Blue light promotes ascorbate synthesis by deactivating the PAS/LOV photoreceptor that inhibits GDP-L-galactose phosphorylase.

Ascorbate (vitamin C) is an essential antioxidant in fresh fruits and vegetables. To gain insight into the regulation of ascorbate metabolism in plants, we studied mutant tomato plants (Solanum lycopersicum) that produce ascorbate-enriched fruits. The causal mutation, identified by a mapping-by-sequencing strategy, corresponded to a knock-out recessive mutation in a class of photoreceptor named PAS/LOV protein (PLP), which acts as a negative regulator of ascorbate biosynthesis. This trait was confirmed by CRISPR/Cas9 gene editing and further found in all plant organs, including fruit that accumulated 2 to 3 times more ascorbate than in the WT. The functional characterization revealed that PLP interacted with the 2 isoforms of GDP-L-galactose phosphorylase (GGP), known as the controlling step of the L-galactose pathway of ascorbate synthesis. The interaction with GGP occurred in the cytoplasm and the nucleus, but was abolished when PLP was truncated. These results were confirmed by a synthetic approach using an animal cell system, which additionally demonstrated that blue light modulated the PLP-GGP interaction. Assays performed in vitro with heterologously expressed GGP and PLP showed that PLP is a noncompetitive inhibitor of GGP that is inactivated after blue light exposure. This discovery provides a greater understanding of the light-dependent regulation of ascorbate metabolism in plants.

Nitrate transport via NRT2.1 mediates NIN-LIKE PROTEIN-dependent suppression of root nodulation in Lotus japonicus.

Legumes have adaptive mechanisms that regulate nodulation in response to the amount of nitrogen in the soil. In Lotus japonicus, two NODULE INCEPTION (NIN)-LIKE PROTEIN (NLP) transcription factors, LjNLP4 and LjNLP1, play pivotal roles in the negative regulation of nodulation by controlling the expression of symbiotic genes in high nitrate conditions. Despite an improved understanding of the molecular basis for regulating nodulation, how nitrate plays a role in the signaling pathway to negatively regulate this process is largely unknown. Here, we show that nitrate transport via NITRATE TRANSPORTER 2.1 (LjNRT2.1) is a key step in the NLP signaling pathway to control nodulation. A mutation in the LjNRT2.1 gene attenuates the nitrate-induced control of nodulation. LjNLP1 is necessary and sufficient to induce LjNRT2.1 expression, thereby regulating nitrate uptake/transport. Our data suggest that LjNRT2.1-mediated nitrate uptake/transport is required for LjNLP4 nuclear localization and induction/repression of symbiotic genes. We further show that LjNIN, a positive regulator of nodulation, counteracts the LjNLP1-dependent induction of LjNRT2.1 expression, which is linked to a reduction in nitrate uptake. These findings suggest a plant strategy in which nitrogen acquisition switches from obtaining nitrogen from the soil to symbiotic nitrogen fixation.

Different DNA-binding specificities of NLP and NIN transcription factors underlie nitrate-induced control of root nodulation.

Leguminous plants produce nodules for nitrogen fixation; however, nodule production incurs an energy cost. Therefore, as an adaptive strategy, leguminous plants halt root nodule development when sufficient amounts of nitrogen nutrients, such as nitrate, are present in the environment. Although legume NODULE INCEPTION (NIN)-LIKE PROTEIN (NLP) transcription factors have recently been identified, understanding how nodulation is controlled by nitrate, a fundamental question for nitrate-mediated transcriptional regulation of symbiotic genes, remains elusive. Here, we show that two Lotus japonicus NLPs, NITRATE UNRESPONSIVE SYMBIOSIS 1 (NRSYM1)/LjNLP4 and NRSYM2/LjNLP1, have overlapping functions in the nitrate-induced control of nodulation and act as master regulators for nitrate-dependent gene expression. We further identify candidate target genes of LjNLP4 by combining transcriptome analysis with a DNA affinity purification-seq approach. We then demonstrate that LjNLP4 and LjNIN, a key nodulation-specific regulator and paralog of LjNLP4, have different DNA-binding specificities. Moreover, LjNLP4-LjNIN dimerization underlies LjNLP4-mediated bifunctional transcriptional regulation. These data provide a basic principle for how nitrate controls nodulation through positive and negative regulation of symbiotic genes.

A Stereoselective Strigolactone Biosynthesis Catalyzed by a 2-Oxoglutarate-Dependent Dioxygenase in Sorghum.

Seeds of root parasitic plants, Striga, Orobanche and Phelipanche spp., are induced to germinate by strigolactones (SLs) exudated from host roots. In Striga-resistant cultivars of Sorghum bicolor, the loss-of-function of the Low Germination Stimulant 1 (LGS1) gene changes the major SL from 5-deoxystrigol (5DS) to orobanchol, which has an opposite C-ring stereochemistry. The biosynthetic pathway of 5DS catalyzed by LGS1 has not been fully elucidated. Since other unknown regulators, in addition to LGS1 encoding a sulfotransferase, appear to be necessary for the stereoselective biosynthesis of 5DS, we examined Sobic.005G213500 (Sb3500), encoding a 2-oxoglutarate-dependent dioxygenase, as a candidate regulator, which is co-expressed with LGS1 and located 5'-upstream of LGS1 in the sorghum genome. When LGS1 was expressed with known SL biosynthetic enzyme genes including the cytochrome P450 SbMAX1a in Nicotiana benthamiana leaves, 5DS and its diastereomer 4-deoxyorobanchol (4DO) were produced in approximately equal amounts, while the production of 5DS was significantly larger than that of 4DO when Sb3500 was also co-expressed. We also confirmed the stereoselective 5DS production in an in vitro feeding experiment using synthetic chemicals with recombinant proteins expressed in Escherichia coli and yeast. This finding demonstrates that Sb3500 is a stereoselective regulator in the conversion of the SL precursor carlactone to 5DS, catalyzed by LGS1 and SbMAX1a, providing a detailed understanding of how different SLs are produced to combat parasitic weed infestations.

Identification of a Prunus MAX1 homolog as a unique strigol synthase.

Strigol is the first identified and one of the most important strigolactones (SLs), but the biosynthetic pathway remains elusive. We functionally identified a strigol synthase (cytochrome P450 711A enzyme) in the Prunus genus through rapid gene screening in a set of SL-producing microbial consortia, and confirmed its unique catalytic activity (catalyzing multistep oxidation) through substrate feeding experiments and mutant analysis. We also reconstructed the biosynthetic pathway of strigol in Nicotiana benthamiana and reported the total biosynthesis of strigol in the Escherichia coli-yeast consortium, from the simple sugar xylose, which paves the way for large-scale production of strigol. As proof of concept, strigol and orobanchol were detected in Prunus persica root extrudes. This demonstrated a successful prediction of metabolites produced in plants through gene function identification, highlighting the importance of deciphering the sequence-function correlation of plant biosynthetic enzymes to more accurately predicate plant metabolites without metabolic analysis. This finding revealed the evolutionary and functional diversity of CYP711A (MAX1) in SL biosynthesis, which can synthesize different stereo-configurations of SLs (strigol- or orobanchol-type). This work again emphasizes the importance of microbial bioproduction platform as an efficient and handy tool to functionally identify plant metabolism.

Chorioallantoic membrane assay revealed the role of TIPARP (2,3,7,8-tetrachlorodibenzo-p-dioxin-inducible poly (ADP-ribose) polymerase) in lung adenocarcinoma-induced angiogenesis.

BACKGROUND: The chorioallantoic membrane (CAM) assay is a well-established technique to evaluate tumor invasion and angiogenesis and may overcome the shortcoming of the patient-derived xenograft (PDX) mouse model. Currently, few reports have described lung cancer invasion and angiogenesis in the CAM assay. We therefore used the CAM assay in the evaluation of lung cancer. METHOD: Lung cancer cell line-derived organoids or lung cancer cell lines were transplanted into the CAM on embryonic development day (EDD) 10, and an analysis was performed on EDD 15. Microscopic and macroscopic images and movies of the grafts on the CAM were captured and analyzed. The relationships between the graft and chick vessels were evaluated using immunohistochemistry. RESULTS: We transplanted lung cancer cell lines and cell line-derived organoid into a CAM to investigate angiogenesis and invasion. They engrafted on the CAM at a rate of 50-83%. A549-OKS cells showed enhanced cell invasion and angiogenesis on the CAM in comparison to A549-GFP cells as was reported in vitro. Next, we found that A549-TIPARP cells promoted angiogenesis on the CAM. RNA-seq identified 203 genes that were upregulated more than twofold in comparison to A549-GFP cells. A pathway analysis revealed many upregulated pathways related to degradation and synthesis of the extracellular matrix in A549-TIPARP cells. CONCLUSIONS: The CAM assay can be used to evaluate and research invasion and angiogenesis in lung cancer. The elevated expression of TIPARP in lung cancer may induce angiogenesis by remodeling the extracellular matrix.

Involvement of Activation of Mast Cells via IgE Signaling and Epithelial Cell-Derived Cytokines in the Pathogenesis of Pollen Food Allergy Syndrome in a Murine Model.

Murine models to elucidate the pathogenesis of pollen food allergy syndrome (PFAS), characterized by oral hypersensitivity symptoms induced by specific foods in patients previously sensitized with a pollen, are lacking. The study aimed to examine PFAS pathogenesis in a novel murine model. Birch pollen-immunized mice were orally administered apple extract, and oral symptoms were evaluated based on oral rubbing frequency following the challenge. The birch pollen-immunized mice orally challenged with apple extract exhibited PFAS-like symptoms, including oral rubbing and positive reaction of swelling by the prick test. The apple extract administered with a protease inhibitor reduced the oral rubbing frequency, which was also significantly reduced in the immunized Fcer1a -/- and mast cell-deficient mice compared with the immunized control mice. The oral rubbing frequency, serum IgE levels, and Th2-cytokine production by the cervical lymph node cells were significantly reduced in the immunized Il-33 -/- and thymic stromal lymphopoietin receptor-deficient (Crlf2 -/-) mice as compared with the immunized wild-type mice. IL-33 and thymic stromal lymphopoietin involve the pathogenesis of PFAS. The apple-extract stimulation did not lead to increased Th2-cytokine production in the oral mucosa or number of group 2 innate lymphoid cells or eosinophils. PFAS involves an early-phase response by mast cell degranulation via IgE signaling after the cross-reactivity of Bet v 1-specific IgE and the food allergen, and exacerbation of allergic symptom via proteases in food; PFAS does not involve a late phase with local Th2/eosinophilic inflammation in the oral mucosa. This novel murine model might be used for elucidating the pathogenesis and assessing new therapeutic strategies for PFAS.

Stimulation of Tomato Drought Tolerance by PHYTOCHROME A and B1B2 Mutations.

Drought stress is a severe environmental issue that threatens agriculture at a large scale. PHYTOCHROMES (PHYs) are important photoreceptors in plants that control plant growth and development and are involved in plant stress response. The aim of this study was to identify the role of PHYs in the tomato cv. 'Moneymaker' under drought conditions. The tomato genome contains five PHYs, among which mutant lines in tomato PHYA and PHYB (B1 and B2) were used. Compared to the WT, phyA and phyB1B2 mutants exhibited drought tolerance and showed inhibition of electrolyte leakage and malondialdehyde accumulation, indicating decreased membrane damage in the leaves. Both phy mutants also inhibited oxidative damage by enhancing the expression of reactive oxygen species (ROS) scavenger genes, inhibiting hydrogen peroxide (H2O2) accumulation, and enhancing the percentage of antioxidant activities via DPPH test. Moreover, expression levels of several aquaporins were significantly higher in phyA and phyB1B2, and the relative water content (RWC) in leaves was higher than the RWC in the WT under drought stress, suggesting the enhancement of hydration status in the phy mutants. Therefore, inhibition of oxidative damage in phyA and phyB1B2 mutants may mitigate the harmful effects of drought by preventing membrane damage and conserving the plant hydrostatus.

CRISPR/Cas9 Technique for Temperature, Drought, and Salinity Stress Responses.

Global warming and climate change have severely affected plant growth and food production. Therefore, minimizing these effects is required for sustainable crop yields. Understanding the molecular mechanisms in response to abiotic stresses and improving agricultural traits to make crops tolerant to abiotic stresses have been going on unceasingly. To generate desirable varieties of crops, traditional and molecular breeding techniques have been tried, but both approaches are time-consuming. Clustered regularly interspaced short palindromic repeat/Cas9 (CRISPR/Cas9) and transcription activator-like effector nucleases (TALENs) are genome-editing technologies that have recently attracted the attention of plant breeders for genetic modification. These technologies are powerful tools in the basic and applied sciences for understanding gene function, as well as in the field of crop breeding. In this review, we focus on the application of genome-editing systems in plants to understand gene function in response to abiotic stresses and to improve tolerance to abiotic stresses, such as temperature, drought, and salinity stresses.

Liquid biopsy for detecting epidermal growth factor receptor mutation among patients with non-small cell lung cancer treated with afatinib: a multicenter prospective study.

BACKGROUND: This study aimed to determine the effectiveness of liquid biopsy in detecting epidermal growth factor receptor (EGFR) mutations at diagnosis, disease progression, and intermediate stages. METHODS: This prospective, multicenter, observational study included 30 patients with non-small cell lung cancer treated with afatinib, harboring a major EGFR mutation confirmed by tumor tissue biopsy. We collected blood samples for liquid biopsy at diagnosis, intermediate stage, and progressive disease. Tissue and liquid biopsies were examined using Cobas ® EGFR Mutation Test v2. RESULTS: Liquid biopsy detected EGFR mutations in 63.6% of the patients at diagnosis. The presence of metastasis in the extrathoracic, brain, and adrenal glands correlated positively with the detection of EGFR mutations. Patients with positive EGFR mutations at diagnosis had significantly shorter overall and progression-free survival than patients with negative EGFR mutations. Four of the 18 patients (22.2%) who reached progressive disease had positive EGFR T790M mutations. Three of 10 patients (30.0%) with progressive disease were positive and negative for T790M using tumor re-biopsy and liquid biopsy, respectively. The results of EGFR mutation by tissue re-biopsy were the same as those of liquid biopsy in the three patients who were positive for significant EGFR mutations but negative for the T790M mutation using liquid biopsy at progressing disease. Only two patients were positive for major EGFR mutations at intermediate levels. CONCLUSIONS: Liquid biopsy can be a prognostic factor in EGFR-tyrosine kinase inhibitor treatments at diagnosis. Tumor re-biopsy can be omitted in patients with positive EGFR mutations by liquid biopsy at PD.

Quantitative evaluation of glycan-binding specificity of recombinant concanavalin A produced in lettuce (Lactuca sativa).

Concanavalin A (ConA), a mannose (Man)-specific leguminous lectin isolated from the jack bean (Canavalia ensiformis) seed extracts, was discovered over a century ago. Although ConA has been extensively applied in various life science research, recombinant mature ConA expression has not been fully established. Here, we aimed to produce recombinant ConA (rConA) in lettuce (Lactuca sativa) using an Agrobacterium tumefaciens-mediated transient expression system. rConA could be produced as a fully active form from soluble fractions of lettuce leaves and purified by affinity chromatography. From 12 g wet weight of lettuce leaves, 0.9 mg rConA could be purified. The glycan-binding properties of rConA were then compared with that of the native ConA isolated from jack bean using glycoconjugate microarray and frontal affinity chromatography. rConA demonstrated a glycan-binding specificity similar to nConA. Both molecules bound to N-glycans containing a terminal Man residue. Consistent with previous reports, terminal Manα1-6Man was found to be an essential unit for the high-affinity binding of rConA and nConA, while bisecting GlcNAc diminished the binding of rConA and nConA to Manα1-6Man-terminated N-glycans. These results demonstrate that the fully active rConA could be produced using the A. tumefaciens-mediated transient expression system and used as a recombinant substitute for nConA.

Correction: LACK OF SYMBIONT ACCOMMODATION controls intracellular symbiont accommodation in root nodule and arbuscular mycorrhizal symbiosis in Lotus japonicus.

[This corrects the article DOI: 10.1371/journal.pgen.1007865.].

LACK OF SYMBIONT ACCOMMODATION controls intracellular symbiont accommodation in root nodule and arbuscular mycorrhizal symbiosis in Lotus japonicus.

Nitrogen-fixing rhizobia and arbuscular mycorrhizal fungi (AMF) form symbioses with plant roots and these are established by precise regulation of symbiont accommodation within host plant cells. In model legumes such as Lotus japonicus and Medicago truncatula, rhizobia enter into roots through an intracellular invasion system that depends on the formation of a root-hair infection thread (IT). While IT-mediated intracellular rhizobia invasion is thought to be the most evolutionarily derived invasion system, some studies have indicated that a basal intercellular invasion system can replace it when some nodulation-related factors are genetically modified. In addition, intracellular rhizobia accommodation is suggested to have a similar mechanism as AMF accommodation. Nevertheless, our understanding of the underlying genetic mechanisms is incomplete. Here we identify a L. japonicus nodulation-deficient mutant, with a mutation in the LACK OF SYMBIONT ACCOMMODATION (LAN) gene, in which root-hair IT formation is strongly reduced, but intercellular rhizobial invasion eventually results in functional nodule formation. LjLAN encodes a protein that is homologous to Arabidopsis MEDIATOR 2/29/32 possibly acting as a subunit of a Mediator complex, a multiprotein complex required for gene transcription. We also show that LjLAN acts in parallel with a signaling pathway including LjCYCLOPS. In addition, the lan mutation drastically reduces the colonization levels of AMF. Taken together, our data provide a new factor that has a common role in symbiont accommodation process during root nodule and AM symbiosis.

Functional Characterization of Tomato Phytochrome A and B1B2 Mutants in Response to Heat Stress.

Heat stress (HS) is a prevalent negative factor affecting plant growth and development, as it is predominant worldwide and threatens agriculture on a large scale. PHYTOCHROMES (PHYs) are photoreceptors that control plant growth and development, and the stress signaling response partially interferes with their activity. PHYA, B1, and B2 are the most well-known PHY types in tomatoes. Our study aimed to identify the role of tomato 'Money Maker' phyA and phyB1B2 mutants in stable and fluctuating high temperatures at different growth stages. In the seed germination and vegetative growth stages, the phy mutants were HS tolerant, while during the flowering stage the phy mutants revealed two opposing roles depending on the HS exposure period. The response of the phy mutants to HS during the fruiting stage showed similarity to WT. The most obvious stage that demonstrated phy mutants' tolerance was the vegetative growth stage, in which a high degree of membrane stability and enhanced water preservation were achieved by the regulation of stomatal closure. In addition, both mutants upregulated the expression of heat-responsive genes related to heat tolerance. In addition to lower malondialdehyde accumulation, the phyA mutant enhanced proline levels. These results clarified the response of tomato phyA and phyB1B2 mutants to HS.

Specific methylation of (11R)-carlactonoic acid by an Arabidopsis SABATH methyltransferase.

MAIN CONCLUSION: An Arabidopsis S-adenosyl-L-methionine-dependent methyltransferase belonging to the SABATH family catalyzes the specific carboxymethylation of (11R)-carlactonoic acid. Methyl carlactonoate (MeCLA), found in Arabidopsis (Arabidopsis thaliana) as a non-canonical strigolactone (SL), may be a biosynthetic intermediate of various non-canonical SLs and biologically active as a plant hormone. MeCLA is formed from carlactonoic acid (CLA), but the methyltransferases (MTs) converting CLA to MeCLA remain unclear. Previous studies have demonstrated that the carboxymethylation of acidic plant hormones is catalyzed by the same protein family, the SABATH family (Wang et al. in Evol Bioinform 15:117693431986086. https://doi.org/10.1177/1176934319860864 , 2019). In the present study, we focused on the At4g36470 gene, an Arabidopsis SABATH MT gene co-expressed with the MAX1 gene responsible for CLA formation for biochemical characterization. The recombinant At4g36470 protein expressed in Escherichia coli exhibited exclusive activity against naturally occurring (11R)-CLA among the substrates, including CLA enantiomers and a variety of acidic plant hormones. The apparent Km value for (11R)-CLA was 1.46 µM, which was relatively smaller than that of the other Arabidopsis SABATH MTs responsible for the carboxymethylation of acidic plant hormones. The strict substrate specificity and high affinity of At4g36470 suggested it is an (11R)-CLA MT. We also confirmed the function of the identified gene by reconstructing MeCLA biosynthesis using transient expression in Nicotiana benthamiana. Phylogenetic analysis demonstrated that At4g36470 and its orthologs in non-canonical SL-producing plants cluster together in an exclusive clade, suggesting that the SABATH MTs of this clade may be involved in the carboxymethylation of CLA and the biosynthesis of non-canonical SLs.

Gene expression of PLAT and ATS3 proteins increases plant resistance to insects.

MAIN CONCLUSION: Genes of the PLAT protein family, including PLAT and ATS3 subfamilies of higher plants and homologs of liverwort, are involved in plant defense against insects. Laticifer cells in plants contain large amounts of anti-microbe or anti-insect proteins and are involved in plant defense against biotic stresses. We previously found that PLAT proteins accumulate in laticifers of fig tree (Ficus carica) at comparable levels to those of chitinases, and the transcript level of ATS3, another PLAT domain-containing protein, is highest in the transcriptome of laticifers of Euphorbia tirucalli. In this study, we investigated whether the PLAT domain-containing proteins are involved in defense against insects. Larvae of the lepidopteran Spodoptera litura showed retarded growth when fed with Nicotiana benthamiana leaves expressing F. carica PLAT or E. tirucalli ATS3 genes, introduced by agroinfiltration using expression vector pBYR2HS. Transcriptome analysis of these leaves indicated that ethylene and jasmonate signaling were activated, leading to increased expression of genes for PR-1, ß-1,3-glucanase, PR5 and trypsin inhibitors, suggesting an indirect mechanism of PLAT- and ATS3-induced resistance in the host plant. Direct cytotoxicity of PLAT and ATS3 to insects was also possible because heterologous expression of the corresponding genes in Drosophila melanogaster caused apoptosis-mediated cell death in this insect. Larval growth retardation of S. litura occurred when they were fed radish sprouts, a good host for agroinfiltration, expressing any of nine homologous genes of dicotyledon Arabidopsis thaliana, monocotyledon Brachypodium distachyon, conifer Picea sitchensis and liverwort Marchantia polymorpha. Of these nine genes, the heterologous expression of A. thaliana AT5G62200 and AT5G62210 caused significant increases in larval death. These results indicated that the PLAT protein family has largely conserved anti-insect activity in the plant kingdom (249 words).

Strigolactone biosynthesis catalyzed by cytochrome P450 and sulfotransferase in sorghum.

Root parasitic plants such as Striga, Orobanche, and Phelipanche spp. cause serious damage to crop production world-wide. Deletion of the Low Germination Stimulant 1 (LGS1) gene gives a Striga-resistance trait in sorghum (Sorghum bicolor). The LGS1 gene encodes a sulfotransferase-like protein, but its function has not been elucidated. Since the profile of strigolactones (SLs) that induce seed germination in root parasitic plants is altered in the lgs1 mutant, LGS1 is thought to be an SL biosynthetic enzyme. In order to clarify the enzymatic function of LGS1, we looked for candidate SL substrates that accumulate in the lgs1 mutants and performed in vivo and in vitro metabolism experiments. We found the SL precursor 18-hydroxycarlactonoic acid (18-OH-CLA) is a substrate for LGS1. CYP711A cytochrome P450 enzymes (SbMAX1 proteins) in sorghum produce 18-OH-CLA. When LGS1 and SbMAX1 coding sequences were co-expressed in Nicotiana benthamiana with the upstream SL biosynthesis genes from sorghum, the canonical SLs 5-deoxystrigol and 4-deoxyorobanchol were produced. This finding showed that LGS1 in sorghum uses a sulfo group to catalyze leaving of a hydroxyl group and cyclization of 18-OH-CLA. A similar SL biosynthetic pathway has not been found in other plant species.

Efficient base editing in tomato using a highly expressed transient system.

KEY MESSAGE: Base editing in tomatoes was achieved by transient expression. The Solanaceae plants, particularly the tomato (Solanum lycopersicum), is of huge economic value worldwide. The tomato is a unique model plant for studying the functions of genes related to fruit ripening. Deeper understanding of tomatoes is of great importance for both plant research and the economy. Genome editing technology, such as CRISPR/Cas9, has been used for functional genetic research. However, some challenges, such as low transformation efficiency, remain with this technology. Moreover, the foreign Cas9 and gRNA expression cassettes must be removed to obtain null-segregants In this study, we used a high-level transient expression system to improve the base editing technology. A high-level transient expression system has been established previously using geminiviral replication and a double terminator. The pBYR2HS vector was used for this transient expression system. nCas9-CDA and sgRNA-SlHWS were introduced into this vector, and the protein and RNA were then transiently expressed in tomato tissues by agroinfiltration. The homozygous mutant produced by base editing was obtained in the next generation with an efficiency of about 18%. nCas9-free next-generation plants were 71%. All the homozygous base-edited plants in next generation are nCas9-free. These findings show that the high-level transient expression system is useful for base editing in tomatoes.

Asymptomatic COVID-19 re-infection in a Japanese male by elevated half-maximal inhibitory concentration (IC50) of neutralizing antibodies.

INTRODUCTION: "Re-infection" with COVID-19 is a growing concern; re-infection cases have reported worldwide. However, the clinical characteristics of SARS-CoV-2 re-infection, including the levels and role of anti-SARS-CoV-2 Spike protein IgG antibodies and the half-maximal concentration (IC50) of neutralizing antibodies remain unknown. METHODS: Both the epidemiological and clinical information has been collected during two episodes of COVID-19 in a patient. Laboratory results, including RT-PCR, Ct values, anti-SARS-CoV-2 Spike protein IgG antibodies, and the IC50 of neutralizing antibodies levels were analyzed on the patient. RESULTS: The patient was a 58-year-old man who developed moderate COVID-19 pneumonia with oxygen demand (cannula 2 L/min) in the first episode. By day 30, he recuperated and was discharged after testing negative for SARS-CoV-2. After two and a half months, his three family members showed COVID-19 symptoms and tested positive for SARS-CoV-2. He tested positive for SARS-CoV-2 once again and was asymptomatic (the second episode). The IC50 of neutralizing antibodies against SARS-CoV-2 greatly increased from 50.0 µg/mL (after the first episode) to 14.8 µg/mL (after the second episode), and remained strongly reactive (20.1 µl/mL) after 47 days of the second episode. CONCLUSIONS: Epidemiological, clinical, and serological analyses confirmed that the patient had re-infection instead of persistent viral shedding from first infection. Our results suggest that SARS-CoV-2 re-infection may manifest as asymptomatic with increased neutralizing antibody levels. Further studies such as the virus characteristics, immunology, and epidemiology on SARS-CoV-2 re-infection are needed.

[Giant Solitary Fibrous Tumor of Pleura Metastasizing Contralateral Lung Following Two Times of Surgery for Ipsilateral Pleural Disseminations].

We report a case of giant solitary fibrous tumor (SFT) of pleura metastatising contralateral lung following 2 times of surgery for ipsilateral pleural disseminations. A 70-year-old woman was carried to our hospital by ambulance because of hypoglycemic attack. A chest X-ray film showed a huge mass in the right lung field. A computed tomography guided biopsy revealed a SFT producing IGF-Ⅱ, which caused hypoglycemic attack. After surgery, she was relieved of hypoglycemic attack and discharged from the hospital 14 days following the surgery. SFT repeatedly relapsed in the ipsilateral pleura. In the follow-up period, 2 times of resection of disseminated nodules were carried out. Finally, SFT developed ipsilateral pleural disseminations and contralateral pulmonary metastases, accompanying hypoglycemic attack. She died 76 months after the initial surgery.