A proteomics approach for the identification and cloning of monoclonal antibodies from serum

We describe a proteomics approach that identifies antigen-specific antibody sequences directly from circulating polyclonal antibodies in the serum of an immunized animal. The approach involves affinity purification of antibodies with high specific activity and then analyzing digested antibody fractions by nano-flow liquid chromatography coupled to tandem mass spectrometry. High-confidence peptide spectral matches of antibody variable regions are obtained by searching a reference database created by next-generation DNA sequencing of the B-cell immunoglobulin repertoire of the immunized animal. Finally, heavy and light chain sequences are paired and expressed as recombinant monoclonal antibodies. Using this technology, we isolated monoclonal antibodies for five antigens from the sera of immunized rabbits and mice. The antigen-specific activities of the monoclonal antibodies recapitulate or surpass those of the original affinity-purified polyclonal antibodies. This technology may aid the discovery and development of vaccines and antibody therapeutics, and help us gain a deeper understanding of the humoral response.     

STED microscopy with optimized labeling density reveals 9-fold arrangement of a centriole protein

Super-resolution fluorescence microscopy can achieve resolution beyond the optical diffraction limit, partially closing the gap between conventional optical imaging and electron microscopy for elucidation of subcellular architecture. The centriole, a key component of the cellular control and division machinery, is 250 nm in diameter, a spatial scale where super-resolution methods such as stimulated emission depletion (STED) microscopy can provide previously unobtainable detail. We use STED with a resolution of 60 nm to demonstrate that the centriole distal appendage protein Cep164 localizes in nine clusters spaced around a ring of ～300 nm in diameter, and quantify the influence of the labeling density in STED immunofluorescence microscopy. We find that the labeling density dramatically influences the observed number, size, and brightness of labeled Cep164 clusters, and estimate the average number of secondary antibody labels per cluster. The arrangements are morphologically similar in centrioles of both proliferating cells and differentiated multiciliated cells, suggesting a relationship of this structure to function. Our STED measurements in single centrioles are consistent with results obtained by electron microscopy, which involve ensemble averaging or very different sample preparation conditions, suggesting that we have arrived at a direct measurement of a centriole protein by careful optimization of the labeling density.     

Trypanosoma cruzi: acute and long-term infection in the vertebrate host can modify the response to benznidazole

We analyzed the influence of Trypanosoma cruzi maintenance in different hosts (dog and mouse) on its susceptibility to benznidazole treatment. Five T. cruzi stocks were isolated from dogs inoculated with Be-62 or Be-78 strain (both sensitive to benznidazole) 2-10 years ago, and the benznidazole sensitivity was then determined using the mouse as experimental model. The different T. cruzi stocks obtained from long-term infected dogs showed 50-90% drug resistance right after isolation. However, maintenance of these T. cruzi stocks in mice, by successive blood passages (2.5 years), led to either a decrease or stability of the drug resistance pattern and an increase in parasite virulence. We also demonstrated the effectiveness of the induction of parasitemia reactivation by cyclophosphamide immunosuppression in the evaluation of the response to the specific drug treatment.     

Trypanosoma cruzi: immunoglobulin isotype profiles during the acute phase of canine experimental infection with metacyclic or blood trypomastigotes

A detailed investigation has been carried out about the serological profiles of groups of dogs experimentally infected with metacyclic (MT) or blood (BT) trypomastigotes of Berenice-78 Trypanosoma cruzi strain. Peripheral blood was collected from infected dogs and uninfected controls, weekly during 35 days following the acute phase of infection, and immunoglobulin profiles were determined by ELISA. Dogs infected with BT exhibited unaltered levels of IgG2, increases in IgM, IgE, IgA, IgG and IgG1. In contrast, dogs infected with MT presented unaltered levels of IgE and IgG1 and an increase in IgM, IgA, IgG and IgG2 levels. Compared with the MT group, animals infected with BT showed significant increases in IgM on days 7, 14 and 28, in IgA on days 7, 14 and 21, in IgE on days 7 and 14, in IgG on days 14 and 28, and in IgG1 on days 7, 14 and 21. Parasitemia levels of the infected animals were measured over the same time period. No correlations were found between the immunoglobulin profiles and the parasitemia levels. The results demonstrated that the inoculum source (BT or MT) influence the immunoglobulin isotype profile that may drive distinct outcome of acute canine Chagas disease.     

Mitochondrial DNA polymorphism A4917G is independently associated with age-related macular degeneration

The objective of this study was to determine if MTND2*LHON4917G (4917G), a specific non-synonymous polymorphism in the mitochondrial genome previously associated with neurodegenerative phenotypes, is associated with increased risk for age-related macular degeneration (AMD). A preliminary study of 393 individuals (293 cases and 100 controls) ascertained at Vanderbilt revealed an increased occurrence of 4917G in cases compared to controls (15.4% vs.9.0%, p = 0.11). Since there was a significant age difference between cases and controls in this initial analysis, we extended the study by selecting Caucasian pairs matched at the exact age at examination. From the 1547 individuals in the Vanderbilt/Duke AMD population association study (including 157 in the preliminary study), we were able to match 560 (280 cases and 280 unaffected) on exact age at examination. This study population was genotyped for 4917G plus specific AMD-associated nuclear genome polymorphisms in CFH, LOC387715 and ApoE. Following adjustment for the listed nuclear genome polymorphisms, 4917G independently predicts the presence of AMD (OR = 2.16, 95%CI 1.20-3.91, p = 0.01). In conclusion, a specific mitochondrial polymorphism previously implicated in other neurodegenerative phenotypes (4917G) appears to convey risk for AMD independent of recently discovered nuclear DNA polymorphisms.     

Ecology and biogeography of the Neotropical otter <Emphasis Type="Italic">Lontra longicaudis</Emphasis>: existing knowledge and open questions

The Neotropical otter, Lontra longicaudis, is considered near threatened by the IUCN, and with declining populations, it is still one of the lesser known species of otter in the world. Like other otter species, L. longicaudis is associated with water courses in a large variety of habitats and biomes across its area of distribution. Here, we present a review of the available literature on L. longicaudis aiming to update information on its distribution, ecology and systematics. We performed online searches using combinations of the words L. longicaudis, river otter, Neotropical otter, nutria and lobito del río, limiting our search to 1972–2016. Recent information on evolutionary history and biogeographic patterns, distribution patterns, as well as on the ecology of the species across a large part of its distribution range, including trophic ecology and intraspecific communication, was retrieved. We found severe gaps in knowledge including aspects of life history, ground validation of new potential distribution areas and long-term population monitoring aiming at designing more efficient conservation measures and techniques. In this context, we conclude by presenting perspectives for future studies.     

A comparison of camera trap and permanent recording video camera efficiency in wildlife underpasses

In the current context of biodiversity loss through habitat fragmentation, the effectiveness of wildlife crossings, installed at great expense as compensatory measures, is of vital importance for ecological and socio‐economic actors. The evaluation of these structures is directly impacted by the efficiency of monitoring tools (camera traps…), which are used to assess the effectiveness of these crossings by observing the animals that use them. The aim of this study was to quantify the efficiency of camera traps in a wildlife crossing evaluation. Six permanent recording video systems sharing the same field of view as six Reconyx HC600 camera traps installed in three wildlife underpasses were used to assess the exact proportion of missed events (event being the presence of an animal within the field of view), and the error rate concerning underpass crossing behavior (defined as either Entry or Refusal). A sequence of photographs was triggered by either animals (true trigger) or artefacts (false trigger). We quantified the number of false triggers that had actually been caused by animals that were not visible on the images (“false” false triggers). Camera traps failed to record 43.6% of small mammal events (voles, mice, shrews, etc.) and 17% of medium‐sized mammal events. The type of crossing behavior (Entry or Refusal) was incorrectly assessed in 40.1% of events, with a higher error rate for entries than for refusals. Among the 3.8% of false triggers, 85% of them were “false” false triggers. This study indicates a global underestimation of the effectiveness of wildlife crossings for small mammals. Means to improve the efficiency are discussed.