Engineering branched ionizable lipid for hepatic delivery of clustered regularly interspaced short palindromic repeat-Cas9 ribonucleoproteins.

The delivery of the CRISPR/Cas ribonucleoprotein (RNP) has received attention for clinical applications owing to its high efficiency with few off-target effects. Lipid nanoparticles (LNPs) are potential non-viral vectors for the delivery of RNPs. Herein, we report the engineering of a branched scaffold structure of ionizable lipids for the hepatic delivery of RNPs. Both the total carbon number and branching position were critical for the functional delivery of RNPs. The optimal ionizable lipid exhibited a more than 98% reduction in transthyretin protein after a single dose with no obvious signs of toxicity. The mechanistic study has revealed that optimal LNPs have a unique "flower-like structure" that depends on both the lipid structure and the payload and that these LNPs accumulate in hepatocytes in an apolipoprotein E-independent manner. These results represent a major step toward the realization of in vivo genome editing therapy via RNP delivery using chemically synthesizable LNP formulations.

Spatiotemporal control of transgene expression using an infrared laser in the crustacean Daphnia magna.

The crustacean Daphnia magna is an emerging model for ecological and toxicological genomics. However, the lack of methods for spatial and temporal control of gene expression has impaired the elucidation of molecular mechanisms underlying responses to environments in vivo. Here we report local activation of the hsp70 promoter-driven gene cassette in D. magna by the infrared laser-evoked gene operator (IR-LEGO), a method for heating the target cells with infrared irradiation. We identified the heat-inducible promoter upstream of the D. magna hsp70-A gene. Using this promoter, we generated a transgenic Daphnia harboring the heat-shock responsive GFP reporter gene and confirmed that the GFP gene responds to heat treatment not only in juveniles and adults but also in embryos. We collected embryos from the reporter line and irradiated four different regions of interest in the embryos: a proximal region of the third thoracic segment, a part of the midline, a second maxilla, and a distal region of the endopodite of the second antenna, all of which increased GFP fluorescence with an infrared laser. Our results suggest that the IR-LEGO method is useful for spatial and temporal control of gene expression and would advance the functional genomics in D. magna.

Sum of variance for quantifying the variation of multiple sequential data for the crispness evaluation of chicken nugget.

Crispness is one of the words most frequently used to describe the texture of fried or dried food in addition to being a key to the determination of freshness for many non-fried foods. In this study, a new feature value called the sum of variance was assessed for its contribution to the estimation of crispness. Dynamic time warping and its averaging algorithms were employed to determine the sum of variance from a set of sequential force data measured using an instrument. The sum of variance is a feature value that expresses the variance of multiple sequential data. In an experiment, seven chicken nugget samples were prepared, and five panels evaluated their texture according to six Japanese word descriptors. An instrument experiment determined the six feature values, including the sum of variance from the measurement data, whereas multiple linear regression was applied to determine the relationship between the sensory values and feature values. For three of the six textures, the sum of variance reduced the error between the sensory values and their estimated values by up to 50%, confirming that this feature contributes to the textural estimation of food crispness.

Cholangiocyte senescence caused by lysophosphatidylcholine as a potential implication in carcinogenesis.

BACKGROUND: The incidence of biliary tract cancer in patients with pancreaticobiliary maljunction or intrahepatic cholelithiasis is markedly high with undefined mechanism. In these diseases, biliary lysophosphatidylcholine (LPC) level is reportedly increased. This study investigated the influence of LPC on cholangiocytes focusing on cellular senescence and its potential contribution to carcinogenesis. METHODS: Cultured MMNK-1, an immortalized human cholangiocyte was treated with LPC in vitro and its effect was evaluated. RESULTS: Lysophosphatidylcholine demonstrated cytotoxicity with generation of intracellular reactive oxygen species. Accordingly, LPC provoked oxidative DNA injury, whereas the gene expressions of DNA repair enzyme (OGG1, MUTYH, MTH1) remained unchanged. Interestingly, LPC caused global DNA hypomethylation, which is frequently observed in cancer tissues. Microarray analysis identified differentially regulated genes in response to LPC, which included the components of senescence-associated secretory phenotype (SASP) including interleukin-8 (IL-8), IL-6, transforming growth factor-ß and plasminogen activator inhibitor-1. Significant induction of these genes was further confirmed by quantitative real-time polymerase chain reaction. In addition to upregulation of p21 gene expression, senescence-associated beta-galactosidase activity, a widely used marker of cellular senescence was significantly induced by the treatment of LPC. CONCLUSIONS: Based on these data, cholangiocyte senescence and SASP caused by LPC are potential pathogenic mechanisms in the development of biliary tract cancer.

p53 directly regulates the transcription of the human frataxin gene and its lack of regulation in tumor cells decreases the utilization of mitochondrial iron.

Mitochondrial frataxin functions in iron homeostasis, biogenesis of iron-sulfur clusters, protection from oxidative stress and apoptosis, and as a tumor suppressor protein. We examined regulation of the expression of the human frataxin by p53. Pifithrin-α, an inhibitor of p53 function, and knockdown of p53 decreased the level of frataxin mRNA in human kidney HEK 293T cells. The transcriptional activity of the human frataxin gene is enhanced by the proximal promoter containing the p53-responsive element (p53RE) on the gene. Chromatin immunoprecipitation assay and electrophoretic mobility shift assay confirmed the binding of p53 to the human frataxin p53RE. The expression of wild-type p53 in human cancer HeLa cells increased the reporter activity carrying p53RE at the region of -209 to -200bp of the frataxin promoter. Finally, when the HeLa cells overexpressing frataxin were treated with 5-aminolevulinic acid (ALA), there was less accumulation of protoporphyrin than HeLa control cells, and it was sharply decreased by the addition of iron citrate, suggesting that the utilization of mitochondrial iron for heme biosynthesis can be dependent on the level of frataxin. Alternatively, the low expression of frataxin not regulated by p53 in tumor cells lowers the utilization of iron in mitochondria, causing the tumor-specific ALA-induced accumulation of protoporphyrin.