Distinct phosphorylation states of mammalian CaMKIIß control the induction and maintenance of sleep.

The reduced sleep duration previously observed in Camk2b knockout mice revealed a role for Ca2+/calmodulin-dependent protein kinase II (CaMKII)ß as a sleep-promoting kinase. However, the underlying mechanism by which CaMKIIß supports sleep regulation is largely unknown. Here, we demonstrate that activation or inhibition of CaMKIIß can increase or decrease sleep duration in mice by almost 2-fold, supporting the role of CaMKIIß as a core sleep regulator in mammals. Importantly, we show that this sleep regulation depends on the kinase activity of CaMKIIß. A CaMKIIß mutant mimicking the constitutive-active (auto)phosphorylation state promotes the transition from awake state to sleep state, while mutants mimicking subsequent multisite (auto)phosphorylation states suppress the transition from sleep state to awake state. These results suggest that the phosphorylation states of CaMKIIß differently control sleep induction and maintenance processes, leading us to propose a "phosphorylation hypothesis of sleep" for the molecular control of sleep in mammals.

A Novel Three-Dimensional Imaging System Based on Polysaccharide Staining for Accurate Histopathological Diagnosis of Inflammatory Bowel Diseases.

BACKGROUND & AIMS: Tissue-clearing and three-dimensional (3D) imaging techniques aid clinical histopathological evaluation; however, further methodological developments are required before use in clinical practice. METHODS: We sought to develop a novel fluorescence staining method based on the classical periodic acid-Schiff stain. We further attempted to develop a 3D imaging system based on this staining method and evaluated whether the system can be used for quantitative 3D pathological evaluation and deep learning-based automatic diagnosis of inflammatory bowel diseases. RESULTS: We successfully developed a novel periodic acid-FAM hydrazide (PAFhy) staining method for 3D imaging when combined with a tissue-clearing technique (PAFhy-3D). This strategy enabled clear and detailed imaging of the 3D architectures of crypts in human colorectal mucosa. PAFhy-3D imaging also revealed abnormal architectural changes in crypts in ulcerative colitis tissues and identified the distributions of neutrophils in cryptitis and crypt abscesses. PAFhy-3D revealed novel pathological findings including spiral staircase-like crypts specific to inflammatory bowel diseases. Quantitative analysis of crypts based on 3D morphologic changes enabled differential diagnosis of ulcerative colitis, Crohn's disease, and non-inflammatory bowel disease; such discrimination could not be achieved by pathologists. Furthermore, a deep learning-based system using PAFhy-3D images was used to distinguish these diseases The accuracies were excellent (macro-average area under the curve = 0.94; F1 scores = 0.875 for ulcerative colitis, 0.717 for Crohn's disease, and 0.819 for non-inflammatory bowel disease). CONCLUSIONS: PAFhy staining and PAFhy-3D imaging are promising approaches for next-generation experimental and clinical histopathology.