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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.

Abnormal expression of histone deacetylases (HDACs) is reported to be associated with angiogenesis, metastasis and chemotherapy resistance regarding cancer in a wide range of previous studies. Suberoylanilide hydroxamic acid (SAHA) is well known to function as a pan-inhibitor for HDACs and recognized as one of the therapeutic drug candidates to epigenetically coordinate cancer cell fate regulation on a genomic scale. Here, we established a Real-Time Search (RTS)-assisted mass spectrometric platform for system-wide quantification of translated products encoded by non-canonical short open reading frames (ORFs) as well as already annotated protein coding sequences (CDSs) on the human transciptome and applied this methodology to quantitative proteomic analyses of suberoylanilide hydroxamic acid (SAHA)-treated human HeLa cells to evaluate proteome-wide regulation in response to drug perturbation. Very intriguingly, our RTS-based in-depth proteomic analysis enabled us to identify approximately 5000 novel peptides from the ribosome profiling-based short ORFs encoded in the diversified regions on presumed 'non-coding' nucleotide sequences of mRNAs as well as lncRNAs and nonsense mediated decay (NMD) transcripts. Furthermore, TMT-based multiplex large-scale quantification of the whole proteome changes upon differential SAHA treatment unveiled dose-dependent selective translational regulation of a limited fraction of the non-canonical short ORFs in addition to key cell cycle/proliferation-related molecules such as UBE2C, CENPF and PRC1. Our study provided the first system-wide landscape of drug-perturbed translational modulation on both canonical and non-canonical proteome dynamics in human cancer cells. [ABSTRACT FROM AUTHOR]

Rotavirus-induced diarrhea is a life-threatening disease in immunocompromised individuals and in children in developing countries. We have developed a system for prophylaxis and therapy against rotavirus disease using transgenic rice expressing the neutralizing variable domain of a rotavirus-specific llama heavy-chain antibody fragment (MucoRice-ARP1). MucoRice-ARP1 was produced at high levels in rice seeds using an overexpression system and RNAi technology to suppress the production of major rice endogenous storage proteins. Orally administered MucoRice-ARP1 markedly decreased the viral load in immunocompetent and immunodeficient mice. The antibody retained in vitro neutralizing activity after long-term storage (>1 yr) and boiling and conferred protection in mice even after heat treatment at 94°C for 30 minutes. High-yield, water-soluble, and purification-free MucoRice-ARP1 thus forms the basis for orally administered prophylaxis and therapy against rotavirus infections., Introduction Rotavirus is the leading cause of diarrhea in infants and young children worldwide, causing more than 114 million episodes of diarrhea annually in children under the age of 5 [...]