Heterologous production of novel and rare C30-carotenoids using Planococcus carotenoid biosynthesis genes.

BACKGROUND: Members of the genus Planococcus have been revealed to utilize and degrade solvents such as aromatic hydrocarbons and alkanes, and likely to acquire tolerance to solvents. A yellow marine bacterium Planococcus maritimus strain iso-3 was isolated from an intertidal sediment that looked industrially polluted, from the Clyde estuary in the UK. This bacterium was found to produce a yellow acyclic carotenoid with a basic carbon 30 (C30) structure, which was determined to be methyl 5-glucosyl-5,6-dihydro-4,4'-diapolycopenoate. In the present study, we tried to isolate and identify genes involved in carotenoid biosynthesis from this marine bacterium, and to produce novel or rare C30-carotenoids with anti-oxidative activity in Escherichia coli by combinations of the isolated genes. RESULTS: A carotenoid biosynthesis gene cluster was found out through sequence analysis of the P. maritimus genomic DNA. This cluster consisted of seven carotenoid biosynthesis candidate genes (orf1-7). Then, we isolated the individual genes and analyzed the functions of these genes by expressing them in E. coli. The results indicated that orf2 and orf1 encoded 4,4'-diapophytoene synthase (CrtM) and 4,4'-diapophytoene desaturase (CrtNa), respectively. Furthermore, orf4 and orf5 were revealed to code for hydroxydiaponeurosporene desaturase (CrtNb) and glucosyltransferase (GT), respectively. By utilizing these carotenoid biosynthesis genes, we produced five intermediate C30-carotenoids. Their structural determination showed that two of them were novel compounds, 5-hydroxy-5,6-dihydro-4,4'-diaponeurosporene and 5-glucosyl-5,6-dihydro-4,4'-diapolycopene, and that one rare carotenoid 5-hydroxy-5,6-dihydro-4,4'-diapolycopene is included there. Moderate singlet oxygen-quenching activities were observed in the five C30-carotenoids including the two novel and one rare compounds. CONCLUSIONS: The carotenoid biosynthesis genes from P. maritimus strain iso-3, were isolated and functionally identified. Furthermore, we were able to produce two novel and one rare C30-carotenoids in E. coli, followed by positive evaluations of their singlet oxygen-quenching activities.

Violaxanthin: natural function and occurrence, biosynthesis, and heterologous production.

Violaxanthin is biosynthesized from zeaxanthin with zeaxanthin epoxidase (ZEP) by way of antheraxanthin only in photosynthetic eukaryotes including higher plants and involved in the xanthophyll cycle to eliminate excessive light energy. Violaxanthin and antheraxanthin have commercially been unavailable, in contrast to commercial production of other carotenoids contained in higher plants, e.g., lycopene, ß-carotene, lutein, zeaxanthin, ß-cryptoxanthin, and capsanthin. One of the reasons is considered that resource plants or other resource organisms do not exist for enabling efficient supply of the epoxy-carotenoids, which are expected to be produced through (metabolic) pathway engineering with heterologous microbial hosts such as Escherichia coli and Saccharomyces cerevisiae. In this Mini-Review, we show heterologous production of violaxanthin with the two microorganisms that have exhibited significant advances these days. We further describe natural function and occurrence, and biosynthesis involving violaxanthin, antheraxanthin, and their derivatives that include auroxanthin and mutatoxanthin. KEY POINTS: • A comprehensive review on epoxy-carotenoids violaxanthin and antheraxanthin. • Pathway engineering for the epoxy-carotenoids in heterologous microbes. • Our new findings on violaxanthin production with the budding yeast.

Tapentadol in Cancer Patients with Neuropathic Pain: A Comparison of Methadone, Oxycodone, Fentanyl, and Hydromorphone.

Tapentadol has µ-opioid receptor stimulating and noradrenaline reuptake inhibiting properties, and should be effective for neuropathic pain (NP). However, the efficacy of tapentadol for NP in cancer patients is unclear. Ashiya Municipal Hospital (Hyogo, Japan) enrolled five groups of Japanese cancer patients between January 1, 2013, and December 31, 2019. Patients with NP were administered tapentadol (n = 29), methadone (n = 32), oxycodone (n = 20), fentanyl (n = 26), or hydromorphone (n = 20). The primary endpoint was the difference in the verbal rating scale (VRS) scores between days 0 and 7. The secondary endpoint was the tolerability of each opioid. Before administering opioids among the five groups, there was no significant difference in the VRS score (p = 0.99). The mean reduction in the VRS score on day 7 was significantly greater in the tapentadol group than in the oxycodone group (p = 0.0024) and was larger than that of the methadone, fentanyl, and hydromorphone groups. Regarding safety, the discontinuation rate in the tapentadol group was the lowest of all groups (tapentadol vs. methadone vs. oxycodone vs. fentanyl vs. hydromorphone, 0.0% vs. 6.3% vs. 5.0% vs. 3.8% vs. 10.0%, respectively). This study suggests that tapentadol could be efficacious for cancer patients with NP, and a preferred option in cases that require immediate dose adjustment or for those at high risk for adverse effects. However, the pain intensity was evaluated without pain assessment scales specific to NP. Thus, we think that it is desirable to validate our findings using assessment scales, such as the painDETECT questionnaire in future.

Carotenoid Biosynthesis in Liverworts.

In higher plants, there are many studies on carotenoid biosynthetic pathways and their relevant genes. On the other hand, few researches exist on carotenoid biosynthesis in early-land plants containing liverworts, mosses, and ferns. Thus, the evolutionary history of carotenoid biosynthesis genes in land plants has remained unclear. A liverwort Marchantia polymorpha is thought to be one of the first land plants, since this plant remains a primitive figure. Moreover, this liverwort is regarded as the model plant of bryophytes due to several reasons. In this chapter, we review carotenoid biosynthesis in liverworts and discuss the functional evolution and evolutionary history of carotenogenic genes in land plants.

Carotenoid Biosynthesis in Animals: Case of Arthropods.

All the organisms that belong to the animal kingdom had been believed not to synthesize carotenoids de novo. However, several groups of arthropods, which contain aphids, spider mites, and flies belonging to the family Cecidomyiidae, have been unexpectedly shown to possess carotenoid biosynthesis genes of fungal origin since 2010. On the other hand, few reports have shown direct evidence corroborating the catalytic functions of the enzymes that the carotenogenic genes encode. In the present review, we want to overview the carotenoid biosynthetic pathway of the pea aphid (Acyrthosiphon pisum), which was elucidated through functional analysis of carotenogenic genes that exist on its genome using Escherichia coli that accumulates carotenoid substrates, in addition to carotenoid biosynthesis in the other carotenogenic arthropods.

Pathway engineering for efficient biosynthesis of violaxanthin in Escherichia coli.

Carotenoids are naturally synthesized in some species of bacteria, archaea, and fungi (including yeasts) as well as all photosynthetic organisms. Escherichia coli has been the most popular bacterial host for the heterologous production of a variety of carotenoids, including even xanthophylls unique to photosynthetic eukaryotes such as lutein, antheraxanthin, and violaxanthin. However, conversion efficiency of these epoxy-xanthophylls (antheraxanthin and violaxanthin) from zeaxanthin remained substantially low. We here examined several factors affecting their productivity in E. coli. Two sorts of plasmids were introduced into the bacterial host, i.e., a plasmid to produce zeaxanthin due to the presence of the Pantoea ananatis crtE, crtB, crtI, crtY, and crtZ genes in addition to the Haematococcus pluvialis IDI gene, and one containing each of zeaxanthin epoxidase (ZEP) genes originated from nine photosynthetic eukaryotes. It was consequently found that paprika (Capsicum annuum) ZEP (CaZEP) showed the highest conversion activity. Next, using the CaZEP gene, we performed optimization experiments in relation to E. coli strains as the production hosts, expression vectors, and ribosome-binding site (RBS) sequences. As a result, the highest productivity of violaxanthin (231 µg/g dry weight) was observed, when the pUC18 vector was used with CaZEP preceded by a RBS sequence of score 5000 in strain JM101(DE3).

Production of prostaglandin F2α by molecular breeding of an oleaginous fungus Mortierella alpina.

Cyclooxygenases are responsible for the production of prostaglandin H2 (PGH2) from arachidonic acid. PGH2 can be converted into some bioactive prostaglandins, including prostaglandin F2α (PGF2α), a potent chemical messenger used as a biological regulator in the fields of obstetrics and gynecology. The chemical messenger PGF2α has been industrially produced by chemical synthesis. To develop a biotechnological process, in which PGF2α can be produced by a microorganism, we transformed an oleaginous fungus, Mortierella alpina 1S-4, rich in triacylglycerol consisting of arachidonic acid using a cyclooxygenase gene from a red alga, Gracilaria vermiculophylla. PGF2α was accumulated not only in the mycelia of the transformants but also in the extracellular medium. After 12 days of cultivation approximately 860 ng/g and 6421 µg/L of PGF2α were accumulated in mycelia and the extracellular medium, respectively. The results could facilitate the development of novel fermentative methods for the production of prostanoids using an oleaginous fungus.

Structural and functional analysis of the carotenoid biosynthesis genes of a Pseudomonas strain isolated from the excrement of Autumn Darter.

There are many reports about carotenoid-producing bacteria and carotenoid biosynthesis genes. In databases for Pseudomonas genome sequences, there are genes homologous to carotenoid biosynthesis genes, but the function of these genes in Pseudomonas has not been elucidated. In this study, we cloned the carotenoid biosynthesis genes from a Pseudomonas sp. strain, named Akiakane, which was isolated from the excrement of the Autumn Darter dragonfly. Using an Escherichia coli functional expression system, we confirmed that the idi, crtE, crtB, crtI, and crtY gene products of the Akiakane strain show predictable catalytic activities. A cluster of six genes was also found, which was comparable to other carotenoid-producing bacteria that belong to the α-Proteobacteria or γ-Proteobacteria class.

Pathway engineering for the production of ß-amyrin and cycloartenol in Escherichia coli-a method to biosynthesize plant-derived triterpene skeletons in E. coli.

Cycloartenol is biosynthetically the first sterol skeleton, which is metabolized to phytosterols such as ß-sitosterol and stigmasterol. ß-Amyrin is the most commonly occurring aglycone skeleton for oleanane-type saponins such as glycyrrhizin and saikosaponins. It has been regarded that these cyclic triterpenes are unable to be produced in Escherichia coli, while no reports are available on their production with E. coli. Here, we describe a method to synthesize triterpene skeletons from higher plants, including cycloartenol and ß-amyrin. We introduced into E. coli the biosynthetic pathway genes from farnesyl diphosphate (FPP) to cycloartenol or ß-amyrin, which contained Arabidopsis (Arabidopsis thaliana)-derived squalene synthase (AtSQS) and squalene epoxidase (AtSQE) genes in addition to the Arabidopsis cycloartenol synthase (AtCAS1) gene, or the ß-amyrin synthase (EtAS) gene of the petroleum plant Euphorbia tirucalli, along with the isopentenyl diphosphate isomerase (HpIDI) gene from a green algae Haematococcus pluvialis. The order of genes, HpIDI, AtSQS, AtSQE, driven by transcriptional read-through from a tac promoter to an rrnB terminator, was crucial for their functional expression in E. coli to produce cycloartenol or ß-amyrin. The co-expression of a bacterial NADPH-regenerating gene (zwf or gdh) as well as bacterial redox partner protein genes (camA and camB, or NsRED and NsFER) was found to increase the amounts of these triterpenes several fold. The present study could open up opportunities not only to carry out functional analysis of a higher-plant-derived oxidosqualene cyclase (OSC) gene in E. coli but also to produce functional triterpenes that originate from medicinal or herbal plants.

Carotenoid analysis of sweetpotato Ipomoea batatas and functional identification of its lycopene ß- and ε-cyclase genes.

Sweetpotato Ipomoea batatas is known as a hexaploid species. Here, we analyzed carotenoids contained in the leaves and tubers of sweetpotato cultivars 'White Star' (WS) and W71. These cultivars were found to contain several carotenoids unique to sweetpotato tubers such as ß-carotene-5,6,5',8'-diepoxide and ß-carotene-5,8-epoxide. Next, we isolated two kinds of carotene cyclase genes that encode lycopene ß- and ε-cyclases from the WS and W71 leaves, by RT-PCR and subsequent RACE. Two and three lycopene ß-cyclase gene sequences were, respectively, isolated from WS, named IbLCYb1, 2, and from W71, IbLCYb3, 4, 5. Meanwhile, only a single lycopene ε-cyclase gene sequence, designated IbLCYe, was isolated from both WS and W71. These genes were separately introduced into a lycopene-synthesizing Escherichia coli transformed with the Pantoea ananatis crtE, crtB and crtI genes, followed by HPLC analysis. ß-Carotene was detected in E. coli cells that carried IbLCYb1-4, indicating that the IbLCYb1-4 genes encode lycopene ß-cyclase. Meanwhile, the introduction of IbLCYe into the lycopene-synthesizing E. coli led to efficient production of δ-carotene with a monocyclic ε-ring, providing evidence that the IbLCYe gene codes for lycopene ε-(mono)cyclase. Expression of the ß- and ε-cyclase genes was analyzed as well.

Biosynthetic routes of hydroxylated carotenoids (xanthophylls) in Marchantia polymorpha, and production of novel and rare xanthophylls through pathway engineering in Escherichia coli.

MAIN CONCLUSION: MpBHY codes for a carotene ß-ring 3(,3')-hydroxylase responsible for both zeaxanthin and lutein biosynthesis in liverwort. MpCYP97C functions as an ε-ring hydroxylase (zeinoxanthin 3'-hydroxylase) to produce lutein in liverwort. Xanthophylls are oxygenated or hydroxylated carotenes that are most abundant in the light-harvesting complexes of plants. The plant-type xanthophylls consist of α-xanthophyll (lutein) and ß-xanthophylls (zeaxanthin, antheraxanthin, violaxanthin and neoxanthin). The α-xanthophyll and ß-xanthophylls are derived from α-carotene and ß-carotene by carotene hydroxylase activities, respectively. ß-Ring 3,3'-hydroxylase that mediates the route of zeaxanthin from ß-carotene via ß-cryptoxanthin is present in higher plants and is encoded by the BHY (BCH) gene. On the other hand, CYP97A (or BHY) and CYP97C genes are responsible for ß-ring 3-hydroxylation and ε-ring 3'-hydroxylation, respectively, in routes from α-carotene to lutein. To elucidate the evolution of the biosynthetic routes of such hydroxylated carotenoids from carotenes in land plants, we identified and functionally analyzed carotenoid hydroxylase genes of liverwort Marchantia polymorpha L. Three genes homologous to higher plants, BHY, CYP97A, and CYP97C, were isolated and named MpBHY, MpCYP97A, and MpCYP97C, respectively. MpBHY was found to code for ß-ring hydroxylase, which is responsible for both routes starting from ß-carotene and α-carotene. MpCYP97C functioned as an ε-ring hydroxylase not for α-carotene but for zeinoxanthin, while MpCYP97A showed no hydroxylation activity for ß-carotene or α-carotene. These findings suggest the original functions of the hydroxylation enzymes of carotenes in land plants, which are thought to diversify in higher plants. In addition, we generated recombinant Escherichia coli cells, which produced rare and novel carotenoids such as α-echinenone and 4-ketozeinoxanthin, through pathway engineering using bacterial carotenogenic genes that include crtW, in addition to the liverwort MpLCYb, MpLCYe and MpBHY genes.

Carotenoid analysis of a liverwort Marchantia polymorpha and functional identification of its lycopene ß- and ε-cyclase genes.

Carotenoid biosynthesis in bryophytes has yet to be clarified. The liverwort Marchantia polymorpha L. is known to be an early land plant and is an emerging bryophyte model. In order to gain insight into the evolution of carotenoid biosynthesis in plants, we studied carotenoid biosynthesis in this liverwort. As is the case in higher plants, liverwort thalli contain lutein and ß-carotene, as major carotenoids, as well as zeaxanthin, antheraxanthin, violaxanthin and 9'-cis-neoxanthin. Based on liverwort expressed sequence tag (EST)/cDNA and genome sequences, we isolated two cyclase genes encoding lycopene ß-cyclase (LCYb) and lycopene ε-cyclase (LCYe), which were involved in the synthesis of ß-carotene and α-carotene. These enzymes were phylogenetically positioned between corresponding proteins of a green alga (Chlorophyta) and higher plants. Functional analysis of the two genes was performed using a heterologous Escherichia coli expression system, in which the Pantoea ananatis lycopene biosynthesis genes were co-expressed. The results indicated liverwort LCYb activity for the synthesis of ß-carotene from lycopene, which was the same as that of higher plants. On the other hand, liverwort LCYe was able to form two ε-rings from lycopene to ε-carotene via δ-carotene, which was different from the Arabidopsis LCYe enzyme which generates only one ε-ring from lycopene.

Comparison of the Effects of OPRM1 A118G Polymorphism Using Different Opioids: A Prospective Study.

CONTEXT: µ-opioid receptor gene (OPRM1) A118G polymorphism (rs1799971) causes loss of N-glycosylation sites at the extracellular domain of µ-opioid receptors. G-allele carriers show a limited response to morphine; however, studies investigating the impact of A118G polymorphism on the efficacy of opioids other than morphine are limited. OBJECTIVE: To compare the impact of A118G polymorphism on the efficacy of various opioids. METHODS: This prospective cohort study enrolled 222 in-patients administered one of the following opioid therapies for cancer pain as part of an opioid introduction or rotation strategy: tapentadol extended-release tablets, methadone tablets, hydromorphone controlled-release tablets, oxycodone controlled-release tablets, or transdermal fentanyl patches. The impact of A118G polymorphism on the difference in the Brief Pain Inventory-Short Form score on days three, seven, and 14 from baseline was compared among the groups. RESULTS: Overall, 81, 74, and 67 patients had the AA, AG, and GG genotypes, respectively, with an OPRM1 A118G G-allele variant frequency of 0.47. The reduction in the Brief Pain Inventory-Short Form score after opioid therapy initiation did not differ significantly among the patients with the three A118G genotypes treated with tapentadol (p = 0.84) or methadone (p = 0.97), whereas it was significantly smaller in G-allele carriers than that in AA homozygous patients treated with hydromorphone (p < 0.001), oxycodone (p = 0.031), or fentanyl (p < 0.001). CONCLUSION: Tapentadol and methadone may be more suitable than hydromorphone, oxycodone, and fentanyl for G-allele carriers due to their dual mechanism of action and low susceptibility to OPRM1 A118G polymorphism.

Cleavage of Hsp70.1 causes lysosomal cell death under stress conditions.

Autophagy mediates the degradation of intracellular macromolecules and organelles within lysosomes. There are three types of autophagy: macroautophagy, microautophagy, and chaperone-mediated autophagy. Heat shock protein 70.1 (Hsp70.1) exhibits dual functions as a chaperone protein and a lysosomal membrane stabilizer. Since chaperone-mediated autophagy participates in the recycling of ∼30% cytosolic proteins, its disorder causes cell susceptibility to stress conditions. Cargo proteins destined for degradation such as amyloid precursor protein and tau protein are trafficked by Hsp70.1 from the cytosol into lysosomes. Hsp70.1 is composed of an N-terminal nucleotide-binding domain (NBD) and a C-terminal domain that binds to cargo proteins, termed the substrate-binding domain (SBD). The NBD and SBD are connected by the interdomain linker LL1, which modulates the allosteric structure of Hsp70.1 in response to ADP/ATP binding. After the passage of the Hsp70.1-cargo complex through the lysosomal limiting membrane, high-affinity binding of the positive-charged SBD with negative-charged bis(monoacylglycero)phosphate (BMP) at the internal vesicular membranes activates acid sphingomyelinase to generate ceramide for stabilizing lysosomal membranes. As the integrity of the lysosomal limiting membrane is critical to ensure cargo protein degradation within the acidic lumen, the disintegration of the lysosomal limiting membrane is lethal to cells. After the intake of high-fat diets, however, ß-oxidation of fatty acids in the mitochondria generates reactive oxygen species, which enhance the oxidation of membrane linoleic acids to produce 4-hydroxy-2-nonenal (4-HNE). In addition, 4-HNE is produced during the heating of linoleic acid-rich vegetable oils and incorporated into the body via deep-fried foods. This endogenous and exogenous 4-HNE synergically causes an increase in its serum and organ levels to induce carbonylation of Hsp70.1 at Arg469, which facilitates its conformational change and access of activated µ-calpain to LL1. Therefore, the cleavage of Hsp70.1 occurs prior to its influx into the lysosomal lumen, which leads to lysosomal membrane permeabilization/rupture. The resultant leakage of cathepsins is responsible for lysosomal cell death, which would be one of the causative factors of lifestyle-related diseases.

Bioproduction of prostaglandins in a transgenic liverwort, Marchantia polymorpha.

Prostaglandins are biologically active substances used in a wide range of medical treatments. Prostaglandins have been supplied mainly by chemical synthesis; nevertheless, the high cost of prostaglandin production remains a factor. To lower the cost of prostaglandin production, we attempted to produce prostaglandins using a liverwort, Marchantia polymorpha L., which accumulates arachidonic acid, which is known as a substrate of prostaglandins. Here we report the first bioproduction of prostaglandins in plant species by introducing a cyclooxygenase gene from a red alga, Gracilaria vermiculophylla into the liverwort. The transgenic liverworts accumulated prostaglandin F2α, prostaglandin E2 and prostaglandin D2 which were not detected in the wild-type liverwort. Moreover, we succeeded in drastically increasing the bioproduction of prostaglandins using an in vitro reaction system with the extracts of transgenic liverworts.

Reconstruction of the Native Biosynthetic System of Carotenoids in E. coli─Biosynthesis of a Series of Carotenoids Specific to Paprika Fruit.

Capsanthin, capsorubin, cucurbitaxanthin A, and capsanthin 3,6-epoxide, a series of carotenoids specific to the red fruit of paprika (Capsicum annuum), were produced in pathway-engineered Escherichia coli cells. These cells functionally expressed multiple genes for eight carotenogenic enzymes, two of which, paprika capsanthin/capsorubin synthase (CaCCS) and zeaxanthin epoxidase (CaZEP), were designed to be located adjacently. The biosynthesis of these carotenoids, except for capsanthin, was the first successful attempt in E. coli. In a previous study, the levels of capsanthin synthesized were low despite the high expression of the CaCCS gene, which may have been due to the dual activity of CaCCS as a lycopene ß-cyclase and CCS. An enhanced interaction between CaCCS and CaZEP that supplies antheraxanthin and violaxanthin, substrates for CaCCS, was considered to be crucial for an efficient reaction. To achieve this, we adapted S·tag and S-protein binding. The S·tag Thrombin Purification Kit (Novagen) is merchandized for in vitro affinity purification, and S·tag-fused proteins in the E. coli lysate are specifically trapped by S-proteins fixed on the agarose carrier. Furthermore, S-proteins have been reported to oligomerize via C-terminal swapping. In the present study, CaCCS and CaZEP were individually fused to the S·tag and designed to interact on oligomerized S-protein scaffolds in E. coli, which led to the biosynthesis of not only capsanthin and capsorubin but also cucurbitaxanthin A and capsanthin 3,6-epoxide. The latter reaction by CaCCS was assigned for the first time. This approach reinforces the scaffold's importance for multienzyme pathways when native biosynthetic systems are reconstructed in microorganisms.

Identification of Factors Contributing to Methadone-Induced Daytime Sleepiness in Cancer Patients and Proposal of the Conversion Ratio from Other Opioids to Oral Methadone: A Retrospective Cohort Study.

Background: When methadone is used to treat cancer pain, the Japanese health insurance system recommends to determine the starting dose according to the equivalency conversion table based on the morphine-equivalent daily dose (MEDD) of prior opioids proposed by the National Comprehensive Cancer Network. Owing to the wide range in variability of the conversion table, methadone increases the incidence of daytime sleepiness. Objective: To identify the factors associated with daytime sleepiness and propose a conversion ratio from pretreatment MEDD to oral methadone that decreases the risk of daytime sleepiness. Design: Retrospective cohort study. Setting/Subjects: One hundred patients who started oral methadone to relieve cancer pain at Ashiya Municipal Hospital (Hyogo, Japan) from January 1, 2013, to August 31, 2022, were enrolled. Measurements: The primary endpoint, the conversion ratio from pretreatment MEDD to oral methadone without daytime sleepiness, was determined using receiver operator characteristic (ROC) curve analysis. Results: The incidence of daytime sleepiness within seven days of methadone initiation was 40.0%. The factors identified as contributing to daytime sleepiness were pretreatment MEDD (odds ratio [OR]: 0.941, 95% confidence interval [CI]: 0.916-0.966, p <0.001) and methadone dose (OR: 1.395, 95% CI: 1.178-1.652, p <0.001). The conversion ratio from pretreatment MEDD to oral methadone was 0.24, with an area under the ROC curve of 0.909 (p <0.001). Conclusions: Daytime sleepiness developed when methadone dose is high relative to pretreatment MEDD. To the best of our knowledge, this is the first study to suggest the conversion ratio from pretreatment MEDD to oral methadone without causing daytime sleepiness.

Synthetic-biological approach for production of neoxanthin in Escherichia coli.

Carotenoids are isoprenoid pigments produced typically in plants, algae, and part of bacteria and fungi. Violaxanthin, neoxanthin, and lutein are xanthophylls biosynthesized specifically in land plants and part of algae. Nowadays, it is feasible to produce violaxanthin and lutein in Escherichia coli by pathway engineering, whereas there is no report to synthesize neoxanthin in E. coli. So far, several genes have been reported to code for neoxanthin synthases, e.g., NSY (NXS), ABA4 and VDL, which were assigned to catalyze a reaction for forming neoxanthin from violaxanthin. However, neither gene of these was common in plants or algae that biosynthesize neoxanthin, nor was confirmed by the E. coli complementation system. This study showed that the algal VDL gene (PtVDL1) was functional in recombinant E. coli cells accumulating violaxanthin to produce neoxanthin, whereas the E. coli cells failed to generate neoxanthin, when the NSY or ABA4 gene was introduced there instead of VDL. This result notes that VDL is one of veritable neoxanthin synthase genes.

Differences in the Analgesic Effect of Opioids on Pain in Cancer Patients With Spinal Metastases.

Background: Spinal metastasis pain includes both inflammatory and neuropathic pain, and opioids, which have only a µ-opioid receptor-stimulating effect, are generally less effective in neuropathic pain. However, no previous study has been conducted for the comparisons of the efficacy of opioids in treating spinal metastasis pain. Objective: To compare the efficacy of tapentadol and methadone with other opioids for back pain caused by a metastatic spinal tumor. Design: Retrospective cohort study. Setting/Subjects: A total of 274 patients were enrolled, who started a tapentadol extended-release tablet, methadone tablet, hydromorphone extended-release tablet, oxycodone extended-release tablet, or transdermal fentanyl patch for cancer pain due to spinal metastasis in Japan from January 1, 2013 to October 31, 2021. Measurements: The primary endpoint, the difference in the numerical rating scale (NRS) scores before and seven days after each opioid administration, was compared among the five groups. Results: In patients with numbness, a decrease of the NRS score on day seven compared with before starting each opioid was significantly higher in the tapentadol group than those in the hydromorphone, oxycodone, and fentanyl groups and comparable to that in the methadone group. In patients without numbness, no significant differences were observed in decreases of the NRS scores on day seven among the five groups. Conclusions: Tapentadol and methadone may be more effective than hydromorphone, oxycodone, and fentanyl for cancer pain due to spinal metastasis with numbness.

Cold-induced accumulation of ω-3 polyunsaturated fatty acid in a liverwort, Marchantia polymorpha L.

The liverwort Marchantia polymorpha L. synthesizes various long-chain polyunsaturated fatty acids including arachidonic acid and eicosapentaenoic acid, neither of which is produced by higher plants. Here we report the effects of temperature on long-chain polyunsaturated fatty acid accumulation in the liverwort. The accumulation of ω-3 polyunsaturated fatty acids increased significantly as the growth temperature decreased. Specifically, the relative content of eicosapentaenoic acid to total fatty acids at 5 °C was approximately 3-fold higher than at 25 °C. On the other hand, the accumulation of ω-6 polyunsaturated fatty acids decreased at low temperatures. An analysis of gene expression indicated that the mRNA of the MpFAD3 gene for ER ω-3 desaturase increased significantly at 5 °C. These results indicate that in the liverwort the n-3 pathway was enhanced at low temperature, mainly via expression of the cold-induced ω-3 desaturase gene, leading to increased accumulation of eicosapentaenoic acid.