUORE, via the double read-out of the light and the heat released by particles interacting in the bolometers. In this work, we present the characterization of the first light detectors developed by CALDER. We describe the analysis tools to evaluate the resonator parameters (resonant frequency and quality factors) taking into account simultaneously all the resonance distortions introduced by the read-out chain (as the feed-line impedance and its mismatch) and by the power stored in the resonator itself. We detail the method for the selection of the optimal point for the detector operation (maximizing the signal-to-noise ratio). Finally, we present the response of the detector to optical pulses in the energy range of 0-30 keV