Quantitative Chemical Analysis of Defensive Secretion of Megacrania tsudai (Phasmatidae) and Effect of Actinidine on its Potential Predators.

Insects avoid predation in various ways, and some use multiple ways to avoid predation. However, the effects of comprehensive avoidance methods and the differences in avoidance methods among different life stages of insects have not been sufficiently discussed. The big head stick insect Megacrania tsudai uses background matching as its primary defense and chemical defense as its secondary defense. The aims of this study were to identify and isolate the chemical components of M. tsudai using repeatable methods, determine the amount of the main chemical compound, and reveal the effect of the main chemical compound on its predators. We established a repeatable gas chromatography-mass spectrometry (GC-MS) method to identify the chemical compounds of these secretions, and identified actinidine as the main compound. Actinidine was identified by nuclear magnetic resonance (NMR), and the amount of actinidine in each instar was calculated by constructing a calibration curve using pure actinidine. Mass ratios did not drastically change among instars. Furthermore, experiments involving dropping an aqueous solution of actinidine demonstrated removal behavior in geckos, frogs, and spiders. These results indicated that M. tsudai conducts secondary defenses using defensive secretions consisting mainly of actinidine.

A method of producing genetically manipulated mouse mammary gland.

BACKGROUND: To obtain a deep understanding of the mechanism by which breast cancer develops, the genes involved in tumorigenesis should be analyzed in vivo. Mouse mammary gland can regenerate completely from a mammary stem cell (MaSC), which enables us to analyze the effect of gene expression and repression on tumorigenesis in mammary gland regenerated from genetically manipulated MaSCs. Although lentiviral and retroviral systems have usually been applied for gene transduction into MaSCs, they are associated with difficulty in introducing long, repeated, or transcriptional termination sequences. There is thus a need for an easier and quicker gene delivery system. METHODS: We devised a new system for gene delivery into MaSCs using the piggyBac transposon vectors and electroporation. Compared with viral systems, this system enables easier and quicker transfection of even long, repeated, or transcriptional termination DNA sequences. We designed gene expression vectors of the transposon system, equipped with a luciferase (Luc) expression cassette for monitoring gene transduction into regenerative mammary gland in mice by in-vivo imaging. A doxycycline (Dox)-inducible system was also integrated for expressing the target gene after mammary regeneration to mimic the actual mechanism of tumorigenesis. RESULTS: With this new gene delivery system, genetically manipulated mammary glands were successfully reconstituted even though the vector size was > 200 kb and even in the presence of DNA elements such as promoters and transcription termination sequences, which are major obstacles to viral vector packaging. They differentiated correctly into both basal and luminal cells, and showed normal morphological change and milk production after pregnancy, as well as self-renewal capacity. Using the Tet-On system, gene expression can be controlled by the addition of Dox after mammary reconstitution. In a case study using polyoma-virus middle T antigen (PyMT), oncogene-induced tumorigenesis was achieved. The histological appearance of the tumor was highly similar to that of the mouse mammary tumor virus-PyMT transgenic mouse model. CONCLUSIONS: With this system, gene transduction in the mammary gland can be easily and quickly achieved, and gene expression can be controlled by Dox administration. This system for genetic manipulation could be useful for analyzing genes involved in breast cancer.

The expression of IGF-1R in Helicobacter pylori-infected intestinal metaplasia and gastric cancer.

Overexpression of IGF-1R has been demonstrated in gastrointestinal cancers, and its expression is reported as the result of the loss of tumor suppressors. IL-16 is involved in the pathophysiological process of chronic inflammatory diseases. The aim of this study is to determine the changes in the expression of IGF-1R in intestinal metaplasia (IM) and gastric cancer (GC) as well as the effect of Helicobacter pylori (H. pylori) and IL-16 on cell proliferation and IGF-1R expression in gastric cells. AGS cells were incubated with combinations of IL-16 and H. pylori. Gastric cell proliferation was studied by BrdU uptake. In H. pylori infected mucosa, IGF-1R was significantly higher in IM than chronic gastritis (CG), and also higher in GC than CG and IM. H. pylori significantly decreased BrdU uptake. IL-16 increased BrdU uptake and IGF-1R on AGS cells which had been decreased by H. pylori. Co-incubation with IL-16 increased the expression of IGF-1R mRNA in H. pylori infected cells. We conclude that the expression of IGF-1R in H. pylori infected gastric mucosa may indicate an early stage of carcinogenesis. The IL-16 secretion by H. pylori can be a trigger for the expression of IGF-1R, and it may also be a factor for gastric carcinogenesis.

Diastereo- and regioselective addition of thioamide dianions to imines and aziridines: synthesis of N-thioacyl-1,2-diamines and N-thioacyl-1,3-diamines.

Addition reactions of thioamide dianions that were derived from N-arylmethyl thioamides to imines and aziridines were carried out. The reactions of imines gave the addition products of N-thioacyl-1,2-diamines in a highly diastereoselective manner in good-to-excellent yields. The diastereomeric purity of these N-thioacyl-1,2-diamines could be enriched by simple recrystallization. The reduction of N-thioacyl-1,2-diamines with LiAlH(4) gave their corresponding 1,2-diamines in moderate-to-good yields with retention of their stereochemistry. The oxidative-desulfurization/cyclization of an N-thioacyl-1,2-diamine in CuCl(2)/O(2) and I(2)/pyridine systems gave the cyclized product in moderate yield and the trans isomer was obtained as the sole product. On the other hand, a similar cyclization reaction with antiformin (aq. NaClO) as an oxidant gave the cis isomer as the major product. The reactions of N-tosylaziridines gave the addition products of N-thioacyl-1,3-diamines with low diastereoselectivity but high regioselectivity and in good-to-excellent yields. The use of AlMe(3) as an additive improved the efficiency and regioselectivity of the reaction. The stereochemistry of the obtained products was determined by X-ray diffraction.

Effect of filler load on structural coloration and color adjustment potential of resin composites.

The color-matching ability of single-shade composites Omnichroma (OMN) and Omnichroma Flow (OCF) is ensured by structural color, which arises from monodisperse 260 nm spherical fillers. This study evaluated how filler load influences the color adjustment potential of composite restorations to human teeth of various shades. The performance of composites containing the 260 nm spherical fillers -OMN (79 wt%), OCF (71 wt%), and experimental composites R1 (65 wt%), and R2 (60 wt%)- was compared to two conventional A2-shade composites and a transparent composite. Additionally, the translucency parameter, spectral reflectance, and light transmission properties were assessed. Composites with a lower load of the 260 nm spherical fillers exhibited lower light diffusion and lower reflectance in the yellow-to-red range (580-650 nm), which is characteristic for the structural color of OMN and OCF. The best color adjustment was achieved with OCF, presumably due to its high translucency and uniform spectral reflectance.

Does the thickness of universal-shade composites affect the ability to reflect the color of background dentin?

The recently introduced resin composites with a universal shade are claimed to match any tooth color. In this study, it was examined how composite thickness affects the color adjustment of conventional and universal-shade composites to background dentin. Thirty sound human central incisors of various shade were used, and color differences between their intact labial surfaces, exposed dentin surfaces and composite restorations (thickness 1-3 mm) were evaluated. In addition, the translucency, light transmission characteristics, and spectral reflectance of the composites were measured. The results showed that universal-shade composites outperformed conventional composites of A2 shade in the adjustment of hue and chroma (p<0.05) but not in lightness (p>0.05). The color adjustment potential of all composites significantly decreased as their thickness increased (p<0.05). The effect of thickness on Omnichroma and Omnichroma Flow was less marked, presumably because of their higher translucency (p<0.05) and structural coloration which induces light reflectance in the yellow-to-red range.

First record of non-flying mammalian contributors to pollination in a tropical montane forest in Asia.

This study aims to identify the flower visitors of Mucuna thailandica (Fabaceae), endemic plant species in montane forests in Thailand, to determine their potential pollinators. The genus Mucuna produces papilionaceous flowers and has an explosive flower-opening step. Explosive opening rapidly exposes stamens and pistil from keel petals and releases pollen. The flower of this species depends completely on animals to perform this step, essential for pollination success. Using a camera trap survey, we revealed that non-flying mammals, such as squirrels (Callosciurus sp.) and masked palm civets (Paguma larvata), opened flowers explosively. Thus, these mammals contribute to the pollination of M. thailandica. This is the first report of non-flying mammals contributing to pollination in montane forests in tropical Asia.

Color adjustment potential of single-shade resin composite to various-shade human teeth: Effect of structural color phenomenon.

This study evaluated the effect of the structural color phenomenon in resin composites (RCs) on the color adjustment of restorations by investgating their color reproduction performance in human incisors of various shade. Cervical cavities were filled with a single-shade RC with 260 nm spherical fillers (Omnichroma (OMN)), conventional A2-shade RCs (Estelite Σ Quick or Clearfil AP-X), or experimental RCs with 5-50 nm fumed silica fillers (R1) and 100 nm spherical fillers (R2). Color parameters (L*C*h*) were measured using a CIE XYZ camera along the centerline of the restorations, and the color difference (∆E00) between corresponding areas of intact and restored teeth was calculated. Additionally, the reflectance spectra of OMN, R1, and R2 were investigated. OMN exhibited significantly lower ∆E00 than other tested RCs (p<0.05) and its reflection spectrum ranged from blue to red, while a blue peak was observed with R1 and R2, indicating a higher color adjustment potential of OMN.

Trapped radical behavior of electron beam irradiated polytetrafluoroethylene fine powder at various temperatures.

Polytetrafluoroethylene (PTFE) fine powder with 93% crystallinity was irradiated by an electron beam (EB) at various temperatures under a nitrogen atmosphere. Trapped free radicals in PTFE were studied using electron spin resonance (ESR) spectroscopy. The observed spectra of the samples exposed to air after irradiation at various temperatures showed asymmetrical signals, which are middle-chain type peroxide macroradicals derived from fluoroalkyl radicals. The radical yields at each irradiation temperature increased with increasing absorbed dose, and eventually saturated. The higher irradiation temperature resulted in higher radical yields when compared at the same exposed dose. Furthermore, the G-value of the radicals (G(R·)) increases with increasing irradiation temperatures corresponding to each relaxation and transition temperature. It is concluded that the chain reaction by the fluorine extraction from the main chain due to the end-chain radical generated via ß-scission after dissociative electron attachment (DEA) is enhanced by the synergistic effect of heat and radiation.

The pollination system of the widely distributed mammal-pollinated Mucuna macrocarpa (Fabaceae) in the tropics.

Although the pollinators of some plant species differ across regions, only a few mammal-pollinated plant species have regional pollinator differences in Asia. Mucuna macrocarpa (Fabaceae) is pollinated by squirrels, flying foxes, and macaques in subtropical and temperate islands. In this study, the pollination system of M. macrocarpa was identified in tropical Asia, where the genus originally diversified. This species requires "explosive opening" of the flower, where the wing petals must be pressed down and the banner petal pushed upward to fully expose the stamens and pistil. A bagging experiment showed that fruits did not develop in inflorescences (n = 66) with unopened flowers, whereas fruits developed in 68.7% of inflorescences (n = 131) with opened flowers. This indicated that the explosive opening is needed for the species to reproduce. Four potential pollinator mammals were identified by a video camera-trap survey, and 78.3% and 60.1% of monitored inflorescences (n = 138) were opened by gray-bellied squirrels (Callosciurus caniceps) and Finlayson's squirrels (C. finlaysonii), respectively, even though more than 10 mammal species visited flowers. Nectar was surrounded by the calyx, and the volume and sugar concentration of secreted nectar did not change during the day. This nectar secretion pattern is similar to those reported by previous studies in other regions. These results showed that the main pollinators of M. macrocarpa in the tropics are squirrels. However, the species' nectar secretion pattern is not specifically adapted to this particular pollinator. Pollinators of M. macrocarpa differ throughout the distribution range based on the fauna present, but there might not have been no distinctive changes in the attractive traits that accompanied these changes in pollinators.

Full Factorial Microfluidic Designs and Devices for Parallelizing Human Pluripotent Stem Cell Differentiation.

Human pluripotent stem cells (hPSCs) are promising therapeutic tools for regenerative therapies and disease modeling. Differentiation of cultured hPSCs is influenced by both exogenous factors added to the cultures and endogenously secreted molecules. Optimization of protocols for the differentiation of hPSCs into different cell types is difficult because of the many variables that can influence cell fate. We present microfluidic devices designed to perform three- and four-factor, two-level full factorial experiments in parallel for investigating and directly optimizing hPSC differentiation. These devices feature diffusion-isolated, independent culture wells that allow for control of both exogenous and endogenous cellular signals and that allow for immunocytochemistry (ICC) and confocal microscopy in situ. These devices are fabricated by soft lithography in conjunction with 3D-printed molds and are operable with a single syringe pump, eliminating the need for specialized equipment or cleanroom facilities. Their utility was demonstrated by on-chip differentiation of hPSCs into the auditory neuron lineage. More broadly, these devices enable multiplexing for experimentation with any adherent cell type or even multiple cell types, allowing efficient investigation of the effects of medium conditions, pharmaceuticals, or other soluble reagents.

Efficacy and Safety of Adsorptive Granulocyte and Monocyte Apheresis in Elderly and Pregnant Patients With Ulcerative Colitis.

In patients with active ulcerative colitis (UC), adsorptive granulocyte/monocyte apheresis (GMA) is expected to promote remission. We conducted a retrospective cohort study to evaluate the efficacy and safety of GMA in patients with active UC. Twenty-one UC patients including five pregnant or lactating mothers and four elderly patients (aged >60 years) received up to 10 GMA sessions. UC severity was evaluated at baseline and after GMA therapy according to Lichtiger's Clinical Activity Index (CAI). We defined clinical remission as CAI ≤4. Overall, the median CAI score after GMA therapy had decreased from 9 to 4 (P < 0.001). The clinical remission rate was 62%, but in the elderly and pregnant or lactating mothers, the remission rates were 100% and 60%, respectively. No severe adverse effects were seen in this study. Our results may support GMA as an effective and safe treatment for active UC patients, including elderly patients and pregnant cases.

Endoscope images of Schönlein-Henoch purpura.

BACKGROUND/AIMS: Schönlein-Henoch purpura (SHP) is a systemic condition characterized by purpura associated with leukocytoclastic vasculitis. SHP diagnosis is more difficult in infrequent cases where gastrointestinal (GI) symptoms precede purpura. This report examines 11 cases of SHP at our hospital with specific regard to the incidences and details of GI symptoms. METHODS: The clinical manifestations and endoscopic findings were investigated for their utility in SHP diagnosis. RESULTS: Among the 11 cases, 3 showed GI symptoms prior to other manifestations. In terms of GI symptoms, abdominal pain was reported in all 11 cases, diarrhea in 4 cases, and bloody stools in 3 cases. Endoscopic findings were seen in the stomach in 7/10 cases, in the small intestine including the duodenum in 10/11 cases, and in the large intestine in 6/10 cases. The frequency of ulcer formation was significantly higher in the small intestine (including the duodenum) than in the stomach. Multiple specific erythematosus lesions were observed in the stomach and large intestine. CONCLUSION: Familiarity with characteristic endoscopic findings and careful observation of all GI findings are essential for diagnosing SHP in cases in which GI tract symptoms precede cutaneous findings.

Floral traits of mammal-pollinated Mucuna macrocarpa (Fabaceae): Implications for generalist-like pollination systems.

Floral traits are adapted by plants to attract pollinators. Some of those plants that have different pollinators in different regions adapt to each pollinator in each region to maximize their pollination success. Mucuna macrocarpa (Fabaceae) limits the pollinators using its floral structure and is pollinated by different mammals in different regions. Here, we examine the relationships between floral traits of M. macrocarpa and the external morphology of mammalian pollinators in different regions of its distribution. Field surveys were conducted on Kyushu and Okinawajima Island in Japan, and in Taiwan, where the main pollinators are the Japanese macaque Macaca fuscata, Ryukyu flying fox Pteropus dasymallus, and red-bellied squirrel Callosciurus erythraeus, respectively. We measured the floral shapes, nectar secretion patterns, sugar components, and external morphology of the pollinators. Results showed that floral shape was slightly different among regions and that flower sizes were not correlated with the external morphology of the pollinators. Volume and sugar rate of nectar were not significantly different among the three regions and did not change throughout the day in any of the regions. However, nectar concentration was higher in Kyushu than in the other two regions. These results suggest that the floral traits of M. macrocarpa are not adapted to each pollinator in each region. Although this plant limits the number of pollinators using its flower structure, it has not adapted to specific mammals and may attract several species of mammals. Such generalist-like pollination system might have evolved in the Old World.

Nitric Oxide Production from Nitrite Reduction and Hydroxylamine Oxidation by Copper-containing Dissimilatory Nitrite Reductase (NirK) from the Aerobic Ammonia-oxidizing Archaeon, Nitrososphaera viennensis.

Aerobic ammonia-oxidizing archaea (AOA) play a crucial role in the global nitrogen cycle by oxidizing ammonia to nitrite, and nitric oxide (NO) is a key intermediate in AOA for sustaining aerobic ammonia oxidation activity. We herein heterologously expressed the NO-forming, copper-containing, dissimilatory nitrite reductase (NirK) from Nitrososphaera viennensis and investigated its enzymatic properties. The recombinant protein catalyzed the reduction of 15NO2- to 15NO, the oxidation of hydroxylamine (15NH2OH) to 15NO, and the production of 14-15N2O from 15NH2OH and 14NO2-. To the best of our knowledge, the present study is the first to document the enzymatic properties of AOA NirK.

Atomically Flat Pt Skin and Striking Enrichment of Co in Underlying Alloy at Pt3Co(111) Single Crystal with Unprecedented Activity for the Oxygen Reduction Reaction.

By the use of in situ scanning tunneling microscopy and surface X-ray scattering techniques, we have clarified the surface structure and the layer-by-layer compositions of a Pt skin/Pt3Co(111) single-crystal electrode, which exhibited extremely high activity for the oxygen reduction reaction. The topmost layer was found to be an atomically flat Pt skin with (1 × 1) structure. Cobalt was enriched in the second layer up to 98 atom %, whereas the Co content in the third and fourth layers was slightly smaller than that in the bulk. By X-ray photoelectron spectroscopy, the Co in the subsurface layers was found to be positively charged, which is consistent with an electronic modification of the Pt skin. The extremely high activity at the Pt skin/Pt3Co(111) single crystal is correlated with this specific surface structure.

Suspected Hepatically Localized Granulomatosis with Polyangiitis.

The patient was a 72-year-old woman whose alkaline phosphatase levels had been elevated since she was 56 years old. Liver biopsies obtained when the patient was 64 and 66 years of age led to a suspicion of cholangitis caused by vasculitis. Furthermore, proteinase-3 anti-neutrophil cytoplasmic antibody positivity led to a suspicion of granulomatosis with polyangiitis, but subjective symptoms and disorders in other organs were absent, so this suspicion was not confirmed. Cholangitis caused by vasculitis rarely occurs without vasculitis in other organs. We herein report this case in which we obtained distinctive laparoscopic and imaging findings that raised suspicions of liver circulatory failure.

Directed Differentiation of Human Embryonic Stem Cells Toward Placode-Derived Spiral Ganglion-Like Sensory Neurons.

The ability to generate spiral ganglion neurons (SGNs) from stem cells is a necessary prerequisite for development of cell-replacement therapies for sensorineural hearing loss. We present a protocol that directs human embryonic stem cells (hESCs) toward a purified population of otic neuronal progenitors (ONPs) and SGN-like cells. Between 82% and 95% of these cells express SGN molecular markers, they preferentially extend neurites to the cochlear nucleus rather than nonauditory nuclei, and they generate action potentials. The protocol follows an in vitro stepwise recapitulation of developmental events inherent to normal differentiation of hESCs into SGNs, resulting in efficient sequential generation of nonneuronal ectoderm, preplacodal ectoderm, early prosensory ONPs, late ONPs, and cells with cellular and molecular characteristics of human SGNs. We thus describe the sequential signaling pathways that generate the early and later lineage species in the human SGN lineage, thereby better describing key developmental processes. The results indicate that our protocol generates cells that closely replicate the phenotypic characteristics of human SGNs, advancing the process of guiding hESCs to states serving inner-ear cell-replacement therapies and possible next-generation hybrid auditory prostheses. © Stem Cells Translational Medicine 2017;6:923-936.

Creating a stem cell niche in the inner ear using self-assembling peptide amphiphiles.

The use of human embryonic stem cells (hESCs) for regeneration of the spiral ganglion will require techniques for promoting otic neuronal progenitor (ONP) differentiation, anchoring of cells to anatomically appropriate and specific niches, and long-term cell survival after transplantation. In this study, we used self-assembling peptide amphiphile (PA) molecules that display an IKVAV epitope (IKVAV-PA) to create a niche for hESC-derived ONPs that supported neuronal differentiation and survival both in vitro and in vivo after transplantation into rodent inner ears. A feature of the IKVAV-PA gel is its ability to form organized nanofibers that promote directed neurite growth. Culture of hESC-derived ONPs in IKVAV-PA gels did not alter cell proliferation or viability. However, the presence of IKVAV-PA gels increased the number of cells expressing the neuronal marker beta-III tubulin and improved neurite extension. The self-assembly properties of the IKVAV-PA gel allowed it to be injected as a liquid into the inner ear to create a biophysical niche for transplanted cells after gelation in vivo. Injection of ONPs combined with IKVAV-PA into the modiolus of X-SCID rats increased survival and localization of the cells around the injection site compared to controls. Human cadaveric temporal bone studies demonstrated the technical feasibility of a transmastoid surgical approach for clinical intracochlear injection of the IKVAV-PA/ONP combination. Combining stem cell transplantation with injection of self-assembling PA gels to create a supportive niche may improve clinical approaches to spiral ganglion regeneration.

Population Dynamics and the Effects of Temperature on the Eggs of the Seawater-dispersed Stick Insect Megacrania tsudai (Phasmida: Phasmatidae).

Shun Kobayashi, Ryota Usui, Kouta Nomoto, Mineyuki Ushirokita, Tetsuo Denda, and Masako Izawa (2016) Although the stick insect Megacrania tsudai cannot fly, its eggs are dispersed by seawater, which allows the species to distribute itself widely via the ocean. The life history of this non-volant insect that establishes settlements by egg dispersal remains poorly understood. We aimed to clarify the population dynamics and effects of temperature on the eggs of M. tsudai through field observation in near the northern limits of the distribution and laboratory experiments. In the wild, all instars appeared over a period of several months, with a larger proportion of first instars emerging from December to March, and a greater number of adults being observed from June to August. Laboratory experiments showed that the hatching rate of M. tsudai was not different between conditions of 25 and 30°C, whereas it was significantly lower at 20°C. The egg-development period was shorter when eggs were maintained under high temperature conditions, and longer for eggs kept at 20°C. The effective cumulative temperature was estimated as 1561.5-2000.0°C, and the developmental zero was estimated as 12.2-13.9°C. The peak appearance of the first instar in the laboratory, which was used to obtain an estimate for the effective cumulative temperature and peak appearance of adults in the wild, was not entirely accurate because the peak appearance of the first instars in the wild was not consistently observed when the estimation indicated that they should appear. Megacrania tsudai is a univoltine near the northern limit of its distribution, and temperature has a strong effect on its egg development. Our estimation of population dynamics by laboratory experiments did not exactly predict what was observed in the field and it may be controlled by other factors. However, our findings indicate that the limiting factor of the distribution of this species is likely the effect of temperature on egg hatching.