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Course-based undergraduate research experiences (CUREs) integrate authentic research into undergraduate chemistry laboratories, introducing students to research while simultaneously reinforcing fundamental concepts. Despite their ubiquitous nature in bioanalytical research, few CUREs have been published applying the fundamental techniques of separations, spectroscopy, quantification, and mass spectrometry. To engage students in learning these increasingly essential bioanalytical techniques, we designed and implemented a semester-long project-based course centered around the purification, quantification, and identification of heterologously expressed proteins in five succinct and adaptable modules. Instructors can use these modules to form the foundation of a CURE specific to their research interests. The extensive commercial availability of plasmids for transformation combined with the modular approach to laboratory experiments enables convenient customization to accommodate diverse research goals. Instructors can tailor the modules to meet the curricular requirements and instrumentation capabilities of their institutions and can easily extend the research goals to incorporate more specialized analytical techniques, as needed. Through the implementation of the five modules, students apply the fundaments of acid−base chemistry, statistics, quantification strategies, spectrophotometry, separations, and mass spectrometry, thus covering the material required in most undergraduate introductory analytical courses. Instructors can then use these modules as a backbone to support student-led discovery-based investigations for the remainder of the course. Students demonstrate their understanding through the completion of a comprehensive, publication-style laboratory report as well as a poster presentation at a university-wide undergraduate research symposium. Since first offering this course in 2016, student evaluations have been exceedingly positive, with over 75% of students indicating that the course both increased their scientific skills as well as their confidence in their ability to succeed in further science courses. Furthermore, 22% of students reported they were "much more" or "extremely more" likely to enroll in a Ph.D. program in science, math, or engineering following the courses, emphasizing the impact that project-based laboratories can have on undergraduate chemists' career trajectories.

Melanoma, a type of cancer that develops in melanocytes, is usually caused by direct exposure of skin to ultraviolet (UV) radiation resulting in cellular damage. In this study, a procedure to determine the effects of various commercial sunscreens with SPF values ranging from 15 to 100 was developed using pig skin to mimic human skin. These sunscreens contain inorganic filters, such as zinc oxide and titanium dioxide; active organic ingredients, such as octocrylene and oxybenzone; or both. As a model for human skin, pig skin was analyzed before and after UV exposure, and the presence of free radicals was measured using electron paramagnetic resonance (EPR) spectroscopy. Using this method, students were able to quantify radical formation following irradiation and use this as a basis to compare the efficacies of sunscreens against UVA. This experiment allowed undergraduate students to characterize a complex chemical process (light-induced radical formation) and relate it to something they experience every day (sun damage). Interestingly, students found higher levels of postillumination radical formation in sunscreen-treated samples, perhaps indicating sunscreen-induced stabilization of these species. Student outcomes included learning how to collect and interpret EPR data, statistical analysis of these data, and the preparation of reproducible biological samples. Students also consulted literature sources to properly display their measurements.

Cysteine is the most intrinsically nucleophilic residue in proteins and serves as a mediator against increasing reactive oxygen species (ROS) via reversible thiol oxidation. Despite the importance of cysteine oxidation in understanding biological stress response, cysteine sites most reactive toward ROS remain largely unknown and are a major analytical challenge. Herein, a chemical proteomic method to quantify site-specific cysteine reactivity using a maleimide-activated, thiol-reactive probe (N-propargylmaleimide, NPM) is described. Implementation of a gel-based approach via conjugation of rhodamine-azide to NPM-labeled cysteine residues by copper-catalyzed azide−alkyne cycloaddition (CuAAC) click chemistry allowed simple and highly sensitive fluorescence profiling. Relative quantification of >1500 unique cysteine sites from greater than 800 proteins was achieved by conjugating dialkoxydiphenylsilane (DADPS) biotin-azide by the CuAAC reaction and subsequently performing biotin−streptavidin affinity purification and mass-spectrometry-based proteomics. Taken together, this work defines a novel role for the NPM probe in chemical proteomics and presents a robust method for determination of cysteine reactivity during oxidative stress response.

The target of rapamycin (TOR) kinase is a master metabolic regulator with roles in nutritional sensing, protein translation, and autophagy. In Chlamydomonas reinhardtii, a unicellular green alga, TOR has been linked to the regulation of increased triacylglycerol (TAG) accumulation, suggesting that TOR or a downstream target(s) is responsible for the elusive “lipid switch” in control of increasing TAG accumulation under nutrient limitation. However, while TOR has been well characterized in mammalian systems, it is still poorly understood in photosynthetic systems, and little work has been done to show the role of oxidative signaling in TOR regulation. In this study, the TOR inhibitor AZD8055 was used to relate reversible thiol oxidation to the physiological changes seen under TOR inhibition, including increased TAG content. Using oxidized cysteine resin-assisted capture enrichment coupled with label-free quantitative proteomics, 401 proteins were determined to have significant changes in oxidation following TOR inhibition. These oxidative changes mirrored characterized physiological modifications, supporting the role of reversible thiol oxidation in TOR regulation of TAG production, protein translation, carbohydrate catabolism, and photosynthesis through the use of reversible thiol oxidation. The delineation of redox-controlled proteins under TOR inhibition provides a framework for further characterization of the TOR pathway in photosynthetic eukaryotes.

Cannabis consumer products are a $4.6 billion industry in the U.S. that is projected to exceed $14 billion by 2025. Despite an absence of U.S. Food and Drug Administration (FDA) regulation or clinical data, thousands of nutraceuticals, topical consumer products, and beauty products claim benefits of hemp or cannabidiol. However, a lack of required quality control measures prevents consumers from knowing the true concentration or purities of cannabis-labeled products. Thirteen over-the-counter consumer products were examined for the presence of cannabidiol (CBD), cannabinol (CBN), Δ9-tetrahydrocannabinol (THC), cannabidiolic acid (CBDA), and Δ9-tetrahydrocannabinolic acid A (THCA). Additionally, the efficacy of topical applications was investigated using a porcine skin model, in which particle size and zeta potential relate to skin permeability. Skin permeation was correlated to particle size and relative stability in skin-like conditions but not directly related to the CBD content, suggesting that topical products can be designed to enhance overall skin permeation. Of the products analyzed, all products have some traceable amount of cannabinoids, while seven products had multiple cannabinoids with quantifiable amounts. Overall, the need for further regulation is clear, as most products have apparent distinctions between their true and labeled contents.

Despite the continuous emergence of multi-drug resistant pathogens, the number of new antimicrobials reaching the market is critically low. Natural product peptides are a rich source of bioactive compounds, and advances in mass spectrometry have achieved unprecedented capabilities for the discovery and characterization of novel molecular species. However, traditional bioactivity assay formats hinder the discovery and biochemical characterization of natural product antimicrobial peptides (AMPs), necessitating large sample quantities and significant optimization of experimental parameters to achieve accurate/consistent activity measurements. Microfluidic devices offer a promising alternative to bulk assay systems. Herein, a microfluidics-based bioassay was compared to the traditional 96-well plate format in respective commercially-available hardware. Bioactivity in each assay type was compared using a Viola inconspicua peptide library screened against E. coli ATCC 25922. Brightfield microcopy was used to determine bioactivity in microfluidic channels while both common optical and fluorescence-based measurements of cell viability were critically assessed in plate-based assays. Exhibiting some variation in optical density and fluorescence-based measurements, all plate-based assays conferred bioactivity in late eluting V. inconspicua library fractions. However, significant differences in the bioactivity profiles of plate-based and microfluidic assays were found, and may be derived from the materials comprising each assay device or the growth/assay conditions utilized in each format. While new technologies are necessary to overcome the limitations of traditional bioactivity assays, we demonstrate that off-the-shelf implementation of microfluidic devices is non-trivial and significant method development/optimization is required before conventional use can be realized for sensitive and rapid detection of AMPs in natural product matrices.

Summary Stress and nutrient availability influence cell proliferation through complex intracellular signalling networks. In a previous study it was found that pyro‐inositol polyphosphates (InsP7 and InsP8) produced by VIP1 kinase, and target of rapamycin (TOR) kinase signalling interacted synergistically to control cell growth and lipid metabolism in the green alga Chlamydomonas reinhardtii. However, the relationship between InsPs and TOR was not completely elucidated. We used an in vivo assay for TOR activity together with global proteomic and phosphoproteomic analyses to assess differences between wild‐type and vip1‐1 in the presence and absence of rapamycin. We found that TOR signalling is more severely affected by the inhibitor rapamycin in a vip1‐1 mutant compared with wild‐type, indicating that InsP7 and InsP8 produced by VIP1 act independently but also coordinately with TOR. Additionally, among hundreds of differentially phosphorylated peptides detected, an enrichment for photosynthesis‐related proteins was observed, particularly photosystem II proteins. The significance of these results was underscored by the finding that vip1‐1 strains show multiple defects in photosynthetic physiology that were exacerbated under high light conditions. These results suggest a novel role for inositol pyrophosphates and TOR signalling in coordinating photosystem phosphorylation patterns in Chlamydomonas cells in response to light stress and possibly other stresses.