Nerves of Steel: How the Gut Nervous System Promotes a Strong Barrier.

In this issue of Cell, Jarret et al., Lai et al., and Matheis et al. demonstrate the extensive interplay between the nervous system and immune and epithelial cells of the gut to orchestrate host defense in homeostasis and following Salmonella infection.

Co-transcriptional genome surveillance by HUSH is coupled to termination machinery.

The HUSH complex recognizes and silences foreign DNA such as viruses, transposons, and transgenes without prior exposure to its targets. Here, we show that endogenous targets of the HUSH complex fall into two distinct classes based on the presence or absence of H3K9me3. These classes are further distinguished by their transposon content and differential response to the loss of HUSH. A de novo genomic rearrangement at the Sox2 locus induces a switch from H3K9me3-independent to H3K9me3-associated HUSH targeting, resulting in silencing. We further demonstrate that HUSH interacts with the termination factor WDR82 and-via its component MPP8-with nascent RNA. HUSH accumulates at sites of high RNAPII occupancy including long exons and transcription termination sites in a manner dependent on WDR82 and CPSF. Together, our results uncover the functional diversity of HUSH targets and show that this vertebrate-specific complex exploits evolutionarily ancient transcription termination machinery for co-transcriptional chromatin targeting and genome surveillance.

No pain, no gain: Sensory neurons heal a sunburn.

In a recent issue of Nature, Hoeffel et al. describe a novel pathway of sterile tissue repair utilizing a mouse model of sunburn. This wound healing pathway is coordinated by sensory neuron-derived TAFA4 that induces IL-10 production from Tim4+ dermal macrophages to prevent sustained inflammation and the emergence of tissue fibrosis.

Substance P Release by Sensory Neurons Triggers Dendritic Cell Migration and Initiates the Type-2 Immune Response to Allergens.

Dendritic cells (DCs) of the cDC2 lineage initiate allergic immunity and in the dermis are marked by their expression of CD301b. CD301b+ dermal DCs respond to allergens encountered in vivo, but not in vitro. This suggests that another cell in the dermis may sense allergens and relay that information to activate and induce the migration of CD301b+ DCs to the draining lymph node (dLN). Using a model of cutaneous allergen exposure, we show that allergens directly activated TRPV1+ sensory neurons leading to itch and pain behaviors. Allergen-activated sensory neurons released the neuropeptide Substance P, which stimulated proximally located CD301b+ DCs through the Mas-related G-protein coupled receptor member A1 (MRGPRA1). Substance P induced CD301b+ DC migration to the dLN where they initiated T helper-2 cell differentiation. Thus, sensory neurons act as primary sensors of allergens, linking exposure to activation of allergic-skewing DCs and the initiation of an allergic immune response.

Npas4 regulates excitatory-inhibitory balance within neural circuits through cell-type-specific gene programs.

The nervous system adapts to experience by inducing a transcriptional program that controls important aspects of synaptic plasticity. Although the molecular mechanisms of experience-dependent plasticity are well characterized in excitatory neurons, the mechanisms that regulate this process in inhibitory neurons are only poorly understood. Here, we describe a transcriptional program that is induced by neuronal activity in inhibitory neurons. We find that, while neuronal activity induces expression of early-response transcription factors such as Npas4 in both excitatory and inhibitory neurons, Npas4 activates distinct programs of late-response genes in inhibitory and excitatory neurons. These late-response genes differentially regulate synaptic input to these two types of neurons, promoting inhibition onto excitatory neurons while inducing excitation onto inhibitory neurons. These findings suggest that the functional outcomes of activity-induced transcriptional responses are adapted in a cell-type-specific manner to achieve a circuit-wide homeostatic response.

AP-1 Transcription Factors and the BAF Complex Mediate Signal-Dependent Enhancer Selection.

Enhancer elements are genomic regulatory sequences that direct the selective expression of genes so that genetically identical cells can differentiate and acquire the highly specialized forms and functions required to build a functioning animal. To differentiate, cells must select from among the ∼106 enhancers encoded in the genome the thousands of enhancers that drive the gene programs that impart their distinct features. We used a genetic approach to identify transcription factors (TFs) required for enhancer selection in fibroblasts. This revealed that the broadly expressed, growth-factor-inducible TFs FOS/JUN (AP-1) play a central role in enhancer selection. FOS/JUN selects enhancers together with cell-type-specific TFs by collaboratively binding to nucleosomal enhancers and recruiting the SWI/SNF (BAF) chromatin remodeling complex to establish accessible chromatin. These experiments demonstrate how environmental signals acting via FOS/JUN and BAF coordinate with cell-type-specific TFs to select enhancer repertoires that enable differentiation during development.

A tool for monitoring cell type-specific focused ultrasound neuromodulation and control of chronic epilepsy.

Focused ultrasound (FUS) is a powerful tool for noninvasive modulation of deep brain activity with promising therapeutic potential for refractory epilepsy; however, tools for examining FUS effects on specific cell types within the deep brain do not yet exist. Consequently, how cell types within heterogeneous networks can be modulated and whether parameters can be identified to bias these networks in the context of complex behaviors remains unknown. To address this, we developed a fiber Photometry Coupled focused Ultrasound System (PhoCUS) for simultaneously monitoring FUS effects on neural activity of subcortical genetically targeted cell types in freely behaving animals. We identified a parameter set that selectively increases activity of parvalbumin interneurons while suppressing excitatory neurons in the hippocampus. A net inhibitory effect localized to the hippocampus was further confirmed through whole brain metabolic imaging. Finally, these inhibitory selective parameters achieved significant spike suppression in the kainate model of chronic temporal lobe epilepsy, opening the door for future noninvasive therapies.

Assessing the Toxicity of Benzotriazole Ultraviolet Stabilizers to Fishes: Insights into Aryl Hydrocarbon Receptor-Mediated Effects.

Benzotriazole ultraviolet stabilizers (BUVSs) are chemicals used to mitigate UV-induced damage to manufactured goods. Their presence in aquatic environments and biota raises concerns, as certain BUVSs activate the aryl hydrocarbon receptor (AhR), which is linked to adverse effects in fish. However, potencies of BUVSs as AhR agonists and species sensitivities to AhR activation are poorly understood. This study evaluated the toxicity of three BUVSs using embryotoxicity assays. Zebrafish (Danio rerio) embryos exposed to BUVSs by microinjection suffered dose-dependent increases in mortality, with LD50 values of 4772, 11 608, and 56 292 ng/g-egg for UV-P, UV-9, and UV-090, respectively. The potencies and species sensitivities to AhR2 activation by BUVSs were assessed using a luciferase reporter gene assay with COS-7 cells transfected with the AhR2 of zebrafish and eight other fishes. The rank order of potency for activation of the AhR2 from all nine species was UV-P > UV-9 > UV-090. However, AhR2s among species differed in sensitivities to activation by up to 100-fold. An approximate reversed rank order of species sensitivity was observed compared to the rank order of sensitivity to 2,3,7,8-tetrachlorodibenzo[p]dioxin, the prototypical AhR agonist. Despite this, a pre-existing quantitative adverse outcome pathway linking AhR activation to embryo lethality could predict embryotoxicities of BUVSs in zebrafish.

Selective silencing of euchromatic L1s revealed by genome-wide screens for L1 regulators.

Transposable elements, also known as transposons, are now recognized not only as parasitic DNA, the spread of which in the genome must be controlled by the host, but also as major players in genome evolution and regulation. Long interspersed element-1 (LINE-1, also known as L1), the only currently autonomous mobile transposon in humans, occupies 17% of the genome and generates inter- and intra-individual genetic variation, in some cases resulting in disease. However, how L1 activity is controlled and the function of L1s in host gene regulation are not completely understood. Here we use CRISPR-Cas9 screening strategies in two distinct human cell lines to provide a genome-wide survey of genes involved in the control of L1 retrotransposition. We identify functionally diverse genes that either promote or restrict L1 retrotransposition. These genes, which are often associated with human diseases, control the L1 life cycle at the transcriptional or the post-transcriptional level in a manner that can depend on the endogenous L1 nucleotide sequence, underscoring the complexity of L1 regulation. We further investigate the restriction of L1 by the protein MORC2 and by the human silencing hub (HUSH) complex subunits MPP8 and TASOR. HUSH and MORC2 can selectively bind evolutionarily young, full-length L1s located within transcriptionally permissive euchromatic environments, and promote deposition of histone H3 Lys9 trimethylation (H3K9me3) for transcriptional silencing. Notably, these silencing events often occur within introns of transcriptionally active genes, and lead to the downregulation of host gene expression in a HUSH-, MORC2-, and L1-dependent manner. Together, these results provide a rich resource for studies of L1 retrotransposition, elucidate a novel L1 restriction pathway and illustrate how epigenetic silencing of transposable elements rewires host gene expression programs.

Amine-Functionalized Polybutadiene Synthesis by Tunable Postpolymerization Hydroaminoalkylation.

Early transition metal-catalyzed hydroaminoalkylation is a powerful single-step method to selectively add amines to polybutadienes, offering an efficient strategy to access amine-functionalized polyolefins. Aryl and alkyl secondary amines were used with a tantalum catalyst to functionalize both 28 wt% (PBD13) and 70 wt% (PBD50) 1,2-polybutadiene polymers. The degree of amination was controlled by modifying amine and catalyst loading in both small- and multigram-scale reactions. The vinyl groups of 1,2-polybutadiene were aminated with ease, and unexpectedly the hydroaminoalkylation of challenging internal alkenes of the 1,4-polybutadiene unit was observed. This unanticipated reactivity was proposed to be due to a directing group effect. This hypothesis was supported with small-molecule model substrates, which also showed directed internal alkene amination. Increasing degrees of amination resulted in materials with dramatically higher and tunable glass transition temperature (Tg) values, due to the dynamic cross-linking accessible to hydrogen-bonding, amine-containing materials. Primary amine-functionalized polybutadiene was also prepared, demonstrating that a broad new class of amine-containing polyolefins can be accessed by postpolymerization hydroaminoalkylation.

Halide Noninnocence and Direct Photoreduction of Ni(II) Enables Coupling of Aryl Chlorides in Dual Catalytic, Carbon-Heteroatom Bond-Forming Reactions.

Recent mechanistic studies of dual photoredox/Ni-catalyzed, light-driven cross-coupling reactions have found that the photocatalyst (PC) operates through either reductive quenching or energy transfer cycles. To date, reports invoking oxidative quenching cycles are comparatively rare and direct observation of such a quenching event has not been reported. However, when PCs with highly reducing excited states are used (e.g., Ir(ppy)3), photoreduction of Ni(II) to Ni(I) is thermodynamically feasible. Recently, a unified reaction system using Ir(ppy)3 was developed for forming C-O, C-N, and C-S bonds under the same conditions, a prospect that is challenging with PCs that can photooxidize these nucleophiles. Herein, in a detailed mechanistic study of this system, we observe oxidative quenching of the PC (Ir(ppy)3 or a phenoxazine) via nanosecond transient absorption spectroscopy. Speciation studies support that a mixture of Ni-bipyridine complexes forms under the reaction conditions, and the rate constant for photoreduction increases when more than one ligand is bound. Oxidative addition of an aryl iodide was observed indirectly via oxidation of the resulting iodide by Ir(IV)(ppy)3. Intriguingly, the persistence of the Ir(IV)/Ni(I) ion pair formed in the oxidative quenching step was found to be necessary to simulate the observed kinetics. Both bromide and iodide anions were found to reduce the oxidized form of the PC back to its neutral state. These mechanistic insights inspired the addition of a chloride salt additive, which was found to alter Ni speciation, leading to a 36-fold increase in the initial turnover frequency, enabling the coupling of aryl chlorides.

Altered sleep during spontaneous cannabinoid withdrawal in male mice.

Cessation of cannabinoid use in humans often leads to a withdrawal state that includes sleep disruption. Despite important health implications, little is known about how cannabinoid abstention affects sleep architecture, in part because spontaneous cannabinoid withdrawal is difficult to model in animals. In concurrent work we report that repeated administration of the high-efficacy cannabinoid 1 (CB1) receptor agonist AM2389 to mice for 5 days led to heightened locomotor activity and paw tremor following treatment discontinuation, potentially indicative of spontaneous cannabinoid withdrawal. Here, we performed parallel studies to examine effects on sleep. Using implantable electroencephalography (EEG) and electromyography (EMG) telemetry we examined sleep and neurophysiological measures before, during, and after 5 days of twice-daily AM2389 injections. We report that AM2389 produces decreases in locomotor activity that wane with repeated treatment, whereas discontinuation produces rebound increases in activity that persist for several days. Likewise, AM2389 initially produces profound increases in slow-wave sleep (SWS) and decreases in rapid eye movement (REM) sleep, as well as consolidation of sleep. By the third AM2389 treatment, this pattern transitions to decreases in SWS and total time sleeping. This pattern persists following AM2389 discontinuation and is accompanied by emergence of sleep fragmentation. Double-labeling immunohistochemistry for hypocretin/orexin (a sleep-regulating peptide) and c-Fos (a neuronal activity marker) in lateral hypothalamus revealed decreases in c-Fos/orexin+ cells following acute AM2389 and increases following discontinuation, aligning with the sleep changes. These findings indicate that AM2389 profoundly alters sleep in mice and suggest that sleep disruption following treatment cessation reflects spontaneous cannabinoid withdrawal.

Translation readthrough mitigation.

A fraction of ribosomes engaged in translation will fail to terminate when reaching a stop codon, yielding nascent proteins inappropriately extended on their C termini. Although such extended proteins can interfere with normal cellular processes, known mechanisms of translational surveillance are insufficient to protect cells from potential dominant consequences. Here, through a combination of transgenics and CRISPR­Cas9 gene editing in Caenorhabditis elegans, we demonstrate a consistent ability of cells to block accumulation of C-terminal-extended proteins that result from failure to terminate at stop codons. Sequences encoded by the 3' untranslated region (UTR) were sufficient to lower protein levels. Measurements of mRNA levels and translation suggested a co- or post-translational mechanism of action for these sequences in C. elegans. Similar mechanisms evidently operate in human cells, in which we observed a comparable tendency for translated human 3' UTR sequences to reduce mature protein expression in tissue culture assays, including 3' UTR sequences from the hypomorphic 'Constant Spring' haemoglobin stop codon variant. We suggest that 3' UTRs may encode peptide sequences that destabilize the attached protein, providing mitigation of unwelcome and varied translation errors.

Characterization of long G4-rich enhancer-associated genomic regions engaging in a novel loop:loop 'G4 Kissing' interaction.

Mammalian antibody switch regions (∼1500 bp) are composed of a series of closely neighboring G4-capable sequences. Whereas numerous structural and genome-wide analyses of roles for minimal G4s in transcriptional regulation have been reported, Long G4-capable regions (LG4s)-like those at antibody switch regions-remain virtually unexplored. Using a novel computational approach we have identified 301 LG4s in the human genome and find LG4s prone to mutation and significantly associated with chromosomal rearrangements in malignancy. Strikingly, 217 LG4s overlap annotated enhancers, and we find the promoters regulated by these enhancers markedly enriched in G4-capable sequences suggesting G4s facilitate promoter-enhancer interactions. Finally, and much to our surprise, we also find single-stranded loops of minimal G4s within individual LG4 loci are frequently highly complementary to one another with 178 LG4 loci averaging >35 internal loop:loop complements of >8 bp. As such, we hypothesized (then experimentally confirmed) that G4 loops within individual LG4 loci directly basepair with one another (similar to characterized stem-loop kissing interactions) forming a hitherto undescribed, higher-order, G4-based secondary structure we term a 'G4 Kiss or G4K'. In conclusion, LG4s adopt novel, higher-order, composite G4 structures directly contributing to the inherent instability, regulatory capacity, and maintenance of these conspicuous genomic regions.

A decision tree model for neuroimmune guidance of allergic immunity.

The immune system defends the body from infectious and non-infectious threats. Distinct recognition strategies have evolved to generate antigen-specific immunity against pathogens or toxins versus antigen-independent tissue repair. Structural recognition, or the sensing of conserved motifs, guides the immune response to viruses, bacteria, fungi, and unicellular parasites. Functional recognition, which is sensing that is based on the activities of an input, guides antigen-independent tissue healing and antigen-specific Type 2 immunity to toxins, allergens, and helminth parasites. Damage-associated molecular patterns (DAMPs), released from damaged and dying cells, permit functional recognition by immune cells. However, the DAMP paradigm alone does not explain how functional recognition can lead to such disparate immune responses, namely wound healing and Type 2 immunity. Recent work established that sensory neurons release neuropeptides in response to a variety of toxins and allergens. These neuropeptides act on local innate immune cells, stimulating or inhibiting their activities. By integrating our knowledge on DAMP function with new information on the role of neuropeptides in innate immune activation in Type 2 immunity, we describe a decision tree model of functional recognition. In this model, neuropeptides complement or antagonize DAMPs to guide the development of antigen-specific Type 2 immunity through the activation of innate immune cells. We discuss why this decision tree system evolved and its implications to allergic diseases.

Improved Testing and Design of Intubation Boxes During the COVID-19 Pandemic.

STUDY OBJECTIVE: Throughout the coronavirus disease 2019 pandemic, many emergency departments have been using passive protective enclosures ("intubation boxes") during intubation. The effectiveness of these enclosures remains uncertain. We sought to quantify their ability to contain aerosols using industry standard test protocols. METHODS: We tested a commercially available passive protective enclosure representing the most common design and compared this with a modified enclosure that incorporated a vacuum system for active air filtration during simulated intubations and negative-pressure isolation. We evaluated the enclosures by using the same 3 tests air filtration experts use to certify class I biosafety cabinets: visual smoke pattern analysis using neutrally buoyant smoke, aerosol leak testing using a test aerosol that mimics the size of virus-containing particulates, and air velocity measurements. RESULTS: Qualitative evaluation revealed smoke escaping from all passive enclosure openings. Aerosol leak testing demonstrated elevated particle concentrations outside the enclosure during simulated intubations. In contrast, vacuum-filter-equipped enclosures fully contained the visible smoke and test aerosol to standards consistent with class I biosafety cabinet certification. CONCLUSION: Passive enclosures for intubation failed to contain aerosols, but the addition of a vacuum and active air filtration reduced aerosol spread during simulated intubation and patient isolation.

Ozone-induced enhancement of airway hyperreactivity in rhesus macaques: Effects of antioxidant treatment.

BACKGROUND: Ozone (O3) inhalation elicits airway inflammation and impairs treatment responsiveness in asthmatic patients. The underlying immune mechanisms have been difficult to study because of the lack of relevant experimental models. Rhesus macaques spontaneously have asthma and have a similar immune system to human subjects. OBJECTIVES: We sought to investigate mucosal immune changes after O3 inhalation in a clinically relevant nonhuman primate asthma model and to study the effects of an antioxidant synthetic lignan (synthetic secoisolariciresinol diglucoside [LGM2605]). METHODS: A cohort of macaques (n = 17) previously characterized with airway hyperreactivity (AHR) to methacholine was assessed (day 1). Macaques were treated (orally) with LGM2605 (25 mg/kg) or placebo twice per day for 7 days, exposed to 0.3 ppm O3 or air for 6 hours (on day 7), and studied 12 hours later (day 8). Lung function, blood and bronchoalveolar lavage (BAL) fluid immune cell profile, and bronchial brushing and blood cell mRNA expression were assessed. RESULTS: O3 induced significant BAL fluid neutrophilia and eosinophilia and increased AHR and expression of IL6 and IL25 mRNA in the airway epithelium together with increased BAL fluid group 2 innate lymphoid cell (ILC2s), CD1c+ myeloid dendritic cell, and CD4+ T-cell counts and diminished surfactant protein D expression. Although LGM2605 attenuated some of the immune and inflammatory changes, it completely abolished O3-induced AHR. CONCLUSION: ILC2s, CD1c+ myeloid dendritic cells, and CD4+ T cells are selectively involved in O3-induced asthma exacerbation. The inflammatory changes were partially prevented by antioxidant pretreatment with LGM2605, which had an unexpectedly disproportionate protective effect on AHR.

Humidity Sensing through Reversible Isomerization of a Covalent Organic Framework.

Here we report that a covalent organic framework (COF), which contains 2,5-di(imine)-substituted 1,4-dihydroxybenzene (diiminol) moieties, undergoes color changes in the presence of solvents or solvent vapor that are rapid, passive, reversible, and easily detectable by the naked eye. A new visible absorption band appears in the presence of polar solvents, especially water, suggesting reversible conversion to another species. This reversibility is attributed to the ability of the diiminol to rapidly tautomerize to an iminol/cis-ketoenamine and its inability to doubly tautomerize to a diketoenamine. Density functional theory (DFT) calculations suggest similar energies for the two tautomers in the presence of water, but the diiminol is much more stable in its absence. Time-dependent DFT calculations confirm that the iminol/cis-ketoenamine absorbs at longer wavelength than the diiminol and indicate that this absorption has significant charge-transfer character. A colorimetric humidity sensing device constructed from an oriented thin film of the COF responded quickly to water vapor and was stable for months. These results suggest that tautomerization-induced electronic structure changes can be exploited in COF platforms to give rapid, reversible sensing in systems that exhibit long-term stability.

Sudden Cardiac Arrest during Participation in Competitive Sports.

BACKGROUND: The incidence of sudden cardiac arrest during participation in sports activities remains unknown. Preparticipation screening programs aimed at preventing sudden cardiac arrest during sports activities are thought to be able to identify at-risk athletes; however, the efficacy of these programs remains controversial. We sought to identify all sudden cardiac arrests that occurred during participation in sports activities within a specific region of Canada and to determine their causes. METHODS: In this retrospective study, we used the Rescu Epistry cardiac arrest database (which contains records of every cardiac arrest attended by paramedics in the network region) to identify all out-of-hospital cardiac arrests that occurred from 2009 through 2014 in persons 12 to 45 years of age during participation in a sport. Cases were adjudicated as sudden cardiac arrest (i.e., having a cardiac cause) or as an event resulting from a noncardiac cause, on the basis of records from multiple sources, including ambulance call reports, autopsy reports, in-hospital data, and records of direct interviews with patients or family members. RESULTS: Over the course of 18.5 million person-years of observation, 74 sudden cardiac arrests occurred during participation in a sport; of these, 16 occurred during competitive sports and 58 occurred during noncompetitive sports. The incidence of sudden cardiac arrest during competitive sports was 0.76 cases per 100,000 athlete-years, with 43.8% of the athletes surviving until they were discharged from the hospital. Among the competitive athletes, two deaths were attributed to hypertrophic cardiomyopathy and none to arrhythmogenic right ventricular cardiomyopathy. Three cases of sudden cardiac arrest that occurred during participation in competitive sports were determined to have been potentially identifiable if the athletes had undergone preparticipation screening. CONCLUSIONS: In our study involving persons who had out-of-hospital cardiac arrest, the incidence of sudden cardiac arrest during participation in competitive sports was 0.76 cases per 100,000 athlete-years. The occurrence of sudden cardiac arrest due to structural heart disease was uncommon during participation in competitive sports. (Funded by the National Heart, Lung, and Blood Institute and others.).

Designing High-Triplet-Yield Phenothiazine Donor-Acceptor Complexes for Photoredox Catalysis.

Phenothiazine, owing to its ease of oxidation and modularity with respect to facile functionalization, is an attractive central chemical unit from which to construct highly reducing organic photoredox catalysts. While design improvements have been made in the community, the yield of intersystem crossing (ΦISC), which determines access to the long-lived triplet excited state, has yet to be systematically optimized. Herein, we explore the impacts of N-aryl substituent variation on excited-state dynamics using picosecond to millisecond transient absorption and emission spectroscopies. Design principles are uncovered that center on controlling the energy of an intermediate charge transfer (CT) state within the singlet excited-state manifold, which, in turn, dictates the yield of CT-state formation and the rate constants for its depletion. Ultimately, we find ΦISC to be highly sensitive to the electron-withdrawing character of the N-aryl electron acceptor in the aforementioned CT state, with ΦISC ranging from ∼0 to 0.96.