Non-lethal Inhibition of Gut Microbial Trimethylamine Production for the Treatment of Atherosclerosis.

Trimethylamine (TMA) N-oxide (TMAO), a gut-microbiota-dependent metabolite, both enhances atherosclerosis in animal models and is associated with cardiovascular risks in clinical studies. Here, we investigate the impact of targeted inhibition of the first step in TMAO generation, commensal microbial TMA production, on diet-induced atherosclerosis. A structural analog of choline, 3,3-dimethyl-1-butanol (DMB), is shown to non-lethally inhibit TMA formation from cultured microbes, to inhibit distinct microbial TMA lyases, and to both inhibit TMA production from physiologic polymicrobial cultures (e.g., intestinal contents, human feces) and reduce TMAO levels in mice fed a high-choline or L-carnitine diet. DMB inhibited choline diet-enhanced endogenous macrophage foam cell formation and atherosclerotic lesion development in apolipoprotein e(-/-) mice without alterations in circulating cholesterol levels. The present studies suggest that targeting gut microbial production of TMA specifically and non-lethal microbial inhibitors in general may serve as a potential therapeutic approach for the treatment of cardiometabolic diseases.

Genetic identification and separation of innate and experience-dependent courtship behaviors in Drosophila.

Wild-type D. melanogaster males innately possess the ability to perform a multistep courtship ritual to conspecific females. The potential for this behavior is specified by the male-specific products of the fruitless (fru(M)) gene; males without fru(M) do not court females when held in isolation. We show that such fru(M) null males acquire the potential for courtship when grouped with other flies; they apparently learn to court flies with which they were grouped, irrespective of sex or species and retain this behavior for at least a week. The male-specific product of the doublesex gene (dsx(M)) is necessary and sufficient for the acquisition of the potential for such experience-dependent courtship. These results reveal a process that builds, via dsx(M) and social experience, the potential for a more flexible sexual behavior, which could be evolutionarily conserved as dsx-related genes that function in sexual development are found throughout the animal kingdom.

Genetic and neural mechanisms that inhibit Drosophila from mating with other species.

Genetically hard-wired neural mechanisms must enforce behavioral reproductive isolation because interspecies courtship is rare even in sexually naïve animals of most species. We find that the chemoreceptor Gr32a inhibits male D. melanogaster from courting diverse fruit fly species. Gr32a recognizes nonvolatile aversive cues present on these reproductively dead-end targets, and activity of Gr32a neurons is necessary and sufficient to inhibit interspecies courtship. Male-specific Fruitless (Fru(M)), a master regulator of courtship, also inhibits interspecies courtship. Gr32a and Fru(M) are not coexpressed, but Fru(M) neurons contact Gr32a neurons, suggesting that these genes influence a shared neural circuit that inhibits interspecies courtship. Gr32a and Fru(M) also suppress within-species intermale courtship, but we show that distinct mechanisms preclude sexual displays toward conspecific males and other species. Although this chemosensory pathway does not inhibit interspecies mating in D. melanogaster females, similar mechanisms appear to inhibit this behavior in many other male drosophilids.

Siglecs as potential targets of therapy in human mast cell- and/or eosinophil-associated diseases.

Siglecs (sialic acid-binding immunoglobulin-like lectins) are a family of vertebrate glycan-binding cell-surface proteins. The majority mediate cellular inhibitory activity once engaged by specific ligands or ligand-mimicking molecules. As a result, Siglec engagement is now of interest as a strategy to therapeutically dampen unwanted cellular responses. When considering allergic inflammation, human eosinophils and mast cells express overlapping but distinct patterns of Siglecs. For example, Siglec-6 is selectively and prominently expressed on mast cells while Siglec-8 is highly specific for both eosinophils and mast cells. This review will focus on a subset of Siglecs and their various endogenous or synthetic sialoside ligands that regulate eosinophil and mast cell function and survival. It will also summarize how certain Siglecs have become the focus of novel therapies for allergic and other eosinophil- and mast cell-related diseases.

Correction: An allele-sharing, moment-based estimator of global, population-specific and population-pair FST under a general model of population structure.

[This corrects the article DOI: 10.1371/journal.pgen.1010871.].

Detecting inbreeding depression in structured populations.

Measuring inbreeding and its consequences on fitness is central for many areas in biology including human genetics and the conservation of endangered species. However, there is no consensus on the best method, neither for quantification of inbreeding itself nor for the model to estimate its effect on specific traits. We simulated traits based on simulated genomes from a large pedigree and empirical whole-genome sequences of human data from populations with various sizes and structures (from the 1,000 Genomes project). We compare the ability of various inbreeding coefficients ([Formula: see text]) to quantify the strength of inbreeding depression: allele-sharing, two versions of the correlation of uniting gametes which differ in the weight they attribute to each locus and two identical-by-descent segments-based estimators. We also compare two models: the standard linear model and a linear mixed model (LMM) including a genetic relatedness matrix (GRM) as random effect to account for the nonindependence of observations. We find LMMs give better results in scenarios with population or family structure. Within the LMM, we compare three different GRMs and show that in homogeneous populations, there is little difference among the different [Formula: see text] and GRM for inbreeding depression quantification. However, as soon as a strong population or family structure is present, the strength of inbreeding depression can be most efficiently estimated only if i) the phenotypes are regressed on [Formula: see text] based on a weighted version of the correlation of uniting gametes, giving more weight to common alleles and ii) with the GRM obtained from an allele-sharing relatedness estimator.

The skin I live in: Pathogenesis of white-nose syndrome of bats.

The emergence of white-nose syndrome (WNS) in North America has resulted in mass mortalities of hibernating bats and total extirpation of local populations. The need to mitigate this disease has stirred a significant body of research to understand its pathogenesis. Pseudogymnoascus destructans, the causative agent of WNS, is a psychrophilic (cold-loving) fungus that resides within the class Leotiomycetes, which contains mainly plant pathogens and is unrelated to other consequential pathogens of animals. In this review, we revisit the unique biology of hibernating bats and P. destructans and provide an updated analysis of the stages and mechanisms of WNS progression. The extreme life history of hibernating bats, the psychrophilic nature of P. destructans, and its evolutionary distance from other well-characterized animal-infecting fungi translate into unique host-pathogen interactions, many of them yet to be discovered.

Vaccination with O-linked Mannans Protects against Systemic Candidiasis through Innate Lymphocyte Populations.

Candida spp. are the fourth leading cause of bloodstream infections in hospitalized patients and the most common cause of invasive fungal infection. No vaccine against Candida spp. or other fungal pathogens of humans is available. We recently discovered the Blastomyces Dectin-2 ligand endoglucanase 2 that harbors antigenic and adjuvant functions and can function as a protective vaccine against that fungus. We also reported that the adjuvant activity, which is mediated by O-mannans decorating the C terminus of Blastomyces Dectin-2 ligand endoglucanase 2, can augment peptide Ag-induced vaccine immunity against heterologous agents, including Cryptococcus, Candida, and influenza. In this article, we report that the O-linked mannans alone, in the absence of any antigenic peptide, can also protect against systemic candidiasis, reducing kidney fungal load and increasing survival in a Dectin-2-dependent manner. We found that this long-term glycan-induced protection is mediated by innate lymphocyte populations including TCR-γδ+ T cells, innate lymphoid cells, and NK cells that subsequently activate and release reactive oxygen species from neutrophils and monocytes. Our findings suggest that Blastomyces O-mannan displayed by Eng2 induces a form of protective trained immunity mediated by innate lymphocyte populations.

Sensitization with Fungal Protease Allergen Establishes Long-Lived, Allergenic Th Cell Memory in the Lung.

Allergic asthma is a chronic inflammatory disease that affects millions of individuals worldwide. Exposure to allergens produced by a variety of otherwise harmless microbes, including fungi, predisposes individuals to immunopathologic disease upon subsequent encounters with allergen. We developed a mouse model that employs a purified protease produced by Aspergillus (Asp f 13) to investigate the contributions of CD4+ Th cells to recurrent lung inflammation. Notably, memory CD4+ T cells enhanced the eosinophil response of sensitized/rechallenged animals. In addition, memory CD4+ T cells maintained allergenic features, including expression of GATA-binding protein 3 and IL-5. Th2 memory T cells persisted in the peribronchiolar interstitium of the lung and expressed markers of tissue residence, such as CD69, CCR8, and IL-33R. Lastly, we identified a peptide epitope contained within Asp f 13 and generated a peptide-MHC class II tetramer. Using these tools, we further demonstrated the durability and exquisite sensitivity of memory T cells in promoting lung eosinophilia. Our data highlight important features of memory T cells that strengthen the notion that memory T cells are principal drivers of eosinophilic disease in murine models of allergic sensitization and episodic airway inflammation.

An allele-sharing, moment-based estimator of global, population-specific and population-pair FST under a general model of population structure.

Being able to properly quantify genetic differentiation is key to understanding the evolutionary potential of a species. One central parameter in this context is FST, the mean coancestry within populations relative to the mean coancestry between populations. Researchers have been estimating FST globally or between pairs of populations for a long time. More recently, it has been proposed to estimate population-specific FST values, and population-pair mean relative coancestry. Here, we review the several definitions and estimation methods of FST, and stress that they provide values relative to a reference population. We show the good statistical properties of an allele-sharing, method of moments based estimator of FST (global, population-specific and population-pair) under a very general model of population structure. We point to the limitation of existing likelihood and Bayesian estimators when the populations are not independent. Last, we show that recent attempts to estimate absolute, rather than relative, mean coancestry fail to do so.

Novel insecticidal proteins from ferns resemble insecticidal proteins from Bacillus thuringiensis.

Lepidopterans affect crop production worldwide. The use of transgenes encoding insecticidal proteins from Bacillus thuringiensis (Bt) in crop plants is a well-established technology that enhances protection against lepidopteran larvae. Concern about widespread field-evolved resistance to Bt proteins has highlighted an urgent need for new insecticidal proteins with different modes or sites of action. We discovered a new family of insecticidal proteins from ferns. The prototype protein from Pteris species (Order Polypodiales) and variants from two other orders of ferns, Schizaeales and Ophioglossales, were effective against important lepidopteran pests of maize and soybean in diet-based assays. Transgenic maize and soybean plants producing these proteins were more resistant to insect damage than controls. We report here the crystal structure of a variant of the prototype protein to 1.98 Å resolution. Remarkably, despite being derived from plants, the structure resembles the 3-domain Cry proteins from Bt but has only two out of three of their characteristic domains, lacking the C-terminal domain which is typically required for their activities. Two of the fern proteins were effective against strains of fall armyworm that were resistant to Bt 3-domain Cry proteins Cry1Fa or Cry2A.127. This therefore represents a novel family of insecticidal proteins that have the potential to provide future tools for pest control.

Prevention of allergic reactions during oxaliplatin desensitization through inhibition of Bruton tyrosine kinase.

BACKGROUND: Acute infusion reactions to oxaliplatin, a chemotherapeutic used to treat gastrointestinal cancers, are observed in about 20% of patients. Rapid drug desensitization (RDD) protocols often allow the continuation of oxaliplatin in patients with no alternative options. Breakthrough symptoms, including anaphylaxis, can still occur during RDD. OBJECTIVE: Our aim was to evaluate whether pretreatment with acalabrutinib, a Bruton tyrosine kinase inhibitor, can prevent anaphylaxis during RDD in a patient sensitized to oxaliplatin. METHODS: A 52-year-old male with locally advanced gastric carcinoma developed anaphylaxis during his fifth cycle of oxaliplatin. As he required 6 additional cycles to complete his curative-intent treatment regimen, he underwent RDD to oxaliplatin but still developed severe acute reactions. The risks and benefits of adding acalabrutinib before and during RDD were reviewed, and the patient elected to proceed. RESULTS: With acalabrutinib taken before and during the RDD, the patient was able to tolerate oxaliplatin RDD without complication. Consistent with its mechanism of action, acalabrutinib completely blocked the patient's positive skin prick response to oxaliplatin. Acalabrutinib did not alter the percentage of circulating basophils (1.24% vs 0.98%) before the RDD but did protect against basopenia (0.74% vs 0.09%) after the RDD. Acalabrutinib was associated with a drastic reduction in the ability of basophils to upregulate CD63 in vitro following incubation with oxaliplatin (0.11% vs 2.38%) or polyclonal anti-human IgE antibody (0.08% vs 44.2%). CONCLUSIONS: Five doses of acalabrutinib, 100 mg, orally twice daily starting during the evening 2 days before and continuing through RDD allowed a sensitized patient to receive oxaliplatin successfully and safely.

Single cell RNA sequencing of human eosinophils from nasal polyps reveals eosinophil heterogeneity in chronic rhinosinusitis tissue.

BACKGROUND: Chronic rhinosinusitis with nasal polyps (CRSwNP) is characterized by type 2 inflammation in the United States, but the actual roles that eosinophils play in CRSwNP remain largely unclear. OBJECTIVE: To reveal the roles and heterogeneity of eosinophils in nasal polyp (NP) tissue, we performed single cell RNA sequencing (scRNA-Seq) analysis of NP tissue. METHODS: Sinonasal tissues (NP and control sinus tissue) and patient matched peripheral blood (PB) samples were obtained from 5 control patients and 5 patients with CRSwNP. Eosinophils were enriched before processing for scRNA-Seq. The gene expression profiles in eosinophils were determined by microwell-based scRNA-Seq technology (BD Rhapsody platform). We predicted the overall function of NP eosinophils by Gene Ontology (geneontology.org) enrichment and pathway analyses and confirmed expression of selected genes by flow cytometry. RESULTS: After filtering out contaminating cells, we detected 5,542 eosinophils from control PB, 3,883 eosinophils from CRSwNP PB, 101 eosinophils from control sinus tissues (not included in further analyses), and 9,727 eosinophils from NPs by scRNA-Seq. We found that 204 genes were downregulated and 354 genes upregulated in NP eosinophils compared to all PB eosinophils (>1.5-fold, Padj < .05). Upregulated genes in NP eosinophils were associated with activation, cytokine-mediated signaling, growth factor activity, NF-κB signaling, and antiapoptotic molecules. NP eosinophils displayed 4 clusters revealing potential heterogeneity of eosinophils in NP tissue. CONCLUSIONS: Elevated eosinophils in NP tissue appear to exist in several subtypes that may play important pathogenic roles in CRSwNP, in part by controlling inflammation and hyperproliferation of other cells.

Self-administered intranasal etripamil using a symptom-prompted, repeat-dose regimen for atrioventricular-nodal-dependent supraventricular tachycardia (RAPID): a multicentre, randomised trial.

BACKGROUND: Etripamil is a fast-acting, intranasally administered calcium-channel blocker in development for on-demand therapy outside a health-care setting for paroxysmal supraventricular tachycardia. We aimed to evaluate the efficacy and safety of etripamil 70 mg nasal spray using a symptom-prompted, repeat-dose regimen for acute conversion of atrioventricular-nodal-dependent paroxysmal supraventricular tachycardia to sinus rhythm within 30 min. METHODS: RAPID was a multicentre, randomised, placebo-controlled, event-driven trial, conducted at 160 sites in North America and Europe as part 2 of the NODE-301 study. Eligible patients were aged at least 18 years and had a history of paroxysmal supraventricular tachycardia with sustained, symptomatic episodes (≥20 min) as documented by electrocardiogram. Patients were administered two test doses of intranasal etripamil (each 70 mg, 10 min apart) during sinus rhythm; those who tolerated the test doses were randomly assigned (1:1) using an interactive response technology system to receive either etripamil or placebo. Prompted by symptoms of paroxysmal supraventricular tachycardia, patients self-administered a first dose of intranasal 70 mg etripamil or placebo and, if symptoms persisted beyond 10 min, a repeat dose. Continuously recorded electrocardiographic data were adjudicated, by individuals masked to patient assignment, for the primary endpoint of time to conversion of paroxysmal supraventricular tachycardia to sinus rhythm for at least 30 s within 30 min after the first dose, which was measured in all patients who administered blinded study drug for a confirmed atrioventricular-nodal-dependent event. Safety outcomes were assessed in all patients who self-administered blinded study drug for an episode of perceived paroxysmal supraventricular tachycardia. This trial is registered at ClinicalTrials.gov, NCT03464019, and is complete. FINDINGS: Between Oct 13, 2020, and July 20, 2022, among 692 patients randomly assigned, 184 (99 from the etripamil group and 85 from the placebo group) self-administered study drug for atrioventricular-nodal-dependent paroxysmal supraventricular tachycardia, with diagnosis and timing confirmed. Kaplan-Meier estimates of conversion rates by 30 min were 64% (63/99) with etripamil and 31% (26/85) with placebo (hazard ratio 2·62; 95% CI 1·66-4·15; p<0·0001). Median time to conversion was 17·2 min (95% CI 13·4-26·5) with the etripamil regimen versus 53·5 min (38·7-87·3) with placebo. Prespecified sensitivity analyses of the primary assessment were conducted to test robustness, yielding supporting results. Treatment-emergent adverse events occurred in 68 (50%) of 99 patients treated with etripamil and 12 (11%) of 85 patients in the placebo group, most of which were located at the administration site and were mild or moderate, and all of which were transient and resolved without intervention. Adverse events occurring in at least 5% of patients treated with etripamil were nasal discomfort (23%), nasal congestion (13%), and rhinorrhea (9%). No serious etripamil-related adverse events or deaths were reported. INTERPRETATION: Using a symptom-prompted, self-administered, initial and optional-repeat-dosing regimen, intranasal etripamil was well tolerated, safe, and superior to placebo for the rapid conversion of atrioventricular-nodal-dependent paroxysmal supraventricular tachycardia to sinus rhythm. This approach could empower patients to treat paroxysmal supraventricular tachycardia themselves outside of a health-care setting, and has the potential to reduce the need for additional medical interventions, such as intravenous medications given in an acute-care setting. FUNDING: Milestone Pharmaceuticals.

Adjuvant Nivolumab versus Placebo in Muscle-Invasive Urothelial Carcinoma.

BACKGROUND: The role of adjuvant treatment in high-risk muscle-invasive urothelial carcinoma after radical surgery is not clear. METHODS: In a phase 3, multicenter, double-blind, randomized, controlled trial, we assigned patients with muscle-invasive urothelial carcinoma who had undergone radical surgery to receive, in a 1:1 ratio, either nivolumab (240 mg intravenously) or placebo every 2 weeks for up to 1 year. Neoadjuvant cisplatin-based chemotherapy before trial entry was allowed. The primary end points were disease-free survival among all the patients (intention-to-treat population) and among patients with a tumor programmed death ligand 1 (PD-L1) expression level of 1% or more. Survival free from recurrence outside the urothelial tract was a secondary end point. RESULTS: A total of 353 patients were assigned to receive nivolumab and 356 to receive placebo. The median disease-free survival in the intention-to-treat population was 20.8 months (95% confidence interval [CI], 16.5 to 27.6) with nivolumab and 10.8 months (95% CI, 8.3 to 13.9) with placebo. The percentage of patients who were alive and disease-free at 6 months was 74.9% with nivolumab and 60.3% with placebo (hazard ratio for disease recurrence or death, 0.70; 98.22% CI, 0.55 to 0.90; P<0.001). Among patients with a PD-L1 expression level of 1% or more, the percentage of patients was 74.5% and 55.7%, respectively (hazard ratio, 0.55; 98.72% CI, 0.35 to 0.85; P<0.001). The median survival free from recurrence outside the urothelial tract in the intention-to-treat population was 22.9 months (95% CI, 19.2 to 33.4) with nivolumab and 13.7 months (95% CI, 8.4 to 20.3) with placebo. The percentage of patients who were alive and free from recurrence outside the urothelial tract at 6 months was 77.0% with nivolumab and 62.7% with placebo (hazard ratio for recurrence outside the urothelial tract or death, 0.72; 95% CI, 0.59 to 0.89). Among patients with a PD-L1 expression level of 1% or more, the percentage of patients was 75.3% and 56.7%, respectively (hazard ratio, 0.55; 95% CI, 0.39 to 0.79). Treatment-related adverse events of grade 3 or higher occurred in 17.9% of the nivolumab group and 7.2% of the placebo group. Two treatment-related deaths due to pneumonitis were noted in the nivolumab group. CONCLUSIONS: In this trial involving patients with high-risk muscle-invasive urothelial carcinoma who had undergone radical surgery, disease-free survival was longer with adjuvant nivolumab than with placebo in the intention-to-treat population and among patients with a PD-L1 expression level of 1% or more. (Funded by Bristol Myers Squibb and Ono Pharmaceutical; CheckMate 274 ClinicalTrials.gov number, NCT02632409.).

Modeling the Evolution of Rates of Continuous Trait Evolution.

Rates of phenotypic evolution vary markedly across the tree of life, from the accelerated evolution apparent in adaptive radiations to the remarkable evolutionary stasis exhibited by so-called "living fossils." Such rate variation has important consequences for large-scale evolutionary dynamics, generating vast disparities in phenotypic diversity across space, time, and taxa. Despite this, most methods for estimating trait evolution rates assume rates vary deterministically with respect to some variable of interest or change infrequently during a clade's history. These assumptions may cause underfitting of trait evolution models and mislead hypothesis testing. Here, we develop a new trait evolution model that allows rates to vary gradually and stochastically across a clade. Further, we extend this model to accommodate generally decreasing or increasing rates over time, allowing for flexible modeling of "early/late bursts" of trait evolution. We implement a Bayesian method, termed "evolving rates" (evorates for short), to efficiently fit this model to comparative data. Through simulation, we demonstrate that evorates can reliably infer both how and in which lineages trait evolution rates varied during a clade's history. We apply this method to body size evolution in cetaceans, recovering substantial support for an overall slowdown in body size evolution over time with recent bursts among some oceanic dolphins and relative stasis among beaked whales of the genus Mesoplodon. These results unify and expand on previous research, demonstrating the empirical utility of evorates. [cetacea; macroevolution; comparative methods; phenotypic diversity; disparity; early burst; late burst].

T cell receptor cross-reactivity between similar foreign and self peptides influences naive cell population size and autoimmunity.

T cell receptor (TCR) cross-reactivity between major histocompatibility complex II (MHCII)-binding self and foreign peptides could influence the naive CD4(+) T cell repertoire and autoimmunity. We found that nonamer peptides that bind to the same MHCII molecule only need to share five amino acids to cross-react on the same TCR. This property was biologically relevant because systemic expression of a self peptide reduced the size of a naive cell population specific for a related foreign peptide by deletion of cells with cross-reactive TCRs. Reciprocally, an incompletely deleted naive T cell population specific for a tissue-restricted self peptide could be triggered by related microbial peptides to cause autoimmunity. Thus, TCR cross-reactivity between similar self and foreign peptides can reduce the size of certain foreign peptide-specific T cell populations and might allow T cell populations specific for tissue-restricted self peptides to cause autoimmunity after infection.

α-Cleavage of 2,2-Diazido-2,3-dihydroinden-1-one in Solution and Cryogenic Matrices.

The Norrish type I (α-cleavage) reaction is an excellent photochemical method for radical-pair formation in solution. However, in cryogenic matrices, the starting material typically re-forms before the radical pair diffuses apart. This study focused on N2 extrusion from an azido alkyl radical to prevent radical-pair recombination. Irradiation of 2,2-diazido-2,3-dihydroinden-1-one (1) in methanol mainly yielded methyl 2-cyanomethylbenzoate (2) and 2-cyanomethylbenzoic acid (3) via α-cleavage. Laser flash photolysis of 1 in argon-saturated acetonitrile resulted in α-cleavage to form triplet biradical 31Br1 (λmax ∼ 410 nm, τ ∼ 400 ns). In contrast, upon irradiation in glassy 2-methyltetrahydrofuran matrices, triplet alkylnitrene 31N was directly detected using electron spin resonance (D/hc = 1.5646 cm-1, E/hc = 0.00161 cm-1) and absorption spectroscopy (λmax = 276 and 341 nm). Irradiation of 1 in argon matrices generated 31N, benzoyl azide 4, singlet benzoylnitrene 14N, and isocyanide 5, as revealed by IR spectroscopy. The experimental results supported by density functional theory calculations [B3PW91/6-311++G(d,p)] suggest that irradiation of 1 in matrices results in α-cleavage to form biradical 31Br1, which extrudes N2 to yield 31Br2. Rearrangement of 31Br2 into 31N competes with cleavage of a N3 radical to form radical 1Ra3. The N3/1Ra3 radical pair combines to form 4, which upon irradiation yields 14N and 5.

The MT1 receptor as the target of ramelteon neuroprotection in ischemic stroke.

Stroke is the leading cause of death and disability worldwide. Novel and effective therapies for ischemic stroke are urgently needed. Here, we report that melatonin receptor 1A (MT1) agonist ramelteon is a neuroprotective drug candidate as demonstrated by comprehensive experimental models of ischemic stroke, including a middle cerebral artery occlusion (MCAO) mouse model of cerebral ischemia in vivo, organotypic hippocampal slice cultures ex vivo, and cultured neurons in vitro; the neuroprotective effects of ramelteon are diminished in MT1-knockout (KO) mice and MT1-KO cultured neurons. For the first time, we report that the MT1 receptor is significantly depleted in the brain of MCAO mice, and ramelteon treatment significantly recovers the brain MT1 losses in MCAO mice, which is further explained by the Connectivity Map L1000 bioinformatic analysis that shows gene-expression signatures of MCAO mice are negatively connected to melatonin receptor agonist like Ramelteon. We demonstrate that ramelteon improves the cerebral blood flow signals in ischemic stroke that is potentially mediated, at least, partly by mechanisms of activating endothelial nitric oxide synthase. Our results also show that the neuroprotection of ramelteon counteracts reactive oxygen species-induced oxidative stress and activates the nuclear factor erythroid 2-related factor 2/heme oxygenase-1 pathway. Ramelteon inhibits the mitochondrial and autophagic death pathways in MCAO mice and cultured neurons, consistent with gene set enrichment analysis from a bioinformatics perspective angle. Our data suggest that Ramelteon is a potential neuroprotective drug candidate, and MT1 is the neuroprotective target for ischemic stroke, which provides new insights into stroke therapy. MT1-KO mice and cultured neurons may provide animal and cellular models of accelerated ischemic damage and neuronal cell death.

Siglec-F Promotes IL-33-Induced Cytokine Release from Bone Marrow-Derived Eosinophils Independently of the ITIM and ITIM-like Motif Phosphorylation.

Eosinophils are potent innate effector cells associated mainly with type 2 immune responses elicited by helminths and allergens. Their activity needs to be tightly controlled to prevent severe inflammation and tissue damage. Eosinophil degranulation and secretion of inflammatory effector molecules, including cytokines, chemokines, and lipid mediators, can be regulated by activating and inhibitory receptors on the cell surface. In this study, we investigated the modulation of proliferation, apoptosis, gene expression, and cytokine/chemokine secretion from IL-33-activated Mus musculus eosinophils on cross-linking of the transmembrane receptor Sialic acid-binding Ig-like lectin F (Siglec-F). Siglec-F contains an ITIM plus an ITIM-like motif in its intracellular tail and is mainly regarded as an inhibitory and apoptosis-inducing receptor. In vitro costimulation of bone marrow-derived eosinophils with anti-Siglec-F and IL-33 compared with treatment with either alone led to enhanced STAT6 phosphorylation, stronger induction of hypoxia/glycolysis-related proinflammatory genes, and elevated secretion of type 2 cytokines (IL-4, IL-13) and chemokines (CCL3, CCL4) with only minor effects on proliferation and apoptosis. Using a competitive mixed bone marrow chimera approach with wild-type and Siglec-F-deficient eosinophils, we observed no evidence for Siglec-F-regulated inhibition of Aspergillus fumigatus-elicited lung eosinophilia. Truncation of the Siglec-F cytoplasmic tail, but not mutation of the ITIM and ITIM-like motifs, ablated the effect of enhanced cytokine/chemokine secretion. This provides evidence for an ITIM phosphorylation-independent signaling pathway from the cytoplasmic tail of the Siglec-F receptor that enhances effector molecule release from activated eosinophils.