Orientation of the tyrosyl radical in Salmonella typhimurium class Ib ribonucleotide reductase determined by high field EPR of R2F single crystals

The R2 protein of class I ribonucleotide reductase (RNR) generates and stores a tyrosyl radical, located next to a diferric iron center, which is essential for ribonucleotide reduction and thus DNA synthesis. X-ray structures of class Ia and Ib proteins from various organisms served as bases for detailed mechanistic suggestions. The active site tyrosine in R2F of class Ib RNR of Salmonella typhimurium is located at larger distance to the diiron site, and shows a different side chain orientation, as compared with the tyrosine in R2 of class Ia RNR from Escherichia coli.No structural information has been available for the active tyrosyl radical in R2F. Here we report on high field EPR experiments of single crystals of R2F from S. typhimurium, containing the radical Tyr-105*. Full rotational pattern of the spectra were recorded, and the orientation of the g-tensor axes were determined, which directly reflect the orientation of the radical Tyr-105* in the crystal frame. Comparison with the orientation of the reduced tyrosine Tyr-105-OH from the x-ray structure reveals a rotation of the tyrosyl side chain, which reduces the distance between the tyrosyl radical and the nearest iron ligands toward similar values as observed earlier for Tyr-122* in E. coli R2. Presence of the substrate binding subunit R1E did not change the EPR spectra of Tyr-105*, indicating that binding of R2E alone induces no structural change of the diiron site. The present study demonstrates that structural and functional information about active radical states can be obtained by combining x-ray and high-field-EPR crystallography.     

Lambda interferon (IFN-lambda) in serum is decreased in hantavirus-infected patients, and in vitro-established infection is insensitive to treatment with all IFNs and inhibits IFN-gamma-induced nitric oxide production

Hantaviruses, causing hemorrhagic fever with renal syndrome (HFRS) and hantavirus cardiopulmonary syndrome (HCPS), are known to be sensitive to nitric oxide (NO) and to pretreatment with type I and II interferons (alpha interferon [IFN-alpha]/IFN-beta and IFN-gamma, respectively). Elevated serum levels of NO and IFN-gamma have been observed in HFRS patients, but little is known regarding the systemic levels of other IFNs and the possible effects of hantaviruses on innate antiviral immune responses. In Puumala virus-infected HFRS patients (n = 18), we report that the levels of IFN-alpha and IFN-beta are similar, whereas the level of IFN-lambda (type III IFN) is significantly decreased, during acute (day of hospitalization) compared to the convalescent phase. The possible antiviral effects of IFN-lambda on the prototypic hantavirus Hantaan virus (HTNV) replication was then investigated. Pretreatment of A549 cells with IFN-lambda alone inhibited HTNV replication, and IFN-lambda combined with IFN-gamma induced additive antiviral effects. We then studied the effect of postinfection treatment with IFNs. Interestingly, an already-established HTNV infection was insensitive to subsequent IFN-alpha, -beta, -gamma, and -lambda stimulation, and HTNV-infected cells produced less NO compared to noninfected cells when stimulated with IFN-gamma and IL-1beta. Furthermore, less phosphorylated STAT1 after IFN treatment was observed in the nuclei of infected cells than in those of noninfected cells. The results suggest that hantavirus can interfere with the activation of antiviral innate immune responses in patients and inhibit the antiviral effects of all IFNs. We believe that future studies addressing the mechanisms by which hantaviruses interfere with the activation and shaping of immune responses may bring more knowledge regarding HFRS and HCPS pathogenesis.     

The offshore-ring: A new system design for the open ocean aquaculture of macroalgae

Mass culture of benthic macroalgae under rough offshore conditions in the North Sea requires rigid culture support systems that cannot only withstand rough weather conditions but can also be effectively handled while at the same time retain the cultured species. Various carrier constructions and different mooring systems were tested. Laminaria saccharina grew on all of these carriers with initially high (up to 14.5% per day) and later decreasing length increments. Longlines, ladder and grid systems had certain disadvantages and these are discussed. The study results led to a new ring carrier (patent pending), first used in 1994/1995, which was gradually improved until 2002. This system now emerges as being superior, since it resists not only rough weather conditions (2 m s^–1 current velocity, 6 m wave height) but also permits ease of handling when compared to other constructions. The ring allows various operational modes and can be equipped with culture lines that can be collected offshore or transported to shore facilities for harvesting. The modular nature of the tested ring system lends itself for future use in integrated aquaculture systems located in or attached to offshore wind farms.     

Surrogate antigens as targets for proteome-wide binder selection

In the past decade, many initiatives were taken for the development of antibodies for proteome-wide studies, as well as characterisation and validation of clinically relevant disease biomarkers. Phage display offers many advantages compared to antibody generation by immunisation because it is an unlimited resource of affinity reagents without batch-to-batch variation and is also amendable for high throughput in contrast to conventional hybridoma technology. One of the major bottlenecks to proteome-wide binder selection is the limited supply of suitable target antigens representative of the human proteome. Here, we provide proof of principle of using easily accessible, cancer-associated protein epitope signature tags (PrESTs), routinely generated within the Human Protein Atlas project, as surrogate antigens for full-length proteins in phage selections for the retrieval of target-specific binders. These binders were subsequently tested in western blot, immunohistochemistry and protein microarray application to demonstrate their functionality.     

Production of dissociated sensory neuron cultures and considerations for their use in studying neuronal function and plasticity

Dissociated primary sensory neurons are commonly used to study growth factor-dependent cell survival, axon outgrowth, differentiation and basic mechanisms of sensory physiology and pain. Spinal or trigeminal sensory neurons can be collected from embryos, neonates or adults, treated with enzymes that degrade the extracellular matrix, triturated and grown in defined media with or without growth factors and additional animal sera. Production of cultures can take as little as 2.5 h. Cells can be used almost immediately or maintained for as long as 1 month. Ease of production and the ability to control growth conditions make sensory neuron culture a powerful model system for studying basic neurobiology of central and peripheral nervous systems.     

Modeling the response of a population of olfactory receptor neurons to an odorant

We modeled the firing rate of populations of olfactory receptor neurons (ORNs) responding to an odorant at different concentrations. Two cases were considered: a population of ORNs that all express the same olfactory receptor (OR), and a population that expresses many different ORs. To take into account ORN variability, we replaced single parameter values in a biophysical ORN model with values drawn from statistical distributions, chosen to correspond to experimental data. For ORNs expressing the same OR, we found that the distributions of firing frequencies are Gaussian at all concentrations, with larger mean and standard deviation at higher concentrations. For a population expressing different ORs, the distribution of firing frequencies can be described as the superposition of a Gaussian distribution and a lognormal distribution. Distributions of maximum value and dynamic range of spiking frequencies in the simulated ORN population were similar to experimental results.     

A rotating bed system bioreactor enables cultivation of primary osteoblasts on well‐characterized sponceram® regarding structural and flow properties

The development of bone tissue engineering depends on the availability of suitable biomaterials, a well‐defined and controlled bioreactor system, and on the use of adequate cells. The biomaterial must fulfill chemical, biological, and mechanical requirements. Besides biocompatibility, the structural and flow characteristics of the biomaterial are of utmost importance for a successful dynamic cultivation of osteoblasts, since fluid percolation within the microstructure must be assured to supply to cells nutrients and waste removal. Therefore, the biomaterial must consist of a three‐dimensional structure, exhibit high porosity and present an interconnected porous network. Sponceram®, a ZrO₂ based porous ceramic, is characterized in the presented work with regard to its microstructural design. Intrinsic permeability is obtained through a standard Darcy's experiment, while Young's modulus is derived from a two plates stress–strain test in the linear range. Furthermore, the material is applied for the dynamic cultivation of primary osteoblasts in a newly developed rotating bed bioreactor. © 2010 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

Individual variation in pup vocalizations and absence of behavioral signs of maternal vocal recognition in Weddell seals (Leptonychotes weddellii)

Individually stereotyped vocalizations often play an important role in relocation of offspring in gregarious breeders. In phocids, mothers often alternate between foraging at sea and attending their pup. Pup calls are individually distinctive in various phocid species. However, experimental evidence for maternal recognition is rare. In this study, we recorded Weddell seal (Leptonychotes weddellii) pup vocalizations at two whelping patches in Atka Bay, Antarctica, and explored individual vocal variation based on eight vocal parameters. Overall, 58% of calls were correctly classified according to individual. For males (n= 12) and females (n= 9), respectively, nine and seven individuals were correctly identified based on vocal parameters. To investigate whether mothers respond differently to calls of familiar vs. unfamiliar pups, we conducted playback experiments with 21 mothers. Maternal responses did not differ between playbacks of own, familiar, and unfamiliar pup calls. We suggest that Weddell seal pup calls may need to contain only a critical amount of individually distinct information because mothers and pups use a combination of sensory modalities for identification. However, it cannot be excluded that pup developmental factors and differing environmental factors between colonies affect pup acoustic behavior and the role of acoustic cues in the relocation process.     

Effect of concentration and hydraulic reaction time on the removal of pharmaceutical compounds in a membrane bioreactor inoculated with activated sludge

Pharmaceuticals are often not fully removed in wastewater treatment plants (WWTPs) and are thus being detected at trace levels in water bodies all over the world posing a risk to numerous organisms. These organic micropollutants (OMPs) reach WWTPs at concentrations sometimes too low to serve as growth substrate for microorganisms; thus, co-metabolism is thought to be the main conversion mechanism. In this study, the microbial removal of six pharmaceuticals was investigated in a membrane bioreactor at increasing concentrations (4–800 nM) of the compounds and using three different hydraulic retention times (HRT; 1, 3.5 and 5 days). The bioreactor was inoculated with activated sludge from a municipal WWTP and fed with ammonium, acetate and methanol as main growth substrates to mimic co-metabolism. Each pharmaceutical had a different average removal efficiency: acetaminophen (100%) > fluoxetine (50%) > metoprolol (25%) > diclofenac (20%) > metformin (15%) > carbamazepine (10%). Higher pharmaceutical influent concentrations proportionally increased the removal rate of each compound, but surprisingly not the removal percentage. Furthermore, only metformin removal improved to 80–100% when HRT or biomass concentration was increased. Microbial community changes were followed with 16S rRNA gene amplicon sequencing in response to the increment of pharmaceutical concentration: Nitrospirae and Planctomycetes 16S rRNA relative gene abundance decreased, whereas Acidobacteria and Bacteroidetes increased. Remarkably, the Dokdonella genus, previously implicated in acetaminophen metabolism, showed a 30-fold increase in abundance at the highest concentration of pharmaceuticals applied. Taken together, these results suggest that the incomplete removal of most pharmaceutical compounds in WWTPs is dependent on neither concentration nor reaction time. Accordingly, we propose a chemical equilibrium or a growth substrate limitation as the responsible mechanisms of the incomplete removal. Finally, Dokdonella could be the main acetaminophen degrader under activated sludge conditions, and non-antibiotic pharmaceuticals might still be toxic to relevant WWTP bacteria.