Serological and virological survey and resighting of marked wild geese in Germany

In order to investigate the potential role of arctic geese in the epidemiology, the spatial and temporal spread of selected avian diseases, in autumn 2002, a virological and serological survey designed as capture-mark-resighting study was conducted in one of the most important coastal resting sites for migratory waterfowl in Germany. Oropharyngeal, cloacal swabs and blood samples were collected from a total of 147 birds comprising of three different arctic geese species including White-fronted Goose (Anser albifrons), Tundra Bean Goose (Anser fabalis rossicus), Pink-footed Goose (Anser brachyrhynchus) as well as from 29 non-migratory Canada Geese (Branta canadensis). Altogether, six adeno-like viruses (ALV; 95% CI, 1.74?C9.92%) and two avian paramyxoviruses (APMV-4; 95% CI, 0.19?C5.53%) were isolated mainly from juvenile White-fronted Geese. In addition, four Canada Geese were infected with lentogenic APMV-1 (95% CI, 3.89?C31.66%) at the date of sampling. No avian influenza viruses, reo-like viruses could be isolated despite serological evidence. Likewise, no evidence of current or previous infection by West Nile virus was found. Of the 147 birds tagged in the following years, 137 birds were re-sighted between 2002 and 2008 accumulating to 1925 sightings. About 90% of all sightings were reported from the main wintering and resting sites in Germany and The Netherlands. Eight of the resighted geese were virus positive (ALV and APMV-4) at the time point of sampling in 2002.     

Surface-enhanced vibrational spectroscopy for probing transient interactions of proteins with biomimetic interfaces: electric field effects on structure, dynamics and function of cytochrome c

Most of the biochemical and biophysical processes of proteins take place at membranes, and are thus under the influence of strong local electric fields, which are likely to affect the structure as well as the reaction mechanism and dynamics. To analyse such electric field effects, biomimetic interfaces may be employed that consist of membrane models deposited on nanostructured metal electrodes. For such devices, surface-enhanced resonance Raman and IR absorption spectroscopy are powerful techniques to disentangle the complex interfacial processes of proteins in terms of rotational diffusion, electron transfer, and protein and cofactor structural changes. The present article reviews the results obtained for the haem protein cytochrome c, which is widely used as a model protein for studying the various reaction steps of interfacial redox processes in general. In addition, it is shown that electric field effects may be functional for the natural redox processes of cytochrome c in the respiratory chain, as well as for the switch from the redox to the peroxidase function, one of the key events preceding apoptosis.     

Spatial distribution of cryptic species diversity in european freshwater amphipods (Gammarus fossarum) as revealed by pyrosequencing

In order to understand and protect ecosystems, local gene pools need to be evaluated with respect to their uniqueness. Cryptic species present a challenge in this context because their presence, if unrecognized, may lead to serious misjudgement of the distribution of evolutionarily distinct genetic entities. In this study, we describe the current geographical distribution of cryptic species of the ecologically important stream amphipod Gammarus fossarum (types A, B and C). We use a novel pyrosequencing assay for molecular species identification and survey 62 populations in Switzerland, plus several populations in Germany and eastern France. In addition, we compile data from previous publications (mainly Germany). A clear transition is observed from type A in the east (Danube and Po drainages) to types B and, more rarely, C in the west (Meuse, Rhone, and four smaller French river systems). Within the Rhine drainage, the cryptic species meet in a contact zone which spans the entire G. fossarum distribution range from north to south. This large-scale geographical sorting indicates that types A and B persisted in separate refugia during Pleistocene glaciations. Within the contact zone, the species rarely co-occur at the same site, suggesting that ecological processes may preclude long-term coexistence. The clear phylogeographical signal observed in this study implies that, in many parts of Europe, only one of the cryptic species is present.     

Characterization of novel cutaneous human papillomavirus genotypes HPV-150 and HPV-151

DNA from two novel HPV genotypes, HPV-150 and HPV-151, isolated from hair follicles of immuno-competent individuals, was fully cloned, sequenced and characterized. The complete genomes of HPV-150 and HPV-151 are 7,436-bp and 7,386-bp in length, respectively. Both contain genes for at least six proteins, namely E6, E7, E1, E2, L2, L1, as well as a non-coding upstream regulatory region located between the L1 and E6 genes: spanning 416-bp in HPV-150 (genomic positions 7,371 to 350) and 322-bp in HPV-151 (genomic positions 7,213 to 148). HPV-150 and HPV-151 are phylogenetically placed within the Betapapillomavirus genus and are most closely related to HPV-96 and HPV-22, respectively. As in other members of this genus, the intergenic E2-L2 region is very short and does not encode for an E5 gene. Both genotypes contain typical zinc binding domains in their E6 and E7 proteins, but HPV-151 lacks the regular pRb-binding core sequence within its E7 protein. In order to assess the tissue predilection and clinical significance of the novel genotypes, quantitative type-specific real-time PCR assays were developed. The 95% detection limits of the HPV-150 and HPV-151 assays were 7.3 copies/reaction (range 5.6 to 11.4) and 3.4 copies/reaction (range 2.5 to 6.0), respectively. Testing of a representative collection of HPV-associated mucosal and cutaneous benign and malignant neoplasms and hair follicles (total of 540 samples) revealed that HPV-150 and HPV-151 are relatively rare genotypes with a cutaneous tropism. Both genotypes were found in sporadic cases of common warts and SCC and BCC of the skin as single or multiple infections usually with low viral loads. HPV-150 can establish persistent infection of hair follicles in immuno-competent individuals. A partial L1 sequence of a putative novel HPV genotype, related to HPV-150, was identified in a squamous cell carcinoma of the skin obtained from a 64-year old immuno-compromised male patient.     

Artificial skin--culturing of different skin cell lines for generating an artificial skin substitute on cross-weaved spider silk fibres

Background

In the field of Plastic Reconstructive Surgery the development of new innovative matrices for skin repair is in urgent need. The ideal biomaterial should promote attachment, proliferation and growth of cells. Additionally, it should degrade in an appropriate time period without releasing harmful substances, but not exert a pathological immune response. Spider dragline silk from Nephila spp meets these demands to a large extent.

Methodology/Principal Findings

Native spider dragline silk, harvested directly out of Nephila spp spiders, was woven on steel frames. Constructs were sterilized and seeded with fibroblasts. After two weeks of cultivating single fibroblasts, keratinocytes were added to generate a bilayered skin model, consisting of dermis and epidermis equivalents. For the next three weeks, constructs in co-culture were lifted on an originally designed setup for air/liquid interface cultivation. After the culturing period, constructs were embedded in paraffin with an especially developed program for spidersilk to avoid supercontraction. Paraffin cross- sections were stained in Haematoxylin & Eosin (H&E) for microscopic analyses.

Conclusion/Significance

Native spider dragline silk woven on steel frames provides a suitable matrix for 3 dimensional skin cell culturing. Both fibroblasts and keratinocytes cell lines adhere to the spider silk fibres and proliferate. Guided by the spider silk fibres, they sprout into the meshes and reach confluence in at most one week. A well-balanced, bilayered cocultivation in two continuously separated strata can be achieved by serum reduction, changing the medium conditions and the cultivation period at the air/liquid interphase. Therefore spider silk appears to be a promising biomaterial for the enhancement of skin regeneration.     

Effect of constitution on mass of individual organs and their association with metabolic rate in humans--a detailed view on allometric scaling

Resting energy expenditure (REE)-power relationships result from multiple underlying factors including weight and height. In addition, detailed body composition, including fat free mass (FFM) and its components, skeletal muscle mass and internal organs with high metabolic rates (i.e. brain, heart, liver, kidneys), are major determinants of REE. Since the mass of individual organs scales to height as well as to weight (and, thus, to constitution), the variance in these associations may also add to the variance in REE. Here we address body composition (measured by magnetic resonance imaging) and REE (assessed by indirect calorimetry) in a group of 330 healthy volunteers differing with respect to age (17-78 years), sex (61% female) and BMI (15.9-47.8 kg/m(2)). Using three dimensional data interpolation we found that the inter-individual variance related to scaling of organ mass to height and weight and, thus, the constitution-related variances in either FFM (model 1) or kidneys, muscle, brain and liver (model 2) explained up to 43% of the inter-individual variance in REE. These data are the first evidence that constitution adds to the complexity of REE. Since organs scale differently as weight as well as height the "fit" of organ masses within constitution should be considered as a further trait.     

Characterization of changes in serum anti-glycan antibodies in Crohn's disease--a longitudinal analysis

Introduction

Anti-glycan antibodies are a promising tool for differential diagnosis and disease stratification of patients with Crohn''s disease (CD). We longitudinally assessed level and status changes of anti-glycan antibodies over time in individual CD patients as well as determinants of this phenomenon.

Methods

859 serum samples derived from a cohort of 253 inflammatory bowel disease (IBD) patients (207 CD, 46 ulcerative colitis (UC)) were tested for the presence of anti-laminarin (Anti-L), anti-chitin (Anti-C), anti-chitobioside (ACCA), anti-laminaribioside (ALCA), anti-mannobioside (AMCA) and anti-Saccharomyces cerevisiae (gASCA) antibodies by ELISA. All patients had at least two and up to eleven serum samples taken during the disease course.

Results

Median follow-up time for CD was 17.4 months (Interquartile range (IQR) 8.0, 31.6 months) and for UC 10.9 months (IQR 4.9, 21.0 months). In a subgroup of CD subjects marked changes in the overall immune response (quartile sum score) and levels of individual markers were observed over time. The marker status (positive versus negative) remained widely stable. Neither clinical phenotype nor NOD2 genotype was associated with the observed fluctuations. In a longitudinal analysis neither changes in disease activity nor CD behavior led to alterations in the levels of the glycan markers. The ability of the panel to discriminate CD from UC or its association with CD phenotypes remained stable during follow-up. In the serum of UC patients neither significant level nor status changes were observed.

Conclusions

While the levels of anti-glycan antibodies fluctuate in a subgroup of CD patients the antibody status is widely stable over time.     

Clinically translatable cell tracking and quantification by MRI in cartilage repair using superparamagnetic iron oxides

BACKGROUND: Articular cartilage has very limited intrinsic regenerative capacity, making cell-based therapy a tempting approach for cartilage repair. Cell tracking can be a major step towards unraveling and improving the repair process of these therapies. We studied superparamagnetic iron oxides (SPIO) for labeling human bone marrow-derived mesenchymal stem cells (hBMSCs) regarding effectivity, cell viability, long term metabolic cell activity, chondrogenic differentiation and hBMSC secretion profile. We additionally examined the capacity of synovial cells to endocytose SPIO from dead, labeled cells, together with the use of magnetic resonance imaging (MRI) for intra-articular visualization and quantification of SPIO labeled cells. METHODOLOGY/PRINICIPAL FINDINGS: Efficacy and various safety aspects of SPIO cell labeling were determined using appropriate assays. Synovial SPIO re-uptake was investigated in vitro by co-labeling cells with SPIO and green fluorescent protein (GFP). MRI experiments were performed on a clinical 3.0T MRI scanner. Two cell-based cartilage repair techniques were mimicked for evaluating MRI traceability of labeled cells: intra-articular cell injection and cell implantation in cartilage defects. Cells were applied ex vivo or in vitro in an intra-articular environment and immediately scanned. SPIO labeling was effective and did not impair any of the studied safety aspects, including hBMSC secretion profile. SPIO from dead, labeled cells could be taken up by synovial cells. Both injected and implanted SPIO-labeled cells could accurately be visualized by MRI in a clinically relevant sized joint model using clinically applied cell doses. Finally, we quantified the amount of labeled cells seeded in cartilage defects using MR-based relaxometry. CONCLUSIONS: SPIO labeling appears to be safe without influencing cell behavior. SPIO labeled cells can be visualized in an intra-articular environment and quantified when seeded in cartilage defects.