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Isolation and characterization of circular replicase-encoding single-stranded (ss) DNA from animal, plant and environmental samples are rapidly evolving in virology. We detected 21 circular DNA elements, including one genomoviral sequence, in individual milk samples from domesticated Asian water buffaloes (Bubalus arnee f. bubalis). Most of the obtained genomes are related to Sphinx 1.76 and Sphinx 2.36 sequences and share a high degree of similarity to recently published circular DNAs—named BMMF (bovine meat and milk factors)—that have been isolated from commercial milk, as well as from bovine serum. Characteristic features such as rep genes, tandem repeats and inverted repeats were detected. These BMMF have recently been found to be present in taurine-type dairy cattle breeds descending from the aurochs (Bos primigenius). Importantly, the occurrence of BMMF has been linked to the higher incidence of colorectal and breast cancer in North America and Western Europe compared with Asia. This is the first report of circular ssDNA detected in milk from the domesticated form of the wild Asian water buffalo (B. arnee) belonging to the subfamily Bovinae. This novelty should be taken into account in view of the above-mentioned cancer hypothesis.
Marie-T. König; Robert Fux; Ellen Link; Gerd Sutter; Erwin Märtlbauer; Andrea Didier. Circular Rep-Encoding Single-Stranded DNA Sequences in Milk from Water Buffaloes (Bubalus arnee f. bubalis). Viruses 2021, 13, 1088 .
AMA StyleMarie-T. König, Robert Fux, Ellen Link, Gerd Sutter, Erwin Märtlbauer, Andrea Didier. Circular Rep-Encoding Single-Stranded DNA Sequences in Milk from Water Buffaloes (Bubalus arnee f. bubalis). Viruses. 2021; 13 (6):1088.
Chicago/Turabian StyleMarie-T. König; Robert Fux; Ellen Link; Gerd Sutter; Erwin Märtlbauer; Andrea Didier. 2021. "Circular Rep-Encoding Single-Stranded DNA Sequences in Milk from Water Buffaloes (Bubalus arnee f. bubalis)." Viruses 13, no. 6: 1088.
A major virulence factor involved in Bacillus cereus food poisoning is the three-component enterotoxin hemolysin BL. It consists of the binding component B and the two lytic components L1 and L2. Studying its mode of action has been challenging, as natural culture supernatants additionally contain Nhe, the second three-component enterotoxin, and purification of recombinant (r) Hbl components has been difficult. In this study, we report on pore-forming, cytotoxic, cell binding and hemolytic activity of recently generated rHbl components expressed in E. coli. It is known that all three Hbl components are necessary for cytotoxicity and pore formation. Here we show that an excess of rHbl B enhances, while an excess of rHbl L1 hinders, the velocity of pore formation. Most rapid pore formation was observed with ratios L2:L1:B = 1:1:10 and 10:1:10. It was further verified that Hbl activity is due to sequential binding of the components B - L1 - L2. Accordingly, all bioassays proved that binding of Hbl B to the cell surface is the crucial step for pore formation and cytotoxic activity. Binding of Hbl B took place within minutes, while apposition of the following L1 and L2 occurred immediately. Further on, applying toxin components simultaneously, it seemed that Hbl L1 enhanced binding of B to the target cell surface. Overall, these data contribute significantly to the elucidation of the mode of action of Hbl, and suggest that its mechanism of pore formation differs substantially from that of Nhe, although both enterotoxin complexes are sequentially highly related.
Nadja Jessberger; Richard Dietrich; Stefanie Schwemmer; Franziska Tausch; Valerie Schwenk; Andrea Didier; Erwin Märtlbauer. Binding to The Target Cell Surface Is The Crucial Step in Pore Formation of Hemolysin BL from Bacillus cereus. Toxins 2019, 11, 281 .
AMA StyleNadja Jessberger, Richard Dietrich, Stefanie Schwemmer, Franziska Tausch, Valerie Schwenk, Andrea Didier, Erwin Märtlbauer. Binding to The Target Cell Surface Is The Crucial Step in Pore Formation of Hemolysin BL from Bacillus cereus. Toxins. 2019; 11 (5):281.
Chicago/Turabian StyleNadja Jessberger; Richard Dietrich; Stefanie Schwemmer; Franziska Tausch; Valerie Schwenk; Andrea Didier; Erwin Märtlbauer. 2019. "Binding to The Target Cell Surface Is The Crucial Step in Pore Formation of Hemolysin BL from Bacillus cereus." Toxins 11, no. 5: 281.
The non-hemolytic enterotoxin complex (Nhe) is supposed to be the main virulence factor of B. cereus causing a diarrheal outcome of food poisoning. This tripartite toxin consists of the single components NheA, -B and -C all of them being necessary for maximum toxicity. In the past, research activities aiming to elucidate the mode-of-action of Nhe were mostly focused on the B- and C-component. In this study the generation of novel monoclonal antibodies (mAb) and their thorough characterization enabled the determination of key features for NheA. By the means of immunoaffinity chromatography it could be shown that NheA does not interact with -B and -C in solution. Additionally, the establishment of a highly sensitive sandwich-EIA now enables the detection of NheA in B. cereus supernatants down to 20 pg ml-1.Peptide-based epitope mapping in combination with partially deleted recombinant NheA fragments allowed the allocation of the binding regions for the three mAbs under study. Furthermore, by different EIA set-ups the conformational flexibility of NheA could be shown. For two of the antibodies under study different mechanisms of NheA neutralization were proven. Due to prevention of complete pore formation by one of the antibodies, NheA could be detected in an intermediate stage of the tripartite complex on the cell surface. Taken together, the results obtained in the present study allow a refinement of the mode-of-action for the Nhe toxin-complex.
A. Didier; R. Dietrich; Erwin Märtlbauer. Antibody Binding Studies Reveal Conformational Flexibility of the Bacillus cereus Non-Hemolytic Enterotoxin (Nhe) A-Component. PLOS ONE 2016, 11, e0165135 -e0165135.
AMA StyleA. Didier, R. Dietrich, Erwin Märtlbauer. Antibody Binding Studies Reveal Conformational Flexibility of the Bacillus cereus Non-Hemolytic Enterotoxin (Nhe) A-Component. PLOS ONE. 2016; 11 (10):e0165135-e0165135.
Chicago/Turabian StyleA. Didier; R. Dietrich; Erwin Märtlbauer. 2016. "Antibody Binding Studies Reveal Conformational Flexibility of the Bacillus cereus Non-Hemolytic Enterotoxin (Nhe) A-Component." PLOS ONE 11, no. 10: e0165135-e0165135.
The non-hemolytic enterotoxin (Nhe) of Bacillus cereus is a three-partite toxin formed of the components NheA, -B and -C. Pore formation and subsequent lysis of target cells caused by Nhe is an orchestrated process comprising three steps: (i) formation of NheB/C oligomers in solution, (ii) attachment of the oligomers to the cell membrane, (iii) binding of NheA to the oligomers. The present study aimed to characterize the properties of the NheB/C complex and the fate of the target cell upon binding. An enzyme immunoassay allowing kinetic measurements and surface plasmon resonance revealed the fast and high affinity formation of the NheB/C oligomers. The benefit of these complexes is a more stable cell binding as well as stronger and earlier cytotoxic effect. High molecular mass hetero-oligomers (620 kDa) probably consisting of one NheC and up to 15 NheB were detected by size-exclusion chromatography and on native PAGE immunoblots. Due to the NheBC application the morphology and membrane permeability of Vero cells is partly disturbed. Formation of stable transmembrane channels with a conductance of about 870 pS and a diameter of about 2 nm due to the application of NheBC could be demonstrated in lipid bilayer experiments. Thus, the NheBC complex itself has a tendency to increase the membrane permeability prior to the emergence of full pores containing also NheA.
Kui Zhu; Andrea Didier; Richard Dietrich; Uta Heilkenbrinker; Eva Waltenberger; Nadja Jessberger; Erwin Märtlbauer; Roland Benz. Formation of small transmembrane pores: An intermediate stage on the way to Bacillus cereus non-hemolytic enterotoxin (Nhe) full pores in the absence of NheA. Biochemical and Biophysical Research Communications 2016, 469, 613 -618.
AMA StyleKui Zhu, Andrea Didier, Richard Dietrich, Uta Heilkenbrinker, Eva Waltenberger, Nadja Jessberger, Erwin Märtlbauer, Roland Benz. Formation of small transmembrane pores: An intermediate stage on the way to Bacillus cereus non-hemolytic enterotoxin (Nhe) full pores in the absence of NheA. Biochemical and Biophysical Research Communications. 2016; 469 (3):613-618.
Chicago/Turabian StyleKui Zhu; Andrea Didier; Richard Dietrich; Uta Heilkenbrinker; Eva Waltenberger; Nadja Jessberger; Erwin Märtlbauer; Roland Benz. 2016. "Formation of small transmembrane pores: An intermediate stage on the way to Bacillus cereus non-hemolytic enterotoxin (Nhe) full pores in the absence of NheA." Biochemical and Biophysical Research Communications 469, no. 3: 613-618.
The ability of Bacillus cereus to cause foodborne toxicoinfections leads to increasing concerns regarding consumer protection. For the diarrhea-associated enterotoxins, the assessment of the non-hemolytic enterotoxin B (NheB) titer determined by a sandwich enzyme immunoassay (EIA) correlates best with in vitro cytotoxicity. In general, the regulation of enterotoxin expression of B. cereus is a coordinately-regulated process influenced by environmental, and probably also by host factors. As long as these factors are not completely understood, the currently-applied diagnostic procedures are based on indirect approaches to assess the potential virulence of an isolate. To date, sandwich EIA results serve as a surrogate marker to categorize isolates as either potentially low or highly toxic. Here, we report on a single amino acid exchange in the NheB sequence leading to an underestimation of the cytotoxic potential in a limited number of strains. During the screening of a large panel of B. cereus isolates, six showed uncommon features with low sandwich EIA titers despite high cytotoxicity. Sequence analysis revealed the point-mutation Glu151Asp in the potential binding region of the capture antibody. Application of this antibody also results in low titers in an indirect EIA format and shows variable detection intensities in Western-immunoblots. A commercially-available assay based on a lateral flow device detects all strains correctly as NheB producers in a qualitative manner. In conclusion, isolates showing low NheB titers should additionally be assayed in an indirect EIA or for their in vitro cytotoxicity to ensure a correct classification as either low or highly toxic.
Andrea Didier; Nadja Jeßberger; Victoria Krey; Richard Dietrich; Siegfried Scherer; Erwin Märtlbauer. The Mutation Glu151Asp in the B-Component of the Bacillus cereus Non-Hemolytic Enterotoxin (Nhe) Leads to a Diverging Reactivity in Antibody-Based Detection Systems. Toxins 2015, 7, 4655 -4667.
AMA StyleAndrea Didier, Nadja Jeßberger, Victoria Krey, Richard Dietrich, Siegfried Scherer, Erwin Märtlbauer. The Mutation Glu151Asp in the B-Component of the Bacillus cereus Non-Hemolytic Enterotoxin (Nhe) Leads to a Diverging Reactivity in Antibody-Based Detection Systems. Toxins. 2015; 7 (11):4655-4667.
Chicago/Turabian StyleAndrea Didier; Nadja Jeßberger; Victoria Krey; Richard Dietrich; Siegfried Scherer; Erwin Märtlbauer. 2015. "The Mutation Glu151Asp in the B-Component of the Bacillus cereus Non-Hemolytic Enterotoxin (Nhe) Leads to a Diverging Reactivity in Antibody-Based Detection Systems." Toxins 7, no. 11: 4655-4667.
Considering the urgent demand for rapid and accurate determination of bacterial toxins and the recent promising developments in nanotechnology and microfluidics, this review summarizes new achievements of the past five years. Firstly, bacterial toxins will be categorized according to their antibody binding properties into low and high molecular weight compounds. Secondly, the types of antibodies and new techniques for producing antibodies are discussed, including poly- and mono-clonal antibodies, single-chain variable fragments (scFv), as well as heavy-chain and recombinant antibodies. Thirdly, the use of different nanomaterials, such as gold nanoparticles (AuNPs), magnetic nanoparticles (MNPs), quantum dots (QDs) and carbon nanomaterials (graphene and carbon nanotube), for labeling antibodies and toxins or for readout techniques will be summarized. Fourthly, microscale analysis or minimized devices, for example microfluidics or lab-on-a-chip (LOC), which have attracted increasing attention in combination with immunoassays for the robust detection or point-of-care testing (POCT), will be reviewed. Finally, some new materials and analytical strategies, which might be promising for analyzing toxins in the near future, will be shortly introduced.
Kui Zhu; Richard Dietrich; Andrea Didier; Dominik Doyscher; Erwin Märtlbauer. Recent Developments in Antibody-Based Assays for the Detection of Bacterial Toxins. Toxins 2014, 6, 1325 -1348.
AMA StyleKui Zhu, Richard Dietrich, Andrea Didier, Dominik Doyscher, Erwin Märtlbauer. Recent Developments in Antibody-Based Assays for the Detection of Bacterial Toxins. Toxins. 2014; 6 (4):1325-1348.
Chicago/Turabian StyleKui Zhu; Richard Dietrich; Andrea Didier; Dominik Doyscher; Erwin Märtlbauer. 2014. "Recent Developments in Antibody-Based Assays for the Detection of Bacterial Toxins." Toxins 6, no. 4: 1325-1348.
The present study shows that PrPc is expressed in the mammary gland and milk fractions of domestic ruminants in a species-specific manner. By applying immunohistochemistry, Western blot and ELISA, clear expression differences between bovine, ovine and caprine mammary gland, skimmed milk, acid whey and cream could be demonstrated, the highest relative PrPc levels being associated with the cream fraction. In the bovine gland PrPc was preferentially detectable at the basolateral surface of mammary gland epithelial cells, whereas in ovine and caprine samples the prion protein was more homogeneously distributed. Moreover, in ovine and caprine bovine mammary gland epithelial cells, apocrine secretory vesicles were strongly stained. Ovine and caprine milk proved to contain PrPc in all fractions with an additional truncated form at 12 kDa in Western blot. This truncated isoform is the predominate one in caprine acid whey. These results support the hypothesis that the apocrine secretion mode of milk fat globules is a major way of PrPc transport into the milk.
A. Didier; R. Gebert; R. Dietrich; M. Schweiger; M. Gareis; Erwin Märtlbauer; W.M. Amselgruber. Cellular prion protein in mammary gland and milk fractions of domestic ruminants. Biochemical and Biophysical Research Communications 2008, 369, 841 -844.
AMA StyleA. Didier, R. Gebert, R. Dietrich, M. Schweiger, M. Gareis, Erwin Märtlbauer, W.M. Amselgruber. Cellular prion protein in mammary gland and milk fractions of domestic ruminants. Biochemical and Biophysical Research Communications. 2008; 369 (3):841-844.
Chicago/Turabian StyleA. Didier; R. Gebert; R. Dietrich; M. Schweiger; M. Gareis; Erwin Märtlbauer; W.M. Amselgruber. 2008. "Cellular prion protein in mammary gland and milk fractions of domestic ruminants." Biochemical and Biophysical Research Communications 369, no. 3: 841-844.
The programmed cell death—so-called apoptosis—is a physiological process occurring in all multicellular organisms to control cell-number homeostasis. Nevertheless, increase of apoptotic cell death in different organs can lead to pathological alterations. As zinc is a potent inhibitor of apoptosis, we investigated the influence of zinc deficiency on mRNA expression levels of caspase-3 and Fas in adult rats. For this purpose, 24 adult rats fed a Zn-deficient diet for up to 29 days were compared to seven animals in the control group. After 1, 2, 4, 7, 11, 16, 22 and 29 days of treatment three animals were sacrificed (n=24). Total RNA extraction from thymus, liver, jejunum and colon was carried out. Samples were reverse transcribed and subjected to real-time PCR. Relative quantification of caspase-3 and Fas mRNA expression was achieved on the basis of normalisation by glycerolaldehyd-3-phosphate-dehydrogenase mRNA expression levels in all samples. In jejunum, up to day 11 the relative mRNA expression of the respective genes decreased. A significant increase in caspase-3 and Fas expression was found from day 11 of zinc deficiency onward. In contrast, mRNA expression in liver and colon remained unaffected, whereas thymus showed a slight but not significant increase in the expression of these genes. This study provides the first evidence that even moderate zinc deficiency in an adult, non-growing rat model is able to elevate mRNA expression levels of factors involved in early stages of apoptosis.
Andrea Didier; Wilhelm Windisch; Michael W. Pfaffl. Elevated caspase-3 and Fas mRNA expression in jejunum of adult rats during subclinical zinc deficiency. Journal of Trace Elements in Medicine and Biology 2004, 18, 41 -45.
AMA StyleAndrea Didier, Wilhelm Windisch, Michael W. Pfaffl. Elevated caspase-3 and Fas mRNA expression in jejunum of adult rats during subclinical zinc deficiency. Journal of Trace Elements in Medicine and Biology. 2004; 18 (1):41-45.
Chicago/Turabian StyleAndrea Didier; Wilhelm Windisch; Michael W. Pfaffl. 2004. "Elevated caspase-3 and Fas mRNA expression in jejunum of adult rats during subclinical zinc deficiency." Journal of Trace Elements in Medicine and Biology 18, no. 1: 41-45.