Sugar-seeking insects as a source of diverse bread-making yeasts with enhanced attributes.

Insects represent a particularly interesting habitat in which to search for novel yeasts of value to industry. Insect-associated yeasts have the potential to have traits relevant to modern food and beverage production due to insect-yeast interactions, with such traits including diverse carbohydrate metabolisms, high sugar tolerance, and general stress tolerance. Here, we consider the potential value of insect-associated yeasts in the specific context of baking. We isolated 63 yeast strains from 13 species of hymenoptera from the United States, representing 37 yeast species from 14 genera. Screening for the ability to ferment maltose, a sugar important for bread production, resulted in the identification of 13 strains of Candida, Lachancea, and Pichia species. We assessed their ability to leaven dough. All strains produced baked loaves comparable to a commercial baking strain of Saccharomyces cerevisiae. The same 13 strains were also grown under various sugar and salt conditions relevant to osmotic challenges experienced in the manufacturing processes and the production of sweet dough. We show that many of these yeast strains, most notably strains of Lachancea species, grow at a similar or higher rate and population size as commercial baker's yeast. We additionally assessed the comparative phenotypes and genetics of insect-associated S. cerevisiae strains unable to ferment maltose and identified baking-relevant traits, including variations in the HOG1 signaling pathway and diverse carbohydrate metabolisms. Our results suggest that non-conventional yeasts have high potential for baking and, more generally, showcase the success of bioprospecting in insects for identifying yeasts relevant for industrial uses.

Systematic analysis of F-box proteins reveals a new branch of the yeast mating pathway.

The mating pathway in yeast Saccharomyces cerevisiae has long been used to reveal new mechanisms of signal transduction. The pathway comprises a pheromone receptor, a heterotrimeric G protein, and intracellular effectors of morphogenesis and transcription. Polarized cell growth, in the direction of a potential mating partner, is accomplished by the G-protein ßγ subunits and the small G-protein Cdc42. Transcription induction, needed for cell-cell fusion, is mediated by Gßγ and the mitogen-activated protein kinase (MAPK) scaffold protein Ste5. A potential third pathway is initiated by the G-protein α subunit Gpa1. Gpa1 signaling was shown previously to involve the F-box adaptor protein Dia2 and an endosomal effector protein, the phosphatidylinositol 3-kinase Vps34. Vps34 is also required for proper vacuolar sorting and autophagy. Here, using a panel of reporter assays, we demonstrate that mating pheromone stimulates vacuolar targeting of a cytoplasmic reporter protein and that this process depends on Vps34. Through a systematic analysis of F-box deletion mutants, we show that Dia2 is required to sustain pheromone-induced vacuolar targeting. We also found that other F-box proteins selectively regulate morphogenesis (Ydr306, renamed Pfu1) and transcription (Ucc1). These findings point to the existence of a new and distinct branch of the pheromone-signaling pathway, one that likely leads to vacuolar engulfment of cytoplasmic proteins and recycling of cellular contents in preparation for mating.

Transfer of CD8+ T cell memory using Bcl-2 as a marker.

The processes that regulate T cell memory generation are important for therapeutic design and the immune response to disease. However, what allows a subset of effector T cells to survive the contraction period to become memory cells is incompletely understood. The Bcl-2 family is critical for T cell survival, and Bcl-2 has been proposed to be important for the survival of memory cells. However, previous studies have relied on double-knockout models, potentially skewing the role of Bcl-2, and the use of Bcl-2 as a marker in adoptive transfer experiments, a method required to confirm the memory potential of cell subsets, has not been possible because of the intracellular localization of the protein. In this study, we present a novel Bcl-2 reporter mouse model and, to our knowledge, show for the first time that a distinct subset of effector T cells, and also a subset within the CD127(hi)KLRG1(lo) memory precursor effector cell population, retains high Bcl-2 expression at the peak of the CD8(+) T cell response to Listeria monocytogenes. Furthermore, we show that Bcl-2 correlates with memory potential in adoptive transfer experiments using both total responding CD8(+) T cells and memory precursor effector cells. These results show that even within the memory precursor effector cell population, Bcl-2 confers a survival advantage in a subset of effector CD8(+) T cells that allows differentiation into memory cells and cement Bcl-2 as a critical factor for T cell memory.

Regulation of steady-state neutrophil homeostasis by macrophages.

The timely clearance of apoptotic neutrophils from inflammation sites is an important function of macrophages; however, the role of macrophages in maintaining neutrophil homeostasis under steady-state conditions is less well understood. By conditionally deleting the antiapoptotic gene cellular FLICE-like inhibitory protein (C-FLIP) in myeloid cells, we have generated a novel mouse model deficient in marginal zone and bone marrow stromal macrophages. These mice develop severe neutrophilia, splenomegaly, extramedullary hematopoiesis, decreased body weight, and increased production of granulocyte colony-stimulating factor (G-CSF) and IL-1ß, but not IL-17. c-FLIP(f/f) LysM-Cre mice exhibit delayed clearance of circulating neutrophils, suggesting that failure of macrophages to efficiently clear apoptotic neutrophils causes production of cytokines that drive excess granulopoiesis. Further, blocking G-CSF but not IL-1R signaling in vivo rescues this neutrophilia, suggesting that a G-CSF-dependent, IL-1ß-independent pathway plays a role in promoting neutrophil production in mice with defective clearance of apoptotic cells.

Hands-on immunology: Engaging learners of all ages through tactile teaching tools.

Ensuring the public has a fundamental understanding of human-microbe interactions, immune responses, and vaccines is a critical challenge in the midst of a pandemic. These topics are commonly taught in undergraduate- and graduate-level microbiology and immunology courses; however, creating engaging methods of teaching these complex concepts to students of all ages is necessary to keep younger students interested when science seems hard. Building on the Tactile Teaching Tools with Guided Inquiry Learning (TTT-GIL) method we used to create an interactive lac operon molecular puzzle, we report here two TTT-GIL activities designed to engage diverse learners from middle schoolers to masters students in exploring molecular interactions within the immune system. By pairing physical models with structured activities built on the constructivist framework of Process-Oriented Guided Inquiry Learning (POGIL), TTT-GIL activities guide learners through their interaction with the model, using the Learning Cycle to facilitate construction of new concepts. Moreover, TTT-GIL activities are designed utilizing Universal Design for Learning (UDL) principles to include all learners through multiple means of engagement, representation, and action. The TTT-GIL activities reported here include a web-enhanced activity designed to teach concepts related to antibody-epitope binding and specificity to deaf and hard-of-hearing middle and high school students in a remote setting and a team-based activity that simulates the evolution of the Major Histocompatibility Complex (MHC) haplotype of a population exposed to pathogens. These activities incorporate TTT-GIL to engage learners in the exploration of fundamental immunology concepts and can be adapted for use with learners of different levels and educational backgrounds.

Using Metabolic Engineering to Connect Molecular Biology Techniques to Societal Challenges.

Genetically modified organisms (GMOs) are a topic of broad interest and are discussed in classes ranging from introductory biology to bioethics to more advanced methods-focused molecular biology courses. In most cases, GMOs are discussed in the context of introducing a single protein-coding gene to produce a single desired trait in a crop. For example, a commercially available kit allows students to test whether food products contain GMOs by detecting the Bacillus thuringiensis delta-endotoxin gene, which confers resistance to European corn borers. We have developed an 8-week laboratory module for upper-division undergraduates and graduate students that builds upon students' basic understanding of GMOs to introduce them to the techniques used to sustainably produce commercially valuable products in yeast through metabolic engineering. In this course, students use recombination-based methods to assemble genes encoding entire metabolic pathways in Saccharomyces cerevisiae, perform genetic screens to identify yeast genes that impact metabolite yield, and use error-prone PCR to optimize metabolic pathway function. In parallel to these laboratory-based activities, students engage with the societal impact of these approaches through case studies of products made via yeast metabolic engineering, such as opioids, omega-3 fatty acids, and the Impossible Burger. In this report, we focus on these case studies as well as an individual sustainability project assignment created for this course. This assignment, which spans the 8-week module, asks students to find examples of yeast metabolic engineering that could be used to address current sustainability challenges in their communities. By the end of the course, students synthesize this information to create a case study that could be used to teach concepts related to metabolic engineering and sustainability to their peers. Student approaches to this project have varied from literature reviews, to news searches, to directly contacting and interviewing researchers using novel metabolic engineering approaches. These student-produced projects are used as case studies in future semesters, amplifying student voices and contributing to student ownership. While developed in the context of this course, the sustainability project and case studies are broadly applicable and could be adapted for use in biology or bioethics courses at the undergraduate or graduate level. Through this report, we hope to gain collaborators interested in implementing a version of the course at their institutions, allowing for robust assessment of the impact of the course on a larger group of students.

STEM BUILD: An Online Community To Decrease Barriers to Implementation of Inclusive Tactile Teaching Tools.

Access to 3D printing and other "maker" technologies has opened new doors for the creation of classroom activities using physical models. Multiple strategies for implementing 3D-printed models exist, and work to define best practices is ongoing. We outline the strengths and weaknesses of common strategies for employing physical models in undergraduate biology courses and describe a novel strategy that we have developed to pair 3D-printed models with guided inquiry learning to create inclusive and interactive learning experiences. We further introduce the STEM BUILD website, a resource that we have developed to facilitate collaboration among instructors, makers, researchers, and Universal Design for Learning experts and reduce barriers to broad implementation of inclusive kinesthetic learning activities.

Building the lac Operon: A Guided-Inquiry Activity Using 3D-Printed Models.

Undergraduate biology courses rely heavily on visual representation of information. Students view images of plants, animals, and microbes, interpret data presented in graphs, and use drawings to understand how cells and molecules interact in three dimensions. Traditional teaching approaches exclude students with visual impairments and disadvantage students with disabilities that affect their interpretation and processing of visual and spatial information, and also students who simply do not identify as "visual learners." By using new technologies to develop tactile teaching tools (TTTs) that can be employed in classrooms, we aim to create inclusive learning environments and more effectively instruct diverse learners. The advent of affordable and accessible 3D printing technology makes it possible to create tactile models that represent molecules, cells, and entire organisms more accurately than traditional visual representations. We describe the assessment of a 3D gene expression puzzle as a guided inquiry learning activity in which students must correctly assemble a series of components in order to achieve an output. Upon completion of the puzzle, the TTT provides tactile feedback through vibration to signal transcriptional activation. Analysis of pre- and postassessment performance demonstrated statistically significant increases in individual students' paired assessment scores in two different classroom implementations, with a greater effect size at a rural minority-serving institution than an urban R1 university. These encouraging preliminary data suggest that TTTs with guided-inquiry learning disproportionately benefit disadvantaged student populations and could serve as a tool in leveling the playing field when teaching abstract biological concepts in diverse educational settings.

Undergraduate journal club as an intervention to improve student development in applying the scientific process.

Active learning improves student performance in STEM courses. Exposure to active learning environments generally occurs through traditional laboratory courses and independent research, both of which require access to resources that are limited at many universities. A previously reported active learning-based undergraduate journal club improved student achievement in communicating science. Here, we expanded on this previous journal club to improve student performance in the process of science. We developed and implemented a series of workshops and seminars referred to as "CASL Club," an undergraduate journal club targeted at improving student development in applying the scientific process. Students were surveyed before and after CASL club about their confidence in accessing, analyzing, and reporting scientific research. Post-CASL club, the students reported increases in confidence in their abilities to access and present scientific articles and write scientific abstracts. Additionally, the students reported improved confidence and performance in their courses. Compared to the previous journal club study, the majority of sampled journal club participants were not exposed to primary literature as part of their general coursework. Our results illustrate active-learning based undergraduate journal clubs as a way to expose students to primary literature and improve students' ability to apply scientific process in an active-learning environment at resource-limited universities.

c-FLIP protects eosinophils from TNF-α-mediated cell death in vivo.

Understanding the signals that regulate eosinophil survival and death is critical to developing new treatments for asthma, atopy, and gastrointestinal disease. Previous studies suggest that TNF-α stimulation protects eosinophils from apoptosis, and this TNF-α-mediated protection is mediated by the upregulation of an unknown protein by NF-κB. Here, we show for the first time that eosinophils express the caspase 8-inhibitory protein c-FLIP, and c-FLIP expression is upregulated upon TNF-α stimulation. Considering that c-FLIP expression is regulated by NF-κB, we hypothesized that c-FLIP might serve as the "molecular switch" that converts TNFRI activation to a pro-survival signal in eosinophils. Indeed, we found that one c-FLIP isoform, c-FLIPL, is required for mouse eosinophil survival in the presence of TNF-α both in vitro and in vivo. Importantly, our results suggest c-FLIP as a potential therapeutic target for the treatment of eosinophil-mediated disease.

The crosstalk between autophagy and apoptosis: where does this lead?

Recent advances in the understanding of the molecular processes contributing to autophagy have provided insight into the relationship between autophagy and apoptosis. In contrast to the concept of "autophagic cell death," accumulating evidence suggests that autophagy serves a largely cytoprotective role in physiologically relevant conditions. The cytoprotective function of autophagy is mediated in many circumstances by negative modulation of apoptosis. Apoptotic signaling, in turn, serves to inhibit autophagy. While the mechanisms mediating the complex counter-regulation of apoptosis and autophagy are not yet fully understood, important points of crosstalk include the interactions between Beclin-1 and Bcl-2/Bcl-xL and between FADD and Atg5, caspase- and calpain-mediated cleavage of autophagy-related proteins, and autophagic degradation of caspases. Continued investigation of these and other means of crosstalk between apoptosis and autophagy is necessary to elucidate the mechanisms controlling the balance between survival and death both under normal conditions and in diseases including cancer.

Regulation of CD8(+) T cell functions by RARgamma.

Retinoic acid plays a key role in the development and function of the immune system; however, the contribution of each of the three retinoic acid receptors (RARs) to the T cell immune response is not yet well understood. Of these receptors, both RARalpha and RARgamma are expressed in T lymphocytes. While possible functional redundancy thus complicates understanding of the role of each receptor in T cells, emerging data suggest that RARalpha and RARgamma function differently in thymocyte development and that RARgamma is required for both primary and secondary CD8(+) T cell immune responses.

Visualization of antigen presentation by actin-mediated targeting of glycolipid-enriched membrane domains to the immune synapse of B cell APCs.

Glycolipid-enriched membrane (GEM) domains, or lipid rafts, function in signaling in immune cells, but their properties during Ag presentation are less clear. To address this question, GEM domains were studied using fluorescence cell imaging of mouse CH27 B cells presenting Ag to D10 T cells. Our experiments showed that APCs were enriched with GEM domains in the immune synapse, and this occurred in an actin-dependent manner. This enrichment was specific to GEM domains, because a marker for non-GEM regions of the membrane was excluded from the immune synapse. Furthermore, fluorescence photobleaching experiments showed that protein in the immune synapse was dynamic and rapidly exchanged with that in other compartments of CH27 cells. To identify the signals for targeting GEM domains to the immune synapse in APCs, capping of the domains was measured in cells after cross-linking surface molecules. This showed that co-cross-linking CD48 with MHC class II was required for efficient capping and intracellular signaling. Capping of GEM domains by co-cross-linking CD48 and MHC class II occurred with co-capping of filamentous actin, and both domain capping and T cell-CH27 cell conjugation were inhibited by pretreating CH27 cells with latrunculin B. Furthermore, disruption of the actin cytoskeleton of the CH27 cells also inhibited formation of a mature immune synapse in those T cells that did conjugate to APCs. Thus, Ag presentation and efficient T cell stimulation occur by an actin-dependent targeting of GEM domains in the APC to the site of T cell engagement.