First description of giant Archaea (Thaumarchaeota) associated with putative bacterial ectosymbionts in a sulfidic marine habitat

Archaea may be involved in global energy cycles, and are known for their ability to interact with eukaryotic species (sponges, corals and ascidians) or as archaeal-bacterial consortia. The recently proposed phylum Thaumarchaeota may represent the deepest branching lineage in the archaeal phylogeny emerging before the divergence between Euryarchaeota and Crenarchaeota. Here we report the first characterization of two marine thaumarchaeal species from shallow waters that consist of multiple giant cells. One species is coated with sulfur-oxidizing γ-Proteobacteria. These new uncultured thaumarchaeal species are able to live in the sulfide-rich environments of a tropical mangrove swamp, either on living tissues such as roots or on various kinds of materials such as stones, sunken woods, etc. These archaea and archaea/bacteria associations have been studied using light microscopy, transmission electron microscopy and scanning electron microscopy. Species identification of archaeons and the putative bacterial symbiont have been assessed by 16S small subunit ribosomal RNA analysis. The sulfur-oxidizing ability of the bacteria has been assessed by genetic investigation on alpha-subunit of the adenosine-5'-phosphosulfate reductase/oxidase's (AprA). Species identifications have been confirmed by fluorescence in situ hybridization using specific probes designed in this study. In this article, we describe two new giant archaeal species that form the biggest archaeal filaments ever observed. One of these species is covered by a specific biofilm of sulfur-oxidizing γ-Proteobacteria. This study highlights an unexpected morphological and genetic diversity of the phylum Thaumarchaeota.     

A fast and robust hepatocyte quantification algorithm including vein processing

Background  

Quantification of different types of cells is often needed for analysis of histological images. In our project, we compute the relative number of proliferating hepatocytes for the evaluation of the regeneration process after partial hepatectomy in normal rat livers.     

On competition of predators and prey infection

A class of prey–predator models with infected prey is investigated. Predation terms are either of Holling type II or III, infection is either modelled by mass action or standard incidence. It is shown that the key for understanding the model behaviour is the competition of predators versus infection. In the presented models the predator is not susceptible to the infection and the infection of the prey has no influence on the ability of the predator of catching the prey. However, the prey population can be seen as a resource which both the predators and the infection depend on. The competition for this resource is strong—the principle of competitive exclusion holds for biologically meaningful choices of parameters as long as there is no destabilisation by a Hopf bifurcation. The representation of models in competition diagrams which are introduced in this article can be used for a wide range of competition models which seems to be a promising method with many potential applications.     

An Unconventional Copper Protein Required for Cytochrome c Oxidase Respiratory Function under Extreme Acidic Conditions

Very little is known about the processes used by acidophile organisms to preserve stability and function of respiratory pathways. Here, we reveal a potential strategy of these organisms for protecting and keeping functional key enzymes under extreme conditions. Using Acidithiobacillus ferrooxidans, we have identified a protein belonging to a new cupredoxin subfamily, AcoP, for “acidophile CcO partner,” which is required for the cytochrome c oxidase (CcO) function. We show that it is a multifunctional copper protein with at least two roles as follows: (i) as a chaperone-like protein involved in the protection of the Cu_A center of the CcO complex and (ii) as a linker between the periplasmic cytochrome c and the inner membrane cytochrome c oxidase. It could represent an interesting model for investigating the multifunctionality of proteins known to be crucial in pathways of energy metabolism.     

Crystal Structure of the Entire Ectodomain of gp130: INSIGHTS INTO THE MOLECULAR ASSEMBLY OF THE TALL CYTOKINE RECEPTOR COMPLEXES*

gp130 is the shared signal-transducing receptor subunit for the large and important family of interleukin 6-like cytokines. Previous x-ray structures of ligand-receptor complexes of this family lack the three membrane-proximal domains that are essential for signal transduction. Here we report the crystal structure of the entire extracellular portion of human gp130 (domains 1–6, D1–D6) at 3.6 Å resolution, in an unliganded form, as well as a higher resolution structure of the membrane-proximal fibronectin type III domains (D4–D6) at 1.9 Å. This represents the first atomic resolution structure of the complete ectodomain of any “tall” cytokine receptor. These structures show that other than a reorientation of the D1 domain, there is little structural change in gp130 upon ligand binding. They also reveal that the interface between the D4 and D5 domains forms an acute bend in the gp130 structure. Key residues at this interface are highly conserved across the entire tall receptor family, suggesting that this acute bend may be a common feature of these receptors. Importantly, this geometry positions the C termini of the membrane-proximal fibronectin type III domains of the tall cytokine receptors in close proximity within the transmembrane complex, favorable for receptor-associated Janus kinases to trans-phosphorylate and activate each other.     

A new protocol for functional analysis of adipogenesis using reverse transfection technology and time‐lapse video microscopy

Since the worldwide increase in obesity represents a growing challenge for healthcare systems, research focusing on fat cell metabolism has become a focal point of interest. Here, we describe a small interfering RNA (siRNA)‐technology‐based screening method to study fat cell differentiation in human primary preadipocytes that could be further developed towards an automated middle‐throughput screening procedure. First, we established optimal conditions for the reverse transfection of human primary preadipocytes demonstrating that an efficient reverse transfection of preadipocytes is technically feasible. Aligning the processes of reverse transfection and fat cell differentiation utilizing peroxisome proliferator‐activated receptor γ (PPARγ)‐siRNA, we showed that preadipocyte differentiation was suppressed by knock‐down of PPARγ, the key regulator of fat cell differentiation. The use of fluorescently labelled fatty acids in combination with fluorescence time‐lapse microscopy over a longer period of time enabled us to quantify the PPARγ phenotype. Additionally, our data demonstrate that reverse transfection of human cultured preadipocytes with TIP60 (HIV‐1 Tat‐interacting protein 60)–siRNA lead to a TIP60 knock‐down and subsequently inhibits fat cell differentiation, suggesting a role of this protein in human adipogenesis. In conclusion, we established a protocol that allows for an efficient functional and time‐dependent analysis by quantitative time‐lapse microscopy to identify novel adipogenesis‐associated genes.     

Simultaneous quantification of the organophosphorus pesticides dimethoate and omethoate in porcine plasma and urine by LC–ESI-MS/MS and flow-injection-ESI-MS/MS

Dimethoate is an organophosphorus toxicant used in agri- and horticulture as a systemic broad-spectrum insecticide. It also exhibits toxic activity towards mammalian organism provoked by catalytic desulfuration in the liver producing its oxon-derivative omethoate thus inhibiting acetylcholinesterase, initiating cholinergic crisis and ultimately leading to death by respiratory paralysis and cardiovascular collapse. Pharmaco- and toxicokinetic studies in animal models help to broaden basic understanding of medical intervention by antidotes and supportive care. Therefore, we developed and validated a LC–ESI-MS/MS method suitable for the simultaneous, selective, precise (RSD_intra-day 1–8%; RSD_inter-day 5–14%), accurate (intra-day: 95–107%; inter-day: 90–115%), and robust quantification of both pesticides from porcine urine and plasma after deproteinization by precipitation and extensive dilution (1:11,250 for plasma and 1:40,000 for urine). Accordingly, lower limits of quantification (0.24–0.49 μg/ml plasma and 0.78–1.56 μg/ml urine) and lower limits of detection (0.12–0.24 μg/ml plasma and 0.39–0.78 μg/ml urine) were equivalent to quite low absolute on-column amounts (1.1–2.1 pg for plasma and 2.0–3.9 pg for urine). The calibration range (0.24–250 μg/ml plasma and 0.78–200 μg/ml urine) was subdivided into two linear ranges (r² ≥ 0.998) each covering nearly two orders of magnitude. The lack of any interfering peak in 6 individual blank specimens from plasma and urine demonstrated the high selectivity of the method. Furthermore, extensive sample dilution causing lowest concentration of potentially interfering matrix ingredients prompted us to develop and validate an additional flow-injection method (FI-ESI-MS/MS). Validation characteristics were as good as for the chromatographic method but sample throughput was enhanced by a factor of 6. Effects on ionization provoked by plasma and urine matrix from 6 individuals as well as in the presence of therapeutics (antidotes) administered in an animal study were investigated systematically underling in the reliability of the presented methods. Both methods were applied to porcine samples derived from an in vivo animal study.     

Effects of thawing,refreezing and storage conditions of tissue samples and protein extracts on 2‐DE spot intensity

We report that reliable quantitative proteome analyses can be performed with tissue samples stored at ?80°C for up to 10 years. However, storing protein extracts at 4°C for 24 h and freezing protein extracts at ?80°C and thawing them significantly altered 41.6 and 17.5% of all spot intensities on 2‐DE gels, respectively. Fortunately, these storing effects did not impair the reliability of quantifying 2‐DE experiments. Nonetheless, the results show that freezing and storage conditions should be carefully controlled in proteomic experiments.