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Neuromedin U Deficiency Disrupts Daily Testosterone Fluctuation and Reduces Wheel-running Activity in Rats
Full text linkThe objective of this study was to elucidate the role of endogenous Neuromedin U (NMU) in rats by performing NMU knockout (KO). Male, but not female NMU KO rats exhibited decreased wheel-running activity vs wildtype (WT), although overall home cage activity was not affected. Plasma testosterone in WT rats varied significantly over the course of a day, with a peak at ZT1 and a nadir at ZT18, whereas in NMU KO rats testosterone remained stable throughout the day. Chronic administration of testosterone restored wheel-running activity in NMU KO rats to the same level as in WT rats, suggesting that the decrease in wheel-running activity in NMU KO rats is due to the disruption of the diurnal change of testosterone. Accordingly, expression of the luteinizing hormone beta subunit (Lhb) mRNA in the pars distalis of anterior pituitary was significantly lower in NMU KO rats; immunostaining revealed that the size of luteinizing hormone (LH)–expressing cells was also relatively small in those animals. In the brain of male WT rats, Nmu was highly expressed in the pars tuberalis, and the NMU receptor Nmur2 was highly expressed in the ependymal cell layer of the third ventricle. This study reveals a novel function of NMU and indicates that endogenous NMU in rats plays a role in the regulation of motivated activity via regulation of testosterone
A repertoire of visible light–sensitive opsins in the deep-sea hydrothermal vent shrimp Rimicaris hybisae
Full text linkUnlike terrestrial environments, where humans reside, there is no sunlight in the deep sea. Instead, dim visible light from black-body radiation and bioluminescence illuminates hydrothermal vent areas in the deep sea. A deep-sea hydrothermal vent shrimp, Rimicaris hybisae, is thought to detect this dim light using its enlarged dorsal eye; however, the molecular basis of its photoreception remains unexplored. Here, we characterized the molecular properties of opsins, universal photoreceptive proteins in animals, found in R. hybisae. Transcriptomic analysis identified six opsins: three Gq-coupled opsins, one Opn3, one Opn5, and one peropsin. Functional analysis revealed that five of these opsins exhibited light-dependent G protein activity, whereas peropsin exhibited the ability to convert all-trans-retinal to 11-cis-retinal like photoisomerases. Notably, all the R. hybisae opsins, including Opn5, convergently show visible light sensitivity (around 457–517 nm), whereas most opsins categorized as Opn5 have been demonstrated to be UV sensitive. Mutational analysis revealed that the unique visible light sensitivity of R. hybisae Opn5 is achieved through the stabilization of a protonated Schiff base by a counterion residue at position 83 (Asp83), which differs from the position identified in other opsins. These findings suggest that the vent shrimp R. hybisae has adapted its photoreceptive devices to dim deep-sea hydrothermal light by selectively maintaining a repertoire of visible light–sensitive opsins, including the uniquely tuned Opn5
Photochemical internalization of mRNA using a photosensitizer and nucleic acid carriers
Full text linkmRNA has great potential for therapeutic applications because it can encode a variety of proteins and antigens, in addition to advantages over DNA in terms of gene expression without genomic integration, nuclear localization, or transcription. However, therapeutic applications of mRNA require safe and effective delivery into target cells. Therefore, we aimed to investigate photochemical internalization (PCI) as a promising strategy for delivering mRNA to target cells. In this strategy, mRNA is taken up into cells by endocytosis, accumulates in endosomes, and is released in a light-dependent manner from the endosomes using an endosome-accumulating photosensitizer, aluminum phthalocyanine disulfonate (AlPcS2a), in combination with nucleic acid carrier molecules. We compared the efficacy of various nucleic acid carriers, including branched polyethyleneimine (bPEI) and poly{N'-[N-(2-aminoethyl)-2-aminoethyl] aspartamide} (PAsp(DET)) under the same conditions for PCI-based mRNA delivery. Our results indicated that bPEI and PAsp(DET) at low N/P ratios exhibited efficient light-enhancement of mRNA expression by PCI with AlPcS2a. Notably, bPEI exhibited the highest light-dependent mRNA delivery among the carriers evaluated (including cationic polymers, cationic peptides, and lipids), whereas PAsp(DET) showed promise for clinical use because of its lower toxicity compared with bPEI. This PCI strategy allows effective cytosolic mRNA delivery at low N/P ratios, thereby reducing cationic carrier molecule-induced cytotoxicity. This method allows spatiotemporal control of protein expression and holds potential for novel light-dependent mRNA therapies. Overall, this study provided valuable insights into optimizing mRNA delivery systems for therapeutic applications
Neurotransmitter and Receptor Mapping in Drosophila Circadian Clock Neurons via T2A-GAL4 Screening
Full text linkThe circadian neuronal network in the brain comprises central pacemaker neurons and associated input and output pathways. These components work together to generate coherent rhythmicity, synchronize with environmental time cues, and convey circadian information to downstream neurons that regulate behaviors such as the sleep/wake cycle. To mediate these functions, neurotransmitters and neuromodulators play essential roles in transmitting and modulating signals between neurons. In Drosophila melanogaster, approximately 240 brain neurons function as clock neurons. Previous studies have identified several neurotransmitters and neuromodulators, including the Pigment-dispersing factor (PDF) neuropeptide, along with their corresponding receptors in clock neurons. However, our understanding of the neurotransmitters and receptors involved in the circadian system remains incomplete. In this study, we conducted a T2A-GAL4-based screening for neurotransmitter and receptor genes expressed in clock neurons. We identified 2 neurotransmitter-related genes and 22 receptor genes. Notably, while previous studies had reported the expression of 6 neuropeptide receptor genes in large ventrolateral neurons (l-LNv), we also found that 14 receptor genes—including those for dopamine, serotonin, and γ-aminobutyric acid—are expressed in l-LNv neurons. These findings suggest that l-LNv neurons serve as key integrative hubs within the circadian network, receiving diverse external signals
Tumor Microvessels with Specific Morphology as a Prognostic Factor in Esophageal Squamous Cell Carcinoma
Full text linkBackground Angiogenesis is essential for tumor progression. Microvessel density (MVD) is a widely used histological method to assess angiogenesis using immunostained sections, but its prognostic significance in esophageal cancer remains controversial. Recently, the evaluation of microvascular architecture has gained importance as a method to assess tumor aggressiveness. The present study aimed to identify the histological characteristics of tumor microvessels that are associated with the aggressiveness of esophageal squamous cell carcinoma.
Patients and Methods A total of 108 esophageal squamous cell carcinoma tissues were immunohistochemically stained with blood vessel markers and angiogenesis-related markers, including CD31, alpha smooth muscle actin, vascular endothelial growth factor A (VEGF-A), CD206, and D2-40. MVD, microvessel pericyte coverage index (MPI), and tumor vascular morphology were evaluated by microscopy.
Results MVD was significantly associated with patient outcomes, whereas neither MPI nor VEGF-A expression throughout the tumor showed a significant correlation. In addition, the presence of blood vessels encircling clusters of tumor cells, termed C-shaped microvessels, and excessively branching microvessels, termed X-shaped microvessels, was significantly associated with poor prognosis. These vessel types were also correlated with clinicopathological parameters, including deeper invasion of the primary tumor, presence of lymph node metastasis, advanced pathological stage, and distant metastasis. Focal VEGF-A immunoexpression in tumor cells was higher in areas containing C-shaped or X-shaped microvessels compared with areas lacking these vessel morphologies.
Conclusions The data suggest that tumor microvessels with specific morphologies (C-shaped and X-shaped microvessels) may serve as a promising prognostic factor in esophageal squamous cell carcinoma
Automated identification of the origin of energy loss in nonoriented electrical steel by feature extended Ginzburg–Landau free energy framework
Full text linkThis study presents the automated identification of the complex magnetization reversal process in nonoriented electrical steel (NOES) using the feature extended Ginzburg–Landau (eX-GL) free energy framework. eX-GL provides a robust connection between microscopic magnetic domains and macroscopic magnetic hysteresis using a data science perspective. This method employs physically meaningful features to analyze the energy landscape, providing insights into the mechanisms behind function. We obtained features representing both the microstructure and energy of the domain wall. The causes of iron loss were traced to the original domain structure, through which we could successfully distinguish and visualize the role of pinning as a promoting and resisting factor. We found that the reversal process was governed not only by general grain boundary pinning but also by segmented magnetic domains within the grain. This method revealed the complex interplay between magnetism and metallography and introduced a new means for transformative material design, bridging structures and functions
Longitudinal changes and tracking of in-school physical activity in primary school children: four-year longitudinal study
Full text linkBackground There is little evidence on the tracking of physical activity during school hours. In addition, tracking physical activity in schools provides important evidence for understanding children’s physical activity and conducting intervention studies. Therefore, this study examined longitudinal changes and tracking of in-school physical activity in primary school children.
Methods In this study, physical activity was investigated longitudinally in primary school children for 4 years. The baseline participants consisted of 103 second-grade students (7–8 years old) who participated. Step counts and moderate-to-vigorous physical activity (MVPA) in school and during first recess and lunch/second recess were examined using an accelerometer (Kenz Lifecorder GS 4-second version; Suzuken Co. Ltd, Nagoya, Japan).
Results After excluding missing data (moving school; n = 8, physical activity; n = 8), 87 (43 boys and 44 girls) of whom were included in the final analysis. Step counts and MVPA during school and physical education in boys did not decrease across the school years. By contrast, in girls, step counts during school did not decrease across the school years, however MVPA did decrease. In addition, for both sexes, step counts and MVPA during first recess decrease across the school years. During lunch/second recess, only step counts decrease across the school years in both sexes. In addition, the tracking coefficients for step counts and MVPA for boys in school and during first recess and lunch/second recess were found across many school years. Contrarily, girls had fewer significant tracking coefficients between school years than boys. There were also few significant tracking coefficients between grades for physical education step counts and MVPA for both boys and girls.
Conclusions Our results suggested that in-school step counts for both boys and girls does not decrease across the school years. However, given that girls demonstrated reduced levels of in-school MVPA across the school years, it is important to promote strategies to increase MVPA in this group
An ultra-simplified protocol for PCR template preparation from both unsporulated and sporulated Eimeria oocysts
Full text linkMolecular biological techniques have enabled the accurate identification of the avian Eimeria parasite, however, the preparation of PCR template remains a bottleneck due to contaminants from feces and the robust oocyst's wall resistant to chemical and mechanical force. Generally, the preparation of PCR template involves three main steps: (1) pretreatment of oocysts; (2) disruption of oocysts; and (3) purification of genomic DNA. We prepared PCR templates from both unsporulated and sporulated E. tenella oocysts using various protocols, followed by species-specific PCR to define the limit of detection. Our data revealed that whereas neither pretreatment of oocysts with sodium hypochlorite nor purification of genomic DNA with commercial kits improved the limit of detection of PCR, disruption of oocysts was a critical step in the preparation of PCR templates. The most sensitive PCR assay was achieved with the template prepared by disrupting oocysts suspended in distilled water, followed by bead-beating and heating at 99°C for 5 min, which detected 0.16 oocysts per PCR. This ultra-simplified protocol for preparation of PCR template, which does not require expensive reagents or equipment, will significantly enhance the sensitive and efficient molecular identification of Eimeria. It will improve our understanding of the prevalence of this parasite at the species level and contribute to the development of techniques for the control in the field