High-throughput isotopic analysis of RNA microarrays to quantify microbial resource use

Most microorganisms remain uncultivated, and typically their ecological roles must be inferred from diversity and genomic studies. To directly measure functional roles of uncultivated microbes, we developed Chip-stable isotope probing (SIP), a high-sensitivity, high-throughput SIP method performed on a phylogenetic microarray (chip). This approach consists of microbial community incubations with isotopically labeled substrates, hybridization of the extracted community rRNA to a microarray and measurement of isotope incorporation—and therefore substrate use—by secondary ion mass spectrometer imaging (NanoSIMS). Laboratory experiments demonstrated that Chip-SIP can detect isotopic enrichment of 0.5 atom % ¹³C and 0.1 atom % ¹⁵N, thus permitting experiments with short incubation times and low substrate concentrations. We applied Chip-SIP analysis to a natural estuarine community and quantified amino acid, nucleic acid or fatty acid incorporation by 81 distinct microbial taxa, thus demonstrating that resource partitioning occurs with relatively simple organic substrates. The Chip-SIP approach expands the repertoire of stable isotope-enabled methods available to microbial ecologists and provides a means to test genomics-generated hypotheses about biogeochemical function in any natural environment.     

Cathepsins X and B can be differentiated through their respective mono- and dipeptidyl carboxypeptidase activities

Several new cysteine proteases of the papain family have been discovered in the past few years. To help in the assignment of physiological roles and in the design of specific inhibitors, a clear picture of the specificities of these enzymes is needed. One of these novel enzymes, cathepsin X, displays a unique specificity, cleaving single amino acid residues at the C-terminus of substrates very efficiently. In this study, the carboxypeptidase activities and substrate specificity of cathepsins X and B have been investigated in detail and compared. Using quenched fluorogenic substrates and HPLC measurements, it was shown that cathepsin X preferentially cleaves substrates through a monopeptidyl carboxypeptidase pathway, while cathepsin B displays a preference for the dipeptidyl pathway. The preference for one or the other pathway is about the same for both enzymes, i.e., approximately 2 orders of magnitude, a result supported by molecular modeling of enzyme-substrate complexes. Cleavage of a C-terminal dipeptide of a substrate by cathepsin X can become more important under conditions that preclude efficient monopeptidyl carboxypeptidase activity, e.g., nonoptimal interactions in subsites S(2)-S(1). These results confirm that cathepsin X is designed to function as a monopeptidyl carboxypeptidase. Contrary to a recent report [Klemencic, I., et al. (2000) Eur. J. Biochem. 267, 5404-5412], it is shown that cathepsins X and B do not share similar activity profiles, and that reagents are available to clearly distinguish the two enzymes. In particular, CA074 was found to inactivate cathepsin B at least 34000-fold more efficiently than cathepsin X. The insights obtained from this and previous studies have been used to produce an inhibitor designed to exploit the unique structural features responsible for the carboxypeptidase activity of cathepsin X. Although of moderate potency, this E-64 derivative is the first reported example of a cathepsin X-specific inhibitor.     

Sandfly-Borne Phlebovirus Isolations from Turkey: New Insight into the Sandfly fever Sicilian and Sandfly fever Naples Species

BackgroundMany studies have presented virus sequences which suggest the existence of a variety of putative new phleboviruses transmitted by sandflies in the Old World. However, in most of these studies, only partial sequences in the polymerase or the nucleoprotein genes were characterised. Therefore to further our understand of the presence and potential medical importance of sandfly-borne phleboviruses that circulate in southern Anatolia, we initiated field campaigns in 2012 and 2013 designed to identify, isolate and characterise phleboviruses in sandflies in this regionConclusions/SignificanceThe results indicate that a variety of phleboviruses are co-circulating in this region of southern Anatolia. Based on our studies, these new viruses clearly belong to genetic groups that include several human pathogens. However, whether or not Toros and Zerdali viruses can infect humans and cause diseases such as sandfly fever remains to be investigated.     

High‐Throughput DNA sequencing of ancient wood

Reconstructing the colonization and demographic dynamics that gave rise to extant forests is essential to forecasts of forest responses to environmental changes. Classical approaches to map how population of trees changed through space and time largely rely on pollen distribution patterns, with only a limited number of studies exploiting DNA molecules preserved in wooden tree archaeological and subfossil remains. Here, we advance such analyses by applying high‐throughput (HTS) DNA sequencing to wood archaeological and subfossil material for the first time, using a comprehensive sample of 167 European white oak waterlogged remains spanning a large temporal (from 550 to 9,800 years) and geographical range across Europe. The successful characterization of the endogenous DNA and exogenous microbial DNA of 140 (~83%) samples helped the identification of environmental conditions favouring long‐term DNA preservation in wood remains, and started to unveil the first trends in the DNA decay process in wood material. Additionally, the maternally inherited chloroplast haplotypes of 21 samples from three periods of forest human‐induced use (Neolithic, Bronze Age and Middle Ages) were found to be consistent with those of modern populations growing in the same geographic areas. Our work paves the way for further studies aiming at using ancient DNA preserved in wood to reconstruct the micro‐evolutionary response of trees to climate change and human forest management.     

Neisseria meningitidis infection of human endothelial cells interferes with leukocyte transmigration by preventing the formation of endothelial docking structures

Neisseria meningitidis elicits the formation of membrane protrusions on vascular endothelial cells, enabling its internalization and transcytosis. We provide evidence that this process interferes with the transendothelial migration of leukocytes. Bacteria adhering to endothelial cells actively recruit ezrin, moesin, and ezrin binding adhesion molecules. These molecules no longer accumulate at sites of leukocyte-endothelial contact, preventing the formation of the endothelial docking structures required for proper leukocyte diapedesis. Overexpression of exogenous ezrin or moesin is sufficient to rescue the formation of docking structures on and leukocyte migration through infected endothelial monolayers. Inversely, expression of the dominant-negative NH(2)-terminal domain of ezrin markedly inhibits the formation of docking structures and leukocyte diapedesis through noninfected monolayers. Ezrin and moesin thus appear as pivotal endothelial proteins required for leukocyte diapedesis that are titrated away by N. meningitidis. These results highlight a novel strategy developed by a bacterial pathogen to hamper the host inflammatory response by interfering with leukocyte-endothelial cell interaction.     

Flow cytometric identification and enumeration of photosynthetic sulfur bacteria and potential for ecophysiological studies at the single-cell level

We show the potential of flow cytometry as a fast tool for population identification and enumeration of photosynthetic sulfur bacteria. Purple (PSB) and green sulfur bacteria (GSB) oxidize hydrogen sulfide to elemental sulfur that can act as storage compound to be further oxidized to sulfate generating the reducing power required for growth. Both groups have different elemental sulfur allocation strategies: whereas PSB store elemental sulfur as intracellular inclusions, GSB allocate sulfur globules externally. We used well-characterized laboratory strains and complex natural photosynthetic populations developing in a sharply stratified meromictic lake to show that PSB and GSB could be detected, differentiated and enumerated in unstained samples using a blue laser-based flow cytometer. Variations in cell-specific pigment content and the dynamics of sulfur accumulation, both intra- and extracellularly, were also detected in flow cytometric plots as sulfur accumulation changed the light scatter characteristics of the cells. These data were used to show the potential for studies on the metabolic status and the rate of activity at the single-cell level. Flow cytometric identification and enumeration resulted in faster and more precise analyses than previous approaches, and may open the door to more complex ecophysiological experiments with photosynthetic sulfur bacteria in mixed cultures and natural environments.     

Comparison of the Belgian Rheumatoid Arthritis Disability Assessment and Health Assessment Questionnaires as Tools to Predict the Need for Support Measures in Patients with Rheumatoid Arthritis

Objective

Scores on the Health Assessment Questionnaire (HAQ) predict the need for support measures in patients with rheumatoid arthritis (RA). In this study we compare the performance of the HAQ in this context with that of the more disease-specific Belgian Rheumatoid Arthritis Disability Assessment (BRADA) questionnaire.

Methods

In this multicenter observational study, patients with RA and disease duration of at least one year who consulted their rheumatologist for a routine follow-up visit filled out the HAQ, and BRADA questionnaires. The performance of HAQ and BRADA to predict the need for support measures available to patients with RA was evaluated using Receiver Operator Characteristic (ROC) curves, with the expert opinion of the rheumatologist as a reference.

Results

The study analyzed data of 301 patients with RA (70.8% females) with mean age 59.8±12.8, disease duration 11.4±9.3 years, and DAS28 values of 2.84±1.18. HAQ scores averaged 0.97±0.73 and BRADA scores were 3.92±3.49 over the last week and 3.89±3.50 over the last 3 months. The area under the ROC curves for the BRADA scores for the support measures investigated ranged from 0.702 to 0.862 and did not differ significantly from those of the HAQ (range 0.725–0.860).

Conclusion

The disease-specific BRADA questionnaire is equivalent to the HAQ in predicting the need for support measures in patients with stable RA.