Effect of Indoor Forest Bathing on Reducing Feelings of Fatigue Using Cerebral Activity as an Indicator.

We created an indoor forest bathing environment in a sunlight-type environmentally controlled chamber and both physiological and psychological measurements were conducted for the evaluation of mental fatigue reduction. At first, a working memory load experiment was performed among 10 participants in a space without plants to identify an indicator correlating with feelings of fatigue, using the cerebral activity of the prefrontal cortex. Then, the indicator was used to evaluate whether a 20-min exposure to an indoor forest bathing environment reduced the level of the feeling of fatigue. The working memory load experiment demonstrated that, when mental fatigue increased, the amount of oxygenated hemoglobin (oxy-Hb) in the right prefrontal cortex and the right-left difference in oxy-Hb (ΔRL oxy-Hb) in the prefrontal cortex increased. These were proposed as indicators of mental fatigue. In the indoor forest bathing experiment, staying in an indoor green space showed that the subjective values of feeling of fatigue decreased and ΔRL oxy-Hb decreased. Since these results demonstrated an opposite effect to the increase in ΔRL oxy-Hb related to the feeling of fatigue, it was inferred that the decrease in ΔRL oxy-Hb reflected the fatigue reduction in the indoor forest bathing environment.

Seq2seq Fingerprint with Byte-Pair Encoding for Predicting Changes in Protein Stability upon Single Point Mutation.

The engineering of stable proteins is crucial for various industrial purposes. Several machine learning methods have been developed to predict changes in the stability of proteins corresponding to single point mutations. To improve the prediction accuracy, we propose a new unsupervised descriptor for protein sequences, which is based on a sequence-to-sequence (seq2seq) neural network model combined with a sequence-compression method called byte-pair encoding (BPE). Our results demonstrate that BPE can encode a protein sequence into a sequence of shorter length, thereby enabling efficient training of the seq2seq model. Furthermore, we implement a basic predictor using the proposed descriptor, and our experimental results demonstrate that the predictor achieves state-of-the-art accuracy in tests for proteins that are not included in the training data.

Combinatorial Screening for Transgenic Yeasts with High Cellulase Activities in Combination with a Tunable Expression System.

Combinatorial screening used together with a broad library of gene expression cassettes is expected to produce a powerful tool for the optimization of the simultaneous expression of multiple enzymes. Recently, we proposed a highly tunable protein expression system that utilized multiple genome-integrated target genes to fine-tune enzyme expression in yeast cells. This tunable system included a library of expression cassettes each composed of three gene-expression control elements that in different combinations produced a wide range of protein expression levels. In this study, four gene expression cassettes with graded protein expression levels were applied to the expression of three cellulases: cellobiohydrolase 1, cellobiohydrolase 2, and endoglucanase 2. After combinatorial screening for transgenic yeasts simultaneously secreting these three cellulases, we obtained strains with higher cellulase expressions than a strain harboring three cellulase-expression constructs within one high-performance gene expression cassette. These results show that our method will be of broad use throughout the field of metabolic engineering.

Overexpression of a novel Arabidopsis PP2C isoform, AtPP2CF1, enhances plant biomass production by increasing inflorescence stem growth.

In contrast to mammals, higher plants have evolved to express diverse protein phosphatase 2Cs (PP2Cs). Of all Arabidopsis thaliana PP2Cs, members of PP2C subfamily A, including ABI1, have been shown to be key negative regulators of abscisic acid (ABA) signalling pathways, which regulate plant growth and development as well as tolerance to adverse environmental conditions. However, little is known about the enzymatic and signalling roles of other PP2C subfamilies. Here, we report a novel Arabidopsis subfamily E PP2C gene, At3g05640, designated AtPP2CF1. AtPP2CF1 was dramatically expressed in response to exogenous ABA and was expressed in vascular tissues and guard cells, similar to most subfamily A PP2C genes. In vitro enzymatic activity assays showed that AtPP2CF1 possessed functional PP2C activity. However, yeast two-hybrid analysis revealed that AtPP2CF1 did not interact with PYR/PYL/RCAR receptors or three SnRK2 kinases, which are ABI1-interacting proteins. This was supported by homology-based structural modelling demonstrating that the putative active- and substrate-binding site of AtPP2CF1 differed from that of ABI1. Furthermore, while overexpression of ABI1 in plants induced an ABA-insensitive phenotype, Arabidopsis plants overexpressing AtPP2CF1 (AtPP2CF1oe) were weakly hypersensitive to ABA during seed germination and drought stress. Unexpectedly, AtPP2CF1oe plants also exhibited increased biomass yield, mainly due to accelerated growth of inflorescence stems through the activation of cell proliferation and expansion. Our results provide new insights into the physiological significance of AtPP2CF1 as a candidate gene for plant growth production and for potential application in the sustainable supply of plant biomass.

Characterization of five terminator regions that increase the protein yield of a transgene in Saccharomyces cerevisiae.

Strong terminator regions could be used to improve metabolically engineered yeasts by increasing the target enzyme protein yields above those achieved with traditional terminator regions. We recently identified five strong terminator regions (RPL41Bt, RPL15At, DIT1t, RPL3t, and IDP1t) in a comprehensive analysis of Saccharomyces cerevisiae. The effect of the terminator regions was analyzed by measuring the protein production of a linked transgene, and was shown to be twice that of a traditional terminator region (PGK1t). Here, we investigated whether the activity of the terminator regions is affected by exchange of a strong promoter or reporter in the linked transgene, carbon source for cell growth, stress factors, host yeast strain, or stage of the growth phase. Our results indicate that the activities of all five terminator regions were twice that of PGK1t in all conditions tested. In addition, we demonstrated that the strong activity of these terminator regions could be used to improve secretory production of endoglucanase II derived from Tricoderma ressei, and that the DIT1t strain was the best of the five strains for this purpose. We therefore propose that DIT1t, and the four other terminator regions, could be applied to the development of improved metabolically engineered yeasts.

A genome-wide activity assessment of terminator regions in Saccharomyces cerevisiae provides a ″terminatome″ toolbox.

The terminator regions of eukaryotes encode functional elements in the 3' untranslated region (3'-UTR) that influence the 3'-end processing of mRNA, mRNA stability, and translational efficiency, which can modulate protein production. However, the contribution of these terminator regions to gene expression remains unclear, and therefore their utilization in metabolic engineering or synthetic genetic circuits has been limited. Here, we comprehensively evaluated the activity of 5302 terminator regions from a total of 5880 genes in the budding yeast Saccharomyces cerevisiae by inserting each terminator region downstream of the P TDH3 - green fluorescent protein (GFP) reporter gene and measuring the fluorescent intensity of GFP. Terminator region activities relative to that of the PGK1 standard terminator ranged from 0.036 to 2.52, with a mean of 0.87. We thus could isolate the most and least active terminator regions. The activities of the terminator regions showed a positive correlation with mRNA abundance, indicating that the terminator region is a determinant of mRNA abundance. The least active terminator regions tended to encode longer 3'-UTRs, suggesting the existence of active degradation mechanisms for those mRNAs. The terminator regions of ribosomal protein genes tended to be the most active, suggesting the existence of a common regulator of those genes. The ″terminatome″ (the genome-wide set of terminator regions) thus not only provides valuable information to understand the modulatory roles of terminator regions on gene expression but also serves as a useful toolbox for the development of metabolically and genetically engineered yeast.

Enhancement of thermal stabilization of formaldehyde dehydrogenase from Pseudomonas putida by directed evolution.

We used directed evolution to enhance the thermostability of formaldehyde dehydrogenase from Pseudomonas putida. At 50 degrees C, the wild-type enzyme was inactivated within 30 min, but the variants obtained retained 80% activity for at least 300 min. At room temperature (30 degrees C), the variants obtained retained <80% activity for at least 500 h (21 d).

Improvement of H2O2 stability of manganese peroxidase by combinatorial mutagenesis and high-throughput screening using in vitro expression with protein disulfide isomerase.

A functional expression system for a heme protein of Phanerochaete chrysosporium, manganese peroxidase (MnP), was developed using the Escherichia coli in vitro coupled transcription/translation system in the presence of hemin and fungal protein disulfide isomerase. This system has allowed the high-throughput construction and screening of a large diversity of mutant heme enzymes and has made it possible to improve the enzymatic function efficiently. Here we increased the H2O2 stability of MnP using this system; a mutant MnP library containing three randomized amino acid residues located in the H2O2-binding pocket of MnP was designed and constructed on a 384-well plate using SIMPLEX (single-molecule-PCR-linked in vitro expression). The screening of 10(4) samples independently expressed for improved H2O2 stability led to four positive mutants, the H2O2 stability of which was nine times higher than that of the wild-type.