Inhibitory Effect on RT-PCR and Restriction Enzyme Activity by Ommochrome and Its Mechanism.

To explore the physiological role and/or pharmacological effects of ommochrome, which is a natural organic pigment widely distributed in Protostomia, we attempted to investigate the influence of ommochrome on RT-PCR and activities of restriction enzymes. It was found that ommin, an ommochrome purified from the diapause eggs of Bombyx mori, inhibited the RT-PCR and restriction enzyme activities. The mechanism of these inhibitory reactions is assumed to be the direct binding of ommochrome to DNA rather than acting against the enzymes because, similarly to actinomycin D, there is a phenoxazine ring in the structure of ommin that is known to be intercalated to DNA. To reveal the ommin/DNA interaction, it was investigated by computational approaches such as molecular docking, molecular dynamics simulation, and free energy calculation. From the computational analyses, it was expected that ommin would bind to DNA with almost the same strength as actinomycin D and intercalate into DNA. This is the first report on the pharmacological effect of ommochrome and its inhibitory mechanism obtained from biochemical and computational analyses.

Comparative Studies on Electrodes for Rumen Bacteria Microbial Fuel Cells.

Microbial fuel cells (MFCs) using rumen bacteria have been proposed as a power source for running devices inside cattle. In this study, we explored the key parameters of the conventional bamboo charcoal electrode in an attempt to improve the amount of electrical power generated by the microbial fuel cell. We evaluated the effects of the electrode's surface area, thickness, and rumen content on power generation and determined that only the electrode's surface area affects power generation levels. Furthermore, our observations and bacterial count on the electrode revealed that rumen bacteria concentrated on the surface of the bamboo charcoal electrode and did not penetrate the interior, explaining why only the electrode's surface area affected power generation levels. A Copper (Cu) plate and Cu paper electrodes were also used to evaluate the effect of different electrodes on measuring the rumen bacteria MFC's power potential, which had a temporarily higher maximum power point (MPP) compared to the bamboo charcoal electrode. However, the open circuit voltage and MPP decreased significantly over time due to the corrosion of the Cu electrodes. The MPP for the Cu plate electrode was 775 mW/m2 and the MPP for the Cu paper electrode was 1240 mW/m2, while the MPP for bamboo charcoal electrodes was only 18.7 mW/m2. In the future, rumen bacteria MFCs are expected to be used as the power supply of rumen sensors.

Demonstration of a spherical plasma mirror for the counter-propagating kilojoule-class petawatt LFEX laser system.

A counter-propagating laser-beam platform using a spherical plasma mirror was developed for the kilojoule-class petawatt LFEX laser. The temporal and spatial overlaps of the incoming and redirected beams were measured with an optical interferometer and an x-ray pinhole camera. The plasma mirror performance was evaluated by measuring fast electrons, ions, and neutrons generated in the counter-propagating laser interaction with a Cu-doped deuterated film on both sides. The reflectivity and peak intensity were estimated as ∼50% and ∼5 × 1018 W/cm2, respectively. The platform could enable studies of counter-streaming charged particles in high-energy-density plasmas for fundamental and inertial confinement fusion research.

Molecular Characterization and Tissue Distribution of Calcineurin Regulatory B Subunit during the Prevention of Diapause in the Silkworm, Bombyx mori.

To clarify the molecular mechanism of prevention of entry into diapause in Bombyx mori by HCl treatment, we biochemically analyzed calcineurin regulatory B subunit (CNB) in diapause eggs treated with HCl solution. Our previous studies revealed that HCl treatment causes Ca2+ to efflux from diapause eggs. Therefore, we attempted to analyze CNB, which is known to associate with Ca2+. The gene expression level of CNB was increased by HCl treatment and the changes of the gene expression were almost the same as that in the non-diapause eggs. As for diapause eggs, almost no gene expression of CNB was confirmed except just after oviposition. In the assay for phosphorylation by protein kinase CK2, recombinant CNB (rCNB) was phosphorylated in vitro. Additionally, a Ca2+ binding assay indicated that rCNB shows affinity for Ca2+. The distribution of CNB was investigated with an immunohistochemical technique using antiserum against rCNB in diapause eggs and HCl-treated diapause eggs. CNB was localized in serosa cells and yolk cells in both eggs. These data may suggest that CNB is activated by intracellular Ca2+ or efflux Ca2+ resulting from HCl treatment, and that it plays a role in the molecular mechanisms of artificial diapause prevention or the breaking of diapause in the silkworm.

Quantum transport evidence of Weyl fermions in an epitaxial ferromagnetic oxide.

Magnetic Weyl semimetals have novel transport phenomena related to pairs of Weyl nodes in the band structure. Although the existence of Weyl fermions is expected in various oxides, the evidence of Weyl fermions in oxide materials remains elusive. Here we show direct quantum transport evidence of Weyl fermions in an epitaxial 4d ferromagnetic oxide SrRuO3. We employ machine-learning-assisted molecular beam epitaxy to synthesize SrRuO3 films whose quality is sufficiently high to probe their intrinsic transport properties. Experimental observation of the five transport signatures of Weyl fermions-the linear positive magnetoresistance, chiral-anomaly-induced negative magnetoresistance, π phase shift in a quantum oscillation, light cyclotron mass, and high quantum mobility of about 10,000 cm2V-1s-1-combined with first-principles electronic structure calculations establishes SrRuO3 as a magnetic Weyl semimetal. We also clarify the disorder dependence of the transport of the Weyl fermions, which gives a clear guideline for accessing the topologically nontrivial transport phenomena.

Chronic pancreatitis in farmed pigs fed excessive zinc oxide.

Most of the pigs on a farm in Aichi Prefecture, Japan had chronic diarrhea and severe wasting. The pigs had consumed 8,000 ppm zinc oxide (ZnO) as a feed additive. The pancreas of each of 4 autopsied pigs was less than half the normal size. Acinar cells were considerably decreased. Epithelial duct-like cells were increased and tested positive for cytokeratin AE1/AE3, Ki67, PGP9.5, and Sox9. Pancreatic islet cells were decreased and shrunken. The α and δ cells were relatively decreased, and their distribution was abnormal. Islet cells were positive for PGP9.5. The livers and kidneys had high accumulations of zinc (Zn; 788 µg/g and 613 µg/g, respectively). Copper was deficient in the liver, likely as a result of Zn poisoning. Our immunohistologic examination suggested that the high dose of ZnO could influence the function of islet cells in addition to that of acinar cells. Given that colistin sulfate has been banned as a feed additive in order to reduce antimicrobial use in Japan, the use of ZnO in the livestock industry is expected to increase. Zn supplementation of pig feed must be monitored to prevent Zn poisoning and contamination of soil and water.

Petapascal Pressure Driven by Fast Isochoric Heating with a Multipicosecond Intense Laser Pulse.

Fast isochoric laser heating is a scheme to heat matter with a relativistic intensity (>10^{18} W/cm^{2}) laser pulse for producing an ultrahigh-energy-density (UHED) state. We have demonstrated an efficient fast isochoric heating of a compressed dense plasma core with a multipicosecond kilojoule-class petawatt laser and an assistance of externally applied kilotesla magnetic fields for guiding fast electrons to the dense plasma. A UHED state of 2.2 PPa is achieved experimentally with 4.6 kJ of total laser energy that is one order of magnitude lower than the energy used in the conventional implosion scheme. A two-dimensional particle-in-cell simulation confirmed that diffusive heating from a laser-plasma interaction zone to the dense plasma plays an essential role to the efficient creation of the UHED state.

Evaluation of ruminal motility in cattle by a bolus-type wireless sensor.

We evaluated the relationship between ruminal motility measured by a force transducer and acceleration measured by bolus sensor, and we assessed the detection of ruminal motility in cattle by a bolus-type wireless sensor. The bolus sensor can be orally administered to cattle and was placed in the reticulum for continuous measurements. The probe was almost horizontal to the longitudinal axis. The bolus sensor's basic y-axis acceleration movement appeared to have a very distinct vertical pattern, occurring roughly 1-1.5 times/min with a duration of approximately 8 sec, displaying at around 500 mG. A significant positive correlation was observed between the ruminal contraction revealed by the force transducer and the acceleration shown by the bolus sensor (P<0.01). The contraction of the dorsal sac of the rumen and the acceleration signals in the reticulum occurred at practically the same time. The frequency and amplitude of ruminal contraction demonstrated by the bolus sensor and the force transducer in feeding were significantly higher than those at rest (P<0.01). The bolus sensor could also detect ruminal atony in the cattle after the administration of xylazine. A bolus-type wireless sensor may thus be useful for the measurement of ruminal motility in cattle and for detecting rumen dysfunction (e.g., ruminal atony).

Effect of mosapride on ruminal motility in cattle.

This study aimed to assess the effects of mosapride at various dosages on ruminal motility in cattle and the absorption kinetics of mosapride in cattle. Mosapride was rapidly absorbed after oral administration in cattle. Oral administration at dosages of 1, 3, and 10 mg/kg resulted in a dose-dependent increase in the detection time. At the 1 mg/kg dose, the motility index of rumen in cattle significantly increased at 60, 70, and 80 min (134 ± 20, 168 ± 37, 173 ± 45%, respectively), compared with that of the control. The administration of mosapride in cattle did not cause any subsequent adverse clinical signs or blood test abnormalities. It was suggested that mosapride enhanced ruminal motility without adverse effects in cattle.

Unique arrangement of bone matrix orthogonal to osteoblast alignment controlled by Tspan11-mediated focal adhesion assembly.

During tissue construction, cells coordinate extracellular matrix (ECM) assembly depending on the cellular arrangement. The traditional understanding of the relationship between the ECM and cells is limited to the orientation-matched interaction between them. Indeed, it is commonly accepted that the bone matrix (collagen/apatite) is formed along osteoblast orientation. Nonetheless, our recent findings are contrary to the above theory; osteoblasts on nanogrooves organize formation of the bone matrix perpendicular to cell orientation. However, the precise molecular mechanisms underlying the orthogonal organization of bone matrix are still unknown. Here, we show that mature fibrillar focal adhesions (FAs) facilitate the perpendicular arrangement between cells and bone matrix. The osteoblasts aligned along nanogrooves expressed highly mature fibrillar FAs mediated by integrin clustering. Microarray analysis revealed that Tspan11, a member of the transmembrane tetraspanin protein family, was upregulated in cells on the nanogrooved surface compared with that in cells on isotropic, flat, or rough surfaces. Tspan11 silencing significantly disrupted osteoblast alignment and further construction of aligned bone matrix orthogonal to cell orientation. Our results demonstrate that the unique bone matrix formation orthogonal to cell alignment is facilitated by FA maturation. To the best of our knowledge, this report is the first to show that FA assembly mediated by Tspan11 determines the direction of bone matrix organization.

Molecular Characterization and Tissue Distribution of Mitochondrial Ca2+ -Dependent Solute Carrier Protein during Prevention of Diapause by HCl Treatment in the Silkworm, Bombyx mori.

To clarify the molecular mechanism of prevention of entry into diapause in Bombyx mori by HCl treatment, we biochemically analyzed mitochondrial Ca2+ -dependent solute carrier protein (MCSC) in diapause eggs treated with HCl solution. Our previous studies revealed that HCl treatment causes Ca2+ to efflux from diapause eggs. Therefore, we attempted to analyze MCSC, which is known to associate with Ca2+ . The isolated cDNA of B. mori MCSC (BmMCSC) had an open reading flame (ORF) of 667 amino acid residues, and the ORF contained two EF-hand calcium-binding domains and three characteristic features of the mitochondrial solute carrier superfamily. The gene expression level of BmMCSC increased by HCl treatment. A Ca2+ binding assay indicated that recombinant BmMCSC (rBmMCSC) shows an affinity with Ca2 + . The distribution of BmMCSC was investigated with an immunohistochemical technique using antisera against BmMCSC in diapause eggs and HCl-treated diapause eggs. BmMCSC was localized in serosa cells in both eggs. These data may suggest that BmMCSC is activated by intracellular Ca2+ or efflux Ca2+ by HCl treatment, and that it plays a role in the molecular mechanisms of artificial diapause prevention or the breaking of diapause in the silkworm.

Magnetized fast isochoric laser heating for efficient creation of ultra-high-energy-density states.

Fast isochoric heating of a pre-compressed plasma core with a high-intensity short-pulse laser is an attractive and alternative approach to create ultra-high-energy-density states like those found in inertial confinement fusion (ICF) ignition sparks. Laser-produced relativistic electron beam (REB) deposits a part of kinetic energy in the core, and then the heated region becomes the hot spark to trigger the ignition. However, due to the inherent large angular spread of the produced REB, only a small portion of the REB collides with the core. Here, we demonstrate a factor-of-two enhancement of laser-to-core energy coupling with the magnetized fast isochoric heating. The method employs a magnetic field of hundreds of Tesla that is applied to the transport region from the REB generation zone to the core which results in guiding the REB along the magnetic field lines to the core. This scheme may provide more efficient energy coupling compared to the conventional ICF scheme.

Biologic Response of Colorectal Cancer Xenograft Tumors to Sequential Treatment with Panitumumab and Bevacizumab.

Recent studies in RAS wild-type (WT) metastatic colorectal cancer (mCRC) suggest that the survival benefits of therapy using anti-epidermal growth factor receptor (anti-EGFR) and anti-vascular endothelial growth factor (anti-VEGF) antibodies combined with chemotherapy are maximized when the anti-EGFR antibody is given as first-line, followed by subsequent anti-VEGF antibody therapy. We report reverse-translational research using LIM1215 xenografts of RAS WT mCRC to elucidate the biologic mechanisms underlying this clinical observation. Sequential administration of panitumumab then bevacizumab (PB) demonstrated a stronger tendency to inhibit tumor growth than bevacizumab then panitumumab (BP). Cell proliferation was reduced significantly with PB (P < .01) but not with BP based on Ki-67 index. Phosphoproteomic analysis demonstrated reduced phosphorylation of EGFR and EPHA2 with PB and BP compared with control. Western blotting showed reduced EPHA2 expression and S897-phosphorylation with PB; RSK phosphorylation was largely unaffected by PB but increased significantly with BP. In quantitative real-time PCR analyses, PB significantly reduced the expression of both lipogenic (FASN, MVD) and hypoxia-related (CA9, TGFBI) genes versus control. These results suggest that numerous mechanisms at the levels of gene expression, protein expression, and protein phosphorylation may explain the improved clinical activity of PB over BP in patients with RAS WT mCRC.

Li-ion diffusion in Li intercalated graphite C6Li and C12Li probed by µ+SR.

In order to study a diffusive behavior of Li+ in Li intercalated graphites, we have measured muon spin relaxation (µ+SR) spectra for C6Li and C12Li synthesized with an electrochemical reaction between Li and graphite in a Li-ion battery. For both compounds, it was found that Li+ ions start to diffuse above 230 K and the diffusive behavior obeys a thermal activation process. The activation energy (Ea) for C6Li is obtained as 270(5) meV, while Ea = 170(20) meV for C12Li. Assuming a jump diffusion of Li+ in the Li layer of C6Li and C12Li, a self-diffusion coefficient DLi at 310 K was estimated as 7.6(3) × 10-11 (cm2 s-1) in C6Li and 14.6(4) × 10-11 (cm2 s-1) in C12Li.

Panitumumab interaction with TAS-102 leads to combinational anticancer effects via blocking of EGFR-mediated tumor response to trifluridine.

Panitumumab is a monoclonal antibody developed against the human epidermal growth factor receptor (EGFR). TAS-102 is a novel chemotherapeutic agent containing trifluridine (FTD) as the active cytotoxic component. Both panitumumab and TAS-102 have been approved for the treatment of metastatic colorectal cancer. In this study, we revealed the mechanism underlying the anticancer effects of panitumumab/TAS-102 combination using preclinical models. Panitumumab/FTD cotreatment showed additive antiproliferative effects in LIM1215 and synergistic antiproliferative effects in SW48 colon cancer cells. Consistent with the in vitro effects, panitumumab/TAS-102 combination caused tumor regression in LIM1215 and COL-01-JCK colon cancer patient-derived xenograft models. In LIM1215 cells, FTD induced extracellular signal-regulated kinase (ERK)/protein kinase B (AKT)/signal transducer and activator of transcription 3 (STAT3) phosphorylation and subsequent serine/threonine phosphorylation of EGFR, while it had no effects on EGFR tyrosine phosphorylation. Panitumumab and the tyrosine kinase inhibitor erlotinib reduced the basal level of EGFR tyrosine phosphorylation and reversed FTD-induced ERK/AKT/STAT3 and EGFR serine/threonine phosphorylation. These results suggested that FTD in combination with the basal activity of EGFR tyrosine kinase induced downstream prosurvival signaling through ERK/AKT/STAT3 phosphorylation. Collectively, we propose that panitumumab interacts with FTD by targeting EGFR-mediated adaptive responses, thereby exerting anticancer effects when used in combination with TAS-102. These preclinical findings provide a compelling rationale for evaluating the combination of anti-EGFR antibodies with TAS-102 against metastatic colorectal cancer.

Analysis of gene expression profiles of Lactobacillus paracasei induced by direct contact with Saccharomyces cerevisiae through recognition of yeast mannan.

Co-culture of lactic acid bacteria (LAB) and yeast induces specific responses that are not observed in pure culture. Gene expression profiles of Lactobacillus paracasei ATCC 334 co-cultured with Saccharomyces cerevisiae IFO 0216 were analyzed by DNA microarray, and the responses induced by direct contact with the yeast cells were investigated. Coating the LAB cells with recombinant DnaK, which acts as an adhesive protein between LAB and yeast cells, enhanced the ratio of adhesion of the LAB cells to the yeast cells. The signals induced by direct contact were clarified by removal of the LAB cells unbound to the yeast cells. The genes induced by direct contact with heat-inactivated yeast cells were very similar to both those induced by the intact yeast cells and those induced by a soluble mannan. The top 20 genes upregulated by direct contact with the heat-inactivated yeast cells mainly encoded proteins related to exopolysaccharide synthesis, modification of surface proteins, and transport systems. In the case of the most upregulated gene, LSEI_0669, encoding a protein that has a region homologous to polyprenyl glycosylphosphotransferase, the expression level was upregulated 7.6-, 11.0-, and 8.8-fold by the heat-inactivated yeast cells, the intact yeast cells, and the soluble mannan, respectively, whereas it was only upregulated 1.8-fold when the non-adherent LAB cells were not removed before RNA extraction. Our results indicated that the LAB responded to direct contact with the yeast cells through recognition of mannan on the surface of the yeast.

Nitric oxide synthase during early embryonic development in silkworm Bombyx mori: Gene expression, enzyme activity, and tissue distribution.

To elucidate the mechanism for embryonic diapause or the breakdown of diapause in Bombyx mori, we biochemically analyzed nitric oxide synthase (NOS) during the embryogenesis of B. mori. The gene expression and enzyme activity of B. mori NOS (BmNOS) were examined in diapause, non-diapause, and HCl-treated diapause eggs. In the case of HCl-treated diapause eggs, the gene expression and enzyme activity of BmNOS were induced by HCl treatment. However, in the case of diapause and non-diapause eggs during embryogenesis, changes in the BmNOS activity and gene expressions did not coincide except 48-60 h after oviposition in diapause eggs. The results imply that changes in BmNOS activity during the embryogenesis of diapause and non-diapause eggs are regulated not only at the level of transcription but also post-transcription. The distribution and localization of BmNOS were also investigated with an immunohistochemical technique using antibodies against the universal NOS; the localization of BmNOS was observed mainly in the cytoplasm of yolk cells in diapause eggs and HCl-treated diapause eggs. These data suggest that BmNOS has an important role in the early embryonic development of the B. mori.

Mechanism of the inhibition of leukemia cell growth and induction of apoptosis through the activation of ATR and PTEN by the topoisomerase inhibitor 3EZ, 20Ac-ingenol.

The PI3K/Akt signaling pathway is constitutively activated in various leukemias. In the present study, the topoisomerase inhibitor, 3EZ, 20Ac-ingenol, was more effective in inhibiting the growth of BALL-1 cells than that of normal lymphocyte cells. ATM/ATR protein levels were increased, PTEN protein was upregulated, and p-Akt protein was downregulated at early time points after treatment with 3EZ, 20Ac-ingenol. In further experiments, p53 protein expression was increased, and H2AX phosphorylation and p21 protein expression were induced after treatment with 3EZ, 20Ac-ingenol. Moreover, the activation of caspase 3 followed decrease in the Bcl-2/Bax ratio after treatment with 3EZ, 20Ac-ingenol, and accumulation of sub-G1 phase cells was observed in flow cytometry analyses. These data suggest that 3EZ, 20Ac-ingenol-induced DNA damage downregulates p-Akt and upregulates ATR leading to cell cycle arrest and increased apoptosis in BALL-1 cells.

HCl Treatment for Preventing Diapause Causes Ca2+ Efflux in Bombyx mori Eggs.

To elucidate the mechanism for preventing entry into embryonic diapause or breakdown of diapause in Bombyx mori by HCl and dimethyl sulfoxide (DMSO) treatment or a combination of cold and HCl treatment, we performed quantitative analysis of Ca2+ and Mg2+ in the chorion and egg content using inductively coupled plasma atomic emission spectrometry (ICP-AES). When diapause eggs that had been incubated at 25°C for 2 days from oviposition and at 4°C for an additional six days were treated with HCl solution, the amount of Ca2+ in the chorion and egg content after HCl treatment was reduced to one-seventh, as compared with the amount before treatment. In contrast, there was no change in the amount of Mg2+ with HCl treatment. The amount of Ca2+ in the HCl solution after the diapause eggs were treated increased 7.5-fold, as compared with that of eggs treated with water. Even when 17-day-old diapausing eggs were treated with HCl, which did not break diapause, the amount of Ca2+ in the chorion and egg content was reduced to one-fifth, as compared with the control. Meanwhile, changes in Ca2+ and Mg2+ contents were not observed in 12-hr-old diapause-destined eggs before or after treatment with DMSO, which effectively prevents diapause. These data may suggest that Ca2+ efflux from diapause eggs by HCl is not directly associated with preventing entry into diapause or breaking of diapause. In addition, we discovered that the amount of Ca2+ in diapause-destined eggs was more than 2.4-fold larger than in non-diapause-destined eggs.

Abnormal arrangement of a collagen/apatite extracellular matrix orthogonal to osteoblast alignment is constructed by a nanoscale periodic surface structure.

Morphological and directional alteration of cells is essential for structurally appropriate construction of tissues and organs. In particular, osteoblast alignment is crucial for the realization of anisotropic bone tissue microstructure. In this article, the orientation of a collagen/apatite extracellular matrix (ECM) was established by controlling osteoblast alignment using a surface geometry with nanometer-sized periodicity induced by laser ablation. Laser irradiation induced self-organized periodic structures (laser-induced periodic surface structures; LIPSS) with a spatial period equal to the wavelength of the incident laser on the surface of biomedical alloys of Ti-6Al-4V and Co-Cr-Mo. Osteoblast orientation was successfully induced parallel to the grating structure. Notably, both the fibrous orientation of the secreted collagen matrix and the c-axis of the produced apatite crystals were orientated orthogonal to the cell direction. To the best of our knowledge, this is the first report demonstrating that bone tissue anisotropy is controllable, including the characteristic organization of a collagen/apatite composite orthogonal to the osteoblast orientation, by controlling the cell alignment using periodic surface geometry.