Regional synapse gain and loss accompany memory formation in larval zebrafish

Defining the structural and functional changes in the nervous system underlying learning and memory represents a major challenge for modern neuroscience. Although changes in neuronal activity following memory formation have been studied [B. F. Grewe et al., Nature 543, 670–675 (2017); M. T. Rogan, U. V. Stäubli, J. E. LeDoux, Nature 390, 604–607 (1997)], the underlying structural changes at the synapse level remain poorly understood. Here, we capture synaptic changes in the midlarval zebrafish brain that occur during associative memory formation by imaging excitatory synapses labeled with recombinant probes using selective plane illumination microscopy. Imaging the same subjects before and after classical conditioning at single-synapse resolution provides an unbiased mapping of synaptic changes accompanying memory formation. In control animals and animals that failed to learn the task, there were no significant changes in the spatial patterns of synapses in the pallium, which contains the equivalent of the mammalian amygdala and is essential for associative learning in teleost fish [M. Portavella, J. P. Vargas, B. Torres, C. Salas, Brain Res. Bull. 57, 397–399 (2002)]. In zebrafish that formed memories, we saw a dramatic increase in the number of synapses in the ventrolateral pallium, which contains neurons active during memory formation and retrieval. Concurrently, synapse loss predominated in the dorsomedial pallium. Surprisingly, we did not observe significant changes in the intensity of synaptic labeling, a proxy for synaptic strength, with memory formation in any region of the pallium. Our results suggest that memory formation due to classical conditioning is associated with reciprocal changes in synapse numbers in the pallium.

Targeted vaccination and the speed of SARS-CoV-2 adaptation

The limited supply of vaccines against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) raises the question of targeted vaccination. Many countries have opted to vaccinate older and more sensitive hosts first to minimize the disease burden. However, what are the evolutionary consequences of targeted vaccination? We clarify the consequences of different vaccination strategies through the analysis of the speed of viral adaptation measured as the rate of change of the frequency of a vaccine-adapted variant. We show that such a variant is expected to spread faster if vaccination targets individuals who are likely to be involved in a higher number of contacts. We also discuss the pros and cons of dose-sparing strategies. Because delaying the second dose increases the proportion of the population vaccinated with a single dose, this strategy can both speed up the spread of the vaccine-adapted variant and reduce the cumulative number of deaths. Hence, strategies that are most effective at slowing viral adaptation may not always be epidemiologically optimal. A careful assessment of both the epidemiological and evolutionary consequences of alternative vaccination strategies is required to determine which individuals should be vaccinated first.

A functional division of Drosophila sweet taste neurons that is value-based and task-specific

Sucrose is an attractive feeding substance and a positive reinforcer for Drosophila. But Drosophila females have been shown to robustly reject a sucrose-containing option for egg-laying when given a choice between a plain and a sucrose-containing option in specific contexts. How the sweet taste system of Drosophila promotes context-dependent devaluation of an egg-laying option that contains sucrose, an otherwise highly appetitive tastant, is unknown. Here, we report that devaluation of sweetness/sucrose for egg-laying is executed by a sensory pathway recruited specifically by the sweet neurons on the legs of Drosophila. First, silencing just the leg sweet neurons caused acceptance of the sucrose option in a sucrose versus plain decision, whereas expressing the channelrhodopsin CsChrimson in them caused rejection of a plain option that was “baited” with light over another that was not. Analogous bidirectional manipulations of other sweet neurons did not produce these effects. Second, circuit tracing revealed that the leg sweet neurons receive different presynaptic neuromodulations compared to some other sweet neurons and were the only ones with postsynaptic partners that projected prominently to the superior lateral protocerebrum (SLP) in the brain. Third, silencing one specific SLP-projecting postsynaptic partner of the leg sweet neurons reduced sucrose rejection, whereas expressing CsChrimson in it promoted rejection of a light-baited option during egg-laying. These results uncover that the Drosophila sweet taste system exhibits a functional division that is value-based and task-specific, challenging the conventional view that the system adheres to a simple labeled-line coding scheme.

CD169+ macrophages in lymph node and spleen critically depend on dual RANK and LTbetaR signaling

CD169+ macrophages reside in lymph node (LN) and spleen and play an important role in the immune defense against pathogens. As resident macrophages, they are responsive to environmental cues to shape their tissue-specific identity. We have previously shown that LN CD169+ macrophages require RANKL for formation of their niche and their differentiation. Here, we demonstrate that they are also dependent on direct lymphotoxin beta (LTβ) receptor (R) signaling. In the absence or the reduced expression of either RANK or LTβR, their differentiation is perturbed, generating myeloid cells expressing SIGN-R1 in LNs. Conditions of combined haploinsufficiencies of RANK and LTβR revealed that both receptors contribute equally to LN CD169+ macrophage differentiation. In the spleen, the Cd169-directed ablation of either receptor results in a selective loss of marginal metallophilic macrophages (MMMs). Using a RANKL reporter mouse, we identify splenic marginal zone stromal cells as a source of RANKL and demonstrate that it participates in MMM differentiation. The loss of MMMs had no effect on the splenic B cell compartments but compromised viral capture and the expansion of virus-specific CD8+ T cells. Taken together, the data provide evidence that CD169+ macrophage differentiation in LN and spleen requires dual signals from LTβR and RANK with implications for the immune response.

Inhibition of EZH2 transactivation function sensitizes solid tumors to genotoxic stress

Drugs that block the activity of the methyltransferase EZH2 are in clinical development for the treatment of non-Hodgkin lymphomas harboring EZH2 gain-of-function mutations that enhance its polycomb repressive function. We have previously reported that EZH2 can act as a transcriptional activator in castration-resistant prostate cancer (CRPC). Now we show that EZH2 inhibitors can also block the transactivation activity of EZH2 and inhibit the growth of CRPC cells. Gene expression and epigenomics profiling of cells treated with EZH2 inhibitors demonstrated that in addition to derepressing gene expression, these compounds also robustly down-regulate a set of DNA damage repair (DDR) genes, especially those involved in the base excision repair (BER) pathway. Methylation of the pioneer factor FOXA1 by EZH2 contributes to the activation of these genes, and interaction with the transcriptional coactivator P300 via the transactivation domain on EZH2 directly turns on the transcription. In addition, CRISPR-Cas9–mediated knockout screens in the presence of EZH2 inhibitors identified these BER genes as the determinants that underlie the growth-inhibitory effect of EZH2 inhibitors. Interrogation of public data from diverse types of solid tumors expressing wild-type EZH2 demonstrated that expression of DDR genes is significantly correlated with EZH2 dependency and cellular sensitivity to EZH2 inhibitors. Consistent with these findings, treatment of CRPC cells with EZH2 inhibitors dramatically enhances their sensitivity to genotoxic stress. These studies reveal a previously unappreciated mechanism of action of EZH2 inhibitors and provide a mechanistic basis for potential combination cancer therapies.

Volatile-consuming reactions fracture rocks and self-accelerate fluid flow in the lithosphere

Hydration and carbonation reactions within the Earth cause an increase in solid volume by up to several tens of vol%, which can induce stress and rock fracture. Observations of naturally hydrated and carbonated peridotite suggest that permeability and fluid flow are enhanced by reaction-induced fracturing. However, permeability enhancement during solid-volume–increasing reactions has not been achieved in the laboratory, and the mechanisms of reaction-accelerated fluid flow remain largely unknown. Here, we present experimental evidence of significant permeability enhancement by volume-increasing reactions under confining pressure. The hydromechanical behavior of hydration of sintered periclase [MgO + H2O → Mg(OH)2] depends mainly on the initial pore-fluid connectivity. Permeability increased by three orders of magnitude for low-connectivity samples, whereas it decreased by two orders of magnitude for high-connectivity samples. Permeability enhancement was caused by hierarchical fracturing of the reacting materials, whereas a decrease was associated with homogeneous pore clogging by the reaction products. These behaviors suggest that the fluid flow rate, relative to reaction rate, is the main control on hydromechanical evolution during volume-increasing reactions. We suggest that an extremely high reaction rate and low pore-fluid connectivity lead to local stress perturbations and are essential for reaction-induced fracturing and accelerated fluid flow during hydration/carbonation.

Ribosomally derived lipopeptides containing distinct fatty acyl moieties

Lipopeptides represent a large group of microbial natural products that include important antibacterial and antifungal drugs and some of the most-powerful known biosurfactants. The vast majority of lipopeptides comprise cyclic peptide backbones N-terminally equipped with various fatty acyl moieties. The known compounds of this type are biosynthesized by nonribosomal peptide synthetases, giant enzyme complexes that assemble their products in a non–gene-encoded manner. Here, we report the genome-guided discovery of ribosomally derived, fatty-acylated lipopeptides, termed selidamides. Heterologous reconstitution of three pathways, two from cyanobacteria and one from an arctic, ocean-derived alphaproteobacterium, allowed structural characterization of the probable natural products and suggest that selidamides are widespread over various bacterial phyla. The identified representatives feature cyclic peptide moieties and fatty acyl units attached to (hydroxy)ornithine or lysine side chains by maturases of the GCN5-related N-acetyltransferase superfamily. In contrast to nonribosomal lipopeptides that are usually produced as congener mixtures, the three selidamides are selectively fatty acylated with C10, C12, or C16 fatty acids, respectively. These results highlight the ability of ribosomal pathways to emulate products with diverse, nonribosomal-like features and add to the biocatalytic toolbox for peptide drug improvement and targeted discovery.

Genome-wide screens identify specific drivers of mutant hTERT promoters

Cancer-specific hTERT promoter mutations reported in 19% of cancers result in enhanced telomerase activity. Understanding the distinctions between transcriptional regulation of wild-type (WT) and mutant (Mut) hTERT promoters may open up avenues for development of inhibitors which specially block hTERT expression in cancer cells. To comprehensively identify physiological regulators of WT- or Mut-hTERT promoters, we generated several isogenic reporter cells driven by endogenous hTERT loci. Genome-wide CRISPR-Cas9 and small interfering RNA screens using these isogenic reporter lines identified specific regulators of Mut-hTERT promoters. We validate and characterize one of these hits, namely, MED12, a kinase subunit of mediator complex. We demonstrate that MED12 specifically drives expression of hTERT from the Mut-hTERT promoter by mediating long-range chromatin interaction between the proximal Mut-hTERT promoter and T-INT1 distal regulatory region 260 kb upstream. Several hits identified in our screens could serve as potential therapeutic targets, inhibition of which may specifically block Mut-hTERT promoter driven telomerase reactivation in cancers.

Floxed exon (Flexon): A flexibly positioned stop cassette for recombinase-mediated conditional gene expression

Conditional gene expression is a powerful tool for genetic analysis of biological phenomena. In the widely used “lox-stop-lox” approach, insertion of a stop cassette consisting of a series of stop codons and polyadenylation signals flanked by lox sites into the 5′ untranslated region (UTR) of a gene prevents expression until the cassette is excised by tissue-specific expression of Cre recombinase. Although lox-stop-lox and similar approaches using other site-specific recombinases have been successfully used in many experimental systems, this design has certain limitations. Here, we describe the Floxed exon (Flexon) approach, which uses a stop cassette composed of an artificial exon flanked by artificial introns, designed to cause premature termination of translation and nonsense-mediated decay of the mRNA and allowing for flexible placement into a gene. We demonstrate its efficacy in Caenorhabditis elegans by showing that, when promoters that cause weak and/or transient cell-specific expression are used to drive Cre in combination with a gfp(flexon) transgene, strong and sustained expression of green fluorescent protein (GFP) is obtained in specific lineages. We also demonstrate its efficacy in an endogenous gene context: we inserted a flexon into the Argonaute gene rde-1 to abrogate RNA interference (RNAi), and restored RNAi tissue specifically by expression of Cre. Finally, we describe several potential additional applications of the Flexon approach, including more precise control of gene expression using intersectional methods, tissue-specific protein degradation, and generation of genetic mosaics. The Flexon approach should be feasible in any system where a site-specific recombination-based method may be applied.