How to choose a biodiversity indicator – Redundancy and complementarity of biodiversity metrics in a freshwater ecosystem

A range of biodiversity metrics are available to assess the ecological integrity of aquatic ecosystems. However, performance varies considerably among different types of metrics and provides different information regarding ecosystem conditions, thus making difficult the selection of appropriate metrics for biomonitoring. The present study evaluated the robustness of six biodiversity metrics to assess environmental change and determine their utility as relevant indicators of ecosystem biodiversity and functionality. Traditional metrics such as species richness and Shannon diversity were considered along with alternative metrics such as functional diversity, size diversity and taxonomic distinctness. To that end, invertebrate assemblages in a river floodplain were used as a case study to evaluate the performance of metrics using Generalized Additive Models (GAM). GAM explained between eight and 49% of the variability in biodiversity. The regression models exhibited differences in the response of biodiversity indicators to environmental factors, suggesting that intermediate levels of turbidity and low salinity are conditions favouring increased biodiversity in the study area. Based on correlations among metrics and responses to primary environmental factors, it is concluded that Shannon and functional diversity, and rarefied species richness generated similar information regarding ecosystem conditions (i.e., the metrics were redundant); while size diversity and distinctness provided useful additional data characterizing ecosystem quality (i.e., the metrics were complementary). Functional diversity indicated not only number and dominance of species, but also each species functional role in the community, and was therefore the most informative biodiversity metric. Nevertheless, the use of a combination of metrics, for example functional and size diversity, and variation in taxonomic distinctness, provides complementary data that will serve to achieve a more thorough understanding of ecosystem structure and function, and response to primary environmental influences.     

Tyrosine 656 in topoisomerase IIβ is important for the catalytic activity of the enzyme: Identification based on artifactual +80-Da modification at this site

Topoisomerase (topo) II catalyzes topological changes in DNA. Although both human isozymes, topo IIα and β are phosphorylated, site‐specific phosphorylation of topo IIβ is poorly characterized. Using LC‐MS/MS analysis of topo IIβ, cleaved with trypsin, Arg C or cyanogen bromide (CNBr) plus trypsin, we detected four +80‐Da modified sites: tyr656, ser1395, thr1426 and ser1545. Phosphorylation at ser1395, thr1426 and ser1545 was established based on neutral loss of H₃PO₄ (?98 Da) in the CID spectra and on differences in 2‐D‐phosphopeptide maps of ³²P‐labeled wild‐type (WT) and S1395A or T1426A/S1545A mutant topo IIβ. However, phosphorylation at tyr656 could not be verified by 2‐D‐phosphopeptide mapping of ³²P‐labeled WT and Y656F mutant protein or by Western blotting with phosphotyrosine‐specific antibodies. Since the +80‐Da modification on tyr656 was observed exclusively during cleavage with CNBr and trypsin, this modification likely represented bromination, which occurred during CNBr cleavage. Re‐evaluation of the CID spectra identified +78/+80‐Da fragment ions in CID spectra of two peptides containing tyr656 and tyr711, confirming bromination. Interestingly, mutation of only tyr656, but not ser1395, thr1326 or ser1545, decreased topo IIβ activity, suggesting a functional role for tyr656. These results, while identifying an important tyrosine in topo IIβ, underscore the importance of careful interpretation of modifications having the same nominal mass.     

Using time-resolved fluorescence to measure serum venom-specific IgE and IgG

We adapted DELFIA™ (dissociation-enhanced lanthanide fluoroimmunoassay), a time resolved fluorescence method, to quantitate whole venom specific and allergenic peptide-specific IgE (sIgE), sIgG₁ and sIgG₄ in serum from people clinically allergic to Australian native ant venoms, of which the predominant cause of allergy is jack jumper ant venom (JJAV). Intra-assay CV was 6.3% and inter-assay CV was 13.7% for JJAV sIgE. DELFIA and Phadia CAP JJAV sIgE results correlated well and had similar sensitivity and specificity for the detection of JJAV sIgE against intradermal skin testing as the gold standard. DELFIA was easily adapted for detecting sIgE to a panel of other native ant venoms.     

Mycobacterium tuberculosis lipolytic enzymes as potential biomarkers for the diagnosis of active tuberculosis

Background

New diagnosis tests are urgently needed to address the global tuberculosis (TB) burden and to improve control programs especially in resource-limited settings. An effective in vitro diagnostic of TB based on serological methods would be regarded as an attractive progress because immunoassays are simple, rapid, inexpensive, and may offer the possibility to detect cases missed by standard sputum smear microscopy. However, currently available serology tests for TB are highly variable in sensitivity and specificity. Lipolytic enzymes have recently emerged as key factors in lipid metabolization during dormancy and/or exit of the non-replicating growth phase, a prerequisite step of TB reactivation. The focus of this study was to analyze and compare the potential of four Mycobacterium tuberculosis lipolytic enzymes (LipY, Rv0183, Rv1984c and Rv3452) as new markers in the serodiagnosis of active TB.

Methods

Recombinant proteins were produced and used in optimized ELISA aimed to detect IgG and IgM serum antibodies against the four lipolytic enzymes. The capacity of the assays to identify infection was evaluated in patients with either active TB or latent TB and compared with two distinct control groups consisting of BCG-vaccinated blood donors and hospitalized non-TB individuals.

Results

A robust humoral response was detected in patients with active TB whereas antibodies against lipolytic enzymes were infrequently detected in either uninfected groups or in subjects with latent infection. High specifity levels, ranging from 93.9% to 97.5%, were obtained for all four antigens with sensitivity values ranging from 73.4% to 90.5%, with Rv3452 displaying the highest performances. Patients with active TB usually exhibited strong IgG responses but poor IgM responses.

Conclusion

These results clearly indicate that the lipolytic enzymes tested are strongly immunogenic allowing to distinguish active from latent TB infections. They appear as potent biomarkers providing high sensitivity and specificity levels for the immunodiagnosis of active TB.     

Development and characterisation of 20 microsatellite loci isolated from the large bent-wing bat, Miniopterus schreibersii (Chiroptera: Miniopteridae) and their cross-taxa utility in the family Miniopteridae

The large bent-wing bat, Miniopterus schreibersii (Kuhl 1819), has a long history of taxonomic uncertainty and many populations are known to be in a state of decline. Microsatellite loci were developed for the taxonomic and population genetic assessment of the Australian complex of this species. Of the 33 primer sets designed for this research, seven (21%) were deemed suitably polymorphic for population-level analyses of the Australian taxa, with five (71%) of these loci revealing moderate to high levels of polymorphism (PIC = 0.56 to 0.91). The cross-taxa utility of the M. schreibersii microsatellite markers was assessed in the microbat (Chiroptera) family Miniopteridae. Sub-species and species covering the Miniopteridae's global distribution (with the exception of the Middle East) were selected, numbering 25 taxa in total. Amplification was successful for 26 loci, of which 20 (77%) were polymorphic. High cross-taxa utility of markers was observed with amplification achieved for all taxa for between four (20%) and 20 (100%) loci, and polymorphism was considered moderate to high (PIC = 0.47-0.91) for 12 (60%) of these loci. The high cross-taxa utility of the microsatellites reported herein reveal versatile and cost-effective molecular markers, contributing an important genetic resource for the research and conservation of Miniopteridae species worldwide.     

RET mutational spectrum in Hirschsprung disease: evaluation of 601 Chinese patients

Rare (RVs) and common variants of the RET gene contribute to Hirschsprung disease (HSCR; congenital aganglionosis). While RET common variants are strongly associated with the commonest manifestation of the disease (males; short-segment aganglionosis; sporadic), rare coding sequence (CDS) variants are more frequently found in the lesser common and more severe forms of the disease (females; long/total colonic aganglionosis; familial).Here we present the screening for RVs in the RET CDS and intron/exon boundaries of 601 Chinese HSCR patients, the largest number of patients ever reported. We identified 61 different heterozygous RVs (50 novel) distributed among 100 patients (16.64%). Those include 14 silent, 29 missense, 5 nonsense, 4 frame-shifts, and one in-frame amino-acid deletion in the CDS, two splice-site deletions, 4 nucleotide substitutions and a 22-bp deletion in the intron/exon boundaries and 1 single-nucleotide substitution in the 5' untranslated region. Exonic variants were mainly clustered in RET the extracellular domain. RET RVs were more frequent among patients with the most severe phenotype (24% vs. 15% in short-HSCR). Phasing RVs with the RET HSCR-associated haplotype suggests that RVs do not underlie the undisputable association of RET common variants with HSCR. None of the variants were found in 250 Chinese controls.     

DNA-based faecal dietary analysis: a comparison of qPCR and high throughput sequencing approaches

The genetic analysis of faecal material represents a relatively non-invasive way to study animal diet and has been widely adopted in ecological research. Due to the heterogeneous nature of faecal material the primary obstacle, common to all genetic approaches, is a means to dissect the constituent DNA sequences. Traditionally, bacterial cloning of PCR amplified products was employed; less common has been the use of species-specific quantitative PCR (qPCR) assays. Currently, with the advent of High-Throughput Sequencing (HTS) technologies and indexed primers it has become possible to conduct genetic audits of faecal material to a much greater depth than previously possible. To date, no studies have systematically compared the estimates obtained by HTS with that of qPCR. What are the relative strengths and weaknesses of each technique and how quantitative are deep-sequencing approaches that employ universal primers? Using the locally threatened Little Penguin (Eudyptula minor) as a model organism, it is shown here that both qPCR and HTS techniques are highly correlated and produce strikingly similar quantitative estimates of fish DNA in faecal material, with no statistical difference. By designing four species-specific fish qPCR assays and comparing the data to the same four fish in the HTS data it was possible to directly compare the strengths and weaknesses of both techniques. To obtain reproducible quantitative data one of the key, and often overlooked, steps common to both approaches is ensuring that efficient DNA isolation methods are employed and that extracts are free of inhibitors. Taken together, the methodology chosen for long-term faecal monitoring programs is largely dependent on the complexity of the prey species present and the level of accuracy that is desired. Importantly, these methods should not be thought of as mutually exclusive, as the use of both HTS and qPCR in tandem will generate datasets with the highest fidelity.     

Microcolony cultivation on a soil substrate membrane system selects for previously uncultured soil bacteria

Traditional microbiological methods of cultivation recover less than 1% of the total bacterial species, and the culturable portion of bacteria is not representative of the total phylogenetic diversity. Classical cultivation strategies are now known to supply excessive nutrients to a system and therefore select for fast-growing bacteria that are capable of colony or biofilm formation. New approaches to the cultivation of bacteria which rely on growth in dilute nutrient media or simulated environments are beginning to address this problem of selection. Here we describe a novel microcultivation method for soil bacteria that mimics natural conditions. Our soil slurry membrane system combines a polycarbonate membrane as a growth support and soil extract as the substrate. The result is abundant growth of uncharacterized bacteria as microcolonies. By combining microcultivation with fluorescent in situ hybridization, previously "unculturable" organisms belonging to cultivated and noncultivated divisions, including candidate division TM7, can be identified by fluorescence microscopy. Successful growth of soil bacteria as microcolonies confirmed that the missing culturable majority may have a growth strategy that is not observed when traditional cultivation indicators are used.