Cystine and tyrosine feed reduces oxidative and ER stress in CHO cells.

Multi-omics analyses was performed to compare the conditions of adding Tyr and Cystine in CHO cells. The addition of cystine resulted in decreased viability and productivity owing to endoplasmic reticulum (ER) stress and the promotion of ER-associated degradation (ERAD) and apoptosis. In contrast, addition of Tyr suppressed ER stress and apoptosis. This effect could be due to the increase in ubiquinone (Coenzyme Q10) biosynthesized from Tyr. To inhibit apoptosis caused by cystine addition, Tyr was added simultaneously with cystine, which improved growth, viability, and mAb productivity owing to the activation of GSH metabolism, suppression of ER stress and oxidative stress, reduction of ERAD, and activation of the tricarboxylic acid cycle.

Effect of alcohol on productivity and quality of adeno-associated virus 2 in HEK293 cells.

Investigation of enhancers to improve recombinant adeno-associated virus 2 (rAAV2) productivity by human embryonic kidney 293 cells (HEK293) suspension culture showed that the addition of ethanol improved the productivity and packaged genome integrity of rAAV2. Further optimization showed that adding ethanol in the range of 0.09%-1.11% (v/v) during rAAV2 production effectively improved rAAV2 productivity and quality. In addition, ethanol addition improved cell viability. Furthermore, proteome and pathway analysis of the cells during rAAV2 production showed that the addition of ethanol resulted in the upregulation of pathways related to intercellular signaling, gene expression, cell morphology, intercellular maintenance, and others. In contrast, pathways related to cell death, immunity, and reactions to infection were downregulated. These changes in pathway regulation were responsible for the improvement in rAAV2 productivity, packaged genome integrity, and cell viability during rAAV2 production. The results of this study can be applied to the production of viral vectors for in vivo gene therapy in an inexpensive and safe manner.

PiggyBac Transposon-Mediated CD19 Chimeric Antigen Receptor-T Cells Derived From CD45RA-Positive Peripheral Blood Mononuclear Cells Possess Potent and Sustained Antileukemic Function.

The quality of chimeric antigen receptor (CAR)-T cell products, namely, memory and exhaustion markers, affects the long-term functionality of CAR-T cells. We previously reported that piggyBac (PB) transposon-mediated CD19 CAR-T cells exhibit a memory-rich phenotype that is characterized by the high proportion of CD45RA+/C-C chemokine receptor type 7 (CCR7)+ T-cell fraction. To further investigate the favorable phenotype of PB-CD19 CAR-T cells, we generated PB-CD19 CAR-T cells from CD45RA+ and CD45RA- peripheral blood mononuclear cells (PBMCs) (RA+ CAR and RA- CAR, respectively), and compared their phenotypes and antitumor activity. RA+ CAR-T cells showed better transient gene transfer efficiency 24 h after transduction and superior expansion capacity after 14 days of culture than those shown by RA- CAR-T cells. RA+ CAR-T cells exhibited dominant CD8 expression, decreased expression of the exhaustion marker programmed cell death protein-1 (PD-1) and T-cell senescence marker CD57, and enriched naïve/stem cell memory fraction, which are associated with the longevity of CAR-T cells. Transcriptome analysis showed that canonical exhaustion markers were downregulated in RA+ CAR-T, even after antigen stimulation. Although antigen stimulation could increase CAR expression, leading to tonic CAR signaling and exhaustion, the expression of CAR molecules on cell surface after antigen stimulation in RA+ CAR-T cells was controlled at a relatively lower level than that in RA- CAR-T cells. In the in vivo stress test, RA+ CAR-T cells achieved prolonged tumor control with expansion of CAR-T cells compared with RA- CAR-T cells. CAR-T cells were not detected in the control or RA- CAR-T cells but RA+ CAR-T cells were expanded even after 50 days of treatment, as confirmed by sequential bone marrow aspiration. Our results suggest that PB-mediated RA+ CAR-T cells exhibit a memory-rich phenotype and superior antitumor function, thus CD45RA+ PBMCs might be considered an efficient starting material for PB-CAR-T cell manufacturing. This novel approach will be beneficial for effective treatment of B cell malignancies.

Genome sequencing of the NIES Cyanobacteria collection with a focus on the heterocyst-forming clade.

Cyanobacteria are a diverse group of Gram-negative prokaryotes that perform oxygenic photosynthesis. Cyanobacteria have been used for research on photosynthesis and have attracted attention as a platform for biomaterial/biofuel production. Cyanobacteria are also present in almost all habitats on Earth and have extensive impacts on global ecosystems. Given their biological, economical, and ecological importance, the number of high-quality genome sequences for Cyanobacteria strains is limited. Here, we performed genome sequencing of Cyanobacteria strains in the National Institute for Environmental Studies microbial culture collection in Japan. We sequenced 28 strains that can form a heterocyst, a morphologically distinct cell that is specialized for fixing nitrogen, and 3 non-heterocystous strains. Using Illumina sequencing of paired-end and mate-pair libraries with in silico finishing, we constructed highly contiguous assemblies. We determined the phylogenetic relationship of the sequenced genome assemblies and found potential difficulties in the classification of certain heterocystous clades based on morphological observation. We also revealed a bias on the sequenced strains by the phylogenetic analysis of the 16S rRNA gene including unsequenced strains. Genome sequencing of Cyanobacteria strains deposited in worldwide culture collections will contribute to understanding the enormous genetic and phenotypic diversity within the phylum Cyanobacteria.

Distribution of Phototrophic Purple Nonsulfur Bacteria in Massive Blooms in Coastal and Wastewater Ditch Environments.

The biodiversity of phototrophic purple nonsulfur bacteria (PNSB) in comparison with purple sulfur bacteria (PSB) in colored blooms and microbial mats that developed in coastal mudflats and pools and wastewater ditches was investigated. For this, a combination of photopigment and quinone profiling, pufM gene-targeted quantitative PCR, and pufM gene clone library analysis was used in addition to conventional microscopic and cultivation methods. Red and pink blooms in the coastal environments contained PSB as the major populations, and smaller but significant densities of PNSB, with members of Rhodovulum predominating. On the other hand, red-pink blooms and mats in the wastewater ditches exclusively yielded PNSB, with Rhodobacter, Rhodopseudomonas, and/or Pararhodospirillum as the major constituents. The important environmental factors affecting PNSB populations were organic matter and sulfide concentrations and oxidation‒reduction potential (ORP). Namely, light-exposed, sulfide-deficient water bodies with high-strength organic matter and in a limited range of ORP provide favorable conditions for the massive growth of PNSB over co-existing PSB. We also report high-quality genome sequences of Rhodovulum sp. strain MB263, previously isolated from a pink mudflat, and Rhodovulum sulfidophilum DSM 1374T, which would enhance our understanding of how PNSB respond to various environmental factors in the natural ecosystem.

Involvement of the response regulator CtrA in the extracellular DNA production of the marine bacterium Rhodovulum sulfidophilum.

The marine bacterium Rhodovulum sulfidophilum is a nonsulfur phototrophic bacterium, which is known to produce extracellular nucleic acids in soluble form in culture medium. In the present paper, constructing the response regulator ctrA-deficient mutant of R. sulfidophilum, we found that this mutation causes a significant decrease in the extracellular DNA production. However, by the introduction of a plasmid containing the wild type ctrA gene into the mutant, the amount of extracellular DNA produced was recovered. This is the first and clear evidence that the extracellular DNA production is actively controlled by the CtrA in R. sulfidophilum.

Characterization of the genuine type 2 chromatic acclimation in the two Geminocystis cyanobacteria.

Certain cyanobacteria can adjust the wavelengths of light they absorb by remodeling their photosynthetic antenna complex phycobilisome via a process called chromatic acclimation (CA). Although several types of CA have been reported, the diversity of the molecular mechanisms of CA among the cyanobacteria phylum is not fully understood. Here, we characterized the molecular process of CA of Geminocystis sp. strains National Institute of Environmental Studies (NIES)-3708 and NIES-3709. Absorption and fluorescence spectroscopy revealed that both strains dramatically alter their phycoerythrin content in response to green and red light. Whole-genome comparison revealed that the two strains share the typical phycobilisome structure consisting of a central core and peripheral rods, but they differ in the number of rod linkers of phycoerythrin and thus have differing capacity for phycoerythrin accumulation. RNA sequencing analysis suggested that the length of phycoerythrin rods in each phycobilisome is strictly regulated by the green light and red light-sensing CcaS/R system, whereas the total number of phycobilisomes is governed by the excitation-balancing system between phycobilisomes and photosystems. We reclassify the conventional CA types based on the genome information and designate CA of the two strains as genuine type 2, where components of phycoerythrin, but not rod-membrane linker of phycocyanin, are regulated by the CcaS/R system.

Complete Genome Sequence of Rhodovulum sulfidophilum DSM 2351, an Extracellular Nucleic Acid-Producing Bacterium.

Rhodovulum sulfidophilum DSM 2351 is the nonsulfur photosynthetic bacterium that efficiently releases nucleic acids into the extracellular milieu, which leads to flocculation. In this study, we determined the complete genome sequence of R. sulfidophilum DSM 2351, which will provide new insights into the mechanism of its unique nucleic acid release.

The gene transfer agent-like particle of the marine phototrophic bacterium Rhodovulum sulfidophilum.

Gene transfer agents (GTAs) are shaped like bacteriophage particles but have many properties that distinguish them from bacteriophages. GTAs play a role in horizontal gene transfer in nature and thus affect the evolution of prokaryotic genomes. In the course of studies on the extracellular production of designed RNAs using the marine bacterium Rhodovulum sulfidophilum, we found that this bacterium produces a GTA-like particle. The particle contains DNA fragments of 4.5 kb, which consist of randomly fragmented genomic DNA from the bacterium. This 4.5-kb DNA production was prevented while quorum sensing was inhibited. Direct observation of the particle by transmission electron microscopy revealed that the particle resembles a tailed phage and has a head diameter of about 40 nm and a tail length of about 60 nm. We also identified the structural genes for the GTA in the genome. Translated amino acid sequences and gene positions are closely related to those of the genes that encode the Rhodobacter capsulatus GTA. This is the first report of a GTA-like particle from the genus Rhodovulum. However, gene transfer activity of this particle has not yet been confirmed. The differences between this particle and other GTAs are discussed.

Short hairpin RNAs of designed sequences can be extracellularly produced by the marine bacterium Rhodovulum sulfidophilum.

Previously, we proposed a new method for production of RNA aptamers using the marine bacterium Rhodovulum sulfidophilum. A streptavidin RNA aptamer (an RNA which binds to streptavidin) was extracellularly produced by this bacterium containing engineered plasmid. The aptamer had full biological function. As a next step we attempted to produce another functional RNA, short hairpin RNAs (shRNAs) using this bacterial system. We have designed two types of shRNAs targeted to the luciferase gene. Here we report that shRNAs are successfully produced extracellularly by this system. Even if the shRNA has a long stem-loop structure which is thought to interfere with transcription in bacterial cells, the yield of the shRNA is almost the same as that of the streptavidin RNA aptamer. During the course of these experiments, we also found a new type of RNA processing for the double-stranded region of the shRNA.