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We previously established the selection-marker-free rice-based oral cholera vaccine (MucoRice-CTB) line 51A for human use by Agrobacterium-mediated co-transformation and conducted a double-blind, randomized, placebo-controlled phase I trial in Japan and the United States. Although MucoRice-CTB 51A was acceptably safe and well tolerated by healthy Japanese and U.S. subjects and induced CTB-specific antibodies neutralizing cholera toxin secreted by Vibrio cholerae, we were limited to a 6-g cohort in the U.S. trial because of insufficient production of MucoRice-CTB. Since MucoRice-CTB 51A did not grow in sunlight, we re-examined the previously established marker-free lines and selected MucoRice-CTB line 19A. Southern blot analysis of line 19A showed a single copy of the CTB gene. We resequenced the whole genome and detected the transgene in an intergenic region in chromosome 1. After establishing a master seed bank of MucoRice-CTB line 19A, we established a hydroponic production facility with LED lighting to reduce electricity consumption and to increase production capacity for clinical trials. Shotgun MS/MS proteomics analysis of MucoRice-CTB 19A showed low levels of α-amylase/trypsin inhibitor-like proteins (major rice allergens), which was consistent with the data for line 51A. We also demonstrated that MucoRice-CTB 19A had high oral immunogenicity and induced protective immunity against cholera toxin challenge in mice. These results indicate that MucoRice-CTB 19A is a suitable oral cholera vaccine candidate for Phase I and II clinical trials in humans, including a V. cholerae challenge study.

BACKGROUND: We have previously developed a rice-based oral vaccine against cholera diarrhea, MucoRice-CTB. Using Agrobacterium-mediated co-transformation, we produced the selection marker-free MucoRice-CTB line 51A, which has three copies of the cholera toxin B subunit (CTB) gene and two copies of an RNAi cassette inserted into the rice genome. We determined the sequence and location of the transgenes on rice chromosomes 3 and 12. The expression of alpha-amylase/trypsin inhibitor, a major allergen protein in rice, is lower in this line than in wild-type rice. Line 51A was self-pollinated for five generations to fix the transgenes, and the seeds of the sixth generation produced by T5 plants were defined as the master seed bank (MSB). T6 plants were grown from part of the MSB seeds and were self-pollinated to produce T7 seeds (next seed bank; NSB). NSB was examined and its whole genome and proteome were compared with those of MSB. RESULTS: We re-sequenced the transgenes of NSB and MSB and confirmed the positions of the three CTB genes inserted into chromosomes 3 and 12. The DNA sequences of the transgenes were identical between NSB and MSB. Using whole-genome sequencing, we compared the genome sequences of three NSB with three MSB samples, and evaluated the effects of SNPs and genomic structural variants by clustering. No functionally important mutations (SNPs, translocations, deletions, or inversions of genic regions on chromosomes) between NSB and MSB samples were detected. Analysis of salt-soluble proteins from NSB and MSB samples by shot-gun MS/MS detected no considerable differences in protein abundance. No difference in the expression pattern of storage proteins and CTB in mature seeds of NSB and MSB was detected by immuno-fluorescence microscopy. CONCLUSIONS: All analyses revealed no considerable differences between NSB and MSB samples. Therefore, NSB can be used to replace MSB in the near future.

Airflow plays a crucial role in plant growth because it supplies CO2, O2, and energy to plants in a plant factory with artificial light (PFAL). Therefore, understanding how various factors affect airflow in and around a plant canopy is essential. In this study, we developed a computational fluid dynamics (CFD) model with realistic plant structures created using structure-from-motion imaging to investigate airflow in and around a plant canopy. The averages of the absolute percentage errors of simulated air velocity in three conditions were 6.7%, 10.1%, 12.7%, respectively. The simulated and measured air velocities agreed well, confirming the accuracy of the developed CFD model. The effects of inflow velocities and plant canopy structures on the airflow in and around the plant canopy were analysed using the validated CFD model. The inflow velocities significantly decreased stagnant zones (from 62.4% to 7.2%) and increased the airflow uniformity in and around the plant canopy. A staggered layout of the plant canopy slightly decreased stagnant zones (from 16.4% to 13.2%) and increased the airflow uniformity. The airflow in and around the plant canopy was further inhibited by a large plant structure. This CFD model provided a basis for improving the airflow status in and around a plant canopy in a PFAL. [ABSTRACT FROM AUTHOR]

Improving edible biomass space use efficacy (EBSUE) is important for sustainably producing edamame and dwarf tomatoes in plant factories with artificial light. Photosynthetic photon flux density (PPFD) may increase EBSUE and space use efficacy (SUE). However, no study has quantitatively explained how PPFD affects EBSUE in edamame and dwarf tomatoes. This study aimed to quantitatively validate the effects of PPFD on EBSUE in dwarf tomatoes and edamame and verify whether this effect differs between these crops. The edamame and dwarf tomato cultivars 'Enrei' and 'Micro-Tom', respectively, were cultivated under treatments with PPFDs of 300, 500, and 700 µmol m−2 s−1. The results showed that the EBSUE and SUE increased with increasing PPFD in both crops. The EBSUE increased depending on the increase in SUE, the dry mass ratio of the edible part to the total plant in the edamame, and the SUE only in the dwarf tomatoes. In conclusion, a high PPFD can improve the EBSUE and SUE of edamame and dwarf tomatoes in different ways at the reproductive growth stage. The findings from this study offer valuable information on optimizing space and resource usage in plant factories with artificial light and vertical farms. Additionally, they shed light on the quantitative impact of PPFD on both EBSUE and SUE. [ABSTRACT FROM AUTHOR]

LUBAC (linear ubiquitin chain assembly complex) activates the canonical NF-κB pathway through linear polyubiquitination of NEMO (NF-κB essential modulator, also known as IKKγ) and RIP1. However, the regulatory mechanism of LUBAC-mediated NF-κB activation remains elusive. Here, we show that A20 suppresses LUBAC-mediated NF-κB activation by binding linear polyubiquitin via the C-terminal seventh zinc finger (ZF7), whereas CYLD suppresses it through deubiquitinase (DUB) activity. We determined the crystal structures of A20 ZF7 in complex with linear diubiquitin at 1.70-1.98 Å resolutions. The crystal structures revealed that A20 ZF7 simultaneously recognizes the Met1-linked proximal and distal ubiquitins, and that genetic mutations associated with B cell lymphomas map to the ubiquitin-binding sites. Our functional analysis indicated that the binding of A20 ZF7 to linear polyubiquitin contributes to the recruitment of A20 into a TNF receptor (TNFR) signalling complex containing LUBAC and IκB kinase (IKK), which results in NF-κB suppression. These findings provide new insight into the regulation of immune and inflammatory responses.

Summary: We construct a latent employment estimate for the United States, which both reconciles the information from separate payroll and household surveys and incorporates the preliminary data revision process of the payroll data. We find that our reconciled latent employment series looks somewhat different than the initial release of payroll employment and is closer to the fully revised data that is benchmarked to a near census of employment. A real‐time exercise, however, suggests that the reconciled employment estimate is remarkably similar to the initial release of payroll employment with near zero weight on the household survey information. [ABSTRACT FROM AUTHOR]

Airflow parameters play a vital role in regulating the microclimate of the plant canopy and significantly affect plant development and growth in plant factories with artificial light (PFALs). A computational fluid dynamics (CFD) model with a realistic soybean canopy was developed to predict the distribution of radiation (W m−2), leaf temperature (°C), air velocity (m s−1), air temperature (°C), and relative humidity (%) within a plant canopy. The accuracy of the CFD model was confirmed by comparing mean absolute percentage errors of these variables which were 9.9 %, 1.4 %, 11.4 %, 0.8 %, and 1.8 %, respectively. The validated CFD model was used to simulate the lamp temperature and microclimate distribution of the soybean canopy under different inflow parameter levels on a cultivation shelf. The results revealed that the microclimate was primarily influenced by lamp radiation rather than convective heat when the inflow velocity was below 0.57 m s−1. Inflow velocity, air temperature, and relative humidity exerted more significant effects on air velocity, air temperature, and relative humidity, respectively, than the other variables in the plant canopy. The increase of inflow velocity significantly improved the uniformity of air velocity, temperature, and relative humidity in the plant canopy. However, note that this study investigated the lamp temperature and microclimate distribution in plant canopies under different airflow parameters using the CFD model without coupling plant transpiration to the surrounding climate. This provides valuable insights into the control of the plant canopy microclimate in general and more particularly on the shelves of PFALs. [Display omitted] • CFD model with realistic plants was developed to simulate plant canopy microclimate. • A realistic plant model is established using structure-from-motion imaging. • The coupling of convective and radiative heat transfer from lamps was analyzed. [ABSTRACT FROM AUTHOR]