The impact of point mutations in the human androgen receptor : classification of mutations on the basis of transcriptional activity

Peer reviewedPublisher PD

Almost There: Transmission Routes of Bacterial Symbionts between Trophic Levels

Many intracellular microbial symbionts of arthropods are strictly vertically transmitted and manipulate their host's reproduction in ways that enhance their own transmission. Rare horizontal transmission events are nonetheless necessary for symbiont spread to novel host lineages. Horizontal transmission has been mostly inferred from phylogenetic studies but the mechanisms of spread are still largely a mystery. Here, we investigated transmission of two distantly related bacterial symbionts – Rickettsia and Hamiltonella – from their host, the sweet potato whitefly, Bemisia tabaci, to three species of whitefly parasitoids: Eretmocerus emiratus, Eretmocerus eremicus and Encarsia pergandiella. We also examined the potential for vertical transmission of these whitefly symbionts between parasitoid generations. Using florescence in situ hybridization (FISH) and transmission electron microscopy we found that Rickettsia invades Eretmocerus larvae during development in a Rickettsia-infected host, persists in adults and in females, reaches the ovaries. However, Rickettsia does not appear to penetrate the oocytes, but instead is localized in the follicular epithelial cells only. Consequently, Rickettsia is not vertically transmitted in Eretmocerus wasps, a result supported by diagnostic polymerase chain reaction (PCR). In contrast, Rickettsia proved to be merely transient in the digestive tract of Encarsia and was excreted with the meconia before wasp pupation. Adults of all three parasitoid species frequently acquired Rickettsia via contact with infected whiteflies, most likely by feeding on the host hemolymph (host feeding), but the rate of infection declined sharply within a few days of wasps being removed from infected whiteflies. In contrast with Rickettsia, Hamiltonella did not establish in any of the parasitoids tested, and none of the parasitoids acquired Hamiltonella by host feeding. This study demonstrates potential routes and barriers to horizontal transmission of symbionts across trophic levels. The possible mechanisms that lead to the differences in transmission of species of symbionts among species of hosts are discussed

Characterisation of Peptide Microarrays for Studying Antibody-Antigen Binding Using Surface Plasmon Resonance Imagery

BACKGROUND: Non-specific binding to biosensor surfaces is a major obstacle to quantitative analysis of selective retention of analytes at immobilized target molecules. Although a range of chemical antifouling monolayers has been developed to address this problem, many macromolecular interactions still remain refractory to analysis due to the prevalent high degree of non-specific binding. We describe how we use the dynamic process of the formation of self assembling monolayers and optimise physical and chemical properties thus reducing considerably non-specific binding and allowing analysis of specific binding of analytes to immobilized target molecules. METHODOLOGY/PRINCIPAL FINDINGS: We illustrate this approach by the production of specific protein arrays for the analysis of interactions between the 65kDa isoform of human glutamate decarboxylase (GAD65) and a human monoclonal antibody. Our data illustrate that we have effectively eliminated non-specific interactions with the surface containing the immobilised GAD65 molecules. The findings have several implications. First, this approach obviates the dubious process of background subtraction and gives access to more accurate kinetic and equilibrium values that are no longer contaminated by multiphase non-specific binding. Second, an enhanced signal to noise ratio increases not only the sensitivity but also confidence in the use of SPR to generate kinetic constants that may then be inserted into van't Hoff type analyses to provide comparative DeltaG, DeltaS and DeltaH values, making this an efficient, rapid and competitive alternative to ITC measurements used in drug and macromolecular-interaction mechanistic studies. Third, the accuracy of the measurements allows the application of more intricate interaction models than simple Langmuir monophasic binding. CONCLUSIONS: The detection and measurement of antibody binding by the type 1 diabetes autoantigen GAD65 represents an example of an antibody-antigen interaction where good structural, mechanistic and immunological data are available. Using SPRi we were able to characterise the kinetics of the interaction in greater detail than ELISA/RIA methods. Furthermore, our data indicate that SPRi is well suited to a multiplexed immunoassay using GAD65 proteins, and may be applicable to other biomarkers

Generation of tumour-specific cytotoxic T-cell clones from histocompatibility leucocyte antigen-identical siblings of patients with melanoma

Lymphodepletion and infusion of autologous expanded tumour-infiltrating lymphocytes is effective therapy for patients with malignant melanoma. Antitumour responses are likely to be mediated by HLA class I- and II-restricted immune responses directed at tumour antigens. We assessed whether the peripheral blood of normal HLA-matched siblings of patients with melanoma could be used to generate lymphocytes with antimelanoma activity for adoptive immunotherapy after allogeneic blood or marrow transplantation. Melanoma cell lines were derived from two donors and were used to stimulate the mononuclear cells of three HLA-identical siblings. CD4+ clones dominated cultures. Of these, approximately half were directly cytotoxic towards recipient melanoma cells and secreted interferon-γ in response to tumour stimulation. More than half of the noncytotoxic clones also secreted interferon-γ after melanoma stimulation. No CD4+ clones responded to stimulation with recipient haemopoietic cells. The majority of CD8+ clones directly lysed recipient melanoma, but did not persist in long-term culture in vitro. No crossreactivity with recipient haemopoietic cells was observed. The antigenic target of one CD4+ clone was determined to be an HLA-DR11-restricted MAGE-3 epitope. Antigenic targets of the remaining clones were not elucidated, but appeared to be restricted through a non-HLA-DR class II molecule. We conclude that the blood of allogeneic HLA-matched sibling donors contains melanoma-reactive lymphocyte precursors directed at tumour-associated antigens. Adoptive immunotherapy with unselected or ex vivo-stimulated donor lymphocytes after allogeneic stem cell transplantation has a rational basis for the treatment of malignant melanoma

Anti-tumour activity and toxicity of the new prodrug9-aminocamptothecin glucuronide (9ACG) in mice

Cancer chemotherapy is limited by the modest therapeutic index of most antineoplastic drugs. Some glucuronide prodrugs may display selective anti-tumour activity against tumours that accumulate β-glucuronidase. We examined the toxicity and anti-tumour activity of 9-aminocamptothecin glucuronide, a new glucuronide prodrug of 9-aminocamptothecin, to evaluate its potential clinical utility. 9-aminocamptothecin glucuronide was 25–60 times less toxic than 9-aminocamptothecin to five human cancer cell lines. β-glucuronidase activated 9-aminocamptothecin glucuronide to produce similar cell killing as 9-aminocamptothecin or topotecan. The in vivo toxicity of 9-aminocamptothecin glucuronide in BALB/c mice was dose-, route-, sex- and age-dependent. 9-aminocamptothecin glucuronide was significantly less toxic to female than to male mice but the difference decreased with age. 9-aminocamptothecin glucuronide and 9-aminocamptothecin produced similar inhibition (∼80%) of LS174T human colorectal carcinoma tumours. 9-aminocamptothecin glucuronide cured a high percentage of CL1-5 human lung cancer xenografts with efficacy that was similar to or greater than 9-aminocamptothecin, irinotecan and topotecan. The potent anti-tumour activity of 9-aminocamptothecin glucuronide suggests that this prodrug should be further evaluated for cancer treatment

Preconditioning Involves Selective Mitophagy Mediated by Parkin and p62/SQSTM1

Autophagy-dependent mitochondrial turnover in response to cellular stress is necessary for maintaining cellular homeostasis. However, the mechanisms that govern the selective targeting of damaged mitochondria are poorly understood. Parkin, an E3 ubiquitin ligase, has been shown to be essential for the selective clearance of damaged mitochondria. Parkin is expressed in the heart, yet its function has not been investigated in the context of cardioprotection. We previously reported that autophagy is required for cardioprotection by ischemic preconditioning (IPC). In the present study, we used simulated ischemia (sI) in vitro and IPC of hearts to investigate the role of Parkin in mediating cardioprotection ex vivo and in vivo. In HL-1 cells, sI induced Parkin translocation to mitochondria and mitochondrial elimination. IPC induced Parkin translocation to mitochondria in Langendorff-perfused rat hearts and in vivo in mice subjected to regional IPC. Mitochondrial depolarization with an uncoupling agent similarly induced Parkin translocation to mitochondria in cells and Langendorff-perfused rat hearts. Mitochondrial loss was blunted in Atg5-deficient cells, revealing the requirement for autophagy in mitochondrial elimination. Consistent with previous reports indicating a role for p62/SQSTM1 in mitophagy, we found that depletion of p62 attenuated mitophagy and exacerbated cell death in HL-1 cardiomyocytes subjected to sI. While wild type mice showed p62 translocation to mitochondria and an increase in ubiquitination, Parkin knockout mice exhibited attenuated IPC-induced p62 translocation to the mitochondria. Importantly, ablation of Parkin in mice abolished the cardioprotective effects of IPC. These results reveal for the first time the crucial role of Parkin and mitophagy in cardioprotection

Genetics of decayed sexual traits in a parasitoid wasp with endosymbiont-induced asexuality.

Trait decay may occur when selective pressures shift, owing to changes in environment or life style, rendering formerly adaptive traits non-functional or even maladaptive. It remains largely unknown if such decay would stem from multiple mutations with small effects or rather involve few loci with major phenotypic effects. Here, we investigate the decay of female sexual traits, and the genetic causes thereof, in a transition from haplodiploid sexual reproduction to endosymbiont-induced asexual reproduction in the parasitoid wasp Asobara japonica. We take advantage of the fact that asexual females cured of their endosymbionts produce sons instead of daughters, and that these sons can be crossed with sexual females. By combining behavioral experiments with crosses designed to introgress alleles from the asexual into the sexual genome, we found that sexual attractiveness, mating, egg fertilization and plastic adjustment of offspring sex ratio (in response to variation in local mate competition) are decayed in asexual A. japonica females. Furthermore, introgression experiments revealed that the propensity for cured asexual females to produce only sons (because of decayed sexual attractiveness, mating behavior and/or egg fertilization) is likely caused by recessive genetic effects at a single locus. Recessive effects were also found to cause decay of plastic sex-ratio adjustment under variable levels of local mate competition. Our results suggest that few recessive mutations drive decay of female sexual traits, at least in asexual species deriving from haplodiploid sexual ancestors

Integration of Sensory and Reward Information during Perceptual Decision-Making in Lateral Intraparietal Cortex (LIP) of the Macaque Monkey

Single neurons in cortical area LIP are known to carry information relevant to both sensory and value-based decisions that are reported by eye movements. It is not known, however, how sensory and value information are combined in LIP when individual decisions must be based on a combination of these variables. To investigate this issue, we conducted behavioral and electrophysiological experiments in rhesus monkeys during performance of a two-alternative, forced-choice discrimination of motion direction (sensory component). Monkeys reported each decision by making an eye movement to one of two visual targets associated with the two possible directions of motion. We introduced choice biases to the monkeys' decision process (value component) by randomly interleaving balanced reward conditions (equal reward value for the two choices) with unbalanced conditions (one alternative worth twice as much as the other). The monkeys' behavior, as well as that of most LIP neurons, reflected the influence of all relevant variables: the strength of the sensory information, the value of the target in the neuron's response field, and the value of the target outside the response field. Overall, detailed analysis and computer simulation reveal that our data are consistent with a two-stage drift diffusion model proposed by Diederich and Bussmeyer [1] for the effect of payoffs in the context of sensory discrimination tasks. Initial processing of payoff information strongly influences the starting point for the accumulation of sensory evidence, while exerting little if any effect on the rate of accumulation of sensory evidence

Genetics of decayed sexual traits in a parasitoid wasp with endosymbiont-induced asexuality

Trait decay may occur when selective pressures shift, owing to changes in environment or life style, rendering formerly adaptive traits non-functional or even maladaptive. It remains largely unknown if such decay would stem from multiple mutations with small effects or rather involve few loci with major phenotypic effects. Here, we investigate the decay of female sexual traits, and the genetic causes thereof, in a transition from haplodiploid sexual reproduction to endosymbiont-induced asexual reproduction in the parasitoid wasp Asobara japonica. We take advantage of the fact that asexual females cured of their endosymbionts produce sons instead of daughters, and that these sons can be crossed with sexual females. By combining behavioral experiments with crosses designed to introgress alleles from the asexual into the sexual genome, we found that sexual attractiveness, mating, egg fertilization and plastic adjustment of offspring sex ratio (in response to variation in local mate competition) are decayed in asexual A. japonica females. Furthermore, introgression experiments revealed that the propensity for cured asexual females to produce only sons (because of decayed sexual attractiveness, mating behavior and/or egg fertilization) is likely caused by recessive genetic effects at a single locus. Recessive effects were also found to cause decay of plastic sex-ratio adjustment under variable levels of local mate competition. Our results suggest that few recessive mutations drive decay of female sexual traits, at least in asexual species deriving from haplodiploid sexual ancestors