In vitro metabolic studies and machine learning analysis of mass spectrometry data: A dual strategy for differentiating alpha-pyrrolidinohexiophenone (α-PHP) and alpha-pyrrolidinoisohexanophenone (α-PiHP) in urine analysis.

Synthetic cathinones are some of the most prevalent new psychoactive substances (NPSs) globally, with alpha-pyrrolidinoisohexanophenone (α-PiHP) being particularly noted for its widespread use in the United States, Europe, and Taiwan. However, the analysis of isomeric NPSs such as α-PiHP and alpha-pyrrolidinohexiophenone (α-PHP) is challenging owing to similarities in their retention times and mass spectra. This study proposes a dual strategy based on in vitro metabolic experiments and machine learning-based classification modelling for differentiating α-PHP and α-PiHP in urine samples: (1) in vitro metabolic experiments using pooled human liver microsomes and liquid chromatography tandem quadrupole time-of-flight mass spectrometry (LC-QTOF-MS) were conducted to identify the key metabolites of α-PHP and α-PiHP from the high-resolution MS/MS spectra. After 5 h incubation, 71.4 % of α-PHP and 64.7 % of α-PiHP remained unmetabolised. Nine phase I metabolites were identified for each compound, including primary ß-ketone reduction (M1) metabolites. Comparing the metabolites and retention times confirmed the efficacy of in vitro metabolic experiments for differentiating NPS isomers. Subsequently, analysis of seven real urine samples revealed the presence for various metabolites, including M1, that could be used as suitable detection markers at low concentrations. The aliphatic hydroxylation (M2) metabolite peak counts and metabolite retention times were used to determine α-PiHP use. (2) Classification models for the parent compounds and M1 metabolites were developed using principal component analysis for feature extraction and logistic regression for classification. The training and test sets were devised from the spectra of standard samples or supernatants from in vitro metabolism experiments with different incubation times. Both models had classification accuracies of 100 % and accurately identified α-PiHP and its M1 metabolite in seven real urine samples. The proposed methodology effectively distinguished between such isomers and confirmed their presence at low concentrations. Overall, this study introduces a novel concept that addresses the complexities in analysing isomeric NPSs and suggests a path towards enhancing the accuracy and reliability of NPS detection.

Chemical constituents and their anti-neuroinflammatory activities from the bark of Taiwan incense cedar, Calocedrus formosana.

One undescribed C40 terpenoid, calomacroquinoic acid; four undescribed diterpenes, 5α,6α-epoxy-7α-hydroxyferruginol, 15-ethoxysugiol, 7-methoxy-6,7-secoabieta-8,11,13-triene-6,12-diol, and ethyl 7,8-secoabieta-11,14-dioxo-7-ate; two compounds isolated from Nature for the first time, 6ß,7α-dihydroxyferruginol and 12-O-methyltaxochinon; and six known compounds were successfully identified from the bark of Taiwan incense cedar Calocedrus formosana. Structures of all isolates were elucidated by physical data (appearance, ultraviolet, infrared, specific rotation, and X-ray) and spectroscopic data (1D and 2D nuclear magnetic resonance, and high-resolution electron ionization mass spectrometry). The biosynthetic pathway of calomacroquinoic acid is also described in the current study. Nitric oxide production in lipopolysaccharide (LPS)-stimulated BV-2 microglia cells was inhibited by 6,7-dehydroferruginol, 7α,11-dihydroxy-12-methoxy-8,11,13-abietriene, and trans-communic acid. Altogether, the bark of C. formosana possessed several potential natural therapeutics against inflammation-related neuronal diseases.

Leptospira borgpetersenii Leucine-Rich Repeat Proteins Provide Strong Protective Efficacy as Novel Leptospiral Vaccine Candidates.

Leucine-rich repeat (LRR) proteins are advocated for being assessed in vaccine development. Leptospiral LRR proteins were identified recently in silico from the genome of Leptospira borgpetersenii serogroup Sejroe, the seroprevalence of leptospiral infections of cattle in Thailand. Two LRR recombinant proteins, rKU_Sej_LRR_2012M (2012) and rhKU_Sej_LRR_2271 (2271), containing predicted immunogenic epitopes, were investigated for their cross-protective efficacies in an acute leptospirosis model with heterologous Leptospira serovar Pomona, though, strains from serogroup Sejroe are host-adapted to bovine, leading to chronic disease. Since serovar Pomona is frequently reported as seropositive in cattle, buffaloes, pigs, and dogs in Thailand and causes acute and severe leptospirosis in cattle by incidental infection, the serogroup Sejroe LRR proteins were evaluated for their cross-protective immunity. The protective efficacies were 37.5%, 50.0%, and 75.0% based on the survival rate for the control, 2012, and 2271 groups, respectively. Sera from 2012-immunized hamsters showed weak bactericidal action compared to sera from 2271-immunized hamsters (p < 0.05). Therefore, bacterial tissue clearances, inflammatory responses, and humoral and cell-mediated immune (HMI and CMI) responses were evaluated only in 2271-immunized hamsters challenged with virulent L. interrogans serovar Pomona. The 2271 protein induced prompt humoral immune responses (p < 0.05) and leptospiral tissue clearance, reducing tissue inflammation in immunized hamsters. In addition, protein 2271 and its immunogenic peptides stimulated splenocyte lymphoproliferation and stimulated both HMI and CMI responses by activating Th1 and Th2 cytokine gene expression in vaccinated hamsters. Our data suggest that the immunogenic potential renders rhKU_Sej_LRR_2271 protein a promising candidate for the development of a novel cross-protective vaccine against animal leptospirosis.

Anti-lymphangiogenic diterpenes from the bark of Calocedrus macrolepis var. formosana.

Eight new diterpenes, 6α,7ß-dihydroxyferruginol (1), 6α,7α-dihydroxyferruginol (2), 6α-hydroxyhinokiol (3), 4α-hydroxy-7-oxo-18-norabieta-8,11,13-trien-4α-ol (4a), 15,16-dehydrosugiol (5), 7-methoxy-6,7-secoabieta-8,11,13-triene-6,12-diol (6), 7α-acetoxyabieta-8,12-diene-11,14-dione (7), 7α-butyloxyethyloxyabieta-8,12-diene-11,14-dione (8), along with four known compounds, 6,7-dehydroferruginol (9), 12-hydroxy-6,7-secoabieta-8,11,13-triene-6,7-dial (10), 7α-11-dihydroxy-12-methoxy-8,11,13-abietatriene (11), and 11,14-dihydroxy-8,11,13-abietatrien-7-one (12) were successfully isolated from the bark of Calocedrus macrolepis var. formosana. The structures of all isolates were elucidated by physical data (appearance, UV, IR, optical rotation) and spectroscopic data (1D, 2D NMR, and HREIMS). Compounds 9, 10, 11, and 12 showed promising growth-inhibitory effect on human lymphatic endothelial cells (LECs). Among these compounds, compound 10 exerted the most potent anti-lymphangiogenesis property by suppressing cell growth and tube formation of LECs. In conclusion, the results revealed the anti-lymphangiogenic potentials of Formosan C. macrolepis var. formosana.

Trend and geographic analysis of the prevalence of dengue in Taiwan, 2010-2015.

BACKGROUND: Dengue is the most rapidly spreading disease caused by a mosquito-borne virus in the world. The incidence of dengue globally has increased 30-fold in the last 50 years. Understanding the prevalence of dengue and its longitudinal trends can improve prevention and control strategies. This study assessed the trends in prevalence of dengue in Taiwan by population characteristics and geographical region. METHODS: Dengue and population data for the years 2010-2015 were obtained from the public statistics databases of the Taiwan Centers for Disease Control and Department of Statistics, Taiwan Ministry of Interior. Yearly prevalence rates of dengue were calculated by age group, sex, and administrative area within five geographic regions (northern, mid-western, southern, and eastern regions, and outer islands). RESULTS: The national prevalence rate of dengue decreased gradually from 8 to 4 per 100000 population between 2010 and 2013, but it increased substantially in 2014 and 2015 to 67 and 187 per 100000 population, respectively. There was no significant difference in prevalence rate between males and females. People aged 60-69 years had a significantly higher prevalence rate than those in the other age groups during 2010-2014, and people aged over 70 years had the highest rate in 2015 (309 per 100000 population). The southern region had the highest yearly dengue prevalence rate (the rate decreased from 23 to 9 per 100000 population between 2010 and 2013, but increased to 220 and 616 per 100000 population in 2014 and 2015, respectively). Three unexpected outbreaks of dengue were observed during the study period: the first outbreak occurred in Penghu County in 2011 (prevalence rate 101 per 100000 population), the second in Kaohsiung City in 2014 (prevalence rate 540 per 100000 population), and the third in Tainan City in 2015 (prevalence rate 1208 per 100000 population). CONCLUSIONS: More efforts are still needed to prevent and control dengue in Taiwan. The government should direct resources and interventions to southern Taiwan, which has a tropical climate and a high population density, and should target older people who are more likely to be infected. Strategies are also needed to respond quickly to unexpected incidents to prevent dengue outbreaks.

Modified method for determination of sulfur metabolites in plant tissues by stable isotope dilution-based liquid chromatography-electrospray ionization-tandem mass spectrometry.

A wide variety of sulfur metabolites play important roles in plant functions. We have developed a precise and sensitive method for the simultaneous measurement of several sulfur metabolites based on liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) and (34)S metabolic labeling of sulfur-containing metabolites in Arabidopsis thaliana seedlings. However, some sulfur metabolites were unstable during the extraction procedure. Our proposed method does not allow for the detection of the important sulfur metabolite homocysteine because of its instability during sample extraction. Stable isotope-labeled sulfur metabolites of A. thaliana shoot were extracted and utilized as internal standards for quantification of sulfur metabolites with LC-MS/MS using S-adenosylmethionine (SAM), S-adenosylhomocysteine (SAH), methionine (Met), glutathione (GSH), and glutathione disulfide (GSSG) as example metabolites. These metabolites were detected using electrospray ionization in positive mode. Standard curves were linear (r(2)>0.99) over a range of concentrations (SAM 0.01-2.0µM, SAH 0.002-0.10µM, Met 0.05-4.0µM, GSH 0.17-20.0µM, GSSG 0.07-20.0µM), with limits of detection for SAM, SAH, Met, GSH, and GSSG of 0.83, 0.67, 10, 0.56, and 1.1nM, respectively; and the within-run and between-run coefficients of variation based on quality control samples were less than 8%.

Determination of nimetazepam and 7-aminonimetazepam in human urine by using liquid chromatography-tandem mass spectrometry.

We report determination of metabolites of popular drugs of abuse, including nimetazepam and nitrazepam, in urine by using liquid chromatography/mass spectrometry. Nimetazepam and its metabolites, 7-aminonimetazepam and nitrazepam, were extracted by solid-phase extraction using a DAU cartridge. An ammonium acetate buffer solution (pH 4) and a Luna polar-RP column were selected as the mobile and stationary phase, respectively, for liquid chromatography. Mass spectrometry was used for analysis and was optimized for operation in the positive mode for all analytes. The urine specimens were screened for the presence of nimetazepam and its metabolites nitrazepam and 7-aminonimetazepam at a concentration of 0.1ng/mL. Presence of 7-aminonimetazepam in the urine was an indicator of the subject being a probable abuser of nimetazepam.

Simultaneous determination of sulphur metabolites in Arabidopsis thaliana via LC-ESI-MS/MS and ³4S-metabolic labelling.

INTRODUCTION: Sulphur-containing metabolites play an important role in metabolism and homeostasis. Determination of these metabolites is challenging owing to their low concentrations and the interference in mass spectrometry analysis. OBJECTIVE: To develop a sensitive and accurate method based on liquid chromatography, electrospray ionisation, tandem mass spectrometry (LC-ESI-MS/MS) and ³4S-metabolic labelling for quantification of methionine, reduced glutathione, oxidised glutathione in Arabidopsis thaliana. METHODOLOGY: A hydroponic set-up was used for the in vivo ³4S-metabolic labelling of A. thaliana. The ³4S-labelled metabolites biosynthesised in plant were extracted and used as internal standards. Tissue was extracted with perchloric acid (PCA) or PCA containing a known amount of the analytes for recovery analysis. Tissue extract mixed with extract of ³4S-labelled A. thaliana in an appropriate ratio was subjected to a LC system and electrospray ionisation-mass spectrometric (ESI-MS) analysis. Quantification of metabolites was measured by comparing the ³4S/³4S ratios obtained for samples with the calibration curves. RESULTS: Calibration curves showed linearity with regression coefficients in the range of 0.9994-0.9999. Analyte recoveries were approximately 100%. The coefficients of variation of intra-assay and inter-assay were less than 4.2% and 5%, respectively. The ranges for the limits of detection determined for Met, GSSG and GSH were 10 fmol, < 10 fmol and 1.12 fmol and the limits of quantification determined for Met, GSSG and GSH were 0.44 pmol, 0.16 pmol and 34 fmol, respectively. CONCLUSION: The validated method for determination of methionine, reduced glutathione and oxidised glutathione was effectively applied to measure metabolite dynamics of sulphur-containing metabolites at the whole-plant level.

Novel terpenoids from Calocedrus macrolepis var. formosana.

Calocetriol (1), diacetylcalocediol (2), and ferrugimenthenol (3) were isolated from the bark of Calocedrus macrolepis var. formosana. Among them, 1 and 2 are secoabietane-type diterpenoids, and 3, with a novel C(20)-C(10) skeleton, is classified as a meroterpenoid. The structures of 1-3 were elucidated by spectroscopic analyses, and their biological activities were also evaluated. Compound 3 exhibited significant cytotoxic activity against human oral epidermoid carcinoma KB cells with an IC(50) value of 9.0±0.1 µM.

Isolation and identification of cucurbitane-type triterpenoids with partial agonist/antagonist potential for estrogen receptors from Momordica charantia.

This study aims at investigating the estrogenic activity and active cucurbitane-type triterpenoid compounds of bitter gourd (Momordica charantia, MC) using a transactivation assay for estrogen receptors (ER) α and ß. The lyophilized fruits of MC were exhaustively extracted with ethyl acetate (EA) and 95% ethanol (EtOH), sequentially. The nonsaponifiable fraction (NS) of the EA extract as well as the acid hydrolyzed EtOH extract (AH) was fractionated and isolated by repeated column chromatography and further purified by preparative HPLC or RP-HPLC. One known compound, 5ß,19-epoxycucurbita-6,24-diene-3ß,23ξ-diol (6), was isolated from the NS, and five new compounds (1-5) were isolated from AH and identified as cucurbita-6,22(E),24-trien-3ß-ol-19,5ß-olide (1), 5ß,19-epoxycucurbita-6,22(E),24-triene-3ß,19-diol (2), 3ß-hydroxycucurbita-5(10),6,22(E),24-tetraen-19-al (3), 19-dimethoxycucurbita-5(10),6,22(E),24-tetraen-3ß-ol (4), and 19-nor-cucurbita-5(10),6,8,22(E),24-pentaen-3ß-ol (5). In the noncytotoxic concentration range, compounds 1, 2, 5 and 6 showed weak agonistic activity via ER α and ß. Compounds 1, 2, 3 and 6 significantly antagonized the transactvation of 17ß-estradiol (E(2)) via both ER α and ß. In conclusion, this study demonstrates, for the first time as far as we know, the partial agonist/antagonist activity via ER of four new and one known cucurbitane-type triterpenoids from MC. Further studies are worthy to explore the selective estrogen receptor modulator (SERM) activity of MC.

Allelopathy on bark of downed logs of Chamaecyparis Obtusa sieb. and Zucc. var. formosana (Hayata) Rehder.

Chamaecyparis obtusa Sieb. and Zucc. var. formosana (Hayata) Rehder is the dominant species in the temperate forest of Yuanyang Lake Nature Reserve (YYL), Taiwan. Although downed logs of C. obstusa var. formosana occupy only a small percentage of the forest floor area in YYL, they are important regeneration substrates. Seedlings of this species often grow without competition on the new downed logs, and a few broadleaf trees grow with them. We hypothesized that the bark of the newly fallen logs possesses allelopathic potential that provides a habitat especially suitable for seedling establishment. Eight different seeds including those from Lactuca sativa L. (lettuce), Bidens pilosa (an invasive weed), and six species in YYL were planted on the bark of the downed logs in an incubator for germination tests. Two dominant species in the forest of YYL, C. obtusa var. formosana and Rhododendron formosanum, were able to grow normally, but the others, Pieris taiwanensis, Barthea formosana, Chamaecyparis formosensis, Miscanthus transmorrisonensis, lettuce, and B. pilosa were growth inhibited. A bioactivity-guided isolation was designed to isolate allelochemicals from the bark. Salicylic acid, one of the inhibiting substances, was isolated and identified by gas chromatography/mass spectroscopy (GC/MS), proton nuclear magnetic resonance ((1)H NMR), and infrared (IR). Bioassay of salicylic acid confirmed a phytotoxic effect. The results suggest that the dominance of C. obtusa var. formosana seedlings on bark could be partly due to allelopathy.

C35 terpenoids from the bark of Calocedrus macrolepis var. formosana with activity against human cancer cell lines.

Ferrugicadinol (1) and ferrugieudesmol (2), two new compounds with a unique C35 terpene skeleton, were isolated from the bark of Calocedrus macrolepis var. formosana. Their structures were elucidated principally from spectroscopic evidence. The two new C35 terpenes showed cytotoxicity against human oral epidermoid carcinoma KB cells with IC50 values ranging from 11 to 14 microM.

Calocedimers A, B, C, and D from the bark of Calocedrus macrolepis var. formosana.

Calocedimers A, B, C, and D, together with four known compounds, platydiol, alpha-cadinol, ferrugiol, and 6,7-dehydroferrugiol, were isolated from the bark of Calocedrus macrolepis var. formosana. Calocedimers A, B, C, and D are the dimers of abietane-O-camphane, abietane-O-cadinane, abietane-O-abietane, and abietane-O-abietane, respectively. Their structures were elucidated from spectroscopic data.

Formosadimers A, B, and C from the Bark of Calocedrus macrolepis var. formosana.

Formosadimers A, B, and C, together with one known compound, sugikurojin B, have been isolated from the bark of Calocedrus macrolepis var. formosana. Formosadimers A, B, and C, and sugikurojin B are dimers of the abietane-O-abietane type. Their structures were elucidated principally based on spectroscopic data.