Abstract The probiotic yeast Saccharomyces boulardii ( Sb ) is a promising chassis to deliver therapeutic proteins to the gut due to Sb ’s innate therapeutic properties, resistance to phage and antibiotics, and high protein secretion capacity. To maintain therapeutic efficacy in the context of challenges such as washout, low rates of diffusion, weak target binding, and/or high rates of proteolysis, it is desirable to engineer Sb strains with enhanced levels of protein secretion. In this work, we explored genetic modifications in both cis - (i.e., to the expression cassette of the secreted protein) and trans - (i.e., to the Sb genome) that enhance Sb ’s ability to secrete proteins, taking a Clostridioides difficile Toxin A neutralizing peptide (NPA) as our model therapeutic. First, by modulating the copy number of the NPA expression cassette, we found NPA concentrations in the supernatant could be varied by 6-fold (76-458 mg/L) in microbioreactor fermentations. In the context of high NPA copy number, we found a previously-developed collection of native and synthetic secretion signals could further tune NPA secretion between 121 - 463 mg/L. Then, guided by prior knowledge of S. cerevisiae ’s secretion mechanisms, we generated a library of homozygous single gene deletion strains, the most productive of which achieved 2297 mg/L secretory production of NPA. We then expanded on this library by performing combinatorial gene deletions, supplemented by proteomics experiments. We ultimately constructed a quadruple protease-deficient Sb strain that produces 5045 mg/L secretory NPA, an improvement of >10-fold over wild-type Sb . Overall, this work systematically explores a broad collection of engineering strategies to improve protein secretion in Sb and highlights the ability of proteomics to highlight under-explored mediators of this process. In doing so, we created a set of probiotic strains that are capable of delivering a wide range of protein titers and therefore furthers the ability of Sb to deliver therapeutics to the gut and other settings to which it is adapted.
Vinyl chloride (VC) is an industrial chemical that is known to be carcinogenic to animals and humans. VC primarily induces hepatic angiosarcomas following high exposures (≥50 ppm). VC is also found in Superfund sites at ppb concentrations as a result of microbial metabolism of trichloroethylene and perchloroethylene. Here, we report a new sensitive LC-MS/MS method to analyze the major DNA adduct formed by VC, 7-(2-oxoethylguanine) (7-OEG). We used this method to analyze tissue DNA from both adult and weanling rats exposed to 1100 ppm [(13)C(2)]-VC for 5 days. After neutral thermal hydrolysis, 7-OEG was derivatized with O-t-butyl hydroxylamine to an oxime adduct, followed by LC-MS/MS analysis. The limit of detection was 1 fmol, and the limit of quantitation was 1.5 fmol on the column. The use of stable isotope VC allowed us to demonstrate for the first time that endogenous 7-OEG was present in tissue DNA. We hypothesized that endogenous 7-OEG was formed from lipid peroxidation and demonstrated the formation of [(13)C(2)]-7-OEG from the reaction of calf thymus DNA with [(13)C(18)]-ethyl linoleate (EtLa) under peroxidizing conditions. The concentrations of endogenous 7-OEG in liver, lung, kidney, spleen, testis, and brain DNA from adult and weanling rats typically ranged from 1.0 to 10.0 adducts per 10(6) guanine. The exogenous 7-OEG in liver DNA from adult rats exposed to 1100 ppm [(13)C(2)]-VC for 5 days was 104.0 ± 23.0 adducts per 10(6) guanine (n = 4), while concentrations in other tissues ranged from 1.0 to 39.0 adducts per 10(6) guanine (n = 4). Although endogenous concentrations of 7-OEG in tissues in weanling rats were similar to those of adult rats, exogenous [(13)C(2)]-7-OEG concentrations were higher in weanlings, averaging 300 adducts per 10(6) guanine in liver. Studies on the persistence of [(13)C(2)]-7-OEG in adult rats sacrificed 2, 4, and 8 weeks postexposure to [(13)C(2)]-VC demonstrated a half-life of 7-OEG of 4 days in both liver and lung.
Per- and polyfluorinated alkyl substances (PFASs) contaminate groundwater, surface water, and finished drinking water internationally. Their ecological persistence and adverse human health effects demand effective remediation approaches. Motivated by the limitations in selectivity and performance of current PFAS removal technologies, we report a platform approach for the development of ionic fluorogel resins that effectively remove a chemically diverse mixture of PFAS from water. The synthesis of a material library with systematic variation in fluorous and ionic components led to the identification of a resin that demonstrated rapid removal of PFASs with high affinity and selectivity in the presence of nonfluorous contaminants commonly found in groundwater. The material can be regenerated and reused multiple times. We demonstrate ionic fluorogels as effective adsorbents for the removal of 21 legacy and emerging PFASs from settled water collected at the Sweeney Water Treatment Plant in Wilmington, North Carolina.
Numerous experiments have demonstrated the genotoxic and mutagenic effects of formaldehyde, including DNA−protein cross-links (DPC). Histone was reported to be involved in the formation of DPC in which the ε-amino groups of lysine and exocyclic amino groups of DNA were thought to be cross-linked through multiple step reactions. Using mass spectrometry, the N-terminus of histone and lysine residues located in both the histone N-terminal tail and the globular fold domain were identified as binding sites for formaldehyde in the current study. The observation that only lysine residues without post-translational modification (PTM) can be attacked by formaldehyde indicates that PTM blocks the reaction between lysine and formaldehyde. Additionally, we found that formaldehyde-induced Schiff bases on lysine residues could inhibit the formation of PTM on histone, raising the possibility that formaldehyde might alter epigenetic regulation.
The AMCP Managed Care & Specialty Pharmacy Annual Meeting 2019 in San Diego, California, is expected to attract more than 4,000 managed care pharmacists and other health care professionals who manage and evaluate drug therapies, develop and manage networks, and work with medical managers and information specialists to improve the care of all individuals enrolled in managed care programs. The AMCP Abstracts program provides a forum through which authors can share their insights and outcomes of advanced managed care practice. Poster presentations are scheduled for Wednesday, March 27, from 11:30 am to 1:00 pm. Posters will also be displayed on Tuesday, March 26, from 5:30 pm to 7:00 pm, during the opening night reception in the Expo, and on Thursday, March 28, from 9:30 am to 11:00 am. Podium presentations for the Platinum award-winning abstracts are Tuesday, March 26, from 2:30 pm to 3:45 pm. Professional abstracts that have been reviewed are published in the Journal of Managed Care & Specialty Pharmacy's Meeting Abstracts supplement. Abstracts were submitted in the following categories.
