Precise regulation of stem cell activity is crucial for tissue homeostasis and necessary to prevent overproliferation. In the Drosophila adult gut, high levels of reactive oxygen species (ROS) has been detected with different types of tissue damage, and oxidative stress has been shown to be both necessary and sufficient to trigger intestinal stem cell (ISC) proliferation. However, the connection between oxidative stress and mitogenic signals remains obscure. In a screen for genes required for ISC proliferation in response to oxidative stress, we identified two regulators of cytosolic Ca²⁺ levels, transient receptor potential A1 (TRPA1) and ryanodine receptor (RyR). Characterization of TRPA1 and RyR demonstrates that Ca²⁺ signaling is required for oxidative stress-induced activation of the Ras/MAPK pathway, which in turns drives ISC proliferation. Our findings provide a link between redox regulation and Ca²⁺ signaling and reveal a novel mechanism by which ISCs detect stress signals.

Many types of retinal neuron modulate the distribution of their processes to ensure a uniform coverage of the retinal surface. Dendritic field area, for instance, is inversely related to the variation in cellular density for many cell types, observed either across retinal eccentricity or between different strains of mice that differ in cell number. Dopaminergic amacrine (DA) cells, by contrast, have dendritic arbors that bear no spatial relationship to the presence of their immediate homotypic neighbors, yet it remains to be determined whether their coverage upon the retina, as a population, is conserved across variation in their total number. The present study assessed the overall density of the dopaminergic plexus in the inner plexiform layer in the presence of large variation in the total number of DA cells, as well as their retinal dopamine content, to determine whether either of these features is conserved. We first compared these traits between two strains of mice (C57BL/6J and A/J) that exhibit a two-fold difference in DA cell number. We subsequently examined these same traits in littermate mice for which the pro-apoptotic Bax gene was either intact or knocked out, yielding a five-fold difference in DA cell number. In both comparisons, we found greater plexus density and DA content in the strain or condition with the greater number of DA cells. The population of DA cells, therefore, does not appear to self-regulate its process coverage to achieve a constant density as the DA mosaic is established during development, nor its functional dopamine content in maturity.

Border cell migration in the Drosophila ovary has emerged as a genetically tractable model for studying collective cell movement. Over many years border cell migration was exclusively studied in fixed samples due to the inability to culture stage 9 egg chambers in vitro. Although culturing late-stage egg chambers was long feasible, stage 9 egg chambers survived only briefly outside the female body. We identified culture conditions that support stage 9 egg chamber development and sustain complete migration of border cells ex vivo. This protocol enables one to compare the dynamics of egg chamber development in wild-type and mutant egg chambers using time-lapse microscopy and taking advantage of a multiposition microscope with a motorized imaging stage. In addition, this protocol has been successfully used in combination with fluorescence resonance energy transfer biosensors, photo-activatable proteins, and pharmacological agents and can be used with wide-field or confocal microscopes in either an upright or an inverted configuration.

Opioid drugs are widely used analgesics that activate the G protein-coupled µ-opioid receptor, whose endogenous neuropeptide agonists, endorphins and enkephalins, are potent pain relievers. The therapeutic utility of opioid drugs is hindered by development of tolerance to the analgesic effects, requiring dose escalation for persistent pain control and leading to overdose and fatal respiratory distress. The prevailing hypothesis is that the intended analgesic effects of opioid drugs are mediated by µ-opioid receptor signaling to G protein, while the side-effects of respiratory depression and analgesic tolerance are caused by engagement of the receptor with the arrestin-3 protein. Consequently, opioid drug development has focused exclusively on identifying agonists devoid of arrestin-3 engagement. Here, we challenge the prevailing hypothesis with a panel of six clinically relevant opioid drugs and mice of three distinct genotypes with varying abilities to promote morphine-mediated arrestin-3 engagement. With this genetic and pharmacological approach, we demonstrate that arrestin-3 recruitment does not impact respiratory depression, and effective arrestin-3 engagement reduces, rather than exacerbates, the development of analgesic tolerance. These studies suggest that future development of safer opioids should focus on identifying opioid ligands that recruit both G protein and arrestin-3, thereby mimicking the signaling profile of most endogenous µ-opioid receptor agonists.

E-cadherin is a major homophilic cell-cell adhesion molecule that inhibits motility of individual cells on matrix. However, its contribution to migration of cells through cell-rich tissues is less clear. We developed an in vivo sensor of mechanical tension across E-cadherin molecules, which we combined with cell-type-specific RNAi, photoactivatable Rac, and morphodynamic profiling, to interrogate how E-cadherin contributes to collective migration of cells between other cells. Using the Drosophila ovary as a model, we found that adhesion between border cells and their substrate, the nurse cells, functions in a positive feedback loop with Rac and actin assembly to stabilize forward-directed protrusion and directionally persistent movement. Adhesion between individual border cells communicates direction from the lead cell to the followers. Adhesion between motile cells and polar cells holds the cluster together and polarizes each individual cell. Thus, E-cadherin is an integral component of the guidance mechanisms that orchestrate collective chemotaxis in vivo.

Opioid therapeutics are excellent analgesics, whose utility is compromised by dependence. Morphine (1) and its clinically relevant derivatives such as OxyContin (2), Vicodin (3), and Dilaudid (4) are "biased" agonists at the μ opioid receptor (OR), wherein they engage G protein signaling but poorly engage β-arrestin and the endocytic machinery. In contrast, endorphins, the endogenous peptide agonists for ORs, are potent analgesics, show reduced liability for tolerance and dependence, and engage both G protein and β-arrestin pathways as "balanced" agonists. We set out to determine if marine-derived alkaloids could serve as novel OR agonist chemotypes with a signaling profile distinct from morphine and more similar to the endorphins. Screening of 96 sponge-derived extracts followed by LC-MS-based purification to pinpoint the active compounds and subsequent evaluation of a mini library of related alkaloids identified two structural classes that modulate the ORs. These included the following: aaptamine (10), 9-demethyl aaptamine (11), demethyl (oxy)-aaptamine (12) with activity at the δ-OR (EC₅₀: 5.1, 4.1, 2.3 μM, respectively) and fascaplysin (17), and 10-bromo fascaplysin (18) with activity at the μ-OR (EC₅₀: 6.3, 4.2 μM respectively). An in vivo evaluation of 10 using δ-KO mice indicated its previously reported antidepressant-like effects are dependent on the δ-OR. Importantly, 17 functioned as a balanced agonist promoting both G protein signaling and β-arrestin recruitment along with receptor endocytosis similar to the endorphins. Collectively these results demonstrate the burgeoning potential for marine natural products to serve as novel lead compounds for therapeutic targets in neuroscience research.

Water reclamation and ecological reuse is gradually becoming a popular solution to address the high pollutant loads and insufficient ecological flow of many urban rivers. However, emerging contaminants in water reuse system and associated human health and ecological risks need to be assessed. This study determined the occurrence and human health and ecological risk assessments of 35 emerging contaminants during one year, including 5 types of persistent organic pollutants (POPs), 5 pharmaceutical and personal care products (PPCPs), 7 endocrine disrupting chemicals (EDCs) and 18 disinfection by-products (DBPs), in a wastewater treatment plant (WWTP) and receiving rivers, as well as an unimpacted river for comparison. Results showed that most of PPCPs and EDCs, especially antibiotics, triclosan, estrogens and bisphenol A, occurred frequently at relatively high concentrations, and they were removed from 20.5% to 88.7% with a mean of 58.9% via WWTP. The highest potential noncarcinogenic and carcinogenic risks in different reuse scenarios were assessed using maximal detected concentrations, all below the acceptable risk limits, with the highest total combined risk value of 9.21 × 10^-9 and 9.98 × 10^-7, respectively. Ecological risk assessment was conducted using risk quotient (RQ) method and indicated that several PPCPs, EDCs and haloacetonitriles (HANs) pose high risk (RQ > 1) to aquatic ecology in the rivers, with the highest RQ up to 83.8. The study suggested that ecological risks need to be urgently addressed by updating and optimizing the process in WWTPs to strengthen the removal efficiencies of emerging contaminants. The study can serve as a reference for safer water reuse in the future, while further studies could be conducted on the health risk of specific groups of people, exposure parameters in water reuse, as well as more emerging contaminants.

The gut microbiome composition is influenced by many factors including environmental exposures. Here, we investigated the effect of thirdhand cigarette smoke (THS) and exposure age on gut microbiome diversity. C57BL/6 mice were exposed to THS at human exposure relevant levels for three weeks during three different life stages: postnatal (0-3 weeks of age), pubescent (4-7 weeks of age), and adult (9-12 weeks of age), respectively. Cecal microbiome profiles were assessed at 17 weeks of age by 16S rRNA gene sequencing. We found that age at THS exposure strongly influenced the cecal microbiome composition. Although postnatal THS exposure significantly influenced the microbial composition, pubescent and adulthood exposures only had minor effects. The microbiome of postnatally THS-exposed mice significantly increased several degradation pathways that regulate glycolysis and pyruvate decarboxylation, and significantly decreased coenzyme A biosynthesis and pyrimidine deoxyribonucleoside salvage. Our results indicate that mouse postnatal development is particularly susceptible to persistent THS exposure effects on the gut microbiome.

Psoriasis is histologically characterized by keratinocytes (KC) hyperproliferation, inflammation, and increased angiogenesis, but the pathological factor responsible for these symptoms is unknown. Here, a neuroendocrine peptide (prokineticin 2, PK2), is highly expressed in human and mouse psoriatic skins but no significant change in other autoimmune diseases, suggesting that PK2 is a psoriasis-specific factor. Bacterial products significantly up-regulated PK2, implying that infection induces PK2 over-expression. PK2 promoted KC and macrophage to produce interleukin-1 (IL-1), the central player of inflammation and psoriasis, which acts on adjacent fibroblast to induce inflammatory cascades and KC hyperproliferation. IL-1 feeds back on macrophages to induce PK2 production to perpetuate PK2-IL-1 positive feedback loop. PK2 also promoted angiogenesis, another psoriatic symptom. In mouse models, PK2 over-expression aggravated psoriasis while its knock-down inhibited pathological development. The results indicate that PK2 over-production perpetuates psoriatic symptoms by creating PK-2-IL-1 vicious loop. PK2 is a central player in psoriasis and a promising psoriasis-specific target.

Transmembrane (TM) domains as simple as a single span can perform complex biological functions using entirely lipid-embedded chemical features. Computational design has the potential to generate custom tool molecules directly targeting membrane proteins at their functional TM regions. Thus far, designed TM domain-targeting agents have been limited to mimicking the binding modes and motifs of natural TM interaction partners. Here, we demonstrate the design of de novo TM proteins targeting the erythropoietin receptor (EpoR) TM domain in a custom binding topology competitive with receptor homodimerization. The TM proteins expressed in mammalian cells complex with EpoR and inhibit erythropoietin-induced cell proliferation. In vitro, the synthetic TM domain complex outcompetes EpoR homodimerization. Structural characterization reveals that the complex involves the intended amino acids and agrees with our designed molecular model of antiparallel TM helices at 1:1 stoichiometry. Thus, membrane protein TM regions can now be targeted in custom-designed topologies.