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Antibacterial activities against Staphylococcus aureus and Bacillus subtilis were found in an ethanol fraction of tempe, an Indonesian fermented soybean produced using Rhizopus oligosporus. The ethanol fraction contained free fatty acids, monoglycerides, and fatty acid ethyl esters. Among these substances, linoleic acid and α-linolenic acid exhibited antibacterial activities against S. aureus and B. subtilis, whereas 1-monolinolenin and 2-monolinolenin exhibited antibacterial activity against B. subtilis. The other free fatty acids, 1-monoolein, monolinoleins, ethyl linoleate, and ethyl linolenate did not exhibit bactericidal activities. These results revealed that R. oligosporus produced the long-chain polyunsaturated fatty acids and monolinolenins as antibacterial substances against the Gram-positive bacteria during the fungal growth and fermentation of heat-processed soybean.

Intravenous immunoglobulin (IVIg) therapy is widely used to treat autoimmune and infectious disorders. Despite the clinical efficacy of IVIg therapy, its precise immunosuppressive mechanisms remain unclear. Here, we provide evidence that IVIg acts directly on T cells to suppress their activation upon T cell receptor (TCR) ligation. IVIg suppressed the proliferation of murine splenocytes upon stimulation with anti-CD3 antibody and T cell-tropic mitogens. These immunosuppressive effects of IVIg were still intact against purified T cells, and the depletion of naturally-occurring regulatory T cells (nTreg) had no effect on T cell regulatory activity. Instead, we found that IVIg negatively regulated TCR signaling; IVIg co-stimulation impaired IκB degradation, nuclear translocation of the nuclear factor of activated T cells (NFAT), and the activation of mitogen-activated protein kinase (MAPK, Erk1/2). These results suggest an additional new immunosuppressive role of IVIg, which acts directly on conventional T cells to suppress the TCR signaling pathway.

A 70-year-old woman presented dysphagia and postprandial vomiting, and weight loss of about 15 kg in one year. She was markedly emaciated with a body mass index of 12.4 kg/m

Biopharmaceutical proteins are usually produced by culturing recombinant Chinese hamster ovary (CHO) cells. High producer cell lines are screened from transfected cells with random integration of target genes. Since transgene expression is susceptible to the surrounding environment of the integrated genomic locus, producer cell lines should be selected from a large number of recombinant cells with heterogeneous transgene insertion. In contrast, targeted integration into a characterized genomic locus allows for predictable transgene expression and less clonal variability, and thus stable production of target proteins can be expected. Genome editing technology based on programmable nucleases has recently emerged as a versatile tool for precise editing of target locus in the cell genome. Here, we demonstrated targeted knock-in of transgenes into the hypoxanthine phosphoribosyltransferase (hprt) locus of CHO cells using CRISPR/Cas9 and CRISPR-mediated precise integration into target chromosome (PITCh) systems. We also generated knock-in CHO cells based on the homology-independent targeted integration (HITI) system. We evaluated the knock-in efficiency of transgenes into the hprt locus using these systems.

Antibacterial activities against

Chinese hamster ovary (CHO) cells have been used as host cells for the production of pharmaceutical proteins. For the high and stable production of target proteins, the transgene should be integrated into a suitable genomic locus of host cells. Here, we generated knock-in CHO cells, in which transgene cassettes without a vector backbone sequence were integrated into the hprt locus of the CHO genome using clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 and CRISPR-mediated precise integration into target chromosome (CRIS-PITCh) systems. We investigated the efficiency of targeted knock-in of transgenes using these systems. As a practical example, we generated knock-in CHO cells producing an scFv-Fc antibody using the CRIS-PITCh system mediated by microhomology sequences for targeting. We found that the CRIS-PITCh system can facilitate targeted knock-in for CHO cell engineering.

Ikarugamycin (IK) is an antibiotic which has been reported to have a variety of functions, such as inhibition of clathrin-mediated endocytosis (CME), anti-tumor effects and regulation of the immune system. Whether IK influences cytokine production is poorly understood. We have investigated the relationship between IK and production of tumor necrosis factor-α (TNF). TNF plays a pivotal role in pathogenesis of many diseases. Although the dynamics of soluble TNF (sTNF) has been widely explored so far, the functions of the membrane form of TNF (mTNF) have not been fully elucidated. We demonstrated that IK increases the amount of mTNF and prolongs the duration of TNF expression. This effect is unrelated to the shedding activity of disintegrin and metalloproteinase domain-containing protein 17 (ADAM 17). Our results revealed that there is a mechanism to terminate inflammation at the cellular level which IK dysregulates. Furthermore, IK can be a tool to study TNF signaling due to its effect of increasing mTNF expression.

Background Retrotransposons, such as long interspersed element-1 (LINE-1), can copy themselves to other genomic loci via a transposition event (termed retrotransposition). Retrotransposons, therefore, have potential use as an efficient gene delivery tool to integrate multiple copies of a target gene into a host genome. Here, we developed a retrotransposon vector based on LINE-1 that achieves target gene integration of multiple transgene copies. Methods The retrotransposon vector contains a neomycin resistance gene split by an intron as a marker gene, and a gene encoding an antibody single-chain variable fragment (Fv) fused with the constant antibody region (Fc) (scFv-Fc) as a model target gene. G418-resistant Chinese hamster ovary cells were generated using this retrotransposon vector, and scFv-Fc was produced in the culture medium. Results To regulate retrotransposition, we developed a retrotransposon vector system that separately expressed the two open reading frames (ORF1 and ORF2) of LINE-1. Genomic PCR analysis detected the transgene sequence in almost all tested clones. Compared with clones established using the intact LINE-1 vector, clones generated with the split ORF1 and ORF2 system showed similar specific scFv-Fc productivity and retrotransposition efficiency. Conclusion This approach of using a retrotransposon-based vector system has the potential to provide a new gene delivery tool for mammalian cells. This article is protected by copyright. All rights reserved.

Gene delivery methods for animal cells are one of the most important tools in biotechnology fields such as pharmaceutical protein production, generation of transgenic animals and gene therapy. Because retrotransposons can move their own sequences to new genomic locations by a “copy-and-paste” process known as retrotransposition, we attempted to develop a novel gene transfer system based on retrotransposon. A full-length long interspersed element-1 (LINE-1) contains a 5’ untranslated region (5’UTR), two non-overlapping open reading frames (ORFs) separated by a short inter-ORF sequence, and a 3’UTR terminating in an adenosine-rich tract. We constructed a LINE-1 vector plasmid including components necessary for retrotransposition. An intron-disruptedNeoreporter gene and a scFv-Fc expression unit under the control of CMV promoter were added into 3’UTR in order to evaluate retrotransposition and express scFv-Fc. CHO-K1 cells transfected with the plasmids were screened with G418. The established cell clones produced scFv-Fc proteins in the culture medium. To control retrotransposition steadily, we also established retrotransposon systems that supply ORF2 or ORF1–2 separately. Genomic PCR analysis revealed that transgene sequences derived from the LINE-1 vector were positive in all clones. All the clones tested produced scFv-Fc in the culture medium.

The isotopic analysis of atmospheric volatile organic compounds (VOCs), and in particular of their stable carbon isotope ratio (δ13C), could potentially be used as an effective tool for identifying the sources of VOCs. However, to date, there have been very few such analyses. In this work, we analyze the δ13C values of VOCs using thermal desorption coupled with chromatography, combustion, and isotope ratio mass spectrometry (TD–GC/C/IRMS). The measured peak shapes were of high quality and 36 compounds in a standard gas containing 58 VOCs (C5–C11) were detected. The measured δ13C varied widely, from −49.7‰ to −22.9‰, while the standard deviation of the δ13C values varied from 0.07‰ to 0.85‰ (n = 5). We then measured samples from two passenger cars in hot and cold modes, three gas stations, roadside air, and ambient air. In comparison with existing studies, the analytical precision for the 36 compounds in this study was reasonable. By comparing the δ13C values obtained from the cars and gas stations, we could identify some degree of the sources of VOCs in the roadside and ambient air samples.

We demonstrated mode-switching of self-motion coupled with diffusion of molecules at a solid/liquid interface. A camphor boat moved spontaneously on water and the mode of self-motion depended on the setup of the boat. When a camphor disk was connected to the center of a larger plastic plate, intermittent motion (alternating between rest and rapid motion) was observed. When the position of the camphor disk was changed from the center to one of its edges, the period of intermittent motion decreased, and intermittent motion changed to continuous motion. The features of this self-motion and mode-switching were qualitatively reproduced by a numerical calculation using a mathematical model that incorporates the distribution of camphor molecules at the solid/liquid interface.

A change in the mode of self-motion was investigated for a camphor disk on water upon the addition of sulfate surfactants with alkyl chains of different lengths as a simple autonomous system. With an increase in the concentration of surfactant with a longer alkyl chain (number of hydrocarbons: 14 or 16), two mode changes (continuous --intermittent (alternating between motion and rest) --no motion) were observed. With an increase in the concentration of surfactant with a shorter alkyl chain (number of hydrocarbon: 10 or 12), four mode changes (continuous --intermittent --continuous --intermittent --no motion) were observed. These two types of mode changes are discussed in relation to the solubility of the surfactant and camphor in the water phase and the surface tension of the surfactant, camphor, and a mixture of surfactant and camphor as the driving force of motion.

The periodic motion of a camphor float on water was investigated as a simple example of an autonomous motor. When a camphor disk that was connected to the center of a larger plastic disk was floated on water, intermittent motion (alternating between rest and rapid motion) was observed. The period of intermittent motion decreased with an increase in the hydrophobicity of the plastic film, which depended on the density of carbon powder that had been adhered to the plastic film with a laser printer. When a square plastic film with half-hydrophobic and half-hydrophilic surfaces was used as the camphor float, the direction of intermittent motion was mostly toward the hydrophilic side. These phenomena are discussed in relation to the diffusion of camphor molecules at the film/water interface and the surface tension around the film as the driving force.

As a simple example of an autonomous motor, the characteristic features of self-motion coupled with the acid-base reaction were numerically and experimentally investigated at the air/aqueous interface. Oscillatory and uniform motion were categorized as a function of the reaction order by numerical computations using a mathematical model that incorporates both the distribution of the surface active layer developed from a material particle as the driving force and the kinetics of the acid-base reaction. The nature of the self-motion was experimentally observed for a boat adhered to a camphor derivative with a mono- or di-carboxylic acid on a phosphate aqueous phase as the base. © the Owner Societies.

The length of Ag nanorods is uniquely controlled by irradiation with visible light using anatase TiO2-containing tubular SiO2 mesoporous templates. Spherical Ag nanoparticles and Ag nanorods of controlled length were fabricated by irradiation with visible light of selected wavelengths.