The in vitro real-time oscillation monitoring system identifies potential entrainment factors for circadian clocks

BACKGROUND: Circadian rhythms are endogenous, self-sustained oscillations with approximately 24-hr rhythmicity that are manifested in various physiological and metabolic processes. The circadian organization of these processes in mammals is governed by the master oscillator within the suprachiasmatic nuclei (SCN) of the hypothalamus. Recent findings revealed that circadian oscillators exist in most organs, tissues, and even in immortalized cells, and that the oscillators in peripheral tissues are likely to be coordinated by SCN, the master oscillator. Some candidates for endogenous entrainment factors have sporadically been reported, however, their details remain mainly obscure. RESULTS: We developed the in vitro real-time oscillation monitoring system (IV-ROMS) by measuring the activity of luciferase coupled to the oscillatory gene promoter using photomultiplier tubes and applied this system to screen and identify factors able to influence circadian rhythmicity. Using this IV-ROMS as the primary screening of entrainment factors for circadian clocks, we identified 12 candidates as the potential entrainment factor in a total of 299 peptides and bioactive lipids. Among them, four candidates (endothelin-1, all-trans retinoic acid, 9-cis retinoic acid, and 13-cis retinoic acid) have already been reported as the entrainment factors in vivo and in vitro. We demonstrated that one of the novel candidates, 15-deoxy-Δ(12,14)-prostaglandin J(2 )(15d-PGJ(2)), a natural ligand of the peroxisome proliferator-activated receptor-γ (PPAR-γ), triggers the rhythmic expression of endogenous clock genes in NIH3T3 cells. Furthermore, we showed that 15d-PGJ(2 )transiently induces Cry1, Cry2, and Rorα mRNA expressions and that 15d-PGJ(2)-induced entrainment signaling pathway is PPAR-γ – and MAPKs (ERK, JNK, p38MAPK)-independent. CONCLUSION: Here, we identified 15d-PGJ(2 )as an entrainment factor in vitro. Using our developed IV-ROMS to screen 299 compounds, we found eight novel and four known molecules to be potential entrainment factors for circadian clocks, indicating that this assay system is a powerful and useful tool in initial screenings

Asynchronous Rhythm of Steroidogenic Factor 1 and Period Homolog 2 mRNA Expression in Mouse Y1 Adrenocorticol Tumor Cells

The relationship between the expression of Steroidogenic factor 1 (Sf1) and the circadian-related gene, period homolog 2 (Per2), in the adrenal cortex is still unknown. We show here that in Y1 adrenocortical tumor cells, expression of steroidogenic-related genes such as P450scc mRNA and Sf1 mRNA were asynchronous with Per2 mRNA. SF1 promoter analyses showed that the E-box element functions in a rhythmic pattern. Rhythmic expression of Upstream factor 1 mRNA, correlated well with Sf1 mRNA expression. We propose that tumorigenesis of adrenocortical lesions cause disruption of synchronous expression of steroidogenic-related and circadian-related genes

Selective vulnerability of human-induced pluripotent stem cells to dihydroorotate dehydrogenase inhibition during mesenchymal stem/stromal cell purification

間葉系幹細胞から未分化iPS細胞を選択除去する方法を開発: iPS細胞由来間葉系幹細胞の再生医療利用に期待. 京都大学プレスリリース. 2023-03-01.The use of induced mesenchymal stem/stromal cells (iMSCs) derived from human induced pluripotent stem cells (hiPSCs) in regenerative medicine involves the risk of teratoma formation due to hiPSCs contamination in iMSCs. Therefore, eradicating the remaining undifferentiated hiPSCs is crucial for the effectiveness of the strategy. The present study demonstrates the Brequinar (BRQ)-induced inhibition of dihydroorotate dehydrogenase (DHODH), a key enzyme in de novo pyrimidine biosynthesis, selectively induces apoptosis, cell cycle arrest, and differentiation; furthermore, it promotes transcriptional changes and prevents the growth of 3-dimensional hiPSC aggregates. Contrastingly, BRQ-treated iMSCs showed no changes in survival, differentiation potential, or gene expression. The results suggest that BRQ is a potential agent for the effective purification of iMSCs from a mixed population of iMSCs and hiPSCs, which is a crucial step in successful iMSC-based therapy

Measurements of Individual Radiation Doses in Residents Living Around the Fukushima Nuclear Power Plant

At the outset of the accident at Fukushima Daiichi Nuclear Power Plant in March 2011, the radiation doses experienced by residents were calculated from the readings at monitoring posts, with several assumptions being made from the point of view of protection and safety. However, health effects should also be estimated by obtaining measurements of the individual radiation doses. The individual external radiation doses, determined by a behavior survey in the "evacuation and deliberate evacuation area" in the first 4 months, were <5 mSv in 97.4% of residents (maximum: 15 mSv). Doses in Fukushima Prefecture were <3 mSv in 99.3% of 386,572 residents analyzed. External doses in Fukushima City determined by personal dosimeters were <1 mSv/3 months (September-November, 2011) in 99.7% of residents (maximum: 2.7 mSv). Thyroid radiation doses, determined in March using a NaI (TI) scintillation survey meter in children in the evacuation and deliberate evacuation area, were <10 mSv in 95.7% of children (maximum: 35 mSv). Therefore, all doses were less than the intervention level of 50 mSv proposed by international organizations. Internal radiation doses determined by cesium-134 (134C) and cesium-137 ( 137C) whole-body counters (WBCs) were <1 mSv in 99% of the residents, and the maximum thyroid equivalent dose by iodine-131 WBCs was 20 mSv. The exploratory committee of the Fukushima Health Management Survey mentions on its website that radiation from the accident is unlikely to be a cause of adverse health effects in the future. In any event, sincere scientific efforts must continue to obtain individual radiation doses that are as accurate as possible. However, observation of the health effects of the radiation doses described above will require reevaluation of the protocol used for determining adverse health effects. The dose-response relationship is crucial, and the aim of the survey should be to collect sufficient data to confirm the presence or absence of radiation health effects. In particular, the schedule of decontamination needs reconsideration. The decontamination map is determined based on the results of airborne monitoring and the radiation dose calculated from readings taken at the monitoring posts at the initial period of the accident. The decontamination protocol should be reevaluated based on the individual doses of the people who desire to live in those areas

A detection method for latent circadian rhythm sleep-wake disorder

Background Individuals with typical circadian rhythm sleep-wake disorders (CRSWDs) have a habitual sleep timing that is desynchronized from social time schedules. However, it is possible to willfully force synchronisation against circadian-driven sleepiness, which causes other sleep problems. This pathology is distinguishable from typical CRSWDs and is referred to here as latent CRSWD (LCRSWD). Conventional diagnostic methods for typical CRSWDs are insufficient for detecting LCRSWD because sufferers have an apparently normal habitual sleep timing. Methods We first evaluated the reliability of circadian phase estimation based on clock gene expression using hair follicles collected at three time points without sleep interruption. Next, to identify detection criteria for LCRSWD, we compared circadian and sleep parameters according to estimated circadian phases, at the group and individual level, between subjects with low and high Pittsburgh Sleep Quality Index (PSQI) scores. To validate the reliability of identified detection criteria, we investigated whether the same subjects could be reproducibly identified at a later date and whether circadian amelioration resulted in sleep improvement. Findings We successfully validated the reliability of circadian phase estimation at three time points and identified potential detection criteria for individuals with LCRSWD attributed to delayed circadian-driven sleepiness. In particular, a criterion based on the interval between the times of the estimated circadian phase of clock gene expression and getting out of bed on work or school days was promising. We also successfully confirmed the reproducibility of candidate screening and sleep improvement by circadian amelioration, supporting the reliability of the detection criteria. Interpretation Although several limitations remain, our present study demonstrates a promising prototype of a detection method for LCRSWD attributed to delayed circadian-driven sleepiness. More extensive trials are needed to further validate this method

Establishment of a monoclonal antibody for human LXRα: Detection of LXRα protein expression in human macrophages

Liver X activated receptor alpha (LXRα) forms a functional dimeric nuclear receptor with RXR that regulates the metabolism of several important lipids, including cholesterol and bile acids. As compared with RXR, the LXRα protein level in the cell is low and the LXRα protein itself is very hard to detect. We have previously reported that the mRNA for LXRα is highly expressed in human cultured macrophages. In order to confirm the presence of the LXRα protein in the human macrophage, we have established a monoclonal antibody against LXRα, K-8607. The binding of mAb K-8607 to the human LXRα protein was confirmed by a wide variety of different techniques, including immunoblotting, immunohistochemistry, and electrophoretic mobility shift assay (EMSA). By immunoblotting with this antibody, the presence of native LXR protein in primary cultured human macrophage was demonstrated, as was its absence in human monocytes. This monoclonal anti-LXRα antibody should prove to be a useful tool in the analysis of the human LXRα protein

An out-of-lab trial: a case example for the effect of intensive exercise on rhythms of human clock gene expression

Background: Although out-of-lab investigation of the human circadian clock at the clock gene expression level remains difficult, a recent method using hair follicle cells might be useful. While exercise may function as an entrainment cue for circadian rhythms, it remains unclear whether exercise affects human circadian clock gene expression. Methods: Efforts to observe apparent effects of exercise on clock gene expression require that several specific conditions be met: intense exercise should be habitually performed at a relatively uncommon time of day over an extended period; and any relative phase shift thereby observed should be validated by comparison of exercise and no-exercise periods. Wake-up and meal times should be kept almost constant over the experimental period. The present study was conducted using a professional fighter who met these strict criteria as subject. Facial hair samples were collected at 4-h intervals around the clock to ascertain rhythms of clock gene expression. Results: During a period in which nighttime training (from 20:00 to 22:00) was habitually performed, circadian clock gene expression was phase-delayed by 2 to 4 h compared with that during a no-exercise period. Maximum level and circadian amplitude of clock gene expression were not affected by the nighttime training. Conclusion: Our trial observations illustrate the possibility that heavy physical exercise might strongly affect the circadian phase of clock gene expression. Exercise might be therefore effective for the clinical care of circadian disorders. The results also suggest that athletes may require careful scheduling of heavy physical exercise to maintain normal circadian phase and ensure optimal athletic performance

Intermittent parathyroid hormone 1-34 induces oxidation and deterioration of mineral and collagen quality in newly formed mandibular bone

Intermittent parathyroid hormone (PTH) administration is known to promote bone healing after surgical procedures. However, the mechanism and influence of PTH on the mineral and collagen quality of the jaw are not well understood. Most studies have focused on analyzing the bone density and microstructure of the mandible, and have insufficiently investigated its mineral and collagen quality. Oxidative stress activates osteoclasts, produces advanced glycation end products, and worsens mineral and collagen quality. We hypothesized that PTH induces oxidation and affects the mineral and collagen quality of newly formed mandibular bone. To test this, we examined the mineral and collagen quality of newly formed mandibular bone in rats administered PTH, and analyzed serum after intermittent PTH administration to examine the degree of oxidation. PTH administration reduced mineralization and worsened mineral and collagen quality in newly formed bone. In addition, total anti-oxidant capacity in serum was significantly decreased and the oxidative-INDEX was increased among PTH-treated compared to vehicle-treated rats, indicating serum oxidation. In conclusion, intermittent administration of PTH reduced mineral and collagen quality in newly formed mandibular bone. This effect may have been induced by oxidation

Enhanced chondrogenic differentiation of iPS cell-derived mesenchymal stem/stromal cells via neural crest cell induction for hyaline cartilage repair

iPS細胞由来の間葉系幹細胞から高品質な軟骨を作製. 京都大学プレスリリース. 2023-06-08.Generation of high-quality cartilage from iPS cell-derived mesenchymal stem cells. 京都大学プレスリリース. 2023-06-15.Background: To date, there is no effective long-lasting treatment for cartilage tissue repair. Primary chondrocytes and mesenchymal stem/stromal cells are the most commonly used cell sources in regenerative medicine. However, both cell types have limitations, such as dedifferentiation, donor morbidity, and limited expansion. Here, we report a stepwise differentiation method to generate matrix-rich cartilage spheroids from induced pluripotent stem cell-derived mesenchymal stem/stromal cells (iMSCs) via the induction of neural crest cells under xeno-free conditions. Methods: The genes and signaling pathways regulating the chondrogenic susceptibility of iMSCs generated under different conditions were studied. Enhanced chondrogenic differentiation was achieved using a combination of growth factors and small-molecule inducers. Results: We demonstrated that the use of a thienoindazole derivative, TD-198946, synergistically improves chondrogenesis in iMSCs. The proposed strategy produced controlled-size spheroids and increased cartilage extracellular matrix production with no signs of dedifferentiation, fibrotic cartilage formation, or hypertrophy in vivo. Conclusion: These findings provide a novel cell source for stem cell-based cartilage repair. Furthermore, since chondrogenic spheroids have the potential to fuse within a few days, they can be used as building blocks for biofabrication of larger cartilage tissues using technologies such as the Kenzan Bioprinting method