Delineation of individual human chromosomes in metaphase and interphase cells by in situ suppression hybridization using recombinant DNA libraries

A method of in situ hybridization for visualizing individual human chromosomes from pter to qter, both in metaphase spreads and interphase nuclei, is reported. DNA inserts from a single chromosomal library are labeled with biotin and partially preannealed with a titrated amount of total human genomic DNA prior to hybridization with cellular or chromosomal preparations. The cross-hybridization of repetitive sequences to nontargeted chromosomes can be markedly suppressed under appropriate preannealing conditions. The remaining single-stranded DNA is hybridized to specimens of interest and detected with fluorescent or enzymelabeled avidin conjugates following post-hybridization washes. DNA inserts from recombinant libraries for chromosomes 1, 4, 7, 8, 13, 14, 18, 20, 21, 22, and X were assessed for their ability to decorate specifically their cognate chromosome; most libraries proved to be highly specific. Quantitative densitometric analyses indicated that the ratio of specific to nonspecific hybridization signal under optimal preannealing conditions was at least 8:1. Interphase nuclei showed a cohesive territorial organization of chromosomal domains, and laserscanning confocal fluorescence microscopy was used to aid the 3-D visualization of these domains. This method should be useful for both karyotypic studies and for the analysis of chromosome topography in interphase cells

Sex chromosome positions in human interphase nuclei as studied by in situ hybridization with chromosome specific DNA probes

Two cloned repetitive DNA probes, pXBR and CY1, which bind preferentially to specific regions of the human X and Y chromosome, respectively, were used to study the distribution of the sex chromosomes in human lymphocyte nuclei by in situ hybridization experiments. Our data indicate a large variability of the distances between the sex chromosomes in male and female interphase nuclei. However, the mean distance observed between the X and Y chromosome was significantly smaller than the mean distance observed between the two X-chromosomes. The distribution of distances determined experimentally is compared with three model distributions of distances, and the question of a non-random distribution of sex chromosomes is discussed. Mathematical details of these model distributions are provided in an Appendix to this paper. In the case of a human translocation chromosome (XqterXp22.2::Yq11Y qter) contained in the Chinese hamster x human hybrid cell line 445 x 393, the binding sites of pXBR and CY1 were found close to each other in most interphase nuclei. These data demonstrate the potential use of chromosome-specific repetitive DNA probes to study the problem of interphase chromosome topography

Three-dimensional super-resolution microscopy of the inactive X chromosome territory reveals a collapse of its active nuclear compartment harboring distinct Xist RNA foci

Background: A Xist RNA decorated Barr body is the structural hallmark of the compacted inactive X territory in female mammals. Using super resolution three-dimensional structured illumination microscopy (3D-SIM) and quantitative image analysis, we compared its ultrastructure with active chromosome territories (CTs) in human and mouse somatic cells, and explored the spatio-temporal process of Barr body formation at onset of inactivation in early differentiating mouse embryonic stem cells (ESCs). Results: We demonstrate that all CTs are composed of structurally linked chromatin domain clusters (CDCs). In active CTs the periphery of CDCs harbors low-density chromatin enriched with transcriptionally competent markers, called the perichromatin region (PR). The PR borders on a contiguous channel system, the interchromatin compartment (IC), which starts at nuclear pores and pervades CTs. We propose that the PR and macromolecular complexes in IC channels together form the transcriptionally permissive active nuclear compartment (ANC). The Barr body differs from active CTs by a partially collapsed ANC with CDCs coming significantly closer together, although a rudimentary IC channel system connected to nuclear pores is maintained. Distinct Xist RNA foci, closely adjacent to the nuclear matrix scaffold attachment factor-A (SAF-A) localize throughout Xi along the rudimentary ANC. In early differentiating ESCs initial Xist RNA spreading precedes Barr body formation, which occurs concurrent with the subsequent exclusion of RNA polymerase II (RNAP II). Induction of a transgenic autosomal Xist RNA in a male ESC triggers the formation of an `autosomal Barr body' with less compacted chromatin and incomplete RNAP II exclusion. Conclusions: 3D-SIM provides experimental evidence for profound differences between the functional architecture of transcriptionally active CTs and the Barr body. Basic structural features of CT organization such as CDCs and IC channels are however still recognized, arguing against a uniform compaction of the Barr body at the nucleosome level. The localization of distinct Xist RNA foci at boundaries of the rudimentary ANC may be considered as snap-shots of a dynamic interaction with silenced genes. Enrichment of SAF-A within Xi territories and its close spatial association with Xist RNA suggests their cooperative function for structural organization of Xi

Tuning the energetics and tailoring the optical properties of silver clusters confined in zeolites

The integration of metal atoms and clusters in well-defined dielectric cavities is a powerful strategy to impart new properties to them that depend on the size and geometry of the confined space as well as on metal-host electrostatic interactions. Here, we unravel the dependence of the electronic properties of metal clusters on space confinement by studying the ionization potential of silver clusters embedded in four different zeolite environments over a range of silver concentrations. Extensive characterization reveals a strong influence of silver loading and host environment on the cluster ionization potential, which is also correlated to the cluster's optical and structural properties. Through fine-tuning of the zeolite host environment, we demonstrate photoluminescence quantum yields approaching unity. This work extends our understanding of structure property relationships of small metal clusters and applies this understanding to develop highly photoluminescent materials with potential applications in optoelectronics and bioimaging

Impact of Low-Level-Viremia on HIV-1 Drug-Resistance Evolution among Antiretroviral Treated-Patients

to determine the emergence and evolution of DRAM during LLV in HIV-1-infected patients while receiving antiretroviral therapy (ART).Retrospective analysis of patients presenting a LLV episode defined as pVL between 40 and 500 c/mL on at least 3 occasions during a 6-month period or longer while on the same ART. Resistance genotypic testing was performed at the onset and at the end of LLV period. Emerging DRAM was defined during LLV if never detected on baseline genotype or before.48 patients including 4 naive and 44 pretreated (median 9 years) presented a LLV episode with a median duration of 11 months. Current ART included 2NRTI (94%), ritonavir-boosted PI (94%), NNRTI (23%), and/or raltegravir (19%). Median pVL during LLV was 134 c/mL. Successful resistance testing at both onset and end of the LLV episode were obtained for 37 patients (77%), among who 11 (30%) acquired at least 1 DRAM during the LLV period: for NRTI in 6, for NNRTI in 1, for PI in 4, and for raltegravir in 2. During the LLV period, number of drugs with genotypic resistance increased from a median of 4.5 to 6 drugs. Duration and pVL level of LLV episode, duration of previous ART, current and nadir CD4 count, number of baseline DRAM and GSS were not identified as predictive factors of resistance acquisition during LLV, probably due to limited number of patients.Persistent LLV episodes below 500 c/ml while receiving ART is associated with emerging DRAM for all drug classes and a decreasing in further therapeutic options, suggesting to earlier consider resistance monitoring and ART optimization in this setting

Changes in chromatin structure during processing of wax-embedded tissue sections

The use of immunofluorescence (IF) and fluorescence in situ hybridisation (FISH) underpins much of our understanding of how chromatin is organised in the nucleus. However, there has only recently been an appreciation that these types of study need to move away from cells grown in culture and towards an investigation of nuclear organisation in cells in situ in their normal tissue architecture. Such analyses, however, especially of archival clinical samples, often requires use of formalin-fixed paraffin wax-embedded tissue sections which need addition steps of processing prior to IF or FISH. Here we quantify the changes in nuclear and chromatin structure that may be caused by these additional processing steps. Treatments, especially the microwaving to reverse fixation, do significantly alter nuclear architecture and chromatin texture, and these must be considered when inferring the original organisation of the nucleus from data collected from wax-embedded tissue sections

Increased Resin Collection after Parasite Challenge: A Case of Self-Medication in Honey Bees?

The constant pressure posed by parasites has caused species throughout the animal kingdom to evolve suites of mechanisms to resist infection. Individual barriers and physiological defenses are considered the main barriers against parasites in invertebrate species. However, behavioral traits and other non-immunological defenses can also effectively reduce parasite transmission and infection intensity. In social insects, behaviors that reduce colony-level parasite loads are termed “social immunity.” One example of a behavioral defense is resin collection. Honey bees forage for plant-produced resins and incorporate them into their nest architecture. This use of resins can reduce chronic elevation of an individual bee's immune response. Since high activation of individual immunity can impose colony-level fitness costs, collection of resins may benefit both the individual and colony fitness. However the use of resins as a more direct defense against pathogens is unclear. Here we present evidence that honey bee colonies may self-medicate with plant resins in response to a fungal infection. Self-medication is generally defined as an individual responding to infection by ingesting or harvesting non-nutritive compounds or plant materials. Our results show that colonies increase resin foraging rates after a challenge with a fungal parasite (Ascophaera apis: chalkbrood or CB). Additionally, colonies experimentally enriched with resin had decreased infection intensities of this fungal parasite. If considered self-medication, this is a particularly unique example because it operates at the colony level. Most instances of self-medication involve pharmacophagy, whereby individuals change their diet in response to direct infection with a parasite. In this case with honey bees, resins are not ingested but used within the hive by adult bees exposed to fungal spores. Thus the colony, as the unit of selection, may be responding to infection through self-medication by increasing the number of individuals that forage for resin

Conditional Transgenesis Using Dimerizable Cre (DiCre)

Cre recombinase is extensively used to engineer the genome of experimental animals. However, its usefulness is still limited by the lack of an efficient temporal control over its activity. We have recently developed a conceptually new approach to regulate Cre recombinase, that we have called Dimerizable Cre or DiCre. It is based on splitting Cre into two inactive moieties and fusing them to FKBP12 (FK506-binding protein) and FRB (binding domain of the FKBP12-rapamycin associated protein), respectively. These latter can be efficiently hetero-dimerized by rapamycin, leading to the reinstatement of Cre activity. We have been able to show, using in vitro approaches, that this ligand-induced dimerization is an efficient way to regulate Cre activity, and presents a low background activity together with a high efficiency of recombination following dimerization. To test the in vivo performance of this system, we have, in the present work, knocked-in DiCre into the Rosa26 locus of mice. To evaluate the performance of the DiCre system, mice have been mated with indicator mice (Z/EG or R26R) and Cre-induced recombination was examined following activation of DiCre by rapamycin during embryonic development or after birth of progenies. No recombination could be observed in the absence of treatment of the animals, indicating a lack of background activity of DiCre in the absence of rapamycin. Postnatal rapamycin treatment (one to five daily injection, 10 mg/kg i.p) induced recombination in a number of different tissues of progenies such as liver, heart, kidney, muscle, etc. On the other hand, recombination was at a very low level following in utero treatment of DiCre×R26R mice. In conclusion, DiCre has indeed the potentiality to be used to establish conditional Cre-deleter mice. An added advantage of this system is that, contrary to other modulatable Cre systems, it offers the possibility of obtaining regulated recombination in a combinatorial manner, i.e. induce recombination at any desired time-point specifically in cells characterized by the simultaneous expression of two different promoters

Susceptibility of the human retrovirus XMRV to antiretroviral inhibitors

<p>Abstract</p> <p>Background</p> <p>XMRV (xenotropic murine leukemia virus-related virus) is the first known example of an exogenous gammaretrovirus that can infect humans. A limited number of reports suggest that XMRV is intrinsically resistant to many of the antiretroviral drugs used to treat HIV-1 infection, but is sensitive to a small subset of these inhibitors. In the present study, we used a novel marker transfer assay to directly compare the antiviral drug sensitivities of XMRV and HIV-1 under identical conditions in the same host cell type.</p> <p>Results</p> <p>We extend the findings of previous studies by showing that, in addition to AZT and tenofovir, XMRV and HIV-1 are equally sensitive to AZddA (3'-azido-2',3'-dideoxyadenosine), AZddG (3'-azido-2',3'-dideoxyguanosine) and adefovir. These results indicate that specific 3'-azido or acyclic nucleoside analog inhibitors of HIV-1 reverse transcriptase (RT) also block XMRV infection with comparable efficacy <it>in vitro</it>. Our data confirm that XMRV is highly resistant to the non-nucleoside RT inhibitors nevirapine and efavirenz and to inhibitors of HIV-1 protease. In addition, we show that the integrase inhibitors raltegravir and elvitegravir are active against XMRV, with EC₅₀values in the nanomolar range.</p> <p>Conclusions</p> <p>Our analysis demonstrates that XMRV exhibits a distinct pattern of nucleoside analog susceptibility that correlates with the structure of the pseudosugar moiety and that XMRV is sensitive to a broader range of antiretroviral drugs than has previously been reported. We suggest that the divergent drug sensitivity profiles of XMRV and HIV-1 are partially explained by specific amino acid differences in their respective protease, RT and integrase sequences. Our data provide a basis for choosing specific antiretroviral drugs for clinical studies in XMRV-infected patients.</p

Beyond the particular and universal:dependence, independence, and interdependence of context, justice, and ethics

This article reflects on context effects in the study of behavioral ethics and organizational justice. After a general overview, we review three key challenges confronting research in these two domains. First, we consider social scientific versus normative approaches to inquiry. The former aims for a scientific description, while the latter aims to provide prescriptive advice for moral conduct. We argue that the social scientific view can be enriched by considering normative paradigms. The next challenge we consider, involves the duality of morally upright versus morally inappropriate behavior. We observe that there is a long tradition of categorizing behavior dichotomously (e.g., good vs. bad) rather than continuously. We conclude by observing that more research is needed to compare the dichotomous versus continuous perspectives. Third, we examine the role of “cold” cognitions and “hot” affect in making judgments of ethicality. Historically speaking, research has empathized cognition, though recent work has begun to add greater balance to affective reactions. We argue that both cognition and affect are important, but more research is needed to determine how they work together. After considering these three challenges, we then turn to our special issue, providing short reviews of each contribution and how they help in better addressing the three challenges we have identified