Straightforward Fabrication of Double Roughness Structures on a Microcrystalline Film of a Diarylethene Derivative.

Double roughness structure mimicking the surface of a lotus leaf was prepared using a newly synthesized diarylethene having a six-membered perfluorocyclohexene ring. The cubic-shaped crystals of the open-ring isomer, with sizes of approximately 7 µm, appeared immediately following solution casting. Upon UV irradiation, each cubic crystal was covered with needle-shaped crystals of the closed-ring isomer to form double roughness structures within 1 h. This structure could bear the continuous impact of water droplets.

Three-dimensional morphology of bovine blastocysts hatched against lipopolysaccharide exposure in vitro.

Embryo transfer in cattle is globally becoming more ubiquitous, but the pregnancy rate is lower than that of artificial insemination. The uterus contains its own bacteria, and concentrations of lipopolysaccharides (LPS) from gram-negative bacteria are higher in uteri affected by endometritis than in healthy uteri and they suppress embryogenesis. The purpose of this study was to investigate the morphological characteristics of bovine embryos with a higher viability and implantability, by analyzing the morphology of bovine blastocysts that successfully hatched under challenge of LPS, using an optical coherence tomography (OCT) system. Developing embryos produced by in vitro fertilization that had reached the blastocyst stage on Day 7 were three-dimensionally scanned using an OCT system, then were continued to culture with or without LPS until Day 9, when the presence or absence of hatching was determined. The OCT-captured three-dimensional images were used to quantify 20 different metrics, including inner cell mass (ICM), trophectoderm, blastocoel, and total embryo volume; each of the parameters was compared between the hatched and unhatched embryos. Under the LPS challenge, hatched embryos had higher ICM thickness and volume, and lower trophectoderm thickness than unhatched embryos. Furthermore, hatched embryos under LPS challenge had higher ICM thickness and ICM volume than hatched embryos without LPS challenge. The present results suggest the possibility that ICM thickness and ICM volume calculated by OCT system could be indices for good quality bovine embryos.

Hatchability evaluation of bovine IVF embryos using OCT-based 3D image analysis.

Although embryo transfer is widely applied in cattle, many of the transferred embryos do not result in pregnancy. To determine a new parameter for bovine embryo evaluation, we investigated the relationships between in vitro hatchability and embryo morphological parameters using optical coherence tomography (OCT) that we established recently. Bovine embryos were obtained from Japanese Black cattle by in vitro fertilization (IVF). The quality of the blastocysts was examined under an inverted microscope and confirmed as Codes 1-3 according to the IETS standards for embryo evaluation. The OCT images of the embryos were captured on Day 7 after IVF, and the embryos were cultured until Day 9 to determine their hatchability. During OCT, the embryos were irradiated with near-infrared light for a few minutes to obtain three-dimensional images. In total, 22 parameters were assessed for each of the 42 embryos, of which 25 hatched (H embryos) and 17 did not (NH embryos). The thickness of the trophectoderm (TE) and TE+zona pellucida (ZP) was lesser, and the volumes of the TE, ZP, blastocoel, and whole embryo and blastocoel diameter were greater in the H embryos than in the NH embryos. PCA identified that the increase in the blastocoel-related value along with the decrease in the thickness-related value of the TE and/or ZP could be indicators for evaluating the hatchability of bovine IVF embryos. These results support the idea that OCT-captured structural data of blastocyst-stage embryos can be used as a potential model to predict the quality of bovine embryos.

Lipopolysaccharide (LPS) suppresses follicle development marker expression and enhances cytokine expressions, which results in fail to granulosa cell proliferation in developing follicle in cows.

Postpartum endometritis is known to be associated with ovarian dysfunction in cows. Lipopolysaccharide (LPS) generated by Gram-negative bacteria is recognized by toll-like receptor 4 (TLR4), which leads to an inflammatory response by the generation of cytokines such as tumor necrosis factor-α (TNF-α) and interleukins. In this study, we investigated the effect of endometrial LPS on granulosa cell functions during early follicular development in cows. Uteri and follicles were obtained from a slaughterhouse and classified into either clinical endometritis (CE) or normal groups by vaginal mucus test. TLR4 mRNA and protein in normal cows were expressed in granulosa cells collected from follicles measuring 1-3 and 4-7 mm in a diameter, respectively. LPS content in endometrium and follicular fluid of CE cows was significantly higher than that in normal cows. Compared to normal cows, CE cows showed lower expression of follicular development markers (FSHR, CYP19A1, CCND2, and LHCGR) in granulosa cells, lower estradiol-17ß concentrations in follicular fluid, and lower granulosa cell proliferation. CE contraction significantly increased cytokine expressions (TNF, IL-1A, and IL-1B) in granulosa cells and suppressed apoptosis of granulosa cells compared to normal cows. LPS significantly suppressed the expression of follicular development markers and the production of estradiol-17ß in granulosa cells and reduced granulosa cells proliferation compared to cells cultured without LPS. LPS significantly increased cytokine expressions and suppressed granulosa cell apoptosis. Thus, the present results suggest that the existence of LPS in developing follicles is one of the causes of ovarian quiescence in cows.

Turn-on mode fluorescent diarylethene containing neopentyl substituents that undergoes all-visible-light switching.

This paper presents a strategy for improving the all-visible-light switching response of turn-on mode fluorescent diarylethene derivatives. Introduction of neopentyl or isobutyl substituents at the reactive carbons (2- and 2'-positions) of an oxidized bis(benzothienyl)perfluorocyclopentene derivative, which undergoes both cyclization and cycloreversion reactions upon irradiation with visible light, was effective in increasing the cycloreversion quantum yield by one or two orders of magnitude in comparison with the yield of an ethyl-substituted derivative. Any significant influence on the cyclization and fluorescence quantum yields was not observed by the introduction of neopentyl or isobutyl substituents.

Molecular crystalline capsules that release their contents by light.

Here, we present single crystalline capsules of a photoresponsive molecule produced by simple recrystallization from organic solutions without direct human processing. During the crystal growth process, a movie was taken of the capsule taking in the organic solution. The capsules responded rapidly (<1 s) to the UV light stimuli and released the captured solution or solute. In principle, they can take in any substance dissolved in organic solvents, and their size can be controlled. Moreover, the capsule can be broken by multi-photon excitation using a near-infrared laser within the biological window. Furthermore, because the molecular packing in the crystal is unidirectional, the response can be controlled by the polarization of the light. This study shows the new potential of photoresponsive molecules.

Spontaneous Combustion of 2-Bromo-3-Methoxythiophene: A Study on Reaction Pathways and Energetics by Quantum Chemical Calculations.

Reaction pathways and energetics for the dimerization and trimerization reactions of 2-bromo-3-methoxythiophene (2Br-3Met) molecules are investigated using hybrid density functional theory (DFT) calculations to obtain insight into the oligomerization reaction observed in the spontaneous combustion of pure liquid 2Br-3Met. The calculations show that the carbon-bromine bond in a 2Br-3Met molecule elongates easily, and the trans addition of this C-Br bond to a double bond in the neighboring 2Br-3Met molecule occurs easily at room temperature, reflecting the evaluated activation energy of ΔHa = 12.46 kcal/mol (enthalpy) or ΔGa = 35.68 kcal/mol (Gibbs free energy, 298.150 K and 1 atm). The formation process of trimers is calculated in a similar way. A model for the explanation of spontaneous combustion is proposed; large oligomers of the 2Br-3Met molecule are produced spontaneously following the initial formation of dimers or trimers. UV-vis spectra and vibration spectra are obtained for related molecular species, which show reasonable agreement with the experimental results.

Three-Dimensional Live Imaging of Bovine Preimplantation Embryos: A New Method for IVF Embryo Evaluation.

Conception rates for transferred bovine embryos are lower than those for artificial insemination. Embryo transfer (ET) is widely used in cattle but many of the transferred embryos fail to develop, thus, a more effective method for selecting bovine embryos suitable for ET is required. To evaluate the developmental potential of bovine preimplantation embryos (2-cell stage embryos and blastocysts), we have used the non-invasive method of optical coherence tomography (OCT) to obtain live images. The images were used to evaluate 22 parameters of blastocysts, such as the volume of the inner cell mass and the thicknesses of the trophectoderm (TE). Bovine embryos were obtained by in vitro fertilization (IVF) of the cumulus-oocyte complexes aspirated by ovum pick-up from Japanese Black cattle. The quality of the blastocysts was examined under an inverted microscope and all were confirmed to be Code1 according to the International Embryo Transfer Society standards for embryo evaluation. The OCT images of embryos were taken at the 2-cell and blastocyst stages prior to the transfer. In OCT, the embryos were irradiated with near-infrared light for a few minutes to capture three-dimensional images. Nuclei of the 2-cell stage embryos were clearly observed by OCT, and polynuclear cells at the 2-cell stage were also clearly found. With OCT, we were able to observe embryos at the blastocyst stage and evaluate their parameters. The conception rate following OCT (15/30; 50%) is typical for ETs and no newborn calves showed neonatal overgrowth or died, indicating that the OCT did not adversely affect the ET. A principal components analysis was unable to identify the parameters associated with successful pregnancy, while by using hierarchical clustering analysis, TE volume has been suggested to be one of the parameters for the evaluation of bovine embryo. The present results show that OCT imaging can be used to investigate time-dependent changes of IVF embryos. With further improvements, it should be useful for selecting high-quality embryos for transfer.

Three-dimensional live imaging of bovine embryos by optical coherence tomography.

While embryo transfer (ET) is widely practiced, many of the transferred embryos fail to develop in cattle. To establish a more effective method for selecting bovine embryos for ET, here we quantified morphological parameters of living embryos using three-dimensional (3D) images non-invasively captured by optical coherence tomography (OCT). Seven Japanese Black embryos produced by in vitro fertilization that had reached the expanded blastocyst stage after 7 days of culture were transferred after imaged by OCT. Twenty-two parameters, including thickness and volumes of the inner cell mass, trophectoderm, and zona pellucida, and volumes of blastocoel and whole embryo, were quantified from 3D images. Four of the seven recipients became pregnant. We suggest that these 22 parameters can be potentially employed to evaluate the quality of bovine embryos before ET.

Identification of a repressor for the two iol operons required for inositol catabolism in Geobacillus kaustophilus.

Geobacillus kaustophilus HTA426, a thermophilic Gram-positive bacterium, feeds on inositol as its sole carbon source, and an iol gene cluster required for inositol catabolism has been postulated with reference to the iol genes in Bacillus subtilis. The iol gene cluster of G. kaustophilus comprises two tandem operons induced in the presence of inositol; however, the mechanism underlying this induction remains unclear. B. subtilis iolQ is known to be involved in the regulation of iolX encoding scyllo-inositol dehydrogenase, and its homologue in HTA426 was found two genes upstream of the first gene (gk1899) of the iol gene cluster and was termed iolQ in G. kaustophilus. When iolQ was inactivated in G. kaustophilus, not only cellular myo-inositol dehydrogenase activity due to gk1899 expression but also the transcription of the two iol operons became constitutive. IolQ was produced and purified as a C-terminal histidine (His)-tagged fusion protein in Escherichia coli and subjected to an in vitro gel electrophoresis mobility shift assay to examine its DNA-binding property. It was observed that IolQ bound to the DNA fragments containing each of the two iol promoter regions and that DNA binding was antagonized by myo-inositol. Moreover, DNase I footprinting analyses identified two tandem binding sites of IolQ within each of the iol promoter regions. By comparing the sequences of the binding sites, a consensus sequence for IolQ binding was deduced to form a palindrome of 5'-RGWAAGCGCTTSCY-3' (where R=A or G, W=A or T, S=G or C, and Y=C or T). IolQ functions as a transcriptional repressor regulating the induction of the two iol operons responding to myo-inositol.

Identification of methionine -rich insoluble proteins in the shell of the pearl oyster, Pinctada fucata.

The molluscan shell is a biomineral that comprises calcium carbonate and organic matrices controlling the crystal growth of calcium carbonate. The main components of organic matrices are insoluble chitin and proteins. Various kinds of proteins have been identified by solubilizing them with reagents, such as acid or detergent. However, insoluble proteins remained due to the formation of a solid complex with chitin. Herein, we identified these proteins from the nacreous layer, prismatic layer, and hinge ligament of Pinctada fucata using mercaptoethanol and trypsin. Most identified proteins contained a methionine-rich region in common. We focused on one of these proteins, NU-5, to examine the function in shell formation. Gene expression analysis of NU-5 showed that NU-5 was highly expressed in the mantle, and a knockdown of NU-5 prevented the formation of aragonite tablets in the nacre, which suggested that NU-5 was required for nacre formation. Dynamic light scattering and circular dichroism revealed that recombinant NU-5 had aggregation activity and changed its secondary structure in the presence of calcium ions. These findings suggest that insoluble proteins containing methionine-rich regions may be important for scaffold formation, which is an initial stage of biomineral formation.

Triple nostrils in a calf.

BACKGROUND: Nasal abnormalities are rare in bovines. In humans, nasal deformities are mainly classified as proboscis lateralis or supernumerary nostrils. This report discusses the etiology of triple nostrils in a calf, based on computed tomography, magnetic resonance imaging, and endoscopy. CASE PRESENTATION: A female Holstein calf presented with triple nostrils. The following abnormalities were observed: (1) formation of a small and flat blind-ended middle nostril between the right and left nostrils; (2) presence of a hair-bearing surface on the muzzle; (3) abnormal curvature of the nasal septum, resulting in a narrower right nasal cavity due to transformation of the nasal bones; and (4) formation of a bone-like structure within the nasal septum. These findings were similar to those of supernumerary nostrils in humans. CONCLUSIONS: To the best of our knowledge, this represents the first description of a calf with triple nostrils. The use of imaging modalities is necessary for investigating the etiology of triple nostrils.

Multiple roles of hypoxia in bovine corpus luteum.

There has been increasing interest in the role of hypoxia in the microenvironment of organs, because of the discovery of hypoxia-inducible factor-1 (HIF1), which acts as a transcription factor for many genes activated specifically under hypoxic conditions. The ovary changes day by day during the estrous cycle as it goes through phases of follicular growth, ovulation, and formation and regression of the corpus luteum (CL). These phenomena are regulated by hypothalamic and pituitary hormones, sex steroids, peptides and cytokines, as well as oxygen conditions. Hypoxia strongly induces angiogenesis via transcription of a potent angiogenic factor, vascular endothelial growth factor (VEGF), that is regulated by HIF1. A CL forms with a rapid increase of angiogenesis that is mainly induced by HIF1-VEGF signaling. Hypoxia also contributes to luteolysis by down-regulating progesterone synthesis and by up-regulating apoptosis of luteal cells. This review focuses on recent studies on the roles of hypoxia- and HIF1-regulated genes in the regulation of bovine CL function.

Research of postpartum endometritis in Japanese Black cattle with cystic ovarian disease by vaginal mucus test and endometrial cytology.

The relationship between endometritis and cystic ovarian disease (COD) is still unclear in Japanese Black cattle. Endometritis is classified into clinical endometritis (CE) and subclinical endometritis (SE). The objective of this study was to clarify the interaction between postpartum endometritis (CE and SE) and COD in Japanese Black cattle. Twenty-six suckled cows with COD (COD group) and 16 suckled cows with cyclical ovarian activity (CA group) were submitted for the experiment. Uterine conditions of cows were classified into three groups (normal, CE, and SE) with vaginal mucus test and endometrial cytology. The combined data of CE and SE were represented as data for total endometritis (EMT total). The prevalence of EMT total in the COD group (42.3 %, 11 / 26 ) was significantly higher than that of the CA group (12.5 %, 2 / 16 ). The mean percentage of polymorphonuclear neutrophils (PMN %) in the COD group was significantly higher than that of the CA group at 40-60 DPP (days postpartum). Compared to 61-295 DPP, the mean PMN % at 40-60 DPP was significantly higher in the COD group. The diameters of uterine horn and cervix did not differ among normal uterine condition, CE and SE in the COD group, and they did not differ between normal uterine condition and SE in the CA group. However, endometrial thickness during both 40-60 and 61-295 DPP were greater in the COD group than in the CA group. In conclusion, Japanese Black cattle with COD have a potential implication on endometritis at 40-60 DPP compared to the normal ovarian cycle. As a specific symptom was not observed by transrectal ultrasonography, endometrial cytology is effective for diagnosis of SE in Japanese Black cattle.

Photoinduced swing of a diarylethene thin broad sword shaped crystal: a study on the detailed mechanism.

We report a swinging motion of photochromic thin broad sword shaped crystals upon continuous irradiation with UV light. By contrast in thick crystals, photosalient phenomena were observed. The bending and swinging mechanisms are in fact due to molecular size changes as well as phase transitions. The first slight bending away from the light source is due to photocyclization-induced surface expansion, and the second dramatic bending toward UV incidence is due to single-crystal-to-single-crystal (SCSC) phase transition from the original phase I to phase IIUV. Upon visible light irradiation, the crystal returned to phase I. A similar SCSC phase transition with a similar volume decrease occurred by lowering the temperature (phase IIItemp). For both photoinduced and thermal SCSC phase transitions, the symmetry of the unit cell is lowered; in phase IIUV the twisting angle of disordered phenyl groups is different between two adjacent molecules, while in phase IIItemp, the population of the phenyl rotamer is different between adjacent molecules. In the case of phase IIUV, we found thickness dependent photosalient phenomena. The thin broad sword shaped crystals with a 3 µm thickness showed no photosalient phenomena, whereas photoinduced SCSC phase transition occurred. In contrast, large crystals of several tens of µm thickness showed photosalient phenomena on the irradiated surface where SCSC phase transition occurred. The results indicated that the accumulated strain, between isomerized and non-isomerized layers, gave rise to the photosalient phenomenon.

A unique methionine-rich protein-aragonite crystal complex: Structure and mechanical functions of the Pinctada fucata bivalve hinge ligament.

The bivalve hinge ligament holds the two shells together. The ligament functions as a spring to open the shells after they were closed by the adductor muscle. The ligament is a mineralized tissue that bears no resemblance to any other known tissue. About half the ligament is composed of a protein-rich matrix, and half of long and extremely thin segmented aragonite crystals. Here we study the hinge ligament of the pearl oyster Pinctada fucata. FIB SEM shows that the 3D organization is remarkably ordered. The full sequence of the major protein component contains a continuous segment of 30 repeats of MMMLPD. There is no known homologous protein. Knockdown of this protein prevents crystal formation, demonstrating that the integrity of the matrix is necessary for crystals to form. X-ray diffraction shows that the aragonite crystals are more aligned in the compressed ligament, indicating that the crystals may be actively contributing to the elastic properties. The fusion interphase that joins the ligament to the shell nacre is composed of a prismatic mineralized tissue with a thin organic-rich layer at its center. Nanoindentation of the dry interphase shows that the elastic modulus of the nacre adjacent to the interphase gradually decreases until it approximates that of the interphase. The interphase modulus slightly increases until it matches the ligament. All these observations demonstrate that the ligament shell complex is a remarkable biological tissue that has evolved unique properties that enable bivalves to open their shell effectively innumerable times during the lifetime of the animal. STATEMENT OF SIGNIFICANCE: The hinge ligament shell complex is a unique functioning structural tissue whose elastic properties enable the shell to open without expending energy. Methionine-rich proteins are not known elsewhere raising fundamental questions about secondary and tertiary structures contributing to its elastic properties. The segmented and extremely thin aragonite crystals embedded in this matrix may also have unexpected elastic materials properties as they flex during compression. The structure of the interphase comprises a fascinating biological joint that connects two very different materials. The interphase materials, including the nacre, are graded with respect to elastic modulus so as to approximately match the connecting components. The interphase incorporates a thin organic rich layer that presumably functions as a gasket. This study raises many fundamental questions relevant to the diverse fields of protein chemistry, biomineralization and biological materials.

Crystal Growth Technique for Formation of Double Roughness Structures Mimicking Lotus Leaf.

Bio-inspired functional materials have received much attention for their potential to provide sustainable and advanced materials. The lotus effect has proven to be one of the most remarkable biomimetic effects since it was discovered by Barthlott. A superhydrophobic surface with the ability to bounce water droplets is the origin of the self-cleaning mechanism that keeps the surface clean by removing dust using water droplets moving with momentum. We have developed a crystal growth technique (CGT) of photochromic diarylethenes over the past decade, and from this, we fabricated a surface structure that closely resembles the natural lotus leaf's characteristic of controlling the Laplace pressure and clarified the importance of the double roughness structure of the surface. The bouncing ability is also discussed in terms of the characteristic size of the double roughness structure theoretically. Moreover, this work clarifies the exquisiteness of the double roughness structure of the leaf. We also show that the CGT is a versatile technique with the potential to fabricate desired structured surfaces.

Aggregation-induced emission effect on turn-off fluorescent switching of a photochromic diarylethene.

Background: Diarylethenes are well-known photochromic compounds, which undergo cyclization and cycloreversion reactions between open- and closed-ring isomers. Recently, diarylethene derivatives with photoswitchable fluorescent properties were prepared. They are applicable for fluorescence imaging including bio-imaging. On the other hand, a new system called "excited state intramolecular proton transfer (ESIPT)" is reported. In the system, absorption and emission bands are largely separated due to the proton transfer, hence it showed strong fluorescence even in the crystalline state. We aimed to construct the photochromic system incorporating the ESIPT mechanism. Results: A diarylethene incorporating a fluorescent moiety that exhibit ESIPT behavior was prepared. The ESIPT is one of the examples which express the mechanisms of aggregation-induced emission (AIE). This compound emits orange fluorescence with a large Stokes shift derived from ESIPT in aprotic solvents such as THF or hexane, while it exhibits only a photochromic reaction in protic solvents such as methanol. In addition, it shows turn-off type fluorescence switching in an aprotic solvent and in crystals. The fluorescence is quenched as the content of closed-ring isomers increases upon UV light irradiation. Conclusions: A diarylethene containing an ESIPT functional group was prepared. It showed fluorescent turn-off behavior during photochromism in aprotic solvents as well as in crystalline state upon UV light irradiation. Furthermore, it showed AIE in THF/water mixtures with blue-shift of the emission.

Object Transportation System Mimicking the Cilia of Paramecium aurelia Making Use of the Light-Controllable Crystal Bending Behavior of a Photochromic Diarylethene.

The design of an object transportation system exploiting the bending behavior of surface-assembled diarylethene crystals is reported. A photoactuated smart surface based on this system can transport polystyrene beads to a desired area depending on the direction of the incident light. Two main challenges were addressed to accomplish directional motion along a surface: first, the preparation of crystals whose bending behavior depends on the direction of incident light; second, the preparation of a film on which these photochromic crystal plates are aligned. Nuclei generation and nuclear growth engineering were achieved by using a roughness-controlled dotted microstructured substrate. This system demonstrates how to achieve a mechanical function as shown by remote-controlled motion along a surface.

Effect of hypoxia on progesterone production by cultured bovine early and mid luteal cells.

A major role of the corpus luteum (CL) is to produce progesterone (P4). The CL has immature vasculature shortly after ovulation, suggesting it exists under hypoxic conditions. To elucidate the mechanism involved in regulation of luteal cell function during CL development, we compared the effect of hypoxia on P4 production by cultured bovine early and mid luteal cells. Luteal cells obtained from early and mid CL were incubated under different O2 concentrations (20% and 3%) with or without hCG (1 U/ml) for 6 h and 24 h. After 6 h of culture in the presence of hCG, P4 production was not affected by hypoxia whereas decrease in its production by mid luteal cells was observed. After 24 h of culture, P4 production was significantly decreased by hypoxia in both stages of luteal cells regardless of the use of hCG. At 6 h of culture, hypoxia increased mRNA expression of hydroxyl-Δ-5-steroid dehydrogenase, 3ß- and steroid Δ-isomerase 1 (HSD3B1) in early luteal cells, and decreased mRNA expression of cytochrome P450 cholesterol side chain cleavage (CYP11A1) enzyme in mid luteal cells. At 24 h of culture, mRNA expressions of steroidogenic acute regulatory protein (STAR), CYP11A1, and HSD3B1 were not affected by hypoxia in both stages of luteal cells. The overall results suggest that early luteal cells maintain P4 production under hypoxic conditions, and hypoxia-induced HSD3B1 may support this P4 production in the bovine early CL.