Melatonin regulates diurnal changes in locomotor activity in vertebrates, but the molecular mechanism for this neurohormonal regulation of behavior is poorly understood. Here we show that 7␣-hydroxypregnenolone, a previously undescribed avian neurosteroid, mediates melatonin action on diurnal locomotor rhythms in quail. In this study, we first identified 7␣-hydroxypregnenolone and its stereoisomer 7␤-hydroxypregnenolone in quail brain. These neurosteroids have not been described previously in avian brain. We then demonstrated that 7␣-hydroxypregnenolone acutely increased quail locomotor activity. To analyze the production of 7␣-hydroxypregnenolone, cytochrome P450 7␣ , a steroidogenic enzyme of this neurosteroid, was also identified. Subsequently, we demonstrated diurnal changes in 7␣-hydroxypregnenolone synthesis in quail. 7␣-Hydroxypregnenolone synthesis and locomotor activity in males were much higher than in females. This is the first demonstration in any vertebrate of a clear sex difference in neurosteroid synthesis. This sex difference in 7␣-hydroxypregnenolone synthesis corresponded to the sex difference in locomotion. We show that only males exhibited marked diurnal changes in 7␣-hydroxypregnenolone synthesis, and these changes occurred in parallel with changes in locomotor activity. Finally, we identified melatonin as a key component of the mechanism regulating 7␣-hydroxypregnenolone synthesis. Increased synthesis of 7␣-hydroxypregnenolone occurred in males in vivo after melatonin removal via pinealectomy and orbital enucleation (Px plus Ex). Conversely, decreased synthesis of this neurosteroid occurred after melatonin administration to Px plus Ex males. This study demonstrates that melatonin regulates synthesis of 7␣-hydroxypregnenolone, a key factor for induction of locomotor activity, thus inducing diurnal locomotor changes in male birds. This is a previously undescribed role for melatonin.

Many enveloped viruses induce multinucleated cells (syncytia), reflective of membrane fusion events caused by the same machinery that underlies viral entry. These syncytia are thought to facilitate replication and evasion of the host immune response. Here, we report that co-culture of human cells expressing the receptor ACE2 with cells expressing SARS-CoV-2 spike, results in synapse-like intercellular contacts that initiate cell-cell fusion, producing syncytia resembling those we identify in lungs of COVID-19 patients. To assess the mechanism of spike/ACE2-driven membrane fusion, we developed a microscopy-based, cell-cell fusion assay to screen ~6000 drugs and >30 spike variants. Together with quantitative cell biology approaches, the screen reveals an essential role for biophysical aspects of the membrane, particularly cholesterol-rich regions, in spike-mediated fusion, which extends to replication-competent SARS-CoV-2 isolates. Our findings potentially provide a molecular basis for positive outcomes reported in COVID-19 patients taking statins and suggest new strategies for therapeutics targeting the membrane of SARS-CoV-2 and other fusogenic viruses.

In late 2022, the SARS-CoV-2 Omicron subvariants have highly diversified, and XBB is spreading rapidly around the world. Our phylogenetic analyses suggested that XBB emerged by recombination of two co-circulating BA.2 lineages, BJ.1 and BM.1.1.1 (a progeny of BA.2.75), during the summer of 2022 around India. In vitro experiments revealed that XBB is the most profoundly resistant variant to BA.2/5 breakthrough infection sera ever and is more fusogenic than BA.2.75. Notably, the recombination breakpoint is located in the receptor-binding domain of spike, and each region of recombined spike conferred immune evasion and augmented fusogenicity to the XBB spike. Finally, the intrinsic pathogenicity of XBB in hamsters is comparable to or even lower than that of BA.2.75. Our multiscale investigation provided evidence suggesting that XBB is the first documented SARS-CoV-2 variant increasing its fitness through recombination rather than single mutations.

The activation of tumor suppressor p53 induces apoptosis or cell cycle arrest depending on the state and type of cell, but it is not fully understood how these different responses are regulated. Here, we show that Puma and Noxa, the well-known p53-inducible proapoptotic members of the Bcl-2 family, differentially participate in dual pathways of the induction of apoptosis. In normal cells, Puma but not Noxa induces mitochondrial outer membrane permeabilization (MOMP), and this function is mediated in part by a pathway that involves calcium release from the endoplasmic reticulum (ER) and the subsequent caspase activation. However, upon E1A oncoprotein expression, cells also become susceptible to MOMP induction by Noxa, owing to their sensitization to the ER-independent pathway. These findings offer a new insight into differential cellular responses induced by p53, and may have therapeutic implications in cancer.

The PIWI-interacting RNA (piRNA) pathway preserves genomic integrity by repressing transposable elements (TEs) in animal germ cells. Among PIWI-clade proteins in Drosophila, Piwi transcriptionally silences its targets through interactions with cofactors, including Panoramix (Panx) and forms heterochromatin characterized by H3K9me3 and H1. Here, we identified Nxf2, a nuclear RNA export factor (NXF) variant, as a protein that forms complexes with Piwi, Panx, and p15. Panx-Nxf2-P15 complex formation is necessary in the silencing by stabilizing protein levels of Nxf2 and Panx. Notably, ectopic targeting of Nxf2 initiates co-transcriptional repression of the target reporter in a manner independent of H3K9me3 marks or H1. However, continuous silencing requires HP1a and H1. In addition, Nxf2 directly interacts with target TE transcripts in a Piwidependent manner. These findings suggest a model in which the Panx-Nxf2-P15 complex enforces the association of Piwi with target transcripts to trigger co-transcriptional repression, prior to heterochromatin formation in the nuclear piRNA pathway. Our results provide an unexpected connection between an NXF variant and small RNA-mediated co-transcriptional silencing. The EMBO JournalKensaku Murano et al F Density plots for normalized H3K9me3 ChIP-seq signals over the consensus sequence from mdg1, gypsy (targeted by Piwi-piRNA, orange), and roo (not targeted by Piwi-piRNA, blue) TEs in EGFP-, Piwi-, Panx-, and Nxf2-KD OSCs. G Boxplots as in (D) showing fold changes in the H3K9me3 levels of Piwi-piRNA-targeted and un-targeted TEs upon the indicated KD. H Northern blotting for Idefix-piRNA, traffic jam (tj)-piRNA, and esiRNA sl-1 (control) on total RNA isolated from OSCs with the indicated KD.

1The PIWI-interacting RNA (piRNA) pathway preserves genomic integrity by 2 repressing transposable elements (TEs) in animal germ cells. Among PIWI-clade 3 proteins in Drosophila, Piwi transcriptionally silences its targets through interactions 4 with cofactors, including Panoramix (Panx) and forms heterochromatin characterized by 5H3K9me3 and H1. Here, we identified Nxf2, a nuclear RNA export factor (NXF) 6 variant, as a protein that forms complexes with Piwi, Panx, and p15. Panx-Nxf2-p15 7 complex formation is necessary in the silencing by stabilizing protein levels of Nxf2 8 and Panx. Notably, ectopic targeting of Nxf2 initiates co-transcriptional repression of 9 the target reporter in a manner independent of H3K9me3 marks or H1. However, 10 continuous silencing requires HP1a and H1. In addition, Nxf2 directly interacts with 11 target TE transcripts in a Piwi-dependent manner. These findings suggest a model in 12 which the Nxf2-Panx-p15 complex enforces the association of Piwi with target 13

Superoxide dismutase 1 (SOD1) is an important antioxidative enzyme that protects skin from oxidative stress. SOD1 (-/-) mice with a genetic background of b129Sv mice showed facial skin damage after 15 weeks of age. Eyelid swelling occurred as the initial symptom and caused impairment by triggering self-scratching. The period required for wound healing in the back was markedly delayed in 20-week SOD1 (-/-) mice. Oxidative stress markers, 4-hydroxynonenal and thiobarbituric acid-reactive substances, were unexpectedly lower in SOD1 (-/-) mice at day 1 after wounding. The decay rate of electron paramagnetic resonance signal intensity of intravenously injected nitroxide radical indicated that the half-life of the signal intensity was significantly prolonged in the wounded skin of SOD1 (+/+) mice. However, while the half-life of the signal intensity in control skin was a little longer in SOD1 (-/-) mice, it did not change in wounded skin. Taken together, these data suggest that the skin of SOD1 (-/-) mice is in redox imbalance and prone to damage by wounding.

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