One-Step Generation of Multiple Gene-Edited Pigs by Electroporation of the CRISPR/Cas9 System into Zygotes to Reduce Xenoantigen Biosynthesis

Xenoantigens cause hyperacute rejection and limit the success of interspecific xenografts. Therefore, genes involved in xenoantigen biosynthesis, such as GGTA1, CMAH, and B4GALNT2, are key targets to improve the outcomes of xenotransplantation. In this study, we introduced a CRISPR/Cas9 system simultaneously targeting GGTA1, CMAH, and B4GALNT2 into in vitro-fertilized zygotes using electroporation for the one-step generation of multiple gene-edited pigs without xenoantigens. First, we optimized the combination of guide RNAs (gRNAs) targeting GGTA1 and CMAH with respect to gene editing efficiency in zygotes, and transferred electroporated embryos with the optimized gRNAs and Cas9 into recipient gilts. Next, we optimized the Cas9 protein concentration with respect to the gene editing efficiency when GGTA1, CMAH, and B4GALNT2 were targeted simultaneously, and generated gene-edited pigs using the optimized conditions. We achieved the one-step generation of GGTA1/CMAH double-edited pigs and GGTA1/CMAH/B4GALNT2 triple-edited pigs. Immunohistological analyses demonstrated the downregulation of xenoantigens; however, these multiple gene-edited pigs were genetic mosaics that failed to knock out some xenoantigens. Although mosaicism should be resolved, the electroporation technique could become a primary method for the one-step generation of multiple gene modifications in pigs aimed at improving pig-to-human xenotransplantation.     

Reduced Claudin-12 Expression Predicts Poor Prognosis in Cervical Cancer

Background: Within the claudin (CLDN) family, CLDN12 mRNA expression is altered in various types of cancer, but its clinicopathological relevance has yet to be established due to the absence of specific antibodies (Abs) with broad applications. Methods: We generated a monoclonal Ab (mAb) against human/mouse CLDN12 and verified its specificity. By performing immunohistochemical staining and semiquantification, we evaluated the relationship between CLDN12 expression and clinicopathological parameters in tissues from 138 cases of cervical cancer. Results: Western blot and immunohistochemical analyses revealed that the established mAb selectively recognized the CLDN12 protein. Twenty six of the 138 cases (18.8%) showed low CLDN12 expression, and the disease-specific survival (DSS) and recurrence-free survival rates were significantly decreased compared with those in the high CLDN12 expression group. We also demonstrated, via univariable and multivariable analyses, that the low CLDN12 expression represents a significant prognostic factor for the DSS of cervical cancer patients (HR 3.412, p = 0.002 and HR 2.615, p = 0.029, respectively). Conclusions: It can be concluded that a reduced CLDN12 expression predicts a poor outcome for cervical cancer. The novel anti-CLDN12 mAb could be a valuable tool to evaluate the biological relevance of the CLDN12 expression in diverse cancer types and other diseases.     

The mechanism of sequence non-specific DNA binding of HMG1/2-box B in HMG1 with DNA

The DNA binding mechanism of box B in HMG1, a member of thesequence non-specific DNA binding HMG1/2-box family of proteins,has been examined by both mutation analyses and molecular modelingtechniques. Substitution of the residue 102F, which is characteristicallyexposed to solvent, with a small hydrophobic amino acid affectedits DNA binding activity. However, no additional effect wasobserved by the further mutation of flanking 101F. Moleculardynamics simulation and modeling studies revealed that 102Fintercalates into DNA base-pairs, being supported by the flanking101F. The mutants with a small hydrophobic residue at position102 tolerated the substitution for 101F because the side chainat position 102 is too short to intercalate. Thus the intercalationof 102F and the positive effect of the flanking 101F residueare important for the sequence non-specific DNA binding of theHMG1/2-box. The conserved basic residues of 95K, 96R and 109Rwere also examined for their roles in DNA binding. These residuesinteracted with DNA mainly by electrostatic interaction andmaintained the location of the box on the DNA, which prescribedthe intercalation of 102F. The DNA intercalation by HMG1 consistsof an ingenious mechanism which brings DNA conformational changesnecessary for biological functions.     

Characterization of microspherical semi-transparent solar cells and modules

Microspherical solar cells and modules have been fabricated. The spherical nature of these semi-transparent devices allows the microspherical cells to harvest both directly incident and diffuse components of sunlight thereby improving the solar energy conversion efficiency. Indoor and outdoor characterizations of these three dimensional semi-transparent cells and modules are carried out using a Lambertian reflector in order to assess the maximum efficiency of the devices. In the absence of the reflector the cell efficiency is 13.5% under standard illumination (100 mW cm⁻², A.M. 1.5, 25 °C). However, this is significantly enhanced in the presence of the reflector. Microspherical modules with the reflector are directly compared to similar semi-transparent modules comprised of traditional planar devices, in outdoor tests at low light intensity (2.5–25 mW cm⁻²) to further demonstrate the benefits of the design particularly at low angle of incident radiation.     

Proteolytic activity and recombinant protein production in virus-infected Sf-9 insect cell cultures supplemented with carboxyl and cysteine protease inhibitors

In insect cell-baculovirus expression systems for recombinant protein production, it is sometimes necessary to supplement cultures with protease inhibitors to protect recombinant proteins against proteolysis. To date, however, there is no information available concerning protease activities in inhibitor-supplemented cultures. The aim of the present study was to investigate intracellular and extracellular protease activities in cultures of virus-infected Sf-9 insect cells which were supplemented with inhibitors against carboxyl and cysteine proteases produced during culture. Prior to the supplementation culture, the cell toxicity of several protease inhibitors was determined. As a result, pepstatin A (carboxyl protease inhibitor) and E64, cystatin, leupeptin, and antipain (cysteine protease inhibitors) tested in this study showed no apparent negative effects on the growth and viability of noninfected Sf-9 insect cells at low concentrations. In addition, E64 and pepstatin A could rapidly permeate virus-infected Sf-9 cells and inhibit the respective intracellular protease activities. A virus-infected culture with a multiplicity of infection of 1 was carried out with E64 and pepstatin A which were added to the culture medium at 2 d post-infection. As a result of inhibitor supplementation, the cellular activity for recombinant protein biosynthesis was reduced by 5-30%. However, a significant reduction in carboxyl and cysteine protease activities was observed not only in the medium but also intracellularly. This is the first study that directly demonstrates a reduction in extracellular and intracellular protease activities in protease inhibitor-supplemented cultures of virus-infected insect cells.     

Internal oxidation of Fe-Al alloys in the α-phase region

The internal oxidation behavior of Fe-0.069, 0.158, and 0.274 wt% Al alloys was investigated in the -phase region. The internal oxidation experiments have been made over the temperature range from 1023 to 1123 K using a mixture of iron and its oxide powders. A parabolic rate law holds in the present alloys, where the rate constant, K_p, depends upon the oxidation temperature as well as the aluminum content. The internal oxidation of Fe-Al alloys is, therefore, controlled by a diffusion process of oxygen in the alloy. The oxide formed in the oxidation layer is the stoichiometric FeAl₂O₄ (hercynite). The aluminum concentration, N _Al ^Io , in the oxidation layer was calculated by taking account of counterdiffusion of aluminum. Furthermore, the oxygen concentration, N _O ^S , at the specimen surface was evaluated on the basis of thermodynamics. Using these estimated values of K_p, N _Al ^IO , and N _O ^S , the diffusion coefficient of oxygen, D _O ^IO , in the oxidation layer, where the oxide particles were dispersed, was also calculated. D _O ^IO increases as the volume fraction of the oxide, f^IO, increases. The diffusion coefficient of oxygen, D_O, in -iron was determined by extrapolating D _O ^IO to f^IO=0.     

Steam reforming on Ni-samaria-doped ceria cermet anode for practical size solid oxide fuel cell at intermediate temperatures

Direct internal and external reforming operations on Ni-samaria-doped ceria (SDC) anode with the practical size solid oxide fuel cell (SOFC) at intermediate temperatures from 600 to 750 °C are carried out to reveal the reforming activities and the electrochemical activities, being compared with the hydrogen-fueled power generation. The cell performance with direct internal and external steam reforming of methane and their limiting current densities were almost the same irrespective of the progress of reaction in the methane reformate at 700 and 750 °C. The durability test for 5.5 h at 750 °C with direct internal reforming operation confirmed that the cell performance did not deteriorate. The operation temperature of the cell controlled the reforming activities on the anode, and the large size electrode gave rise to high conversion due to the slow space velocity of the steam reforming. Direct internal steam reforming attained sufficient level of conversion for SOFC power generation with methane at 700 and 750 °C on the large Ni-SDC cermet anode.     

A method to modify coordinates of detectors in positron emission tomography systems

A new method to modify coordinates of detectors in any positron emission tomography (PET) system using a radioactive point source is developed. This method is based on selecting a centered detector in each detector block of PET and measuring coincidence counts between the two centered detectors in opposite detector blocks to find the coordinates of their LOR (Line of Response). Due to slight misalignment of detector positions, measured LORs may not intersect at a single point. Based on the proposed method, the coordinates of detectors can be measured with very high accuracy and the coordinate of the center of the gantry (which is normally the same as the center of field of view) can be defined correctly. The results of the application of our method to a small animal PET system (FinePET), which was recently developed at Tohoku University, Japan, are shown here. The method is expected to contribute to the design and development of PET systems which can realize a very high spatial resolution of less than 1 mm FWHM.     

Synergistic effect of Pt or Pd and perovskite oxide for water gas shift reaction

The water gas shift (WGS) reaction over Pt and Pd catalysts supported on various perovskite oxides has been investigated at 573 K without catalyst pretreatment. The Pt and Pd catalysts on LaCoO₃ support showed high catalytic activity. Interaction between Pt or Pd and the support is considered to promote the WGS reaction: Pt/LaCoO₃ had high initial activity but deactivated immediately; Pd/LaCoO₃ was less active than Pt/LaCoO₃, but had superior stability. Catalysts were characterized using XRD, STEM, XPS, and H₂-temperature programmed reduction (TPR). Results of this study showed that reduction of the support decreased the CO conversion on Pt/LaCoO₃. On the other hand, Pd/LaCoO₃ showed stable activity for the WGS reaction. Therefore, Pd was added to Pt/LaCoO₃ for stabilizing the catalyst activity, and 0.5 wt.% Pd/1 wt.% Pt/LaCoO₃ catalyst showed higher activity and stability.     

Molecular recognition by wall-assembling-type nanocavity in aqueous media

Steroid cyclophanes, each having a macrocyclic ring attached to four bile acid moieties via chiral lysine connectors, were synthesized, and the binding of the 2-naphthylphenylketone (guest) to the steroid cyclophanes in water was investigated. The circular dichroism spectra of the steroid cyclophane with cholic acid and L-lysine were significantly affected by the binding of the guest, and the induced circular dichroism based on the absorption of the achiral guest was also observed. The binding of the guest to the steroid cyclophane with cholic acids and D-lysines induced changes in the circular dichroism spectra with the opposite sign of the molecular ellipticities. An induced circular dichroism spectral change was not observed upon binding of the guest to the analogous host without OH sites. These results strongly suggest that the guest is conformationally fixed through hydrogen bonding between the carbonyl group of the guest and the steroidal hydroxyl group of the host. The assembly of only four steroid residues on the macrocyclic ring probably provided a hydrophobic nanocavity for hydrogen bonding.