Physico-chemical foundations underpinning microarray and next-generation sequencing experiments

Hybridization of nucleic acids on solid surfaces is a key process involved in high-throughput technologies such as microarrays and, in some cases, next-generation sequencing (NGS). A physical understanding of the hybridization process helps to determine the accuracy of these technologies. The goal of a widespread research program is to develop reliable transformations between the raw signals reported by the technologies and individual molecular concentrations from an ensemble of nucleic acids. This research has inputs from many areas, from bioinformatics and biostatistics, to theoretical and experimental biochemistry and biophysics, to computer simulations. A group of leading researchers met in Ploen Germany in 2011 to discuss present knowledge and limitations of our physico-chemical understanding of high-throughput nucleic acid technologies. This meeting inspired us to write this summary, which provides an overview of the state-of-the-art approaches based on physico-chemical foundation to modeling of the nucleic acids hybridization process on solid surfaces. In addition, practical application of current knowledge is emphasized.     

Precision mapping of the human O‐GalNAc glycoproteome through SimpleCell technology

Glycosylation is the most abundant and diverse posttranslational modification of proteins. While several types of glycosylation can be predicted by the protein sequence context, and substantial knowledge of these glycoproteomes is available, our knowledge of the GalNAc‐type O‐glycosylation is highly limited. This type of glycosylation is unique in being regulated by 20 polypeptide GalNAc‐transferases attaching the initiating GalNAc monosaccharides to Ser and Thr (and likely some Tyr) residues. We have developed a genetic engineering approach using human cell lines to simplify O‐glycosylation (SimpleCells) that enables proteome‐wide discovery of O‐glycan sites using ‘bottom‐up’ ETD‐based mass spectrometric analysis. We implemented this on 12 human cell lines from different organs, and present a first map of the human O‐glycoproteome with almost 3000 glycosites in over 600 O‐glycoproteins as well as an improved NetOGlyc4.0 model for prediction of O‐glycosylation. The finding of unique subsets of O‐glycoproteins in each cell line provides evidence that the O‐glycoproteome is differentially regulated and dynamic. The greatly expanded view of the O‐glycoproteome should facilitate the exploration of how site‐specific O‐glycosylation regulates protein function.     

Differences in Fecal Androgen Patterns of Breeding and Nonbreeding Kori Bustards (Ardeotis kori)

To better understand breeding conditions to promote reproduction in captive kori bustards, fundamental endocrine studies measuring fecal androgen metabolites in male and female kori bustards were conducted. Feces collected weekly from males and females were analyzed for testosterone using enzyme‐linked immunoassay. Results from adult males (n = 5), adult females (n = 10), immature males (n = 10), and immature females (n = 10) revealed seasonally elevated testosterone concentrations in fertile, but not nonfertile adult males and females (P > 0.05). Adult females that were not maintained in a breeding group, or that did not produce eggs, did not demonstrate increases in testosterone compared to egg laying counterparts. In males, but not females, seasonal testosterone increases were accompanied by weight gain. Peaks in male fecal androgen metabolites ranged from 10‐ to 22‐fold higher than nonbreeding season (181.5 ± 19.1 vs. 17.0 ± 0.94 ng/g; P < 0.05). Mean breeding season values for adult males were 83.6 ± 6.1 ng/g vs. nonbreeding season values of 12.3 ± 0.73 ng/g (P < 0.05). In females, average breeding season testosterone concentrations were approximately 4‐fold higher than nonbreeding season (55.9 ± 6.0 vs. 14.5 ± 1.8 ng/g), with peaks 10‐ to 30‐fold higher. Results show that noninvasive fecal androgen metabolite analysis can provide a means of predicting fertility potential of male and female kori bustards and might be utilized to assess effects of modifying captive environments to promote reproduction in this species. Zoo Biol. 32:54‐62, 2013. © 2012 Wiley Periodicals, Inc.     

Carbon allocation under light and nitrogen resource gradients in two model marine phytoplankton1

Marine phytoplankton have conserved elemental stoichiometry, but there can be significant deviations from this Redfield ratio. Moreover, phytoplankton allocate reduced carbon (C) to different biochemical pools based on nutritional status and light availability, adding complexity to this relationship. This allocation influences physiology, ecology, and biogeochemistry. Here, we present results on the physiological and biochemical properties of two evolutionarily distinct model marine phytoplankton, a diatom (cf. Staurosira sp. Ehrenberg) and a chlorophyte (Chlorella sp. M. Beijerinck) grown under light and nitrogen resource gradients to characterize how carbon is allocated under different energy and substrate conditions. We found that nitrogen (N)‐replete growth rate increased monotonically with light until it reached a threshold intensity (~200 μmol photons · m^?2 · s^?1). For Chlorella sp., the nitrogen quota (pg · μm^?3) was greatest below this threshold, beyond which it was reduced by the effect of N‐stress, while for Staurosira sp. there was no trend. Both species maintained constant maximum quantum yield of photosynthesis (mol C · mol photons^?1) over the range of light and N‐gradients studied (although each species used different photophysiological strategies). In both species, C:chl a (g · g^?1) increased as a function of light and N‐stress, while C:N (mol · mol^?1) and relative neutral lipid:C (rel. lipid · g^?1) were most strongly influenced by N‐stress above the threshold light intensity. These results demonstrated that the interaction of substrate (N‐availability) and energy gradients influenced C‐allocation, and that general patterns of biochemical responses may be conserved among phytoplankton; they provided a framework for predicting phytoplankton biochemical composition in ecological, biogeochemical, or biotechnological applications.     

Identification of Legionella spp. in Environmental Water Samples by ScanVIT-Legionella™ Method in Spain

Rapid and more sensitive methods for the detection and quantification of viable Legionella cells have been developed. In this paper, a comparative analysis of environmental water samples using the ScanVIT-Legionella? method and the traditional “gold standard” method of culturing is realised indicating the usefulness of the ScanVIT method. The ScanVIT-Legionella? method was performed on environmental water samples from different locations of Huesca region (Spain). Legionella micro-colonies should appear green colour and Legionella pneumophila micro-colonies appear red. Twenty-one environmental water samples analysed by standard culture plus five control samples (Two sterile water samples with Legionella as positive controls and three sterile water samples as negative controls). All of them were used to apply ScanVIT-Legionella? method. From of 21 environmental samples eleven were positive, six negative with both methods and four samples were negative for culture method and positive for ScanVIT-Legionella? method. The positive control samples were positive and the negative were negative for both methods. A comparative analysis of the results obtained with two methods showed a strong positive determination coefficient (R² = 0.99753). The results demonstrate the usefulness of the ScanVIT-Legionella? method as a rapid diagnostic tool in order to provide a diagnosis as quick as possible. ScanVIT-Legionella? method offers a series of advantages such as quickly diagnosis, higher sensitivity and the possibility to identify Legionella spp. and L. pneumophila simultaneously.     

Serologic survey of West Nile virus in horses from Central-West,Northeast and Southeast Brazil

Since the emergence of West Nile virus (WNV) in North America in 1999, there have been several reports of WNV activity in Central and South American countries. To detect WNV in Brazil, we performed a serological survey of horses from different regions of Brazil using recombinant peptides from domain III of WNV. Positive samples were validated with the neutralisation test. Our results showed that of 79 ELISA-positive horses, nine expressed WNV-specific neutralising antibodies. Eight of the infected horses were from the state of Mato Grosso do Sul and one was from the state of Paraíba. Our results provide additional evidence for the emergence of WNV in Brazil and for its circulation in multiple regions of the country.     

Changes in biofilm structure during the colonization of chalcopyrite by Acidithiobacillus thiooxidans

Biofilms of Acidithiobacillus thiooxidans were grown on the surface of massive chalcopyrite electrodes (MCE) where different secondary sulfur phases were previously formed by potentiostatic oxidation of MCE at 0.780?≤?E _an?≤?0.965 V (electrooxidized MCE, eMCE). The formation of mainly S⁰ and minor amounts of CuS and S _n ^2? were detected on eMCEs. The eMCEs were incubated with A. thiooxidans cells for 1, 12, 24, 48, and 120 h in order to temporally monitor changes in eMCE's secondary phases, biofilm structure, and extracellular polymeric substance (EPS) composition (lipids, proteins, and polysaccharides) using microscopic, spectroscopic, electrochemical, and biochemical techniques. The results show significant cell attachments with stratified biofilm structure since the first hour of incubation and EPS composition changes, the most important being production after 48–120 h when the highest amount of lipids and proteins were registered. During 120 h, periodic oxidation/formation of S⁰/S _n ^2? was recorded on biooxidized eMCEs, until a stable CuS composition was formed. In contrast, no evidence of CuS formation was observed on the eMCEs of the abiotic control, confirming that CuS formation results from microbial activity. The surface transformation of eMCE induces a structural transformation of the biofilm, evolving directly to a multilayered biofilm with more hydrophobic EPS and proteins after 120 h. Our results suggest that A. thiooxidans responded to the spatial and temporal distribution and chemical reactivity of the S _n ^2?/S⁰/CuS phases throughout 120 h. These results suggested a strong correlation between surface speciation, hydrophobic domains in EPS, and biofilm organization during chalcopyrite biooxidation by A. thiooxidans.     

Causes of death in different areas for Bonelli's Eagle Hieraaetus fasciatus in Spain

The Spanish Bonelli's Eagle populations have decreased markedly because of high mortality. We recorded 424 cases of dead eagles between 1990 and 1998 in Spain which after cross-comparison corresponded to 377 individuals. Electrocution (55% of deaths), followed by direct persecution (26%) were the main causes of death. No differences in the cause of death were found between sexes. Non-adult eagles mostly died of electrocution whereas adults were mainly the victims of persecution. A log-linear model showed that these differences were associated with a difference in the spatial distribution of age classes, rather than to age or experience per se. Persecution was the main cause of death in breeding areas and electrocution in non-breeding areas. There were differences between regions: electrocution was the main cause in Catalonia and Central Spain (50% and 86% respectively) whereas direct persecution was the main cause in Levant and Northern Spain (52% and 43% respectively). We recommend that steps are taken in order to reduce eagle mortality, taking into account the differences between regions and areas.     

Using DNA-barcoding for sorting out protist species complexes: A case study of the Nebela tincta–collaris–bohemica group (Amoebozoa; Arcellinida,Hyalospheniidae)

Species identification by means of morphology is often problematic in protists. Nebela tincta–collaris–bohemica (Arcellinida) is a species complex of small to medium-sized (ca.100 μm) testate amoebae common in peat bogs and forest soils. The taxonomic validity of characters used to define species within this group is debated and causes confusion in studies of biogeography, and applications in palaeoecology.We examined the relationship between morphological and genetic diversity within this species complex by combined analyses of light microscopy imaging and Cytochrome Oxidase Subunit 1(COI) sequences obtained from the same individual amoeba cells. Our goals were (1) to clarify the taxonomy and the phylogenetic relationships within this group, and (2) to evaluate if individual genotypes corresponded to specific morphotypes and the extent of phenotypic plasticity.We show here that small variations in test morphology that have been often overlooked by traditional taxonomy correspond to distinct haplotypes. We therefore revise the taxonomy of the group. We redefine Nebela tincta (Leidy) Kosakyan et Lara and N. collaris (Ehrenberg 1848) Kosakyan et Gomaa, change N. tincta var. rotunda Penard to N. rotunda (Penard 1890), describe three new species: N. guttata n. sp. Kosakyan et Lara, N. pechorensis n. sp. Kosakyan et Mitchell, and N. aliciae n. sp. Mitchell et Lara.