Development and assessment of a novel Arxula adeninivorans androgen screen (A-YAS) assay and its application in analysis of cattle urine

Gerlach, Torsten; Knaust, Jacqueline; Kaiser, Christian; Körner, Martina; Hettwer, Karina; Uhlig, Steffen; Simon, Kirsten; Baronian, Keith; Kunze, Gotthard

doi:10.1016/j.scitotenv.2014.05.100

Cited by 17 publications

(10 citation statements)

References 63 publications

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“…ASR1 results in the binding of progesterone receptor to its response element which induces the extracellular production of phytase K, which then catalyzes a reaction to produce the chromogenic substrate, pnitrophenol. This system has been successfully demonstrated in the detection of other steroids [28,43]. When dsRED is used as reporter gene, the binding of the progesterone receptor to its response element induces the production of the intracellular dsRED protein, which is fluorescent and can be excited at 422 nm to give an emission signal at 488 nm.…”

Section: Discussionmentioning

confidence: 99%

“…The reaction was stopped by adding 80 μl 3 M NaOH and the absorbance of the solution was measured at 405 nm with a Sunrise™ reader M200 pro (Tecan Trading AG, Zürich, Switzerland). Determination of anti-progesterone effects was performed as described in previous work [28]. All experiments were performed in triplicate.…”

Section: Biochemical Detection Of Progesterone Receptor Interactionmentioning

confidence: 99%

“…Some in vitro whole cell biosensors have focused on estrogen-or androgen-related compounds, for example the YES assay (using S. cerevisiae as the detection element) [24] or the A-YES assay (using A. adeninivorans) [25] and their counterparts such as YAS [26,27] and A-YAS assays for androgen binding substances [28]. For progesterone detection, similar assays have been developed using mammalian or yeast cells as host for the production of the progesterone receptor and an enzyme or a fluorescent protein as reporter [29][30][31][32][33][34].…”

Section: Introductionmentioning

confidence: 99%

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Development of a recombinant Arxula adeninivorans cell bioassay for the detection of molecules with progesterone activity in wastewater

et al. 2015

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This study describes the development of a bioassay to detect the presence of progesterone and progesterone-like molecules in wastewater samples. The basis of the bioassay is the integration of the human progesterone receptor gene into the yeast Arxula adeninivorans for the constitutive synthesis of the receptor. After incubation, binding of the analyte to the receptor induces the production of a reporter protein. Two reporter proteins were compared for detection parameters such as half-maximal activity (EC50), limit of detection (LoD) and limit of quantification (LoQ). When the extracellular phytase K was used, an EC50 value of 155 ng L(-1) and a LoD of 27 ng L(-1) progesterone were obtained after 4 h incubation, while use of the fluorescent dsRED as the reporter protein, resulted in an EC50 of 320 ng L(-1) and a LoD of 65 ng L(-1) after 20 h incubation. Use of phytase K as the reporter protein offers decreased incubation time and increased sensitivity; however the dsRED reporter system is less labor-intensive. Additionally, the affinity of known agonists and antagonists of the human progesterone receptor was determined. The utility of this bioassay was confirmed by measuring total progesterone equivalent concentration of samples from a wastewater treatment plant. The A. adeninivorans-based transactivation assay was able to measure concentrations of about 311 ng L(-1) in the influent stream but could not detect progesterone activity in effluent. One key feature of the assay is the robustness of A. adeninivorans, which allows sample measurement without any sample preparation.

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Section: Discussionmentioning

confidence: 99%

Section: Biochemical Detection Of Progesterone Receptor Interactionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Development of a recombinant Arxula adeninivorans cell bioassay for the detection of molecules with progesterone activity in wastewater

et al. 2015

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“…The screenings of the transgenic A. adeninivorans G1212/YRC102-hAhRhARNT-phyK strains were assessed for activation with and without 10 g/l 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), which is the most active agonist for hAhR. Phytase activity assay was performed as previously described [30,31,33].…”

Section: Construction Of a Adeninivorans Transformantsmentioning

confidence: 99%

“…Other widely used methods are based on biosensors which focus most on the determination of hormones (estrogenic, androgenic, gestagenic molecules) for example the YES-assay [27], E-Screen assay [28], A-YES assay [29], nAES assay [30], EstraMonitor apparatus [31,32], A-YAS [33] and recombinant cell bioassays [34]. There are several bacterial and yeast-based assays for pharmaceutical screening [35,36] which are used for detecting genotoxicity.…”

Section: Introductionmentioning

confidence: 99%

The determination of pharmaceuticals in wastewater using a recombinant Arxula adeninivorans whole cell biosensor

Pham¹,

Giersberg²,

Gehrmann³

et al. 2015

Sensors and Actuators B: Chemical

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Simultaneous detection of three sex steroid hormone classes using a novel yeast‐based biosensor

Chamas

Pham

Jähne³

et al. 2017

Biotech & Bioengineering

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A biosensor detecting estrogens, progestogens, and androgens in complex samples and in a single step is described. Three Arxula adeninivorans yeast strains were created, each strain producing a different recombinant human hormone receptor and a different fluorescent reporter protein. These strains were then mixed to create G1212/YRC102-hHR-fluo, the biological component of the biosensor. During incubation with G1212/YRC102-hHR-fluo, hormones present in a sample bind to their target receptor, which leads to the production of a specific fluorescent protein. Three fluorescence scans of the yeast suspension determine which fluorescence protein has been produced, thus revealing which hormone receptor (estrogen, progesterone, and androgen) has been activated by the hormones or hormone mimics present in the sample. The biosensor has similar sensitivities to the existing A. adeninivorans cell-based assays. The detection of the three hormone classes in one single experiment reduces the labor and time required to assay for the three hormone classes. The biosensor was also trialed with animal serum samples for the detection of progestogens, androgens, and estrogens and gave results that correlated well with ELISA analysis in case of progestogens. These results highlight the potential usefulness of the biosensor for comprehensive determination of hormone status in samples from veterinary origin. Biotechnol. Bioeng. 2017;114: 1539-1549. © 2017 Wiley Periodicals, Inc.

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Development and assessment of a novel Arxula adeninivorans androgen screen (A-YAS) assay and its application in analysis of cattle urine

Cited by 17 publications

References 63 publications

Development of a recombinant Arxula adeninivorans cell bioassay for the detection of molecules with progesterone activity in wastewater

Development of a recombinant Arxula adeninivorans cell bioassay for the detection of molecules with progesterone activity in wastewater

The determination of pharmaceuticals in wastewater using a recombinant Arxula adeninivorans whole cell biosensor

Simultaneous detection of three sex steroid hormone classes using a novel yeast‐based biosensor

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