Human Salivary Amylase Gene Copy Number Impacts Oral and Gut Microbiomes

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Geographically contrasting biodiversity reductions in a widespread New Zealand seabird

Unravelling prehistoric anthropogenic impacts on biodiversity represents a key challenge for biologists and archaeologists. New Zealand's endemic Stewart Island Shag (Leucocarbo chalconotus) comprises two distinct phylogeographic lineages, currently restricted to the country's south and southeast. However, fossil and archaeological remains suggest a far more widespread distribution at the time of Polynesian settlement ca. 1280 AD, encompassing much of coastal South Island. We used modern and ancient DNA, radiocarbon dating, and Bayesian modelling, to assess the impacts of human arrival on this taxon. Our analyses show that the southeast South Island (Otago) lineage was formerly widespread across coastal South Island, but experienced dramatic population extinctions, range retraction and lineage loss soon after human arrival. By comparison, the southernmost (Foveaux Strait) lineage has experienced a relatively stable demographic and biogeographic history since human arrival, retaining much of its mitochondrial diversity. Archaeological data suggest that these contrasting demographic histories (retraction vs. stability) reflect differential human impacts in mainland South Island vs. Foveaux Strait, highlighting the importance of testing for temporal and spatial variation in human‐driven faunal declines.     

Neuronal Antibodies in Children with or without Narcolepsy following H1N1-AS03 Vaccination

Type 1 narcolepsy is caused by deficiency of hypothalamic orexin/hypocretin. An autoimmune basis is suspected, but no specific antibodies, either causative or as biomarkers, have been identified. However, the AS03 adjuvanted split virion H1N1 (H1N1-AS03) vaccine, created to protect against the 2009 Pandemic, has been implicated as a trigger of narcolepsy particularly in children. Sera and CSFs from 13 H1N1-AS03-vaccinated patients (12 children, 1 young adult) with type 1 narcolepsy were tested for autoantibodies to known neuronal antigens including the N-methyl-D-aspartate receptor (NMDAR) and contactin-associated protein 2 (CASPR2), both associated with encephalopathies that include disordered sleep, to rodent brain tissue including the lateral hypothalamus, and to live hippocampal neurons in culture. When sufficient sample was available, CSF levels of melanin-concentrating hormone (MCH) were measured. Sera from 44 H1N1-ASO3-vaccinated children without narcolepsy were also examined. None of these patients’ CSFs or sera was positive for NMDAR or CASPR2 antibodies or binding to neurons; 4/13 sera bound to orexin-neurons in rat brain tissue, but also to other neurons. MCH levels were a marginally raised (n = 8; p = 0.054) in orexin-deficient narcolepsy patients compared with orexin-normal children (n = 6). In the 44 H1N1-AS03-vaccinated healthy children, there was no rise in total IgG levels or in CASPR2 or NMDAR antibodies three weeks following vaccination. In conclusion, there were no narcolepsy-specific autoantibodies identified in type 1 narcolepsy sera or CSFs, and no evidence for a general increase in immune reactivity following H1N1-AS03 vaccination in the healthy children. Antibodies to other neuronal specific membrane targets, with their potential for directing use of immunotherapies, are still an important goal for future research.     

Fecal Volatile Organic Ccompound Profiles from White-Tailed Deer (Odocoileus virginianus) as Indicators of Mycobacterium bovis Exposure or Mycobacterium bovis Bacille Calmette-Guerin (BCG) Vaccination

White-tailed deer (Odocoileus virginianus) serve as a reservoir for bovine tuberculosis, caused by Mycobacterium bovis, and can be a source of infection in cattle. Vaccination with M. bovis Bacille Calmette Guerin (BCG) is being considered for management of bovine tuberculosis in deer. Presently, no method exists to non-invasively monitor the presence of bovine tuberculosis in deer. In this study, volatile organic compound profiles of BCG-vaccinated and non-vaccinated deer, before and after experimental challenge with M. bovis strain 95–1315, were generated using solid phase microextraction fiber head-space sampling over suspended fecal pellets with analysis by gas chromatography/mass spectrometry. Chromatograms were processed using XCMS Online to characterize ion variation among treatment groups. The principal component scores resulting from significant (α = 0.05) ion responses were used to build linear discriminant analysis models. The sensitivity and specificity of these models were used to evaluate the feasibility of using this analytical approach to distinguish within group comparisons between pre- and post-M. bovis challenge: non-vaccinated male or female deer, BCG-vaccinated male deer, and the mixed gender non-vaccinated deer data. Seventeen compounds were identified in this analysis. The peak areas for these compounds were used to build a linear discriminant classification model based on principal component analysis scores to evaluate the feasibility of discriminating between fecal samples from M. bovis challenged deer, irrespective of vaccination status. The model best representing the data had a sensitivity of 78.6% and a specificity of 91.4%. The fecal head-space sampling approach presented in this pilot study provides a non-invasive method to discriminate between M. bovis challenged deer and BCG-vaccinated deer. Additionally, the technique may prove invaluable for BCG efficacy studies with free-ranging deer as well as for use as a non-invasive monitoring system for the detection of tuberculosis in captive deer and other livestock.     

Ectopic Expression of a Neospora caninum Kazal Type Inhibitor Triggers Developmental Defects in Toxoplasma and Plasmodium

Regulated proteolysis is known to control a variety of vital processes in apicomplexan parasites including invasion and egress of host cells. Serine proteases have been proposed as targets for drug development based upon inhibitor studies that show parasite attenuation and transmission blockage. Genetic studies suggest that serine proteases, such as subtilisin and rhomboid proteases, are essential but functional studies have proved challenging as active proteases are difficult to express. Proteinaceous Protease Inhibitors (PPIs) provide an alternative way to address the role of serine proteases in apicomplexan biology. To validate such an approach, a Neospora caninum Kazal inhibitor (NcPI-S) was expressed ectopically in two apicomplexan species, Toxoplasma gondii tachyzoites and Plasmodium berghei ookinetes, with the aim to disrupt proteolytic processes taking place within the secretory pathway. NcPI-S negatively affected proliferation of Toxoplasma tachyzoites, while it had no effect on invasion and egress. Expression of the inhibitor in P. berghei zygotes blocked their development into mature and invasive ookinetes. Moreover, ultra-structural studies indicated that expression of NcPI-S interfered with normal formation of micronemes, which was also confirmed by the lack of expression of the micronemal protein SOAP in these parasites. Our results suggest that NcPI-S could be a useful tool to investigate the function of proteases in processes fundamental for parasite survival, contributing to the effort to identify targets for parasite attenuation and transmission blockage.     

Selection of antibodies against a single rare cell present in a heterogeneous population using phage display

Here we describe a new method applying phage-displayed antibody libraries to the selection of antibodies against a single identified cell on a glass slide. This is the only described method that has successfully achieved selection of antibodies against a single rare cell in a heterogeneous population of cells. The phage library is incubated with the slide containing the identified rare cell of interest; incubation is followed by UV irradiation while protecting the target cell with a minute disc. The UV light inactivates all phages outside the shielded area by cross-linking the DNA constituting their genomes. The expected yield is between one and ten phage particles from a single cell selection. The encoded antibodies are subsequently produced monoclonally and tested for specificity. This method can be applied within a week to carry out ten or more individual cell selections. Including subsequent testing of antibody specificity, a specific antibody can be identified within 2 months.     

Structure Distinctions of Pelagic Rotifer Plankton in Stratified Lakes With Different Human Impact

Pelagic rotifer plankton was studied in four stratified lakes with different degrees of human impact from June to July 2001 and throughout 2002. Rotifer species diversity was closely correlated to temperature and oxygen concentration (correlation coefficients were 0.90 and 0.87, respectively) in the water column of the hypertrophic Lake Kruglik. In the mesotrophic lakes, the correlation coefficients were much lower and their reduction was related to decreasing human impact on the lakes. Species richness was similar in Lakes Kruglik and S. Volos, but the spatial structure of the community differed greatly. The maximum rotifer density was observed in the epilimnion of Lake Kruglik, with densities dropping sharply towards the hypolimnion. In the mesotrophic lakes, the highest rotifer density was recorded in the meta- and hypolimnion. A comparative analysis of the morphometric characteristics of Keratella cochlearis showed that (1)␣the lorica length of ovigerous females increased in all four lakes with decreasing temperature; (2) the shortest lorica length was in Lake Kruglik at the same temperature; (3) in the mesotrophic lakes a significant increase in lorica length occurred as the temperature decreased from 14.2 °C to 4.2 °C. There is the similar relationship in rotifers of the genus Filinia. Hypoxia in the clino- and hypolimnion of Lake Kruglik reduced the diversity of spatial niches created by thermal stratification. As a result, the number of non-overlapping niches for rotifers in Lake Kruglik is reduced by a factor of 2–5 compared to that in mesotrophic lakes, but the mean value of the overlapping index is significantly higher.