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Establishment of mammalian cell lines with high protein productivity is an important object in the field of biopharmaceutics. Toward this end, Tol2 transposon-based expression systems have been developed as effective means to facilitate protein productivity. Here, we proposed novel strategies to improve conventional Tol2 transposon systems. The use of Tol2 transposase mRNA as a helper vector improved the efficiency of transgene integration and protein production. Moreover, the use of the Tol2 transposon vector containing the minimum cis-sequences essential for transposition (mini-TP) also served as one of the efficient means to generate recombinant cells that enable higher protein production. Furthermore, mini-TP showed a more beneficial response to DNA methylation inhibitors, suggesting that the use of mini-TP with DNA methylation inhibitors could be used as a means of commercial production. Taken together, our results provide effective strategies to improve the Tol2 transposon-based expression system. These strategies will be applicable to the production of therapeutic proteins and open new avenues in biopharmaceutical research.

Traditional cell line development is based on random genomic integration of transgenes. Random integration leads to unpredictable expression and results in clonal heterogeneity requiring a tedious screening procedure. Therefore, a new strategy is needed to establish clones that exhibit stable transgene expression. Here, we performed CRISPR/Cas9-mediated site-specific integration (SSI) to incorporate a landing pad (LP; containing mCherry) at a genomic hotspot (Fer1L4) allowing stable and strong expression. Site-specific integration of LP on Fer1L4 was demonstrated by sequencing results representing the swapped sequences in mCherry-expressing cells. We then performed Cre/Lox-based recombinase-mediated cassette exchange (RMCE) to exchange LP with a targeting vector (TV; containing GFP) in clones established by CRISPR/Cas9-mediated SSI. The success of Cre/Lox-based RMCE was evidenced by sequencing results representing the swapped sequences in GFP-expressing cells. Furthermore, the swapped clones expressing GFP was enriched up to 80%, indicating that the efficiency of Cre/Lox-based RMCE would be sufficient to swap pre-existing cassettes with gene-of-interest (GOI). Taken together, our study provides a new platform for Cre/Lox-based RMCE to iteratively establish stable clones from existing ones previously established by SSI at a genomic hotspot.

Cet article met l’accent sur la notion de création, entendue comme cercle vertueux dans l’œuvre de Bergson. La figure du cercle vertueux désigne un processus qui tire de soi rétrospectivement son identité à travers les événements présents. Nous montrerons que ce schème est à l’œuvre dans l’acte libre, puis dans l’action des vivants, et surtout au niveau du processus évolutif lui-même. Nous verrons ensuite s’il est possible d’élargir la perspective au-delà de ce schème traditionnel amené par le spiritualisme bergsonien. Nous montrerons que, contrairement à ce que souhaitait Bergson, nous pouvons en élargir l’extension au monde matériel lui-même.

In this paper, we understand the emergence of life as a pure individuation process. Individuation already occurs in open thermodynamics systems near equilibrium. We understand such open systems, as already recursively characterized (R1) by the relation between their internal properties, and their boundary conditions. Second, global properties emerge in such physical systems. We interpret this change as the fact that their structure is the recursive result of their operations (R2). We propose a simulation of the emergence of life in Earth by a mapping (R) through which (R1R2) operators are applied to themselves, so that RN = (R1R2)N. We suggest that under specific thermodynamic (open systems out of equilibrium) and chemical conditions (autocatalysis, kinetic dynamic stability), this mapping can go up to a limit characterized by a fixed-point equation: [Formula: see text]. In this equation, ([Formula: see text]) symbolizes a regime of permanent resonance characterizing the biosphere, as open from inside, by the recursive differential relation between the biosphere and all its holobionts. As such the biosphere is closed on itself as a pure differential entity. ([Formula: see text]) symbolizes the regime of permanent change characterizing the emergence of evolution in the biosphere. As such the biosphere is closed on itself, by the principle of descent with modifications, and by the fact that every holobiont evolves in a niche, while evolving with it.

Lysosomal storage disease (LSD) is an inherited metabolic disorder caused by enzyme deficiency in lysosomes. Some treatments for LSD can slow progression, but there are no effective treatments to restore the pathological phenotype to normal levels. Lysosomes and mitochondria interact with each other, and this crosstalk plays a role in the maintenance of cellular homeostasis. Deficiency of lysosome enzymes in LSD impairs the turnover of mitochondrial defects, leading to deterioration of the mitochondrial respiratory chain (MRC). Cells with MRC impairment are associated with reduced lysosomal calcium homeostasis, resulting in impaired autophagic and endolysosomal function. This malicious feedback loop between lysosomes and mitochondria exacerbates LSD. In this review, we assess the interactions between mitochondria and lysosomes and propose the mitochondrial–lysosomal axis as a research target to treat LSD. The importance of the mitochondrial–lysosomal axis has been systematically characterized in several studies, suggesting that proper regulation of this axis represents an important investigative guide for the development of therapeutics for LSD. Therefore, studying the mitochondrial–lysosomal axis will not only add knowledge of the essential physiological processes of LSD, but also provide new strategies for treatment of LSD.

In this paper, we insist on stressing the epistemic and metaphysical difference between individual and individuation, a distinction originally developed by Gilbert Simondon. Individuation occurs in complex physical systems by the coupling (R1) between the system and its outside conditions. As such the system is not well defined by its sole constituents. Let's characterize (R2) as follows: the system is not entirely defined by its structure at a given time because this structure will change and global emergent properties will appear, as in the paradigmatic example of phase transition. Thus physical individuation is defined both by the coupling of a physical system with its environment (R1) and by its diachronic dynamics taking place (R2). We interpret biological individuation as a second order one, i.e. as a recursive procedure through which physical individuation is also acting on "its own theatre". We represent this procedure like a mapping through which (R1R2) are applied to themselves, so that: RN = (R1R2)N. We highlight the relation between this assumption and the concept of extended criticality developed by Bailly, Longo and Montevil.

Acaiberry(Euterpe oleracea Mart.) was partially substituted from the formula for juak and the subsequent effects on the physiochemical and physical characteristics. And this study was carried out to standardize the recipe of acaiberry-juak. Acaiberry powder was added in 0(control), 1, 3 and 5% substitution ratios by weight per glutinous rice flour. Also, acaiberry puree was added in 3, 6 and 9% substitution ratios by weight water. The moisture contents showed juak with addition of acaiberry contents were lower then control. The diameter of the juak decreased with increasing levels of the added acaiberry powder and puree, whereas the height increased with increasing amount of the powder and puree. The Hunter colorimeteric L-values of the dough and acaiberry-juak decreased as the amount of acaiberry powder and puree increased. Hunter a and b-values both increased with increasing levels of the acaiberry powder and puree. Textural characteristics there were increase in hardness, springiness, chewiness as the amount of acaiberry powder and puree increased(p

We explore the concept of “biological individuation” (l’individuation biologique) which was introduced by a French philosopher: Simondon in his doctorate thesis initially defended in 1958 and entitled “l'individuation a la lumiere des notions de formes et d'information. More precisely, we will focus on the assertion according to which within the living, there is “a regime of internal resonance” that requires “permanent communication” and through which individuation is acting on itself. We will provide a formal representation of this assertion.

The effects of addition of turmeric powder (0%, 2%, 4%, 6% and 8%) were examined in order to obtain an antioxidant-enriched cake with good physico-chemical and sensorial properties. The rheological properties of doughs were evaluated using dynamic rheological measurements. Physical properties, curcumin content, radical scavenging activity (RSA-DPPH assay) and sensory analysis (hedonic test) of the supplemented cake were determined. Addition of turmeric powder up to 8% caused significant changes on dough characteristics and on cake rheological properties. The highest curcumin (203 mg/kg) and RSA-DPPH activity (45%) were achieved in the cake having the highest percentage of turmeric powder (8%); however, this sample showed the worst results regarding the rheological properties. Moreover, by sensory evaluation this cake sample was not acceptable. A 6% substitution of wheat flour with turmeric powder showed acceptable sensory scores which were comparable to those of 0–4% turmeric cakes. This indicated that up to 6% level of turmeric powder might be included in cake formulation.

Le probleme de l’identite personnelle est une preoccupation essentielle des philosophes modernes depuis que la conscience est mise en scene philosophiquement. Cependant parmi eux il n’y en a pas beaucoup qui considerent la memoire comme le fondement de l’identite humaine, bien qu’aujourd’hui, et grace aux neurosciences, on sache pourtant qu’elle joue un role capital. D’une maniere generale, les empiristes s’y interessent davantage que les rationnalistes. Ceux-ci ayant comme ideal normatif les systemes mathematiques ne pensent pas qu’elle puisse contribuer a elargir nos connaissances du monde. C’est ce qui explique que le systeme des idees claires et distinctes chez Descartes, s’etablit directement par la conscience presente. De son cote, l’empiriste sceptique Hume, bien qu’il ait souligne l’importance de la memoire dans notre personnalite, n’a pas pu mettre en pleine lumiere sa nature et son fonctionnement. L’esprit considere comme l’ensemble des perceptions et des idees est loin de constituer l’identite de l’homme normal. On voit dans les theories bergsoniennes de la duree et de la memoire une toute autre perspective qui fonde l’identite personnelle sur l’equi libre mental de la conscience presente et de l’inconscient, c’est-a-dire du moi superficiel et du moi profond.

Service products are consumed by customers as soon as they are produced and delivered. In the service operations, customer contact is an essential part of the service delivery process. Often, the mode and degree of customer contact affects the service quality, and thus customer contact could be an important determinant of service quality. Customer contact is an important element of service process design and management for such industries as airline, hotel, restaurant, etc. In customer contact, there are two aspects: quantity and quality of contact. In this paper, we focused on the quantity aspect of customer contact and investigated its impact on service quality. This paper examines the relationships between the degree of customer contact, i.e., a quantitative measure, and the service quality, and attempts: 1) to assess the impacts of customer contact on the service quality; 2) to identify important elements of customer contact that has significant impacts on the service quality. Data were collected from the customers who use airlines. Survey questionnaire was developed based on the previous research in this field, and used for data collection.