Comparison of the morphology of oocysts and the phylogenetic analysis of four Ascogregarina species (Eugregarinidae: Lecudinidae) as inferred from small subunit ribosomal DNA sequences

This study on the ultrastructure of the oocysts of four isolated species of Ascogregarina (A. taiwanensis (Lien and Levine) (Eugregarinidae: Lecudinidae) from Aedes albopictus (Skuse), A. culicis (Ross) (Eugregarinidae: Lecudinidae) from Aedes aegypti (L.), A. armigerei (Eugregarinidae: Lecudinidae) from Armigeres subalbatus (Coquillet), and Ascogregarina sp. (Eugregarinidae: Lecudinidae) from Ochlerotatus japonicus japonicus (Theobald)) using a scanning electron microscope revealed significant differences in size and in surface structure. The average length of the oocyst was greatest in A. armigerei (13.2+/-0.2 microm) (mean+/-SD) and least in A. culicis (8.8+/-0.4 microm). Oocysts were of moderate length in A. taiwanensis (9.9+/-0.6 microm) and in Ascogregarina sp. (10.7+/-1.1 microm) isolated from O. j. japonicus. The ultrastructure of the surface of the A. culicis oocyst was rough in texture with numerous dense spots and was easily distinguishable from the oocysts of the other three Ascogregarina spp. The maximum likelihood tree inferred from small subunit ribosomal DNA (SSU rDNA) sequences indicated that the four Ascogregarina spp. form a monophyletic cluster among other gregarine parasites. Within the Ascogregarina clade, A. culicis, A. taiwanensis, and Ascogregarina sp. from O. j. japonicus showed a close relationship, whereas A. armigerei was a distantly related species.     

The responses of HT22 cells to the blockade of mitochondrial complexes and potential protective effect of selenium supplementation

Mitochondria are the major reactive oxygen species (ROS)--generating sites in mammalian cells. Blockade of complexes in the electron transport chain (ETC) increases the leakage of single electrons to O(2) and therefore increases ROS levels. Complexes I and III have been reported to be the major ROS-generating sites in mitochondria. In this study, using mouse hippocampal HT22 cells as in vitro model, we monitored the change of intracellular ROS level in response to the blockade of ETC at different complex, and measured changes of gene expression of antioxidant enzymes and phase II enzymes, also evaluated potential protective effect of selenium (Se) supplementation to the cells under this oxidative stress. In summary, our results showed that complex I was the major ROS-generating site in HT22 cells. Complex I blockade upregulated the mRNA levels of glutamylcysteine synthetase heavy and light chains, glutathione-S-transferases omega1 and alpha 2, hemoxygenase 1, thioredoxin reductase 1, and selenoprotein H. Unexpectedly, the expression of the enzymes that directly scavenge ROS decreased, including superoxide dismutases 1 and 2, glutathione peroxidase 1, and catalase. Se supplementation increased glutathione levels and glutathione peroxidase activity, indicating a potential protective role in oxidative stress caused by ETC blockade.     

Nanoneedle Surface Modification with 2-Methacryloyloxyethyl Phosphorylcholine Polymer to Reduce Nonspecific Protein Adsorption in a Living Cell

Previously, we developed a new molecular delivery system to target single living cells by using atomic force microscope and ultrathin needle referred to as nanoneedle. This system delivers molecules into the cell by attaching them to the surface of nanoneedle. However, nonspecific protein adsorption on the nanoneedle surface inside the living cells limits the range of application of this system. In the present study, we focused on nonspecific protein adsorption onto the nanoneedle surface inside the cells and examined whether this protein adsorption was reduced by modifying the nanoneedle surface with a biocompatible phospholipid polymer containing 2-methacryloyloxyethyl phosphorylcholine (MPC) unit. MPC polymer coating of the surface of silicon wafer reduced nonspecific adsorption of proteins from liver extracts and prevented the formation of clot-like protein aggregates. MPC polymer also decreased nonspecific adsorption of cytosolic protein onto the nanoneedle surface inside the living cell. On the other hand, MPC polymer showed no effect on nonspecific mechanical interaction between nanoneedle and the cell components. Surface modification with MPC polymer is a useful technique to modify the surface properties of nanoneedle.     

2-Aminoethyl diphenylborinate analogues: selective inhibition for store-operated Ca2+ entry

Capacitative calcium entry (CCE), the mechanism that replenishes the internal Ca2+ stores with Ca2+ from the extracellular milieu in response to depletion of the store, is mediated by Ca2+ channels in the plasma membrane generally referred to as store-operated channels (SOCs). However, the roles of SOCs in the more physiological context have been fully elucidated. 2-Aminoethyl diphenylborinate (2-APB) strongly inhibits SOCs, as well as inositol-1,4,5 trisphosphate (IP3) receptors. In the present study, we screened a library of 166 2-APB analogues for effects on CCE and IP3-induced Ca2+ release in order to discover specific SOC inhibitors, and found that some blocked both store-operated and receptor-operated Ca2+ influx more strongly and selectively than 2-APB. Indeed, these new compounds ceased the prolonged intracellular Ca2+ oscillations induced by a low concentration of ATP in CHO-K1 cells. These novel SOC inhibitors will be valuable pharmacological and biochemical tools for elucidating the physiological roles.     

The regulation of human blastoid research: A bioethical discussion of the limits of regulation

The creation of human blastoids holds great potential for research on early human development but also raises considerations about the ethics of such research and its regulation. Subject Categories: Development, Economics, Law & Politics

Developmental research has made considerable progress modeling either part of or the entire embryonic development of both humans and non‐human animals. A major step forward was the ability to grow blastocyst‐like structures from pluripotent stem cells: these structures, known as “blastoids,” mimic early embryonic development up to and potentially beyond the blastocyst stage 5–6 days after the first cell division. Blastoids have attracted considerable attention as an effective research tool to understand early human development and to elucidate the causes of infertility, teratogenesis, and other developmental abnormalities.

… many scientists see the use of human blastoids as an exciting scientific opportunity, as it may help to reduce the need for human embryos in research.

Until now, research with blastoids has mainly studied early development in mice, but, as of 2021, research results are also being reported from human blastoids (see “Further Reading”). Indeed, many scientists see the use of human blastoids as an exciting scientific opportunity, as it may help to reduce the need for human embryos in research (Ravindran, ²⁰²¹). However, as with any research that uses human embryos or human stem cells derived from embryos, human blastoid research raises ethical questions and is subject to regulation and approval. The latest ISSCR guidelines state that “[f]orms of research with embryos … and stem cell‐based embryo models … are permissible only after review and approval through a specialized scientific and ethics review process” (ISSCR, ²⁰²¹). Thus, although blastoids are models of embryonic development, they are currently considered to require the same or similar ethical considerations as blastocysts or cells derived from human embryos. In fact, Australia made a decision to regulate blastoid research in the same manner as research on human embryos (Australia NHMRC, ²⁰²¹).     

Environmental risk assessment of transgenic miraculin-accumulating tomato in a confined field trial in Japan

The commercial use of genetically modified (GM) crops requires prior assessment of the risks to the environment when these crops are grown in the field or distributed. Assessments protocols vary across countries and GM crop events, but there is a common need to assess environmental biosafety. In this study, we conducted an environmental risk assessment in a confined field of GM tomato plants that can produce miraculin, a taste-altering protein that causes sour tastes to be perceived as sweet, for practical use in Japan. The evaluation was conducted for 1) competitiveness (the ability to compete with wild plants for nutrients, sunlight, and growing areas and prevent their growth) and 2) the production of toxic substances (the ability to produce substances that interfere with the habitat and growth of wild plants, animals, and microorganisms). Investigations of plant morphology and growth characteristics as well as tolerance to low temperature during early growth and overwintering for assessment endpoints related to competitiveness showed no biologically meaningful difference between GM tomato and non-GM tomato. In addition, harmful substances in plant residues and root secretions were assessed by the plow-in method, succeeding crop test and soil microflora tests, and it was determined that GM tomato does not exhibit an increase in harmful substances. Based on these results, it was concluded that GM miraculin-accumulating tomato is comparable to conventional tomato and is unlikely to have unintended adverse effects in the natural environment of Japan.     

Mechanistic study on the reaction of boronic acids with H-resorcinol

Kinetics for the reaction of m-nitrophenyl- (m-NO₂Ph-), phenyl- (Ph-), methyl(Me-), and n-butyl- (n-Bu-) boronic acids with H-resorcinol has been studied at various temperatures and pressures. The pseudo first-order rate constant (k_obs) exhibited saturation in the plot of k_obs against the total boronic acid concentration (C_B) for the reactions of m-NO₂PhB(OH)₂ and PhB(OH)₂, while the plot was linear for the reactions of MeB(OH)₂ and n-BuB(OH)₂, which leads to the following equations, respectively: k_obs=k*KC_B/(1+KC_B) and k_obs=k*KC_B=k_fC_B. It was experimentally shown that the reaction of boronic acids with H-resorcinol proceeds with rate determining second chelate-ring closure.     

Uptake of inorganic ions and compatible solutes in cultured mangrove cells during salt stress

Summary Callus of the mangrove plant, Sonneratia alba J. Smith, established from pistils of flower buds were cultured on solid Murashige and Skoog medium supplemented with 0 to 500 mM NaCl. Maximum growth was observed with 50 mM NaCl, and net growth of callus occurred for concentrations up to 200 mM NaCl. At 500 mM NaCl, growth of callus was completely inhibited, although a part of the tissue was still alive after 30 d. Cellular levels of Na⁺ and Cl⁻ were greatly increased by the treatment with NaCl. Uptake of K⁺ was also enhanced and was accompanied by increasing levels of Na⁺ and Cl⁻ so that the Na⁺/K⁺ ratio was almost constant (4.1–4.2) in callus grown with 50–200 mM NaCl. Levels of Mg²⁺ and Ca²⁺ were not changed significantly with 50–200 mM NaCl, whereas levels of free NH ₄ ⁺ , NO ₃ ⁻ and SO ₄ ²⁻ ions, which are convertible to organic compounds, were lowest in callus grown with 50 mM NaCl. The rate of conversion of ¹⁵NH ₄ ⁺ into macromolecules during 30 d culture with 0–100 mM NaCl did not vary greatly, but 200 mM NaCl reduced the biosynthesis of macromolecules from this ion. The highest rate of conversion of ¹⁵NO ₃ ⁻ into macromolecules was observed at 50 mM NaCl. Identification of compatible solutes with NMR-spectroscopy indicated that mannitol is the compatible solute for intact plants of Sonneratia alba, but no accumulation of mannitol was found in calluses, not even in those grown at high concentrations of NaCl.     

On‐demand killing of adherent cells on photo‐acid‐generating culture substrates

As a powerful tool of cell screening and cell purification, we developed a novel method to kill adherent cells as cultured on a substrate by micro‐projection of incoherent visible light. To kill the cells by the mild light irradiated by electrically controllable micro‐projection systems currently available, we introduced the assist of the photo‐responsive culture substrates functionalized with a photo‐acid‐generating polymer. In clear contrast to the existing laser‐based methods requiring point scanning, areal micro‐prjection of blue light with the wavelength 436 nm killed many CHO‐K1 cells at a time in the irradiated area on the substrate. The effect of the photo‐generated acid was so confined that selective killing of targeted cells was achieved without critical damage to the neighboring cells. Further, we demonstrated the photo‐selective killing of the adherent cells after preliminarily patterning through the photo‐induced removal of cell adhesion‐inhibiting polymer. Biotechnol. Bioeng. 2013; 110: 348–352. © 2012 Wiley Periodicals, Inc.