Direct Injection of CRISPR/Cas9-Related mRNA into Cytoplasm of Parthenogenetically Activated Porcine Oocytes Causes Frequent Mosaicism for Indel Mutations

Some reports demonstrated successful genome editing in pigs by one-step zygote microinjection of mRNA of CRISPR/Cas9-related components. Given the relatively long gestation periods and the high cost of housing, the establishment of a single blastocyst-based assay for rapid optimization of the above system is required. As a proof-of-concept, we attempted to disrupt a gene (GGTA1) encoding the α-1,3-galactosyltransferase that synthesizes the α-Gal epitope using parthenogenetically activated porcine oocytes. The lack of α-Gal epitope expression can be monitored by staining with fluorescently labeled isolectin BS-I-B₄ (IB4), which binds specifically to the α-Gal epitope. When oocytes were injected with guide RNA specific to GGTA1 together with enhanced green fluorescent protein (EGFP) and human Cas9 mRNAs, 65% (24/37) of the developing blastocysts exhibited green fluorescence, although almost all (96%, 23/24) showed a mosaic fluorescent pattern. Staining with IB4 revealed that the green fluorescent area often had a reduced binding activity to IB4. Of the 16 samples tested, six (five fluorescent and one non-fluorescent blastocysts) had indel mutations, suggesting a correlation between EGFP expression and mutation induction. Furthermore, it is suggested that zygote microinjection of mRNAs might lead to the production of piglets with cells harboring various mutation types.     

Competitive inhibition by procaine of carbachol-induced stimulus-secretion coupling in rat pancreatic acini

1. Procaine (0.03-10 mM) inhibited carbachol (CCh)-induced amylase release from rat isolated pancreatic acini in a competitive manner. Kinetic analysis of the relation between CCh concentrations and the amount of amylase released in the presence of various procaine concentrations indicated that procaine caused competitive inhibition with the affinity constant (pA2) value of 5.00 +/- 0.08. 2. Receptor binding assay confirmed that procaine (0.01-10 mM) competitively inhibited [N-methyl-3H]-scopolamine chloride ([3H]-NMS) binding to its receptor with binding affinity (pKi) of 4.63 +/- 0.10. 3. Procaine transformed CCh-evoked [Ca2+]i dynamics: the initial rise in [Ca2+]i followed by a gradual decay during continuous stimulation with 3 microM CCh was transformed by 0.3 mM procaine to the oscillatory [Ca2+]i dynamics, which resembled the response to 0.3 microM CCh in the absence of procaine. The initial phase of [Ca2+]i oscillation corresponded to the initial phase of CCh-induced amylase release in isolated perfused acini. 4. Procaine (0.3-3 mM) did not inhibit the secretory response to cholecystokinin octapeptide (CCK-8) in isolated incubated acini. A higher concentration of procaine (10 mM) caused weak but significant inhibition of the response to only limited concentrations of CCK-8, 30 and 100 pM. Procaine lower than 10 mM was ineffective on [125I]-BH-CCK-8 binding, although procaine (10 mM) caused weak but significant inhibition of the binding.     

Surgical pitfalls in pinealoma surgery

The author has treated 30 cases of pinealoma by the infratentorial supracerebellar approach with the patient in the sitting position. Long-term outcome has been excellent in 26 cases, poor in 1, and death in 3 cases. Postoperative deaths were due to: (i) high venous pressure in the jugular vein caused by a tourniquet placed around the neck to prevent air-embolism, making hemostasis more difficulty, (ii) the absence of retraction resulting in a narrow surgical field when acute brain swelling developed, and (iii) high coagulation and ablation of the precentral cerebellar vein causing diffusion thrombosis of deep veins. The details of these cases are presented in this article.     

An IR study on ion-specific and solvent-specific swelling of poly(N-vinyl-2-pyrrolidone) gel

Swelling degrees of poly(N-vinyl-2-pyrrolidone) (PVP) gel were measured in aqueous salt solutions and in water/organic solvent mixtures to find marked ion- and solvent-specificities. In order to investigate any correlation of those specificities with hydration or solvation of PVP, IR spectra band of the CO group was monitored by means of ATR method both for PVP gel and the relevant solution systems. Dependence of the peak frequency on the swelling ratio suggested that hydration of PVP carbonyl group in deswollen gel systems is different from that in the corresponding solution systems. In the solution systems, PVP carbonyl band showed a high-wavenumber shift for deswollen systems, which can be well correlated with changes in water proton charge through ionic hydration and with Gutmann's acceptor number of organic solvents. In the deswollen gel systems, the CO band showed a low-wavenumber shift, suggesting a strong hydration or doubly hydrated state. This unexpected behavior was interpreted by assuming an intermolecular hydrogen bond of two carbonyl groups intermediated by water molecules.     

Stereoscopic synthesized brain-surface imaging with MR angiography for localization of a peripheral mycotic aneurysm: case report

A patient with subacute bacterial endocarditis and a peripheral mycotic aneurysm is presented. We used a combined multi-slice surface anatomy scanning (SAS) and contrast-enhanced MR angiography image to determine the exact location of the small lesion by applying a skin marker on the scalp and visualizing the relationship of the marker to the brain surface structures and to the lesion. This technique was useful for the removal of a small peripheral aneurysm using only a limited craniotomy.     

Estimation of the functional reserve of the human liver by urinary D-glucaric acid excretion after vitamin C administration

Increased monoamine metabolism in experimental herpes simplex virus (HSV) encephalitis is well established. Both serotonin (5-HT) and dopamine (DA) systems are affected. HSV invades the raphe nuclei after its entry into the brain stem. However, no studies have been published concerning influences of HSV on the neurotransmitters in the raphe. In the present study, concentrations of 5-HT and DA and their metabolites in the raphe nuclei and related brain regions in rabbits with fulminant HSV encephalitis have been analyzed using high-pressure liquid chromatography. Encephalitis was induced by corneal inoculation with HSV. Homovanillic acid (HVA) and dihydroxyphenyl acetic acid (DOPAC) concentrations and HVA/DA ratios were increased in the raphe nuclei suggesting increased DA turnover. The most substantial changes were bilaterally decreased 5-HT and 5-hydroxyindoleacetic acid (5-HIAA) concentrations in the raphe nuclei. The decrease in the raphe 5-HT was reflected also to the projection areas in the hemispheres, where 5-HT concentrations were lower in HSV-inoculated rabbits than in controls. The changes strongly suggest a direct influence of HSV on serotoninergic neurons. Since the ventral parts of the limbic system have rich serotoninergic afferents from the raphe, this also suggests that HSV may reach hemispheres in HSV encephalitis from the brain stem via the ascending serotoninergic system.     

Tyrosine phosphorylation as a convergent pathway of heterotrimeric G protein- and rho protein-mediated Ca2+ sensitization of smooth muscle of rabbit mesenteric artery

1. The aim of this study was to determine whether different signal transduction mechanisms underlie the Ca2+ sensitizing effects of guanosine 5'-O-(3-thiotriphosphate) (GTP(gamma)S) and receptor agonists on beta-escin-skinned smooth muscle of rabbit mesenteric artery. 2. In the homogenate of the beta-escin-skinned arterial strip, C3 exoenzyme of Clostridium botulinum catalyzed the [32P]-ADP-ribosylation of only one protein that had the same molecular mass as the protein detected in Western blots with anti-rho p21 antibody. Pretreatment of preparations with C3 resulted in great inhibition of GTP(gamma)S-induced Ca2+ sensitization, although the effect of GTP(gamma)S at higher concentrations (> or = 30 microM) was not completely blocked by this treatment. In contrast, the enhancement by phenylephrine and histamine, in the presence of guanosine 5'-triphosphate, of the Ca2+-induced contraction was not affected by C3 pretreatment. 3. The protein kinase C (PKC) inhibitors calphostin C and staurosporine completely eliminated the enhancement by phorbol ester 12,13-dibutyrate of the Ca2+-induced contraction. However, these PKC inhibitors had no effect on GTP(gamma)S- and receptor agonist-induced Ca2+ sensitization. 4. The tyrosine kinase inhibitors genistein and tyrphostin 25 caused an irreversible and complete block of the enhancement by GTP(gamma)S of the Ca2+-induced contraction without affecting this Ca2+ contraction. The inactive genistein analogue daidzein did not modify the effect of GTP(gamma)S. The Ca2+ sensitizing effects of phenylephrine and histamine were also blocked by these tyrosine kinase inhibitors. 5. These results suggest that rho p21 predominantly mediates GTP(gamma)S-induced Ca2+ sensitization of beta-escin-skinned smooth muscle of rabbit mesenteric artery, while the Ca2+ sensitizing actions of heterotrimeric G protein-coupled receptor agonists do not involve this small G protein. However, it seems that tyrosine phosphorylation, but not PKC activation, plays an important role in both of the rho p21 protein- and heterotrimeric G protein-mediated Ca2+ sensitization mechanisms.     

Stability of metastable tetragonal ZrO2 in compound powders and nucleation arguments

ZrO₂-SiO₂ (11) mixtures and ZrO₂ particles were prepared by a sol-gel method from the solutions of ZrOCl₂·8H₂O (ZOC) + Si(OC₂H₅)₄ (TEOS) + C₂H₅OH and ZOC + C₂H₅OH + H₂O + NH₄Cl systems, respectively. Quantitative changes of phase crystallized by heating were compared with those of ZOC + TEOS + H₂O and/or ZrO(NO₃)₂ · 2H₂O + H₂O systems, respectively. The stability of metastable tetragonal (mt)-ZrO₂ particles depends on the Young's modulus of the SiO₂ matrix. Transition metal oxides existing on the interface assisted in stabilizing mt-ZrO₂. The order of the assistance agreed with that of the relative field strengths of their oxides. A relationship between the ionic radius and the volatility of anionic groups, and the difficulty of nucleation for the martensitic transformation accompanying a shear stress, is suggested.     

Cognitive model of team cooperation in en-route air traffic control

Since controller teams are in charge of en-route air traffic control, team cooperation is a key issue for good control performance. We conducted ethnographic field observation at the Tokyo Area Control Center and then analyzed the obtained data to develop a cognitive model of team cooperation in en-route air traffic control. We segmented conversational records, behavioral records, and so on by control unit, and then clarified relations between the segments and identified expert knowledge and judgment behind them. Cognitive processes of controller teams were reconstructed based on a concept of distributed cognition. The analysis revealed that the mutual belief model is applicable to team cooperation processes, the role assignment of tasks within a controller team is implicit and that control plans are implemented smoothly once team situation awareness has been established. A cognitive model of controllers?? team cooperation has been constructed based on these findings.     

Unmanned Ground Vehicle Navigation in Coordinate-Free and Localization-Free Wireless Sensor and Actuator Networks

We present a set of algorithms for the navigation of Unmanned Ground Vehicles (UGVs) towards a set of pre-identified target nodes in coordinate-free and localization-free wireless sensor and actuator networks. The UGVs are equipped with a set of wireless listeners that provide sensing information about the potential field generated by the network of actuators. Two main navigation scenarios are considered: single-UGV, single-destination navigation and multi-UGV, multi-destination navigation. For the single-UGV, single-destination case, we present both centralized and distributed navigation algorithms. Both algorithms share a similar two-phase concept. In the first phase, the system assigns level numbers to individual nodes based on their hop distance from the target nodes. In the second phase, the UGV uses the potential field created by the network of actuators to move towards the target nodes, requiring cooperation between triplets of actuator nodes and the UGV. The hop distance to the target nodes is used to control the main moving direction while the potential field, which can be measured by listeners on the UGV, is used to determine the UGV’s movement. For the multi-UGV, multi-destination case, we present a decentralized allocation algorithm such that multiple UGVs avoid converging to the same destination. After each UGV determines its destination, the proposed navigation scheme is applied. The presented algorithms do not attempt to localize UGVs or sensor nodes and are therefore suitable for operating in GPS-free/denied environments. We also present a study of the communication complexity of the algorithms as well as simulation examples that verify the proposed algorithms and compare their performances.