Clinical outcomes of scalp or face angiosarcoma treatment with intensity-modulated radiotherapy: a multicenter study.

Combined modality therapy, including radiotherapy (RT), is a common treatment for scalp or face angiosarcoma. Although intensity-modulated radiotherapy (IMRT) can deliver homogeneous doses to the scalp or face, clinical data are limited. This multicenter study aimed to evaluate scalp or face angiosarcoma treated with definitive or post-operative IMRT. We retrospectively analyzed data from patients who received IMRT for scalp or face angiosarcoma at three institutions between January 2015 and March 2020. Local control (LC) rate, overall survival (OS), progression-free survival (PFS), recurrence patterns and toxicity were evaluated. Fifteen patients underwent IMRT during the study period. Definitive RT was performed on 10 patients and post-operative RT was performed on 5 patients. The 1-year LC rate was 85.7% (95% confidence interval [CI], 53.9-96.2%). The 1-year OS and PFS rates were 66.7% (95% CI, 37.5-84.6%) and 53.3% (95% CI, 26.3%-74.4%), respectively. Univariate analysis revealed that a clinical target volume over 500 cm3 was associated with poor LC. Distant metastasis was the most common recurrence pattern. All patients experienced Grade 2 or 3 radiation dermatitis, and five patients experienced grade ≥ 3 skin ulceration. One patient who underwent maintenance therapy with pazopanib developed Grade 5 skin ulceration. Fisher's exact test showed that post-operative RT was significantly associated with an increased risk of skin ulceration of grade ≥ 3. These results demonstrate that IMRT is a feasible and effective treatment for scalp or face angiosarcoma, although skin ulceration of grade ≥ 3 is a common adverse event in patients who receive post-operative RT.

In vitro metabolic profiling of new synthetic cannabinoids, ADB-FUBIATA, AFUBIATA, CH-FUBIATA, and CH-PIATA.

In the recreational drug market, synthetic cannabinoids with a new acetamide linker structure emerged, most likely to circumvent the law. As the knowledge of drug metabolites is vital for proving drug consumption, the phase I metabolism of the newly emerging cannabinoids, ADB-FUBIATA, AFUBIATA, CH-FUBIATA, and CH-PIATA, was investigated. Each drug (10 µmol/L) was incubated with human liver microsomes for 1 h, and the samples, after dilution, were analyzed by liquid chromatography-high-resolution mass spectrometry. All drugs were metabolized via hydroxylation and N-dealkylation, while AFUBIATA and CH-PIATA additionally underwent ketone formation. The metabolites AF7 (hydroxylated at the indole/adjacent methylene) of ADB-FUBIATA, A16 (hydroxylated at the adamantane) of AFUBIATA, CF15 (hydroxylated at the cyclohexane) of CH-FUBIATA, and CP9 (hydroxylated at the pentane) of CH-PIATA were the most abundant metabolites by considering the peak areas on the chromatograms, and are recommended for urinalysis. The structure-metabolism relationship was also discussed, which generally agreed well with previously reported metabolic pathways of other synthetic cannabinoids. However, the preferred hydroxylation site of ADB-FUBIATA, the indole/adjacent methylene, clearly differed from that of ADB-FUBICA, the 3,3-dimethylbutanamide moiety, despite their structures differing only by a methylene group, emphasizing that metabolic predictions of new drugs should not replace in vitro experimental analyses, albeit helpful.

Impact of planning organ at risk volume margins and matching method on late gastrointestinal toxicity in moderately hypofractionated IMRT for locally advanced pancreatic ductal adenocarcinoma.

BACKGROUND: This study examined the differences in late gastrointestinal (GI) toxicities in moderately hypofractionated intensity-modulated radiation therapy (IMRT) for locally advanced pancreatic ductal adenocarcinoma (LA-PDAC) by changing the planning organs at risk volume (PRV) margin and the target matching method and assessed the causes of adverse events. METHODS: We examined 37 patients with LA-PDAC who underwent moderately hypofractionated IMRT between 2016 and 2020 at our institution; 23 patients were treated with wide PRV margins and soft tissue matching (Protocol A) and 14 with narrow PRV margins and fiducial marker matching (Protocol B). The GI toxicities, local control (LC) rate, and overall survival (OS) were assessed for each protocol. The initially planned and daily doses to the gross tumor volume (GTV), stomach, and duodenum, reproduced from cone-beam computed tomography, were evaluated. RESULTS: The late GI toxicity rate of grades 3-4 was higher in Protocol B (42.9%) than in Protocol A (4.3%). Although the 2-year LC rates were significantly higher in Protocol B (90.0%) than in Protocol A (33.3%), no significant difference was observed in OS rates. In the initial plan, no deviations were found for the stomach and duodenum from the dose constraints in either protocol. In contrast, daily dose evaluation for the stomach to duodenal bulb revealed that the frequency of deviation of V3 Gy per session was 44.8% in Protocol B, which was significantly higher than the 24.3% in Protocol A. CONCLUSIONS: Reducing PRV margins with fiducial marker matching increased GI toxicities in exchange for improved LC. Daily dose analysis indicated the trade-off between the GTV dose coverage and the irradiated doses to the GI. This study showed that even with strict matching methods, the PRV margin could not be reduced safely because of GI inter-fractional error, which is expected to be resolved with online adaptive radiotherapy.

Systematic In Vitro Metabolic Profiling of the OXIZID Synthetic Cannabinoids BZO-4en-POXIZID, BZO-POXIZID, 5F-BZO-POXIZID, BZO-HEXOXIZID and BZO-CHMOXIZID.

A new class of synthetic cannabinoids termed OXIZIDs has recently emerged on the recreational drug market. In order to continue the detection of new drugs in biological specimens, the identification of metabolites is essential. The aim of this study was to elucidate the metabolites of BZO-4en-POXIZID produced in human liver microsomes (HLMs) and human hepatocyte incubations and to compare the results with closely related analogs using the same experimental setup. Each drug was incubated for 1 h in HLM and BZO-4en-POXIZID was also incubated in human hepatocytes for up to 3 h. Subsequently, the incubates were analyzed by liquid chromatography-high-resolution mass spectrometry. BZO-4en-POXIZID metabolites were obtained in the incubation with HLMs and human hepatocytes, via the metabolic pathways of dihydrodiol formation, hydroxylation, reduction of the alkene bond and glucuronidation. The major metabolic pathway was found to be dihydrodiol formation at the pentenyl tail moiety. BZO-POXIZID, 5 F-BZO-POXIZID, BZO-HEXOXIZID and BZO-CHMOXIZID underwent similar metabolism to those reported in the literature, via the metabolic pathways of N-dealkylation, hydroxylation, ketone formation and oxidative defluorination (to alcohol or carboxylic acid). The results suggest that OXIZIDs are mainly metabolized at the N-alkyl moiety and the major metabolic pathways are hydroxylation when the N-alkyl moiety is a simple hydrocarbon, whereas functional-group-specific pathways (dihydrodiol formation and oxidative defluorination) are preferred when the moiety contains specific functional groups (alkene or fluoro), as has been observed for other synthetic cannabinoids. The major metabolites generated via these major metabolic pathways should serve as useful analytical targets for urine analysis. Furthermore, the higher abundance of glucuronidated metabolite suggests that enzymatic hydrolysis of glucuronides may be necessary for urine analysis to increase phase I metabolite concentration and improve detection.

Forensic Discrimination of Drug Powder Based on Drug Mixing Condition Determined Using Micro Fourier Transform Infrared Spectroscopy.

The quantitative evaluation of the drug mixing condition was conducted for application in the forensic discrimination of drug powders using micro Fourier transform infrared (FT-IR) spectroscopy. Bromhexine hydrochloride (BHCl) and p-hydroxybenzoic acid (PHBA) were used as the simulated drug and additive, respectively. Equal masses of two chemicals were (1) simply mixed, (2) homogenized using agate mortar, or (3) dissolved in methanol and dried, and then (4) homogenized using agate mortar. The mixed powders dispersed on BaF2 plates were subjected to mapping analysis of micro FT-IR spectroscopy using synchrotron radiation (SR) or globar light in transmission mode with aperture sizes of 2.5 x 2.5 and 10 x 10µm2, and x-y scanning steps of 2.5 and 10 µm, respectively. The areas of the vibration bands specific to BHCl (C-N bending) and PHBA (C=O stretching) were converted to the molar contents (CBHCl, CPHBA), and the relative content ratio (RCR: CPHBA/[CBHCl + CPHBA]) was used as one mixing parameter. The resulting two-dimensional distribution map provided the relative spatial localizations of the two species, and frequency histograms with a horizontal axis of RCR were plotted to evaluate the RCR distribution. The percentage frequency of the extreme value in which RCR was 0 or 1 (%EV) was used as one mixing index. After excluding the extreme values, the coefficient of variation (CV) of the RCR distribution was used as another mixing index. The differentiation among four mixing modes could be evaluated from the standpoint of %EV and CV, and the discrimination capacity by SR instrument was superior to that by globe light instrument.

Perianal Bowen's disease treated with radiotherapy preserving anal function with a unique skin reaction considered as 'tumoritis'.

Bowen's disease (BD) is a form of intraepidermal squamous cell carcinoma (SCC), and it occasionally occurs on the perianal site. BD is often treated with surgical excision; however, sometimes surgical excision for perianal BD cannot preserve anal function. We report the case of a 72-year-old man presenting with perianal pain and BD. He was treated with Radiotherapy (RT) and preserved his normal anal sphincter function without any recurrence or late adverse event. Moreover, we observed the unique skin reaction called 'tumoritis', which is characterized by mucosal inflammation. Tumoritis indicates the true extent of the tumor and evaluating the tumor or lesion size based on the extent of tumoritis when performing RT is important.

Comparison between human liver microsomes and the fungus Cunninghamella elegans for biotransformation of the synthetic cannabinoid JWH-424 having a bromo-naphthyl moiety analysed by high-resolution mass spectrometry.

PURPOSE: JWH-424, (8-bromo-1-naphthyl)(1-pentyl-1H-indol-3-yl)methanone, is a synthetic cannabinoid, which is a brominated analogue of JWH-018, one of the best-known synthetic cannabinoids. Despite the structural similarity to JWH-018, little is known about JWH-424 including its metabolism. The aim of the study was to compare human liver microsomes (HLM) and the fungus Cunninghamella elegans as the metabolism catalysts for JWH-424 to better understand the characteristic actions of the fungus in the synthetic cannabinoid metabolism. METHODS: JWH-424 was incubated with HLM for 1 h and Cunninghamella elegans for up to 72 h. The HLM incubation mixtures were diluted with methanol and fungal incubation mixtures were extracted with dichloromethane and reconstituted in methanol before analyses by liquid chromatography-high-resolution mass spectrometry (LC-HRMS). RESULTS: HLM incubation resulted in production of ten metabolites through dihydrodiol formation, hydroxylation, and/or ipso substitution of the bromine with a hydroxy group. Fungal incubation led to production of 23 metabolites through carboxylation, dihydrodiol formation, hydroxylation, ketone formation, glucosidation and/or sulfation. CONCLUSIONS: Generally, HLM models give good predictions of human metabolites and structural analogues are metabolised in a similar fashion. However, major hydroxy metabolites produced by HLM were those hydroxylated at naphthalene instead of pentyl moiety, the major site of hydroxylation for JWH-018. Fungal metabolites, on the other hand, had undergone hydroxylation mainly at pentyl moiety. The metabolic disagreement suggests the necessity to verify the human metabolites in authentic urine samples, while H9 and H10 (hydroxynaphthalene), H8 (ipso substitution), F22 (hydroxypentyl), and F17 (dihydroxypentyl) are recommended for monitoring of JWH-424 in urinalysis.

Effects of Machine Washing on the Chromatography Parameters of Polyester Fiber Gel Permeation.

Fiber examination is frequently performed in forensics, and gel permeation chromatography (GPC) is one candidate method for discriminating polyester fibers. Here, the effects of machine washing on weight-average molecular weight (M w), polydispersity index (PDI), and the percentage peak area of cyclic ethylene terephthalate trimer (PPAL) of commercial polyester shirts and manufactured poly(ethylene terephthalate) (PET) yarns were investigated using GPC. GPC was performed using a 1,1,1,3,3,3-hexafluoro-propan-2-ol polymer solubilizer, styrene-divinylbenzene copolymer GPC columns, a chloroform mobile phase, and a 254 nm absorbance monitor. The statistical change in the polyester fibers during machine washing was evaluated by comparing three GPC parameters of the same fiber samples before and after machine washing. Among the commercial polyester shirts examined, the GPC parameters changed significantly after machine washing with a considerable PPAL decrease. In contrast, the GPC parameters of manufactured PET yarns changed significantly with a moderate increase in M w. This work elucidates the change on GPC parameters of polyester fibers by machine washing.

Concurrent Chemoradiotherapy With Weekly Low-Dose Cisplatin for Japanese Patients With Head and Neck Squamous Cell Carcinoma.

BACKGROUND: Concurrent chemoradiotherapy (CCRT) with tri-weekly high-dose cisplatin (HDC) is considered the standard regimen. However, due to significant toxicity, various weekly low-dose schedules have been increasingly used. We investigated the tolerability and survival of patients with head and neck squamous cell carcinoma (HNSCC) who underwent CCRT with low-dose weekly cisplatin (LDC) for Japanese population. METHODS: A retrospective review was conducted among patients with HNSCC who were treated with CCRT/LDC in our institute. Ninety-five patients who met the criteria were enrolled in this study. We evaluated the cycle and cumulative cisplatin dose, completion rate of radiotherapy, adverse events, and survival outcome. RESULTS: The mean cycles and cumulative cisplatin dose were 4.7 cycles and 187 mg/m2. All patients completed planned dose of radiation without prolonged breaks. Leucopoenia was the most frequent dose-limiting factor and 44% patients developed grade 3 or 4 toxicity. The 2-year overall survival and recurrence-free survival were 93% and 74%, respectively. The significant differences of survival outcomes between the patients with total cisplatin dose (⩾200 mg and <200 mg) or among age distribution (35-55, 56-75, and ⩾76) were not observed. CONCLUSIONS: Concurrent chemoradiotherapy/LDC can be safely administered with acceptable toxicity and survival outcome even if the patients with higher age, lower eGFR, and so on.

Evaluation of covalent binding of flutamide and its risk assessment using 19F-NMR.

The formation of reactive metabolites (RMs) is a problem in drug development that sometimes results in severe hepatotoxicity. As detecting RMs themselves is difficult, a covalent binding assay using expensive radiolabelled tracers is usually performed for candidate selection. This study aimed to provide a practical approach toward the risk assessment of hepatotoxicity induced by covalent binding before candidate selection. We focused on flutamide because it contains a trifluoromethyl group that shows a strong singlet peak by 19F nuclear magnetic resonance (NMR) spectrometry. The covalent binding of flutamide was evaluated using quantitative NMR and its risk for hepatotoxicity was assessed by estimating the RM burden, an index that reflects the body burden associated with RM exposure by determining the extent of covalent binding, clinical dose and in vivo clearance. The extent of covalent binding and RM burden was 296 pmol/mg/h and 37.9 mg/day, respectively. Flutamide was categorised as high risk with an RM burden >10 mg/day consistent with its clinical hepatotoxicity. These results indicate that a combination of covalent binding assay using 19F-NMR and RM burden is useful for the risk assessment of RMs without using radiolabelled compounds.

Speciation analysis of Gadolinium-based contrast agents using aqueous eluent-hydrophilic interaction liquid chromatography hyphenated with inductively coupled plasma-mass spectrometry.

Gadolinium chelates are widely used as contrast agents for magnetic resonance imaging (MRI). In recent decades, the amount of Gd in river water has been increasing owing to the input of Gd-based contrast agents. To identify and quantify the Gd-based contrast agents in river water, the novel technique of hydrophilic interaction liquid chromatography (HILIC) hyphenated with inductively coupled plasma-mass spectrometry (ICP-MS) was developed. To avoid deposition of carbon on the ICP-MS interface, a mobile phase consisting of an ammonium acetate buffer diluted with pure water was applied to separate Gd-based contrast agents. Despite the absence of an organic solvent in the mobile phase, six Gd-based contrast agents, Gd-DTPA, Gd-EOB-DTPA, Gd-DOTA, Gd-DTPA-BMA, Gd-BT-DO3A, and Gd-HP-DO3A, were successfully separated. This technique was applied for river water samples. As a result, Gd-DOTA, Gd-BT-DO3A, and Gd-HP-DO3A were observed from the sample near the outfall of a wastewater treatment plant (WWTP), indicating that at least some of the Gd-based contrast agents are passed through treatment in a WWTP. In addition to Gd-based contrast agents, unidentified Gd compounds were found to be present in river water. These results infer that transformation and/or dissociation of Gd chelates may be caused during the treatment procedure in a WWTP.

Hypofractionated intensity-modulated radiotherapy with concurrent chemotherapy for elderly patients with locally advanced pancreatic carcinoma.

BACKGROUND: It is important to understand how elderly patients with locally advanced pancreatic carcinoma (LAPC) should be treated, since the number of elderly cancer patients will increase. However, the optimal treatment for elderly patients with LAPC remains unclear. The purpose of this study was to evaluate the efficacy and safety of hypofractionated intensity-modulated radiotherapy (IMRT) with concurrent gemcitabine for elderly patients with LAPC. METHODS: We retrospectively analysed the data from LAPC patients aged ≥ 75 years treated with hypofractionated IMRT (48 Gy in 15 fractions) with concurrent weekly gemcitabine at our institution from February 2013 to December 2018. Overall survival (OS), progression-free survival (PFS), locoregional progression-free survival (LRPFS), distant metastasis-free survival (DMFS), and the pattern of recurrence and toxicity were analysed. RESULTS: Fifteen patients received treatment during the study period. The median age was 78 years (range 75-86 years), and the Eastern Cooperative Oncology Group (ECOG) performance status (PS) of all patients was 0-1. The median survival time (MST) and median PFS were 20.4 [95% confidence interval (CI) 10.3-36.8] and 13.5 (95% CI 6.4-20.3) months, respectively, and the 1-year OS and PFS rates were 80.0% (95% CI 50-93.1%) and 66.7% (95% CI 37.5-84.6%), respectively. The median LRPFS and median DMFS were 15.6 (95% CI 6.4-36.8) and 14.9 (95% CI 7.0-20.5) months, respectively, and the 1-year LRPFS and DMFS rates were 73.3% (95% CI 43.6-89.1%) and 66.7% (95% CI 37.5-84.6%), respectively. Non-haematologic grade 3 toxicity was observed in three cases, of which only one was induced by radiotherapy, whereas grade 4-5 non-haematologic acute or late toxicities were not observed. CONCLUSIONS: The OS and PFS of elderly patients with LAPC treated using hypofractionated IMRT with concurrent gemcitabine were favourable and without the occurrence of severe toxicity. This treatment strategy is feasible and promising for elderly LAPC patients with good PS.

Therapy-Related Acute Myeloid Leukemia 2 Months after Chemoradiotherapy for Esophageal Cancer: A Case Report.

Therapy-related acute myeloid leukemia (AML) is a rare but potentially fatal adverse event caused by chemotherapy or radiotherapy. Herein we report a patient diagnosed with therapy-related AML 2 months after chemoradiotherapy for esophageal cancer. A 61-year-old man with dysphagia was diagnosed with locally advanced esophageal cancer with para-aortic lymph node metastasis. Laboratory blood test did not reveal any abnormality except mild macrocytic anemia. To alleviate dysphagia due to malignant esophageal stenosis, the patient underwent concurrent chemoradiotherapy of 60 Gy in 30 fractions with cisplatin and 5-fluorouracil at a local area in thoracic esophagus. Dysphagia alleviated during chemoradiotherapy; however, pancytopenia did not recover after the completion of chemoradiotherapy, and general fatigue with fever developed 13 weeks after the last day of chemoradiotherapy. To rule out hematological malignancy, bone marrow biopsy was performed. The bone marrow smear and flow cytometry analysis indicated the development of AML. Chromosomal test revealed a complex karyotype, suggesting that AML was associated with myelodysplastic syndrome. The patient died 1 month after the diagnosis of therapy-related AML. Thus, the findings indicate that therapy-related AML may develop during the acute phase of chemoradiotherapy and bone marrow biopsy is necessary when prolonged pancytopenia exists after chemoradiotherapy.

Simultaneous speciation analysis of inorganic arsenic and methylmercury in edible oil by high-performance liquid chromatography-inductively coupled plasma mass spectrometry.

A simultaneous speciation method of arsenic and mercury species by inductively coupled plasma mass spectrometry with a reversed-phase C18 ODS column was developed. The separation of inorganic arsenic (iAs) from As species and inorganic mercury (iHg) from methylmercury (MeHg) were achieved by HPLC with a single mobile phase containing ion-pair reagent and l-cysteine. The limits of detection of iAs and MeHg were 0.05 ng g-1 as As and 0.09 ng g-1 as Hg, respectively. The simultaneous extraction, i-As and MeHg from in edible oil were achieved using 2% (w/w) tetramethylammonium hydroxide (TMAH) solution. The proposed method was successfully applied to the food matrix type CRMs. When the simultaneous speciation was applied to several kinds of edible oil, iAs and MeHg were founded in the concentration range of 0.001 mg kg-1 to 0.010 mg kg-1 and 1.21 ng g-1 to 10.18 ng g-1, respectively.

A Method for Methylmercury and Inorganic Mercury in Biological Samples Using High Performance Liquid Chromatography-Inductively Coupled Plasma Mass Spectrometry.

A new determination method was developed for the measurement of methylmercury (Me-Hg) and inorganic mercury (i-Hg) in biological samples using high-performance liquid chromatography-inductively coupled plasma-mass spectrometry (HPLC-ICP-MS) following alkaline extraction. Mercury species in biological samples were extracted with 10% (w/w) tetramethylammonium hydroxide (TMAH) solution at 80°C for 2 h. Methylmercury was completely separated from i-Hg by adamantyl type and octadecylsilyl type columns within 6 and 4 min using isocratic elution, respectively. The detection limits (3σ) of adamantyl and octadecylsilyl columns using the proposed system were 0.08 and 0.13 ng g-1 (as Hg), respectively. Inorganic Hg completely separates from Me-Hg without tailing. The proposed determination methods were applied to several biological certified reference materials (CRMs). The measurement results of Me-Hg obtained by the present method were in good agreement within the expanded uncertainties (k = 2) with the certified values. The analytical precision (n = 3) of Me-Hg was less than 2%, and the recoveries of Me-Hg and i-Hg were 101 ± 1 and 103 ± 3%, respectively. In addition, this method enables the determination of Me-Hg and i-Hg for 20 samples in 1 h.

Determination of Inorganic Arsenic in Grape Products Using HPLC-ICP-MS.

The inorganic arsenic (i-As) in grape products, in particular juice, wine and raisins, collected from the Japanese market was investigated. The concentrations of total As in nine grape concentrated juices ranged from 3 to 20 ng g-1, and more than 80% of the As was inorganic according to the results of speciation by HPLC-ICP-MS. Among them, four samples contained more than 10 ng g-1 of i-As, although 10 ng g-1 of i-As is the limit for apple juice recommended by the U.S. Food and Drug Administration. Moreover, more than 10 ng g-1 of i-As was found in some of the wine and raisin samples, with the total As concentrations ranging from 17 to 37 ng g-1. When fresh grapes were analyzed, As was mainly concentrated in the pericarp and a small amount was found in the fruit, although no As was observed in the branches and the juice. When grapes, including the pericarp, are processed, the As concentration in the products may increase during production processes such as drying, concentration, and ripening.

Crystalline-Sponge-Based Structural Analysis of Crude Natural Product Extracts.

Characterization of complex natural product mixtures to the absolute structural level of their components often requires significant amounts of starting materials and lengthy purification process, followed by arduous structure elucidation efforts. The crystalline sponge (CS) method has demonstrated utility in the absolute structure elucidation of isolated organic compounds at miniscule quantities compared to conventional methods. In this work, we developed a new CS-based workflow that greatly expedites the in-depth structural analysis of crude natural product extracts. Using a crude extract of the red alga Laurencia pacifica, we showed that CS affinity screening prior to compound isolation enables prioritization of analytes present in the extract, and we subsequently resolved the molecular structures of six sesquiterpenes with stereochemical clarity from around 10 mg crude extract. This study demonstrates a new chemotyping workflow that can greatly accelerate natural product discovery from complex samples.

Development of a Dual Plasma Desorption/Ionization System for the Noncontact and Highly Sensitive Analysis of Surface Adhesive Compounds.

We developed a dual plasma desorption/ionization system using two plasmas for the semi-invasive analysis of compounds on heat-sensitive substrates such as skin. The first plasma was used for the desorption of the surface compounds, whereas the second was used for the ionization of the desorbed compounds. Using the two plasmas, each process can be optimized individually. A successful analysis of phenyl salicylate and 2-isopropylpyridine was achieved using the developed system. Furthermore, we showed that it was possible to detect the mass signals derived from a sample even at a distance 50 times greater than the distance from the position at which the samples were detached. In addition, to increase the intensity of the mass signal, 0%-0.02% (v/v) of hydrogen gas was added to the base gas generated in the ionizing plasma. We found that by optimizing the gas flow rate through the addition of a small amount of hydrogen gas, it was possible to obtain the intensity of the mass signal that was 45-824 times greater than that obtained without the addition of hydrogen gas.

A Red Algal Bourbonane Sesquiterpene Synthase Defined by Microgram-Scale NMR-Coupled Crystalline Sponge X-ray Diffraction Analysis.

Sesquiterpene scaffolds are the core backbones of many medicinally and industrially important natural products. A plethora of sesquiterpene synthases, widely present in bacteria, fungi, and plants, catalyze the formation of these intricate structures often with multiple stereocenters starting from linear farnesyl diphosphate substrates. Recent advances in next-generation sequencing and metabolomics technologies have greatly facilitated gene discovery for sesquiterpene synthases. However, a major bottleneck limits biochemical characterization of recombinant sesquiterpene synthases: the absolute structural elucidation of the derived sesquiterpene products. Here, we report the identification and biochemical characterization of LphTPS-A, a sesquiterpene synthase from the red macroalga Laurencia pacifica. Using the combination of transcriptomics, sesquiterpene synthase expression in yeast, and microgram-scale nuclear magnetic resonance-coupled crystalline sponge X-ray diffraction analysis, we resolved the absolute stereochemistry of prespatane, the major sesquiterpene product of LphTPS-A, and thereby functionally define LphTPS-A as the first bourbonane-producing sesquiterpene synthase and the first biochemically characterized sesquiterpene synthase from red algae. Our study showcases a workflow integrating multiomics approaches, synthetic biology, and the crystalline sponge method, which is generally applicable for uncovering new terpene chemistry and biochemistry from source-limited living organisms.