Creating Meaningful Connections with the Electron Transport Chain Beyond a Virtual Classroom

At the center of feminist pedagogy is community. Creating community within the classroom and between the class and local communities increases student engagement and access to knowledge. Communities can be a source of solidarity and further decenter authority in the classroom, empowering students, teachers, and the surrounding community to be co-learners. Amid the COVID-19 pandemic, remote teaching posed challenges to student engagement. Here, I describe an assignment used to build an inclusive community of co-learners within a virtual biochemistry class and our local communities. Oxidative phosphorylation is a cellular process used to create energy that depends on the electron transport chain, a series of electron-transfer reactions that can be challenging for students to learn. To help remote students understand the electron transport chain, I designed an assignment for students to 1) learn about electron transport and its connection to oxidative phosphorylation through reenactment and 2) teach classmates and nonscientists within their local communities about this essential process. Through reenactment, students created meaningful connections with this cellular process while educating other nonscientists, extending the virtual classroom into our local communities. Notably, this assignment can be used in in-person teaching and applied to other metabolic pathways to facilitate an intuitive understanding of the biochemical process. @font-face {font-family: Cambria Math ; panose-1:2 4 5 3 5 4 6 3 2 4; mso-font-charset:0; mso-generic-font-family:roman; mso-font-pitch:variable; mso-font-signature:-536870145 1107305727 0 0 415 0;}@font-face {font-family:Calibri; panose-1:2 15 5 2 2 2 4 3 2 4; mso-font-charset:0; mso-generic-font-family:swiss; mso-font-pitch:variable; mso-font-signature:-469750017 -1073732485 9 0 511 0;}@font-face {font-family:Times; panose-1:0 0 5 0 0 0 0 2 0 0; mso-font-charset:0; mso-generic-font-family:auto; mso-font-pitch:variable; mso-font-signature:-536870145 1342185562 0 0 415 0;}p.MsoNormal, li.MsoNormal, div.MsoNormal {mso-style-unhide:no; mso-style-qformat:yes; mso-style-parent: ; margin:0in; line-height:115%; mso-pagination:widow-orphan; font-size:11.0pt; font-family: Arial ,sans-serif; mso-fareast-font-family:Arial; mso-ansi-language:EN;}.MsoChpDefault {mso-style-type:export-only; mso-default-props:yes; font-size:11.0pt; mso-ansi-font-size:11.0pt; mso-bidi-font-size:11.0pt; font-family: Arial ,sans-serif; mso-ascii-font-family:Arial; mso-fareast-font-family:Arial; mso-hansi-font-family:Arial; mso-bidi-font-family:Arial; mso-ansi-language:EN;}.MsoPapDefault {mso-style-type:export-only; line-height:115%;}div.WordSection1 {page:WordSection1;

Discovery, characterization, and rational design of the enzymes involved in monoterpene indole alkaloid biosynthesis in Madagascar periwinkle

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Chemistry, February 2011."February 2011." Vita. Cataloged from PDF version of thesis.Includes bibliographical references.The chemical diversity found in plants has served as a major source of inspiration to many synthetic and biological chemists. Nature has evolved enzyme active sites to catalyze the synthesis of structurally complex compounds that serve as pharmaceuticals, insecticides, dyes, perfumes, and biofuels. In Catharanthus roseus, approximately 130 structurally complex monoterpene indole alkaloids are produced, including the clinically used anti-mitotic drugs, vinblastine and vincristine. The common intermediate to all monoterpene indole alkaloids is strictosidine, the product of an asymmetric Pictet- Spengler condensation of tryptamine and the iridoid monoterpene secologanin. This reaction is catalyzed by the enzyme strictosidine synthase. This thesis describes the use of kinetic isotope effects, the rate dependence on pH, as well as structural and computational data to propose a mechanism by which strictosidine synthase catalyzes the Pictet-Spengler reaction. Notably, the data also shed light on the mechanism of the widely used nonenzymatic reaction. Interestingly, the Pictet-Spenglerase strictosidine synthase belongs to a superfamily of enzymes that have mainly been observed to catalyze ester hydrolysis. Using the [beta]-propeller fold conserved in both strictosidine synthase and the related hydrolase, paraoxonase, rational mutagenesis was used to convert strictosidine synthase into a hydrolase. Intriguingly, during the rational design process, the function of a closely related strictosidine synthase homolog was also functionally characterized as a hydrolase. In addition to reengineering proteins with new catalytic activity, the chemical diversity in plants can also modified using metabolic engineering. However, this approach requires knowledge of the genetic blueprints of the plant to be known. Using the recently released C. roseus transcriptome sequencing data along with co-expression analysis, this thesis describes the functional characterization of a new P450 gene involved in metabolizing a key intermediate in the biosynthesis of bioactive bisindole alkaloids. With the functional characterization of this new gene, a combination of gene silencing and synthetic biology techniques will provide a greater understanding on how to "tune" alkaloid biosynthesis in C. roseus in order to generate more functionally diverse molecules.by Lesley-Ann Giddings.Ph.D

Isolation and Genome Analysis of an Amoeba-Associated Bacterium \u3ci\u3eDyella terrae\u3c/i\u3e Strain Ely Copper Mine From Acid Rock Drainage in Vermont, United States

Protozoa play important roles in microbial communities, regulating populations via predation and contributing to nutrient cycling. While amoebae have been identified in acid rock drainage (ARD) systems, our understanding of their symbioses in these extreme environments is limited. Here, we report the first isolation of the amoeba Stemonitis from an ARD environment as well as the genome sequence and annotation of an associated bacterium, Dyella terrae strain Ely Copper Mine, from Ely Brook at the Ely Copper Mine Superfund site in Vershire, Vermont, United States. Fluorescent in situ hybridization analysis showed this bacterium colonizing cells of Stemonitis sp. in addition to being outside of amoebal cells. This amoeba-resistant bacterium is Gram-negative with a genome size of 5.36Mbp and GC content of 62.5%. The genome of the D. terrae strain Ely Copper Mine encodes de novo biosynthetic pathways for amino acids, carbohydrates, nucleic acids, and lipids. Genes involved in nitrate (1) and sulfate (7) reduction, metal (229) and antibiotic resistance (37), and secondary metabolite production (6) were identified. Notably, 26 hydrolases were identified by RAST as well as other biomass degradation genes, suggesting roles in carbon and energy cycling within the microbial community. The genome also contains type IV secretion system genes involved in amoebae resistance, revealing how this bacterium likely survives predation from Stemonitis sp. This genome analysis and the association of D. terrae strain Ely Copper Mine with Stemonitis sp. provide insight into the functional roles of amoebae and bacteria within ARD environments

Extremophilic Fungi from Marine Environments: Underexplored Sources of Antitumor, Anti-Infective and Other Biologically Active Agents

Marine environments are underexplored terrains containing fungi that produce a diversity of natural products given unique environmental pressures and nutrients. While bacteria are commonly the most studied microorganism for natural products in the marine world, marine fungi are also abundant but remain an untapped source of bioactive metabolites. Given that their terrestrial counterparts have been a source of many blockbuster antitumor agents and anti-infectives, including camptothecin, the penicillins, and cyclosporin A, marine fungi also have the potential to produce new chemical scaffolds as leads to potential drugs. Fungi are more phylogenetically diverse than bacteria and have larger genomes that contain many silent biosynthetic gene clusters involved in making bioactive compounds. However, less than 5% of all known fungi have been cultivated under standard laboratory conditions. While the number of reported natural products from marine fungi is steadily increasing, their number is still significantly lower compared to those reported from their bacterial counterparts. Herein, we discuss many varied cytotoxic and anti-infective fungal metabolites isolated from extreme marine environments, including symbiotic associations as well as extreme pressures, temperatures, salinity, and light. We also discuss cultivation strategies that can be used to produce new bioactive metabolites or increase their production. This review presents a large number of reported structures though, at times, only a few of a large number of related structures are shown

Extremophilic Fungi from Marine Environments: Underexplored Sources of Antitumor, Anti-Infective and Other Biologically Active Agents

Marine environments are underexplored terrains containing fungi that produce a diversity of natural products given unique environmental pressures and nutrients. While bacteria are commonly the most studied microorganism for natural products in the marine world, marine fungi are also abundant but remain an untapped source of bioactive metabolites. Given that their terrestrial counterparts have been a source of many blockbuster antitumor agents and anti-infectives, including camptothecin, the penicillins, and cyclosporin A, marine fungi also have the potential to produce new chemical scaffolds as leads to potential drugs. Fungi are more phylogenetically diverse than bacteria and have larger genomes that contain many silent biosynthetic gene clusters involved in making bioactive compounds. However, less than 5% of all known fungi have been cultivated under standard laboratory conditions. While the number of reported natural products from marine fungi is steadily increasing, their number is still significantly lower compared to those reported from their bacterial counterparts. Herein, we discuss many varied cytotoxic and anti-infective fungal metabolites isolated from extreme marine environments, including symbiotic associations as well as extreme pressures, temperatures, salinity, and light. We also discuss cultivation strategies that can be used to produce new bioactive metabolites or increase their production. This review presents a large number of reported structures though, at times, only a few of a large number of related structures are shown

Microbial Natural Products: Molecular Blueprints for Antitumor Drugs

Microbes from two of the three domains of life, the Prokarya, and Eukarya, continue to serve as rich sources of structurally complex chemical scaffolds that have proven to be essential for the development of anticancer therapeutics. This review describes only a handful of exemplary natural products and their derivatives as well as those that have served as elegant blueprints for the development of novel synthetic structures that are either currently in use or in clinical or preclinical trials together with some of their earlier analogs in some cases whose failure to proceed aided in the derivation of later compounds. In every case, a microbe has been either identified as the producer of secondary metabolites or speculated to be involved in the production via symbiotic associations. Finally, rapidly evolving next-generation sequencing technologies have led to the increasing availability of microbial genomes. Relevant examples of genome mining and genetic manipulation are discussed, demonstrating that we have only barely scratched the surface with regards to harnessing the potential of microbes as sources of new pharmaceutical leads/agents or biological probes

Seasonal Ely Copper Mine Superfund Site Shotgun Metagenomic and Metatranscriptomic Data Analysis

High throughput sequencing data collected from acid rock drainage (ARD) communities can reveal the active taxonomic and functional diversity of these extreme environments, which can be exploited for bioremediation, pharmaceutical, and industrial applications. Here, we report a seasonal comparison of a microbiome and transcriptome in Ely Brook (EB-90M), a confluence of clean water and upstream tributaries that drains the Ely Copper Mine Superfund site in Vershire, VT, USA. Nucleic acids were extracted from EB-90M water and sediment followed by shotgun sequencing using the Illumina NextSeq platform. Approximately 575,933 contigs with a total length of 1.54 Gbp were generated. Contigs of at least a size of 3264 (N50) or greater represented 50% of the sequences and the longest contig was 488,568 bp in length. Using Centrifuge against the NCBI “nt” database 141 phyla, including candidate phyla, were detected. Roughly 380,000 contigs were assembled and ∼1,000,000 DNA and ∼550,000 cDNA sequences were identified and functionally annotated using the Prokka pipeline. Most expressed KEGG-annotated microbial genes were involved in amino acid metabolism and several KEGG pathways were differentially expressed between seasons. Biosynthetic gene clusters involved in secondary metabolism as well as metal- and antibiotic-resistance genes were annotated, some of which were differentially expressed, colocalized, and coexpressed. These data can be used to show how ecological stimuli, such as seasonal variations and metal concentrations, affect the ARD microbiome and select taxa to produce novel natural products. The data reported herein is supporting information for the research article “Characterization of an acid rock drainage microbiome and transcriptome at the Ely Copper Mine Superfund site” by Giddings et al. [1]