Antimutagenic activity of soybeans fermented with basidiomycetes in Ames/Salmonella test

Soybeans fermented with either Phellinus igniarius or Agrocybe cylindracea inhibited the mutagenicity of the directly-acting mutagens: 4-nitro-o-phenylenediamine on Salmonella typhimurium strain TA 98 and NaN₃ on S. typhimurium strain TA 100; and indirectly-acting mutagens, 2-aminofluorene using strain TA 98 and benzo[a]pyrene using strain TA 100, in the presence of a supernatant solution from mammalian hepatic microsomes.     

Structure-guided mutagenesis of a mucin-selective metalloprotease from Akkermansia muciniphila alters substrate preferences

Akkermansia muciniphila, a mucin-degrading microbe found in the human gut, is often associated with positive health outcomes. The abundance of A. muciniphila is modulated by the presence and accessibility of nutrients, which can be derived from diet or host glycoproteins. In particular, the ability to degrade host mucins, a class of proteins carrying densely O-glycosylated domains, provides a competitive advantage in the sustained colonization of niche mucosal environments. Although A. muciniphila is known to rely on mucins as a carbon and nitrogen source, the enzymatic machinery used by this microbe to process mucins in the gut is not yet fully characterized. Here, we focus on the mucin-selective metalloprotease, Amuc_0627 (AM0627), which is known to cleave between adjacent residues carrying truncated core 1 O-glycans. We showed that this enzyme is capable of degrading purified mucin 2 (MUC2), the major protein component of mucus in the gut. An X-ray crystal structure of AM0627 (1.9 Å resolution) revealed O-glycan–binding residues that are conserved between structurally characterized enzymes from the same family. We further rationalized the substrate cleavage motif using molecular modeling to identify nonconserved glycan-interacting residues. We conclude that mutagenesis of these residues resulted in altered substrate preferences down to the glycan level, providing insight into the structural determinants of O-glycan recognition.     

Technical issues in using robots to reproduce joint specific gait

Reproduction of the in vivo motions of joints has become possible with improvements in robot technology and in vivo measuring techniques. A motion analysis system has been used to measure the motions of the tibia and femur of the ovine stifle joint during normal gait. These in vivo motions are then reproduced with a parallel robot. To ensure that the motion of the joint is accurately reproduced and that the resulting data are reliable, the testing frame, the data acquisition system, and the effects of limitations of the testing platform need to be considered. Of the latter, the stiffness of the robot and the ability of the control system to process sequential points on the path of motion in a timely fashion for repeatable path accuracy are of particular importance. Use of the system developed will lead to a better understanding of the mechanical environment of joints and ligaments in vivo.     

Q134R: Small chemical compound with NFAT inhibitory properties improves behavioral performance and synapse function in mouse models of amyloid pathology

Inhibition of the protein phosphatase calcineurin (CN) ameliorates pathophysiologic and cognitive changes in aging rodents and mice with aging‐related Alzheimer''s disease (AD)‐like pathology. However, concerns over adverse effects have slowed the transition of common CN‐inhibiting drugs to the clinic for the treatment of AD and AD‐related disorders. Targeting substrates of CN, like the nuclear factor of activated T cells (NFATs), has been suggested as an alternative, safer approach to CN inhibitors. However, small chemical inhibitors of NFATs have only rarely been described. Here, we investigate a newly developed neuroprotective hydroxyquinoline derivative (Q134R) that suppresses NFAT signaling, without inhibiting CN activity. Q134R partially inhibited NFAT activity in primary rat astrocytes, but did not prevent CN‐mediated dephosphorylation of a non‐NFAT target, either in vivo, or in vitro. Acute (≤1 week) oral delivery of Q134R to APP/PS1 (12 months old) or wild‐type mice (3–4 months old) infused with oligomeric Aβ peptides led to improved Y maze performance. Chronic (≥3 months) oral delivery of Q134R appeared to be safe, and, in fact, promoted survival in wild‐type (WT) mice when given for many months beyond middle age. Finally, chronic delivery of Q134R to APP/PS1 mice during the early stages of amyloid pathology (i.e., between 6 and 9 months) tended to reduce signs of glial reactivity, prevented the upregulation of astrocytic NFAT4, and ameliorated deficits in synaptic strength and plasticity, without noticeably altering parenchymal Aβ plaque pathology. The results suggest that Q134R is a promising drug for treating AD and aging‐related disorders.     

Are Stripes Beneficial? Dazzle Camouflage Influences Perceived Speed and Hit Rates

In the animal kingdom, camouflage refers to patterns that help potential prey avoid detection. Mostly camouflage is thought of as helping prey blend in with their background. In contrast, disruptive or dazzle patterns protect moving targets and have been suggested as an evolutionary force in shaping the dorsal patterns of animals. Dazzle patterns, such as stripes and zigzags, are thought to reduce the probability with which moving prey will be captured by impairing predators'' perception of speed. We investigated how different patterns of stripes (longitudinal—i.e., parallel to movement direction–and vertical–i.e., perpendicular to movement direction) affect the probability with which humans can hit moving objects and if differences in hitting probability are caused by a misperception of speed. A first experiment showed that longitudinally striped objects were hit more often than unicolored objects. However, vertically striped objects did not differ from unicolored objects. A second study examining the link between perceived speed and hitting probability showed that longitudinally and vertically striped objects were both perceived as moving faster and were hit more often than unicolored objects. In sum, our results provide evidence that striped patterns disrupt the perception of speed, which in turn influences how often objects are hit. However, the magnitude and the direction of the effects depend on additional factors such as speed and the task setup.     

Evaluation of cell viability and apoptosis in human amniotic fluid-derived stem cells with natural cryoprotectants

A previous study demonstrated that disaccharides, antioxidants, and caspase inhibitors can be used in freezing solutions to reduce the concentration of Me₂SO from the current standard of 10% (v/v) to 5% (v/v) or 2.5% and to eliminate fetal bovine serum (FBS) for the cryopreservation of human amniotic fluid-derived stem cells (AFSCs). Hence, this study investigated whether an irreversible inhibitor of caspase enzymes, benzyloxycarbonyl-Val-Ala-dl-Asp-fluoromethylketone (zVAD-fmk), could be used in post-thaw culture media to increase the survival rate of AFSCs. Our results showed that AFSCs cryopreserved in freezing solution containing trehalose, catalase, and 5% (v/v) Me₂SO and then supplemented with zVAD-fmk in the post-thaw culture media showed similar post-thawing viability, proliferation, and apoptosis than cells cryopreserved in the control solution (10% (v/v) Me₂SO and 20% FBS). The caspase-3 activity in all the cryopreservation solutions tested was similar to that of the control. Caspase-3, caspase-8, caspase-9, and PARP expression was not found in the cryopreserved cells. In addition, no difference was found in the survival rate and apoptosis between short-term (3 weeks) and long-term (1 year) storage of AFSCs cryopreserved in the solutions used in this study. The results of the present study demonstrate that recovery of cryopreserved cells was enhanced by using a caspase inhibitor in the post-thaw culture media.     

Chemopreventive activity of Cnidii Rhizoma for breast cancer

Chemopreventive potential for human breast cancer was assessed in vitro with Cnidii Rhizoma extract. Cnidii Rhizoma inhibited cell proliferation in estrogen receptor-positive (MCF-7) and estrogen receptor-negative (MDA-MB-231) human breast carcinoma cell lines. Cytochrome P450 (CYP) 1A1-mediated ethoxyresorufin O-deethylase (EROD) activity was inhibited by Cnidii Rhizoma in a concentration-dependent manner. In addition, Cnidii Rhizoma extract caused inhibition of microsomal aromatase (estrogen synthase) activity. Ornithine decarboxylase (ODC) activity was reduced to 40.3% of the control after 6 h treatment with Cnidii Rhizoma (5 mg/mL) in MCF-7 breast cancer cells. Cnidii Rhizoma extract markedly reduced 12-O-tetradecanoylphorbol-13-acetate (TPA)-stimulated matrix metalloproteinase (MMP)-9 activity. These results suggest that Cnidii Rhizoma could be of therapeutic value in preventing human breast cancer.     

Mass Spectrometry-Based Metabolite Profiling in the Mouse Liver following Exposure to Ultraviolet B Radiation

Although many studies have been performed on the effects of ultraviolet (UV) radiation on the skin, only a limited number of reports have investigated these effects on non-skin tissue. This study aimed to describe the metabolite changes in the liver of hairless mice following chronic exposure to UVB radiation. We did not observe significant macroscopic changes or alterations in hepatic cholesterol and triglyceride levels in the liver of UVB-irradiated mice, compared with those for normal mice. In this study, we detected hepatic metabolite changes by UVB exposure and identified several amino acids, fatty acids, nucleosides, carbohydrates, phospholipids, lysophospholipids, and taurine-conjugated cholic acids as candidate biomarkers in response to UVB radiation in the mouse liver by using various mass spectrometry (MS)-based metabolite profiling including ultra-performance liquid chromatography-quadrupole time-of-flight (TOF)-MS, gas chromatography-TOF-MS and nanomate LTQ-MS. Glutamine exhibited the most dramatic change with a 5-fold increase in quantity. The results from altering several types of metabolites suggest that chronic UVB irradiation may impact significantly on major hepatic metabolism processes, despite the fact that the liver is not directly exposed to UVB radiation. MS-based metabolomic approach for determining regulatory hepatic metabolites following UV irradiation will provide a better understanding of the relationship between internal organs and UV light.     

Electron cryotomography sample preparation using the Vitrobot

Electron cryotomography is the highest-resolution structural technique currently available that can be applied to unique objects such as flexible large protein complexes, irregular viruses, organelles and small cells. Specimens are preserved in a near-native, 'frozen-hydrated' state by vitrification. The thickness of the vitreous ice must be optimized for each specimen, and gold fiducials are typically added to facilitate image alignment. Here, we describe in detail our protocols for electron cryotomography sample preparation including (i) introduction of fiducial markers into the sample and (ii) sample vitrification. Because we almost exclusively use an automated, climate-controlled plunge-freezing device (the FEI Vitrobot) to vitrify our samples, we discuss its operation and parameters in detail. A session in which eight grids are prepared takes 1.5-2 h.     

Liquid chromatography/tandem mass spectrometry method for the simultaneous determination of vardenafil and its major metabolite,N-desethylvardenafil,in human plasma: Application to a pharmacokinetic study

A rapid and sensitive LC–MS/MS method for the determination of vardenafil and its major metabolite, N-desethylvardenafil, in human plasma using sildenafil as an internal standard was developed and validated. The analytes were extracted from 0.25-mL aliquots of human plasma by liquid–liquid extraction, using 1 mL of ethyl acetate. Chromatographic separation was carried on a Luna C₁₈ column (50 mm × 2.0 mm, 3 μm) at 40 °C, with an isocratic mobile phase consisting of 10 mM ammonium acetate (pH 5.0) and acetonitrile (10:90, v/v), a flow rate of 0.2 mL/min, and a total run time of 2 min. Detection and quantification were performed using a mass spectrometer in the selected reaction-monitoring mode with positive electrospray ionization at m/z 489.1 → 151.2 for vardenafil, m/z 460.9 → 151.2 for N-desethylvardenafil, and m/z 475.3 → 100.1 for the internal standard (IS), respectively. This assay was linear over a concentration range of 0.5–200 ng/mL with a lower limit of quantification of 0.5 ng/mL for both vardenafil and N-desethylvardenafil. The coefficient of variation for the assay precision was <13.6%, and the accuracy was >93.1%. This method was successfully applied to a pharmacokinetic study after oral administration of vardenafil 20 mg tablet in Korean healthy male volunteers.