Multi-line split DNA synthesis: a novel combinatorial method to make high quality peptide libraries

BACKGROUND: We developed a method to make a various high quality random peptide libraries for evolutionary protein engineering based on a combinatorial DNA synthesis. RESULTS: A split synthesis in codon units was performed with mixtures of bases optimally designed by using a Genetic Algorithm program. It required only standard DNA synthetic reagents and standard DNA synthesizers in three lines. This multi-line split DNA synthesis (MLSDS) is simply realized by adding a mix-and-split process to normal DNA synthesis protocol. Superiority of MLSDS method over other methods was shown. We demonstrated the synthesis of oligonucleotide libraries with 10(16 )diversity, and the construction of a library with random sequence coding 120 amino acids containing few stop codons. CONCLUSIONS: Owing to the flexibility of the MLSDS method, it will be able to design various "rational" libraries by using bioinformatics databases

MARCKSL1 Regulates Spine Formation in the Amygdala and Controls the Hypothalamic-Pituitary-Adrenal Axis and Anxiety-Like Behaviors

Takashi Tanaka, Shoko Shimizu, Masaki Ueno, Yoshitaka Fujihara, Masahito Ikawa, Shingo Miyata, MARCKSL1 Regulates Spine Formation in the Amygdala and Controls the Hypothalamic-Pituitary-Adrenal Axis and Anxiety-Like Behaviors, EBioMedicine, Volume 30, 2018, Pages 62-73, ISSN 2352-3964, https://doi.org/10.1016/j.ebiom.2018.03.018

Fundamental modeling of vehicle power network system using VHDL-AMS

The vehicle system is a multi-domain system that requires many branches of science and engineering. Therefore the development of the vehicle system requires the use of design methodologies that utilize simulations, which have grown increasingly sophisticated in recent years. This paper describes how VHDL-AMS was used to model the powertrain, alternator and battery system that are the basic elements of the vehicle power network system, and examines the simulation results with the aim of realizing a system simulation whose scope covers the entire vehicle.2010 International Symposium on Power Electronics, Electrical Drives, Automation and Motion (SPEEDAM 2010) : Pisa, Italy, 2010.06.14-2010.06.1

Measurement of evaporation-residue cross sections with light beams and deformed lanthanide target nuclei

To obtain a better understanding of the fusion reaction, we have focused on reactions involving deformed nuclei. Evaporation residue cross sections of the 169Tm+20Ne reaction were measured, from which we extracted the fusion excitation function. This is compared with literature data of the 169Tm+16O and 165Ho+20Ne systems. Irradiation with 20Ne ion beam has been carried out at the incident energy near the Coulomb barrier, where the effect of nuclear deformation is prominent. The results are consistent with the idea that the degree of deformation has an effect on the threshold value of the excitation functions near the Coulomb barrier

Comparative study of the work load between one-man buses and two-man buses.

The differences in physiological and safety conditions of one-man buses and two-man buses were examined from the view point of occupational fatigue. This survey consisted of a work load study which included a time study, study of subsidiary behavior, auditory task, memory test, Galvanic Skin Response (GSR) and physiological function tests and a self-administered questionnaire which involved items concerning safety and subjective fatigue complaints. The visual and postural restrictions in the one-man bus were greater than in the two-man bus. The mental capacity of the one-man bus drivers was found to be less. Greater mental fatigue and stress were observed in the one-man bus. More subjective fatigue complaints were observed in the one-man bus. More cases of near accidents were observed in the one-man bus. From these results it was concluded that the one-man bus caused bus drivers a greater mental and physical work load.</p

BCAA catabolism in brown fat controls energy homeostasis through SLC25A44.

Branched-chain amino acid (BCAA; valine, leucine and isoleucine) supplementation is often beneficial to energy expenditure; however, increased circulating&nbsp;levels of BCAA are linked to obesity and diabetes. The mechanisms of this paradox remain unclear. Here we report that, on cold exposure, brown adipose tissue (BAT) actively utilizes BCAA in the mitochondria for thermogenesis and promotes systemic BCAA clearance in mice and humans. In turn, a BAT-specific defect in BCAA catabolism attenuates systemic BCAA clearance, BAT fuel oxidation and thermogenesis, leading to diet-induced obesity and glucose intolerance. Mechanistically, active BCAA catabolism in BAT is mediated by SLC25A44, which transports BCAAs into mitochondria. Our results suggest that BAT serves as a key metabolic filter that controls BCAA clearance via SLC25A44, thereby contributing to the improvement of metabolic health

Isolation and characterization of the TIGA genes, whose transcripts are induced by growth arrest

We report here the isolation of 44 genes that are upregulated after serum starvation and/or contact inhibition. These genes have been termed TIGA, after Transcript Induced by Growth Arrest. We found that there are two kinds of G0 phases caused by serum starvation, namely, the shallow G0 (or G0/G1) and the deep G0 phases. The shallow G0 is induced by only a few hours of serum starvation, while deep G0 is generated after 3 days of serum starvation. We propose that mammalian cells enter deep G0 through a G0 gate, which is only opened on the third day of serum starvation. TIGA1, one of the uncharacterized TIGA genes, encodes a homolog of cyanate permease of bacteria and localizes in mitochondria. This suggests that Tiga1 is involved in the inorganic ion transport and metabolism needed to maintain the deep G0 phase. Ectopic expression of TIGA1 inhibited not only tumor cell proliferation but also anchorage-independent growth of cancer cell lines. A microsatellite marker, ENDL-1, allowed us to detect loss of heterozygosity around the TIGA1 gene region (5q21–22). Further analysis of the TIGA genes we have identified here may help us to better understand the mechanisms that regulate the G0 phase

Guidelines for the use and interpretation of assays for monitoring autophagy (3rd edition)

In 2008 we published the first set of guidelines for standardizing research in autophagy. Since then, research on this topic has continued to accelerate, and many new scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Accordingly, it is important to update these guidelines for monitoring autophagy in different organisms. Various reviews have described the range of assays that have been used for this purpose. Nevertheless, there continues to be confusion regarding acceptable methods to measure autophagy, especially in multicellular eukaryotes. For example, a key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers or volume of autophagic elements (e.g., autophagosomes or autolysosomes) at any stage of the autophagic process versus those that measure fl ux through the autophagy pathway (i.e., the complete process including the amount and rate of cargo sequestered and degraded). In particular, a block in macroautophagy that results in autophagosome accumulation must be differentiated from stimuli that increase autophagic activity, defi ned as increased autophagy induction coupled with increased delivery to, and degradation within, lysosomes (inmost higher eukaryotes and some protists such as Dictyostelium ) or the vacuole (in plants and fungi). In other words, it is especially important that investigators new to the fi eld understand that the appearance of more autophagosomes does not necessarily equate with more autophagy. In fact, in many cases, autophagosomes accumulate because of a block in trafficking to lysosomes without a concomitant change in autophagosome biogenesis, whereas an increase in autolysosomes may reflect a reduction in degradative activity. It is worth emphasizing here that lysosomal digestion is a stage of autophagy and evaluating its competence is a crucial part of the evaluation of autophagic flux, or complete autophagy. Here, we present a set of guidelines for the selection and interpretation of methods for use by investigators who aim to examine macroautophagy and related processes, as well as for reviewers who need to provide realistic and reasonable critiques of papers that are focused on these processes. These guidelines are not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to monitor autophagy. Along these lines, because of the potential for pleiotropic effects due to blocking autophagy through genetic manipulation it is imperative to delete or knock down more than one autophagy-related gene. In addition, some individual Atg proteins, or groups of proteins, are involved in other cellular pathways so not all Atg proteins can be used as a specific marker for an autophagic process. In these guidelines, we consider these various methods of assessing autophagy and what information can, or cannot, be obtained from them. Finally, by discussing the merits and limits of particular autophagy assays, we hope to encourage technical innovation in the field