Two-dimensional distribution of Gi2α in the plasma membrane: a critical evaluation by immunocytochemistry

AbstractCaveolae have been postulated as a center for signal transduction, because many signaling molecules are concentrated in caveolin-rich fractions. We took Gi2α as an example and examined whether it is constitutively concentrated in caveolae. First, the behavior of caveolin and Gi2α in density-equilibrium ultracentrifugation was reexamined. By collecting fractions efficiently, caveolin and Gi2α were found to distribute differently. Secondly, by novel immunocytochemical methods it was found that the labeling density of Gi2α was 2.29 times higher in caveolae than in the non-caveolar plasma membrane. The results indicate that the concentration of Gi2α in caveolae is lower than deduced from most biochemical studies

Intracellular stability of 2′-OMe-4′-thioribonucleoside modified siRNA leads to long-term RNAi effect

Chemically modified siRNAs are expected to have resistance toward nuclease degradation and good thermal stability in duplex formation for in vivo applications. We have recently found that 2′-OMe-4′-thioRNA, a hybrid chemical modification based on 2′-OMeRNA and 4′-thioRNA, has high hybridization affinity for complementary RNA and significant resistance toward degradation in human plasma. These results prompted us to develop chemically modified siRNAs using 2′-OMe-4′-thioribonucleosides for therapeutic application. Effective modification patterns were screened with a luciferase reporter assay. The best modification pattern of siRNA, which conferred duration of the gene-silencing effect without loss of RNAi activity, was identified. Quantification of the remaining siRNA in HeLa-luc cells using a Heat-in-Triton (HIT) qRT–PCR revealed that the intracellular stability of the siRNA modified with 2′-OMe-4′-thioribonucleosides contributed significantly to the duration of its RNAi activity

Effects of Consuming Xylitol on Gut Microbiota and Lipid Metabolism in Mice

The sugar alcohol xylitol inhibits the growth of some bacterial species including Streptococcus mutans. It is used as a food additive to prevent caries. We previously showed that 1.5–4.0 g/kg body weight/day xylitol as part of a high-fat diet (HFD) improved lipid metabolism in rats. However, the effects of lower daily doses of dietary xylitol on gut microbiota and lipid metabolism are unclear. We examined the effect of 40 and 200 mg/kg body weight/day xylitol intake on gut microbiota and lipid metabolism in mice. Bacterial compositions were characterized by denaturing gradient gel electrophoresis and targeted real-time PCR. Luminal metabolites were determined by capillary electrophoresis electrospray ionization time-of-flight mass spectrometry. Plasma lipid parameters and glucose tolerance were examined. Dietary supplementation with low- or medium-dose xylitol (40 or 194 mg/kg body weight/day, respectively) significantly altered the fecal microbiota composition in mice. Relative to mice not fed xylitol, the addition of medium-dose xylitol to a regular and HFD in experimental mice reduced the abundance of fecal Bacteroidetes phylum and the genus Barnesiella, whereas the abundance of Firmicutes phylum and the genus Prevotella was increased in mice fed an HFD with medium-dose dietary xylitol. Body composition, hepatic and serum lipid parameters, oral glucose tolerance, and luminal metabolites were unaffected by xylitol consumption. In mice, 40 and 194 mg/kg body weight/day xylitol in the diet induced gradual changes in gut microbiota but not in lipid metabolism

HFIGMI–VMAT for brain metastases

Volumetric-modulated arc therapy (VMAT) can be used to design hypofractionated radiotherapy treatment plans for multiple brain metastases. The purpose of this study was to evaluate treatment outcomes of hypofractionated image-guided multifocal irradiation using VMAT (HFIGMI–VMAT) for brain metastases. From July 2012 to December 2016, 67 consecutive patients with 601 brain metastases were treated with HFIGMI–VMAT at our institution. The prescribed dose was 50 Gy to a 95% volume of the planning target volume in 10 fractions. Fifty-five of the 67 patients had non-small-cell lung cancer, and the remaining 12 had other types of cancer. The median number of brain metastases was five, and the median maximum diameter was 1.2 cm. The median duration of follow-up was 12.0 months (range, 1.9–44.8 months), and the median survival time 18.7 months. Four patients with six lesions had local recurrences. The local control rate in the 64 assessed patients was 98.4% and 95.3% at 6 and 12 months, respectively (three died before assessment). The local control rate for the 572 assessed lesions was 99.8% and 99.3% at 6 and 12 months, respectively. Thirty-nine patients developed distant brain metastases, the distant brain control rate being 59.7% and 40.5% at 6 and 12 months, respectively. Acute toxicities were generally mild (Grade 1–2). Three patients (4.5%) developed radiation necrosis requiring corticosteroid therapy. The HFIGMI–VMAT technique with flat dose delivery was well tolerated and achieved excellent local control. This technique is a promising treatment option for patients with multiple and large brain metastases

Structure of nitrogen-doped graphene synthesized by combination of imidazole and melamine solid precursors

Here, we demonstrate the synthesis of nitrogen-doped (N-doped) graphene using imidazole and melamine as two different nitrogen containing aromatic rings carbon precursors. Structure of N-doped graphene was investigated at different temperature (800–1020 °C) without changing the precursor quantity. It is observed that higher crystalline N-doped graphene can be obtained from the solid precursors at 1020 °C on Cu foil. X-ray photoelectron spectroscopy (XPS) analysis shows interesting features for the N-doped graphene synthesized from mixture of imidazole and melamine. Overall graphitic nitrogen content is enhanced in the graphene layers using the mixture of precursors, attributing better coordination of carbon and nitrogen atoms on Cu catalyst. Our finding shows that the graphitic and pyridinic nitrogen content in graphene lattice can be tuned by combination of two different nitrogen containing organic molecules

Postnatal liver functional maturation requires Cnot complex-mediated decay of mRNAs encoding cell cycle and immature liver genes

Liver development involves dramatic gene expression changes mediated by transcriptional and post-transcriptional control. Here, we show that the Cnot deadenylase complex plays a crucial role in liver functional maturation. The Cnot3 gene encodes an essential subunit of the Cnot complex. Mice lacking Cnot3 in liver have reduced body and liver masses, and they display anemia and severe liver damage. Histological analyses indicate that Cnot3-deficient (Cnot3(-/-) ) hepatocytes are irregular in size and morphology, resulting in formation of abnormal sinusoids. We observe hepatocyte death, increased abundance of mitotic and mononucleate hepatocytes, and inflammation. Cnot3(-/-) livers show increased expression of immune response-related, cell cycle-regulating and immature liver genes, while many genes relevant to liver functions, such as oxidation-reduction, lipid metabolism and mitochondrial function, decrease, indicating impaired liver functional maturation. Highly expressed mRNAs possess elongated poly(A) tails and are stabilized in Cnot3(-/-) livers, concomitant with an increase of the proteins they encode. In contrast, transcription of liver function-related mRNAs was lower in Cnot3(-/-) livers. We detect efficient suppression of Cnot3 protein postnatally, demonstrating the crucial contribution of mRNA decay to postnatal liver functional maturation

Discovery of anti-inflammatory physiological peptides that promote tissue repair by reinforcing epithelial barrier formation

上皮バリアを形成するペプチドJIPの発見 --JIPは上皮組織修復に貢献する--. 京都大学プレスリリース. 2021-11-18.Epithelial barriers that prevent dehydration and pathogen invasion are established by tight junctions (TJs), and their disruption leads to various inflammatory diseases and tissue destruction. However, a therapeutic strategy to overcome TJ disruption in diseases has not been established because of the lack of clinically applicable TJ-inducing molecules. Here, we found TJ-inducing peptides (JIPs) in mice and humans that corresponded to 35 to 42 residue peptides of the C terminus of alpha 1-antitrypsin (A1AT), an acute-phase anti-inflammatory protein. JIPs were inserted into the plasma membrane of epithelial cells, which promoted TJ formation by directly activating the heterotrimeric G protein G13. In a mouse intestinal epithelial injury model established by dextran sodium sulfate, mouse or human JIP administration restored TJ integrity and strongly prevented colitis. Our study has revealed TJ-inducing anti-inflammatory physiological peptides that play a critical role in tissue repair and proposes a previously unidentified therapeutic strategy for TJ-disrupted diseases

Generating Vegfr3 reporter transgenic mouse expressing membrane-tagged Venus for visualization of VEGFR3 expression in vascular and lymphatic endothelial cells

Vascular endothelial growth factor receptor 3 (Vegfr3) has been widely used as a marker for lymphatic and vascular endothelial cells during mouse embryonic development and in adult mouse, making it valuable for studying angiogenesis and lymphangiogenesis under normal and pathological conditions. Here, we report the generation of a novel transgenic (Tg) mouse that expresses a membrane-localized fluorescent reporter protein, Gap43-Venus, under the control of the Vegfr3 regulatory sequence. Vegfr3-Gap43-Venus BAC Tg recapitulated endogenous Vegfr3 expression in vascular and lymphatic endothelial cells during embryonic development and tumor development. Thus, this Tg mouse line contributes a valuable model to study angiogenesis and lymphangiogenesis in physiological and pathological contexts

CIPRO 2.5: Ciona intestinalis protein database, a unique integrated repository of large-scale omics data, bioinformatic analyses and curated annotation, with user rating and reviewing functionality

The Ciona intestinalis protein database (CIPRO) is an integrated protein database for the tunicate species C. intestinalis. The database is unique in two respects: first, because of its phylogenetic position, Ciona is suitable model for understanding vertebrate evolution; and second, the database includes original large-scale transcriptomic and proteomic data. Ciona intestinalis has also been a favorite of developmental biologists. Therefore, large amounts of data exist on its development and morphology, along with a recent genome sequence and gene expression data. The CIPRO database is aimed at collecting those published data as well as providing unique information from unpublished experimental data, such as 3D expression profiling, 2D-PAGE and mass spectrometry-based large-scale analyses at various developmental stages, curated annotation data and various bioinformatic data, to facilitate research in diverse areas, including developmental, comparative and evolutionary biology. For medical and evolutionary research, homologs in humans and major model organisms are intentionally included. The current database is based on a recently developed KH model containing 36 034 unique sequences, but for higher usability it covers 89 683 all known and predicted proteins from all gene models for this species. Of these sequences, more than 10 000 proteins have been manually annotated. Furthermore, to establish a community-supported protein database, these annotations are open to evaluation by users through the CIPRO website. CIPRO 2.5 is freely accessible at http://cipro.ibio.jp/2.5