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Steroidal glycoalkaloids (SGAs) are specialized metabolites primarily produced by Solanaceae plants such as potatoes and tomatoes. Notably, α-solanine and α-chaconine are recognized as toxic substances in potatoes. While the biosynthetic pathways of SGAs are largely understood, the final steps of α-solanine and α-chaconine biosynthesis remained elusive. In this study, we discovered that two reductase-encoding genes, reductase for potato glycoalkaloid biosynthesis 1 (RPG1) and RPG2, complete SGA biosynthesis in potato. Knockout of both RPG1 and RPG2 in potato hairy roots halted α-solanine production, leading to the accumulation of zwittersolanine. We analyzed the catalytic function of recombinant enzymes and conducted structural determination of the reaction products by nuclear magnetic resonance. As a result, RPG1 converted zwittersolanine to 16-iminiumsolanine, and RPG2 further converted it to α-solanine. RPG2 also transformed zwittersolanine to 22-iminiumsolanine, which RPG1 then converted to α-solanine. Similar processes were observed for α-chaconine synthesis from zwitterchaconine. Due to differences in enzymatic reaction efficiency, the biosynthetic pathway via 16-iminiumsolanine/16-iminiumchaconine was suggested to be predominant in potato. Our results could pave the way for tailoring SGA structures within Solanum plants, enabling the development of Solanum crop varieties with reduced toxicity or enhanced resistance to diseases and pests., (© 2025 The Author(s). New Phytologist © 2025 New Phytologist Foundation.)

Decades of research on the infamous antinutritional steroidal glycoalkaloids (SGAs) in Solanaceae plants have provided deep insights into their metabolism and roles. However, engineering SGAs in heterologous hosts has remained a challenge. We discovered that a protein evolved from the machinery involved in building plant cell walls is the crucial link in the biosynthesis of SGAs. We show that cellulose synthase-like M [GLYCOALKALOID METABOLISM15 (GAME15)] functions both as a cholesterol glucuronosyltransferase and a scaffold protein. Silencing GAME15 depletes SGAs, which makes plants more vulnerable to pests. Our findings illuminate plant evolutionary adaptations that balance chemical defense and self-toxicity and open possibilities for producing steroidal compounds in heterologous systems for food, cosmetics, and pharmaceuticals.

Aflibercept (AFL) plus FOLFIRI prolongs overall survival (OS) in patients with metastatic colorectal cancer (mCRC). However, there is limited evidence on the efficacy and safety of AFL plus FOLFIRI previously treated with anti-epidermal growth factor receptor (EGFR) agents. Therefore, we conducted a prospective open-label phase II trial evaluating the efficacy and safety of AFL plus FOLFIRI in Japanese patients with mCRC failing a prior oxaliplatin-based chemotherapy plus an anti-EGFR agent. AFL (4 mg/kg iv) followed by FOLFIRI (irinotecan 180 mg/m 2 , leucovorin 200 mg/m 2 iv, bolus 5-fluorouracil [5-FU] 400 mg/m 2 , and infusional 5-FU 2400 mg/m 2 /46 h) was given every 2 weeks until progression or unacceptable toxicities. The primary endpoint was progression-free survival (PFS) rate at 6 months. Forty three patients were enrolled between November 2019 and October 2022. The primary endpoint was met: 6-month PFS rate was 58.8% (90% confidence interval [CI], 45.7%-72.0%). Median PFS and OS were 7.3 months (95% CI, 5.5-11.0 months) and 18.8 months (95% CI, 12.9-26.6 months), respectively. The overall response rate was 20.9% (95% CI, 10.0-36.0%) and disease control rate was 88.4% (95% CI, 74.9-96.1%). The main grade ≥3 adverse events included hypertension (62.8%), neutropenia (55.8%), leukopenia (25.6%), febrile neutropenia (11.6%), fatigue (9.3%), anorexia (9.3%), proteinuria (9.3%), and diarrhea (7.0%). No deaths and no new safety signals with a causal relation to the study treatment were observed. This study suggests that AFL plus FOLFIRI shows a high response rate and a manageable safety profile in Japanese patients with mCRC who failed prior oxaliplatin-based chemotherapy plus an anti-EGFR agent., (© 2024 The Author(s). International Journal of Cancer published by John Wiley & Sons Ltd on behalf of UICC.)

Small-bowel diverticulosis is relatively common, but there is no set treatment strategy for duodenal diverticulitis with stone impaction. A woman aged in her 70s presented with a chief complaint of abdominal pain, and she had been reconstructed by the Roux-en-Y method after total gastrectomy. We performed an enhanced computed tomography which revealed edematous wall thickening of the duodenum. We diagnosed her with duodenal diverticulitis and treated them with antibiotics but her disease was not cured, we therefore attempted endoscopic stone removal as a nonoperative treatment. After stone removal with a nonoperative procedure, when we contrasted the duodenal papillary diverticulum, it was found to form a fistula on the other side, and the successful treatment made her discharged on the 17th day. The duodenal diverticulitis with calculus is extremely rare, and there is no report to treat it using double-balloon endoscopy, therefore we report this case with a literature review., Competing Interests: None., (© 2024 The Author(s). DEN Open published by John Wiley & Sons Australia, Ltd on behalf of Japan Gastroenterological Endoscopy Society.)

The period of circadian clocks is maintained at close to 24 hours over a broad range of physiological temperatures due to temperature compensation of period length. Here, we show that the quantitative control of the core clock proteins TIMING OF CAB EXPRESSION 1 [TOC1; also known as PSEUDO-RESPONSE REGULATOR 1 (PRR1)] and PRR5 is crucial for temperature compensation in Arabidopsis thaliana . The prr5 toc1 double mutant has a shortened period at higher temperatures, resulting in weak temperature compensation. Low ambient temperature reduces amounts of PRR5 and TOC1. In low-temperature conditions, PRR5 and TOC1 interact with LOV KELCH PROTEIN 2 (LKP2), a component of the E3 ubiquitin ligase Skp, Cullin, F-box (SCF) complex. The lkp2 mutations attenuate low temperature-induced decrease of PRR5 and TOC1, and the mutants display longer period only at lower temperatures. Our findings reveal that the circadian clock maintains its period length despite ambient temperature fluctuations through temperature- and LKP2 -dependent control of PRR5 and TOC1 abundance.

Plants sense and respond to environmental cues during 24 h fluctuations in their environment. This requires the integration of internal cues such as circadian timing with environmental cues such as light and temperature to elicit cellular responses through signal transduction. However, the integration and transduction of circadian and environmental signals by plants growing in natural environments remains poorly understood. To gain insights into 24 h dynamics of environmental signaling in nature, we performed a field study of signal transduction from the nucleus to chloroplasts in a natural population of Arabidopsis halleri. Using several modeling approaches to interpret the data, we identified that the circadian clock and temperature are key regulators of this pathway under natural conditions. We identified potential time-delay steps between pathway components, and diel fluctuations in the response of the pathway to temperature cues that are reminiscent of the process of circadian gating. We found that our modeling framework can be extended to other signaling pathways that undergo diel oscillations and respond to environmental cues. This approach of combining studies of gene expression in the field with modeling allowed us to identify the dynamic integration and transduction of environmental cues, in plant cells, under naturally fluctuating diel cycles., Competing Interests: Competing interests statement:The authors declare no competing interest.

6-(Methylsulfinyl)hexyl isothiocyanate (6-MSITC), a derivative of glucosinolate with a six-carbon chain, is a compound found in wasabi and has diverse health-promoting properties. The biosynthesis of glucosinolates from methionine depends on a crucial step catalyzed methylthioalkylmalate synthases (MAMs), which are responsible for the generation of glucosinolates with varying chain lengths. In this study, our primary focus was the characterization of two methylthioalkyl malate synthases, MAM1-1 and MAM1-2, derived from Eutrema japonicum, commonly referred to as Japanese wasabi. Eutremajaponicum MAMs (EjMAMs) were expressed in an Escherichiacoli expression system, subsequently purified, and in vitro enzymatic activity was assayed. We explored the kinetic properties, optimal pH conditions, and cofactor preferences of EjMAMs and compared them with those of previously documented MAMs. Surprisingly, EjMAM1-2, categorized as a metallolyase family enzyme, displayed 20% of its maximum activity even in the absence of divalent metal cofactors or under high concentrations of EDTA. Additionally, we utilized AlphaFold2 to generate structural homology models of EjMAMs, and used in silico analysis and mutagenesis studies to investigate the key residues participating in catalytic activity. Moreover, we examined in vivo biosynthesis in E. coli containing Arabidopsis thaliana branched-chain amino acid transferase 3 (AtBCAT3) along with AtMAMs or EjMAMs and demonstrated that EjMAM1-2 exhibited the highest conversion rate among those MAMs, converting l-methionine to 2-(2-methylthio) ethyl malate (2-(2-MT)EM). EjMAM1-2 shows a unique property in vitro and highest activity on converting l-methionine to 2-(2-MT)EM in vivo which displays high potential for isothiocyanate biosynthesis in E. coli platform., (Copyright © 2024 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.)

Unable to move on their own, plants have acquired the ability to produce a wide variety of low molecular weight compounds to survive against various stresses. It is estimated that there are as many as one million different kinds. Plants also have the ability to accumulate high levels of proteins. Although plant-based bioproduction has traditionally relied on classical tissue culture methods, the attraction of bioproduction by plants is increasing with the development of omics and bioinformatics and other various technologies, as well as synthetic biology. This review describes the current status and prospects of these plant-based bioproduction from five advanced research topics, (i) de novo production of plant-derived high value terpenoids in engineered yeast, (ii) biotransformation of plant-based materials, (iii) genome editing technology for plant-based bioproduction, (iv) environmental effect of metabolite production in plant factory, and (v) molecular pharming., (Copyright © 2024. Published by Elsevier B.V.)

Evergreeness is a substantial strategy for temperate and boreal plants and is as common as deciduousness. However, whether evergreen plants switch foliage functions between seasons remains unknown. We conduct an in natura study of leaf senescence control in the evergreen perennial, Arabidopsis halleri. A four-year census of leaf longevity of 102 biweekly cohorts allows us to identify growth season (GS) and overwintering (OW) cohorts characterised by short and extended longevity, respectively, and to recognise three distinct periods in foliage functions, i.e., the growth, overwintering, and reproductive seasons. Photoperiods during leaf expansion separate the GS and OW cohorts, providing primal control of leaf senescence depending on the season, with leaf senescence being shut down during winter. Phenotypic and transcriptomic responses in field experiments indicate that shade-induced and reproductive-sink-triggered senescence are active during the growth and reproductive seasons, respectively. These secondary controls of leaf senescence cause desynchronised and synchronised leaf senescence during growth and reproduction, respectively. Conclusively, seasonal switching of leaf senescence optimises resource production, storage, and translocation for the season, making the evergreen strategy adaptively relevant., (© 2024. The Author(s).)

Background: We clarified the predictive factors for changes in the status of medications for lower urinary tract symptoms (LUTS) 2 years after local radiotherapy for nonmetastatic prostate cancer., Materials and Methods: We retrospectively included patients who underwent local external radiotherapy for nonmetastatic prostate cancer in 8 institutions between April 2001 and March 2016. Patients were divided into the medication and no-medication group based on the use of drugs for LUTS before radiotherapy. We defined improvement of LUTS as when the patient did not require medication for LUTS at 24 months after radiotherapy in the medication group and as deterioration when medication was required in the no-medication group. Logistic regression analysis was used to evaluate predictive factors for changes in medication status., Results: Altogether, 505 patients were divided into a no-medication group (n = 352) and a medication group (n = 153). The number of patients with deterioration and improvement in LUTS was 49 (14%) and 36 (23%), respectively. In the multivariate analysis, the predictive variables for deterioration were the International Prostate Symptom Score (≥8; odds ratio [OR], 2.21; p = 0.014) and the biopsy Gleason score (≤3 + 4 = 7; OR, 2.430; p = 0.008) in the no-medication group, whereas those for improvement were age (<75 years old; OR, 5.81; p = 0.002), the quality of life score (<3; OR, 3.15; p = 0.028), and a positive biopsy core rate (≥50%; OR, 2.530; p = 0.027) in the medication group., Conclusions: These predictive factors for changes in the status of medications for LUTS at 2 years after external radiotherapy may help determine the definitive therapy for nonmetastatic prostate cancer., Competing Interests: The authors declare no conflicts of interest., (Copyright © 2023 The Authors. Published by Wolters Kluwer Health, Inc.)

Glycyrrhizin, a type of the triterpenoid saponin, is a major active ingredient contained in the roots of the medicinal plant licorice (Glycyrrhiza uralensis, G. glabra and G. inflata), and is used worldwide in diverse applications, such as herbal medicines and sweeteners. The growing demand for licorice threatens wild resources and therefore a sustainable method of supplying glycyrrhizin is required. With the goal of establishing an alternative glycyrrhizin supply method not dependent on wild plants, we attempted to produce glycyrrhizin using hairy root culture. We tried to promote glycyrrhizin production by blocking competing pathways using CRISPR/Cas9-based gene editing. CYP93E3 CYP72A566 double-knockout (KO) and CYP93E3 CYP72A566 CYP716A179 LUS1 quadruple-KO variants were generated, and a substantial amount of glycyrrhizin accumulation was confirmed in both types of hairy root. Furthermore, we evaluated the potential for promoting further glycyrrhizin production by simultaneous CYP93E3 CYP72A566 double-KO and CYP88D6-overexpression. This strategy resulted in a 3-fold increase (∼1.4 mg/g) in glycyrrhizin accumulation in double-KO/CYP88D6-overexpression hairy roots, on average, compared with that of double-KO hairy roots. These findings demonstrate that the combination of blocking competing pathways and overexpression of the biosynthetic gene is important for enhancing glycyrrhizin production in G. uralensis hairy roots. Our findings provide the foundation for sustainable glycyrrhizin production using hairy root culture. Given the widespread use of genome editing technology in hairy roots, this combined with gene knockout and overexpression could be widely applied to the production of valuable substances contained in various plant roots., (© The Author(s) 2024. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists.)

Given possible involvement of the central and peripheral angiotensin system in pain processing, we conducted clinical and preclinical studies to test whether pharmacological inhibition of the angiotensin system would prevent diabetic peripheral neuropathy (DPN) accompanying type 2 diabetes mellitus (T2DM). In the preclinical study, the nociceptive sensitivity was determined in leptin-deficient ob/ob mice, a T2DM model. A clinical retrospective cohort study was conducted, using the medical records of T2DM patients receiving antihypertensives at three hospitals for nearly a decade. In the ob/ob mice, daily treatment with perindopril, an angiotensin-converting enzyme inhibitor (ACEI), or telmisartan, an angiotensin receptor blocker (ARB), but not amlodipine, an L-type calcium channel blocker (CaB), significantly inhibited DPN development without affecting the hyperglycemia. In the clinical study, the enrolled 7464 patients were divided into three groups receiving ACEIs, ARBs and the others (non-ACEI, non-ARB antihypertensives). Bonferroni's test indicated significantly later DPN development in the ARB and ACEI groups than the others group. The multivariate Cox proportional analysis detected significant negative association of the prescription of ACEIs or ARBs and β-blockers, but not CaBs or diuretics, with DPN development. Thus, our study suggests that pharmacological inhibition of the angiotensin system is beneficial to prevent DPN accompanying T2DM., (© 2024. The Author(s).)

The symmetry of the right and left bronchi, proposed in a previous comparative anatomical study as the basic model of the mammalian bronchial tree, was examined to determine if it applied to the embryonic human bronchial tree. Imaging data of 41 human embryo specimens at Carnegie stages (CS) 16-23 (equivalent to 6-8 weeks after fertilization) belonging to the Kyoto collection were obtained using phase-contrast X-ray computed tomography. Three-dimensional bronchial trees were then reconstructed from these images. Bronchi branching from both main bronchi were labeled as dorsal, ventral, medial, or lateral systems based on the branching position with numbering starting cranially. The length from the tracheal bifurcation to the branching point of the labeled bronchus was measured, and the right-to-left ratio of the same labeled bronchus in both lungs was calculated. In both lungs, the human embryonic bronchial tree showed symmetry with an alternating pattern of dorsal and lateral systems up to segmental bronchus B9 as the basic shape, with a more peripheral variation. This pattern is similar to that described in adult human lungs. Bronchial length increased with the CS in all labeled bronchi, whereas the right-to-left ratio was constant at approximately 1.0. The data demonstrated that the prototype of the human adult bronchial branching structure is formed and maintained in the embryonic stage. The morphology and branching position of all lobar bronchi and B6, B8, B9, and the subsegmental bronchus of B10 may be genetically determined. On the other hand, no common structures between individual embryos were found in the peripheral branches after the subsegmental bronchus of B10, suggesting that branch formation in this region is influenced more by environmental factors than by genetic factors., (© 2023 Anatomical Society.)

Key Message: CRISPR-Cas9-mediated disruption of a licorice cellulose synthase-derived glycosyltransferase gene, GuCSyGT, demonstrated the in planta role of GuCSyGT as the enzyme catalyzing 3-O-glucuronosylation of triterpenoid aglycones in soyasaponin biosynthesis. Triterpenoid glycosides (saponins) are a large, structurally diverse group of specialized metabolites in plants, including the sweet saponin glycyrrhizin produced by licorice (Glycyrrhiza uralensis) and soyasaponins that occur widely in legumes, with various bioactivities. The triterpenoid saponin biosynthetic pathway involves the glycosylation of triterpenoid sapogenins (the non-sugar part of triterpenoid saponins) by glycosyltransferases (GTs), leading to diverse saponin structures. Previously, we identified a cellulose synthase-derived GT (CSyGT), as a newly discovered class of triterpenoid GT from G. uralensis. GuCSyGT expressed in yeast, which could transfer the sugar glucuronic acid to the C3 position of glycyrrhetinic acid and soyasapogenol B, which are the sapogenins of glycyrrhizin and soyasaponin I, respectively. This suggested that GuCSyGT is involved in the biosynthesis of glycyrrhizin and soyasaponin I. However, the in planta role of GuCSyGT in saponin biosynthesis remains unclear. In this study, we generated GuCSyGT-disrupted licorice hairy roots using CRISPR-Cas9-mediated genome editing and analyzed the saponin content. This revealed that soyasaponin I was completely absent in GuCSyGT-disrupted lines, demonstrating the in planta role of GuCSyGT in saponin biosynthesis., (© 2023. The Author(s).)

Genome editing is highly useful for crop improvement. The method of expressing genome-editing enzymes using a transient expression system in Agrobacterium , called agrobacterial mutagenesis, is a shortcut used in genome-editing technology to improve elite varieties of vegetatively propagated crops, including potato. However, with this method, edited individuals cannot be selected. The transient expression of regeneration-promoting genes can result in shoot regeneration from plantlets, while the constitutive expression of most regeneration-promoting genes does not result in normally regenerated shoots. Here, we report that we could obtain genome-edited potatoes by positive selection. These regenerated shoots were obtained via a method that combined a regeneration-promoting gene with the transient expression of a genome-editing enzyme gene. Moreover, we confirmed that the genome-edited potatoes obtained using this method did not contain the sequence of the binary vector used in Agrobacterium . Our data have been submitted to the Japanese regulatory authority, the Ministry of Education, Culture, Sports, Science and Technology (MEXT), and we are in the process of conducting field tests for further research on these potatoes. Our work presents a powerful method for regarding regeneration and acquisition of genome-edited crops through transient expression of regeneration-promoting gene., Competing Interests: Conflict of interestThe author declares there are no conflicts of interest associated with this manuscript., (© 2023 Japanese Society for Plant Biotechnology.)

The circadian clock is responsible for the temporal regulation of various physiological processes in plants. Individual cells contain a circadian oscillator consisting of a clock gene circuit that coordinates physiological rhythms within the plant body in an orderly manner. The coordination of time information has been studied from the perspective of cell-cell local coupling and long-distance communication between tissues based on the view that the behavior of circadian oscillators represents physiological rhythms. Here, we report the cellular circadian rhythm of bioluminescence reporters that are not governed by the clock gene circuit in expressing cells. We detected cellular bioluminescence rhythms with different free-running periods in the same cells using a dual-color bioluminescence monitoring system in duckweed (Lemna minor) transfected with Arabidopsis CIRCADIAN CLOCK ASSOCIATED 1::luciferace+ (AtCCA1::LUC+) and Cauliflower mosaic virus 35S::modified click-beetle red-color luciferase (CaMV35S::PtRLUC) reporters. Co-transfection experiments with the two reporters and a clock gene-overexpressing effector revealed that the AtCCA1::LUC+ rhythm, but not the CaMV35S::PtRLUC rhythm, was altered in cells with a dysfunctional clock gene circuit. This indicated that the AtCCA1::LUC+ rhythm is a direct output of the cellular circadian oscillator, whereas the CaMV35S::PtRLUC rhythm is not. After plasmolysis, the CaMV35S::PtRLUC rhythm disappeared, whereas the AtCCA1::LUC+ rhythm persisted. This suggests that the CaMV35S::PtRLUC bioluminescence has a symplast/apoplast-mediated circadian rhythm generated at the organismal level. The CaMV35S::PtRLUC-type bioluminescence rhythm was also observed when other bioluminescence reporters were expressed. These results reveal that the plant circadian system consists of both cell-autonomous and noncell-autonomous rhythms that are unaffected by cellular oscillators., Competing Interests: Conflict of interest statement. None declared., (© American Society of Plant Biologists 2023. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Genome editing is a powerful breeding technique that introduces mutations into specific gene sequences in genomes. For genome editing in higher plants, nucleotides for artificial nuclease (e.g. TALEN or CRISPR-Cas9) are transiently or stably introduced into the plant cells. After the introduction of mutations by artificial nucleases, it is necessary to select lines that do not contain the foreign nucleotides to overcome GMO regulation; however, there is still no widely legally authorized and approved method for detecting foreign genes in genome-edited crops. Recently, k-mer analysis based on next-generation sequencing (NGS) was proposed as a new method for detecting foreign DNA in genome-edited agricultural products. Compared to conventional methods, such as PCR and Southern hybridization, in principle, this method can detect short DNA fragments with high accuracy. However, this method has not yet been applied to genome-edited potatoes. In this study, we evaluated the feasibility of k-mer analysis in tetraploid potatoes by computer simulation, and also evaluated whether the k-mer method can detect foreign genes with high accuracy by analyzing samples of genome-edited potatoes. We show that when NGS data (at a depth of × 30 the genome size) are used, the k-mer method can correctly detect foreign genes in the potato genome even with the insertion of DNA fragments of 20 nt in length. Based on these findings, we expect that k-mer analysis will be one of the main methods for detecting foreign genes in genome-edited potatoes., (© 2023. The Author(s).)

Cytochrome P450 monooxygenases (CYPs) are enzymes that play critical roles in the structural diversification of triterpenoids. To perform site-specific oxidations of the triterpene scaffold, CYPs require electrons transferred by NADPH-cytochrome P450 reductase (CPR), which is classified into two main classes, class I and class II, based on their structural difference. Lotus japonicus is a triterpenoids-producing model legume with one CPR class I gene ( LjCPR1 ) and a minimum of two CPR class II genes ( LjCPR2-1 and LjCPR2-2 ). CPR classes I and II from different plants have been reported to be involved in different metabolic pathways. By performing gene expression analyses of L. japonicus hairy root culture treated with methyl jasmonate (MeJA), this study revealed that LjCPR1, CYP716A51 , and LUS were down-regulated which resulted in no change in betulinic acid and lupeol content. In contrast, LjCPR2s, bAS, CYP93E1 , and CYP72A61 were significantly upregulated by MeJA treatment, followed by a significant increase of the precursors for soyasaponins, i.e. β-amyrin, 24-OH β-amyrin, and sophoradiol content. Triterpenoids profile analysis of LORE1 insertion and hairy root mutants showed that the loss of the Ljcpr2-1 gene significantly reduced soyasaponins precursors but not in Ljcpr1 mutants. However, Ljcpr1 and Ljcpr2-1 mutants showed a significant reduction in lupeol and oleanolic, ursolic, and betulinic acid contents. Furthermore, LjCPR1 , but not LjCPR2 , was crucial for seed development, supporting the previous notion that CPR class I might support plant basal metabolism. This study suggests that CPR classes I and II play different roles in L. japonicus triterpenoid biosynthesis., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Istiandari, Yasumoto, Seki, Fukushima and Muranaka.)

Soybeans (Glycine max) develop newly differentiated aerenchymatous phellem (AP) in response to waterlogging stress. AP is formed in the hypocotyl and root, thus contributing to internal aeration and adaptation to waterlogging for several legumes. Extensive accumulation of triterpenoids - lupeol and betulinic acid - has been identified in AP. However, their physiological roles in plants remain unclarified. Lupeol is converted from 2,3-oxidosqualene by lupeol synthase (LUS) and oxidized to betulinic acid. Notably, soybeans have two LUS genes (GmLUS1 and GmLUS2). Functional analysis was performed to reveal the biological and physiological functions of triterpenoids in AP using lus mutants. The AP cells of lus1 mutant lacked triterpenoid accumulation and epicuticular wax. Lupeol and betulinic acid were the major components of epicuticular wax and contributed to tissue hydrophobicity and oxygen transport to the roots. Tissue porosity in AP was lower in the lus1 mutant than in the wild-type, which resulted in reduced oxygen transport to the roots via AP. This reduction in oxygen transport resulted in shallow root systems under waterlogged conditions. Triterpenoid accumulation in AP contributes to effective internal aeration and root development for adaptation to waterlogging, suggesting the significance of triterpenoids in improving waterlogging tolerance., (© 2023 The Authors. New Phytologist © 2023 New Phytologist Foundation.)

Explosive volcanism is a key contributor to climate variability on interannual to centennial timescales 1 . Understanding the far-field societal impacts of eruption-forced climatic changes requires firm event chronologies and reliable estimates of both the burden and altitude (that is, tropospheric versus stratospheric) of volcanic sulfate aerosol 2,3 . However, despite progress in ice-core dating, uncertainties remain in these key factors 4 . This particularly hinders investigation of the role of large, temporally clustered eruptions during the High Medieval Period (HMP, 1100-1300 CE), which have been implicated in the transition from the warm Medieval Climate Anomaly to the Little Ice Age 5 . Here we shed new light on explosive volcanism during the HMP, drawing on analysis of contemporary reports of total lunar eclipses, from which we derive a time series of stratospheric turbidity. By combining this new record with aerosol model simulations and tree-ring-based climate proxies, we refine the estimated dates of five notable eruptions and associate each with stratospheric aerosol veils. Five further eruptions, including one responsible for high sulfur deposition over Greenland circa 1182 CE, affected only the troposphere and had muted climatic consequences. Our findings offer support for further investigation of the decadal-scale to centennial-scale climate response to volcanic eruptions., (© 2023. The Author(s).)

The effects of UGT1A1 gene polymorphisms or prior irinotecan treatment on treatment outcomes of nanoliposomal-irinotecan plus 5-fluorouracil/leucovorin (nal-IRI+5-FU/LV) in patients with unresectable pancreatic ductal adenocarcinoma (PDAC) are not established. This multicenter, retrospective cohort study compared treatment outcomes in patients with UGT1A1*1/*1 and those with UGT1A1*1/*6 or *1/*28 genotypes. We also analyzed the impact of prior irinotecan treatment on survival outcomes in 54 patients treated with nal-IRI+5-FU/LV. Comparable effectiveness was found regardless of the UGT1A1 genotypes. While no significant differences were found, grade ≥3 neutropenia and febrile neutropenia were more frequent in patients with UGT1A1*1/*6 or *1/*28 than in those with UGT1A1*1/*1 genotypes (grade ≥3 neutropenia, 50.0% vs. 30.8%, p = 0.24; febrile neutropenia, 9.1% vs. 0.0%, p = 0.20, respectively). No significant difference in progression-free survival (PFS) and overall survival (OS) was observed between irinotecan-naïve-patients and other patients. However, irinotecan-resistant patients showed significantly shorter PFS (hazard ratio (HR) 2.83, p = 0.017) and OS (HR 2.58, p = 0.033) than other patients. Our study indicated that patients with UGT1A1*1/*6 or *1/*28 may be prone to neutropenia, though further study is needed. The survival benefit of nal-IRI+5-FU/LV could be maintained in patients without disease progression after irinotecan therapy.

Drug-induced thrombocytopenia is associated with bleeding tendency and suggests the need for the immediate suspected drug withdrawal. Patients with drug-induced thrombocytopenia usually experience an acute drop in platelet (PLT) levels a week or two after starting a new medication. Thrombocytopenia has both immune and non-immune mechanisms. Minocycline (MINO)-induced thrombocytopenia is rare; thus, there are few studies of this condition. In the present study, intravenous administration of MINO led to thrombocytopenia. The female patient was 80 years old. She was receiving radiation therapy for tongue cancer and medication for pain control. She had fever and aspiration pneumonia and was being treated with an antibacterial drug. Empiric therapy consisting of intravenous administration of tazobactam/piperacillin was performed; however, inflammation and fever did not improve. The bacterial drug was changed to vancomycin and cefmetazole. Sputum culture was positive for Enterobacter cloacae thus, we changed her treatment to MINO. Seven days after starting MINO, PLT levels were low; however, they recovered when treatment was stopped. Our findings suggest that MINO may rarely cause severe thrombocytopenia; thus, it is necessary to observe the patient's blood collection.

Background and Aims: Plant propagules often possess specialized morphologies that facilitate dispersal across specific landscapes. In the fruit dimorphism of a coastal shrub, Scaevola taccada, individual plants produce either cork-morph or pulp-morph fruits. The former is buoyant and common on sandy beaches, whereas the latter does not float, is bird-dispersed, and is common on elevated sites such as slopes on sea cliffs and behind rocky shores. We hypothesized that beach populations bridge the heterogeneous landscapes by serving as a source of both fruit types, while dispersal is biased for the pulp morph on elevated sites within the islands and for the cork morph between beaches of different islands. Based on this hypothesis, we predicted that populations in elevated sites would diverge genetically over time due to isolation by distance, whereas beach populations would maintain high genetic similarity via current gene flow., Methods: The genetic structure and gene flow in S. taccada were evaluated by investigating genome-wide single nucleotide polymorphisms in plants from 17 sampling sites on six islands (belonging to the Ryukyu, Daito and Ogasawara Islands) in Japan., Key Results: Geographical isolation was detected among the three distant island groups. Analyses within the Ryukyu Islands suggested that sandy beach populations were characterized by genetic admixture, whereas populations in elevated sites were relatively isolated between the islands. Pairwise FST values between islands were lowest between sandy beaches, intermediate between sandy beaches and elevated sites, and highest between elevated sites., Conclusions: Dispersal across the ocean by cork morphs is sufficiently frequent to prevent genetic divergence between beaches of different islands. Stronger genetic isolation of elevated sites between islands suggests that bird dispersal by pulp morphs is restricted mainly within islands. These contrasting patterns of gene flow realized by fruit dimorphism provide evidence that fruit characteristics can strongly mediate genetic structure., (© The Author(s) 2022. Published by Oxford University Press on behalf of the Annals of Botany Company. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Structure-based high-throughput screening of chemical compounds that target protein-protein interactions (PPIs) is a promising technology for gaining insight into how plant development is regulated, leading to many potential agricultural applications. At present, there are no examples of using high-throughput screening to identify chemicals that target plant transcriptional complexes, some of which are responsible for regulating multiple physiological functions. Florigen, a protein encoded by FLOWERING LOCUS T (FT), was initially identified as a molecule that promotes flowering and has since been shown to regulate flowering and other developmental phenomena such as tuber formation in potato (Solanum tuberosum). FT functions as a component of the florigen activation complex (FAC) with a 14-3-3 scaffold protein and FD, a bZIP transcription factor that activates downstream gene expression. Although 14-3-3 is an important component of FAC, little is known about the function of the 14-3-3 protein itself. Here, we report the results of a high-throughput in vitro fluorescence resonance energy transfer (FRET) screening of chemical libraries that enabled us to identify small molecules capable of inhibiting FAC formation. These molecules abrogate the in vitro interaction between the 14-3-3 protein and the OsFD1 peptide, a rice (Oryza sativa) FD, by directly binding to the 14-3-3 protein. Treatment with S4, a specific hit molecule, strongly inhibited FAC activity and flowering in duckweed, tuber formation in potato, and branching in rice in a dose-dependent manner. Our results demonstrate that the high-throughput screening approach based on the three-dimensional structure of PPIs is suitable in plants. In this study, we have proposed good candidate compounds for future modification to obtain inhibitors of florigen-dependent processes through inhibition of FAC formation., (© 2022 Society for Experimental Biology and John Wiley & Sons Ltd.)

A high-quality genome assembly is imperative to explore the evolutionary basis of characteristic attributes that define chemotype and provide essential resources for a molecular breeding strategy for enhanced production of medicinal metabolites. Here, using single-molecule high-fidelity (HiFi) sequencing reads, we report chromosome-scale genome assembly for Chinese licorice (Glycyrrhiza uralensis), a widely used herbal and natural medicine. The entire genome assembly was achieved in eight chromosomes, with contig and scaffold N50 as 36.02 and 60.2 Mb, respectively. With only 17 assembly gaps and half of the chromosomes having no or one assembly gap, the presented genome assembly is among the best plant genomes to date. Our results showed an advantage of using highly accurate long-read HiFi sequencing data for assembling a highly heterozygous genome including its complexed repeat content. Additionally, our analysis revealed that G. uralensis experienced a recent whole-genome duplication at approximately 59.02 million years ago post a gamma (γ) whole-genome triplication event, which contributed to its present chemotype features. The metabolic gene cluster analysis identified 355 gene clusters, which included the entire biosynthesis pathway of glycyrrhizin. The genome assembly and its annotations provide an essential resource for licorice improvement through molecular breeding and the discovery of valuable genes for engineering bioactive components and understanding the evolution of specialized metabolites biosynthesis., (© The Author(s) 2022. Published by Oxford University Press on behalf of Kazusa DNA Research Institute.)

Background: The first-line chemotherapy for patients with RAS and BRAF wild-type metastatic colorectal cancer (mCRC) commonly involves cytotoxic regimens, such as FOLFOX and FOLFIRI, combined with epidermal growth factor receptor (EGFR) antibodies. When progression occurs following anti-EGFR antibody-combined chemotherapy, anti-angiogenic inhibitors can be used as second-line treatment. Although randomized controlled trials have shown that anti-angiogenic inhibitors [bevacizumab, ramucirumab, and aflibercept (AFL)] carry survival benefit when combined with FOLFIRI as second-line chemotherapy, such trials did not provide data on patients with mCRC refractory to anti-EGFR antibody-combined chemotherapy. Therefore, our group planned a multicenter, nonrandomized, single-arm, prospective, phase II study to investigate the safety and efficacy of FOLFIRI plus AFL as a second-line chemotherapy for patients with mCRC refractory to oxaliplatin-based chemotherapy combined with anti-EGFR antibodies., Methods: FOLFIRI (irinotecan 180 mg/m 2 , l -leucovorin 200 mg/m 2 , bolus 5-FU 400 mg/m 2 , and infusional 5-FU 2400 mg/m 2 /46 h) and AFL (4 mg/kg) will be administered every 2 weeks until progression or unacceptable toxicities occur. The primary endpoint will be the 6-month progression-free survival (PFS) rate, whereas the secondary endpoints will include overall survival, PFS, response rate, disease control rate, adverse events, and relative dose intensity for each drug. A sample size of 41 participants will be required. This study will be sponsored by the Non-Profit Organization Hokkaido Gastrointestinal Cancer Study Group and will be supported by a grant from Sanofi., Discussion: There is only an observational study reporting data on FOLFIRI plus AFL for patients with mCRC who previously received anti-EGFR antibodies; therefore, a prospective clinical trial is needed. This study will prospectively evaluate the efficacy and safety of FOLFIRI plus AFL in patients with mCRC who are resistant to anti-EGFR antibodies and have limited data. Moreover, this study will reveal predictive biomarkers for AFL-based chemotherapy., Clinical Trial Registration: Japan Registry of Clinical Trials, jRCTs011190006. Registered 19 November, 2019, https://jrct.niph.go.jp/latest-detail/jRCTs011190006., Competing Interests: HN has received honoraria from Takeda, Sanofi, and Taiho; YoK has received honoraria from Taiho, Yakult Honsha, Takeda, Pfizer, Daiichi Sankyo, Sanofi, and Nipro and has received research funding from Taiho, Yakult Honsha, Daiichi Sankyo, and Sanofi; SY has received honoraria from Takeda, Sanofi, Taiho, and Yakult Honsha; YaK has received honoraria from Takeda, Kyowa Kirin; YT has received honoraria from Taiho, Takeda, and Sawai; MN has received honoraria from Daiichi Sankyo, Taiho, Kyowa Kirin, Takeda and Mochida and has received research funding from Takeda; SS has received honoraria from Sanofi and Daiichi Sankyo; YoS has received honoraria from Yakult Honsha; MKo has received honoraria from Yakult Honsha, Takeda, Sanofi, and Taiho; TA has received honoraria from Takeda and Daiichi Sankyo; NS has received research funding from Takeda; YuSaka has received honoraria from Yakult Honsha and Taiho. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Nakatsumi, Komatsu, Muranaka, Yuki, Kawamoto, Harada, Dazai, Tateyama, Sasaki, Miyagishima, Tsuji, Katagiri, Nakamura, Sogabe, Hatanaka, Meguro, Kobayashi, Ishiguro, Muto, Shindo, Kotaka, Ando, Takagi, Sakamoto and Sakata.)

Background: Small cell carcinoma of the urinary bladder (SCUB) is rare. The optimal treatment for SCUB remains unclear. To address the problem of appropriate treatment for each case, we assessed single-modality and surgery-based multimodality treatments in patients with SCUB., Materials and Methods: We retrospectively reviewed the medical records of 12 patients with SCUB between 1990 and 2013. All patients underwent transurethral resection of the bladder tumor and were diagnosed with SCUB. Their clinicopathological characteristics were assessed, and the outcomes were compared according to the treatment modality., Results: The median (range) age at diagnosis was 66 years (range, 53-85 years). T1-4N0M0 was observed in 8 patients (66%), N1-3M0 in 2 (17%), and NanyM1 in 2 (17%). After transurethral resection of the bladder tumor, 6 patients (50%) underwent cystectomy alone, and 4 (33%) underwent cystectomy and presurgical or adjuvant chemotherapy with etoposide and cisplatin. During the median follow-up period of 20.7 months, 6 patients (50%) died of cancer, and 2 patients (17%) died of other causes. The median overall survival period was 1.9 years. The 5-year overall survival rate in patients who underwent cystectomy and chemotherapy was 75%, whereas that in those who underwent cystectomy alone and transurethral resection alone were 22% and 0%, respectively ( p = 0.012). Recurrence-free survival was significantly correlated with cause-specific survival ( r = 0.95; 95% confidence interval, 0.81-0.99; p < 0.001)., Conclusions: Radical cystectomy with chemotherapy using the etoposide and cisplatin regimen improved the prognosis of patients with SCUB and TxNxM0. The time from initial progression to death due to cancer was very short, indicating that the initial treatment strategy is crucial., (Copyright © 2022 The Authors. Published by Wolters Kluwer Health, Inc.)

Triterpenoids constitute a group of specialized plant metabolites with wide structural diversity and high therapeutic value for human health. Cytochrome P450 monooxygenases (CYP) are a family of enzymes important for generating the structural diversity of triterpenoids by catalyzing the site-specific oxidization of the triterpene backbone. The CYP716 enzyme family has been isolated from various plant families as triterpenoid oxidases; however, their experimental crystal structures are not yet available and the detailed catalytic mechanism remains elusive. Here, we address this challenge by integrating bioinformatics approaches with data from other CYP families. Medicago truncatula CYP716A12, the first functionally characterized CYP716A subfamily enzyme, was chosen as the model for this study. We performed homology modeling, structural alignment, in silico site-directed mutagenesis, and molecular docking analysis to search and screen key amino acid residues relevant to the catalytic activity and substrate specificity of the CYP716A subfamily enzyme in triterpenoid biosynthesis. An in vivo functional analysis using engineered yeast that endogenously produced plant-derived triterpenes was performed to elucidate the results. When the amino acids in the signature region and substrate recognition sites (SRSs) were substituted, the product profile of CYP716A12 was modified. We identified amino acid residues that control the substrate contraction of the enzyme (D292) and engineered the enzyme to improve its catalytic activity and substrate specificity (D122, I212, and Q358) for triterpenoid biosynthesis. In addition, we demonstrated the versatility of this strategy by changing the properties of key residues in SRSs to improve the catalytic activity of Arabidopsis thaliana CYP716A1 (S356) and CYP716A2 (M206, F210) at C-28 on the triterpene backbone. This research has the potential to help in the production of desired triterpenoids in engineered yeast by increasing the catalytic activity and substrate specificity of plant CYP716A subfamily enzymes., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Romsuk, Yasumoto, Seki, Fukushima and Muranaka.)

Oleanolic acid is a pentacyclic triterpenoid found in numerous plant species and is a precursor to several bioactive triterpenoids with commercial potential. However, oleanolic acid accumulates at low levels in plants, and its chemical synthesis is challenging. Here, we established a method for producing oleanolic acid in substantial quantities via heterologous expression of pathway enzymes in Nicotiana benthamiana . The "Tsukuba system" is one of the most efficient agroinfiltration-based transient protein expression systems using the vector pBYR2HS, which contains geminiviral replication machinery and a double terminator for boosting expression. Additionally, the pBYR2HS vector contains an expression cassette for the gene-silencing suppressor p19 protein from tomato bushy stunt virus, which can also contribute to enhancing the expression of target proteins. In this study, we evaluated the applicability of this system to heterologous triterpenoid production in N . benthamiana. Medicago truncatula cytochrome P450 monooxygenase (CYP) 716A12 is the first enzyme to be functionally characterized as β-amyrin C-28 oxidase producing oleanolic acid. A mutant CYP716A12 (D122Q) with improved catalytic activity engineered in our previous study was co-expressed with other enzymes in N . benthamiana leaves. Using pBYR2HS, oleanolic acid yield was increased 13.1-fold compared with that using the conventional binary vector, indicating the advantage of the Tsukuba system. We also demonstrated the efficacy of co-expressing a mutant Arabidopsis thaliana HMGR1 catalytic domain, additional NADPH-cytochrome P450 reductase (CPR) transferring electrons to heterologous CYPs, and application of ascorbic acid for preventing leaf necrosis after agroinfiltration, to improve product yield. As a result, the product yields of both simple (β-amyrin) and oxidized (oleanolic acid and maslinic acid) triterpenoids were significantly improved compared with the previously reported yield in heterologous triterpenoid production in N . benthamiana leaves., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Romsuk, Yasumoto, Fukushima, Miura, Muranaka and Seki.)

Cultivated tomato (Solanum lycopersicum) contains α-tomatine, a steroidal glycoalkaloid (SGA), which functions as a defense compound to protect against pathogens and herbivores; interestingly, wild species in the tomato clade biosynthesize a variety of SGAs. In cultivated tomato, the metabolic detoxification of α-tomatine during tomato fruit ripening is an important trait that aided in its domestication, and two distinct 2-oxoglutarate-dependent dioxygenases (DOXs), a C-23 hydroxylase of α-tomatine (Sl23DOX) and a C-27 hydroxylase of lycoperoside C (Sl27DOX), are key to this process. There are tandemly duplicated DOX genes on tomato chromosome 1, with high levels of similarity to Sl23DOX. While these DOX genes are rarely expressed in cultivated tomato tissues, the recombinant enzymes of Solyc01g006580 and Solyc01g006610 metabolized α-tomatine to habrochaitoside A and (20R)-20-hydroxytomatine and were therefore named as habrochaitoside A synthase (HAS) and α-tomatine 20-hydroxylase (20DOX), respectively. Furthermore, 20DOX and HAS exist in the genome of wild tomato S. habrochaites accession LA1777, which accumulates habrochaitoside A in its fruits, and their expression patterns were in agreement with the SGA profiles in LA1777. These results indicate that the functional divergence of α-tomatine-metabolizing DOX enzymes results from gene duplication and the neofunctionalization of catalytic activity and gene expression, and this contributes to the structural diversity of SGAs in the tomato clade., (© The Author(s) 2022. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Phenotypic variation is the basis for trait adaptation via evolutionary selection. However, the driving forces behind quantitative trait variations remain unclear owing to their complexity at the molecular level. This study focused on the natural variation of the free-running period (FRP) of the circadian clock because FRP is a determining factor of the phase phenotype of clock-dependent physiology. Lemna aequinoctialis in Japan is a paddy field duckweed that exhibits a latitudinal cline of critical day length (CDL) for short-day flowering. We collected 72 strains of L. aequinoctialis and found a significant correlation between FRPs and locally adaptive CDLs, confirming that variation in the FRP-dependent phase phenotype underlies photoperiodic adaptation. Diel transcriptome analysis revealed that the induction timing of an FT gene is key to connecting the clock phase to photoperiodism at the molecular level. This study highlights the importance of FRP as a variation resource for evolutionary adaptation., Competing Interests: The authors declare no competing interests., (© 2022 The Author(s).)

Purpose: Dysgeusia is an adverse event caused by chemotherapy. Although retrospective studies have shown zinc administration improves dysgeusia, there have been no prospective studies. The present study examined effects of zinc therapy on dysgeusia in patients with gastrointestinal cancer., Methods: This multicenter, prospective, observational study enrolled patients with dysgeusia during chemotherapy treatment. Patients received no intervention (control), polaprezinc p.o., or zinc acetate hydrate p.o., and serum zinc levels were measured at 0 (baseline), 6, and 12 weeks. Dysgeusia was assessed using CTCAE v5.0 and subjective total taste acuity (STTA) criteria using questionnaires at baseline and 12 weeks., Results: From February 2020 to June 2021, 180 patients were enrolled from 17 institutes. There were no differences in mean baseline serum zinc levels among the groups (67.3, 66.6, and 67.5 μg/dL in the no intervention, polaprezinc, and zinc acetate hydrate groups, respectively. P = 0.846). The changes in mean serum zinc levels after 12 weeks were - 3.8, + 14.3, and + 46.6 μg/dL, and the efficacy rates of dysgeusia were 33.3%, 36.8%, and 34.6% using CTCAE and 33.3%, 52.6%, 32.7% using STTA in the no intervention, polaprezinc, and zinc acetate hydrate groups, respectively. The STTA scores improved in all groups, with significant improvement observed in the polaprezinc group compared with the no intervention group (P = 0.045)., Conclusion: There was no significant correlation between the degree of serum zinc elevation and improvement in dysgeusia, suggesting that polaprezinc, but not zinc acetate hydrate, was effective in improving chemotherapy-induced dysgeusia., Trial Registration: UMIN000039653. Date of registration: March 2, 2020., (© 2022. The Author(s).)