What Are Essential Skills and Concepts for Teaching Mass Spectrometry to Undergraduates?

Instructors of college-level science courses often have a strong desire to use techniques and concepts from the fields of educational psychology and pedagogy, but it can be difficult to adapt information from those fields to specific technical topics such as mass spectrometry. A useful technique to apply various learning theories to teaching a specific topic is to distinguish between skills and concepts that are needed to understand and apply that topic to various problems. This report summarizes information gathered at the American Society for Mass Spectrometry 2021 Undergraduate Research Interest Group Workshop and seeks to identify essential skills and concepts that need to be taught to undergraduate students learning about mass spectrometry. Some recommendations are made for how members of the mass spectrometry community can engage with their institutions to enhance mass spectrometry education.

Characterization of protein-ligand binding interactions of enoyl-ACP reductase (FabI) by native MS reveals allosteric effects of coenzymes and the inhibitor triclosan.

The enzyme enoyl-ACP reductase (also called FabI in bacteria) is an essential member of the fatty acid synthase II pathway in plants and bacteria. This enzyme is the target of the antibacterial drug triclosan and has been the subject of extensive studies for the past 20 years. Despite the large number of reports describing the biochemistry of this enzyme, there have been no studies that provided direct observation of the protein and its various ligands. Here we describe the use of native MS to characterize the protein-ligand interactions of FabI with its coenzymes NAD+ and NADH and with the inhibitor triclosan. Measurements of the gas-phase affinities of the enzyme for these ligands yielded values that are in close agreement with solution-phase affinity measurements. Additionally, FabI is a homotetramer and we were able to measure the affinity of each subunit for each coenzyme, which revealed that both coenzymes exhibit a positive homotropic allosteric effect. An allosteric effect was also observed in association with the inhibitor triclosan. These observations provide new insights into this well-studied enzyme and suggest that there may still be gaps in the existing mechanistic models that explain FabI inhibition.

Protein Adducts and Protein Oxidation as Molecular Mechanisms of Flavonoid Bioactivity.

There are tens of thousands of scientific papers about flavonoids and their impacts on human health. However, despite the vast amount of energy that has been put toward studying these compounds, a unified molecular mechanism that explains their bioactivity remains elusive. One contributing factor to the absence of a general mechanistic explanation of their bioactivity is the complexity of flavonoid chemistry in aqueous solutions at neutral pH. Flavonoids have acidic protons, are redox active, and frequently auto-oxidize to produce an array of degradation products including electrophilic quinones. Flavonoids are also known to interact with specificity and high affinity with a variety of proteins, and there is evidence that some of these interactions may be covalent. This review summarizes the mechanisms of flavonoid oxidation in aqueous solutions at neutral pH and proposes the formation of protein-flavonoid adducts or flavonoid-induced protein oxidation as putative mechanisms of flavonoid bioactivity in cells. Nucleophilic residues in proteins may be able to form covalent bonds with flavonoid quinones; alternatively, specific amino acid residues such as cysteine, methionine, or tyrosine in proteins could be oxidized by flavonoids. In either case, these protein-flavonoid interactions would likely occur at specific binding sites and the formation of these types of products could effectively explain how flavonoids modify proteins in cells to induce downstream biochemical and cellular changes.

A guided-inquiry investigation of bacterial membrane potential using flow cytometry for an undergraduate biochemistry laboratory course.

Flow cytometry has become an important tool in the life sciences and medical fields, yet there are often few opportunities for undergraduate students to receive training with this type of instrumentation as part of life science curricula at many colleges and universities. We describe a straightforward laboratory exercise designed for a college-level biochemistry course that uses flow cytometry to investigate changes in bacterial membrane potential and cell viability in response to various treatments. Anecdotally, we have noticed that many students often have difficulty understanding the concept of membrane potential and the essential role that it plays in cellular processes. Therefore, this exercise also provides a pedagogical tool for visualizing changes in cellular membrane potential as way to enhancing students' comprehension of this abstract concept. Students' understanding of flow cytometry and membrane potential was assessed using a preactivity and postactivity quiz; the results indicate that the students' understanding of these concepts significantly improved after they completed this laboratory exercise. © 2019 International Union of Biochemistry and Molecular Biology, 48(1):61-66, 2020.

Immunomodulatory and Antibacterial Properties of the Chumash Medicinal Plant Trichostema lanatum.

Background: The woody shrub Trichostema lanatum Benth. (Lamiaceae) is native to Southern California and was reportedly used by the Chumash people as a disinfectant and for the treatment of rheumatism. Based on its traditional uses, this study investigated the antibacterial and immunomodulatory properties of extracts from T. lanatum.Methods: A methanolic extract of the leaves and stems of T. lanatum was tested for immunomodulatory activity by measuring the proliferation of murine macrophage cell cultures and the production of the pro-inflammatory cytokine TNF-α by the cells after treatment with T. lanatum. Antibacterial activity of the extract against a panel of six Gram-positive bacteria and two Gram-negative bacteria was evaluated using a disc-diffusion assay. Results: The T. lanatum extract inhibited the growth of Gram-positive bacteria, but not Gram-negative bacteria. Treatment of activated macrophage cell cultures with T. lanatum extract resulted in decreased proliferation of the activated macrophages and a decrease in the production of TNF-α. Conclusions: These results provide the first pharmacological support for the traditional use of T. lanatum by the Chumash people of Southern California as a disinfectant and treatment for rheumatism.

Antibacterial activity of fractions from three Chumash medicinal plant extracts and in vitro inhibition of the enzyme enoyl reductase by the flavonoid jaceosidin.

We have investigated the in vitro antibacterial bioactivity of dichloromethane-soluble fractions of Artemisia californica, Trichostema lanatum, Salvia apiana, Sambucus nigra ssp. cerulea and Quercus agrifolia Née against a ΔtolC mutant strain of Escherichia coli. These plants are traditional medicinal plants of the Chumash American Indians of Southern California. Bioassay-guided fractionation led to the isolation of three flavonoid compounds from A. californica: jaceosidin (1), jaceidin (2), and chrysoplenol B (3). Compounds 1 and 2 exhibited antibacterial activity against E. coli ΔtolC in liquid cultures. The in vitro activity of 1 against the enoyl reductase enzyme (FabI) was measured using a spectrophotometric assay and found to completely inhibit FabI activity at a concentration of 100 µM. However, comparison of minimum inhibitory concentration values for 1-3 against E. coli ΔtolC and an equivalent strain containing a plasmid constitutively expressing fabI did not reveal any selectivity for FabI in vivo.

4',4‴,7,7″-tetra-O-methylcupressuflavone inhibits seed germination of Lactuca sativa.

Biflavonoids have been isolated from a wide variety of plant species, but little is known about their native biological functions. Here we report a possible ecological role for biflavonoids by describing the isolation of the biflavonoid 4',4‴,7,7″-tetra-O-methylcupressuflavone (1) from Araucaria columnaris and its inhibitory effect on seed germination. Compound 1 was isolated from needles of a single A. columnaris specimen and inhibited germination of Lactuca sativa seeds in a culture-dish assay; it was also detected in soil samples under the canopy where reduced germination was observed, but not in a location away from the canopy where germination was uninhibited.

Briarane diterpenes diminish COX-2 expression in human colon adenocarcinoma cells.

Exploration of a soft coral (Briareum sp.) from Vanuatu led to the isolation of three new briaranes, designated brialalepolides A (1), B (2), and C (3). Compounds 2 and 3 reduced the expression of COX-2 in human colon adenocarcinoma cells, as well as in murine macrophage cells. This is significant because the metabolic products of COX-2 have been implicated in the pathogenesis of colon cancer and other diseases.

Bringing natural products into the fold - exploring the therapeutic lead potential of secondary metabolites for the treatment of protein-misfolding-related neurodegenerative diseases.

A review with 187 references, describing natural products that exhibit bioactive properties relevant to the treatment of protein aggregation-related neurodegenerative diseases (e.g., Huntington's, Parkinson's, and prion diseases) and comparing the chemical properties of these secondary metabolite leads to compounds in clinical use for treating central nervous system conditions.

Mutanobactin A from the human oral pathogen Streptococcus mutans is a cross-kingdom regulator of the yeast-mycelium transition.

The recent investigation of a gene cluster encoding for a hybrid PKS-NRPS metabolite in the oral pathogen Streptococcus mutans UA159 yielded evidence that this natural product might play an important role regulating a range of stress tolerance factors. We have now characterized the major compound generated from this gene cluster, mutanobactin A, and demonstrated that this secondary metabolite is also capable of influencing the yeast-mycelium transition of Candida albicans.

Probing the metabolic aberrations underlying mutant huntingtin toxicity in yeast and assessing their degree of preservation in humans and mice.

Metabolomics is a powerful multiparameter tool for evaluating phenotypic traits associated with disease processes. We have used (1)H NMR metabolome profiling to characterize metabolic aberrations in a yeast model of Huntington's disease that are attributable to the mutant huntingtin protein's gain-of-toxic-function effects. A group of 11 metabolites (alanine, acetate, galactose, glutamine, glycerol, histidine, proline, succinate, threonine, trehalose, and valine) exhibited significant concentration changes in yeast expressing the N-terminal fragment of a mutant human huntingtin gene. Correspondence analysis was used to compare results from our yeast model to data reported from transgenic mice expressing a mutant huntingtin gene fragment and Huntington's disease patients. This technique enabled us to identify a variety of both model-specific (pertaining to a single species) and conserved (observed in multiple species) biomarkers related to mutant huntingtin's toxicity. Among the 59 metabolites identified, four compounds (alanine, glutamine, glycerol, and valine) changed significantly in concentration in all three Huntington's disease systems. We propose that alanine, glutamine, glycerol, and valine should be considered as promising biomarkers for evaluating new Huntington's disease therapies, as well as for providing unique insight into the mechanisms associated with mutant huntingtin toxicity.

A chemical epigenetics approach for engineering the in situ biosynthesis of a cryptic natural product from Aspergillus niger.

A new fungal metabolite, nygerone A (), featuring a unique 1-phenylpyridin-4(1H)-one core that had previously not been reported from any natural source, has been obtained from Aspergillus niger using a chemical epigenetics methodology.

Bioactivity profiling with parallel mass spectrometry reveals an assemblage of green tea metabolites affording protection against human huntingtin and alpha-synuclein toxicity.

Aberrant protein aggregation and misfolding are key pathological features of many neurodegenerative disorders, including Huntington's and Parkinson's diseases. Compounds that offer protection from toxicity associated with aggregation-prone neurodegenerative proteins may have applications for the treatment of a multitude of disorders. A high-throughput bioassay system with parallel electrospray ionization mass spectrometry screening has been designed for critical evaluation of milligram quantities of natural product extracts, including dietary substances, for compounds of pharmacological relevance to the treatment of human neurodegenerative diseases. Using Saccharomyces cerevisiae strains engineered to express mutant human huntingtin and alpha-synuclein, we are able to identify extracts and compounds that protect cells from toxicity associated with these proteins. Applying this screening paradigm, we determined that a bioactive green tea extract contains an assemblage of catechins that were individually characterized for their respective protective effects against huntingtin and alpha-synuclein toxicity.