Identification and evolution of a diterpenoid phytoalexin oryzalactone biosynthetic gene in the genus Oryza.

The natural variation of plant-specialized metabolites represents the evolutionary adaptation of plants to their environments. However, the molecular mechanisms that account for the diversification of the metabolic pathways have not been fully clarified. Rice plants resist attacks from pathogens by accumulating diterpenoid phytoalexins. It has been confirmed that the composition of rice phytoalexins exhibits numerous natural variations. Major rice phytoalexins (momilactones and phytocassanes) are accumulated in most cultivars, although oryzalactone is a cultivar-specific compound. Here, we attempted to reveal the evolutionary trajectory of the diversification of phytoalexins by analyzing the oryzalactone biosynthetic gene in Oryza species. The candidate gene, KSLX-OL, which accounts for oryzalactone biosynthesis, was found around the single-nucleotide polymorphisms specific to the oryzalactone-accumulating cultivars in the long arm of chromosome 11. The metabolite analyses in Nicotiana benthamiana and rice plants overexpressing KSLX-OL indicated that KSLX-OL is responsible for the oryzalactone biosynthesis. KSLX-OL is an allele of KSL8 that is involved in the biosynthesis of another diterpenoid phytoalexin, oryzalexin S and is specifically distributed in the AA genome species. KSLX-NOL and KSLX-bar, which encode similar enzymes but are not involved in oryzalactone biosynthesis, were also found in AA genome species. The phylogenetic analyses of KSLXs, KSL8s, and related pseudogenes (KSL9s) indicated that KSLX-OL was generated from a common ancestor with KSL8 and KSL9 via gene duplication, functional differentiation, and gene fusion. The wide distributions of KSLX-OL and KSL8 in AA genome species demonstrate their long-term coexistence beyond species differentiation, suggesting a balancing selection between the genes.

Molecular identification of a laccase that catalyzes the oxidative coupling of a hydroxycinnamic acid amide for hordatine biosynthesis in barley.

Plants produce dimerized phenolic compounds as secondary metabolites. Hordatine A (HA), a dehydrodimer of p-coumaroylagmatine (pCA), is an antifungal compound accumulated at high levels in young barley (Hordeum vulgare) seedlings. The enzyme responsible for the oxidative dimerization of pCA, which is the final step of the hordatine biosynthetic pathway, has not been identified. In this study, we first verified the presence of this enzyme activity in the crude extract of barley seedlings. Because the enzyme activity was not dependent on H2 O2 , the responsible enzyme was not peroxidase, which was previously implicated in HA biosynthesis. The analysis of the dissection lines of wheat (Triticum aestivum) carrying aberrant barley 2H chromosomes detected HA in the wheat lines carrying the distal part of the 2H short arm. This chromosomal region contains two laccase genes (HvLAC1 and HvLAC2) that are highly expressed at the seedling stage and may encode enzymes that oxidize pCA during the formation of HA. Changes in the HvLAC transcript levels coincided with the changes in the HA biosynthesis-related enzyme activities in the crude extract and the HA content in barley seedlings. Moreover, HvLAC genes were heterologously expressed in Nicotiana benthamiana leaves and in bamboo (Phyllostachys nigra) suspension cells and HA biosynthetic activities were detected in the crude extract of transformed N. benthamiana leaves and bamboo suspension cells. The HA formed by the enzymatic reaction had the same stereo-configuration as the naturally occurring HA. These results demonstrate that HvLAC enzymes mediate the oxidative coupling of pCA during HA biosynthesis.

Suppression of anthracnose disease by orsellinaldehyde isolated from the mushroom Coprinus comatus.

AIMS: Anthracnose caused by Colletotrichum species is one of the most devastating diseases of fruits and crops. We isolated and identified an antifungal compound from the mushroom Coprinus comatus and investigated its inhibitory potential against anthracnose disease-causing fungi with the goal of discovering natural products that can suppress anthracnose-caused plant disease. METHODS AND RESULTS: The culture filtrate of C. comatus was subjected to a bioassay-guided isolation of antifungal compounds. The active compound was identified as orsellinaldehyde (2,4-dihydroxy-6-methylbenzaldehyde) based on mass spectroscopy and nuclear magnetic resonance analyses. Orsellinaldehyde displayed broad-spectrum inhibitory activity against different plant pathogenic fungi. Among the tested Colletotrichum species, it exhibited the lowest IC50 values on conidial germination and germ tube elongation of Colletotrichum orbiculare. The compound also showed remarkable inhibitory activity against Colletotrichum gloeosporiodes. The staining of Colletotrichum conidia with fluorescein diacetate and propidium iodide demonstrated that the compound is fungicidal. The postharvest in-vivo detached fruit assay indicated that orsellinaldehyde suppressed anthracnose lesion symptoms on mango and cucumber fruits caused by C. gloeosporioides and C. orbiculare, respectively. CONCLUSIONS: Orsellinaldehyde was identified as a potent antifungal compound from the culture filtrate of C. comatus. The inhibitory and fungicidal activities of orsellinaldehyde against different Colletotrichum species indicate its potential as a fungicide for protecting various fruits against anthracnose disease-causing fungi.

Isolation of 2,2'-azoxybisbenzyl alcohol from Agaricus subrutilescens and its inhibitory activity against bacterial biofilm formation.

Virulence pathways in pathogenic bacteria are regulated by quorum sensing mechanisms, particularly biofilm formation through autoinducer (AI) production and sensing. In this study, the culture filtrate extracted from an edible mushroom, Agaricus subrutilescens, was fractionated to isolate a compound that inhibits biofilm formation. Four gram-negative bacteria (Klebsiella pneumoniae, Escherichia coli, Proteus mirabilis, and Enterobacter cloacae) and two gram-positive bacteria (Enterococcus faecalis and Staphylococcus aureus) were used for the bioassay. The bioassay-guided chromatographic separations of the culture filtrate extract resulted in the isolation of the compound. Further, spectroscopic analyses revealed the identity of the compound as 2,2'-azoxybisbenzyl alcohol (ABA). The minimum inhibitory and sub-inhibitory concentrations of the compound were also determined. Azoxybisbenzyl alcohol was significantly effective in inhibiting biofilm formation in all tested bacteria, with half-maximal inhibitory concentrations of 3-11 µg/mL. Additionally, the bioactivity of ABA was confirmed through the bioassays for the inhibition of exopolysaccharide matrixes and AI activities.

Inhibition of melanoma cell proliferation by strobilurins isolated from mushrooms and their synthetic analogues.

Strobilurins A and X, isolated from Mucidula venosolamellata culture extracts, demonstrated potent inhibition of human melanoma G-361 cell proliferation. Strobilurin X exhibited milder inhibitory effects on human fibroblast cells (NB1RGB) compared to strobilurin A. Additional strobilurin-related compounds were isolated from the other mushroom species. Oudemansins A and B displayed weaker activities on G-361 cells than strobilurins A and B, respectively, emphasizing the importance of a conjugated double-bond structure. Among isolated compounds, strobilurin G showed the lowest IC50 value for G-361 cells. Additional strobilurins bearing various substituents on the benzene ring were synthesized. Synthetic intermediates lacking the methyl ß-methoxyacrylate group and a strobilurin analogue bearing modified ß-methoxyacrylate moiety showed almost no inhibitory activity against G-361 cells. The introduction of long or bulky substituents at the 4' position of the benzene ring of strobilurins enhanced the activity and selectivity, suggesting differential recognition of the benzene ring by G-361 and NB1RGB cells.

Novel compound, pleuropyronine, and other polyketides isolated from the edible mushroom Pleurotus ostreatus suppress bacterial biofilm formation.

An increase in the number of drug-resistant microbes is a major threat to human health. Bacterial drug resistance is mostly mediated by biofilm formation. In this study, the culture filtrate from the edible mushroom, Pleurotus ostreatus, was fractionated to isolate compounds that inhibit the biofilm formation of six pathogenic bacteria. Notably, we isolated compounds 16 using bioassay-guided chromatographic separations. Spectroscopic and X-ray diffraction analyses identified 1 as a novel fused bicyclic pyrone-furan, named pleuropyronine, whereas 2-6 were known polyketides. Pleuropyronine inhibited biofilm formation in four gram-negative bacteria, with IC50 values ranging from 5.4 to 8.7 µg/mL, whereas 2-6 exhibited IC50 values between 1.0 and 5.3 µg/mL against five bacteria. Additionally, pleuropyronine bioactivity was confirmed by the inhibition of exopolysaccharide and biofilm formation induced by C6-homoserine lactone. Thus, this may serve as a pioneering study on the pharmacological potential of isolated compounds, offering valuable insights for future research.

Identification of sesquiterpene aldehydes as volatile antifungal compounds in Phaeolepiota aurea culture filtrate.

The culture filtrate extract of golden bootleg (Phaeolepiota aurea) exhibited strong antifungal activity in a bioassay for volatile compounds. Purification of active compounds through silica gel column chromatography and HPLC isolated two compounds, 1 and 2, with molecular weights of 234 and 259, respectively, as confirmed by mass spectrometry. NMR analysis identified these compounds as new sesquiterpenoids with the α-bourbonene skeleton. Both compounds had an aldehyde group, while 2 possessed a cyanohydrin group. These compounds were named phaeolep aldehydes A (1) and B (2). Phaeolep aldehyde A inhibited hyphae elongation of Penicillium citrinum, Aspergillus niger, Cladosporium sphaerospermum, and Alternaria brassicicola at 10 ppm, but did not exhibit any antibacterial activity. In contrast, phaeolep aldehyde B exhibited potent inhibitory effects on the spore germination of Pe. citrinum, achieving 58.2% inhibition at 0.25 ppm, outperforming phaeolep aldehyde A, which exhibited 25.7% inhibition at 10 ppm.

NAD+ enhances the activity and thermostability of S-adenosyl-L-homocysteine hydrolase from Pyrococcus horikoshii OT3.

S-Adenosyl-L-methionine (SAM) and S-adenosyl-L-homocysteine (SAH) are important biochemical intermediates. SAM is the major methyl donor for diverse methylation reactions in vivo. The SAM to SAH ratio serves as a marker of methylation capacity. Stable isotope-labeled SAM and SAH are used to measure this ratio with high sensitivity. SAH hydrolase (EC 3.13.2.1; SAHH), which reversibly catalyzes the conversion of adenosine and L-homocysteine to SAH, is used to produce labeled SAH. To produce labeled SAH with high efficiency, we focused on the SAHH of Pyrococcus horikoshii OT3, a thermophilic archaeon. We prepared recombinant P. horikoshii SAHH using Escherichia coli and investigated its enzymatic properties. Unexpectedly, the optimum temperature and thermostability of P. horikoshii SAHH were much lower than its optimum growth temperature. However, addition of NAD+ to the reaction mixture shifted the optimum temperature of P. horikoshii SAHH to a higher temperature, suggesting that NAD+ stabilizes the structure of the enzyme.

Isolation, structure elucidation, and biological activities of sesquiterpenes and phthalides from two edible mushrooms Pleurotus species.

Antimicrobial compounds were purified from culture filtrates from 2 edible Pleurotus species. Using a bioassay-guided fractionation of the culture filtrate extracts, 3 compounds (1-3) were obtained from Pleurotus ostreatus, and another compound (4) was obtained from Pleurotus pulmonarius. Spectroscopic analysis revealed that 1-3 was identified as 5,7-dimethoxyphthalide, 4,6-dimethoxyphthalide, and cheimonophyllon E, respectively, while 4 were identified as pleuroton A. The minimum inhibitory concentration and minimum bactericidal concentration of these compounds were determined against 6 pathogenic bacterial species, Enterococcus faecalis, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter cloacae. Compounds 2 and 4 were inhibitory against all tested bacteria, while 1 and 4 were inhibitory against 3 and 2 species, respectively. In addition, 1-4 inhibited tyrosinase, with IC50 values of 0.10-0.30 mg/mL, and α-glucosidase, with IC50 values of 0.12-0.54 mg/mL. However, their antioxidant capacities were marginal.

(E)-7-phenyl-2-heptene-4,6-diyn-1-ol from Bidens pilosa as a repellent against isopods.

The development of repellents as alternatives to insecticides has expanded in recent years. However, their use in isopod pest control is limited. To develop an isopod repellent, a plant extract library from wild plants native to the Kochi Prefecture was screened for repellent activity against pillbugs, and 82 samples (87%) exhibited repellent activity. Among them, (E)-7-phenyl-2-heptene-4,6-diyn-1-ol was isolated and identified as a repellent from the root of Bidens pilosa. It had a half-maximal effective concentration of 0.20 µm, with a strong repellency. A study of the structure-activity relationship to (E)-7-phenyl-2-heptene-4,6-diyn-1-ol revealed that the presence of a hydroxyl group and an aromatic at both ends of the length of the seven-carbon chain is important for the expression of repellency. These results can potentially lead to a new repellent of phenylalkyl alcohol.

[Surgical Technique for Mesorectal Division in the Robotic Anterior Resection for Rectal Cancer].

Since April 2018, robot-assisted rectal resection has been approved as an insurance medical treatment, and robot- assisted rectal resection is rapidly becoming widespread. Even in robot-assisted laparoscopic surgery, mesorectal division is difficult in a narrow pelvic cavity. At the beginning of the operation, Vessel Sealer ExtendTM(price 89,250 yen)was used, but as the procedure became stable, the mesorectal division was started with bipolar forceps and monopolar scissors. The purpose of this study was to investigate the mesorectal division time and postoperative complications associated with changes in the procedure. 36 patients who underwent robot-assisted anterior resection for rectal cancer by the same surgeon from January 2019 to December 2021. We compared mesorectal division time and postoperative complication. Median operation time were 267 minutes, median console time were 132 minutes. There were no complications such as intestinal obstruction or anastomotic leakage. There was no difference in mesorectal division time time between Vessel Sealer groups and Scissors groups(14 min 55 sec vs 16 min 5 sec). The mesorectal division with bipolar forceps and monopolar scissors could be performed without extending the operation time, and could be performed with cost-benefit and safely.

Genetic mapping identifies a rice naringenin O-glucosyltransferase that influences insect resistance.

Naringenin, the biochemical precursor for predominant flavonoids in grasses, provides protection against UV damage, pathogen infection and insect feeding. To identify previously unknown loci influencing naringenin accumulation in rice (Oryza sativa), recombinant inbred lines derived from the Nipponbare and IR64 cultivars were used to map a quantitative trait locus (QTL) for naringenin abundance to a region of 50 genes on rice chromosome 7. Examination of candidate genes in the QTL confidence interval identified four predicted uridine diphosphate-dependent glucosyltransferases (Os07g31960, Os07g32010, Os07g32020 and Os07g32060). In vitro assays demonstrated that one of these genes, Os07g32020 (UGT707A3), encodes a glucosyltransferase that converts naringenin and uridine diphosphate-glucose to naringenin-7-O-ß-d-glucoside. The function of Os07g32020 was verified with CRISPR/Cas9 mutant lines, which accumulated more naringenin and less naringenin-7-O-ß-d-glucoside and apigenin-7-O-ß-d-glucoside than wild-type Nipponbare. Expression of Os12g13800, which encodes a naringenin 7-O-methyltransferase that produces sakuranetin, was elevated in the mutant lines after treatment with methyl jasmonate and insect pests, Spodoptera litura (cotton leafworm), Oxya hyla intricata (rice grasshopper) and Nilaparvata lugens (brown planthopper), leading to a higher accumulation of sakuranetin. Feeding damage from O. hyla intricata and N. lugens was reduced on the Os07g32020 mutant lines relative to Nipponbare. Modification of the Os07g32020 gene could be used to increase the production of naringenin and sakuranetin rice flavonoids in a more targeted manner. These findings may open up new opportunities for selective breeding of this important rice metabolic trait.

[A Case of Robotic Surgery for Local and Peritoneal Recurrence of Colorectal Cancer].

A 35-year-old women with sigmoid cancer(pT4aN1aM0, pStage Ⅲb)underwent laparoscopic sigmoidectomy. She had 8 courses of CapeOX for adjuvant chemotherapy, but follow up CT scan 1 year after the operation detected intraabdominal nodules in anastomotic site and in left lower quadrant of abdomen. After 10 courses of IRIS plus bevacizumab, the both intraabdominal nodules decreased in size. Robot assisted laparoscopic lower anterior resection and laparoscopic disseminated nodule resection were performed. The patient had no postoperative complications and the postoperative course was good. She remains alive without recurrence at 6 months after the second operation.

Natural variation in the expression and catalytic activity of a naringenin 7-O-methyltransferase influences antifungal defenses in diverse rice cultivars.

Phytoalexins play a pivotal role in plant-pathogen interactions. Whereas leaves of rice (Oryza sativa) cultivar Nipponbare predominantly accumulated the phytoalexin sakuranetin after jasmonic acid induction, only very low amounts accumulated in the Kasalath cultivar. Sakuranetin is synthesized from naringenin by naringenin 7-O-methyltransferase (NOMT). Analysis of chromosome segment substitution lines and backcrossed inbred lines suggested that NOMT is the underlying cause of differential phytoalexin accumulation between Nipponbare and Kasalath. Indeed, both NOMT expression and NOMT enzymatic activity are lower in Kasalath than in Nipponbare. We identified a proline to threonine substitution in Kasalath relative to Nipponbare NOMT as the main cause of the lower enzymatic activity. Expanding this analysis to rice cultivars with varying amounts of sakuranetin collected from around the world showed that NOMT induction is correlated with sakuranetin accumulation. In bioassays with Pyricularia oryzae, Gibberella fujikuroi, Bipolaris oryzae, Burkholderia glumae, Xanthomonas oryzae, Erwinia chrysanthemi, Pseudomonas syringae, and Acidovorax avenae, naringenin was more effective against bacterial pathogens and sakuranetin was more effective against fungal pathogens. Therefore, the relative amounts of naringenin and sakuranetin may provide protection against specific pathogen profiles in different rice-growing environments. In a dendrogram of NOMT genes, those from low-sakuranetin-accumulating cultivars formed at least two clusters, only one of which involves the proline to threonine mutation, suggesting that the low sakuranetin chemotype was acquired more than once in cultivated rice. Strains of the wild rice species Oryza rufipogon also exhibited differential sakuranetin accumulation, indicating that this metabolic diversity predates rice domestication.

Isopentylamine is a novel defence compound induced by insect feeding in rice.

Plants produce a broad variety of defensive metabolites to protect themselves against herbivorous insects. Although polyamines have been implicated in various responses to abiotic and biotic stress, there have been no studies focused on amines in response to insect herbivory. By screening for bioactive amines, we identified isopentylamine as a novel type of herbivory-induced compound in rice leaves, which was derived from the amino acid leucine in stable isotope labelling experiments. Accumulation of isopentylamine increased during herbivory by the brown planthopper (Nilaparvata lugens, BPH) and the rice-feeding armyworm (Mythimna loreyi), as well as in response to treatment with the plant hormone, jasmonic acid. Likewise, isopentylamine accumulation was compromised in rice jasmonate biosynthesis mutants, hebiba and Osjar1. In bio-assays, BPH insects feeding on rice seedlings submerged in 50 mg/L isopentylamine solution had a higher mortality compared with BPH feeding on seedlings submerged in water. Notably, the rice leaves submerged in 50 mg/L solution showed the endogenous concentrations of isopentylamine similar to that induced by BPHs. These results suggest that isopentylamine functions as a new type of plant defence metabolite that is rapidly induced by herbivore attack and deters insect herbivores in rice.

Toward a Systematic Nomenclature for (Neo)Lignanamides.

Phenylalkenoic acid amides, often referred to as phenol amides or hydroxycinnamic acid amides, are bioactive phytochemicals, whose bioactivity can be enhanced by coupling to form dimers or oligomers. Phenylalkenoic acid amides consist of a (hydroxy)cinnamic acid derivative (i.e., the phenylalkenoic acid subunit) linked to an amine-containing compound (i.e., the amine subunit) via an amide bond. The phenylalkenoic acid moiety can undergo oxidative coupling, either catalyzed by oxidative enzymes or due to autoxidation, which leads to the formation of (neo)lignanamides. Dimers described in the literature are often named after the species in which the compound was first discovered; however, the naming of these compounds lacks a systematic approach. We propose a new nomenclature, inspired by the existing system used for hydroxycinnamic acid dimers and lignin. In the proposed systematic nomenclature for (neo)lignanamides, compound names will be composed of three-letter codes and prefixes denoting the subunits, and numbers that indicate the carbon atoms involved in the linkage between the monomeric precursors. The proposed nomenclature is consistent, future-proof, and systematic.

Lemon myrtle extract inhibits lactate production by Streptococcus mutans.

Backhousia citriodora (lemon myrtle) extract has been found to inhibit glucansucrase activity, which plays an important role in biofilm formation by Streptococcus mutans. In addition to glucansucrase, various virulence factors in S. mutans are involved in the initiation of caries. Lactate produced by S. mutans demineralizes the tooth enamel. This study investigated whether lemon myrtle extract can inhibit S. mutans lactate production. Lemon myrtle extract reduced the glycolytic pH drop in S. mutans culture and inhibited lactate production by at least 46%. Ellagic acid, quercetin, hesperetin, and myricetin, major polyphenols in lemon myrtle, reduced the glycolytic pH drop and lactate production, but not lactate dehydrogenase activity. Furthermore, these polyphenols reduced the viable S. mutans cell count. Thus, lemon myrtle extracts may inhibit S. mutans-mediated acidification of the oral cavity, thereby preventing dental caries and tooth decay.

Sugars in an aqueous extract of the spent substrate of the mushroom Hypsizygus marmoreus induce defense responses in rice.

Plant defense responses are activated by various exogenous stimuli. We found that an aqueous extract of spent mushroom substrate used for the cultivation of Hypsizygus marmoreus induced defense responses in rice. Fractionation of the spent mushroom substrate extract indicated that the compounds responsible for this induction were neutral and hydrophilic molecules with molecular weights lower than 3 kDa. Compounds with these characteristics, namely glucose, fructose, and sucrose, were detected in the extract at concentrations of 17.4, 3.3, and 1.6 mM, respectively, and the treatment of rice leaves with these sugars induced defense responses. Furthermore, microarray analysis indicated that the genes involved in defense responses were commonly activated by the treatment of leaves with spent mushroom substrate extract and glucose. These findings indicate that the induction of defense responses by treatment with spent mushroom substrate extract is, at least in part, attributable to the sugar constituents of the extract.

Nucleoside 5'-Phosphoramidates Control the Phenylpropanoid Pathway in Vitis vinifera Suspension-Cultured Cells.

It is known that cells contain various uncommon nucleotides such as dinucleoside polyphosphates (NpnN's) and adenosine 5'-phosphoramidate (NH2-pA) belonging to nucleoside 5'-phosphoramidates (NH2-pNs). Their cellular levels are enzymatically controlled. Some of them are accumulated in cells under stress, and therefore, they could act as signal molecules. Our previous research carried out in Arabidopsis thaliana and grape (Vitis vinifera) showed that NpnN's induced the expression of genes in the phenylpropanoid pathway and favored the accumulation of their products, which protect plants against stress. Moreover, we found that NH2-pA could play a signaling role in Arabidopsis seedlings. Data presented in this paper show that exogenously applied purine (NH2-pA, NH2-pG) and pyrimidine (NH2-pU, NH2-pC) nucleoside 5'-phosphoramidates can modify the expression of genes that control the biosynthesis of both stilbenes and lignin in Vitis vinifera cv. Monastrell suspension-cultured cells. We investigated the expression of genes encoding for phenylalanine ammonia-lyase (PAL1), cinnamate-4-hydroxylase (C4H1), 4-coumarate:coenzyme A ligase (4CL1), chalcone synthase (CHS1), stilbene synthase (STS1), cinnamoyl-coenzyme A:NADP oxidoreductase (CCR2), and cinnamyl alcohol dehydrogenase (CAD1). Each of the tested NH2-pNs also induced the expression of the trans-resveratrol cell membrane transporter VvABCG44 gene and caused the accumulation of trans-resveratrol and trans-piceid in grape cells as well as in the culture medium. NH2-pC, however, evoked the most effective induction of phenylpropanoid pathway genes such as PAL1, C4H1, 4CL1, and STS1. Moreover, this nucleotide also induced at short times the accumulation of N-benzoylputrescine (BenPut), one of the phenylamides that are derivatives of phenylpropanoid and polyamines. The investigated nucleotides did not change either the lignin content or the cell dry weight, nor did they affect the cell viability throughout the experiment. The results suggest that nucleoside 5'-phosphoramidates could be considered as new signaling molecules.

[Passive Orthostatism Using Tilt Table in Critical Patients after Cardiovascular Surgery].

Previously, we performed passive orthostatism using a tilt table for the purpose of early mobilization in intensive care unit patients after cardiovascular surgery. In this study, we introduced VitalGo Total Lift Bed( TLB), which does not require patient-transfer before passive orthostatism, to reduce the burden on patients and medical staff. No obvious adverse events were found throughout the study. In the TLB group, number of medical staff required to perform the passive orthostatism was significantly less compared to the conventional tilt table group.