5G/B5G mmWave Cellular Networks with MEC Prefetching Based on User Context Information.

To deal with recent increasing mobile traffic, ultra-broadband communication with millimeter-wave (mmWave) has been regarded as a key technology for 5G cellular networks. In a previous study, a mmWave heterogeneous network was composed of several mmWave small cells overlaid on the coverage of a macro cell. However, as seen from the optical fiber penetration rate worldwide, it is difficult to say that backhaul with Gbps order is available everywhere. In the case of using mmWave access under a limited backhaul capacity, it becomes a bottleneck at the backhaul; thus, mmWave access cannot fully demonstrate its potential. On the other hand, the concept of multi-access edge computing (MEC) has been proposed to decrease the response latency compared to cloud computing by deploying storage and computation resources to the user side of mobile networks. This paper introduces MEC into mmWave heterogeneous networks and proposes a content prefetching algorithm to resolve such backhaul issues. Context information, such as the destination, mobility, and traffic tendency, is shared through the macro cell to the prefetch application and data that the users request. Prefetched data is stored in the MEC and then transmitted via mmWave without a backhaul bottleneck. The effectiveness is verified through computer simulations where we implement realistic user mobility as well as traffic and backhauling models. The results show that the proposed framework achieved 95% system capacity even under the constraint of a 1 Gbps backhaul link.

Multiple pathways for the formation of the γ-glutamyl peptides γ-glutamyl-valine and γ- glutamyl-valyl-glycine in Saccharomyces cerevisiae.

The role of glutathione (GSH) in eukaryotic cells is well known. The biosynthesis of this γ-glutamine tripeptide is well studied. However, other γ-glutamyl peptides were found in various sources, and the pathways of their formation were not always clear. The aim of the present study was to determine whether Saccharomyces cerevisiae can produce γ-glutamyl tripeptides other than GSH and to identify the pathways associated with the formation of these peptides. The tripeptide γ-Glu-Val-Gly (γ-EVG) was used as a model. Wild-type yeast cells were shown to produce this peptide during cultivation in minimal synthetic medium. Two different biosynthetic pathways for this peptide were identified. The first pathway consisted of two steps. In the first step, γ-Glu-Val (γ-EV) was produced from glutamate and valine by the glutamate-cysteine ligase (GCL) Gsh1p or by the transfer of the γ-glutamyl group from GSH to valine by the γ-glutamyltransferase (GGT) Ecm38p or by the (Dug2p-Dug3p)2 complex. In the next step, γ-EV was combined with glycine by the glutathione synthetase (GS) Gsh2p. The second pathway consisted of transfer of the γ-glutamyl residue from GSH to the dipeptide Val-Gly (VG). This reaction was carried out mainly by the (Dug2p-Dug3p)2 complex, whereas the GGT Ecm38p did not participate in this reaction. The contribution of each of these two pathways to the intracellular pool of γ-EVG was dependent on cultivation conditions. In this work, we also found that Dug1p, previously identified as a Cys-Gly dipeptidase, played an essential role in the hydrolysis of the dipeptide VG in yeast cells. It was also demonstrated that γ-EV and γ-EVG could be effectively imported from the medium and that γ-EVG was imported by Opt1p, known to be a GSH importer. Our results demonstrated that γ-glutamyl peptides, particularly γ-EVG, are produced in yeast as products of several physiologically important reactions and are therefore natural components of yeast cells.

Preparation of a γ-glutamylcysteine-enriched yeast extract from a newly developed GSH2-deficient strain.

Gamma-glutamylcysteine (γ-GC), the precursor of glutathione (GSH), may have significant health benefits as a dietary supplement, but there are few cost-effective methods available for its large-scale production. We developed an efficient method for producing γ-GC in a mutant yeast strain using a three-step breeding procedure and a unique cultivation process. In the first breeding step, we prepared a glutathione synthetase (GSH2)-deficient yeast mutant. In the second step, selenate (SeO(4)(2-)) sensitivity was introduced by crossing the GSH2-deficient mutant with a strain harboring the met30 mutation. In the final step, pantothenic acid auxotrophy was introduced by ethyl methanesulfonate mutagenesis. The isolated strain displayed significantly enhanced cellular γ-GC when cultivated in synthetic medium without pantothenic acid, reaching a maximum level of 4.39% of dry cell weight. Using this strain, we were able to prepare a yeast extract containing approximately 13% γ-GC (w/w), which is markedly higher than the reported value (0.3%) of commercially available yeast extracts. The present method may facilitate large-scale γ-GC production for investigating the nutritive value and other benefits of dietary γ-GC.

Novel method for screening Saccharomyces cerevisiae mutants with increased sulfur-containing compounds: color-based selection of ade1 or ade2 mutants.

We identified Saccharomyces cerevisiae mutants with 100% higher intracellular glutathione using 1-methyl-3-nitro-1-nitrosoguanidine mutagenesis. This method employs visual selection of the most pigmented colonies among met30 strains carrying ade1 and ade2 mutations. Since the method does not involve genetic engineering, the mutants are suitable for use in the food industry.

Novel method for screening Saccharomyces cerevisiae mutants with increased sulfur-containing compounds: color-based selection of colonies using the met30 strain.

Glutathione overproducers were detected by examining the pigmentation intensity of Saccharomyces cerevisiae met30 yeast carrying wild-type alleles for ADE1 and ADE2. Highly pigmented colonies, phenocopies of the ade2 or ade1 mutants, were observed among yeast grown in minimal biotin-free medium with a high methionine content.

A combination of flow cytometry and traditional screening using chemicals to isolate high glutathione-producing yeast mutants.

Traditional screening using chemicals or flow cytometry (FCM) alone is not sufficient to isolate the high glutathione (GSH)-producing yeast strains used in food production. Therefore, to improve screening efficiency, we investigated a combination of both methods. A mutated Saccharomyces cerevisiae strain was labeled with 5-chloromethylfluorescein diacetate and sorted by FCM according to emitted fluorescence intensity. Moderate GSH (1%-2%)-producing mutants were isolated, whereas high GSH (>2%)-producing mutants were not. Traditional screening using cerulenin resulted in similar findings, but a combination of both methods resulted in a 40% increase in the screening yield of high GSH-producing mutants. An analysis of model strains indicated that the ratio of high GSH-producing cells in a sample affected the FCM results. By combining FCM with traditional screening using chemicals, we succeeded in isolating high GSH-producing mutants from several parental strains.

Construction of a Saccharomyces cerevisiae strain with a high level of RNA.

A strategy has been developed for creating Saccharomyces cerevisiae strains with a high RNA content by following a three-step breeding procedure. In the first step, an S. cerevisiae disruptant of the RRN10 gene, one of the components of the UAF (upstream activation factor) complex of rRNA transcription, was constructed and showed severely slow growth. In the second step, seven suppressors were isolated that restored the slow growth of the Δrrn10 disruptant. Genetic analysis revealed that each of the seven suppressors that were isolated appeared to have dominant and multiple mutations. The specific growth rate of those suppressors was increased approximately two-fold as compared with the Δrrn10 parental strain. The absolute RNA content showed that the suppressors had an RNA content 32-56% higher than that of the Δrrn10 parental strain. In the last step, the RRN10 wild-type gene was integrated into chromosome V of each of the original suppressors. The total RNA content of the integrants was also 1.4- to 2.3-fold higher than the wild-type strain. In conclusion, since yeast RNA is the source of 5'-IMP and 5'-GMP that enhance the delicious taste in certain types of food, like soups and sauces, the strategy taken in this study provides effective approach to breed S. cerevisiae strains producing a higher content of RNA that will contribute to yeast food biotechnology.

A new method for repeated "self-cloning" promoter replacement in Saccharomyces cerevisiae.

A method for repeated PCR-mediated promoter replacement in the yeast Saccharomyces cerevisiae is described. It was proposed to use the DNA fragment comprising the marker gene that enables both positive and negative selection (a selectable/counter-selectable marker) surrounded by direct repeats of the desired promoter as a promoter replacement cassette. This fragment is integrated upstream of the target gene because of PCR-added terminal sequences for homologous recombination with the target locus. Subsequent marker excision via homologous recombination between the copies of the two promoters leaves one copy of the desired promoter upstream of the target genes, without any heterologous scar sequence. To test this method, a set of plasmids bearing the S. cerevisiae URA3 gene surrounded by two copies of the ADH1 or PGK1 promoter was constructed. Using these cassettes, the native promoters of the GSH1 and GSH2 genes were replaced in the ura3Δ0 recipient strains. The proposed method is useful for research applications due to simple marker excision, and for construction of "self-cloning" industrial strains, because no heterologous DNA is retained in the genome of the resulting strain after marker excision.