Serotonin Strengthens a Developing Glutamatergic Synapse through a PI3K-Dependent Mechanism.

It is well established that, during neural circuit development, glutamatergic synapses become strengthened via NMDA receptor (NMDAR)-dependent upregulation of AMPA receptor (AMPAR)-mediated currents. In addition, however, it is known that the neuromodulator serotonin is present throughout most regions of the vertebrate brain while synapses are forming and being shaped by activity-dependent processes. This suggests that serotonin may modulate or contribute to these processes. Here, we investigate the role of serotonin in the developing retinotectal projection of the Xenopus tadpole. We altered endogenous serotonin transmission in stage 48/49 (∼10-21 days postfertilization) Xenopus tadpoles and then carried out a set of whole-cell electrophysiological recordings from tectal neurons to assess retinotectal synaptic transmission. Because tadpole sex is indeterminate at these early stages of development, experimental groups were composed of randomly chosen tadpoles. We found that pharmacologically enhancing and reducing serotonin transmission for 24 h up- and downregulates, respectively, AMPAR-mediated currents at individual retinotectal synapses. Inhibiting 5-HT2 receptors also significantly weakened AMPAR-mediated currents and abolished the synapse strengthening effect seen with enhanced serotonin transmission, indicating a 5-HT2 receptor-dependent effect. We also determine that the serotonin-dependent upregulation of synaptic AMPAR currents was mediated via an NMDAR-independent, PI3K-dependent mechanism. Altogether, these findings indicate that serotonin regulates AMPAR currents at developing synapses independent of NMDA transmission, which may explain its role as an enabler of activity-dependent plasticity.

Adaptable, turn-on maturation (ATOM) fluorescent biosensors for multiplexed detection in cells.

A grand challenge in biosensor design is to develop a single-molecule, fluorescent protein-based platform that can be easily adapted to recognize targets of choice. Here, we created a family of adaptable, turn-on maturation (ATOM) biosensors consisting of a monobody (circularly permuted at one of two positions) or a nanobody (circularly permuted at one of three positions) inserted into a fluorescent protein at one of three surface loops. Multiplexed imaging of live human cells coexpressing cyan, yellow and red ATOM sensors detected biosensor targets that were specifically localized to various subcellular compartments. Fluorescence activation involved ligand-dependent chromophore maturation with turn-on ratios of up to 62-fold in cells and 100-fold in vitro. Endoplasmic reticulum- and mitochondria-localized ATOM sensors detected ligands that were targeted to those organelles. The ATOM design was validated with three monobodies and one nanobody inserted into distinct fluorescent proteins, suggesting that customized ATOM sensors can be generated quickly.

Adaptable, Turn-On Monobody (ATOM) Fluorescent Biosensors for Multiplexed Detection in Cells.

A grand challenge in biosensor design is to develop a single molecule, fluorescent protein-based platform that can be easily adapted to recognize targets of choice. Conceptually, this can be achieved by fusing a small, antibody-like binding domain to a fluorescent protein in such a way that target binding activates fluorescence. Although this design is simple to envision, its execution is not obvious. Here, we created a family of adaptable, turn-on monobody (ATOM) biosensors consisting of a monobody, circularly permuted at one of two positions, inserted into a fluorescent protein at one of three surface loops. Multiplexed imaging of live human cells co-expressing cyan, yellow, and red ATOM sensors detected the biosensor targets (WDR5, SH2, and hRAS proteins) that were localized to the nucleus, cytoplasm, and plasma membrane, respectively, with high specificity. ER- and mitochondria-localized ATOM sensors also detected ligands that were targeted to those organelles. Fluorescence activation involved ligand-dependent chromophore maturation with fluorescence turn-on ratios of >20-fold in cells and up to 100-fold in vitro . The sensing mechanism was validated with three arbitrarily chosen monobodies inserted into jellyfish as well as anemone lineages of fluorescent proteins, suggesting that ATOM sensors with different binding specificities and additional colors can be generated relatively quickly.

A circadian-dependent preference for light displayed by Xenopus tadpoles is modulated by serotonin.

Innate visually guided behaviors are thought to promote survival by guiding organisms to sources of food and safety and away from harm without requiring learning. Historically, innate behaviors have been considered hard-wired and invariable, but emerging evidence shows that many innate behaviors are flexible and complex due to modulation. Here, we investigate the modulation of the innate preference for light displayed by the Xenopus laevis tadpole, an exceptionally invasive and well-studied organism that is known to display several different innate visually guided behaviors. We found that tadpoles display a circadian-regulated oscillation in their preference for light over dark which can be altered by experimentally increasing or decreasing levels of serotonin transmission. We also found that endogenous levels of serotonin transmission during the day maintain a consistently moderate preference for light. Theoretically, a moderate preference for light, as opposed to a strong preference, optimizes survival by rendering tadpoles' behavior less predictable.

The bitter end: T2R bitter receptor agonists elevate nuclear calcium and induce apoptosis in non-ciliated airway epithelial cells.

Bitter taste receptors (T2Rs) localize to airway motile cilia and initiate innate immune responses in retaliation to bacterial quorum sensing molecules. Activation of cilia T2Rs leads to calcium-driven NO production that increases cilia beating and directly kills bacteria. Several diseases, including chronic rhinosinusitis, COPD, and cystic fibrosis, are characterized by loss of motile cilia and/or squamous metaplasia. To understand T2R function within the altered landscape of airway disease, we studied T2Rs in non-ciliated airway cell lines and primary cells. Several T2Rs localize to the nucleus in de-differentiated cells that typically localize to cilia in differentiated cells. As cilia and nuclear import utilize shared proteins, some T2Rs may target to the nucleus in the absence of motile cilia. T2R agonists selectively elevated nuclear and mitochondrial calcium through a G-protein-coupled receptor phospholipase C mechanism. Additionally, T2R agonists decreased nuclear cAMP, increased nitric oxide, and increased cGMP, consistent with T2R signaling. Furthermore, exposure to T2R agonists led to nuclear calcium-induced mitochondrial depolarization and caspase activation. T2R agonists induced apoptosis in primary bronchial and nasal cells differentiated at air-liquid interface but then induced to a squamous phenotype by apical submersion. Air-exposed well-differentiated cells did not die. This may be a last-resort defense against bacterial infection. However, it may also increase susceptibility of de-differentiated or remodeled epithelia to damage by bacterial metabolites. Moreover, the T2R-activated apoptosis pathway occurs in airway cancer cells. T2Rs may thus contribute to microbiome-tumor cell crosstalk in airway cancers. Targeting T2Rs may be useful for activating cancer cell apoptosis while sparing surrounding tissue.

"I Like and Prefer to Work Alone": Social Anxiety, Academic Self-Efficacy, and Students' Perceptions of Active Learning.

Although active learning improves student outcomes in science, technology, engineering, and mathematics (STEM) programs, it may provoke anxiety in some students. We examined whether two psychological variables, social anxiety (psychological distress relating to the fear of negative evaluation by others) and academic self-efficacy (confidence in one's ability to overcome academic challenges), interact with student perceptions of evidence-based instructional practices (EBIPs) and associate with their final grades in a STEM-related course. Human anatomy and physiology students in community college courses rated various EBIPs for their perceived educational value and their capacity to elicit anxiety (N = 227). In general, practices causing students the most anxiety (e.g., cold calling) were reported by students as having the least educational value. When controlling for students' self-reported grade point averages, socially anxious students rated several EBIPs as more anxiety inducing, whereas high-efficacy students reported less anxiety surrounding other EBIPs. Furthermore, mediation analysis revealed that individual differences in academic self-efficacy at the beginning of the term explained some of the negative association between students' social anxiety levels and final grades in the course. Our results, obtained in a community college context, support a growing body of evidence that social anxiety and academic self-efficacy are linked with how students perceive and perform in an active-learning environment.

Melanocyte and melanoma cell activation by calprotectin.

Calprotectin, a heterodimer of S100A8 and S100A9, is a proinflammatory cytokine released from ultraviolet radiation-exposed keratinocytes. Calprotectin binds to Toll-like receptor 4, the receptor for advanced glycation end-products, and extracellular matrix metalloproteinase inducer on target cells to stimulate migration. Melanocytes and melanoma cells produce little if any calprotectin, but they do express receptors for the cytokine. Thus, keratinocyte-derived calprotectin has the potential to activate melanocytes and melanoma cells within the epidermis in a paracrine manner. We examined the ability of calprotectin to stimulate proliferation and migration in normal human melanocytes and melanoma cells in vitro. We first showed, by immunofluorescence and quantitative RT-PCR, that the melanocytic cells employed expressed a calprotectin receptor, the receptor for advanced end-products. We then demonstrated that calprotectin significantly enhanced proliferation, migration, and Matrigel invasion in both normal human melanocytes and melanoma cells. Thus, calprotectin is one of the numerous paracrine factors released by ultraviolet radiation-exposed keratinocytes that may promote melanomagenesis and is a potential target for melanoma prevention or therapy.