Bioactivity and Metabolome Mining of Deep-Sea Sediment-Derived Microorganisms Reveal New Hybrid PKS-NRPS Macrolactone from Aspergillus versicolor PS108-62.

Despite low temperatures, poor nutrient levels and high pressure, microorganisms thrive in deep-sea environments of polar regions. The adaptability to such extreme environments renders deep-sea microorganisms an encouraging source of novel, bioactive secondary metabolites. In this study, we isolated 77 microorganisms collected by a remotely operated vehicle from the seafloor in the Fram Strait, Arctic Ocean (depth of 2454 m). Thirty-two bacteria and six fungal strains that represented the phylogenetic diversity of the isolates were cultured using an One-Strain-Many-Compounds (OSMAC) approach. The crude EtOAc extracts were tested for antimicrobial and anticancer activities. While antibacterial activity against methicillin-resistant Staphylococcus aureus (MRSA) and Enterococcus faecium was common for many isolates, only two bacteria displayed anticancer activity, and two fungi inhibited the pathogenic yeast Candida albicans. Due to bioactivity against C. albicans and rich chemical diversity based on molecular network-based untargeted metabolomics, Aspergillus versicolor PS108-62 was selected for an in-depth chemical investigation. A chemical work-up of the SPE-fractions of its dichloromethane subextract led to the isolation of a new PKS-NRPS hybrid macrolactone, versicolide A (1), a new quinazoline (-)-isoversicomide A (3), as well as three known compounds, burnettramic acid A (2), cyclopenol (4) and cyclopenin (5). Their structures were elucidated by a combination of HRMS, NMR, [α]D, FT-IR spectroscopy and computational approaches. Due to the low amounts obtained, only compounds 2 and 4 could be tested for bioactivity, with 2 inhibiting the growth of C. albicans (IC50 7.2 µg/mL). These findings highlight, on the one hand, the vast potential of the genus Aspergillus to produce novel chemistry, particularly from underexplored ecological niches such as the Arctic deep sea, and on the other, the importance of untargeted metabolomics for selection of marine extracts for downstream chemical investigations.

The effect of iron oxide types on the photochemical transformation of organic phosphorus in water.

Iron oxides play an important role in the transport and transformation of organic phosphorus in aquatic environments. However, the effect of different types of iron oxide on the environmental fate of organic phosphorus has remained unclear. In this study, the photodegradation of the organic phosphorus compound adenosine triphosphate (ATP) via the activity of crystalline (goethite) and amorphous (ferrihydrite) iron oxides was investigated. It was found that ATP was photodegraded by goethite, resulting in the release of dissolved inorganic phosphate under simulated sunlight irradiation. The concentration of ATP on goethite decreased by 75% after 6 h of simulated sunlight irradiation, while the concentration of ATP on ferrihydrite decreased by only 22%. ATR-FTIR spectroscopy revealed that the intensity of the peaks for the P-O and PO stretching vibrations in the goethite-ATP complex decreased significantly more after simulated sunlight irradiation than did those for the ferrihydrite treatment. Combined with the higher TOC/TOC0 values for the goethite treatment, the results indicate that a more vigorous photochemical reaction took place in the presence of goethite than with ferrihydrite. Reactive oxygen species analysis also showed that hydroxyl and superoxide anion radicals were generated when goethite was exposed to simulated sunlight irradiation, while ferrihydrite did not exhibit this ability. Overall, this study highlights that the type of iron oxide is an important factor in the transformation of organic phosphorus in aquatic environments.

Molecular Networking-Guided Isolation of New Etzionin-Type Diketopiperazine Hydroxamates from the Persian Gulf Sponge Cliona celata.

The Persian Gulf is a unique and biologically diverse marine environment dominated by invertebrates. In continuation of our research interest in the chemistry and biological activity of marine sponges from the Persian Gulf, we selected the excavating sponge Cliona celata for detailed metabolome analyses, in vitro bioactivity screening, and chemical isolation studies. A UPLC-MS/MS (MS2) molecular-networking-based dereplication strategy allowed annotation and structural prediction of various diketopiperazines (DKPs) and etzionin-type diketopiperazine hydroxamates (DKPHs) in the crude sponge extract. The molecular-networking-guided isolation approach applied to the crude extract afforded the DKPH etzionin (1) and its two new derivatives, clioetzionin A (2) and clioetzionin B (3). Another new modified DKP (4) was identified by MS/MS analyses but could not be isolated in sufficient quantities to confirm its structure. The chemical characterization of the purified DKPHs 1-3 was performed by a combination of 1D and 2D NMR spectroscopy, HRMS, HRMS/MS, and [α]D analyses. Compounds 1 and 2 exhibited broad antibacterial, antifungal, and anticancer activities, with IC50 values ranging from 19.6 to 159.1 µM. This is the first study investigating the chemical constituents of a C. celata specimen from the Persian Gulf. It is also the first report of full spectroscopic data of etzionin based on extensive spectroscopic analyses.

Detection and Fitness of Dicarboximide-Resistant Isolates of Alternaria alternata from Dendrobium officinale, a Chinese Indigenous Medicinal Herb.

Black spot, caused by Alternaria alternata, poses a severe threat to the industry of Dendrobium officinale, a Chinese indigenous medicinal herb. Dicarboximide fungicides (DCFs) have been intensively used to control this disease for decades in China, and offer excellent efficacy. The resistance of phytopathogenic pathogens against DCFs are reportedly selected in fields; however, the DCF resistance of A. alternata from D. officinale is not well understood. The isolates of A. alternata with low procymidone resistance (ProLR) were detected in the commercial orchards of D. officinale in China in 2018 and biochemically characterized in this study. The result showed that the ProLR isolates were selected in the commercial orchards with a resistance frequency of 100%, and no significant difference in mycelial growth, sporulation, and virulence was observed among the ProLR and procymidone-sensitive (ProS) isolates. A positive cross-resistance pattern was exhibited between procymidone and iprodione. Results of amino acid sequence alignment of AaOS-1 from the tested isolates showed that all of the ProLR genotypes could be categorized into two groups, including group I (mutations at AaOs-1) and group II (no mutation). Under procymidone (5.0 µg/ml) treatment conditions, the AaOs-1 expression levels increased in the ProS isolates and ranged from approximately 2.94- to 3.69-fold higher than those under procymidone-free conditions, while the AaOs-1 expressions of the ProLR isolates were significantly lower than those in the ProS isolates under the same conditions. The data indicated that the mutations at AaOs-1 are involved in the DCF resistance of A. alternata selected in the D. officinale orchards.

Detection and Biological Characteristics of Alternaria alternata Resistant to Difenoconazole from Paris polyphylla var. chinensis, an Indigenous Medicinal Herb.

Black spot caused by Alternaria alternata (BSAA) is one of the most common diseases of Paris polyphylla var. chinensis, causing yield losses in China. Demethylation inhibitors (DMIs) have been used to control this disease in China for decades. Some farmers have complained about the decreased efficacy of DMIs against BSAA. The objective of this study was to detect and characterize the resistance of A. alternata against difenoconazole from P. polyphylla var. chinensis during 2018. Of the 22 isolates of A. alternata obtained from Sichuan Province in the southwest of China, 20 were resistant to difenoconazole. Mycelial growth rates and sporulation of the difenoconazole-resistant (DfnR) isolates were not different from those of the difenoconazole-sensitive (DfnS) isolates. No cross resistance between difenoconazole and tebuconazole or propiconazole was observed. Mutations were identified at gene AaCYP51 of DfnR isolates based on the sequence alignment of the DfnR and DfnS isolates. All of the mutations could be divided into three resistant genotypes, I (K715R + Y781C), II (K715R + D1140G + T1628A), and III (no mutation). The docking total score of the DfnS isolates was 5.6020, higher than the resistant genotype I (4.4599) or the resistant genotype II (3.8651), suggesting that the DMI resistance of A. alternata may be caused by the decreased affinity between AaCYP51 and difenoconazole.

[Developing traditional Chinese medicine injection is the need for curing sickness to save patients].

Safety issues of traditional Chinese medicine injections has been heated debate. There are two diametrically opposed views: it should be used reasonable and developed healthily or be forbidden to use. Some people have many misunderstandings and prejudices about the safety of traditional Chinese medicine injections. Compared with western medicine,traditional Chinese medicine has its own particularity. Traditional Chinese medicine has complex components. Its research and clinical application is different from western medicine. Adverse reactions of traditional Chinese medicine injections are related to many factors,such as a large number of irrational use,blind use of traditional Chinese medicine injections and western medicine injections,counterfeit and substandard drugs,incorrect methods of intravenous infusion,toxicity of supplementary materials,drug ingredients. Developing traditional Chinese medicine injection is the need for curing sickness to save patients. The purposeful, targeted, organized and planned systematic research of traditional Chinese medicine injections should be strengthened,especially the safety of traditional Chinese medicine. Strengthen supervision and control of rational drug use.Strengthen the examination and approval,supervision and management of all aspects to ensure the safety of patients.

[Specificity of traditional Chinese medicine's clinical re-evaluation].

Clinical re-evaluation is to verify the drug's safety and effectiveness again,while the drug itself has not been improved. However, due to the complexity of traditional Chinese medicine, ingredients in bulk drugs, prescription, productive processes, quality standards and other aspects need to be enhanced. So improving the quality, safety and effectiveness of traditional Chinese medicine by clinical re-evaluation is also very necessary. Therefore, except for achieving those basic requirements of medicine, it should also be improved on itself and pay full attention to the particularity, then traditional Chinese medicine's clinical re-evaluation will play its due role.

[Effects of bisphenol A exposure during lactation on testicular mitochondria in male mouse offspring].

OBJECTIVE: To study whether maternal bisphenol A (BPA) exposure during lactation stage will induce oxidative stress and apoptosis in male offspring's testis by mitochondrial pathway. METHODS: After delivery, maternal mice were randomly divided into four groups, including high dose group (50 mg/kg), medium dose group (5 mg/kg), low dose group (0.01 mg/kg) and solvent control group. BPA were given daily by gavage to maternal mice during lactation stage. The pups were raised and sacrificed at PND 75. The effect of BPA exposure on the activity of testicular mitochondrial succinate dehydrogenase (SDH), superoxide dismutase (SOD), glutathione peroxidase (GSH-Px) and malondialdehyde (MDA) in male offspring were detected. The expressions of cytochrome C (Cyt C), caspase 3 and apoptosis-inducing factor (AIF) in testes of male offspring were detected by Western blot. Changes of mitochondrial substructure in testicular cells were observed by transmission electron microscopy. RESULTS: The exposed mice had a significant decrease in the activities of testicular mitochondrial SDH (P < 0.05) in all groups and with notable decrease in the activities of GSH-Px and SOD in the high and medium-dose groups (P < 0.05). BPA also caused testicular mitochondrial MDA increased markedly (P < 0.05). Western blot showed that the expression levels of Cyt C, caspase 3 and AIF increased significantly in the testes of BPA-treated groups (P < 0.05). Under transmission electron microscopy, mitochondria swell, crests obscure and disappear were founded in the high and medium-dose groups. CONCLUSION: During lactation stage, maternal exposed to BPA induce oxidative stress and apoptosis of testicular cells by possible mechanisms of mitochondrial pathway,which would cause adverse effects on the early development of testis.

[Comparative on acute myocardial infarction models in beagle dogs and mongrel dogs].

OBJECTIVE: To compare the acute myocardial infarction models in Beagle dogs and mongrel dogs, and study whether the Beagle dog model is sensitive to drug intervention. METHOD: The acute myocardial infarction model of dog was set up through ligation of anterior descending branch of coronary artery in dogs, in order to observe morphological changes of the heart and determine artery length and heart coefficient of exposed anterior descending branch of coronary artery. The epicardium electrocardiogram (sigmaST, N-ST) was used to measure the degree of myocardial ischemia. The quantitative histological assay (nitroblue tetrazolium, N-BT stain) was adopted to determine the area of myocardial infarction. RESULT: There was no significant difference between Beagle dogs and mongrel dogs in terms of sigmaST, N-ST and ischemia area. The diltiazem group of Beagle dogs showed obvious reduction in the ischemia area (P < 0.05 and P < 0.01), with notable decline in sigmaST and N-ST, however, it had no statistical difference compared with the Beagle dog model group. Beagle dogs had clear coronary branches, longer exposed arteries and less difference in organ coefficient, which were suitable for the preparation of the myocardial infarction model, whereas mongrel dogs had irregular coronary branches and exposed arteries, with greater individual difference. CONCLUSION: Beagle dogs are superior to mongrel dogs in the preparation of the acute myocardial infarction model, which is sensitive to for drug intervention.

[Experimental study on anaphylactoid reactions induced by different components of shengmai injection (new production process) on Beagle dogs].

OBJECTIVE: To evaluate the sensitization effect of different components of Shengmai injection (new production process) on Beagle dogs. METHOD: Beagle dogs were randomly divided into 7 groups, 3 in each group. Each group was respectively injected with 5% glucose injection, Ginseng Radix et Rhizoma Rubra extract, Ophiopogonis Radix extract, Schisandrae Chinensis Fructus extract, Schisandrae Chinersis Fructus distillate, Shengmaifang, 0.2% tween 80. The changes of each dog were observed from injection before until 24 hours after injection, and the response level was determined according to the severity of the symptoms. Blood samples were collected before injection and at 10 min after injection for measuring histamine content in plasma by ELISA. Sensitization of the injection was comprehensively determined by combined the response level of symptoms and the histamine level. RESULT: One dog of Ginseng Radix et Rhizoma Rubra extract group showed untypical symptoms of anaphylactoid reactions, and serum histamine of two dogs increased more than doubled. The Beagle dogs administrated with 0.2% tween 80 showed typical symptoms of anaphylactoid reactions, while there was no significant increase of serum histamine. Other groups were observed with no typical anaphylactoid reactions. CONCLUSION: The sensitization effect of Shengmai injection (new production process) may be associated with Ginseng Radix et Rhizoma Rubra extract and 0.2% tween 80.

[Experimental study on anaphylactoid reactions induced by shengmai injection (new production process) on Cynomolgus monkey].

OBJECTIVE: To compare the anaphylactoid effect of old Shengmai injection and new Shengmai injection on Cynomolgus monkey. METHOD: Cynomolgus monkeys were randomly divided into 4 groups, and were respectively injected with 5% glucose injection, old Shengmai injection, new Shengmai injection, positive control drug. The changes of each monkey were observed from injection before until 24 hours after injection, and the response level was determined according to the severity of the symptoms. Blood samples were collected before injection and at 10 min after injection for measuring histamine content in plasma. Blood pressure and heart rates were detected before injection and at 10 min after injection. Sensitization of the injection was comprehensively determined by combined the response level of symptoms and the histamine level. RESULT: The Cynomolgus monkeys administrated with old Shengmai injection showed typical symptoms of anaphylactoid reactions and the content of serum histamine is not more than doubled. The Cynomolgus monkeys administrated with new Shengmai injection showed untypical symptoms of anaphylactoid reactions and the content of serum histamine did not rise. CONCLUSION: The old Shengmai injection can induce typical anaphylactoid reactions on Cynomolgus monkeys, and the sensitization ability is strong. The symptoms of anaphylactoid reactions induced by the new Shengmai injection appeared later and showed lesser degree with the sensitization lower.

[Mechanism studies on hepatotoxicity of rats induced by Sophorae Tonkinensis Radix et Rhizoma in rat].

OBJECTIVE: To study the hepatotoxicity mechanism of rats that induced by Sophorae Tonkinensis Radix et Rhizoma. METHOD: The SD rats were randomly divided into the control and Sophorae Tonkinensis Radix et Rhizoma (RRST) group, given distilled water and RRST 10 g x kg(-1) separately by orally for 7 days, and RRST 20 g x kg(-1) for other days until the 26th day. Blood was drawn from abdominal aorta after the rats were anesthetized by 25% urethane, and then centrifugally processed to get the serum for detection of ALT and AST. The liver tissues of control and experimental group were taken to prepare liver homogenate with cold NS, and centrifugally processed to get the supernatant. The activities of SOD and GSH, the content of gamma-GT and MDA were detected according to the methods of kit. The tumor necosis factor(TNF-alpha) was detected by ABC-ELISA. The expression of ICAM-1 was detected by immunohistochemistry. RESULT: After the rats were given RRST by oral for 26 days, the ALT and AST activities in serum increased, the content of GSH and the ratio of SOD and MDA decreased, the content of gamma-GT and TNF-alpha, the masculine expression of ICAM-1 increased. CONCLUSION: After the rats were given RRST, the liver can be damaged obviously, and the mechanism of hepatotoxicity perhaps related to free radical and inflammatory factor.