Bioactive secondary metabolites from endophytic strains of Neocamarosporium betae collected from desert plants.

Endophytic fungi from desert plants belong to a unique microbial community that has been scarcely investigated chemically and could be a new resource for bioactive natural products. In this study, 13 secondary metabolites (1-13) with diverse carbon skeletons, including a novel polyketide (1) with a unique 5,6-dihydro-4H,7H-2,6-methanopyrano[4,3-d][1,3]dioxocin-7-one ring system and three undescribed polyketides (2, 7, and 11), were obtained from the endophytic fungus Neocamarosporium betae isolated from two desert plant species. Different approaches, including HR-ESI-MS, UV spectroscopy, IR spectroscopy, NMR, and CD, were used to determine the planar and absolute configurations of the compounds. The possible biosynthetic pathways were proposed based on the structural characteristics of compounds 1-13. Compounds 1, 3, 4, and 9 exhibited strong cytotoxicity toward HepG2 cells compared with the positive control. Several metabolites (2, 4-5, 7-9, and 11-13) were phytotoxic to foxtail leaves. The results support the hypothesis that endophytic fungi from special environments, such as desert areas, produce novel bioactive secondary metabolites.

Undescribed diphenyl ethers betaethrins A-I from a desert plant endophytic strain of the fungus Phoma betae A.B. Frank (Didymellaceae).

Ten diphenyl ethers (DPEs), including nine undescribed analogs named betaethrins A-I, were isolated from the desert plant endophytic fungus Phoma betae A.B. Frank (Didymellaceae). Their structures were determined mainly by NMR, HR-ESI-MS spectral and X-ray diffraction experiments. Betaethrins D-I possessed different fatty acid chains connected with the B-ring, which was the first report in all DPEs. The shielding effect of the B-ring on H-6 (A-ring) in methyl barceloneate, betaethrin A and betaethrins D-F (asterric acid analogs) was first observed and analyzed, which could differentiate the 1H-NMR chemical shift values of H-4/H-6 without the assistance of 3-OH. An empirical rule was then suggested: the steric hindrance between the A- and B-rings in asterric acid analogs might prevent these two aromatic rings from rotating freely, which led to the 1H-NMR chemical shift value of H-6 being in the high field zone due to the shielding effect of the B-ring on H-6. Based on the empirical rule, the chemical shift values of the A-ring in methyl barceloneate were revised. The possible biosynthesis of these isolates was postulated. Betaethrin H showed moderate cytotoxicity against MCF-7 and HepG2 cancer cell lines. Betaethrins A-F, H and I displayed strong antioxidant activities. These results further implied that endophytic fungi from unique environments, such as desert plants, with few chemical studies are an important resource of undescribed and bioactive metabolites.

Phaeosphspirone (1/1'), a pair of unique polyketide enantiomers with an unusual 6/5/5/6 tetracyclic ring from the desert plant endophytic fungus Phaeosphaeriaceae sp.

Phaeosphspirone, an undescribed polyketide with a unique 6/5/5/6-fused tetracyclic system, and two known analogues, herbarin and O-methylherbarin, were purified from the endophytic fungus Phaeosphaeriaceae sp. isolated from the desert plant Bassia dasyphylla. The connectivity and relative configuration of phaeosphspirone was elucidated by comprehensive HR-ESI-MS and NMR analysis together with a computer-assisted structure elucidation (CASE) method. A pair of enantiomers existing in phaeosphspirone were separated by HPLC chromatography after reacting with chiral reagents, from which the absolute configuration of phaeosphspirone was simultaneously determined based on Mosher's rule. This tandem strategy provides a useful approach for the separation and stereochemical determination of enantiomers possessing secondary hydroxyl groups. The structural feature of phaeosphspirone, herbarin and O-methylherbarin together with gene cluster analysis suggested their polyketide biosynthetic origin. Herbarin and O-methylherbarin exhibited moderate cytotoxicity against three cancer cell lines.

UPLC-Q-TOF-MS/MS analysis of the guaiane sesquiterpenoids oxytropiols A-J and detection of undescribed analogues from the locoweed endophytic fungus Alternaria oxytropis (Pleosporaceae).

INTRODUCTION: In addition to the mycotoxin swainsonine, the locoweed endophytic fungus Alternaria oxytropis (Pleosporaceae) also produces a series of rarely reported, highly oxygenated bicyclic guaiane sesquiterpenoids. Few investigations on the electrospray tandem mass fragmentation pattern of this sesquiterpenoid have been reported. OBJECTIVES: We aimed to analyze and detect new guaiane sesquiterpenoid analogues from crude extracts of the locoweed endophytic fungus A. oxytropis by UPLC-Q-TOF-MS/MS experiments. MATERIALS AND METHODS: Oxytropiols A-J (1-10) and the extract of the locoweed endophytic fungus A. oxytropis were analyzed by UPLC-Q-TOF-MS/MS in positive mode. RESULTS: Typical neutral losses, McLafferty rearrangement, 1,2-rearrangement, and 1,3-rearrangement were considered to be the main fragmentation patterns for the [M + H]+ /[M + Na]+ ions of 1-10 by UPLC-Q-TOF-MS/MS experiments, and possible fragmentation pathways of 1-10 were suggested. A unique and undescribed analogue named oxytropiol K (11) was found in the extract based on UPLC-Q-TOF-MS/MS analysis. Compound 11 was isolated and elucidated by NMR spectrometry, and its UPLC-Q-TOF-MS/MS analysis was consistent with the fragmentation pathways of 1-10. CONCLUSION: The results further support that UPLC-Q-TOF-MS/MS is a powerful and sensitive tool for the characterization of known compounds (dereplication) and the detection of new analogues from crude extracts and imply that the locoweed endophytic fungus A. oxytropis, with few chemical investigations, is an important resource for undescribed metabolites.

Bioactive metabolites from the desert plant-associated endophytic fungus Chaetomium globosum (Chaetomiaceae).

Globosumin, an undescribed chromene-4,7(4aH)-dione-tetramic acid PKS-PKS-NRPS hybrid, and globosumone, an undescribed azaphilone, together with ten known metabolites, were isolated from the desert plant-associated endophytic fungus Chaetomium globosum (Chaetomiaceae). The planar structures and relative configurations of globosumin and globosumone were determined by high-resolution ESI-MS and NMR data, and the absolute configurations of these two metabolites were determined by electronic circular dichroism (ECD) and circular dichroism (CD) combined with time-dependent density functional theory (TDDFT)-based quantum-chemical calculations. Chaetoglobosin A displayed biological effects against the seedling growth of Arabidopsis thaliana (Brassicaceae) in a dose-dependent manner, and this compound also exhibited biological activity against two cancer cell lines, A549 and HepG2, with IC50 values of 6.82 ± 2.34 and 38.62 ± 7.44 µM, respectively.

New species in Aspergillus section Usti and an overview of Aspergillus section Cavernicolarum.

Aspergillus sections Usti and Cavernicolarum are accommodated in the subgenus Nidulantes. In the present study, a polyphasic approach using morphology and multi-gene phylogeny was applied to investigate the taxonomy of these two sections. Based on the phylogenetic analysis, Aspergillus section Usti includes 25 species, which can be assigned to four series: Calidousti, Deflecti, Monodiorum and Usti. Aspergillus sigarelli is newly described in this section and this species was isolated from a cigarette from PR China and belongs to series Calidousti. It is clearly distinct from other members in this series based on ITS, BenA, CaM and RPB2 sequences. Aspergillus section Usti members like A. calidoustus and A. granulosus are important opportunistic pathogens, it is speculative that more pathogenetic species will be found by using polyphasic taxonomy approaches. Aspergillus section Cavernicolarum includes five species, the growth rates on agar media and size and ornamentation of conidia are important characters for differentiating species in section Cavernicolarum.

New section and species in Talaromyces.

Talaromyces is a monophyletic genus containing seven sections. The number of species in Talaromyces grows rapidly due to reliable and complete sequence data contributed from all over the world. In this study agricultural soil samples from Fujiang, Guangdong, Jiangxi, Shandong, Tibet and Zhejiang provinces of China were collected and analyzed for fungal diversity. Based on a polyphasic approach including phylogenetic analysis of partial ITS, BenA, CaM and RPB2 gene sequences, macro- and micro-morphological analyses, six of them could not be assigned to any described species, and one cannot be assigned to any known sections. Morphological characters as well as their phylogenetic relationship with other Talaromyces species are presented for these putative new species. Penicillium resedanum is combined in Talaromyces section Subinflati as T. resedanus.

Bioactive seco-Sativene Sesquiterpenoids from an Artemisia desertorum Endophytic Fungus, Cochliobolus sativus.

A series of seco-sativene sesquiterpenoids (1-11) including two new natural products (2 and 3), four new analogues (4-7), and six known analogues, helminthosporic acid (1), drechslerine A (8), drechslerine B (9), helminthosporol (10), helminthosporal acid (11), and isosativenediol (12), were purified from the endophytic fungus Cochliobolus sativus isolated from a desert plant, Artemisia desertorum. The stereochemistry of helminthosporic acid (1) was established for the first time by X-ray diffraction, and the structures including relative and absolute configurations of these new compounds were determined by NMR and CD spectra together with biosynthetic considerations. Compounds 5-7 are the first seco-sativene sesquiterpenoids possessing a glucose group on C-15, C-15, and C-14, respectively. Compounds 1, 7, 9, and 11 displayed strong phytotoxic effects on corn leaves by producing visible lesions, and helminthosporic acid (1) was shown to promote division of leaves and roots of Arabidopsis thaliana with a dose-dependent relationship.

Chetocochliodins A-I, Epipoly(thiodioxopiperazines) from Chaetomium cochliodes.

Nine new epipoly(thiodioxopiperazine) (ETP) analogues, chetocochliodins A-I (1-9), along with two known ones, chetoseminudins E and C (10 and 11), were purified from the fungus Chaetomium cochliodes. The planar structures and absolute configurations of these new compounds were determined by extensive NMR spectroscopic analysis, CD spectra, and chemical reactions. Shielding effects from the indole on the 3-SCH3/3-OCH3/3-OCH2- groups facilitated the determination of relative configuration of the analogues. Compound 9 was cytotoxic, suggesting the importance of the sulfide bridge for the diketopiperazine bioactivities.

Bioactive Resorcylic Acid Lactones with Different Ring Systems from Desert Plant Chaetosphaeronema hispidulum. [corrected].

Five new resorcylic acid lactones (RALs) hispidulactones A-E (1, 4, 5, 8, and 9), a new natural product (2), and four known ones (3, 6, 7, and 10) with different ring systems were isolated from the desert plant Chaetosphaeronema hispidulum. [corrected]. The new compounds were characterized by NMR data, CD spectra, and X-ray experiment. The new natural product (2) displayed strongly biological effects on the seedlings growth of Arabidopsis thaliana, Digitaria sanguinalis, and Echinochloa crusgalli with a dose-dependent relationship. Compounds 1, 2, and 6 were also tested cytotoxic activities against three cancer cell lines HCT116, Hela, and MCF7 and only did the new natural product (2) display biological activities with IC50 values at 54.86 ± 1.52, 4. 90 ± 0.02, and 20.04 ± 4.00 µM, respectively, whereas the IC50 values of the positive control cis-platinum were 11.36 ± 0.42, 3.54 ± 0.12, and 14.32 ± 1.01 µM, respectively.

Spiciferone analogs from an endophytic fungus Phoma betae collected from desert plants in West China.

Endophytic fungi from desert, arid, and grassland areas are an ecologically important but unique group with poor chemical investigation. During our ongoing study to mine bioactive secondary metabolites from unique fungal environments, a new shunt product spiciferone F (1) including two new analogs spiciferones G (2) and H (3) together with four known ones spiciferone A (4), spiciferol A (5), 6, and 7 were isolated from endophytic fungus Phoma betae inhabiting in plant Kalidium foliatum (Pall.) Moq from Ningxia Province of West China. The planar, relative, and absolute configurations of these new compounds were elucidated by nuclear magnetic resonance, high-resolution electrospray ionization mass spectrometry, and electronic circular dichroism experiments. According to the shunt products, intermediates and analogs isolated from this endophytic fungus, the possible biosynthetic pathway of spiciferones was reconstructed. Compounds 1-7 were evaluated cytotoxic activities against three cancer cell lines HCT 116, HeLa, and MCF7, and only did 1 display strong biological effect against MCF7 with a half-maximal inhibitory concentration value at 7.73 ± 0.11 µM compared with the cis-platinum (14.32 ± 1.01 µM).

Highly Photosensitive Poly-Sulfur-Bridged Chetomin Analogues from Chaetomium cochliodes.

The highly photosensitive characteristic of poly-sulfide chetomins was first unveiled, and four new unstable analogues, chetomins A-D (1-4), with significant cytotoxicity were successfully purified in darkness. The visible-light-induced desulfurization and intermolecular disproportionation were revealed to initiate the interconversion of chetomin analogues, which explained the long-recognized puzzle of rarity and instability of chetomin analogues.

Hematological Risk Factors for High-Altitude Headache in Chinese Men Following Acute Exposure at 3,700 m.

Background: High-altitude headache (HAH) is a notably common disorder affecting the daily life of travelers ascending to high altitude. Hematological parameters are important clinical examinations for various diseases. Today, hematological characteristics of HAH remain unrevealed. Above all, we aimed to ascertain hematological characteristics and independent risk factors/predictors associated with HAH before and after exposure at 3,700 m. Methods: Forty five healthy men were enrolled in present study. Demographic and clinical data, physiological and hematological parameters were collected 3 days before the ascent and after acute exposure at 3,700 m. Results: HAH patients featured significantly lower white blood cell count (WBC), neutrophil count (NEU#) and percentage (NEU%), and higher percentage of lymphocyte (LYM%) at 3,700 m and significantly lower NEU#, reticulocyte count (RET#) and percentage (RET%) at sea level (all P < 0.05). HAH severity was significantly and negatively associated with WBC, NEU#, and NEU% at 3,700 m and RET# at sea level, whereas was positively associated with LYM% at 3,700 m (all P < 0.05). Moreover, we have found that RET# at sea level and NEU% at 3,700 m was an independent predictor and risk factor for HAH, respectively. Conclusion: The present study is the first to examine the hematological characteristics of HAH. Furthermore, lower RET# at sea level and lower NEU% at 3,700 m is a novel independent predictor and risk factor for HAH, respectively.

Elevated pentose phosphate pathway is involved in the recovery of hypoxia­induced erythrocytosis.

As a typical model of hypoxia­induced excessive erythrocytosis, high altitude polycythemia (HAPC) results in microcirculation disturbance, aggravates tissue hypoxia and results in a severe clinical outcome, without any effective intervention methods except for returning to an oxygen­rich environment. The present study aimed to explore potential therapeutic targets which may participate in the recovery of HAPC by studying the mechanisms of reducing the hemoglobin (HB) concentration during re­oxygenation. A total of 14 and 13 subjects were recruited over a 5,300 m distance and 5,170 m area. The patients were classified into HAPC or control groups based on their HB value. Plasma samples were collected on the day when they finished their stay in plateau for a year, and on the 180th day following their reaching in plain. Metabolic profiling was conducted by UPLC­QTOF/MS. MetaboAnalyst platform was performed to explore the most perturbed metabolic pathways. A panel of differential metabolites were obtained in the recovery phase of HAPC and control groups. The present study identified the uniquely upregulated pentose phosphate pathway in HAPC subjects, along with a significantly decreased HB level. The findings were verified via a direct comparison between HAPC and control subjects at a high altitude. An increased pentose phosphate pathway was identified in control groups compared with HAPC subjects. An elevated pentose phosphate pathway may therefore participate in the recovery of HAPC, whereas a downregulated pentose phosphate pathway may contribute to hypoxia­induced erythrocytosis. The results of the present study provide potential therapeutic strategies and novel insights into the pathogenesis of hypoxia­induced polycythemia.

UPLC­QTOFMS­based metabolomic analysis of the serum of hypoxic preconditioning mice.

Hypoxic preconditioning (HPC) is well­known to exert a protective effect against hypoxic injury; however, the underlying molecular mechanism remains unclear. The present study utilized a serum metabolomics approach to detect the alterations associated with HPC. In the present study, an animal model of HPC was established by exposing adult BALB/c mice to acute repetitive hypoxia four times. The serum samples were collected by orbital blood sampling. Metabolite profiling was performed using ultra­performance liquid chromatography­quadrupole time­of­flight mass spectrometry (UPLC­QTOFMS), in conjunction with univariate and multivariate statistical analyses. The results of the present study confirmed that the HPC mouse model was established and refined, suggesting significant differences between the control and HPC groups at the molecular levels. HPC caused significant metabolic alterations, as represented by the significant upregulation of valine, methionine, tyrosine, isoleucine, phenylalanine, lysophosphatidylcholine (LysoPC; 16:1), LysoPC (22:6), linoelaidylcarnitine, palmitoylcarnitine, octadecenoylcarnitine, taurine, arachidonic acid, linoleic acid, oleic acid and palmitic acid, and the downregulation of acetylcarnitine, malate, citrate and succinate. Using MetaboAnalyst 3.0, a number of key metabolic pathways were observed to be acutely perturbed, including valine, leucine and isoleucine biosynthesis, in addition to taurine, hypotaurine, phenylalanine, linoleic acid and arachidonic acid metabolism. The results of the present study provided novel insights into the mechanisms involved in the acclimatization of organisms to hypoxia, and demonstrated the protective mechanism of HPC.

A Signature of Circulating microRNAs Predicts the Susceptibility of Acute Mountain Sickness.

Background: Acute mountain sickness (AMS) is a common disabling condition in individuals experiencing high altitudes, which may progress to life-threatening high altitude cerebral edema. Today, no established biomarkers are available for prediction the susceptibility of AMS. MicroRNAs emerge as promising sensitive and specific biomarkers for a variety of diseases. Thus, we sought to identify circulating microRNAs suitable for prediction the susceptible of AMS before exposure to high altitude. Methods: We enrolled 109 healthy man adults and collected blood samples before their exposure to high altitude. Then we took them to an elevation of 3648 m for 5 days. Circulating microRNAs expression was measured by microarray and quantitative reverse-transcription polymerase chain reaction (qRT-PCR). AMS was defined as Lake Louise score ≥3 and headache using Lake Louise Acute Mountain Sickness Scoring System. Results: A total of 31 microRNAs were differentially expressed between AMS and Non-AMS groups, 15 up-regulated and 16 down-regulated. Up-regulation of miR-369-3p, miR-449b-3p, miR-136-3p, and miR-4791 in patients with AMS compared with Non-AMS individuals were quantitatively confirmed using qRT-PCR (all, P < 0.001). With multiple logistic regression analysis, a unique signature encompassing miR-369-3p, miR-449b-3p, and miR-136-3p discriminate AMS from Non-AMS (area under the curve 0.986, 95%CI 0.970-1.000, P < 0.001, LR+: 14.21, LR-: 0.08). This signature yielded a 92.68% sensitivity and a 93.48% specificity for AMS vs. Non-AMS. Conclusion: The study here, for the first time, describes a signature of three circulating microRNAs as a robust biomarker to predict the susceptibility of AMS before exposure to high altitude.

Activated corticosterone synthetic pathway is involved in poor responses to re-oxygenation after prolonged hypoxia.

Diverse response patterns to re-oxygenation lead to various physiological or pathological phenotypes, but now lack of systematic research models in vivo. High-altitude de-acclimatization syndrome (HADAS) describes systematic alterations of re-oxygenation returning to plain after a long living in high altitude. In this study, we aim at employing a comprehensive metabolomics to explore the mechanisms for different reactions to re-oxygenation based on systematic quantitation scoring methods of HADAS model. Plasma samples were collected from 22 subjects when they finished their stay in high altitude for 1 year (5300 m), returning plain for 30th day and 180th day. These participants were divided into HADAS-S or HADAS-R group based on HADAS model on the 30th day after their reaching. Metabolic profiling was performed by ultra-performance liquid chromatography-quadrupole time-of-light mass spectrometry (UPLC-QTOFMS) in conjunction with univariate and multivariate statistical analysis. A total of 20 differential metabolites were identified by the comparison between HADAS-S and HADAS-R group. Pathway analysis suggested that the most potential disturbed pathway is sterol synthesis pathway, especially corticosterone synthetic sub-pathway. These molecules detected in this pathway are detailed that they showed a rapid and significant increasing manner in HADAS-S subjects comparing to HADAS-R group in the process of re-oxygenation. In conclusion, we identified that excessive stress responses to re-oxygenation might contribute to the distinctions between HADAS-S and HADAS-R group. These findings provide novel insights for further understanding of the pathogenesis for metabolic abnormalities in re-oxygenation after prolonged hypoxia.

Physiological Adjustments and Circulating MicroRNA Reprogramming Are Involved in Early Acclimatization to High Altitude in Chinese Han Males.

Background: Altitude acclimatization is a physiological process that restores oxygen delivery to the tissues and promotes oxygen use under high altitude hypoxia. High altitude sickness occurs in individuals without acclimatization. Unraveling the molecular underpinnings of altitude acclimatization could help understand the beneficial body responses to high altitude hypoxia as well as the altered biological events in un-acclimatized individuals. This study assessed physiological adjustments and circulating microRNA (cmiRNA) profiles in individuals exposed to high altitude, aiming to explore altitude acclimatization in humans. Methods: Ninety volunteers were enrolled in this study. Among them, 22 individuals provided samples for microRNA arrays; 68 additional individuals constituted the validation set. Un-acclimatized individuals were identified by the Lake Louise Scoring System. Thirty-three phenotypes were recorded pre- and post-exposure to high altitude, including stress hormones, lipid profiles, hematological indices, myocardial enzyme spectrum, and liver and kidney function related enzymes. CmiRNA expression profiles were assessed using miRCURYTM LNA Array (v.18.0) screening, with data validated by quantitative reverse-transcription polymerase chain reaction (qRT-PCR). Then, associations of plasma microRNA expression with physiological adjustments were evaluated. The biological relevance of the main differentially expressed cmiRNAs was explored by bioinformatics prediction. Results: Nineteen of the 33 phenotypes were significantly altered during early altitude acclimatization, including hematological indices, lipid profiles, and stress hormones; meanwhile, 86 cmiRNAs (79 up-regulated and 7 down-regulated) showed differential expression with statistical significance. Among them, 32 and 25 microRNAs were strongly correlated with low-density lipoprotein-cholesterol and total cholesterol elevations, respectively. In addition, 22 microRNAs were closely correlated with cortisol increase. In un-acclimatized individuals, 55 cmiRNAs were up-regulated and 36 down-regulated, compared with acclimatized individuals. The HIF signaling pathway was suppressed in un-acclimatized individuals. Conclusion: Physiological adjustments, including the hematological system, stress hormones, and lipid molecules contributed to early altitude acclimatization, and showed strong correlations with cmiRNA reprogramming. Moreover, acclimatized and un-acclimatized individuals showed different cmiRNA profile. Suppression of the HIF-1 signaling pathway by microRNA regulation may play a key role in the pathogenesis of un-acclimatization with high altitude hypoxia.

Metabolite Modulation in Human Plasma in the Early Phase of Acclimatization to Hypobaric Hypoxia.

The exposure of healthy subjects to high altitude represents a model to explore the pathophysiology of diseases related to tissue hypoxia. We explored a plasma metabolomics approach to detect alterations induced by the exposure of subjects to high altitude. Plasma samples were collected from 60 subjects both on plain and at high altitude (5300 m). Metabolite profiling was performed by gas chromatography-mass spectrometry (GC-MS) and ultra-performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UPLC-QTOFMS) in conjunction with univariate and multivariate statistical analyses. ELISA assays were further employed to measure the levels of several relevant enzymes together with perturbed metabolic pathways. The results showed that hypobaric hypoxia caused significant and comprehensive metabolic changes, as represented by significant changes of 44 metabolites and 4 relevant enzymes. Using MetaboAnalyst 3.0, it was found that several key metabolic pathways were acutely perturbed. In addition, 5 differentially expressed metabolites in pre-exposure samples from the acute mountain sickness-susceptible (AMS-S) group compared with those from the AMS-resistant (AMS-R) group are identified, which warrant further validation as potential predictive biomarkers for AMS-S individuals. These results provide new insights for further understanding the pathophysiological mechanism of early acclimatization to hypobaric hypoxia and other diseases correlated to tissue hypoxia.