The Nef-mediated AIDS-like disease of CD4C/human immunodeficiency virus transgenic mice is associated with increased Fas/FasL expression on T cells and T-cell death but is not prevented in Fas-, FasL-, tumor necrosis factor receptor 1-, or interleukin-1beta-converting enzyme-deficient or Bcl2-expressing transgenic mice

CD4(+)- and CD8(+)-T-cell death is a frequent immunological dysfunction associated with the development of human AIDS. We studied a murine model of AIDS, the CD4C/HIV transgenic (Tg) mouse model, to assess the importance of the apoptotic pathway in human immunodeficiency virus type 1 (HIV-1) pathogenesis. In these Tg mice, Nef is the major determinant of the disease and is expressed in immature and mature CD4(+) T cells and in cells of the macrophage/myeloid lineage. We report here a novel AIDS-like phenotype: enhanced death, most likely by apoptosis (as assessed by 7-aminoactinomycin D and annexin V/propidium iodide staining), of Tg thymic and peripheral CD4(+) and CD8(+) T cells. The Tg CD4(+) and CD8(+) T cells were also more susceptible to cell death after activation in vitro in mixed lymph node (LN) cultures. However, activation-induced cell death was not higher in Tg than in non-Tg-purified CD4(+) T cells. In addition, expression of Fas and FasL, assessed by flow cytometry, was increased in CD4(+) and CD8(+) T cells from Tg mice compared to that of non-Tg littermates. Despite the enhanced expression of Fas and FasL on Tg CD4(+) and CD8(+) T cells, Fas (lpr/lpr) and FasL (gld/gld) mutant CD4C/HIV Tg mice developed an AIDS-like disease indistinguishable from lpr/+ and gld/+ CD4C/HIV Tg mice, including loss of CD4(+) T cells. Similarly, CD4C/HIV Tg mice homozygous for mutations of two other genes implicated in cell death (interleukin-1beta-converting enzyme [ICE], tumor necrosis factor receptor 1 [TNFR-1]) developed similar AIDS-like disease as their respective heterozygous controls. Moreover, the double-Tg mice from a cross between the Bcl2/Wehi25 and CD4C/HIV Tg mice showed no major protection against disease. These results represent genetic evidence for the dispensable role of Fas, FasL, ICE, and TNFR-1 on the development of both T-cell loss and organ disease of these Tg mice. They also provide compelling evidence on the lack of protection by Bcl2 against Tg CD4(+)-T-cell death. In view of the high resemblance between numerous phenotypes observed in the CD4C/HIV Tg mice and in human AIDS, our findings are likely to be relevant for the human disease.     

Attenuation of the neuronal stress responsiveness and corticotrophin releasing hormone synthesis after sexual activity in male rats

Beneficial effects of sexual activity and mating on the responsiveness to environmental stress can be observed in humans and other mammalian species alike, but the underlying neurobiological mechanisms are largely unknown. Sexual activity and mating with a receptive female has recently been shown to reduce the subsequent emotional stress response via activation of the brain oxytocin system. Therefore, we investigated the neuronal and hormonal responses to an acute stressor (forced swimming) after mating in male rats.Attenuation of the stress-induced increase of c-fos and CRH mRNA expression within the hypothalamic paraventricular nucleus 4 h after mating revealed that sexual activity reduced neuronal reactivity in this region. However, this effect was independent of oxytocin as oxytocin receptor blockade, by central administration of an oxytocin receptor antagonist, after mating did not prevent the reduced expression of c-fos mRNA in response to stressor exposure. Mating itself stimulated corticotrophin (ACTH) and corticosterone secretion, which was absent in males after contact with an unreceptive female (non-mated group). However, ACTH and corticosterone responses to forced swimming applied either 45 min or 4 h after female contact were similar between mated and non-mated males. These findings provide evidence for a stress-protective effect of sexual activity and mating in male rats and for dissociation between neuronal and neuroendocrine stress responses.     

Sexual conflict over mating in red-sided garter snakes (Thamnophis sirtalis) as indicated by experimental manipulation of genitalia

Sexual conflict over mating can result in sex-specific morphologies and behaviours that allow each sex to exert control over the outcome of reproduction. Genital traits, in particular, are often directly involved in conflict interactions. Via genital manipulation, we experimentally investigated whether genital traits in red-sided garter snakes influence copulation duration and formation of a copulatory plug. The hemipenes of male red-sided garter snakes have a large basal spine that inserts into the female cloaca during mating. We ablated the spine and found that males were still capable of copulation but copulation duration was much shorter and copulatory plugs were smaller than those produced by intact males. We also anaesthetized the female cloacal region and found that anaesthetized females copulated longer than control females, suggesting that female cloacal and vaginal contractions play a role in controlling copulation duration. Both results, combined with known aspects of the breeding biology of red-sided garter snakes, strongly support the idea that sexual conflict is involved in mating interactions in this species. Our results demonstrate the complex interactions among male and female traits generated by coevolutionary processes in a wild population. Such complexity highlights the importance of simultaneous examination of male and female traits.     

Two-component systems in Pseudomonas aeruginosa: why so many?

Screening the Pseudomonas aeruginosa genome has led to the identification of the highest number of putative genes encoding two-component regulatory systems of all bacterial genomes sequenced to date (64 and 63 encoding response regulators and histidine kinases, respectively). Sixteen atypical kinases, among them 11 devoid of an Hpt domain, and three independent Hpt modules were retrieved. These data suggest that P. aeruginosa possesses complex control strategies with which to respond to environmental challenges.     

Unlocking Short Read Sequencing for Metagenomics

Background

Different high-throughput nucleic acid sequencing platforms are currently available but a trade-off currently exists between the cost and number of reads that can be generated versus the read length that can be achieved.

Methodology/Principal Findings

We describe an experimental and computational pipeline yielding millions of reads that can exceed 200 bp with quality scores approaching that of traditional Sanger sequencing. The method combines an automatable gel-less library construction step with paired-end sequencing on a short-read instrument. With appropriately sized library inserts, mate-pair sequences can overlap, and we describe the SHERA software package that joins them to form a longer composite read.

Conclusions/Significance

This strategy is broadly applicable to sequencing applications that benefit from low-cost high-throughput sequencing, but require longer read lengths. We demonstrate that our approach enables metagenomic analyses using the Illumina Genome Analyzer, with low error rates, and at a fraction of the cost of pyrosequencing.     

Altered NMDA receptor trafficking contributes to sleep deprivation-induced hippocampal synaptic and cognitive impairments

Recent evidence indicates that continuous wakefulness (sleep deprivation, SD) causes impairments in behavioral performance and hippocampal long-term potentiation (LTP) in animals. However, the mechanisms by which SD impairs long-term synaptic plasticity and cognitive function are not clear. Here, we report that 24-h SD in mice results in impaired hippocampus-dependent contextual memory and LTP and, unexpectedly, in reductions of the surface expression of NMDA receptor (NMDAR) subunit NR1 and NMDAR-mediated excitatory post-synaptic currents at hippocampal perforant path-dentate granule cell synapses. The results suggest that the reduction of functional NMDAR in hippocampal neurons may underlie the SD-induced deficits in hippocampus-dependent contextual memory and long-term synaptic plasticity.     

A maximum likelihood framework for protein design

Background  

The aim of protein design is to predict amino-acid sequences compatible with a given target structure. Traditionally envisioned as a purely thermodynamic question, this problem can also be understood in a wider context, where additional constraints are captured by learning the sequence patterns displayed by natural proteins of known conformation. In this latter perspective, however, we still need a theoretical formalization of the question, leading to general and efficient learning methods, and allowing for the selection of fast and accurate objective functions quantifying sequence/structure compatibility.     

A proteomic‐based approach for the identification of immunodominant Cryptococcus neoformans proteins

Cryptococcus neoformans is an opportunistic fungal pathogen that can cause life‐threatening meningoencephalitis in immune compromised patients. Previous, studies in our laboratory have shown that prior exposure to an IFN‐γ‐producing C. neoformans strain (H99γ) elicits protective immunity against a second pulmonary C. neoformans challenge. Here, we characterized the antibody response produced in mice protected against experimental pulmonary C. neoformans infection compared to nonprotected mice. Moreover, we evaluated the efficacy of using serum antibody from protected mice to detect immunodominant C. neoformans proteins. Protected mice were shown to produce significantly more C. neoformans‐specific antibodies following a second experimental pulmonary cryptococcal challenge compared to nonprotected mice. Immunoblot analysis of C. neoformans proteins resolved by 2‐DE using serum from nonprotected mice failed to show any reactivity. In contrast, serum from protected mice was reactive with several cryptococcal protein spots. Analysis of these spots by capillary HPLC‐ESI‐MS/MS identified several cryptococcal proteins shown to be associated with the pathogenesis of cryptococcosis. Our studies demonstrate that mice immunized with C. neoformans strain H99γ produce antibodies that are immune reactive against specific cryptococcal proteins that may provide a basis for the development of immune based therapies that induce protective anticryptococcal immune responses.