Cellular analysis of cleavage-stage chick embryos reveals hidden conservation in vertebrate early development

This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution and reproduction in any medium provided that the original work is properly attributed.Birds and mammals, phylogenetically close amniotes with similar post-gastrula development, exhibit little conservation in their post-fertilization cleavage patterns. Data from the mouse suggest that cellular morphogenesis and molecular signaling at the cleavage stage play important roles in lineage specification at later (blastula and gastrula) stages. Very little is known, however, about cleavage-stage chick embryos, owing to their poor accessibility. This period of chick development takes place before egg-laying and encompasses several fundamental processes of avian embryology, including zygotic gene activation (ZGA) and blastoderm cell-layer increase. We have carried out morphological and cellular analyses of cleavage-stage chick embryos covering the first half of pre-ovipositional development, from Eyal-Giladi and Kochav stage (EGK-) I to EGK-V. Scanning electron microscopy revealed remarkable subcellular details of blastomere cellularization and subgerminal cavity formation. Phosphorylated RNA polymerase II immunostaining showed that ZGA in the chick starts at early EGK-III during the 7th to 8th nuclear division cycle, comparable with the time reported for other yolk-rich vertebrates (e.g. zebrafish and Xenopus). The increase in the number of cell layers after EGK-III is not a direct consequence of oriented cell division. Finally, we present evidence that, as in the zebrafish embryo, a yolk syncytial layer is formed in the avian embryo after EGK-V. Our data suggest that several fundamental features of cleavage-stage development in birds resemble those in yolk-rich anamniote species, revealing conservation in vertebrate early development. Whether this conservation lends morphogenetic support to the anamniote-to-amniote transition in evolution or reflects developmental plasticity in convergent evolution awaits further investigation

microRNA-33 maintains adaptive thermogenesis via enhanced sympathetic nerve activity

褐色脂肪細胞の燃焼を促す新たなメカニズムを解明 --体の熱産生にマイクロRNA-33が関与--. 京都大学プレスリリース. 2021-02-17.Adaptive thermogenesis is essential for survival, and therefore is tightly regulated by a central neural circuit. Here, we show that microRNA (miR)-33 in the brain is indispensable for adaptive thermogenesis. Cold stress increases miR-33 levels in the hypothalamus and miR-33−/− mice are unable to maintain body temperature in cold environments due to reduced sympathetic nerve activity and impaired brown adipose tissue (BAT) thermogenesis. Analysis of miR-33f/f dopamine-β-hydroxylase (DBH)-Cre mice indicates the importance of miR-33 in Dbh-positive cells. Mechanistically, miR-33 deficiency upregulates gamma-aminobutyric acid (GABA)A receptor subunit genes such as Gabrb2 and Gabra4. Knock-down of these genes in Dbh-positive neurons rescues the impaired cold-induced thermogenesis in miR-33f/f DBH-Cre mice. Conversely, increased gene dosage of miR-33 in mice enhances thermogenesis. Thus, miR-33 in the brain contributes to maintenance of BAT thermogenesis and whole-body metabolism via enhanced sympathetic nerve tone through suppressing GABAergic inhibitory neurotransmission. This miR-33-mediated neural mechanism may serve as a physiological adaptive defense mechanism for several stresses including cold stress

Non-destructive Determination of Bovine Milk Progesterone Concentration during Milking Using Near-infrared Spectroscopy

In the current dairy industry, an intensive demand for estrus detection and early diagnosis of pregnancy has been increasing. Progesterone is a steroid hormone that is secreted from corpus luteum into bovine blood and milk, and has a role of maintenance of estrus cycle and pregnancy. Therefore, progesterone concentration in bovine milk is used as an important indicator of estrus detection and early diagnosis of pregnancy. Current method for milk progesterone determination requires a hormone extraction procedure that is time consuming, various types of instruments, reagents management, and various assay methods that are destructive in nature. In contrast, near-infrared spectroscopy (NIRS) is a time saving and non-destructive analytical method that can be used for online real-time determination of milk constituents content such as milk fat, protein, lactose, milk urea nitrogen and somatic cell count. However, there has been limited study on using NIRS for online real-time determination of progesterone concentration in milk during milking. Thus, the objective of this study was to develop an online real-time NIR spectroscopic sensing system for milk progesterone determination during milking by using a specific enzyme immunosorbent assay as a reference (chemical) method. Milk spectra with a wavelength range of 700 to 1050 nm and milk samples were collected every 20 s during milking from four lactating Holstein cows for 28 days using the NIR spectroscopic sensing system. Calibration models were developed using partial least squares analytical method and the precision and accuracy of the models was validated. Milk progesterone concentration for each milking was calculated by taking the progesterone concentration of the milk predicted values and milk yield obtained every 20 s, and was compared with the milk progesterone concentration chemical analysis value for one milking (bucket milk). The results obtained show that the measurement accuracy for one milking of milk progesterone concentrations was reasonably good. By installing the NIR spectroscopic sensing system developed in this study into a milking robot, it could predict milk progesterone concentration for one milking with almost the same accuracy as chemical analysis. Thus, recording this predicted value every milking and monitoring the continuous transition of the milk progesterone concentrations, it becomes possible to use it for the detection of estrus status and for the diagnosis of pregnancy of each cow

EVALUATION AND APPLICATION OF CREATIVITY COLLABORATION SUPPORT SYSTEM GUNGEN DX II FOR CONSENSUS-BUILDING AMONG USERS

We have developed a creativity collaboration support system, called GUNGEN, which supports the KJ method. The KJ method is one of the popular idea generation methods in Japan, like brainstorming. This method consists of four steps: entering ideas, grouping ideas, structuring groups, and writing composition. We have used about one hundred idea labels in the experiments. Several hundred labels are optimal to improve the results of the KJ method. However, long time is necessary to group more labels. Therefore, we have developed GUNGEN DX II for several hundred labels. GUNGEN DX II has a special function to group labels easily and efficiently. The feature for grouping labels is to restrict time and to operate easily. The function generates islands from the common parts of each user's groups automatically by majority decision. We have carried out the KJ method and compared it with the previous system. Even if the number of labels increased, the time required to group labels decreased by about 30%.Creativity collaboration groupware, KJ method, grouping method, consensus among users, automatic arrangement

Potential of Photodynamic Therapy Based on Sugar-Conjugated Photosensitizers

In 2015, the Japanese health insurance approved the use of a second-generation photodynamic therapy (PDT) using talaporfin sodium (TS); however, its cancer cell selectivity and antitumor effects of TS PDT are not comprehensive. The Warburg effect describes the elevated rate of glycolysis in cancer cells, despite the presence of sufficient oxygen. Because cancer cells absorb considerable amounts of glucose, they are visible using positron emission tomography (PET). We developed a third-generation PDT based on the Warburg effect by synthesizing novel photosensitizers (PSs) in the form of sugar-conjugated chlorins. Glucose-conjugated (tetrafluorophenyl) chlorin (G-chlorin) PDT revealed significantly stronger antitumor effects than TS PDT and induced immunogenic cell death (ICD). ICD induced by PDT enhances cancer immunity, and a combination therapy of PDT and immune checkpoint blockers is expected to synergize antitumor effects. Mannose-conjugated (tetrafluorophenyl) chlorin (M-chlorin) PDT, which targets cancer cells and tumor-associated macrophages (TAMs), also shows strong antitumor effects. Finally, we synthesized a glucose-conjugated chlorin e6 (SC-N003HP) that showed 10,000–50,000 times stronger antitumor effects than TS (IC50) in vitro, and it was rapidly metabolized and excreted. In this review, we discuss the potential and the future of next-generation cancer cell-selective PDT and describe three types of sugar-conjugated PSs expected to be clinically developed in the future

A Basic Study of Photodynamic Therapy with Glucose-Conjugated Chlorin e6 Using Mammary Carcinoma Xenografts

By using the Warburg effect—a phenomenon where tumors consume higher glucose levels than normal cells—on cancer cells to enhance the effect of photodynamic therapy (PDT), we developed a new photosensitizer, glucose-conjugated chlorin e6 (G-Ce6). We analyzed the efficacy of PDT with G-Ce6 against canine mammary carcinoma (CMC) in vitro and in vivo. The pharmacokinetics of G-Ce6 at 2, 5, and 20 mg/kg was examined in normal dogs, whereas its intracellular localization, concentration, and photodynamic effects were investigated in vitro using CMC cells (SNP cells). G-Ce6 (10 mg/kg) was administered in vivo at 5 min or 3 h before laser irradiation to SNP tumor-bearing murine models. The in vitro study revealed that G-Ce6 was mainly localized to the lysosomes. Cell viability decreased in a G-Ce6 concentration- and light intensity-dependent manner in the PDT group. Cell death induced by PDT with G-Ce6 was not inhibited by an apoptosis inhibitor. In the in vivo study, 5-min-interval PDT exhibited greater effects than 3-h-interval PDT. The mean maximum blood concentration and half-life of G-Ce6 (2 mg/kg) were 15.19 ± 4.44 μg/mL and 3.02 ± 0.58 h, respectively. Thus, 5-min-interval PDT with G-Ce6 was considered effective against CMC

MicroRNA-33 regulates sterol regulatory element-binding protein 1 expression in mice

脂肪酸とコレステロール合成の切り替えスイッチの発見に成功 -安全な動脈硬化改善薬の開発に期待-. 京都大学プレスリリース. 2013-12-03.MicroRNAs (miRs) are small non-protein-coding RNAs that bind to specific mRNAs and inhibit translation or promote mRNA degradation. Recent reports have indicated that miR-33, which is located within the intron of sterol regulatory element-binding protein (SREBP) 2, controls cholesterol homoeostasis and may be a potential therapeutic target for the treatment of atherosclerosis. Here we show that deletion of miR-33 results in marked worsening of high-fat diet-induced obesity and liver steatosis. Using miR-33−/−Srebf1+/− mice, we demonstrate that SREBP-1 is a target of miR-33 and that the mechanisms leading to obesity and liver steatosis in miR-33−/− mice involve enhanced expression of SREBP-1. These results elucidate a novel interaction between SREBP-1 and SREBP-2 mediated by miR-33 in vivo