Cellular DBP and E4BP4 proteins are critical for determining the period length of the circadian oscillator

AbstractThe phenotypes of mice carrying clock gene mutations have been critical to understanding the mammalian clock function. However, behavior does not necessarily reflect cell-autonomous clock phenotypes, because of the hierarchical dominance of the central clock. We performed cell-based siRNA knockdown and cDNA overexpression and monitored rhythm using bioluminescent reporters of clock genes. We found that knockdown of DBP, D-box positive regulator, in our model led to a short-period phenotype, whereas overexpressing of DBP produced a long-period rhythm when compared to controls. Furthermore, knockdown and overexpressing of E4BP4, D-box negative regulator, led to an opposite effect of DBP. Our experiments demonstrated that D-box regulators play a crucial role in determining the period length of Per1 and Per2 promoter-driven circadian rhythms in Rat-1 fibroblasts

Cytokeratin 13, Cytokeratin 17, and Ki-67 Expression in Human Acquired Cholesteatoma and Their Correlation With Its Destructive Capacity

Objectives Cholesteatoma is a nonneoplastic destructive lesion of the temporal bone with debated pathogenesis and bone resorptive mechanism. Both molecular and cellular events chiefly master its activity. Continued research is necessary to clarify factors related to its aggressiveness. We aimed to investigate the expression of Ki-67, cytokeratin 13 (CK13) and cytokeratin 17 (CK17) in acquired nonrecurrent human cholesteatoma and correlate them with its bone destructive capacity. Methods A prospective quantitative immunohistochemical study was carried out using fresh acquired cholesteatoma tissues (n=19), collected during cholesteatoma surgery. Deep meatal skin tissues from the same patients were used as control (n=8). Cholesteatoma patients were divided into 2 groups and compared (invasive and noninvasive) according to a grading score for bone resorption based upon clinical, radiologic and intraoperative findings. To our knowledge, the role of CK17 in cholesteatoma aggressiveness was first investigated in this paper. Results Both Ki-67 and CK17 were significantly overexpressed in cholesteatoma than control tissues (P<0.001 for both Ki-67 and CK17). In addition, Ki-67 and CK17 were significantly higher in the invasive group than noninvasive group of cholesteatoma (P=0.029, P=0.033, respectively). Furthermore, Ki-67 and CK17 showed a moderate positive correlation with bone erosion scores (r=0.547, P=0.015 and r=0.588, P=0.008, respectively). In terms of CK13, no significant difference was found between cholesteatoma and skin (P=0.766). Conclusion Both Ki-67 and CK17 were overexpressed in cholesteatoma tissue and positively correlated with bone resorption activity. The concept that Ki-67 can be a predictor for aggressiveness of cholesteatoma was supported. In addition, this is the first study demonstrating CK17 as a favoring marker in the aggressiveness of acquired cholesteatoma

Identification of functional clock-controlled elements involved in differential timing of Per1 and Per2 transcription

It has been proposed that robust rhythmic gene expression requires clock-controlled elements (CCEs). Transcription of Per1 was reported to be regulated by the E-box and D-box in conventional reporter assays. However, such experiments are inconclusive in terms of how the CCEs and their combinations determine the phase of the Per1 gene. Whereas the phase of Per2 oscillation was found to be the most delayed among the three Period genes, the phase-delaying regions of the Per2 promoter remain to be determined. We therefore investigated the regulatory mechanism of circadian Per1 and Per2 transcription using an in vitro rhythm oscillation-monitoring system. We found that the copy number of the E-box might play an important role in determining the phase of Per1 oscillation. Based on real-time bioluminescence assays with various promoter constructs, we provide evidence that the non-canonical E-box is involved in the phase delay of Per2 oscillation. Transfection experiments confirmed that the non-canonical E-box could be activated by CLOCK/BMAL1. We also show that the D-box in the third conserved segment of the Per2 promoter generated high amplitude. Our experiments demonstrate that the copy number and various combinations of functional CCEs ultimately led to different circadian phases and amplitudes

Replication of Epstein-Barr Virus Primary Infection in Human Tonsil Tissue Explants

Epstein-Barr virus (EBV) may cause a variety of virus-associated diseases, but no antiviral agents have yet been developed against this virus. Animal models are thus indispensable for the pathological analysis of EBV-related infections and the elucidation of therapeutic methods. To establish a model system for the study of EBV infection, we tested the ability of B95–8 virus and recombinant EBV expressing enhanced green fluorescent protein (EGFP) to replicate in human lymphoid tissue. Human tonsil tissues that had been surgically removed during routine tonsillectomy were sectioned into small blocks and placed on top of collagen sponge gels in culture medium at the air-interface, then a cell-free viral suspension was directly applied to the top of each tissue block. Increasing levels of EBV DNA in culture medium were observed after 12–15 days through 24 days post-infection in tissue models infected with B95–8 and EGFP-EBV. Expression levels of eight EBV-associated genes in cells collected from culture medium were increased during culture. EBV-encoded small RNA-positive cells were detected in the interfollicular areas in paraffin-embedded sections. Flow cytometric analyses revealed that most EGFP+ cells were CD3− CD56− CD19+ HLA-DR+, and represented both naïve (immunoglobulin D+) and memory (CD27+) B cells. Moreover, EBV replication in this model was suppressed by acyclovir treatment in a dose-dependent manner. These data suggest that this model has potential for use in the pathological analysis of local tissues at the time of primary infection, as well as for screening novel antiviral agents

Geographic Information System-Based Analysis of Reclaimable Idle Cropland for Agrivoltaics in Kansai, Japan: Enhancing Energy and Food Security

Decarbonization and idle cropland reclaiming pose critical policy challenges. Agrivoltaic systems (AVSs), which merge agriculture and photovoltaics, offer a promising solution by reducing land use conflicts between agriculture and energy production. This study develops a GIS-based methodology to identify reclaimable idle croplands and assess the AVS’s contribution to agriculture and photovoltaic energy in Japan’s Kansai region. Using official geographic data and excluding high-risk areas, this study employs GIS for quantitative analysis. Our method detected more reclaimable idle cropland than previous studies. Focusing on food security while limiting AVS installations to reclaimable idle croplands could still generate up to 4564.08 GWh of electricity (0.8% of regional consumption) and 930.82 tons of soybeans (6.2% of regional yield). Under a more stringent scenario that excludes areas less suitable for solar power, 5 of 227 municipalities could achieve 15% electricity self-sufficiency through AVS. This research uncovers the existence of reclaimable idle croplands that could not be detected by existing methods and a decentralized power source available alongside food security maintenance. These insights will inform decarbonization and agricultural policy and guide the development of supportive and regulatory AVS frameworks

Integrating Agrivoltaic Systems into Local Industries: A Case Study and Economic Analysis of Rural Japan

The growing number of photovoltaic installations has created competition in land use between the need for electricity and food. Agrivoltaic systems (AVSs) can help solve this problem by increasing land use efficiency through the co-production of electricity and food. However, in Japan, where more than 2000 AVSs have been installed, some undesirable AVS cases have led to new problems. In this study, we developed an AVS installation model that is compatible with a regional society and limits the scale of AVS installation to a low-risk level. AVS projects have also entered local industrial clusters and stimulated the local economy. In this study, we used public information and geographic information systems to ensure quantifiability and applicability. The results revealed that the rural area targeted in this study had an AVS generation potential of 215% (equal to 17.8 GWh) of the region’s annual electricity consumption and an economic ripple effect of 108.9% (EUR 47.8 million) of the region’s gross regional product. Furthermore, the levelized cost of electricity was estimated to be 14.94–25.54 Euro cents/kWh under secure settings. This study provides solutions to food, economic, and energy problems in rural areas by promoting the installation of AVSs

Mechanisms Underlying Improvement in Obstructive Sleep Apnea Syndrome by Uvulopalatopharyngoplasty

In a previous case report, we determined for the first time that uvulopalatopharyngoplasty (UPPP) does not change the volume of the upper airway but causes morphological changes in the entire upper airway. The objective of this study is to elucidate the mechanisms underlying the improvement in obstructive sleep apnea syndrome (OSAS) by UPPP. We present an additional case involving a patient with OSAS treated using UPPP. Morphological and numerical parameter changes after surgery were compared with the corresponding preoperative values. Anatomically accurate upper airway computational models were reconstructed from computed tomographic imaging data. In addition, computed fluid dynamics analysis was performed to reveal inhalation flow characteristics before and after UPPP and clearly assess the effect of UPPP on airflow patterns in the patient’s upper airway. An important benefit of UPPP is the morphological changes in the entire upper airway, in addition to widening the restricted area. These morphological changes induce laminarization of the pharyngeal jet. To obtain sufficient efficacy of UPPP in OSAS, the morphological changes in the upper airway and the airflow pattern after the surgery must be controlled