Ceratinadins E and F, New Bromotyrosine Alkaloids from an Okinawan Marine Sponge Pseudoceratina sp.

Two new bromotyrosine alkaloids, ceratinadins E (1) and F (2), were isolated from an Okinawan marine sponge Pseudoceratina sp. as well as a known bromotyrosine alkaloid, psammaplysin F (3). The gross structures of 1 and 2 were elucidated on the basis of spectroscopic data. The absolute configurations of 1 and 2 were assigned by comparison of the NMR and ECD data with those of a known related bromotyrosine alkaloid, psammaplysin A (4). Ceratinadins E (1) and F (2) are new bromotyrosine alkaloids possessing an 8,10-dibromo-9-methoxy-1,6-dioxa-2-azaspiro[4.6]undeca-2,7,9-trien-4-ol unit with two or three 11-N-methylmoloka’iamine units connected by carbonyl groups, respectively. Ceratinadin E (1) exhibited antimalarial activities against a drug-resistant and a drug-sensitive strains of Plasmodium falciparum (K1 and FCR3 strains, respectively)

Mammalian reverse genetics without crossing reveals Nr3a as a short-sleeper gene

The identification of molecular networks at the system level in mammals is accelerated by next-generation mammalian genetics without crossing, which requires both the efficient production of whole-body biallelic knockout (KO) mice in a single generation and high-performance phenotype analyses. Here, we show that the triple targeting of a single gene using the CRISPR/Cas9 system achieves almost perfect KO efficiency (96%–100%). In addition, we developed a respiration-based fully automated noninvasive sleep phenotyping system, the Snappy Sleep Stager (SSS), for high-performance (95.3% accuracy) sleep/wake staging. Using the triple-target CRISPR and SSS in tandem, we reliably obtained sleep/wake phenotypes, even in double-KO mice. By using this system to comprehensively analyze all of the N-methyl-D-aspartate (NMDA) receptor family members, we found Nr3a as a short-sleeper gene, which is verified by an independent set of triple-target CRISPR. These results demonstrate the application of mammalian reverse genetics without crossing to organism-level systems biology in sleep research

Involvement of Ca2+-dependent hyperpolarization in sleep duration in mammals

The detailed molecular mechanisms underlying the regulation of sleep duration in mammals are still elusive. To address this challenge, we constructed a simple computational model, which recapitulates the electrophysiological characteristics of the slow-wave sleep and awake states. Comprehensive bifurcation analysis predicted that a Ca2+-dependent hyperpolarization pathway may play a role in slow-wave sleep and hence in the regulation of sleep duration. To experimentally validate the prediction, we generate and analyze 21 KO mice. Here we found that impaired Ca2+-dependent K+ channels (Kcnn2 and Kcnn3), voltage-gated Ca2+ channels (Cacna1g and Cacna1h), or Ca2+/calmodulin-dependent kinases (Camk2a and Camk2b) decrease sleep duration, while impaired plasma membrane Ca2+ ATPase (Atp2b3) increases sleep duration. Pharmacological intervention and whole-brain imaging validated that impaired NMDA receptors reduce sleep duration and directly increase the excitability of cells. Based on these results, we propose a hypothesis that a Ca2+-dependent hyperpolarization pathway underlies the regulation of sleep duration in mammals

Robust sleep induction by CaMKIIβ T287D mutant.

(A) Expression levels of endogenous CaMKIIβ and AAV-mediated transduced CaMKIIβ in the brain. Camk2bFLAG/FLAG represents homo knock-in mice in which the FLAG tag was inserted into the endogenous Camk2b locus. PBS: PBS-administrated mice. Immunoblotting against FLAG-tagged protein indicates that AAV-mediated expression of CaMKIIβ is lower than the expression level of endogenous CaMKIIβ. (B) Calculated transduction efficiency plotted against sleep duration. Transduction efficiency is an estimation of the number of AAV vector genomes present per cell in a mouse brain. After the SSS measurements, we purified the AAV vector genomes from the mice brains and then quantified them with a WPRE-specific primer set and normalized to mouse genomes. (C) Sleep transition profiles of mice expressing CaMKIIβ T287-related mutants shown in Fig 1F. The shaded areas represent SEM. (D) Sleep parameters during light or dark period of mice expressing CaMKIIβ T287-related mutants shown in Fig 1F. Multiple comparison tests were performed between all individual groups in each phase. (E, F) Sleep/wake parameters of mice expressing S114-related CaMKIIβ mutants (C) and S109-related CaMKIIβ mutants (D), averaged over 6 days. The shaded areas represent SEM. Multiple comparison tests were performed between all individual groups and resulted in no significant differences. The underlying numerical data can be found in S1 Data, and uncropped or raw image files for S3A Fig are provided in S2 and S3 Data files. Error bars: SEM, *p p p (PDF)</p

Time-of-day analyses for sleep parameters of mice with perturbed CaMKII activity.

(A) Sleep transition profiles of mice expressing the CaMKIIβ del mutant under hSyn1 promoter shown in Fig 2B and 2C. The shaded areas represent SEM. (B) Sleep parameters of mice expressing the CaMKIIβ del mutants shown in Fig 2B and 2C during light or dark period. Multiple comparison tests were performed between all individual groups in each phase. (C) Sleep transition profiles of mice expressing AIP2 or RARA mutant under hSyn1 promoter shown in Fig 2E and 2F. The shaded areas represent SEM. PBS: PBS-injected mice (n = 6). (D) Sleep parameters of mice expressing AIP2 or RARA mutant shown in Fig 2E and 2F during light or dark period. Multiple comparison tests were performed between all individual groups in each phase. The underlying data can be found in S1 Data. Error bars: SEM, *p p p hSyn1, human synapsin-1; (PDF)</p

Expression of the CaMKIIβ throughout the brain by AAV-PHP.eB.

Volume-rendered and single-plane images of the brain expressing H2B-mCherry under hSyn1 promoter by the AAV (mCherry, green) counterstained with RD2 (red). A volume-rendered image is shown in the center. Single-plane and magnified images are shown for cerebral cortex, thalamus, hippocampus, midbrain, cerebellum, striatum, and olfactory bulb. Scale bar in the center image, 3 mm; other scale bars, 100 μm. AAV, adeno-associated virus; CaMKIIβ, calmodulin-dependent protein kinase IIβ; hSyn1, human synapsin-1. (TIFF)</p