Improved initial osteoblast functions on amino-functionalized titanium surfaces

Adhesion and spreading of cells on biomaterials are integrin-mediated processes. But recent findings indicate a key role of the cell membrane associated matrix substance hyaluronan (HA) in interface interactions. Because HA is a negatively charged molecule we assume that a biomaterial surface with an opposed charge could boost the first contact of the cell to the surface. Polished cp titanium (R(a)=0.19 microm) was coated with an amino-group containing plasma polymer (Ti PPA). For this purpose, a microwave excited, pulsed, low-pressure plasma was used. Additionally, collagen was immobilized on Ti PPA with polyethylene glycol diacid (PEG-DA), catalyzed by carbodiimide (CDI). The physico-chemical surface analytical techniques like XPS, FT-IR, water contact angle and zeta-potential verified the retention of the allylamine precursor structure. Human osteoblasts were cultured in serum-free Dulbecco's modified Eagle medium (DMEM). Adhesion and cell cycle phases were calculated by flow cytometry. Spreading and actin cytoskeleton were visualized by confocal microscopy. Gene expression of osteogenic markers was detected by real-time RT-PCR. Ti PPA is significantly advantageous concerning initial adhesion and spreading during the first hours of the cell contact to the surface. The proliferation of osteoblasts is positively influenced. Gene expression of the differentiation marker bone sialoprotein was upregulated after 24h. Our results demonstrate that functionalization of titanium with positively charged amino-groups is sufficiently enough to significantly improve initial steps of the cellular contact to the material surface.     

Influence of a Probiotic Strain of Enterococcus faecium on Salmonella enterica Serovar Typhimurium DT104 Infection in a Porcine Animal Infection Model

The beneficial effects of probiotic Enterococcus spp. in different hosts, such as mice and humans, have previously been reported in several studies. However, studies of large domestic animals, as well as challenge studies with pathogenic microorganisms, are very rare. Here, we investigated the influence of oral treatment of pigs with the probiotic bacterium Enterococcus faecium NCIMB 10415 on Salmonella enterica serovar Typhimurium DT104 infections in weaning piglets. Clinical symptoms, fecal excretion, the organ distribution of Salmonella, and the humoral immune response (immunoglobulin G [IgG], IgM, and IgA levels) in serum were examined. A pool of 89 piglets was randomly divided into probiotic and control groups. The probiotic group received a feed supplement containing E. faecium starting on day 14 postpartum prior to challenge with Salmonella serovar Typhimurium DT104 at 28 days postpartum. After challenge with Salmonella serovar Typhimurium DT104, piglets in both groups showed no severe clinical signs of salmonellosis. However, fecal excretion and colonization of Salmonella in organs were significantly greater in piglets fed E. faecium. Likewise, the humoral immune response against Salmonella (serum IgM and IgA levels) was significantly greater in the probiotic group animals than in control animals. The results of this study suggest that E. faecium NCIMB 10415 treatment enhanced the course of infection in weaning piglets challenged with Salmonella serovar Typhimurium DT104. However, the probiotic treatment also appeared to result in greater production of specific antibodies against Salmonella serovar Typhimurium DT104.The problem of increasing microbial resistance to antibiotics resulting from years of overuse and the resulting ban on the use of antibiotics in animal production have led to increased interest in alternatives to antibiotics in animal production. In recent years, probiotic bacteria have been considered as an alternative means of reducing pathogen loads in animal breeding and production units. However, while a number of studies have focused on the mode of action of probiotics, the mode of action these bacteria is not fully understood yet.A recent interdisciplinary research study of the modes of action of probiotics in swine showed that Enterococcus faecium NCIMB 10415 reduced the pathogenic bacterial load of healthy piglets (20, 26, 30, 36). In vitro studies further demonstrated that this E. faecium probiotic strain decreased the rate of invasion of a porcine intestinal epithelial cell line by Salmonella enterica serovar Typhimurium. To determine whether probiotics also provide a measure of protection during infections, experimental challenge studies with pathogenic bacteria at a defined infectious dose and under comparable conditions seem to be necessary. Field studies could be more representative of the real situation; however, the infection pressure is too low and difficult to define, and systematic sampling cannot be done.Studies of larger domestic and production animals are rare. Most such studies deal with the mode of action of probiotics in the healthy host, and only a few studies have investigated the mode of action in the context of infections with pathogenic bacteria, such as Salmonella.In a related study, weaned piglets were fed a mixture of five probiotic strains (one Pediococcus strain and four Lactobacillus strains) and challenged with Salmonella serovar Typhimurium (7). In that study, reduced incidence, severity, and duration of diarrhea and a reduced microbiological load of Salmonella were observed. Fedorka-Cray et al. (11) observed reduced numbers of Salmonella bacteria in cecal contents and at the ileocolic junction in S. enterica serovar Choleraesuis-challenged weaning piglets fed a competitive exclusion culture. In vitro investigations showed that Enterococcus strains have inhibitory effects on the growth of S. enterica serovar Enteritidis, and these effects were explained by both enterotoxin and nonenterotoxin factors (37). Other studies showed that E. faecium may be beneficial to the adhesion and colonization of Clostridium jejuni in the canine intestine (29) and reduced the rate of carryover infections with obligate intracellular pathogens from infected sows in piglets (26). E. faecium has also been shown to influence the composition of the bacterial community in the avian, swine, and canine gastrointestinal tracts (25, 29, 36).Infections with S. enterica are some of the most important sources of human gastroenteritis (39). In Germany, 52,563 human salmonellosis cases were reported in 2006 (http://www3.rki.de/SurvStat). The consumption of contaminated pork and pork products was found to be associated with 20% of human salmonellosis cases in Germany (33), indicating the importance of meat or meat products as a potential source of infection for consumers. Salmonella serovar Typhimurium, especially phage type DT104, is the Salmonella serotype most frequently isolated from pork (27), and it is of particular concern because of its acquisition of multiple antibiotic resistance (1, 38).In this study, we investigated the effect of E. faecium NCIMB 10415 on the infection dynamics of Salmonella serovar Typhimurium DT104, fecal shedding, and the patterns of Salmonella distribution in internal organs, as well as on the humoral immune response to Salmonella in weaning piglets. To the best of our knowledge, this is the first experimental study of the mode of action of a probiotic strain of E. faecium in which dissemination to different internal organs was investigated using weaned piglets experimentally infected with Salmonella.     

Pluralism and diversity: trends in the use and application of ordination methods 1990‐2007

Question: What are the trends and patterns in the application of ordination techniques in vegetation science since 1990? Location: Worldwide literature analysis. Methods: Evaluation of five major journals of vegetation science; search of all ISI‐listed ecological journals. Data were analysed with ANCOVAs, Spearman rank correlations, GLMs, biodiversity indices and simple graphs. Results: The ISI search retrieved fewer papers that used ordinations than the manual evaluation of five selected journals. Both retrieval methods revealed a clear trend in increasing frequency of ordination applications from 1990 to the present. Canonical Correspondence Analysis was far more frequently detected by the ISI search than any other method. Applications such as Correspondence Analysis/Reciprocal Averaging and Detrended Correspondence Analysis have increasingly been used in studies published in “applied” journals, while Canonical Correspondence Analysis, Redundancy Analysis and Non‐Metric Multidimensional Scaling were more frequently used in journals focusing on more “basic” research. Overall, Detrended Correspondence Analysis was the most commonly applied method within the five major journals, although the number of publications slightly decreased over time. Use of Non‐Metric Multidimensional Scaling has increased over the last 10 years. Conclusion: The availability of suitable software packages has facilitated the application of certain techniques such as Non‐Metric Multidimensional Scaling. However, choices of ordination techniques are currently less driven by the constraints imposed by the software; there is also limited evidence that the choice of methods follows social considerations such as the need to use fashionable methods. Methodological diversity has been maintained or has even increased over time and reflects the researcher's need for diverse analytical tools suitable to address a wide range of questions.     

Rust mite resistance in apple assessed by quantitative trait loci analysis

The aim of this study was to assess the genetic basis of rust mite (Aculus schlechtendali) resistance in apple (Malus × domestica). A. schlechtendali infestation of apple trees has increased as a consequence of reduced side effects of modern fungicides on rust mites. An analysis of quantitative trait loci (QTLs) was carried out using linkage map data available for F₁ progeny plants of the cultivars ‘Fiesta’ × ‘Discovery’. Apple trees representing 160 different genotypes were surveyed for rust mite infestation, each at three different sites in two consecutive years. The distribution of rust mites on the individual apple genotypes was aggregated and significantly affected by apple genotype and site. We identified two QTLs for A. schlechtendali resistance on linkage group 7 of ‘Fiesta’. The AFLP marker E35M42-0146 (20.2 cM) and the RAPD marker AE10-400 (45.8 cM) were closest positioned to the QTLs and explained between 11.0% and 16.6% of the phenotypic variability. Additionally, putative QTLs on the ‘Discovery’ chromosomes 4, 5 and 8 were detected. The SSR marker Hi03a10 identified to be associated to one of the QTLs (AFLP marker E35M42-0146) was traced back in the ‘Fiesta’ pedigree to the apple cultivar ‘Wagener’. This marker may facilitate the breeding of resistant apple cultivars by marker assisted selection. Furthermore, the genetic background of rust mite resistance in existing cultivars can be evaluated by testing them for the identified SSR marker. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Microphytobenthos of Arctic Kongsfjorden (Svalbard, Norway): biomass and potential primary production along the shore line

During summer 2007, Arctic microphytobenthic potential primary production was measured at several stations around the coastline of Kongsfjorden (Svalbard, Norway) at ≤5 m water depth and at two stations at five different water depths (5, 10, 15, 20, 30 m). Oxygen planar optode sensor spots were used ex situ to determine oxygen exchange in the overlying water of intact sediment cores under controlled light (ca. 100 μmol photons m⁻² s⁻¹) and temperature (2–4°C) conditions. Patches of microalgae (mainly diatoms) covering sandy sediments at water depths down to 30 m showed high biomass of up to 317 mg chl a m⁻². In spite of increasing water depth, no significant trend in “photoautotrophic active biomass” (chl a, ratio living/dead cells, cell sizes) and, thus, in primary production was measured at both stations. All sites from ≤5 to 30 m water depth exhibited variable rates of net production from −19 to +40 mg O₂ m⁻² h⁻¹ (−168 to +360 mg C m⁻² day⁻¹) and gross production of about 2–62 mg O₂ m⁻² h⁻¹ (17–554 mg C m⁻² day⁻¹), which is comparable to other polar as well as temperate regions. No relation between photoautotrophic biomass and gross/net production values was found. Microphytobenthos demonstrated significant rates of primary production that is comparable to pelagic production of Kongsfjorden and, hence, emphasised the importance as C source for the zoobenthos.     

Tagging quantitative trait loci for maturity-corrected late blight resistance in tetraploid potato with PCR-based candidate gene markers

Late blight caused by the oomycete Phytophthora infestans is the economically most important and destructive disease in potato cultivation. Quantitative resistance to late blight available in tetraploid cultivars is correlated with late maturity in temperate climates, which is an undesirable characteristic. A total of 30 DNA-based markers known to be linked to loci for pathogen resistance in diploid potato were selected and tested as polymerase chain reaction-based markers for linkage with quantitative trait loci (QTL) for late blight resistance and plant maturity in two half-sib families of tetraploid potatoes. Most markers originated from within or were physically closely linked to candidate genes for quantitative resistance factors. The families were repeatedly evaluated in the field for quantitative resistance to late blight and maturity. Resistance was corrected for the maturity effect. Nine of eleven different map segments tagged by the markers harbored QTL affecting maturity-corrected resistance. Interactions were found between unlinked resistance QTL, providing testable strategies for marker-assisted selection in tetraploid potato. Based on the linkage observed between QTL for resistance and plant maturity and based on the genetic interactions observed between candidate genes tagging resistance QTL, we discuss models for the molecular basis of quantitative resistance and maturity.     

Determination of strongly overlapping signaling activity from microarray data

Background

The Tissue Microarray (TMA) facilitates high-throughput analysis of hundreds of tissue specimens simultaneously. However, bottlenecks in the storage and manipulation of the data generated from TMA reviews have become apparent. A number of software applications have been developed to assist in image and data management; however no solution currently facilitates the easy online review, scoring and subsequent storage of images and data associated with TMA experimentation.

Results

This paper describes the design, development and validation of the Virtual Tissue Matrix (VTM). Through an intuitive HTML driven user interface, the VTM provides digital/virtual slide based images of each TMA core and a means to record observations on each TMA spot. Data generated from a TMA review is stored in an associated relational database, which facilitates the use of flexible scoring forms. The system allows multiple users to record their interpretation of each TMA spot for any parameters assessed. Images generated for the VTM were captured using a standard background lighting intensity and corrective algorithms were applied to each image to eliminate any background lighting hue inconsistencies or vignetting. Validation of the VTM involved examination of inter-and intra-observer variability between microscope and digital TMA reviews. Six bladder TMAs were immunohistochemically stained for E-Cadherin, β-Catenin and PhosphoMet and were assessed by two reviewers for the amount of core and tumour present, the amount and intensity of membrane, cytoplasmic and nuclear staining.

Conclusion

Results show that digital VTM images are representative of the original tissue viewed with a microscope. There were equivalent levels of inter-and intra-observer agreement for five out of the eight parameters assessed. Results also suggest that digital reviews may correct potential problems experienced when reviewing TMAs using a microscope, for example, removal of background lighting variance and tint, and potential disorientation of the reviewer, which may have resulted in the discrepancies evident in the remaining three parameters.     

A three-dimensional colocalization RNA interference screening platform to elucidate the alternative lengthening of telomeres pathway

A high-content colocalization RNA interference screen based on automatic three-color confocal fluorescence microscopy was developed to analyze the alternative lengthening of telomeres (ALT) pathway. Via this pathway telomerase-negative cancer cells can maintain their telomeres and with it their unlimited proliferative potential. A hallmark of ALT cells is the colocalization of promyelocytic leukemia (PML) nuclear bodies with telomeres to form ALT-associated PML nuclear bodies (APBs). In our screen, the presence of APBs was used as a marker to identify proteins required for the ALT mechanism. A cell-based assay and an automatic confocal image acquisition procedure were established. Using automatic image analysis based on 3D parametric intensity models to identify APBs, we conducted an unbiased and quantitative analysis of nine different candidate genes. A comparison with the literature and manual analysis of the gene knockdown demonstrates the reliability of our approach. It extends the available repertoire of high-content screening to studies of cellular colocalizations and allows the identification of candidate genes for the ALT mechanism that represent possible targets for cancer therapy.     

Versican expression during synovial joint morphogenesis

The extracellular matrix (ECM) plays a critical role in governing cell behavior and phenotype during limb skeletogenesis. Chondroitin sulfate proteoglycans (Cspgs) are highly expressed in the ECM of precartilage mesenchymal condensations and are important to limb chondrogenesis and cartilage structure, but little is known regarding their involvement in formation of synovial joints in the embryonic limb. Matrix versican Cspg expression has previously been reported in the epiphysis of developing long bones and presumptive joint; however, detailed analysis has not yet been conducted. In the present study we immunolocalized versican and aggrecan Cspgs during chick elbow joint morphogenesis between HH st25-41 of development. In this study we show that versican and aggrecan expression initially overlapped in the incipient cartilage model of long bones in the wing, but versican was also highly expressed in the perichondrium and presumptive joint interzone during early stages of morphogenesis (HH st25-34). By HH st36-41 versican localization was restricted to the future articular surfaces of the developing joint and surrounding joint capsule while aggrecan localized in an immediately adjacent and predominately non-overlapping region of chondrogenic cells at the epiphyses. These results suggest a potential role for versican proteoglycan in development and maintenance of the synovial joint interzone.