Search

Technical difficulties are occasionally encountered when performing conventional minimally invasive esophagectomy and robot-assisted minimally invasive esophagectomy in patients with a narrow thoracic cavity. Thoracic cavity-to-cage ratio is an indicator of thoracic cavity length. We hypothesized that the thoracic cavity-to-cage ratio could be a predictor of technical difficulties in conventional minimally invasive esophagectomy and robot-assisted minimally invasive esophagectomy.We evaluated 340 patients who underwent minimally invasive esophagectomy for esophageal squamous cell carcinoma between April 2010 and March 2021. Thoracic cavity-to-cage ratio was calculated as the diameter of the thoracic cavity to that of the thoracic cage at the brachiocephalic vein, tracheal bifurcation, and inferior right pulmonary vein levels. Moreover, thoracic cavity-to-cage ratio score, which is an indicator of the whole thoracic cavity length based on the thoracic cavity-to-cage ratio at the 3 levels, was defined. The thoracic procedure time was considered an indicator of surgical difficulty.We divided the patients into the conventional minimally invasive esophagectomy (n = 295) and robot-assisted minimally invasive esophagectomy (n = 45) groups. The patients in each group were divided into 2 cohorts according to median thoracic procedure time. Based on multivariate analysis, body mass index (P = .0007), clinical N stage (P = .0191), and thoracic cavity-to-cage ratio score (P = .0005) were independent factors for thoracic procedure time in the conventional minimally invasive esophagectomy group. Moreover, thoracic cavity-to-cage ratio at the tracheal bifurcation level (P = .0331) was the only independent factor for thoracic procedure time in the robot-assisted minimally invasive esophagectomy group.Thoracic cavity-to-cage ratio could be a predictor of technical difficulties in both conventional minimally invasive esophagectomy and robot-assisted minimally invasive esophagectomy.

Background Retrosternal reconstruction is associated with a lower risk of mediastinitis, gastro-tracheal fistula, and hiatal hernia. Historically, traumatic manual creation of the retrosternal tunnel has been performed using one's fist. We report a novel and atraumatic laparoscopic procedure to create the retrosternal route. Methods We have laparoscopically created the retrosternal route in 25 thoracoscopic, mediastinoscopic, or robot-assisted minimally invasive esophagectomies since August 2019. Specifically, a peritoneal incision is started at the dorsal side of the xiphoid process. Through a 12-mm port inserted slightly to the right of and superior to the umbilical camera port, we dissect loose connective tissues from the caudal to the cranial side using behind the sternum and inside the internal thoracic vessels as landmarks. The time required to create the route was calculated. Then, the cumulative sum (CUSUM) method and the simple moving average of five cases were used to evaluate the learning curve of this novel procedure. Operative outcomes were analyzed according to the learning curve results and also compared with 25 cases of postmediastinal reconstruction counterparts. Results Twenty-five patients were divided into the early group (six patients) and late group (19 patients) based on the peak of the CUSUM chart. The time required for route creation was 28.5 min (median) in the early and 15 min in the late group, indicating a significant difference (P = 0.038). The overall incidence of pleural injury was 20% (5 of 25 patients), with no significant difference between the groups. There was no significant difference in the incidence of perioperative complications. Also, there were no significant differences in perioperative complications or gastric conduit functions 1 year after surgery between the retrosternal and the postmediastinal reconstruction. Conclusion Laparoscopic creation of a retrosternal route for gastric conduit reconstruction is safe and feasible and has a short learning curve.

BACKGROUND Multidisciplinary treatment for esophageal squamous cell carcinoma (ESCC) has improved outcomes, but the prognosis for ESCC remains poor. Nutritional and inflammatory indicators are reported to be associated with cancer prognosis. The combination of albumin and the derived neutrophil-to-lymphocyte ratio (Alb-dNLR) score was established to measure the immune system and nutritional status. The authors hypothesized that the Alb-dNLR score could be a new reliable prognostic factor for ESCC patients. METHODS The study evaluated 269 patients who underwent esophagectomy between April 2010 and March 2018, including 185 patients who received neoadjuvant chemotherapy. The Alb-dNLR score was calculated using serum albumin and the dNLR. The dNLR was calculated as neutrophils to (leukocyte-neutrophil count). The cutoff values of the albumin and dNLR for overall survival (OS) were determined using the receiver operating characteristic curve. Patients were divided into "high" and "low" groups according to the Alb-dNLR score. RESULTS A high Alb-dNLR score was found in 61 cases (22.7%). The 5-year OS was 34% in the high Alb-dNLR group and 66.2% in the low Alb-dNLR group (p < 0.0001). The 5-year cause-specific survival (CSS) was 51.5% in the high Alb-dNLR group and 74.7% in the low Alb-dNLR group (p < 0.0001). Multivariate analyses demonstrated that the Alb-dNLR score was an independent prognostic factor for OS (hazard ratio [HR], 2.198; 95% confidence interval [CI], 1.460-3.263; p = 0.0002) and CSS (HR, 1.733; 95% CI, 1.035-2.835; p = 0.0371). CONCLUSIONS The Alb-dNLR score is an extremely useful, easy-to-use parameter to predict OS and CSS for ESCC patients.

Background Esophagectomy with lymphadenectomy is the principal treatment for localized esophageal cancer. Conventional minimally invasive esophagectomy (C-MIE) in prone position has spread worldwide as it is less invasive. However, its efficacy remains controversial. Robot-assisted MIE (RAMIE) can have more advantages than C-MIE. Therefore, the current study aimed to validate whether RAMIE is associated with lower incidence of left recurrent laryngeal nerve (RLN) palsy compared with C-MIE in prone position. Patients and Methods In total, 404 patients with esophageal carcinoma underwent MIE (353 C-MIEs and 51 RAMIEs) in prone position at Kobe University between 2010 and 2020. Then, propensity score matching was performed, and results showed that 51 patients should be included in each group. Thereafter, the perioperative outcomes between the two groups were compared. Results The RAMIE group had a significantly longer operative time than the C-MIE group (P < 0.0001). However, the number of lymph nodes resected along the left RLN was similar in both groups. Moreover, the incidence of left RLN palsy was significantly lower in the RAMIE group than in the C-MIE [Clavien-Dindo classification grade II; 0 (0%) versus 32 (9%), P = 0.022 in entire cohort, and 0 (0%) versus 5 (10%), P = 0.022 in matched cohort. Esophagectomy Complications Consensus Group definitions type I; 8 (16%) versus 101 (29%), P = 0.041 in entire cohort and 8 (16%) versus 18 (35%) in matched cohort, P = 0.023]. Conclusion RAMIE is superior to C-MIE in prone position in decreasing the incidence of left RLN palsy.

Various nutritional and inflammatory biomarkers have been reported to be associated with cancer prognosis, but most studies have focused on conditions before neoadjuvant chemotherapy (nCT). Developing real-time biomarkers reflecting changes in systemic conditions during nCT is important. We established a novel biomarker, represented as the albumin to derived neutrophil-to-lymphocyte ratio (Alb-dNLR ratio), and calculated the change in Alb-dNLR ratio (ΔAlb-dNLR) during nCT. We aimed to evaluate whether ΔAlb-dNLR is associated with prognosis in ESCC patients. We investigated 172 patients who underwent nCT before esophagectomy between April 2010 and March 2018. dNLR was calculated as the ratio of neutrophil count to (white blood cell count—neutrophil count), Alb-dNLR ratio was calculated by dividing serum albumin level by dNLR, and ΔAlb-dNLR was evaluated by dividing the post-Alb-dNLR ratio by the pre-Alb-dNLR ratio. Patients were divided into ‘high’ and ‘low’ groups according to ΔAlb-dNLR. Thirty-nine patients (22.7%) had a low ΔAlb-dNLR (≤0.8). The 5-year overall survival (OS) rates in patients with low and high ΔAlb-dNLR were 38.1% and 53.6%, respectively (p = 0.0072). Multivariate analyses demonstrated that estimated blood loss (p = 0.044), pathological T stage (p = 0.0005), pathological N stage (p = 0.017), and ΔAlb-dNLR (p = 0.005) were independent prognostic factors for OS. ΔAlb-dNLR is a useful prognostic factor for OS in patients with ESCC receiving nCT.

Recent genomic studies of parasitic plants have revealed that there are numerous footprints indicative of horizontal gene transfer (HGT) to the parasites from their host plants. However, the molecular mechanisms and biological impacts of this phenomenon have remained largely unknown. Here, we made the striking observation that two parasitic dodders, Cuscuta campestris and C. australis, have functional homologues of Si_CYP81Q1, which encodes piperitol/sesamin synthase (PSS) in the phylogenetically remote plant Sesamum indicum (sesame). The apparent lack of sequence similarity between the regions flanking PSS in Sesamum and Cuscuta spp. suggests the occurrence of HGT tightly associated with the PSS gene. Upon parasitism, C. campestris induced expression of the host Si_CYP81Q1 at the parasitic interface and mature and intron-retained Si_CYP81Q1 mRNA was transferred to C. campestris, suggesting that CYP81Q1 was translocated via RNA-mediated HGT. Thus, parasitism-evoked HGT might have had an unexpected role in the metabolic evolution of plants.

Recent advances in treatment for esophageal cancer have improved prognosis after esophagectomy, but they have led to an increased incidence of gastric conduit cancer. In most gastric conduit cancer patients who underwent retrosternal reconstruction, median sternotomy is performed, which is associated with a risk of postoperative bleeding and osteomyelitis; pain often negatively affects respiration. To avoid these problems, we developed thoracoscopic retrosternal gastric conduit resection in the supine position (TRGR-S) as new procedure. Methods We performed the first case of TRGR-S for a 75-year-old male with retrosternal gastric conduit cancer. He was placed in the supine position. Four ports were placed in the left chest wall. The gastric conduit was separated from the epicardium, sternum, and left brachiocephalic vein. Due to adhesions between the gastric tube and the right pleura, combined resection of the right pleura was performed. Next, pediculated jejunal reconstruction via the presternal route was performed. Results Because there were few adhesions in the left thoracic cavity, this approach provided safety and a good surgical view, and it was easy to recognize the landmark including epicardium, sternum, and left brachiocephalic vein leading to appropriate resection of the tissue. Furthermore, there were few restrictions on the operative angle for the forceps and operability was quite ergonomic. Moreover, the lungs can be noninvasively contracted via an artificial pneumothorax. The pathological diagnosis was signet ring cell carcinoma (pT1b, pN0, M0, pStage I), indicating R0 resection. There were no post-operative complications. Conclusion This approach does not require sternotomy, so it has less risk of postoperative bleeding and osteomyelitis. Due to fewer adhesions, this approach is safe and provides a good surgical view. TRGR-S is a safe, ergonomic, and reliable procedure for resection of retrosternal gastric conduit cancer. Video This is the video of the operation ‘TRGR-S’, which is the new procedure for the gastric conduit cancer. https://www.dropbox.com/s/2whnekgp73hw1lz/video%20for%20ISDE2020.mov?dl=0.

Thoracoscopic esophagectomy in the prone position (TEP) for esophageal cancer is reported to have superiority in preserving postoperative respiratory function and reducing postoperative respiratory complications. In Japan, the majority of patients with esophageal cancer are smokers and have obstructive ventilation disorders. But, the feasibirity and safety of TEP for patients with low respiratory function is unclear. Objectives To clarify the feasibirity and safety of TEP for esophageal cancer patients with obstructive respiratory function. Methods The 95 patients with obstructive respiratory disorder who underwent TEP and gastric tube reconstruction via posterior mediastinal route for esophageal cancer from January 2016 to April 2019 were divided into the two groups, low respiratory function (LRF) group and the control group. Short-term outcomes were compared between two groups. Results The control group was 73 cases, and the LRF group was 22 cases. Propensity score matching using age, gender, cT, and cN as covariates was used to identify matched patients (22 per group) in both groups. There were no differences in operation time of overall and intrathoracic part, or blood loss in each group. In the postoperative complications, pneumonia (13.6% vs. 9.1%), recurrent laryngeal palsy (18.2% vs. 22.7%), anastomotic leakage (13.6% vs. 13.6%) and hospital stay (36.3 days vs 27.5 days) were no differences in both groups. Conclusion TEP can be feasible and safe for the patients with obstructive ventilation disorder and low respiratory function.

Reconstruction routes after esophagectomy include posterior mediastinal, retrosternal, and subcutaneous route. We have performed posterior mediastinal reconstruction, but this route has higher risks of gastro-tracheal fistula and hiatal hernia. To avoid these complications, now we take the retrosternal route as our first choice by creating the route laparoscopically before pulling-up gastric conduit. We report the successful and safe procedure. Methods We performed laparoscopic creation of retrosternal route in 13 thoracoscopic/robot-assisted minimally invasive esophagectomies since August 2019. In practice, a peritoneal incision at the dorsal side of the xiphoid process is started. Then, via 12 mm port on the surgeon's right hand inserted slightly to the right and cranial side of the umbilical camera port, we dissect loose connective tissues from the caudal side to the cranial side behind the sternum and inside the internal thoracic vessels as landmarks. The time required to create the route and pleural injury rate during the procedure was examined. Results Thirteen cases were divided into two groups as early period group (seven cases) and later period group (six cases) respectively. The time required for route creation was 31.3 minutes(average) in the early period group, and 16.7 minutes in the later period group. There is tendency towards faster in later period group than in earlier one. The overall pleural injury rate was 15% (2 of 13 cases). Although it was difficult to determine the amount of bleeding, it was visually observed that the bleeding during the route creation was lower in the later period group than in the early period group. Conclusion The entire laparoscopic procedure to create retrosternal route makes it easier to observe and preserve the pleura and internal thoracic vessels compared to blind blunt dissection. As a conclusion, laparoscopic creation of retrosternal route for gastric conduit reconstruction is safe and feasible with good learning curve. Video https://www.dropbox.com/sh/p0wc3x46n33jp23/AADwiWHYIEUNUX6qZsERVIOga?dl=0.

RATIONALE Liquid chromatography/photodiode array atmospheric pressure chemical ionization mass spectrometry (LC/PDA-APCI-MS) is used for the analysis of various carotenoid pigments in plants. Among them, it is difficult to distinguish between the isomeric violaxanthin/neoxanthin esters. METHODS The yellow pigments of tomato petals were extracted with acetone, and the extracts were kept at -30°C to allow the contaminating triacylglycerols to settle out physically. The supernatants were analyzed using LC/PDA-APCI-MS with a high-resolution orbitrap mass spectrometer for their exact masses. The expected carotenoid esters were calculated with the combination of carotenoids and fatty acids, and they were matched with the experimental exact masses. The fatty acid structures in the carotenoid esters were also identified using collision-induced dissociation (CID) tandem mass spectrometry (MS/MS). The isomeric violaxanthin/neoxanthin esters were distinguished using CID MS/MS from their in-source dehydrated product ions as pseudoprecursor ions. RESULTS The in-source dehydrated ions [M - H2 O + H]+ of neoxanthin diesters predominated over their protonated molecules [M + H]+ in LC/MS. By contrast, the protonated molecules of violaxanthin diesters predominated. The 92 u loss product ions [M - H2 O - C7 H8 + H]+ were observed from the dehydrated violaxanthin diesters, but they were not generated from the dehydrated neoxanthin diesters in the CID MS/MS of their dehydrated pseudoprecursor ion [M - H2 O + H]+ . CONCLUSIONS The allene allyl carbocation in neoxanthin diesters was generated from dehydration after preferential protonation at the hydroxy group. The epoxide group of violaxanthin diesters opens easily after protonation; however, the dehydration did not proceed at this stage. The 92 u loss of C7 H8 was explained by an intramolecular [2 + 2] cycloaddition, which proceeded preferentially in dehydrated violaxanthin diesters because the carbocations in the dehydrated species were conjugated to the polyene and those double bonds were depolarized during CID MS/MS. Therefore, the isomeric neoxanthin/violaxanthin diesters were distinguished using LC/PDA-APCI-MS and MS/MS. This method was a practical and useful method of profiling the carotenoid esters of the yellow petals.

Summary Glycyrrhizin, a sweet triterpenoid saponin found in the roots and stolons of Glycyrrhiza species (licorice), is an important active ingredient in traditional herbal medicine. We previously identified two cytochrome P450 monooxygenases, CYP88D6 and CYP72A154, that produce an aglycone of glycyrrhizin, glycyrrhetinic acid, in Glycyrrhiza uralensis. The sugar moiety of glycyrrhizin, which is composed of two glucuronic acids, makes it sweet and reduces its side‐effects. Here, we report that UDP‐glycosyltransferase (UGT) 73P12 catalyzes the second glucuronosylation as the final step of glycyrrhizin biosynthesis in G. uralensis; the UGT73P12 produced glycyrrhizin by transferring a glucuronosyl moiety of UDP‐glucuronic acid to glycyrrhetinic acid 3‐O‐monoglucuronide. We also obtained a natural variant of UGT73P12 from a glycyrrhizin‐deficient (83‐555) strain of G. uralensis. The natural variant showed loss of specificity for UDP‐glucuronic acid and resulted in the production of an alternative saponin, glucoglycyrrhizin. These results are consistent with the chemical phenotype of the 83‐555 strain, and suggest the contribution of UGT73P12 to glycyrrhizin biosynthesis in planta. Furthermore, we identified Arg32 as the essential residue of UGT73P12 that provides high specificity for UDP‐glucuronic acid. These results strongly suggest the existence of an electrostatic interaction between the positively charged Arg32 and the negatively charged carboxy group of UDP‐glucuronic acid. The functional arginine residue and resultant specificity for UDP‐glucuronic acid are unique to UGT73P12 in the UGT73P subfamily. Our findings demonstrate the functional specialization of UGT73P12 for glycyrrhizin biosynthesis during divergent evolution, and provide mechanistic insights into UDP‐sugar selectivity for the rational engineering of sweet triterpenoid saponins., Significance Statement Glycyrrhizin, a licorice‐derived sweet triterpenoid diglycoside, is one of the most important phytoconstituents in the pharmaceutical and food industries. Here, we identified UGT73P12 as the enzyme catalyzing the sugar–sugar linkage to produce glycyrrhizin from its less glycosylated precursor in the presence of UDP‐glucuronic acid, and found that Arg32 of UGT73P12 is essential for the high specificity for UDP‐glucuronic acid, which was probably acquired during evolution to enable the functional specialization of UGT73P12 for glycyrrhizin biosynthesis.

Sesamin is a furofuran-type lignan that is found abundantly in seeds of Sesamum indicum (sesame) and has been widely accepted as a dietary supplement with positive effects on human health. The biological activity of sesamin in human cells and organs has been analysed extensively, although comparatively few studies show biological functions for sesamin in planta. Herein we screened sesamin-binding proteins (SBP) from sesame seedling extracts using sesamin-immobilized nano-beads. In subsequent peptide mass fingerprinting analyses, we identified a SBP, Steroleosin B, which is one of the membrane proteins found in oil bodies. In addition, pull-down assays and saturation transfer difference-nuclear magnetic resonance (STD-NMR) experiments demonstrated that sesamin binds directly to recombinant Steroleosin B in vitro. Finally, ectopic accumulations of sesamin and Steroleosin B in transgenic Arabidopsis thaliana plants induced severe growth defects including suppression of leaf expansion and root elongation. Collectively, these results indicate that sesamin influences tissue development in the presence of Steroleosin B.

A 70-year-old man had undergone thoracoscopic esophagectomy following neoadjuvant chemotherapy for thoracic esophageal squamous cell carcinoma 3 years before presentation. He was undergoing whole-brain irradiation following surgery for a solitary brain metastatic tumor. The chief complaint was left leg pain during irradiation. FDG-PET/CT and MRI revealed metastases in bilateral cauda equina S1 nerve roots. Cerebrospinal fluid examination also revealed malignant cells. He received chemotherapy with 2 courses of 5-fluorouracil and cisplatin following 30 Gy of spinal irradiation. To control neurological symptoms, 4 courses of intrathecal chemotherapy with methotrexate, cytarabine, and betamethasone were performed. However, he gradually weakened and died 8 months after brain metastasis and 7 months after leptomeningeal carcinomatosis. The multidisciplinary treatment using irradiation and systemic and intrathecal chemotherapies could improve the survival of patients with leptomeningeal carcinomatosis of esophageal squamous cell carcinoma.

Summary Sesamum spp. (sesame) are known to accumulate a variety of lignans in a lineage‐specific manner. In cultivated sesame (Sesamum indicum), (+)‐sesamin, (+)‐sesamolin and (+)‐sesaminol triglucoside are the three major lignans found richly in the seeds. A recent study demonstrated that SiCYP92B14 is a pivotal enzyme that allocates the substrate (+)‐sesamin to two products, (+)‐sesamolin and (+)‐sesaminol, through multiple reaction schemes including oxidative rearrangement of α‐oxy‐substituted aryl groups (ORA). In contrast, it remains unclear whether (+)‐sesamin in wild sesame undergoes oxidation reactions as in S. indicum and how, if at all, the ratio of the co‐products is tailored at the molecular level. Here, we functionally characterised SrCYP92B14 as a SiCYP92B14 orthologue from a wild sesame, Sesamum radiatum, in which we revealed accumulation of the (+)‐sesaminol derivatives (+)‐sesangolin and its novel structural isomer (+)‐7´‐episesantalin. Intriguingly, SrCYP92B14 predominantly produced (+)‐sesaminol either through ORA or direct oxidation on the aromatic ring, while a relatively low but detectable level of (+)‐sesamolin was produced. Amino acid substitution analysis suggested that residues in the putative distal helix and the neighbouring heme propionate of CYP92B14 affect the ratios of its co‐products. These data collectively show that the bimodal oxidation mechanism of (+)‐sesamin might be widespread across Sesamum spp., and that CYP92B14 is likely to be a key enzyme in shaping the ratio of (+)‐sesaminol‐ and (+)‐sesamolin‐derived lignans from the biochemical and evolutionary perspectives., Significance Statement In Sesamum indicum (sesame), SiCYP92B14 oxidises (+)‐sesamin, a health‐promoting compound, to either (+)‐sesaminol or (+)‐sesamolin via two distinct reaction mechanisms. The identification and biochemical characterisation of SrCYP92B14 from wild sesame, S. radiatum, revealed that SrCYP92B14 oxidises (+)‐sesamin predominantly to (+)‐sesaminol, which corroborates with the lignan profile of S. radiatum, thus highlighting the relevance of CYP92B14 proteins in shaping the molar ratio of (+)‐sesaminol and (+)‐sesamolin derivatives that accumulate in Sesamum spp.

Sesamin [(7α,7′α,8α,8′α)-3,4:3′,4′-bis(methylenedioxy)-7,9′:7′,9-diepoxylignane] is a major lignan in sesame seeds. Sesamin is converted to the catechol metabolite, SC1 [(7α,7′α,8α,8′α)-3′,4′-methylenedioxy-7,9′:7′,9-diepoxylignane-3,4-diol] with anti-inflammatory effects after oral administration. However, its molecular target remains unknown. Analysis using high-performance affinity nanobeads led to the identification of annexin A1 (ANX A1) as an SC1-binding protein. SC1 was found to bind to the annexin repeat 3 region of ANX A1 with a high-affinity constant (Kd = 2.77 μmol L−1). In U937 cells, SC1 exhibited an anti-inflammatory effect dependent on ANX A1. Furthermore, administration of sesamin or SC1 attenuated carbon tetrachloride-induced liver damage in mice and concurrently suppressed inflammatory responses dependent on ANX A1. The mechanism involved SC1-induced ANX A1 phosphorylation at serine 27 that facilitates extracellular ANX A1 release. Consequently, the ANX A1 released into the extracellular space suppressed the production of tumor necrosis factor α. This study demonstrates that ANX A1 acts as a pivotal target of sesamin metabolites to attenuate inflammatory responses.

The brilliant blue color of the Nemophila menziesii flower is derived from metalloanthocyanin, which consists of anthocyanin {petunidin 3-O-[6-O-(trans-p-coumaroyl)-β-glucoside]-5-O-[6-O-(malonyl)-β-glucoside]}, flavone [apigenin 7-O-β-glucoside-4'-O-(6-O-malonyl)-O-β-glucoside] and metal ions (Mg2+, Fe3+). Although the two glucosyl moieties at the apigenin 7-O and 4'-O positions are essential for metalloanthocyanin formation, the mechanism of glucosylation has not yet been clarified. In this study, we used crude protein extract prepared from N. menziesii petals to determine that apigenin is sequentially glucosylated by the catalysis of UDP-glucose:flavone 4'-O-glucosyltrasferase (F4'GT) and UDP-glucose:flavone 4'-O-glucoside 7-O-glucosyltransferase (F4'G7GT). We identified 150 contigs exhibiting homology with a UDP-glucose-dependent GT in the N. menziesii petal transcriptome and isolated 24 putative full-length GT cDNAs which were then subjected to functional analysis. Two GT cDNAs, NmF4'GT and NmF4'G7GT, which are highly expressed during the early stages of petal development and rarely in leaves, were shown to encode F4'GT and F4'G7GT activities, respectively. Biochemical characterization of the recombinant enzymes revealed that NmF4'GT specifically catalyzed 4'-glucosylation of flavonoids and that NmF4'G7GT specifically catalyzed 7-glucosylation of flavone 4'-O-glucosides and flavones. Apigenin 7,4'-O-diglucoside was efficiently synthesized from apigenin in the presence of recombinant NmF4'GT and NmF4'G7GT. Transgenic tobacco BY-2 cells expressing NmF4'GT and NmF4'G7GT converted apigenin into apigenin 7,4'-O-diglucoside, confirming their activities in vivo. Based on these results, we conclude that these two GTs act co-ordinately to catalyze apigenin 7,4'-O-diglucoside biosynthesis in N. menziesii.

Sesamin is a furofuran-type lignan that is found abundantly in seeds of Sesamum indicum (sesame) and has been widely accepted as a dietary supplement with positive effects on human health. The biological activity of sesamin in human cells and organs has been analysed extensively, although comparatively few studies show biological functions for sesamin in planta. Herein we screened sesamin-binding proteins (SBP) from sesame seedling extracts using sesamin-immobilized nano-beads. In subsequent peptide mass fingerprinting analyses, we identified a SBP, Steroleosin B, which is one of the membrane proteins found in oil bodies. In addition, pull-down assays and saturation transfer difference-nuclear magnetic resonance (STD-NMR) experiments demonstrated that sesamin binds directly to recombinant Steroleosin B in vitro. Finally, ectopic accumulations of sesamin and Steroleosin B in transgenic Arabidopsis thaliana plants induced severe growth defects including suppression of leaf expansion and root elongation. Collectively, these results indicate that sesamin influences tissue development in the presence of Steroleosin B.

Grade 2 or 3 proteinuria was observed in 54 patients out of 158 metastatic colorectal cancer patients receiving anti-VEGF therapy. Patients with diabetes and hypertension were risk for severe proteinuria. ARBs were more frequently used in patients with severe proteinuria. However, they could not reduce proteinuria. The examination of protein/creatinine ratio was useful for continuing anti-VEGF therapy.

Sesame (Sesamum indicum) seeds contain a large number of lignans, phenylpropanoid-related plant specialized metabolites. (+)-Sesamin and (+)-sesamolin are major hydrophobic lignans, whereas (+)-sesaminol primarily accumulates as a water-soluble sesaminol triglucoside (STG) with a sugar chain branched via β1→2 and β1→6-O-glucosidic linkages [i.e. (+)-sesaminol 2-O-β-d-glucosyl-(1→2)-O-β-d-glucoside-(1→6)-O-β-d-glucoside]. We previously reported that the 2-O-glucosylation of (+)-sesaminol aglycon and β1→6-O-glucosylation of (+)-sesaminol 2-O-β-d-glucoside (SMG) are mediated by UDP-sugar-dependent glucosyltransferases (UGT), UGT71A9 and UGT94D1, respectively. Here we identified a distinct UGT, UGT94AG1, that specifically catalyzes the β1→2-O-glucosylation of SMG and (+)-sesaminol 2-O-β-d-glucosyl-(1→6)-O-β-d-glucoside [termed SDG(β1→6)]. UGT94AG1 was phylogenetically related to glycoside-specific glycosyltransferases (GGTs) and co-ordinately expressed with UGT71A9 and UGT94D1 in the seeds. The role of UGT94AG1 in STG biosynthesis was further confirmed by identification of a STG-deficient sesame mutant that predominantly accumulates SDG(β1→6) due to a destructive insertion in the coding sequence of UGT94AG1. We also identified UGT94AA2 as an alternative UGT potentially involved in sugar-sugar β1→6-O-glucosylation, in addition to UGT94D1, during STG biosynthesis. Yeast two-hybrid assays showed that UGT71A9, UGT94AG1, and UGT94AA2 were found to interact with a membrane-associated P450 enzyme, CYP81Q1 (piperitol/sesamin synthase), suggesting that these UGTs are components of a membrane-bound metabolon for STG biosynthesis. A comparison of kinetic parameters of these UGTs further suggested that the main β-O-glucosylation sequence of STG biosynthesis is β1→2-O-glucosylation of SMG by UGT94AG1 followed by UGT94AA2-mediated β1→6-O-glucosylation. These findings together establish the complete biosynthetic pathway of STG and shed light on the evolvability of regio-selectivity of sequential glucosylations catalyzed by GGTs.

A volatile compound was isolated from the fruit of Habanero pepper (Capsicum chinense) and related varieties and identified as 6-methyl-(E)-4-heptenyl 3-methylbutyrate by

Plant specialized metabolites are often found as lineage-specific diastereomeric isomers. For example, Sesamum alatum accumulates the specialized metabolite (+)-2-episesalatin, a furofuran-type lignan with a characteristic diastereomeric configuration rarely found in other Sesamum spp. However, little is known regarding how diastereomeric specificity in lignan biosynthesis is implemented in planta. Here, we show that S. alatum CYP81Q3, a P450 orthologous to S. indicum CYP81Q1, specifically catalyzes methylenedioxy bridge (MDB) formation in (+)-epipinoresinol to produce (+)-pluviatilol. Both (+)-epipinoresinol and (+)-pluviatilol are putative intermediates of (+)-2-episesalatin based on their diastereomeric configurations. On the other hand, CYP81Q3 accepts neither (+)- nor (-)-pinoresinol as a substrate. This diastereomeric selectivity of CYP81Q3 is in clear contrast to that of CYP81Q1, which specifically converts (+)-pinoresinol to (+)-sesamin via (+)-piperitol by the sequential formation of two MDBs but does not accept (+)-epipinoresinol as a substrate. Moreover, (+)-pinoresinol does not interfere with the conversion of (+)-epipinoresinol to (+)-pluviatilol by CYP81Q3. Amino acid substitution and CO difference spectral analyses show that polymorphic residues between CYP81Q1 and CYP81Q3 proximal to their putative substrate pockets are crucial for the functional diversity and stability of these two enzymes. Our data provide clues to understanding how the lineage-specific functional differentiation of respective biosynthetic enzymes substantiates the stereoisomeric diversity of lignan structures.

The original version of of the Supplementary Information associated with this Article inadvertently omitted oligonucleotide primer sequences from Supplementary Table 3 and Supplementary Methods describing the molecular cloning of CYP92B14, CPR1 and CYP81Q cDNA fragments. The HTML has been updated to include a corrected version of the Supplementary Information.

Tea plants (Camellia sinensis) store volatile organic compounds (VOCs; monoterpene, aromatic, and aliphatic alcohols) in the leaves in the form of water-soluble diglycosides, primarily as β-primeverosides (6-O-β-d-xylopyranosyl-β-d-glucopyranosides). These VOCs play a critical role in plant defenses and tea aroma quality, yet little is known about their biosynthesis and physiological roles in planta. Here, we identified two UDP-glycosyltransferases (UGTs) from C. sinensis, UGT85K11 (CsGT1) and UGT94P1 (CsGT2), converting VOCs into β-primeverosides by sequential glucosylation and xylosylation, respectively. CsGT1 exhibits a broad substrate specificity toward monoterpene, aromatic, and aliphatic alcohols to produce the respective glucosides. On the other hand, CsGT2 specifically catalyzes the xylosylation of the 6′-hydroxy group of the sugar moiety of geranyl β-d-glucopyranoside, producing geranyl β-primeveroside. Homology modeling, followed by site-directed mutagenesis of CsGT2, identified a unique isoleucine-141 residue playing a crucial role in sugar donor specificity toward UDP-xylose. The transcripts of both CsGTs were mainly expressed in young leaves, along with β-PRIMEVEROSIDASE encoding a diglycoside-specific glycosidase. In conclusion, our findings reveal the mechanism of aroma β-primeveroside biosynthesis in C. sinensis. This information can be used to preserve tea aroma better during the manufacturing process and to investigate the mechanism of plant chemical defenses.

Sesamin, one of the lignans in sesame seed, was a labile compound in MS and it was reported that the protonated mol- ecule of sesamin decomposed easily in ES ionization process and it cannot be detected (G. Yan, et al., Rapid Commun Mass Spectrom. 2007, 21, 3613-3620). To protect labile compounds, an amino-cyclodextrin (NCyD) was added to the sample to pro- mote the host-guest interaction complex in ESI-MS. As a result, sesamin was ionized as the NCyD-sesamin-NCyD (1:2) com- plex without undesired decomposition, suggesting that the amino-CyDs assist the ionization of the labile molecules capped with CyDs by host-guest interaction and these compounds were ionized without their decomposition, those are like amino-CyD com- plex-assisted ionization. The amino-CyD complexes of sesamin and sesamolin were also analyzed by their ion-mobility MS.

The red wines made from Vitis vinifera were identified as skin-whitening effectors by using in vitro assays. OPCs in the wine were evaluated for tyrosinase activity and melanogenesis. Strong tyrosinase inhibitory activity was observed in fractions with high oligomeric proanthocyanidin (OPC) content. Among OPC dimers, a strong inhibitory effect on tyrosinase was observed with OPCs which contain (+)-catechin as an upper unit. Melanogenesis inhibitory effect was observed with OPCs which have (-)-epicatechin as upper units. Also, OPC trimers, upper and middle units joined with 4 → 8 bonds, showed stronger effects compared to trimers with 4 → 6 linkages. Interestingly, (-)-epicatechin-(4β → 8)-(-)-epicatechin 3-O-gallate, which is a unique component of grapes has potent inhibitory effects on both tyrosinase and melanogenesis. Our data provide structural information about such active compounds. These results suggest that red wines containing OPC, have high melanogenesis inhibitory effect and are supposed to have skin-whitening effect.

Three unknown colors (A1, A2 and A3) in Food red No. 102 (R102) were identified by LC-MS and NMR analysis. Red dye A1 was azo rubine (AZO), which is not designated as a coloring agent in Japan. Red dye A2 was a structural isomer of Fast Red E (FRE), which is also not designated as a coloring agent in Japan. Red dye A3 was predicted to be an overreaction product of R102, possessing the R102 structural framework. Furthermore, AZO, which was assumed to have been produced by dimerization of a diazonium salt used as a synthetic intermediate of R102, was also identified in Food red No. 2 (R2).

Pseudomonas aeruginosa, an opportunistic pathogen, is known to mainly use N-acylhomoserine lactones (AHLs) as autoinducers. Recent progress in this field demonstrated that not only AHLs, but also their degradation products, tetramic acids, may have some biological activities. The present study examined the roles of Pseudomonas autoinducers and tetramic acids in bacterial survival and behavior in ecological niches. P. aeruginosa autoinducers and the tetramic acid derivatives were chemically synthesized, and the structure-activity correlation was investigated. Some tetramic acids derived from AHLs caused a significant reduction in the viability of P. aeruginosa in a concentration dependent manner (30-300 µM). The smaller the inoculum of bacteria, the stronger the bactericidal activity that was observed. The data from tetramic acid derivatives indicated the keto-enol structure of tetramic acid to be critical for the antibacterial activity. Exogenous tetramic acid did not induce significant changes in the formation of biofilm or production of exoproducts such as pyocyanin and elastase. Tetramic acid and disinfectants acted synergistically to kill P. aeruginosa. These results suggest the AHL-degradation product tetramic acid to be useful for bacterial control.

(+)-Sesamin, (+)-sesamolin, and (+)-sesaminol glucosides are phenylpropanoid-derived specialized metabolites called lignans, and are rich in sesame (Sesamum indicum) seed. Despite their renowned anti-oxidative and health-promoting properties, the biosynthesis of (+)-sesamolin and (+)-sesaminol remained largely elusive. Here we show that (+)-sesamolin deficiency in sesame is genetically associated with the deletion of four C-terminal amino acids (Del4C) in a P450 enzyme CYP92B14 that constitutes a novel clade separate from sesamin synthase CYP81Q1. Recombinant CYP92B14 converts (+)-sesamin to (+)-sesamolin and, unexpectedly, (+)-sesaminol through an oxygenation scheme designated as oxidative rearrangement of α-oxy-substituted aryl groups (ORA). Intriguingly, CYP92B14 also generates (+)-sesaminol through direct oxygenation of the aromatic ring. The activity of CYP92B14 is enhanced when co-expressed with CYP81Q1, implying functional coordination of CYP81Q1 with CYP92B14. The discovery of CYP92B14 not only uncovers the last steps in sesame lignan biosynthesis but highlights the remarkable catalytic plasticity of P450s that contributes to metabolic diversity in nature., Sesame seeds contain phenylpropanoid-derived lignans that are potentially beneficial to human health. Here, the authors clone a cytochrome P450 enzyme that is responsible for the last steps of sesame lignan biosynthesis and show that it acts through a novel oxidative rearrangement mechanism.

Flavonoids are most commonly conjugated with various sugar moieties by UDP-sugar:glycosyltransferases (UGTs) in a lineage-specific manner. Generally, the phylogenetics and regiospecificity of flavonoid UGTs are correlated, indicating that the regiospecificity of UGT differentiated prior to speciation. By contrast, it is unclear how the sugar donor specificity of UGTs evolved. Here, we report the biochemical, homology-modeled, and phylogenetic characterization of flavonoid 7-O-glucuronosyltransferases (F7GAT), which is responsible for producing specialized metabolites in Lamiales plants. All of the Lamiales F7GATs were found to be members of the UGT88-related cluster and specifically used UDP-glucuronic acid (UDPGA). We identified an Arg residue that is specifically conserved in the PSPG box in the Lamiales F7GATs. Substitution of this Arg with Trp was sufficient to convert the sugar donor specificity of the Lamiales F7GATs from UDPGA to UDP-glucose. Homology modeling of the Lamiales F7GAT suggested that the Arg residue plays a critical role in the specific recognition of anionic carboxylate of the glucuronic acid moiety of UDPGA with its cationic guanidinium moiety. These results support the hypothesis that differentiation of sugar donor specificity of UGTs occurred locally, in specific plant lineages, after establishment of general regiospecificity for the sugar acceptor. Thus, the plasticity of sugar donor specificity explains, in part, the extraordinary structural diversification of phytochemicals.

A biphenanthryl ether with substituents in the bay regions was efficiently synthesized for the first time. The unique stereostructure of 1 was clarified. Its twisted conformation and optical behavior as well as its characteristic supramolecular helical structure, which is constructed through a C–H⋯F hydrogen bonding network in the solid state, are discussed.

Iron (Fe) is an essential element for plant growth. Gramineous plants have generally developed a distinct strategy to efficiently acquire insoluble Fe, which is characterized by the synthesis and secretion of an Fe-chelating substance, phytosiderophore (PS) such as mugineic acid (MA), and by a specific uptake system for Fe(III)-PS complexes. In a previous study, we identified a gene specifically encoding an Fe(III)-PS transporter (HvYS1) in barley. This gene as well as the encoded protein is specifically expressed in the epidermal cells of the roots, and gene expression is greatly enhanced under Fe-deficient conditions. The localization and substrate specificity of HvYS1 indicate that it is a specific transporter in barley roots. In contrast, ZmYS1, which has been reported as an Fe-PS transporter from maize, possesses broad substrate specificity despite a high homology with HvYS1. By assessing the transport activity of a series of HvYS1-ZmYS1 chimeras, we revealed that the outer membrane loop between the 6th and 7th transmembrane regions is essential for the substrate specificity. We also achieved an efficient short-step synthesis of MA and 2'-deoxymugineic acid (DMA). Our new synthetic method enabled us to use them in a large quantity for biological studies.