Interspecific associations between circulating antioxidant levels and life-history variation in birds

Antioxidants play an important role in protecting tissues against aging-associated oxidative damage and are thus prime candidates for relating physiological mechanisms to variation in life histories. We measured total antioxidant capacity, antioxidant response to stress, and levels of uric acid, vitamin E, and four carotenoids in 95 avian species, mostly passerines from Michigan or Panama. We compared antioxidant measures to seven variables related to life histories (clutch size, survival rate, incubation period, nestling period, basal metabolic rate, body mass, and whether the species lived in a tropical or temperate climate). Life-history-related traits varied over at least three statistically independent axes. Higher antioxidant levels were generally characteristic of more rapid development, lower survival rate, smaller body size, larger clutch size, and higher mass-adjusted metabolic rate, but the relationships of particular antioxidants with individual life-history traits showed considerable complexity. Antioxidant-life history associations differed between tropical and temperate species and varied with respect to taxonomic sampling. Vitamin E showed few relationships with life-history traits. Overall, our results partly support the hypothesis that antioxidant levels evolve to mirror free radical production. Clearly, however, the complex patterns of physiological diversification observed here result from the interplay of many factors, likely including not just investment in somatic maintenance but also phylogenetic constraint, diet, and other aspects of ecology.     

Web building flexibility of an orb‐web spider in a heterogeneous agricultural landscape

Intensification of land‐use in agricultural landscapes is responsible for a decline of biodiversity which provide important ecosystem services like pest‐control. Changes in landscape composition may also induce behavioural changes of predators in response to variation in the biotic or abiotic environment. By controlling for environmentally confounding factors, we here demonstrate that the orb web spider Araneus diadematus alters its web building behaviour in response to changes in the composition of agricultural landscapes. Thereby, the species increases its foraging efficiency (i.e. investments in silk and web asymmetry) with an increase of agricultural land‐use at intermediate spatial scales. This intensification is also related to a decrease in the abundance of larger prey. A negative effect of landscape properties at similar spatial scales on spider fitness was recorded when controlling for relative investments in capture thread length. This study consequently documents the web building flexibility in response to changes in landscape composition, possibly due to changes in prey availability.     

Reduced protein diffusion rate by cytoskeleton in vegetative and polarized dictyostelium cells

Fluorescence recovery after photobleaching measurements with high spatial resolution are performed to elucidate the impact of the actin cytoskeleton on translational mobility of green fluorescent protein (GFP) in aqueous domains of Dictyostelium discoideum amoebae. In vegetative Dictyostelium cells, GFP molecules experience a 3.6-fold reduction of their translational mobility relative to dilute aqueous solutions. In disrupting the actin filamentous network using latrunculin-A, the intact actin cytoskeletal network is shown to contribute an effective viscosity of 1.36 cP, which accounts for 53% of the restrained molecular diffusion of GFP. The remaining 47% of hindered protein motions is ascribed to other mechanical barriers and the viscosity of the cell liquid. A direct correlation between the density of the actin network and its limiting action on protein diffusion is furthermore established from measurements under different osmotic conditions. In highly locomotive polarized cells, the obstructing effect of the actin filamentous network is seen to decline to 0.46 cP in the non-cortical regions of the cell. Our results indicate that the meshwork of actin filaments constitutes the primary mechanical barrier for protein diffusion and that any noticeable reorganization of the network is accompanied by altered intracellular protein mobility.     

Reconstitution of membrane proteins into giant unilamellar vesicles via peptide-induced fusion

In this work, we present a protocol to reconstitute membrane proteins into giant unilamellar vesicles (GUV) via peptide-induced fusion. In principle, GUV provide a well-defined lipid matrix, resembling a close-to-native state for biophysical studies, including optical microspectroscopy, of transmembrane proteins at the molecular level. Furthermore, reconstitution in this manner would also eliminate potential artifacts arising from secondary interactions of proteins, when reconstituted in planar membranes supported on solid surfaces. However, assembly procedures of GUV preclude direct reconstitution. Here, for the first time, a method is described that allows the controlled incorporation of membrane proteins into GUV. We demonstrate that large unilamellar vesicles (LUV, diameter 0.1 microm), to which the small fusogenic peptide WAE has been covalently attached, readily fuse with GUV, as revealed by monitoring lipid and contents mixing by fluorescence microscopy. To monitor contents mixing, a new fluorescence-based enzymatic assay was devised. Fusion does not introduce changes in the membrane morphology, as shown by fluorescence correlation spectroscopy. Analysis of fluorescence confocal imaging intensity revealed that approximately 6 to 10 LUV fused per microm(2) of GUV surface. As a model protein, bacteriorhodopsin (BR) was reconstituted into GUV, using LUV into which BR was incorporated via detergent dialysis. BR did not affect GUV-LUV fusion and the protein was stably inserted into the GUV and functionally active. Fluorescence correlation spectroscopy experiments show that BR inserted into GUV undergoes unrestricted Brownian motion with a diffusion coefficient of 1.2 microm(2)/s. The current procedure offers new opportunities to address issues related to membrane-protein structure and dynamics in a close-to-native state.     

Spatial organization of bacteriorhodopsin in model membranes. Light-induced mobility changes

Bacteriorhodopsin is a proton-transporting membrane protein in Halophilic archaea, and it is considered a prototype of membrane transporters and a model for G-protein-coupled receptors. Oligomerization of the protein has been reported, but it is unknown whether this feature is correlated with, for instance, light activation. Here, we have addressed this issue by reconstituting bacteriorhodopsin into giant unilamellar vesicles. The dynamics of the fully active protein was investigated using fluorescence correlation spectroscopy and freeze fracture electron microscopy. At low protein-to-lipid ratios (<1:10 w/w), a decrease in mobility was observed upon protein photoactivation. This process occurred on a second time scale and was fully reversible, i.e. when the dark-adapted state was reestablished the lateral diffusion rate of the protein was returned to that prior to activation. A similar decrease in lateral mobility as observed upon photoactivation was obtained when bacteriorhodopsin was reconstituted at high protein-to-lipid ratios (>1:10 w/w). We interpret the shifts in mobility during light adaptation as being caused by transient photoinduced oligomerization of bacteriorhodopsin. These observations are fully supported by freeze-fracture electron microscopy, and the size of the clusters during photoactivation was estimated to consist of two or three trimers.     

Preconceptional cystic fibrosis carrier screening: attitudes and intentions of the target population

The aim of this study was to assess the attitudes and intentions of individuals planning a pregnancy with regard to preconceptional cystic fibrosis (CF) carrier screening and to determine factors associated with a positive and negative/neutral intention to have the test. A survey, based on a questionnaire, was conducted among a stratified random sample of 303 recently married couples (606 individuals). Of the eligible individuals, 70% (n = 380) participated. Of the respondents, 73% had a positive attitude toward a routine offer of preconceptional CF carrier screening, and 56% had the intention to participate in a screening program. A positive intention to have the test was associated with high perceived anticipation of regret, intended preconceptional behavior, high perceived pressure from experts, high perceived consequences of the test results, low perceived barriers, and low perceived negative consequences for family members. These results suggest that the offer of routine preconceptional CF carrier screening would lead to substantial acceptance among couples planning a pregnancy. Several variables related with intention were identified.     

Distribution and Impacts of Tasmanian Devil Facial Tumor Disease

The Tasmanian devil, Sarcophilus harrisii, is the largest extant marsupial carnivore. In 1996, a debilitating facial tumor was reported. It is now clear that this is an invariably lethal infectious cancer. The disease has now spread across the majority of the range of the species and is likely to occur across the entire range within 5 to 10 years. The disease has lead to continuing declines of up to 90% and virtual disappearance of older age classes. Mark-recapture analysis and a preliminary epidemiological model developed for the population with the best longitudinal data both project local extinction in that area over a timeframe of 10 to 15 years from disease emergence. However, the prediction of extinction from the model is sensitive to the estimate of the latent period, which is poorly known. As transmission appears to occur by biting, much of which happens during sexual encounters, the dynamics of the disease may be typical of sexually transmitted diseases. This means that transmission is likely to be frequency-dependent with no threshold density for disease maintenance. Extinction over the entire current range of the devil is therefore a real possibility and an unacceptable risk.     

The epithelial polarity regulator LGALS9/galectin-9 induces fatal frustrated autophagy in KRAS mutant colon carcinoma that depends on elevated basal autophagic flux

Oncogenic mutation of KRAS (Kirsten rat sarcoma viral oncogene homolog) in colorectal cancer (CRC) confers resistance to both chemotherapy and EGFR (epidermal growth factor receptor)-targeted therapy. We uncovered that KRAS mutant (KRAS^mut) CRC is uniquely sensitive to treatment with recombinant LGALS9/Galectin-9 (rLGALS9), a recently established regulator of epithelial polarity. Upon treatment of CRC cells, rLGALS9 rapidly internalizes via early- and late-endosomes and accumulates in the lysosomal compartment. Treatment with rLGALS9 is accompanied by induction of frustrated autophagy in KRAS^mut CRC, but not in CRC with BRAF (B-Raf proto-oncogene, serine/threonine kinase) mutations (BRAF^mut). In KRAS^mut CRC, rLGALS9 acts as a lysosomal inhibitor that inhibits autophagosome-lysosome fusion, leading to autophagosome accumulation, excessive lysosomal swelling and cell death. This antitumor activity of rLGALS9 directly correlates with elevated basal autophagic flux in KRAS^mut cancer cells. Thus, rLGALS9 has potent antitumor activity toward refractory KRAS^mut CRC cells that may be exploitable for therapeutic use.     

Analysis of the expressed heavy chain variable-region genes of Macaca fascicularis and isolation of monoclonal antibodies specific for the Ebola virus' soluble glycoprotein

The cynomolgus macaque, Macaca fascicularis, is frequently used in immunological and other biomedical research as a model for man; understanding it's antibody repertoire is, therefore, of fundamental interest. The expressed variable-region gene repertoire of a single M. fascicularis, which was immune to the Ebola virus, was studied. Using 5′ rapid amplification of cDNA ends with immunoglobulin (Ig)G-specific primers, we obtained 30 clones encoding full-length variable, diversity, and joining domains. Similar to the human V_H repertoire, the M. fascicularis repertoire utilized numerous immunoglobulin heavy variable (IGHV) gene fragments, with the V_H3 (41%), V_H4 (39%), and V_H1 (14%) subgroups used more frequently than the V_H5 (3.9%) or V_H7 (1.7%) subgroups. Diverse immunoglobulin heavy joining (IGHJ) fragments also appeared to be utilized, including a putative homolog of JH5β gene segment identified in the related species Macaca mulatta, Rhesus macaque, but not in humans. Although the diverse V region genes in the IgG antibody repertoire of M. fascicularis had likely undergone somatic hypermutations (SHMs), they nevertheless showed high nucleotide identity with the corresponding human germline genes, 80–89% for IGHV and 72–92% for IGHJ. M. fascicularis and human V_H genes were also similar in other aspects: length of complementarity-determining regions and framework regions, and distribution of consensus sites for SHMs. Finally, we demonstrated that monoclonal antibodies (mAbs) specific for an Ebola protein could be obtained from M. fascicularis tissue samples by phage display technology. In summary, the study provides new insight into the M. fascicularis V region gene repertoire and further supports the idea that macaque-derived mAbs may be of therapeutic value to humans.