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Jordan T.,University of Zürich | Seeholzer S.,University of Zürich | Seeholzer S.,Baxter AG | Schwizer S.,University of Zürich | And 4 more authors.
Plant Journal | Year: 2011

The race-specific barley powdery mildew (Blumeria graminis f. sp. hordei) resistance gene Mla occurs as an allelic series and encodes CC-NB-LRR type resistance proteins. Inter-generic allele mining resulted in the isolation and characterisation of an Mla homologue from diploid wheat, designated TmMla1, which shares 78% identity with barley HvMLA1 at the protein level. TmMla1 was found to be a functional resistance gene against Blumeria graminis f. sp. tritici in wheat, hereby providing an example of R gene orthologs controlling the same disease in two different species. TmMLA1 exhibits race-specific resistance activity and its N-terminal coiled-coil domain interacts with the barley transcription factor HvWRKY1. Interestingly, TmMLA1 was not functional in barley transient assays. Replacement of the TmMLA1 LRR domain with that of HvMLA1 revealed that this fusion protein conferred resistance against B. graminis f. sp. hordei isolate K1 in barley. Thus, TmMLA1 not only confers resistance in wheat but possibly also in barley against an as yet unknown barley powdery mildew race. The conservation of functional R gene orthologs over at least 12 million years is surprising given the observed rapid breakdown of Mla-based resistance against barley mildew in agricultural ecosystems. This suggests a high stability of Mla resistance in the natural environment before domestication. © 2011 Blackwell Publishing Ltd.


Seeholzer S.,University of Zürich | Seeholzer S.,Baxter AG | Tsuchimatsu T.,University of Zürich | Tsuchimatsu T.,University of Tokyo | And 10 more authors.
Molecular Plant-Microbe Interactions | Year: 2010

The Mla locus in barley (Hordeum vulgare) conditions iso-late-specific immunity to the powdery mildew fungus (Blumeria graminis f. sp. hordei) and encodes intracellular coiled-coil (CC) domain, nucleotide-binding (NB) site, and leucine-rich repeat (LRR)-containing receptor proteins. Over the last decades, genetic studies in breeding material have identified a large number of functional resistance genes at the Mla locus. To study the structural and func-tional diversity of this locus at the molecular level, we iso-lated 23 candidate MLA cDNAs from barley accessions that were previously shown by genetic studies to harbor different Mla resistance specificities. Resistance activity was detected for 13 candidate MLA cDNAs in a transient gene-expression assay. Sequence alignment of the deduced MLA proteins improved secondary structure predictions, revealing four additional, previously overlooked LRR. Analysis of nucleotide diversity of the candidate and vali-dated MLA cDNAs revealed 34 sites of positive selection. Recombination or gene conversion events were frequent in the first half of the gene but positive selection was also found when this region was excluded. The positively se-lected sites are all, except two, located in the LRR domain and cluster in predicted solvent-exposed residues of the repeats 7 to 15 and adjacent turns on the concave side of the predicted solenoid protein structure. This domain-restricted pattern of positively selected sites, together with the length conservation of individual LRR, suggests direct binding of effectors to MLA receptors. © 2010 The American Phytopathological Society.


Nubling C.M.,Paul Ehrlich Institute | Baylis S.A.,Paul Ehrlich Institute | Hanschmann K.-M.,Paul Ehrlich Institute | Montag-Lessing T.,Paul Ehrlich Institute | And 36 more authors.
Applied and Environmental Microbiology | Year: 2015

Nucleic acid amplification technique (NAT)-based assays (referred to here as NAT assays) are increasingly used as an alternative to culture-based approaches for the detection of mycoplasma contamination of cell cultures. Assay features, like the limit of detection or quantification, vary widely between different mycoplasma NAT assays. Biological reference materials may be useful for harmonization of mycoplasma NAT assays. An international feasibility study included lyophilized preparations of four distantly related mycoplasma species (Acholeplasma laidlawii, Mycoplasma fermentans, M. orale, M. pneumoniae) at different concentrations which were analyzed by 21 laboratories using 26 NAT assays with a qualitative, semiquantitative, or quantitative design. An M. fermentans preparation was shown to decrease the interassay variation when used as a common reference material. The preparation was remanufactured and characterized in a comparability study, and its potency (in NAT-detectable units) across different NATs was determined. The World Health Organization (WHO) Expert Committee on Biological Standardization (ECBS) established this preparation to be the "1st World Health Organization international standard for mycoplasma DNA for nucleic acid amplification technique-based assays designed for generic mycoplasma detection" (WHO Tech Rep Ser 987:42, 2014) with a potency of 200,000 IU/ml. ThisWHOinternational standard is now available as a reference preparation for characterization of NAT assays, e.g., for determination of analytic sensitivity, for calibration of quantitative assays in a common unitage, and for defining regulatory requirements in the field of mycoplasma testing. © 2015, American Society for Microbiology.


Wahl V.,Research Center Pharmaceutical Engineering | Wahl V.,University of Graz | Leitgeb S.,Research Center Pharmaceutical Engineering | Laggner P.,Research Center Pharmaceutical Engineering | And 5 more authors.
European Journal of Pharmaceutics and Biopharmaceutics | Year: 2015

The purpose of this work was to investigate the influence of residual water in freeze-dried protein powders on the dissolution behavior of the solid-state proteins. To that end, six freeze-dried fibrinogen powder lots were stored at four levels of relative humidity and analyzed with regard to the particle size and shape, the specific surface area, the solid state of protein and the inner surface. Furthermore, the dissolution behavior of the powders was investigated. We clearly identified differences in the specific surface area, specific inner surface area, crystallinity, particle size and shape, which we were able to correlate to the dissolution behavior. These differences were triggered due to the different levels of residual moisture during two weeks of storage. Thus, we were able to show that the storage conditions have significant impact on the processing of pharmaceutical protein materials. © 2015 Elsevier B.V. All rights reserved.


Wagner E.M.,Baxter AG
Methods in Molecular Biology | Year: 2013

Two-step quantitative real-time RT-PCR (RT-qPCR), also known as real-time RT-PCR, kinetic RT-PCR, or quantitative fluorescent RT-PCR, has become the method of choice for gene expression analysis during the last few years. It is a fast and convenient PCR method that combines traditional RT-PCR with the phenomenon of fluorescence resonance energy transfer (FRET) using fluorogenic primers. The detection of changes in fluorescence intensity during the reaction enables the user to follow the PCR reaction in real time. RT-qPCR comprises several steps: (1) RNA is isolated from target tissue/cells; (2) mRNA is reverse-transcribed to cDNA; (3) modified gene-specific PCR primers are used to amplify a segment of the cDNA of interest, following the reaction in real time; and (4) the initial concentration of the selected transcript in a specific tissue or cell type is calculated from the exponential phase of the reaction. Relative quantification or absolute quantification compared to standards that are run in parallel can be performed. This chapter describes the entire procedure from isolation of total RNA from liver and fatty tissues/cells to the use of RT-qPCR to study gene expression in these tissues. We perform relative quantification of transcripts to calculate the fold-difference of a certain mRNA level between different samples. In addition, tips for choosing primers and performing analyses are provided to help the beginner in understanding the technique. © Springer Science+Business Media, LLC 2013.


Planitzer C.B.,Baxter AG | Saemann M.D.,Medical University of Vienna | Gajek H.,Baxter AG | Farcet M.R.,Baxter AG | Kreil T.R.,Baxter AG
Transplantation | Year: 2011

Background: Cytomegalovirus (CMV) remains one of the most important pathogens after transplantation, potentially leading to CMV disease, allograft dysfunction, acute, and chronic rejection and opportunistic infections. Immunoglobulin G (IgG) preparations with high antibody titers against CMV are a valuable adjunctive prevention and treatment option for clinicians and apart from standard intravenous immunoglobulin (IVIG), CMV hyperimmune preparations are available. The CMV antibody titer of these preparations is typically determined by Enzyme-linked immunosorbent assay (ELISA), also used for the selection of high titer plasma donors for the production of the CMV Hyperimmune product. However, CMV ELISA titers do not necessarily correlate with CMV antibody function which is determined by virus neutralization tests. Methods: CMV antibody titers were determined by both ELISA and virus neutralization assay and the IgG subclass distribution was compared between a CMV hyperimmune licensed in Europe and standard IVIG preparations. RESULTS.: Although the expected high CMV IgG ELISA antibody titers were confirmed for three lots of a CMV hyperimmune preparation, the functionally more relevant CMV neutralizing antibody titers were significantly higher for 31 lots of standard IVIG preparations. Moreover, considerably lower IgG3 levels were found for the CMV hyperimmune preparation compared with standard IVIG preparations. Conclusions: The higher functional CMV neutralization titers of standard IVIG preparations and the better availability of these preparations, suggest that these products could be a valuable alternative to the CMV hyperimmune preparation. © 2011 by Lippincott Williams & Wilkins.


Friedrich V.,University of Vienna | Gruber C.,University of Vienna | Nimeth I.,University of Vienna | Pabinger S.,AIT Austrian Institute of Technology | And 8 more authors.
Molecular Oral Microbiology | Year: 2015

Tannerella forsythia is the only 'red-complex' bacterium covered by an S-layer, which has been shown to affect virulence. Here, outer membrane vesicles (OMVs) enriched with putative glycoproteins are described as a new addition to the virulence repertoire of T. forsythia. Investigations of this bacterium are hampered by its fastidious growth requirements and the recently discovered mismatch of the available genome sequence (92A2 = ATCC BAA-2717) and the widely used T. forsythia strain (ATCC 43037). T. forsythia was grown anaerobically in serum-free medium and biogenesis of OMVs was analyzed by electron and atomic force microscopy. This revealed OMVs with a mean diameter of ~100 nm budding off from the outer membrane while retaining the S-layer. An LC-ESI-TOF/TOF proteomic analysis of OMVs from three independent biological replicates identified 175 proteins. Of these, 14 exhibited a C-terminal outer membrane translocation signal that directs them to the cell/vesicle surface, 61 and 53 were localized to the outer membrane and periplasm, respectively, 22 were predicted to be extracellular, and 39 to originate from the cytoplasm. Eighty proteins contained the Bacteroidales O-glycosylation motif, 18 of which were confirmed as glycoproteins. Release of pro-inflammatory mediators from the human monocytic cell line U937 and periodontal ligament fibroblasts upon stimulation with OMVs followed a concentration-dependent increase that was more pronounced in the presence of soluble CD14 in conditioned media. The inflammatory response was significantly higher than that caused by whole T. forsythia cells. Our study represents the first characterization of T. forsythia OMVs, their proteomic composition and immunogenic potential. © 2015 John Wiley & Sons A/S.


Lindemann E.,University of Stuttgart | Lindemann E.,Fraunhofer Institute for Interfacial Engineering and Biotechnology | Rohde B.,GATC Biotech. | Rupp S.,Fraunhofer Institute for Interfacial Engineering and Biotechnology | And 3 more authors.
Electrophoresis | Year: 2010

Differential gene expression profiling has become of central importance for the analysis of cellular systems at the transcriptional level. By now, many platform technologies including DNA-microarrays, serial analysis of gene expression or RNA-seq have been established in order to facilitate transcriptional profiling. However, these technologies are all subjected to specific limitations, as they require a priori knowledge of annotated genome sequences or are based on substantial bioinformatic infrastructure, for example. As an unbiased alternative we describe here a multidimensional electrophoretic system of separation for the analysis of gene expression for the global transcriptional profiling in any eukaryotic organism. This approach is compatible with standard laboratory equipment comprising high-resolution separation of complex cDNA-probes using two-dimensional DNA-gel electrophoresis. In this context cDNA fragments are separated using non-denaturing PAGE in the first dimension with subsequent denaturing gradient gel electrophoresis in the second dimension. Two-dimensional spot patterns are quantified by well-established bioinformatic algorithms and selected spots are identified using DNA sequencing. Neither does this method necessarily depend on annotated genome sequences, nor does it require sophisticated instrumentation. Strikingly, quantitative data on differential gene expression derived from multidimensional electrophoretic system of separation for the analysis of gene expression highly correlate with corresponding data from quantitative RT-PCR even for transcriptional profiles of limited amounts of total RNA. © 2010 Wiley-VCH Verlag GmbH & Co. KGaA.


PubMed | Baxter AG
Type: | Journal: Methods in molecular biology (Clifton, N.J.) | Year: 2013

Two-step quantitative real-time RT-PCR (RT-qPCR), also known as real-time RT-PCR, kinetic RT-PCR, or quantitative fluorescent RT-PCR, has become the method of choice for gene expression analysis during the last few years. It is a fast and convenient PCR method that combines traditional RT-PCR with the phenomenon of fluorescence resonance energy transfer (FRET) using fluorogenic primers. The detection of changes in fluorescence intensity during the reaction enables the user to follow the PCR reaction in real time.RT-qPCR comprises several steps: (1) RNA is isolated from target tissue/cells; (2) mRNA is reverse-transcribed to cDNA; (3) modified gene-specific PCR primers are used to amplify a segment of the cDNA of interest, following the reaction in real time; and (4) the initial concentration of the selected transcript in a specific tissue or cell type is calculated from the exponential phase of the reaction. Relative quantification or absolute quantification compared to standards that are run in parallel can be performed.This chapter describes the entire procedure from isolation of total RNA from liver and fatty tissues/cells to the use of RT-qPCR to study gene expression in these tissues. We perform relative quantification of transcripts to calculate the fold-difference of a certain mRNA level between different samples. In addition, tips for choosing primers and performing analyses are provided to help the beginner in understanding the technique.

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