Experimental Infection of Rhesus Macaques and Common Marmosets with a European Strain of West Nile Virus

West Nile virus (WNV) is a mosquito-borne flavivirus that infects humans and other mammals. In some cases WNV causes severe neurological disease. During recent years, outbreaks of WNV are increasing in worldwide distribution and novel genetic variants of the virus have been detected. Although a substantial amount of data exists on WNV infections in rodent models, little is known about early events during WNV infection in primates, including humans. To gain a deeper understanding of this process, we performed experimental infections of rhesus macaques and common marmosets with a virulent European WNV strain (WNV-Ita09) and monitored virological, hematological, and biochemical parameters. WNV-Ita09 productively infected both monkey species, with higher replication and wider tissue distribution in common marmosets compared to rhesus macaques. The animals in this study however, did not develop clinical signs of WNV disease, nor showed substantial deviations in clinical laboratory parameters. In both species, the virus induced a rapid CD56^dimCD16^bright natural killer response, followed by IgM and IgG antibody responses. The results of this study show that healthy rhesus macaques and common marmosets are promising animal models to study WNV-Ita09 infection. Both models may be particularly of use to evaluate potential vaccine candidates or to investigate WNV pathogenesis.     

Increased Osteoclastogenesis in Mice Lacking the Carcinoembryonic Antigen-Related Cell Adhesion Molecule 1

Alterations in bone remodeling are a major public health issue, as therapeutic options for widespread bone disorders such as osteoporosis and tumor-induced osteolysis are still limited. Therefore, a detailed understanding of the regulatory mechanism governing bone cell differentiation in health and disease are of utmost clinical importance. Here we report a novel function of carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1), a member of the immunoglobulin superfamily involved in inflammation and tumorigenesis, in the physiologic regulation of bone remodeling. Assessing the expression of all members of the murine Ceacam family in bone tissue and marrow, we found CEACAM1 and CEACAM10 to be differentially expressed in both bone-forming osteoblasts and bone-resorbing osteoclasts. While Ceacam10-deficient mice displayed no alteration in structural bone parameters, static histomorphometry demonstrated a reduced trabecular bone mass in mice lacking CEACAM1. Furthermore, cellular and dynamic histomorphometry revealed an increased osteoclast formation in Ceacam1-deficient mice, while osteoblast parameters and the bone formation rate remained unchanged. In line with these findings, we detected accelerated osteoclastogenesis in Ceacam1-deficient bone marrow cells, while osteoblast differentiation, as determined by mineralization and alkaline phosphatase assays, was not affected. Therefore, our results provide in vivo and in vitro evidence for a physiologic role of CEACAM1 in the regulation of osteoclastogenesis.     

Patterns of Spatial and Temporal Distribution of Humpback Whales at the Southern Limit of the Southeast Pacific Breeding Area

Understanding the patterns of spatial and temporal distribution in threshold habitats of highly migratory and endangered species is important for understanding their habitat requirements and recovery trends. Herein, we present new data about the distribution of humpback whales (Megaptera novaeangliae) in neritic waters off the northern coast of Peru: an area that constitutes a transitional path from cold, upwelling waters to warm equatorial waters where the breeding habitat is located. Data was collected during four consecutive austral winter/spring seasons from 2010 to 2013, using whale-watching boats as platforms for research. A total of 1048 whales distributed between 487 groups were sighted. The spatial distribution of humpbacks resembled the characteristic segregation of whale groups according to their size/age class and social context in breeding habitats; mother and calf pairs were present in very shallow waters close to the coast, while dyads, trios or more whales were widely distributed from shallow to moderate depths over the continental shelf break. Sea surface temperatures (range: 18.2–25.9°C) in coastal waters were slightly colder than those closer to the oceanic realm, likely due to the influence of cold upwelled waters from the Humboldt Current system. Our results provide new evidence of the southward extension of the breeding region of humpback whales in the Southeast Pacific. Integrating this information with the knowledge from the rest of the breeding region and foraging grounds would enhance our current understanding of population dynamics and recovery trends of this species.     

Development of neutralizing scFv-Fc against botulinum neurotoxin A light chain from a macaque immune library

Botulinum toxins (BoNTs) are among the most toxic substances on earth, with serotype A toxin being the most toxic substance known. They are responsible for human botulism, a disease characterized by flaccid muscle paralysis that occurs naturally through food poisoning or the colonization of the gastrointestinal tract by BoNT-producing clostridia. BoNT has been classified as a category A agent by the Centers for Disease Control, and it is one of six agents with the highest potential risk of use as bioweapons. Human or human-like neutralizing antibodies are thus required for the development of anti-botulinum toxin drugs to deal with this possibility. In this study, Macaca fascicularis was hyperimmunized with a recombinant light chain of BoNT/A. An immune phage display library was constructed and, after multistep panning, several scFv with nanomolar affinities that inhibited the endopeptidase activity of BoNT/A1 in vitro as scFv-Fc, with a molar ratio (ab binding site:toxin) of up to 1:1, were isolated. The neutralization of BoNT/A-induced paralysis by the SEM120-IID5, SEM120-IIIC1 and SEM120-IIIC4 antibodies was demonstrated in mouse phrenic nerve-hemidiaphragm preparations with the holotoxin. The neutralization observed is the strongest ever measured in the phrenic nerve-hemidiaphragm assay for BoNT/A1 for a monoclonal antibody. Several scFv-Fc inhibiting the endopeptidase activity of botulinum neurotoxin A were isolated. For SEM120-IID5, SEM120-IIIC1, and SEM120-IIIC4, inhibitory effects in vitro and protection against the toxin ex vivo were observed. The human-like nature of these antibodies makes them promising lead candidates for further development of immunotherapeutics for this disease.     

Casein Kinase 2 dependent phosphorylation of eIF4B regulates BACE1 expression in Alzheimer’s disease

Alzheimer’s disease (AD) is the most common age-related neurodegenerative disorder. Increased Aβ production plays a fundamental role in the pathogenesis of the disease and BACE1, the protease that triggers the amyloidogenic processing of APP, is a key protein and a pharmacological target in AD. Changes in neuronal activity have been linked to BACE1 expression and Aβ generation, but the underlying mechanisms are still unclear. We provide clear evidence for the role of Casein Kinase 2 in the control of activity-driven BACE1 expression in cultured primary neurons, organotypic brain slices, and murine AD models. More specifically, we demonstrate that neuronal activity promotes Casein Kinase 2 dependent phosphorylation of the translation initiation factor eIF4B and this, in turn, controls BACE1 expression and APP processing. Finally, we show that eIF4B expression and phosphorylation are increased in the brain of APPPS1 and APP-KI mice, as well as in AD patients. Overall, we provide a definition of a mechanism linking brain activity with amyloid production and deposition, opening new perspectives from the therapeutic standpoint.Subject terms: Kinases, Alzheimer''s disease, Neuronal physiology, Pathogenesis     

Allochthonous resources are less important for faunal communities on highly productive,small tropical islands

Ecosystems are interconnected by energy fluxes that provide resources for the inhabiting organisms along the transition zone. Especially where in situ resources are scarce, ecosystems can become highly dependent on external resources. The dependency on external input becomes less pronounced in systems with elevated in situ production, where only consumer species close to the site of external input remain subsidized, whereas species distant to the input site rely on the in situ production of the ecosystem. It is largely unclear though if this pattern is consistent over different consumer species and trophic levels in one ecosystem, and whether consumer species that occur both proximate to and at a distance from the input site differ in their dependency on external resource inputs between sites. Using stable isotope analysis, we investigated the dependency on external marine input for common ground‐associated consumer taxa on small tropical islands with high in situ production. We show that marine input is only relevant for strict beach‐dwelling taxa, while the terrestrial vegetation is the main carbon source for inland‐dwelling taxa. Consumer species that occurred both close (beach) and distant (inland) to the site of marine input showed similar proportions of marine input in their diets. This supports earlier findings that the relevance of external resources becomes limited to species close to the input site in systems with sufficient in situ production. However, it also indicates that the relevance of external input is also species‐dependent, as consumers occurring close and distant to the input site depended equally strong or weak on marine input.     

Effects of temperature-induced variation in anuran larval growth rate on head width and leg length at metamorphosis

We tested whether temperature-induced variation in the growth rate of Rana cascadae tadpoles caused any variation in head width or leg length at metamorphosis, independent of the effects of temperature on body size. Body-size-adjusted head width appears to be insensitive to even large variations in tadpole growth rate. This result mirrors previous observations on the effects of variation in food level and temperature on metric shape in frogs and other ectothermic vertebrates. Leg length, on the other hand, showed a small but statistically significant response to the temperature treatment. Fast-growing tadpoles attained slightly longer legs than slowly growing tadpoles at a common metamorphic body size. This example is the first to show that variation in growth rate per se can influence metric shape (i.e., the rate at which individuals reach a common body size determines their shape at that size). Nevertheless, the induced effects were small, and our results taken together with those of previous studies suggest that environmentally induced variation in growth rate is not a major source of variation in metric shape of skeletal characters in ectothermic vertebrates.     

Tertiary motifs as building blocks for the design of protein‐binding peptides

Despite advances in protein engineering, the de novo design of small proteins or peptides that bind to a desired target remains a difficult task. Most computational methods search for binder structures in a library of candidate scaffolds, which can lead to designs with poor target complementarity and low success rates. Instead of choosing from pre‐defined scaffolds, we propose that custom peptide structures can be constructed to complement a target surface. Our method mines tertiary motifs (TERMs) from known structures to identify surface‐complementing fragments or “seeds.” We combine seeds that satisfy geometric overlap criteria to generate peptide backbones and score the backbones to identify the most likely binding structures. We found that TERM‐based seeds can describe known binding structures with high resolution: the vast majority of peptide binders from 486 peptide‐protein complexes can be covered by seeds generated from single‐chain structures. Furthermore, we demonstrate that known peptide structures can be reconstructed with high accuracy from peptide‐covering seeds. As a proof of concept, we used our method to design 100 peptide binders of TRAF6, seven of which were predicted by Rosetta to form higher‐quality interfaces than a native binder. The designed peptides interact with distinct sites on TRAF6, including the native peptide‐binding site. These results demonstrate that known peptide‐binding structures can be constructed from TERMs in single‐chain structures and suggest that TERM information can be applied to efficiently design novel target‐complementing binders.