Probiotic Treatment Decreases the Number of CD14-Expressing Cells in Porcine Milk Which Correlates with Several Intestinal Immune Parameters in the Piglets

Modulating the mucosal immune system of neonates by probiotic treatment of their mothers is a promising approach which can only be investigated through the use of animal models. Here, we used sows and their piglets to investigate the impact of a bacterial treatment on the sow’s milk and on the neonate piglet intestinal immune system. In previous experiments, feed supplementation of sows with the probiotic Enterococcus faecium NCIMB 10415 during pregnancy and lactation had been shown to affect intestinal microbiota and cytokine expression of the offspring during the suckling and weaning periods. We therefore investigated the composition of the milk from treated sows in comparison to samples from a control group. In treated sows, the amount of lactose increased, and the somatic cell numbers were reduced. In all milk samples, the percentage of cells expressing membranous CD14 (mCD14) was greater than the fractions of immune cells, indicating expression of mCD14 on mammary epithelial cells. However, in the milk of E. faecium-treated sows, mCD14+ cells were reduced. Furthermore, the number of CD14+ milk cells was positively correlated with the percentages of B cells and activated T cells in the ileal MLN of the piglets. This study provides evidence for the expression of mCD14 by the porcine mammary epithelium, and suggests an immunological effect of mCD14+ milk cells on the piglets’ intestinal immune system. Our study further suggests that mCD14+ mammary epithelial cell populations can be modulated by probiotic feed supplementation of the sow.Peer Reviewe

Parallel use of shake flask and microtiter plate online measuring devices (RAMOS and BioLector) reduces the number of experiments in laboratory-scale stirred tank bioreactors

Background Conventional experiments in small scale are often performed in a Black Box fashion, analyzing only the product concentration in the final sample. Online monitoring of relevant process characteristics and parameters such as substrate limitation, product inhibition and oxygen supply is lacking. Therefore, fully equipped laboratory-scale stirred tank bioreactors are hitherto required for detailed studies of new microbial systems. However, they are too spacious, laborious and expensive to be operated in larger number in parallel. Thus, the aim of this study is to present a new experimental approach to obtain dense quantitative process information by parallel use of two small-scale culture systems with online monitoring capabilities: Respiration Activity MOnitoring System (RAMOS) and the BioLector device. Results The same mastermix (medium plus microorganisms) was distributed to the different small-scale culture systems: 1) RAMOS device; 2) 48-well microtiter plate for BioLector device; and 3) separate shake flasks or microtiter plates for offline sampling. By adjusting the same maximum oxygen transfer capacity (OTRmax), the results from the RAMOS and BioLector online monitoring systems supplemented each other very well for all studied microbial systems (E. coli, G. oxydans, K. lactis) and culture conditions (oxygen limitation, diauxic growth, auto-induction, buffer effects). Conclusions The parallel use of RAMOS and BioLector devices is a suitable and fast approach to gain comprehensive quantitative data about growth and production behavior of the evaluated microorganisms. These acquired data largely reduce the necessary number of experiments in laboratory-scale stirred tank bioreactors for basic process development. Thus, much more quantitative information is obtained in parallel in shorter time.Cluster of Excellence “Tailor-Made Fuels from Biomass”, which is funded by the Excellence Initiative by the German federal and state governments to promote science and research at German universities

Practical Atheism as a Challenge to the Salvific Activity of the Church

Atheism as a practice of life is becoming increasingly popular in modern western societies. A life devoid of reference to religious practices is becoming not so much an element of rebellion against the established religious traditions, but more of a certain obviousness that no one contests. Hence the question arises, how is the Church to function in such an environment and how to preach the Gospel? Pope Francis, and whim him modern theology, is increasingly emphasizing the role of the Church as a servant who, in humility and social engagement and through work of mercy, can preach the Gospel to modern man. The Church should proclaim the Gospel not through speculative theology, but through engagement with the poors and most need

Towards the generation of recombinant and pharmaceutically relevant target proteins : phage display based isolation of specific scFv antibodies against metastasizing pancreatic carcinoma for clinical application and development of novel fluorescent reporter tags for on-line monitoring during target protein production

Pancreatic cancer is an aggressive type of neoplasia characterized by its high potential for metastasis with a most devastating prognosis. Initial stages are almost asymptomatic, thus preventing early detection before local tissue invasion due to the lack of reliable diagnostics. Surgical removal in combination with standard first-line chemotherapeutic Gemcitabine treatment and radiation-based therapy are merely life-prolonging options. High resistance towards conventional therapeutics and the huge metastasizing potential leaves minimal residual micrometastasis accountable for an enormously high relapse rate. Therefore, the first aim of this thesis was to develop novel tumor specific human single chain antibody fragments (scFv) for possible future application as cytolytic therapeutics for adjuvant treatment of metastasizing pancreatic cancer, as well as more efficient molecular tools for early diagnosis. Phage display technology was employed to generate pancreas-specific scFv-phage antibodies from the naïve human Tomlinson phage libraries I and J binding against unknown tumor-associated antigen. Highly specific scFv phage ligands were isolated in a two-step panning strategy via depletion on human peripheral blood mononuclear cells (PBMC), followed by a positive selection on the metastatic pancreatic cancer cell line L3.6pl. Monoclonal phage ELISA identified 16 unique L3.6pl positive scFvs, subsequently expressed in eukaryotic HEK293T cells as soluble scFv-SNAP-tag proteins. Additionally, clone 14.1(scFv) SNAP, originally isolated from a laboratory-own murine immunized phage display library, was included into the expression and characterization process. Analysis of binding specificity and cross-reactivity of nine IMAC purified scFv-SNAP proteins was performed by soluble protein ELISA and flow. Of these, four clones displayed internalizing properties during flow cytometric and OPERA-based internalization tests. All positive candidates are clinically relevant pancreatic carcinoma specific scFvs and may provide the prospect of a tumor targeted cancer therapy to eliminate residual cancer cells. Moreover, they are highly promising candidates for diagnostic in vivo imaging tools besides an additional application as theranostics. To produce recombinant pharmaceutically relevant proteins on large scale, highly efficient screening technologies have been developed to characterize optimum cultivation conditions for bacterial growth and production formation. Microtiter plates (MTPs), in combination with measurement systems such as the BioLector®, are a practical tool for non invasive on-line monitoring of product formation of continuously shaken microbial cultures on lab-scale. Conventional reporter proteins for on-line monitoring, such as GFP and its derivatives, or flavin mononucleotide-based fluorescent proteins, are very large which possibly imposes stress on the host organism. Additionally, GFP strongly depends on an oxygen saturated environment for fluorophore formation. To circumvent mentioned drawbacks of conventional reporter tags, short but still optically active reporter tag for on-line detection were designed in this thesis. These novel reporter tags (W-tags) are based on the auto fluorescence of the aromatic amino acid tryptophan. Using in silico techniques, between one and five tryptophan residues (W1–W5) were accumulated in the naturally occuring protein loop of the cold shock protein (Bc Csp), to have equilibrated charges with the tryptophan residues presented on the outer side of the loop. Genetic fusions of these five different W-tags (MW = 3.4 to 5.6 kDa) to the anti-CD30 Ki-4(scFv) as well as the anti-MucI M12(scFv) antibody fragment were produced in a prokaryotic pMT-expression system. Analysis of on-line product fluorescence intensity during fermentation in MTPs followed by molecular biological flow cytometric binding analysis showed that more tryptophan residues within a W-tag generated a stronger tryptophan fluorescence signal gaining intensity corresponding to product formation. Nevertheless, an increase in tryptophan residues also complicated concentration of W-tagged proteins in the cell lysate. Protein recovery was only possible for constructs W1–W3. W4 and W5 remained in the cell pellet due to highly hydrophobic properties of the accumulated tryptophan molecules. Normal and comparative flow cytometry of W-tagged Ki-4(scFv) proteins on L540cy cells, in combination with a Ki-4 full length antibody, confirmed that binding specificity was not influenced whereas W tags with more than one tryptophan residue seemed to have a negative effect on binding activity and affinity. Lacking the main drawbacks of conventional reporter proteins, the novel W tags are an applicable alternative during non-invasive monitoring of recombinant product formation. They present the possibility for rapid and qualitative on line measurement during large-scale production of pharmaceutically relevant target proteins

Summary Report: Hurricane Floyd Symposium

On the morning of Thursday, September 16, 1999, Hurricane Floyd made landfall at the mouth of the Cape Fear River. Rains associated with Hurricanes Dennis, Floyd, and Irene resulted in extensive flooding over a two-month period in eastern North Carolina, with most river basins exceeding the 500-year flood level. Flooding was worst along the Tar River, leaving the towns of Rocky Mount, Tarboro, Princeville, Greenville, and Washington devastated. In North Carolina, Floyd was directly responsible for 35 fatalities and several billions of dollars in property damages. This publication provides a summary of the presentations in the public forum and the research conference. The value of an endeavor like the symposium comes from how the information shapes the way we move forward. Therefore, this publication also serves as a benchmark so that we can measure our progress on hurricane risk mitigation

Short-chain fluorescent tryptophan tags for on-line detection of functional recombinant proteins

<p>Abstract</p> <p>Background</p> <p>Conventional fluorescent proteins, such as GFP, its derivatives and flavin mononucleotide based fluorescent proteins (FbFPs) are often used as fusion tags for detecting recombinant proteins during cultivation. These reporter tags are state-of-the-art; however, they have some drawbacks, which can make on-line monitoring challenging. It is discussed in the literature that the large molecular size of proteins of the GFP family may stress the host cell metabolism during production. In addition, fluorophore formation of GFP derivatives is oxygen-dependent resulting in a lag-time between expression and fluorescence detection and the maturation of the protein is suppressed under oxygen-limited conditions. On the contrary, FbFPs are also applicable in an oxygen-limited or even anaerobic environment but are still quite large (58% of the size of GFP).</p> <p>Results</p> <p>As an alternative to common fluorescent tags we developed five novel tags based on clustered tryptophan residues, called W-tags. They are only 5-11% of the size of GFP. Based on the property of tryptophan to fluoresce in absence of oxygen it is reasonable to assume that the functionality of our W-tags is also given under anaerobic conditions. We fused these W-tags to a recombinant protein model, the anti-CD30 receptor single-chain fragment variable antibody (scFv) Ki-4(scFv) and the anti-MucI single-chain fragment variable M12(scFv). During cultivation in Microtiter plates, the overall tryptophan fluorescence intensity of all cultures was measured on-line for monitoring product formation via the different W-tags. After correlation of the scattered light signal representing biomass concentration and tryptophan fluorescence for the uninduced cultures, the fluorescence originating from the biomass was subtracted from the overall tryptophan signal. The resulting signal, thus, represents the product fluorescence of the tagged and untagged antibody fragments. The product fluorescence signal was increased. Antibodies with W-tags generated stronger signals than the untagged construct.</p> <p>Conclusions</p> <p>Our low-molecular-weight W-tags can be used to monitor the production of antibody fragments on-line. The binding specificity of the recombinant fusion protein is not affected, even though the binding activity decreases slightly with increasing number of tryptophan residues in the W-tags. Thus, the newly designed W-tags offer a versatile and generally applicable alternative to current fluorescent fusion tags.</p

Community wildfire protection plan

207 pp. Bookmarks supplied by UO. Includes maps and figures. Published Novembber, 2007. Captured March 17, 2008.This plan describes Linn County’s risk from wildfires as well as the specific steps that it will take to reduce that risk now and in the future. It is a Community Wildfire Protection Plan (CWPP), a collaborative effort to reduce the potential for future loss of life and property resulting from wildfire.... This CWPP is intended to assist Linn County in reducing its risk from WUI wildfire hazards by identifying resources, information, and strategies for risk reduction. It will also help to guide and coordinate mitigation activities throughout the County. [From the Plan