A case of hypertensive emergency with alveolar hemorrhage and thrombotic microangiopathy.

A 28-year-old woman with a 5-year history of untreated hypertension was admitted for respiratory distress, hemoptysis, and retinopathy. Computed tomography showed diffuse plaques in both lung fields. Acute kidney injury, hemolytic anemia, and thrombocytopenia were noted. Kidney biopsy showed thrombosis with fibrinoid necrosis and edematous intimal thickening and luminal narrowing of the small renal artery, indicating thrombotic microangiopathy; the majority of glomeruli were collapsed. After 8 weeks of treatment with antihypertensive drugs, serum creatinine decreased to 1.0 mg/dL, and the patient recovered. In the absence of any other underlying disease, malignant nephrosclerosis associated with a hypertensive emergency was diagnosed.

Inter-individual variations in circadian misalignment-induced NAFLD pathophysiology in mice.

Pathological consequences of circadian misalignment, such as shift work, show considerable individual differences, but the lack of mechanistic understanding hinders precision prevention to prevent and mitigate disease symptoms. Here, we employed an integrative approach involving physiological, transcriptional, and histological phenotypes to examine inter-individual differences in pre-symptomatic pathological progression, preceding irreversible disease onset, in wild-type mice exposed to chronic jet-lag (CJL). We observed that CJL markedly increased the prevalence of hepatic steatosis with pronounced inter-individual differences. Stratification of individual mice based on CJL-induced hepatic transcriptomic signature, validated by histopathological analysis, pinpoints dysregulation of lipid metabolism. Moreover, the period and power of intrinsic behavioral rhythms were found to significantly correlate with CJL-induced gene signatures. Together, our results suggest circadian rhythm robustness of the animals contributes to inter-individual variations in pathogenesis of circadian misalignment-induced diseases and raise the possibility that these physiological indicators may be available for predictive hallmarks of circadian rhythm disorders.

A Case of Glomerular Microangiopathy-like Nephropathy Caused by Hyperthyroidism.

A 49-year-old Japanese woman was admitted to our hospital with weight loss of 15 kg, nephrotic-range proteinuria (4.5 g/g.Cre), and hematuria over a 6-month period. She had received two doses of the COVID-19 vaccine one year before the onset of the disease, after which the estimated glomerular filtration rate increased. Laboratory tests and other tests led to a diagnosis of hyperthyroidism, and a kidney biopsy showed thrombotic microangiopathy-like glomerular microangiopathy comprising mainly glomerular endothelial cell damage. Thiamazole (30 mg) was started for the hyperthyroidism. Three months later, the thyroid function normalized, and two months later, the proteinuria and hematuria disappeared, suggesting that COVID-19 vaccination and these events were related.

Comprehensive Analysis Identified the Circadian Clock and Global Circadian Gene Expression in Human Corneal Endothelial Cells.

Purpose: To investigate circadian clock oscillation and circadian global gene expression in cultured human corneal endothelial cells (cHCECs) to elucidate and assess the potential function of circadian regulation in HCECs. Methods: In this study, we introduced a circadian bioluminescence reporter, Bmal1:luciferase (Bmal1:luc), into cHCECs and subsequently monitored real-time bioluminescence rhythms. RNA-sequencing data analysis was then performed using sequential time-course samples of the cHCECs to obtain a comprehensive understanding of the circadian gene expression rhythms. The potential relevance of rhythmically expressed genes was then assessed by systematic approaches using functional clustering and individual gene annotations. Results: Bmal1:luc bioluminescence exhibited clear circadian oscillation in the cHCECs. The core clock genes and clock-related genes showed high-amplitude robust circadian messenger RNA (mRNA) expression rhythms in cHCECs after treatment with dexamethasone, and 329 genes that exhibited circadian mRNA expression rhythms were identified (i.e., genes involved in various physiological processes including glycolysis, mitochondrial function, antioxidative systems, hypoxic responses, apoptosis, and extracellular matrix regulation, which represent the physiological functions of HCECs). Conclusions: Our findings revealed that cHCECs have a robust and functional circadian clock, and our discovery that a large number of genes exhibit circadian mRNA expression rhythms in cHCECs suggests a potential contribution of circadian regulation to fine-tune HCEC functions for daily changes in the environment.

Circadian key component CLOCK/BMAL1 interferes with segmentation clock in mouse embryonic organoids.

In mammals, circadian clocks are strictly suppressed during early embryonic stages, as well as in pluripotent stem cells, by the lack of CLOCK/BMAL1-mediated circadian feedback loops. During ontogenesis, the innate circadian clocks emerge gradually at a late developmental stage, and with these, the circadian temporal order is invested in each cell level throughout a body. Meanwhile, in the early developmental stage, a segmented body plan is essential for an intact developmental process, and somitogenesis is controlled by another cell-autonomous oscillator, the segmentation clock, in the posterior presomitic mesoderm (PSM). In the present study, focusing upon the interaction between circadian key components and the segmentation clock, we investigated the effect of the CLOCK/BMAL1 on the segmentation clock Hes7 oscillation, revealing that the expression of functional CLOCK/BMAL1 severely interferes with the ultradian rhythm of segmentation clock in induced PSM and gastruloids. RNA sequencing analysis implied that the premature expression of CLOCK/BMAL1 affects the Hes7 transcription and its regulatory pathways. These results suggest that the suppression of CLOCK/BMAL1-mediated transcriptional regulation during the somitogenesis may be inevitable for intact mammalian development.

Quantitative morphometric analysis of molar teeth and alveolar bone using micro-computed tomography in aged mice.

OBJECTIVES: Irreversible morphological regressions of the teeth or related structures in older people can diminish their overall health. However, research on human aging in dentistry is complicated by several confounding factors. In this study, we conducted a morphometric analysis of the mandibular second molars and surrounding alveolar bone in C57BL/6 mice to evaluate age-related changes in the oral cavity. METHODS: The animals were divided into five groups based on their age: 4 weeks (juvenile mice; n = 5); 20 weeks (n = 7), 50 weeks (n = 5), 77 weeks (n = 7), and 100 weeks (n = 5); changes were evaluated using micro-computed tomography. RESULTS: The molars of juvenile mice had sharp and pointed cusps and presented maximum heights. With age and occlusal wear, the cusp heights demonstrated a significant decrease (up to 75%) until the last stage of life. Conversely, apparent lesions were not observed on the basal portion of the crown, even in the most heavily worn teeth. The roots of the molars continued to grow in length at 4 weeks of age. Alveolar bone resorption begins to occur in middle age and continues throughout life. The proportion of vertical bone loss reached approximately 40% of the entire root length, demonstrating a remarkable increase between weeks 77 and 100. CONCLUSIONS: Overall, these morphological changes were similar to those observed in humans. Therefore, it might be appropriate to use aged mice as an experimental model for basic and clinical research in geriatric dentistry.

Successful Aortic Valve Replacement With Perceval Bioprosthesis for Aortic Stenosis With Membranous Ventricular Septum Aneurysm.

Membranous ventricular septum aneurysm (MVSA) is extremely rare, especially when coexisting with aortic stenosis (AS), and reports regarding the available treatment for MVSA with AS are limited. Aortic valve replacement (AVR) can be challenging because of anatomical reasons. In this case report, a patient with MVSA and severe AS was treated with AVR with the sutureless Perceval bioprosthesis. After implantation, no paravalvular leakage was detected in echocardiography, and no other postoperative complications were observed. Postoperative electrocardiography-gated computed tomography revealed no contrast enhancement for MVSA. The MVSA was closed by the Perceval bioprosthetic valve. Thus, patients with simultaneous MVSA and AS may be effectively treated with AVR using a Perceval bioprosthesis.

Circadian clock and cancer: From a viewpoint of cellular differentiation.

The circadian clock controls and adapts diverse physiological and behavioral processes according to Earth's 24-h cycle of environmental changes. The master pacemaker of the mammalian circadian clock resides in the hypothalamic suprachiasmatic nucleus, but almost all cells throughout the body show circadian oscillations in gene expression patterns and associated functions. Recent studies have shown that the circadian clock gradually develops during embryogenesis. Embryonic stem cells and induced pluripotent stem cells do not show circadian oscillations of gene expression, but gradually develop circadian clock oscillation during differentiation; thus, the developmental program of circadian clock emergence appears closely associated with cellular differentiation. Like embryonic stem cells, certain cancer cell types also lack the circadian clock. Given this similarity between embryonic stem cells and cancer cells, interest is growing in the contributions of circadian clock dysfunction to dedifferentiation and cancer development. In this review, we summarize recent advances in our understanding of circadian clock emergence during ontogenesis, and discuss possible associations with cellular differentiation and carcinogenesis. Considering the multiple physiological functions of circadian rhythms, circadian abnormalities might contribute to a host of diseases, including cancer. Insights on circadian function could lead to the identification of biomarkers for cancer diagnosis and prognosis, as well as novel targets for treatment.

Chronic circadian misalignment accelerates immune senescence and abbreviates lifespan in mice.

Modern society characterized by a 24/7 lifestyle leads to misalignment between environmental cycles and endogenous circadian rhythms. Persisting circadian misalignment leads to deleterious effects on health and healthspan. However, the underlying mechanism remains not fully understood. Here, we subjected adult, wild-type mice to distinct chronic jet-lag paradigms, which showed that long-term circadian misalignment induced significant early mortality. Non-biased RNA sequencing analysis using liver and kidney showed marked activation of gene regulatory pathways associated with the immune system and immune disease in both organs. In accordance, we observed enhanced steatohepatitis with infiltration of inflammatory cells. The investigation of senescence-associated immune cell subsets from the spleens and mesenteric lymph nodes revealed an increase in PD-1+CD44high CD4 T cells as well as CD95+GL7+ germinal center B cells, indicating that the long-term circadian misalignment exacerbates immune senescence and consequent chronic inflammation. Our results underscore immune homeostasis as a pivotal interventional target against clock-related disorders.

Enhanced metastatic growth after local tumor resection in the presence of synchronous metastasis in a mouse allograft model of neuroblastoma.

PURPOSE: We investigated how local tumor resection affects metastatic lesions in neuroblastoma. METHODS: MYCN Tg tumor-derived cells were injected subcutaneously into 129+Ter/SvJcl wild-type mice. First, the frequency of metastasis-bearing mice was investigated immunohistochemically (metastatic ratio) at endpoint or post-injection day (PID) 90. Second, the threshold volume of local tumor in mice bearing microscopic lymph node metastasis (mLNM) was investigated at PID 30. Finally, local tumors were resected after exceeding the threshold. Mice were divided into local tumor resection (Resection) and observation (Observation) groups, and the metastatic ratio and volume of LNM were compared between the groups at endpoint or PID 74. RESULTS: The metastatic ratio without local resection was 88% at PID 78-90. The threshold local tumor volume in the mice with mLNM was 745 mm3 at PID 30, so local tumors were resected after exceeding 700 mm3. The metastatic ratio and LNM volume were significantly greater in the Resection group (n = 16) than in the Observation group (n = 16) (94% vs. 38%, p < 0.001; 2092 ± 2310 vs. 275 ± 218 mm3, p < 0.01; respectively) at PID 50-74. CONCLUSION: Local tumor resection might augment the growth of synchronous microscopic metastases. Our results provide insights into the appropriate timing of local resection for high-risk neuroblastoma.

Human Circadian Molecular Oscillation Development Using Induced Pluripotent Stem Cells.

The mammalian circadian clock, which coordinates various physiological functions, develops gradually during ontogeny. Recently, we have reported the posttranscriptional suppression of CLOCK protein expression as a key mechanism of the emergence of the circadian clock during mouse development. However, whether a common mechanism regulates the development of the human circadian clock remains unclear. In the present study, we show that human induced pluripotent stem cells (iPSCs) have no discernible circadian molecular oscillation. In addition, in vitro differentiation culture of human iPSCs required a longer duration than that required in mouse for the emergence of circadian oscillations. The expression of CLOCK protein in undifferentiated human iPSCs was posttranscriptionally suppressed despite the expression of CLOCK mRNA, which is consistent with our previous observations in mouse embryonic stem cells, iPSCs, and early mouse embryos. These results suggest that CLOCK protein expressions could be posttranscriptionally suppressed in the early developmental stage not only in mice but also in humans.

REV-ERBα and REV-ERBß function as key factors regulating Mammalian Circadian Output.

The circadian clock regulates behavioural and physiological processes in a 24-h cycle. The nuclear receptors REV-ERBα and REV-ERBß are involved in the cell-autonomous circadian transcriptional/translational feedback loops as transcriptional repressors. A number of studies have also demonstrated a pivotal role of REV-ERBs in regulation of metabolic, neuronal, and inflammatory functions including bile acid metabolism, lipid metabolism, and production of inflammatory cytokines. Given the multifunctional role of REV-ERBs, it is important to elucidate the mechanism through which REV-ERBs exert their functions. To this end, we established a Rev-erbα/Rev-erbß double-knockout mouse embryonic stem (ES) cell model and analyzed the circadian clock and clock-controlled output gene expressions. A comprehensive mRNA-seq analysis revealed that the double knockout of both Rev-erbα and Rev-erbß does not abrogate expression rhythms of E-box-regulated core clock genes but drastically changes a diverse set of other rhythmically-expressed output genes. Of note, REV-ERBα/ß deficiency does not compromise circadian expression rhythms of PER2, while REV-ERB target genes, Bmal1 and Npas2, are significantly upregulated. This study highlight the relevance of REV-ERBs as pivotal output mediators of the mammalian circadian clock.

Cell-based screen identifies a new potent and highly selective CK2 inhibitor for modulation of circadian rhythms and cancer cell growth.

Compounds targeting the circadian clock have been identified as potential treatments for clock-related diseases, including cancer. Our cell-based phenotypic screen revealed uncharacterized clock-modulating compounds. Through affinity-based target deconvolution, we identified GO289, which strongly lengthened circadian period, as a potent and selective inhibitor of CK2. Phosphoproteomics identified multiple phosphorylation sites inhibited by GO289 on clock proteins, including PER2 S693. Furthermore, GO289 exhibited cell type-dependent inhibition of cancer cell growth that correlated with cellular clock function. The x-ray crystal structure of the CK2α-GO289 complex revealed critical interactions between GO289 and CK2-specific residues and no direct interaction of GO289 with the hinge region that is highly conserved among kinases. The discovery of GO289 provides a direct link between the circadian clock and cancer regulation and reveals unique design principles underlying kinase selectivity.

Incremental Growth Lines in Mouse Molar Dentin Represent 8-hr Ultradian Rhythm.

Rhythmic incremental growth lines occur in dental hard tissues of vertebrates, and dentinogenesis in rodent incisors is suggested to be controlled by the 24-hr circadian clock. Rodent incisors continue to grow throughout the animal's life; however, similar to human teeth, rodent molars stop growing after crown formation. This similarity suggests that the mouse molar is an excellent model to understand the molecular mechanisms underlying growth of human teeth. However, not much is known about the rhythmic dentinogenesis in mouse molars. Here, we investigated the incremental growth lines in mouse molar dentin using tetracycline as the growth marker. The incremental growth lines were observed to be generated at approximately 8-hr intervals in wild-type mice housed under 12:12 hr light-dark conditions. Moreover, the 8-hr rhythmic increments persisted in the wild-type and Bmal1-/- mice housed in constant darkness, where Bmal1-/- mice become behaviorally arrhythmic. These results revealed that the dentinogenesis in mouse molars underlie the ultradian rhythms with around 8-hr periodicity. Further, the circadian clock does not seem to be involved in this process, providing new insight into the mechanisms involved in the tooth growth.

Disruption of circadian clockwork in in vivo reprogramming-induced mouse kidney tumors.

The circadian clock, which regulates cellular physiology, such as energy metabolism, resides in each cell level throughout the body. Recently, it has been elucidated that the cellular circadian clock is closely linked with cellular differentiation. Moreover, the misregulation of cellular differentiation in mouse embryonic stem cells (ESCs) induced abnormally differentiated cells with impaired circadian clock oscillation, concomitant with the post-transcriptional suppression of CLOCK proteins. Here, we show that the circadian molecular oscillation is disrupted in dysdifferentiation-mediated mouse kidney tumors induced by partial in vivo reprogramming, resembling Wilms tumors. The expression of CLOCK protein was dramatically reduced in the tumor cells despite the Clock mRNA expression. We also showed that a similar loss of CLOCK was observed in human Wilms tumors, suggesting that the circadian molecular clockwork may be disrupted in dysdifferentiation-mediated embryonal tumors such as Wilms tumors, similar to the in vivo reprogramming-induced mouse kidney tumors. These results support our previous reports and may provide a novel viewpoint for understanding the pathophysiological nature of cancers through the correlation between cellular differentiation and circadian clock.

Period2 3'-UTR and microRNA-24 regulate circadian rhythms by repressing PERIOD2 protein accumulation.

We previously created two PER2::LUCIFERASE (PER2::LUC) circadian reporter knockin mice that differ only in the Per2 3'-UTR region: Per2::Luc, which retains the endogenous Per2 3'-UTR and Per2::LucSV, where the endogenous Per2 3'-UTR was replaced by an SV40 late poly(A) signal. To delineate the in vivo functions of Per2 3'-UTR, we analyzed circadian rhythms of Per2::LucSV mice. Interestingly, Per2::LucSV mice displayed more than threefold stronger amplitude in bioluminescence rhythms than Per2::Luc mice, and also exhibited lengthened free-running periods (∼24.0 h), greater phase delays following light pulse, and enhanced temperature compensation relative to Per2::Luc Analysis of the Per2 3'-UTR sequence revealed that miR-24, and to a lesser degree miR-30, suppressed PER2 protein translation, and the reversal of this inhibition in Per2::LucSV augmented PER2::LUC protein level and oscillatory amplitude. Interestingly, Bmal1 mRNA and protein oscillatory amplitude as well as CRY1 protein oscillation were increased in Per2::LucSV mice, suggesting rhythmic overexpression of PER2 enhances expression of Per2 and other core clock genes. Together, these studies provide important mechanistic insights into the regulatory roles of Per2 3'-UTR, miR-24, and PER2 in Per2 expression and core clock function.

Robust circadian clock oscillation and osmotic rhythms in inner medulla reflecting cortico-medullary osmotic gradient rhythm in rodent kidney.

Circadian clocks in mammals function in most organs and tissues throughout the body. Various renal functions such as the glomerular filtration and excretion of electrolytes exhibit circadian rhythms. Although it has been reported that the expression of the clock genes composing molecular oscillators show apparent daily rhythms in rodent kidneys, functional variations of regional clocks are not yet fully understood. In this study, using macroscopic bioluminescence imaging method of the PER2::Luciferase knock-in mouse kidney, we reveal that strong and robust circadian clock oscillation is observed in the medulla. In addition, the osmotic pressure in the inner medulla shows apparent daily fluctuation, but not in the cortex. Quantitative-PCR analysis of the genes contributing to the generation of high osmotic pressure or the water re-absorption in the inner medulla, such as vasopressin receptors (V1aR, V2R), urea transporter (UT-A2) and water channel (Aqp2) show diurnal variations as well as clock genes. Deficiency of an essential clock gene Bmal1 impairs day-night variations of osmotic pressure gradient in the inner medulla, suggesting that circadian clocks in the medulla part of the kidney may regulate the circadian rhythm of cortico-medullary osmotic pressure gradient, and may contribute physiological day-night rhythm of urination.

Involvement of posttranscriptional regulation of Clock in the emergence of circadian clock oscillation during mouse development.

Circadian clock oscillation emerges in mouse embryo in the later developmental stages. Although circadian clock development is closely correlated with cellular differentiation, the mechanisms of its emergence during mammalian development are not well understood. Here, we demonstrate an essential role of the posttranscriptional regulation of Clock subsequent to the cellular differentiation for the emergence of circadian clock oscillation in mouse fetal hearts and mouse embryonic stem cells (ESCs). In mouse fetal hearts, no apparent oscillation of cell-autonomous molecular clock was detectable around E10, whereas oscillation was clearly visible in E18 hearts. Temporal RNA-sequencing analysis using mouse fetal hearts reveals many fewer rhythmic genes in E10-12 hearts (63, no core circadian genes) than in E17-19 hearts (483 genes), suggesting the lack of functional circadian transcriptional/translational feedback loops (TTFLs) of core circadian genes in E10 mouse fetal hearts. In both ESCs and E10 embryos, CLOCK protein was absent despite the expression of Clock mRNA, which we showed was due to Dicer/Dgcr8-dependent translational suppression of CLOCK. The CLOCK protein is required for the discernible molecular oscillation in differentiated cells, and the posttranscriptional regulation of Clock plays a role in setting the timing for the emergence of the circadian clock oscillation during mammalian development.

Effect of Multiple Clock Gene Ablations on the Circadian Period Length and Temperature Compensation in Mammalian Cells.

Most organisms have cell-autonomous circadian clocks to coordinate their activity and physiology according to 24-h environmental changes. Despite recent progress in circadian studies, it is not fully understood how the period length and the robustness of mammalian circadian rhythms are determined. In this study, we established a series of mouse embryonic stem cell (ESC) lines with single or multiplex clock gene ablations using the CRISPR/Cas9-based genome editing method. ESC-based in vitro circadian clock formation assay shows that the CRISPR-mediated clock gene disruption not only reproduces the intrinsic circadian molecular rhythms of previously reported mice tissues and cells lacking clock genes but also reveals that complexed mutations, such as CKIδ(m/m):CKIε(+/m):Cry2(m/m) mutants, exhibit an additively lengthened circadian period. By using these mutant cells, we also investigated the relation between period length alteration and temperature compensation. Although CKIδ-deficient cells slightly affected the temperature insensitivity of period length, we demonstrated that the temperature compensation property is largely maintained in all mutants. These results show that the ESC-based assay system could offer a more systematic and comprehensive approach to the genotype-chronotype analysis of the intracellular circadian clockwork in mammals.

Disruption of MeCP2 attenuates circadian rhythm in CRISPR/Cas9-based Rett syndrome model mouse.

Methyl-CpG-binding protein 2 (Mecp2) is an X-linked gene encoding a methylated DNA-binding nuclear protein which regulates transcriptional activity. The mutation of MECP2 in humans is associated with Rett syndrome (RTT), a neurodevelopmental disorder. Patients with RTT frequently show abnormal sleep patterns and sleep-associated problems, in addition to autistic symptoms, raising the possibility of circadian clock dysfunction in RTT. In this study, we investigated circadian clock function in Mecp2-deficient mice. We successfully generated both male and female Mecp2-deficient mice on the wild-type C57BL/6 background and PER2(Luciferase) (PER2(Luc)) knock-in background using the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system. Generated Mecp2-deficient mice recapitulated reduced activity in mouse models of RTT, and their activity rhythms were diminished in constant dark conditions. Furthermore, real-time bioluminescence imaging showed that the amplitude of PER2(Luc)-driven circadian oscillation was significantly attenuated in Mecp2-deficient SCN neurons. On the other hand, in vitro circadian rhythm development assay using Mecp2-deficient mouse embryonic stem cells (ESCs) did not show amplitude changes of PER2(Luc) bioluminescence rhythms. Together, these results show that Mecp2 deficiency abrogates the circadian pacemaking ability of the SCN, which may be a therapeutic target to treat the sleep problems of patients with RTT.