Yeast grown in continuous culture systems can detect mutagens with improved sensitivity relative to the Ames test.

Continuous culture systems allow for the controlled growth of microorganisms over a long period of time. Here, we develop a novel test for mutagenicity that involves growing yeast in continuous culture systems exposed to low levels of mutagen for a period of approximately 20 days. In contrast, most microorganism-based tests for mutagenicity expose the potential mutagen to the biological reporter at a high concentration of mutagen for a short period of time. Our test improves upon the sensitivity of the well-established Ames test by at least 20-fold for each of two mutagens that act by different mechanisms (the intercalator ethidium bromide and alkylating agent methyl methanesulfonate). To conduct the tests, cultures were grown in small, inexpensive continuous culture systems in media containing (potential) mutagen, and the resulting mutagenicity of the added compound was assessed via two methods: a canavanine-based plate assay and whole genome sequencing. In the canavanine-based plate assay, we were able to detect a clear relationship between the amount of mutagen and the number of canavanine-resistant mutant colonies over a period of one to three weeks of exposure. Whole genome sequencing of yeast grown in continuous culture systems exposed to methyl methanesulfonate demonstrated that quantification of mutations is possible by identifying the number of unique variants across each strain. However, this method had lower sensitivity than the plate-based assay and failed to distinguish the different concentrations of mutagen. In conclusion, we propose that yeast grown in continuous culture systems can provide an improved and more sensitive test for mutagenicity.

When Is an In Silico Representation a Digital Twin? A Biopharmaceutical Industry Approach to the Digital Twin Concept.

Digital twins (DTs) are expected to render process development and life-cycle management much more cost-effective and time-efficient. A DT definition, a brief retrospect on their history and expectations for their deployment in today's business environment, and a detailed financial assessment of their attractive economic benefits are provided in this chapter. The argument that restrictive guidelines set forth by regulatory agencies would hinder the adoption of DTs in the (bio)pharmaceutical industry is revisited, concluding that those companies who collaborate with the agencies to further their technical capabilities will gain significant competitive advantage. The analyzed process development examples show high methodological readiness levels but low systematic adoption of technology. Given the technical feasibilities, financial opportunities, and regulatory encouragement, concerns regarding intellectual property and data sharing, though required to be taken into account, will at best delay an industry-wide adoption of DTs. In conclusion, it is expected that a strategic investment in DTs now will gain an advantage over competition that will be difficult to overcome by late adopters.

"Scentsor": A Whole-Cell Yeast Biosensor with an Olfactory Reporter for Low-Cost and Equipment-Free Detection of Pharmaceuticals.

Portable and inexpensive analytical tools are required to monitor pharmaceutical quality in technology limited settings including low- and middle-income countries (LMICs). Whole cell yeast biosensors have the potential to help meet this need. However, most of the readouts for yeast biosensors require expensive equipment or reagents. To overcome this challenge, we have designed a yeast biosensor that produces a unique scent as a readout. This inducible scent biosensor, or "scentsor", does not require the user to administer additional reagents for reporter development and utilizes only the user's nose to be "read". In this Letter, we describe a scentsor that is responsive to the hormone estradiol (E2). The best estimate threshold (BET) for E2 detection with a panel of human volunteers (n = 49) is 39 nM E2 (15 nM when "non-smellers" are excluded). This concentration of E2 is sensitive enough to detect levels of E2 that would be found in dosage forms. This paper provides evidence that scent has the potential for use in portable yeast biosensors as a readout, particularly for use in LMICs.

A Novel Strategy for Detection and Enumeration of Circulating Rare Cell Populations in Metastatic Cancer Patients Using Automated Microfluidic Filtration and Multiplex Immunoassay.

Size selection via filtration offers an antigen-independent approach for the enrichment of rare cell populations in blood of cancer patients. We evaluated the performance of a novel approach for multiplex rare cell detection in blood samples from metastatic breast (n = 19) and lung cancer patients (n = 21), and healthy controls (n = 30) using an automated microfluidic filtration and multiplex immunoassay strategy. Captured cells were enumerated after sequential staining for specific markers to identify circulating tumor cells (CTCs), circulating mesenchymal cells (CMCs), putative circulating stem cells (CSCs), and circulating endothelial cells (CECs). Preclinical validation experiments using cancer cells spiked into healthy blood demonstrated high recovery rate (mean = 85%) and reproducibility of the assay. In clinical studies, CTCs and CMCs were detected in 35% and 58% of cancer patients, respectively, and were largely absent from healthy controls (3%, p = 0.001). Mean levels of CTCs were significantly higher in breast than in lung cancer patients (p = 0.03). Fifty-three percent (53%) of cancer patients harbored putative CSCs, while none were detectable in healthy controls (p<0.0001). In contrast, CECs were observed in both cancer and control groups. Direct comparison of CellSearch® vs. our microfluidic filter method revealed moderate correlation (R2 = 0.46, kappa = 0.47). Serial blood analysis in breast cancer patients demonstrated the feasibility of monitoring circulating rare cell populations over time. Simultaneous assessment of CTCs, CMCs, CSCs and CECs may provide new tools to study mechanisms of disease progression and treatment response/resistance.