The Hexane Extract of Citrus sphaerocarpa Ameliorates Visceral Adiposity by Regulating the PI3K/AKT/FoxO1 and AMPK/ACC Signaling Pathways in High-Fat-Diet-Induced Obese Mice.

Obesity is an emerging global health issue with an increasing risk of disease linked to lifestyle choices. Previously, we reported that the hexane extract of Citrus sphaerocarpa (CSHE) suppressed lipid accumulation in differentiated 3T3-L1 adipocytes. In this study, we conducted in vivo experiments to assess whether CSHE suppressed obesity in zebrafish and mouse models. We administered 10 and 20 µg/mL CSHE to obese zebrafish juveniles. CSHE significantly inhibited visceral fat accumulation compared to untreated obese fish. Moreover, the oral administration (100 µg/g body weight/day) of CSHE to high-fat-diet-induced obese mice significantly reduced their body weight, visceral fat volume, and hepatic lipid accumulation. The expression analyses of key regulatory genes involved in lipid metabolism revealed that CSHE upregulated the mRNA expression of lipolysis-related genes in the mouse liver (Pparα and Acox1) and downregulated lipogenesis-related gene (Fasn) expression in epididymal white adipose tissue (eWAT). Fluorescence immunostaining demonstrated the CSHE-mediated enhanced phosphorylation of AKT, AMPK, ACC, and FoxO1, which are crucial factors regulating adipogenesis. CSHE-treated differentiated 3T3L1 adipocytes also exhibited an increased phosphorylation of ACC. Therefore, we propose that CSHE suppresses adipogenesis and enhances lipolysis by regulating the PI3K/AKT/FoxO1 and AMPK/ACC signaling pathways. These findings suggested that CSHE is a promising novel preventive and therapeutic agent for managing obesity.

Reorganization of the DNA replication landscape during adipogenesis is closely linked with adipogenic gene expression.

The temporal order of DNA replication along the chromosomes is thought to reflect the transcriptional competence of the genome. During differentiation of mouse 3T3-L1 cells into adipocytes, cells undergo one or two rounds of cell division called mitotic clonal expansion (MCE). MCE is an essential step for adipogenesis; however, little is known about the regulation of DNA replication during this period. Here, we performed genome-wide mapping of replication timing (RT) in mouse 3T3-L1 cells before and during MCE, and identified a number of chromosomal regions shifting toward either earlier or later replication through two rounds of replication. These RT changes were confirmed in individual cells by single-cell DNA-replication sequencing. Coordinate changes between a shift toward earlier replication and transcriptional activation of adipogenesis-associated genes were observed. RT changes occurred before the full expression of these genes, indicating that RT reorganization might contribute to the mature adipocyte phenotype. To support this, cells undergoing two rounds of DNA replication during MCE had a higher potential to differentiate into lipid droplet-accumulating adipocytes, compared with cells undergoing a single round of DNA replication and non-replicating cells.

Camptothecin-Induced Replication Stress Affects DNA Replication Profiling by E/L Repli-Seq.

E/L Repli-seq is a powerful tool for detecting cell type-specific replication landscapes in mammalian cells, but its potential to monitor DNA replication under replication stress awaits better understanding. Here, we used E/L Repli-seq to examine the temporal order of DNA replication in human retinal pigment epithelium cells treated with the topoisomerase I inhibitor camptothecin. We found that the replication profiles by E/L Repli-seq exhibit characteristic patterns after replication-stress induction, including the loss of specific initiation zones within individual early replication timing domains. We also observed global disappearance of the replication timing domain structures in the profiles, which can be explained by checkpoint-dependent suppression of replication initiation. Thus, our results demonstrate the effectiveness of E/L Repli-seq at identifying cells with replication-stress-induced altered DNA replication programs.

The Temporal Order of DNA Replication Shaped by Mammalian DNA Methyltransferases.

Multiple epigenetic pathways underlie the temporal order of DNA replication (replication timing) in the contexts of development and disease. DNA methylation by DNA methyltransferases (Dnmts) and downstream chromatin reorganization and transcriptional changes are thought to impact DNA replication, yet this remains to be comprehensively tested. Using cell-based and genome-wide approaches to measure replication timing, we identified a number of genomic regions undergoing subtle but reproducible replication timing changes in various Dnmt-mutant mouse embryonic stem (ES) cell lines that included a cell line with a drug-inducible Dnmt3a2 expression system. Replication timing within pericentromeric heterochromatin (PH) was shown to be correlated with redistribution of H3K27me3 induced by DNA hypomethylation: Later replicating PH coincided with H3K27me3-enriched regions. In contrast, this relationship with H3K27me3 was not evident within chromosomal arm regions undergoing either early-to-late (EtoL) or late-to-early (LtoE) switching of replication timing upon loss of the Dnmts. Interestingly, Dnmt-sensitive transcriptional up- and downregulation frequently coincided with earlier and later shifts in replication timing of the chromosomal arm regions, respectively. Our study revealed the previously unrecognized complex and diverse effects of the Dnmts loss on the mammalian DNA replication landscape.

Mechanism of aspirin-induced inhibition on the secondary hyperalgesia in osteoarthritis model rats.

AIMS: The daily activity of osteoarthritis (OA) patients is limited by chronic pain and central sensitization. Although non-steroidal anti-inflammatory drugs (NSAIDs) and acetaminophen are the first-line drugs for the treatment of OA-related pain, their efficacy on central sensitization remains unclear. In the present study, we evaluated the effect of acetylsalicylic acid (ASA, Aspirin) using an OA model induced by monosodium iodoacetate (MIA), which has a similar disease progression to human OA. MAIN METHODS: Secondary hyperalgesia was assessed at the plantar surface of the hind paw by Von Frey test. We evaluated the expression of acid-sensing ion channel 3 (ASIC3) in dorsal root ganglia and that of tumor necrosis factor-α (TNF-α) and interleukin-1ß (IL-1ß) in the spinal cord, which may cause secondary hyperalgesia in OA, by immunohistochemical analysis and real-time qPCR. KEY FINDINGS: The administration of ASA attenuated secondary hyperalgesia at 1-3 weeks after MIA, while celecoxib, a selective cyclooxygenase (COX)-2 inhibitor, failed to attenuate secondary hyperalgesia at week 2 after MIA injection, suggesting that ASA exerts its analgesic effect through a COX-2-independent pathway. Immunohistochemical analysis of the dorsal root ganglia indicated that ASA reduced the expression of ASIC3 during OA progression. Expression of TNF-α mRNA, but not IL-1ß mRNA, in the spinal cord following MIA injection was suppressed by ASA administration. SIGNIFICANCE: These findings suggest that ASA may have the ability to attenuate secondary hyperalgesia through suppression of ASIC3 and/or TNF-α expression. ASA is therefore a clinically useful analgesic drug for treatment of secondary hyperalgesia in OA.

A thin layer of sucrose octasulfate protects the oesophageal mucosal epithelium in reflux oesophagitis.

Sucralfate is effective for the treatment of gastric and duodenal ulcers owing to its protective gel-forming ability. However, the mechanism by which sucralfate protects the oesophageal mucosa against reflux oesophagitis has not been clarified. We aimed to investigate the mechanisms of action of sucralfate and sucrose octasulfate (SOS), a component of sucralfate. SOS and sucralfate were administered to oesophagitis-induced rats, and the ulcer lesion size was macroscopically examined and scored. Effective pepsin activity in the gastric juices obtained from the animal model was evaluated by a casein digestion test. Sucralfate and SOS improved the pathology scores in a dose-dependent manner, whereas gastric juice pepsin activity was not impaired by therapeutic doses of SOS. As SOS lacks the ability to form a thick gel layer by polymerisation, we examined the distribution of SOS in the mucosal lumen by imaging mass spectrometry (IMS) to determine whether SOS directly adheres to the mucosal surface. A clear homogeneous thin-layer SOS film (>100 µm thick) was visualized on the oesophageal mucosal surface. Moreover, this SOS film formation was enhanced at ulcer lesion sites. Taken together, SOS appears to protect oesophageal mucosa against reflux oesophagitis via thin-layer formation on the mucosal surface.

SPECT/CT of lung nodules using 111In-DOTA-c(RGDfK) in a mouse lung carcinogenesis model.

OBJECTIVE: Lung cancer is one of the leading causes of cancer-related deaths worldwide, including Japan. Although computed tomography (CT) can detect small lung lesions such as those appearing as ground glass opacity, it cannot differentiate between malignant and non-malignant lesions. Previously, we have shown that single photon emission computed tomography (SPECT) imaging using (111)In-1,4,7,10-tetraazacyclododecane-N,N',N'',N'''-tetraacetic acid-cyclo-(Arg-Gly-Asp-D-Phe-Lys) (DOTA-c(RGDfK)), an imaging probe of αvß3 integrin, is useful for the early detection of pancreatic cancer in a hamster pancreatic carcinogenesis model. In this study, we aimed to assess the usefulness of SPECT/CT with (111)In-DOTA-c(RGDfK) for the evaluation of the malignancy of lung cancer. METHODS: Lung tumors were induced by a single intraperitoneal injection (250 mg/kg) of urethane in male A/J mice. Twenty-six weeks after the urethane treatment, SPECT was performed an hour after injection of (111)In-DOTA-c(RGDfK). Following this, the radioactivity ratios of tumor to normal lung tissue were measured by autoradiography (ARG) in the excised lung samples. We also examined the expression of αvß3 integrin in mouse and human lung samples. RESULTS: Urethane treatment induced 5 hyperplasias, 41 adenomas and 12 adenocarcinomas in the lungs of 8 A/J mice. SPECT with (111)In-DOTA-c(RGDfK) could clearly visualize lung nodules, though we failed to detect small lung nodules like adenoma and hyperplasias (adenocarcinoma: 66.7%, adenoma: 33.6%, hyperplasia: 0.0%). ARG analysis revealed significant uptake of (111)In-DOTA-c(RGDfK) in all the lesions. Moreover, tumor to normal lung tissue ratios increased along with the progression of carcinogenesis. Histopathological examination using human lung tissue samples revealed clear up-regulation of αvß3 integrin in well-differentiated adenocarcinoma (Noguchi type B and C) rather than atypical adenomatous hyperplasia. CONCLUSION: Although there are some limitations in evaluating the malignancy of small lung tumors using (111)In-DOTA-c(RGDfK), SPECT with (111)In-DOTA-c(RGDfK) might be a useful non-invasive imaging approach for evaluating the characteristics of lung tumors in mice, thus showing potential for use in humans.

Syntheses of 2-deoxy-2,3-didehydro-N-acetylneuraminic acid analogues modified by α-acylaminoamido groups at the C-4 position using isocyanide-based four-component coupling and biological evaluation as inhibitors of human parainfluenza virus type 1.

Novel sialidase inhibitors 11 having an α-acylaminoamido group at the C-4 position of Neu5Ac2en 1 against human parainfluenza virus type 1 (hPIV-1) were synthesized using one-pot isocyanide-based four-component condensation, and their inhibitory activities against hPIV-1 sialidase were studied. Compound 11b showed inhibitory activity (IC(50)=5.1 mM) against hPIV-1 sialidase. The degree of inhibition of 11b was much weaker than that of 1 (IC(50)=0.3 mM).

Non-invasive X-ray micro-computed tomographic evaluation of indomethacin on urethane-induced lung carcinogenesis in mice.

BACKGROUND: Lung cancer is the leading cause of cancer-related death worldwide. We previously reported that respiration-gated X-ray micro-computed tomography (micro-CT) is a useful tool for analyzing lung tumor development in animal models. MATERIALS AND METHODS: Lung tumors were induced by a single intraperitoneal injection (250 mg/kg) of urethane in male A/J mice, followed by indomethacin treatment at 5 ppm in the diet. The mice were scanned by micro-CT every 4 weeks from 10 to 26 weeks after urethane administration. RESULTS: Total incidence and multiplicity of lung tumors were not significantly reduced by indomethacin treatment, as compared with untreated mice. However, the incidence of adenocarcinoma tended to be reduced by indomethacin treatment. Moreover, the size of lung tumors, especially adenomas, was suppressed by indomethacin treatment. Micro-CT analysis revealed that indomethacin effectively suppressed tumor development after urethane treatment for 10 weeks. CONCLUSION: These findings indicate that indomethacin suppresses lung carcinogenesis in mice and micro-CT is a useful non-invasive imaging approach for evaluating the characteristics and suppression of lung tumors in mice treated with cancer chemopreventive agents.

Sulfatide negatively regulates the fusion process of human parainfluenza virus type 3.

Sulfatide (HSO(3)-3-galactosylceramide), which enriched in lipid rafts of plasma membranes in various epithelial cell lines, is a critical component of host cells for effective production of influenza A virus. However, the function of sulfatide in other virus infections targeting epithelial cells remains unknown. In this study, the effect of sulfatide on infection of human parainfluenza virus type 3 (hPIV3) was demonstrated by using genetically produced sulfatide-enriched cells and by treatment of hPIV3-infected cells with anti-sulfatide monoclonal antibody (GS-5) as well as by addition of sulfatide to the cells. hPIV3 was found to bind to sulfatide in a virus overlay assay and a solid-phase binding assay. Genetic expression of sulfatide in COS-7 cells defective in sulfatide suppressed initial hPIV3 infection and formation of multinucleate virus-infected cells. Treatment of virus-infected LLC-MK2 cells with GS-5 promoted formation of multinucleate cells. In contrast, exogenous addition of sulfatide to hPIV3-infected COS-7 cells and cells expressing the hPIV3-hemagglutinin-neuraminidase (HN) gene and fusion (F) gene conspicuously reduced the formation of multinucleate cells. The results suggest that sulfatide negatively regulates the fusion process of hPIV3, possibly through interaction with HN or F glycoprotein on the cell surface.

In vivo SPECT imaging with 111In-DOTA-c(RGDfK) to detect early pancreatic cancer in a hamster pancreatic carcinogenesis model.

UNLABELLED: Early detection of pancreatic cancer is key to overcoming its poor prognosis. α(v)ß(3)-integrin is often overexpressed in pancreatic tumor cells, whereas it is scarcely expressed in normal pancreatic cells. In this study, we investigated the usefulness of SPECT imaging with (111)In-1,4,7,10-tetraazacylododecane-N,N',N″,N'''-tetraacetic acid-cyclo-(Arg-Gly-Asp-d-Phe-Lys) [(111)In-DOTA-c(RGDfK)], an imaging probe of α(v)ß(3)-integrin, for the early detection of pancreatic cancer in a hamster pancreatic carcinogenesis model. METHODS: Hamsters were subcutaneously injected with the pancreatic duct carcinogen N-nitrosobis(2-oxopropyl)amine to induce pancreatic cancer. N-nitrosobis(2-oxopropyl)amine-treated hamsters underwent in vivo SPECT with (111)In-DOTA-c(RGDfK). After imaging, the tumor-to-normal pancreatic tissue radioactivity ratios in excised pancreatic samples were measured with autoradiography (ARG) and compared with the immunopathologic findings for α(v)ß(3)-integrin. In a mouse model in which inflammation was induced with turpentine, the uptake of (111)In-DOTA-c(RGDfK) in inflammatory regions was evaluated with ARG and compared with that of (18)F-FDG. RESULTS: (111)In-DOTA-c(RGDfK) was clearly visualized in pancreatic cancer lesions as small as 3 mm in diameter. ARG analysis revealed high tumor-to-normal pancreatic tissue radioactivity ratios (4.6 ± 1.0 [mean ± SD] in adenocarcinoma and 3.3 ± 1.4 in atypical hyperplasia). The uptake of (111)In-DOTA-c(RGDfK) strongly correlated with α(v)ß(3)-integrin expression. In the inflammatory model, inflammation-to-muscle ratios for (18)F-FDG and (111)In-DOTA-c(RGDfK) were 8.37 ± 4.37 and 1.98 ± 0.60, respectively. These results imply that (111)In-DOTA-c(RGDfK) has a lower rate of false-positive tumor detection than (18)F-FDG. CONCLUSION: Our findings suggest that SPECT with (111)In-DOTA-c(RGDfK) has great potential for the early and accurate detection of pancreatic cancer.

Syntheses of 2-deoxy-2,3-didehydro-N-acetylneuraminic acid analogues modified by N-sulfonylamidino groups at the C-4 position and biological evaluation as inhibitors of human parainfluenza virus type 1.

Eleven novel sialidase inhibitors 9 and 10 with an N-sulfonylamidino group at the C-4 position of Neu5Ac2en 1 against human parainfluenza virus type 1 (hPIV-1) were synthesized using copper-catalyzed three-component coupling reactions, and their inhibitory activities against hPIV-1 sialidase were studied.

A single amino acid mutation at position 170 of human parainfluenza virus type 1 fusion glycoprotein induces obvious syncytium formation and caspase-3-dependent cell death.

An escape mutant of human parainfluenza virus type 1 (hPIV1), which was selected by serial passage in the presence of a sialidase inhibitor, 4-O-thiocarbamoylmethyl-2-deoxy-2,3-didehydro-N-acetylneur-aminic acid (TCM-Neu5Ac2en), exhibited remarkable syncytium formation and virus-induced cell death in LLC-MK2 cells but no difference in susceptibility for the sialidase inhibitor TCM-Neu5Ac2en from that of wild-type hPIV1 strain C35 (WT). The mutant virus also had higher replication and plaque formation abilities. The mutant virus acquired two amino acid mutations, Glu to Gly at position 170 and Ala to Glu 442 in fusion (F) glycoprotein, but no mutations in haemaggulutinin-neuraminidase (HN) glycoprotein. Using cells co-expressing F and HN genes with site-specific mutagenesis, we demonstrated that a point mutation of Glu to Gly at position 170, which was estimated to be located in hPIV1 F glycoprotein heptad repeat 1, was required for obvious syncytium formation and caspase-3-dependent cell death. In contrast, wild-type F glycoprotein induced no synctium formation or cell death. The findings suggest that a single amino acid mutation of hPIV1 F glycoprotein promotes syncytium formation that is followed by caspase-3-dependent cell death.