Antibacterial and antioxidant metabolites from the insect-associated fungus Aspergillus fumigatus.

The research on bioactive secondary metabolites from Aspergillus fumigatus afforded six compounds, which were identified by mass spectrometer (MS) and nuclear magnetic resonance (NMR) spectroscopic analysis as cyclopyazonic acid (1), trypacidin A (2), asterric acid (3), methyl asterrate (4), demethylcitreoviranol (5), as well as (5-hydroxy-2-oxo-2H-pyran-4-yl) methyl acetate (6). Cyclopyazonic acid (1) was found to have potent antibacterial effects, especially against Bacillus licheniformis with minimal inhibitory concentration (MIC) value of 3.7µg/mL. Its antibacterial effects were possibly related to the olefinic acid group in the structure. Phenyl ether derivatives 3 and 4, and trypacidin A (2) also exhibited antimicrobial effects. In addition, compound 6 showed significant antioxidant effects with half maximal effective concentration (EC50) value of 10.2µM in the ABTS (2,2-azino-bis-3-ethylbenzothiazoline-6-sulfonic acid) assay, which was better than the positive control.

New Caffeoylquinic Acid Derivatives and Flavanone Glycoside from the Flowers of Chrysanthemum morifolium and Their Bioactivities.

The Chrysanthemum morifolium flower is widely used in China and Japan as a food, beverage, and medicine for many diseases. In our work, two new caffeoylquinic acid derivatives (1, 2), a new flavanone glycoside (3), and six reported flavanones (4⁻9) were isolated and identified from the flowers of C. morifolium. The chemical structures of all isolates were elucidated by the analysis of comprehensive spectroscopic data as well as by comparison with previously reported data. The isolated constituents 1⁻8 were evaluated for their neuroprotective activity, and compounds 3 and 4 displayed neuroprotective effects against hydrogen peroxide-induced neurotoxicity in human neuroblastoma SH-SY5Y cells.

[Establishment of Duplex PCR for Identifying Metagonimus yokogawai and Haplorchis taichui].

OBJECTIVE: To develop a duplex PCR method for identifying Metagonimus yokogawai and Haplorchis taichui. METHODS: ITS1 sequences of M. yokogawai and H. taichui, as well as those of their homologous species were obtained from GenBank, and two sets of specific primer pairs for M. yokogawai and H. taichui were designed accordingly using Primer Premier 5.0 software. PCR reaction system and conditions were optimized. The established duplex PCR method was applied in a pool of M. yokogawai, H. taichui, and 17 related species to examine its specificity. Sensitivity was evaluated through serial dilutions of plasmids containing their specific sequences. Finally, the duplex PCR was applied to identify M. yokogawai and H. taichui among trematodes collected from the viscera of 47 cats and 40 dogs to test its practicality. RESULTS: The duplex PCR method amplified target sequences of M. yokogawai and H. taichui, generating 648 bp and 279 bp products, respectively. No cross reaction was found with the following 17 related species: Haplorchis pumilio, Clonorchis sinensis, Pharyngostomum cordatum, the metacercaria of Metorchis sp. and Exorchis sp., Echinochasmus liliputanus, Echinochasmus perfoliatus, Echinostoma friedi, Hypoderaeum conoideum, Holostephanus sp., Diplodiscus sp., Anisakis sp., Metorchis orientais, Paragonimus westermani, Watsonius watsoni, Notocotylus sp. and Hysterothylacium sp, indicating a high specificity of this method. The detection limits for DNAs of M. yokogawai and H. taichui were 1.49 x 10(-1) pg and 1.14 x 10(-1) pg, suggesting a good sensitivity for this method. Further, the duplex PCR successfully identified M. yokogawai and H. taichui from cat and dog viscera, with no cross amplification of other trematodes. CONCLUSION: The duplex PCR is effective in identifying Metagonimus yokogawai and Haplorchis taichui.

Two new megastigmane glycosides from the leaves of Nicotiana tabacum and their anti-inflammatory activities.

Two new megastigmane glycosides, (6 R,7E,9R)-3-oxo-α-ionyl-9-O-α-L-rhamnopyranosyl-(1''→4')-ß-D-glucopyranoside (1) and (6 R,7E,9R)-3-oxo-α-ionyl-9-O-ß-D-glucopyranosyl-(1''→6')-ß-D-glucopyranoside (2), together with six known analogues (3-8) were isolated from the leaves of Nicotiana tabacum. The structures of all metabolites were determined by comprehensive analysis of NMR and MS spectroscopic data as well as by comparison with those of previously reported. The in vitro anti-inflammatory activity of all isolates was evaluated using a lipopolysaccharide (LPS)-induced RAW264.7 cell inflammatory model, and the compounds 1, 3, 7, and 8 exhibited inhibition of LPS-induced NO production in RAW264.7 macrophage cells with IC50 values of 42.3-61.7 µM (positive control, dexamethasone, IC50 = 21.3 ± 1.2 µM).

A monolithic three-dimensional thermal convective acoustic vector sensor with acoustic-transparent heat sink.

An acoustic vector sensor can directly detect acoustic particle velocity based on the measured temperature difference between closely spaced heated wires. For the detection of velocity in three dimensions, an integrated three-dimensional (3 D) sensor is desired, but it remains challenging in MEMS (Micro-Electro-Mechanical System) manufacturing. Here, a novel monolithic 3 D acoustic vector sensor is proposed, which is constructed using in-plane distributed wires assembled with acoustically transparent heat sink. The planar MEMS structure of the proposed sensor makes it easy to be fabricated and packaged. This work offers a new method for the design of acoustic vector sensors and other thermal convection-based MEMS sensors.

[Detection and identification for Gnathostoma sp. in imported Monopterus albus].

OBJECTIVE: To inspect the third stage larvae of Gnathostoma in imported Monopterus albus, and identify its species. METHODS: Ten batches of M. albus imported to Shanghai were detected for nematode Gnathostoma from January 2010 to March 2011. Fifty-two M. albus imported from the Philippines (25), Indonesia (24) and Bangladesh (3) were sampled (3-10/batch), which were dissected, minced, and digested. The suspension was filtered with 10 mesh screen to take the deposit. The complete parasites were picked out under stereoscope followed by morphological identification. The rate and intensity of infection were calculated. Genomic DNA of Gnathostoma was extracted to amplify internal transcribed spacer region 2 (ITS-2) and cytochrome C oxidase subunit 1 (cox1) by PCR, the product of which was analyzed by electrophoresis and sequencing. The sequences were aligned with corresponding sequences in GenBank. RESULTS: The third stage larvae of Gnathostoma were detected in M. albus from Indonesia and Philippines with infection rate of 36.0% (9/25) and 50.0% (12/24) and average infectiosity of 7.8 (70/9) and 2.8 (34/12), respectively. No Gnathostoma was found in M. albus imported from Bangladesh. Under microscope, the larvae showed one cephalic bulb with 4 rings of hooklets on it, cross striations and small spines on the body surface. The front body spines were bigger and denser, while the rear spines were smaller and sparser. It had 1 cervical papilla and 4 cervical capsules. Morphological characteristics were similar to the third stage larvae of G. spinigerum. PCR results showed that the length of the ITS-2 and cox1 PCR products was 647 bp and 441 bp, respectively. Sequence alignment analysis showed that the two PCR products had 99%-100% consistency with G. spinigerum ITS-2 (GenBank Accession No. AB181155 and Z97175) and cox1 (GenBank Accession No. AY501388, AB180099, and AB551552). CONCLUSION: All the larvae detected in M. albus imported from the Philippines and Indonesia have been identified as G. spinigerum.

Buffalo-Origin Seneca Valley Virus in China: First Report, Isolation, Genome Characterization, and Evolution Analysis.

Pigs are the main host of Seneca Valley virus (SVV), previously known as Senecavirus A (SVA). Pigs affected by SVV have vesicles in the nose, hooves, and limp and may cause death in some severe cases. Occasionally, SVV has also been detected in mice, houseflies, environmental equipment, and corridors in pig farms. Moreover, it was successfully isolated from mouse tissue samples. In this study, an SVV strain (SVA/GD/China/2018) was isolated from a buffalo with mouth ulcers in the Guangdong province of China using seven mammalian cell lines (including BHK-21, NA, PK-15, ST, Vero, Marc-145, and MDBK). The genome of SVA/GD/China/2018 consists of 7,276 nucleotides. Multiple-sequence alignment showed that SVA/GD/China/2018 shared the highest nucleotide similarity (99.1%) with one wild boar-origin SVV strain (Sichuan HS-01) from the Sichuan province of China. Genetic analysis revealed that SVA/GD/China/2018 clustered with those porcine-origin SVV strains. To the best of our knowledge, this is the first report of SVV infection in buffalo, which might expand the host range of the virus. Surveillance should be expanded, and clinical significance of SVV needs to be further evaluated in cattle.

[Response of Soil Respiration and Its Components to Nitrogen and Phosphorus Addition in Farming-Withdrawn Grassland in the Semiarid Loess Hilly-Gully Region].

Understanding the soil respiration characteristics in response to nitrogen and phosphorus addition in farming-withdrawn grasslands within semi-arid loess hilly-gully regions is of great importance for providing a theoretical basis for evaluating the effects of artificial regulation approaches on carbon cycling. We report on a field experiment that was undertaken from May to September 2018 in a farming-withdrawn grassland ecosystem in China, which is dominated by Stipa bungeana and Lespedeza davurica. Three different levels of nitrogen and phosphorus additions were used, including three main plots of N[0, 50, and 100 kg·(hm2·a)-1] and three subplots of P (P2O5)[0,40, and 80 kg·(hm2·a)-1]. The soil respiration rate, heterotrophic respiration rate, soil temperature, and soil moisture were measured monthly in each treatment. Results showed that N and P addition had no effect on soil temperature or moisture content (P>0.05). The soil respiration rate showed an obvious monthly variation and peaked in July. In the treatment without fertilizer addition, the monthly mean soil respiration rate, heterotrophic respiration rate, and autotrophic respiration rate were 0.69, 0.39, and 0.29 g·(m2·h)-1, respectively. P addition had no significant effect on the soil respiration rate and its components without N addition (P>0.05). Under conditions of N addition, P addition significantly increased the soil respiration rate and its component (P<0.05). The monthly mean soil respiration rate, heterotrophic respiration rate, and autotrophic respiration rate were 0.93, 0.50, and 0.47 g·(m2·h)-1, respectively. The Q10 (i.e., temperature sensitivity) values for soil respiration, heterotrophic respiration, and autotrophic respiration in unfertilized soil were 1.86, 2.36, and 2.24, respectively. The addition of N and P reduced the Q10 value of soil respiration and its components. Our findings suggest that the response of soil respiration and its two components to N and P addition in studied farming-withdrawn grassland in the semiarid loess hilly-gully region were closely related to their addition amounts.

[Responses of leaf functional traits of dominant plant species in grassland communities to nitrogen and phosphorus addition in loess hilly-gully region.] / é»„åœŸä¸˜é™µåŒºè‰åœ°æ¤è¢«ç¾¤è½ä¼˜åŠ¿ç§å¶ç‰‡åŠŸèƒ½æ€§çŠ¶å¯¹æ°®ç£·æ·»åŠ çš„å“åº”.

To analyze plant functional traits of dominant species to nitrogen and phosphorus addition, three species (Bothriochloa ischaemum, Stipa bungeana, and Lespedeza davurica) were selected in the loess hilly-gully region. A split-plot experiment which included three N treatments (0, 50, and 100 kg N·hm-2·a-1) and three P treatments (0, 40, and 80 kg P2O5·hm-2·a-1) was conducted. At the fast-growing stage, leaf length, leaf width, specific leaf area, leaf dry matter content, leaf N content, leaf P content, and leaf N:P were measured. Results showed that under 50 and 100 kg N·hm-2·a-1 treatments, leaf length and width of B. ischaemum increased significantly by 35.3% and 64.4%, respectively, while only the leaf length of S. bungeana and the leaf width of L. davurica increased significantly by 58.8% and 33.9%, respectively. Leaf dry matter content of the three species decreased significantly by 10.7%, 15.3% and 11.2%, respectively. Leaf N content and N:P of B. ischaemum and S. bungeana increased significantly by 23.0% and 99.2%, 45.8% and 96.9%, respectively, compared with unfertilized treatments. Under 40 and 80 kg P2O5·hm-2·a-1 treatments, leaf length, leaf width and specific leaf area of L. davurica increased significantly by 56.9%, 41.4% and 19.6%, respectively, while leaf dry matter content decreased significantly by 14.9%. Leaf P content of three species increased significantly by 96.7%, 110.9% and 238.4%, while the N:P decreased significantly by 45.8%, 42.8% and 53.7%, respectively, compared with those under unfertilized. Under 50 kg N·hm-2·a-1 treatment, compared with no P application, leaf length and leaf width of L. davurica and leaf P content of the three species significantly increased, and leaf N content of B. ischaemum and S. bungeana decreased significantly at 40 and 80 kg P2O5·hm-2·a-1 treatments. Under 100 kg N·hm-2·a-1 treatment, leaf length of B. ischaemum and S. bungeana, leaf width of L. davurica and leaf P content of three species significantly increased, while leaf N content of B. ischaemum decreased significantly after P application. In summary, functional traits of dominant species showed significant responses to short-term nitrogen and phosphorus addition, with the different responses were mainly related to species traits and fertilization levels. Such difference reflected plant adaptation to habitat changes. The divergent responses of different species to nitrogen and phosphorus addition played an important role in maintaining diversity and stability of grassland communities.

Nutrient-starved incubation conditions enhance pyrazinamide activity against Mycobacterium tuberculosis.

Pyrazinamide (PZA) is an unconventional frontline tuberculosis drug characterized by high in vivo sterilizing activity, but poor in vitro activity. The study on the mechanism of action of PZA has attracted significant attention because of the peculiarity of PZA and its ability to shorten the tuberculosis chemotherapy. In this study, we examined the effect of nutrient-starved conditions on PZA activity in vitroat acid pH. We also examined the effect of fatty acids, benzoic acid and salicylic acid on PZA activity. The results showed that nutrient-starved conditions lowered the membrane potential of Mycobacterium tuberculosis and enhanced the activity of PZA, with 5- and 10-day starvation conditions resulting in greater enhancement than 3-day starvation. Fatty acids, benzoic acid and salicylic acid enhanced PZA activity in both normal and starved bacilli, especially in starved bacilli. These findings provide further support for the recent model of PZA action and may have implications for developing new drugs that shorten therapy.

[Nutrient starved incubation conditions enhance pyrazinamide activity against Mycobacterium tuberculosis].

OBJECTIVE: To investigate the mechanism of the action and the target of pyrazinamide (PZA), by examining the effect of nutrient-starved conditions on PZA activity. METHODS: M. tuberculosis starved in PBS for 3, 5 or 10 days were treated with PZA, and with benzoic acid, salicylic acid or fatty acid in combination with PZA. The activity of PZA against M.tuberculosis was determined by the number of colony-forming unit (CFU). The membrane potential of M.tuberculosis was determined by flow-cytometry with Rhodamine to investigate the mechanism of PZA activity. RESULTS: The CFU of M. tuberculosis treated with PZA for 0, 3, 5, 10 days nutrient starvation compared with their respective controls, decreased by 23.08%, 37.75%, 82.32%, 81.03%, respectively. The membrane potential of M. tuberculosis in nutrient-starved cultures declined rapidly over the first 5 days' starvation. The starved bacilli treated with PZA had significantly lower level of membrane potential compared with the control. The effect of PZA on lowering the membrane potential was antagonized by addition of glucose, which provided energy for the bacilli. Fatty acids, benzoic acid and salicylic acid-enhanced PZA activity in both normal and starved bacilli, especially in starved bacilli. CONCLUSION: Nutrient starved incubation conditions and weak acids could enhance pyrazinamide activity against Mycobacterium tuberculosis. PZA can impair Mycobacterium tuberculosis cell viability by disrupting the energy supply system and reducing membrane potential of the cells.

Clinical research on navel application of Shehuang Paste combined with Chinese herbal colon dialysis in treatment of refractory cirrhotic ascites complicated with azotemia.

AIM: To explore the efficacy and mechanism of a novel therapeutic method of traditional Chinese medicine in patients with refractory cirrhotic ascites complicated with azotemia. METHODS: Seventy-five cases of refractory cirrhotic ascites complicated with azotemia were randomly divided into 3 groups: comprehensive treatment (n = 29), simple treatment (n = 24), and control (n = 22). The basic treatment methods were the same in all groups, including liver protecting medicines, diuretics and supportive drugs. The control group underwent only the basic treatment. Shehuang Paste (SHP) was applied to the navels of the two treatment groups once a day for 30 d. Colon dialysis with Chinese herbs was administered to the comprehensive treatment group once every two days. Before and after treatment, we measured abdominal circumference, BUN, Cr, serum Na+, urine Na+/K+, liver function, endotoxin content, NO, and ET-1. Color Doppler ultrasonography was conducted to measure the portal vein blood flow. RESULTS: The total effective rate for ascites was 72.4% in the comprehensive treatment group, 45.8% in the simple treatment, contrasting with 18.2% in the controls. Between the two treatment groups and the controls, there were significant differences in the effective rates (P < 0.01, and P < 0.05). There was also a significant difference (P < 0.05) between the two treatment groups. Measurements of Cr and BUN showed higher values for the treatment groups, with the comprehensive better than the simple group (P < 0.05). Sera Na, urine Na/K were different, P < 0.01 between pre- and post-treatment in the comprehensive group, and P < 0.05 in the simple group. The treatment groups' endotoxin content was also significantly reduced (P < 0.01, and P < 0.05), with the comprehensive group better than the simple group (P < 0.05). Portal vein blood flow and NO content significantly reduced (P < 0.05), as did ET-1 content (P < 0.01). There were no significant changes in the control group (P > 0.05). The comprehensive treatment group's pre- and post-treatment portal vein and splenic vein blood flows showed a positive correlation to NO, ET-1 and endotoxin contents. CONCLUSION: When treating refractory cirrhotic ascites complicated with azotemia, Shehuang Paste combined with Chinese herbal dialysis is better than Shehuang Paste alone for ascites resolution, azotemia, and endotoxin elimination. However, both methods on their own were also effective for reducing portal and splenic vein blood flow, and lowering the contents of NO, ET-1 in the two treatment groups.

[Development and application of multiplex-PCR for identification of Actinobacillus pleuropneumoniae].

A multiplex-PCR assay was developed to identify Actinobacillus pleuropneumoniae (App). Two pairs of polymerase chain reaction (PCR) primers were designed for the 16S rRNA and the apxlVA gene, which is specific to all serotypes of App. Two PCR products of 692bp and 363bp were obtained, from the 16S rRNA and the apxlVA gene respectively, for 27 reference A. pleuropneumoniae strains. Only the 692bp fragment was amplified for closely related strains including A. lignieresii. Using the designed primers, the method is capable of detecting A. pleuropneumoniae of as low as 1.3 x 10(3) CFU or 9pg DNA. For 302 suspected isolates, this multiplex-PCR method correctly identified 4 A. pleuropneumoniae strains. The result suggests the use of the multiplex-PCR for routine identification of App.

Screening of single-stranded DNA (ssDNA) aptamers against a zearalenone monoclonal antibody and development of a ssDNA-based enzyme-linked oligonucleotide assay for determination of zearalenone in corn.

A hypotoxic immunosorbent assay for the detection of zearalenone (ZEN) was developed, by identifying a single-stranded DNA (ssDNA) aptamer with high specificity and affinity for a ZEN monoclonal antibody (mAb-ZEN). ssDNA aptamers, which could mimic ZEN epitopes, were identified using the modified systematic evolution of ligands by an exponential enrichment (SELEX) technique. The purified mAb-ZEN was coated on microtiter plates as a target recognized by the random oligonucleotide ssDNA library. The binding affinity between the aptamers and mAb-ZEN during each round was measured by the biotin­streptavidin­horseradish peroxidase system. During 15 rounds of screening, an increasing binding affinity was observed. The enriched ssDNA library binding to mAb-ZEN with high affinity was cloned, sequenced, and analyzed. One aptamer (number 46), which displays the highest affinity and specificity for the mAb-ZEN, was used to establish an indirect competition enzyme-linked oligonucleotide assay (ELONA) to measure the ZEN concentration in corn. Under optimal conditions, the regression equation for quantification of ZEN was y = −0.0778x + 0.713 (R2 = 0.9981). The detection limit and IC50 were 0.01 and 0.2 ng/mL, respectively, with a working range of 0.03­2.5 ng/mL. The recovery rates of the spiked samples in the ELONA ranged from 95 to 105%. Aptamers, which can mimic many types of low-weight analytes in agricultural products, could serve as surrogates for the development of hypotoxic, environmentally friendly immunological detection methods.

The expression of cross-linked elastin by rabbit blood vessel smooth muscle cells cultured in polyhydroxyalkanoate scaffolds.

In order to evaluate the possibility for making artificial blood vessels, a series of microbial copolyesters poly(3-hydroxybutyrate-co-4-hydroxybutyrate)s (P3HB4HB)s containing 0-40 mol% 4-hydroxybutyrate (4HB) were studied for growth and formation of elastin of rabbit blood vessel smooth muscle cells (RaSMCs) incubated on the solvent-casting polyester films. Porous scaffolds of all P3HB4HBs except P(HB-40 mol% 4HB) were used to compare their potentials as materials for tissue engineering blood vessel (TEBV). The polyesters were studied using static contact angles, differential scanning calorimetry (DSC), cell count kit-8 (CCK-8) and Fastin elastin assays. Simultaneously, SEM, H&E and DAPI staining were employed to study cell morphology and cell growth in the polyester scaffolds. Viability of rabbit blood vessel smooth muscle cells (RaSMCs) grown on P(HB-7 mol% 4HB) films was the strongest among all other tested polyester films during the whole growth process. H&E and DAPI staining clearly suggested good cells' growth and even confluent growth in the P3HB4HB scaffolds. Fastin elastin assay demonstrated that 4HB component in the P3HB4HB copolymer benefited the elastin formation and accumulation. P(HB-20 mol% 4HB) showed a 160% more elastin production compared with that on the well-studied poly(3-hydroxybutyrate-co-12 mol% 3-hydroxyhexanoate) (PHBHHx) incubated under the same conditions. Since the P3HB4HB has adjustable elasticity and strength, combined with its ability to induce elastin formation, it can be regarded as a useful material for developing into a material for artificial blood vessels.