槲皮素在介孔分子筛孔道中的组装与缓释研究

采用后组装法将非水溶性的治疗冠心病药物槲皮素组装进入MCM-41介孔分子筛的孔道中,药物组装率达37%[m(药物)/m(药物总量)],用XRD,扫描电镜和IR对药物组装体进行了表征;通过测定组装体在体外模拟人工小肠液中的溶出速率,表明制得了槲皮素/MCM-41缓释体系。     

Materials and Methods

首次描述了葫芦科赤     

Exploring the Antibacterial and Antifungal Potential of Jellyfish-Associated Marine Fungi by Cultivation-Dependent Approaches

Fungi isolated from marine invertebrates are of considerable importance as new promising sources of unique secondary metabolites with significant biomedical potential. However, the cultivable fungal community harbored in jellyfish was less investigated. In this work, we seek to recover symbiotic fungi from different tissues of jellyfish Nemopilema nomurai. A total of seven morphotypes were isolated, which were assigned into four genera (Aspergillus, Cladosporium, Purpureocillium, and Tilletiopsis) from two phyla (Ascomycota and Basidiomycota) by comparing the rDNA-ITS sequences with the reference sequences in GenBank. The most fungi were found in the inner tissues of subumbrella. Two of the cultivation-independent procedures, changing media type and co-cultivation, were employed to maximize the complexity of metabolites. Thus, thirteen EtOAc gum were obtained and fingerprinted by High Performance Liquid Chromatography (HPLC) equipped with a photodiode array (PDA) detector. Antibacterial and antifungal activities of these complex mixtures were tested against a panel of bacterial and fungal pathogens. The antimicrobial results showed that all of the 13 EtOAc extracts displayed different levels of antibacterial activity, three of which exhibited strong to significant antibacterial activity to the bacterial pathogens Staphylococcus aureus and Salmonella entrica. Antifungal activity indicated that the EtOAc extracts from pure culture of Aspergillus versicolor and co-culture of A. versicolor and Tilletiopsis sp. in rice media were promising for searching new compounds, with the maximal mycelial growth inhibition of 82.32% ± 0.61% for Rhizoctonia solani and 48.41% ± 11.02% for Botrytis cinerea at 200 μg/ml, respectively. This study is the first report on the antibacterial and antifungal activity of jellyfish-associated fungi and allows the first sight into cultivable fungal community residing in jellyfish. Induced metabolites by cultivation-dependent approaches provides a new reservoir for drug discovery from jellyfish-derived fungi.     

Quantification of Enteric Viruses,Pathogen Indicators,and Salmonella Bacteria in Class B Anaerobically Digested Biosolids by Culture and Molecular Methods

The most common class B biosolids in the United States are generated by mesophilic anaerobic digestion (MAD), and MAD biosolids have been used for land application. However, the pathogen levels in MAD biosolids are still unclear, especially with respect to enteric viruses. In this study, we determined the occurrence and the quantitative levels of enteric viruses and indicators in 12 MAD biosolid samples and of Salmonella enterica in 6 MAD biosolid samples. Three dewatered biosolid samples were also included in this study for purposes of comparison. Human adenoviruses (HAdV) had the highest gene levels and were detected more frequently than other enteric viruses. The gene levels of noroviruses (NV) reported were comparable to those of enteroviruses (EV) and human polyomaviruses (HPyV). The occurrence percentages of HAdV, HAdV species F, EV, NV GI, NV GII, and HPyV in MAD samples were 83, 83, 42, 50, 75, and 58%, respectively. No hepatitis A virus was detected. Infectious HAdV was detected more frequently than infectious EV, and all infectious HAdV were detected when samples were propagated in A549 cells. Based on most-probable-number (MPN) analysis, A549 cells were more susceptible to biosolid-associated viruses than BGM cells. All indicator levels in MAD biosolids were approximately 10⁴ MPN or PFU per gram (dry), and the dewatered biosolids had significantly higher indicator levels than the MAD biosolids. Only two MAD samples tested positive for Salmonella enterica, where the concentration was below 1.0 MPN/4 g. This study provides a broad comparison of the prevalence of different enteric viruses in MAD biosolids and reports the first detection of noroviruses in class B biosolids. The observed high quantitative and infectivity levels of adenoviruses in MAD biosolids indicate that adenovirus is a good indicator for the evaluation of sludge treatment efficiency.Over the last decade, thousands of people in the United States have been infected with waterborne diseases, a large number of whom were hospitalized. Most of the waterborne disease outbreaks in the United States that occurred between 1991 and 2004 were related to microbial agents, i.e., viruses, bacteria, and parasites (5, 30). The majority of the outbreaks involved unidentified agents, and the Environmental Protection Agency (EPA) suspects that many of the outbreaks due to unidentified sources were caused by enteric viruses (46). Indeed, viruses have a high potential for groundwater pollution due to their small size and low die-off rates. The occurrence of enteric viruses in groundwater has been reported (1, 7, 12, 17). In the United States, approximately 5.6 million dry tons of biosolids are generated annually and 60% of the biosolids are applied to land (36).Several studies have reported the occurrence of enteric viruses in biosolids (18, 35, 47); however, information on the quantity and infectivity of enteric viruses in biosolids is still limited, and most studies focused solely on enteroviruses (41). Few studies have reported the levels of human adenoviruses (HAdV) in biosolids (6, 47), and no quantitative results have yet been reported on some of the emerging viruses, such as hepatitis A virus (HAV) and noroviruses (NV). Also, only one or two types of enteric viruses were quantified in the previous studies; therefore, it is hard to determine and compare the prevalence of different types of enteric virus in biosolids, since the samples and sample processing methods varied from study to study. A few studies focused on the viral infectivity of biosolids, and the results showed that infectious astrovirus and enteroviruses were still present in the treated biosolids (9, 18, 42). However, no results on the occurrence of adenoviruses in biosolids have been reported.PCR techniques have been used in most of the recent environmental virology studies. In comparing these techniques to cell culture, the main advantages of PCR methods for virus detection are fast results, less labor intensiveness, high specificity and sensitivity, and the capability of detecting difficult-to-culture or nonculturable viruses (for examples, human noroviruses and adenovirus 40/41). Quantitative real-time PCR (qPCR), which is considered the latest advancement in PCR technology, can provide both qualitative and quantitative results. However, PCR results may not reflect the infectivity of the samples since PCR only detects the genes of the pathogens; therefore, integrated cell culture-PCR (ICC-PCR) was developed to identify the specific infectious enteric viruses. ICC-PCR has been used to detect infectious enteric viruses in river water, tap water, beach water, and wastewater effluent samples (28, 29, 39, 50). However, Buffalo green monkey (BGM) cell culture, currently recommended by the EPA, has been compared with other cell lines, such as A549 and PLC/PRC/5 (28, 39), and the results showed that enteric viruses were propagated better with these cell lines than with BGM cells.The main objective of this work was to investigate the occurrence and the quantitative levels of the enteric viruses in class B mesophilic anaerobically digested (MAD) biosolid samples by molecular and cell culture methods. These results can be used for risk assessment at biosolid application sites. Also, enteric viruses have been suggested as fecal source tracking indicators (21, 32), and the levels of enteric viruses in biosolids reported in this study would be useful for the determination of which enteric virus is a better fecal source tracking indicator at biosolid application sites. MAD biosolids were chosen since they are the most common type of class B biosolid produced in the United States (47). Three dewatered biosolid samples were also included for comparison purposes. The levels of human adenovirus (HAdV), adenovirus type 40/41 (HAdV 40/41), enterovirus (EV), norovirus GI (NV GI) and NV GII, human polyomavirus (HPyV), and hepatitis A virus (HAV) were quantified by qPCR methods. BGM and A549 cell lines were used to quantify the infectious viruses in the biosolids, and the effectiveness of these two cell lines'' ability to propagate infectious viruses was compared. The occurrence of infectious EV and HAdV in biosolids was determined by ICC-PCR, and the serotypes of the infectious adenoviruses propagated on A549 were further determined. The levels of pathogen indicators and Salmonella enterica are also presented in this study.     

Quantification of Polysaccharides in Vegetable Raw Materials and Lignin Preparations

The phenol-sulfuric acid method has been studied for its capability to analyze vegetable raw materials. This method has made it possible to determine rather simply and with high accuracy polysaccharides in both vegetable raw materials (agricultural vegetables, softwood, and hardwood), and in various lignin preparations (the laboratory-scale and technical). The method is based on the color reaction of monosaccharides with phenol in the presence of concentrated sulfuric acid. The developed modified phenol-sulfuric acid method is universal because allows for the detection of polysaccharides in the samples with both high and low polysaccharide content, i.e., in vegetable raw materials and lignin preparations, respectively. The method is highly sensitive; it is possible to analyze monosaccharides in the mixture at the concentration of 1 × 10^–4 mol L^–1 on average. The hydrolysate volume of 0.25 mL that is ten-times diluted is enough for analysis. The duration of the hydrolysate analysis including the mix preparation, recording of the spectrum, and calculation by the formula does not exceed 30 min. The method can be used for the analysis of the chemical composition of renewable vegetable raw materials when developing technologies for obtaining alternative energy sources.     

Combined Use of Cultivation-Dependent and Cultivation-Independent Methods Indicates that Members of Most Haloarchaeal Groups in an Australian Crystallizer Pond Are Cultivable

Haloarchaea are the dominant microbial flora in hypersaline waters with near-saturating salt levels. The haloarchaeal diversity of an Australian saltern crystallizer pond was examined by use of a library of PCR-amplified 16S rRNA genes and by cultivation. High viable counts (10⁶ CFU/ml) were obtained on solid media. Long incubation times (≥8 weeks) appeared to be more important than the medium composition for maximizing viable counts and diversity. Of 66 isolates examined, all belonged to the family Halobacteriaceae, including members related to species of the genera Haloferax, Halorubrum, and Natronomonas. In addition, isolates belonging to a novel group (the ADL group), previously detected only as 16S rRNA genes in an Antarctic hypersaline lake (Deep Lake), were cultivated for the first time. The 16S rRNA gene library identified the following five main groups: Halorubrum groups 1 and 2 (49%), the SHOW (square haloarchaea of Walsby) group (33%), the ADL group (16%), and the Natronomonas group (2%). There were two significant differences between the organisms detected in cultivation and 16S rRNA sequence results. Firstly, Haloferax spp. were frequently isolated on plates (15% of all isolates) but were not detected in the 16S rRNA sequences. Control experiments indicated that a bias against Haloferax sequences in the generation of the 16S rRNA gene library was unlikely, suggesting that Haloferax spp. readily form colonies, even though they were not a dominant group. Secondly, while the 16S rRNA gene library identified the SHOW group as a major component of the microbial community, no isolates of this group were obtained. This inability to culture members of the SHOW group remains an outstanding problem in studying the ecology of hypersaline environments.     

Cultivation-Dependent and -Independent Approaches for Determining Bacterial Diversity in Heavy-Metal-Contaminated Soil

In recent years, culture-independent methods have been used in preference to traditional isolation techniques for microbial community analysis. However, it is questionable whether uncultured organisms from a given sample are important for determining the impact of anthropogenic stress on indigenous communities. To investigate this, soil samples were taken from a site with patchy metal contamination, and the bacterial community structure was assessed with a variety of approaches. There were small differences in microscopic epifluorescence bacterial counts. Denaturing gradient gel electrophoresis (DGGE) profiles of 16S rRNA gene fragments (16S-DGGE) amplified directly from soil samples were highly similar. A clone library generated from the most contaminated sample revealed a diverse bacterial community, which showed similarities to pristine soil communities from other studies. However, the proportion of bacteria from the soil samples that were culturable on standard plate-counting media varied between 0.08 and 2.2%, and these values correlated negatively with metal concentrations. The culturable communities from each sample were compared by 16S-DGGE of plate washes and by fatty acid profiling of individual isolates. Each approach indicated that there were considerable differences between the compositions of the culturable communities from each sample. DGGE bands from both culture-based and culture-independent approaches were sequenced and compared. These data indicated that metal contamination did not have a significant effect on the total genetic diversity present but affected physiological status, so that the number of bacteria capable of responding to laboratory culture and their taxonomic distribution were altered. Thus, it appears that plate counts may be a more appropriate method for determining the effect of heavy metals on soil bacteria than culture-independent approaches.     

大型真菌分子生物学实验材料的保存方法介绍

介绍了几种保存大型真菌分子生物学实验材料的方法 ,用这几种方法所保存的材料提取的基因组核糖体脱氧核糖核酸 (DNA)质量适于系统生物学及其他分子生物学研究之用。     

Quantification of Nonproteolytic Clostridium botulinum Spore Loads in Food Materials

We have produced data and developed analysis to build representations for the concentration of spores of nonproteolytic Clostridium botulinum in materials that are used during the manufacture of minimally processed chilled foods in the United Kingdom. Food materials are categorized into homogenous groups which include meat, fish, shellfish, cereals, fresh plant material, dairy liquid, dairy nonliquid, mushroom and fungi, and dried herbs and spices. Models are constructed in a Bayesian framework and represent a combination of information from a literature survey of spore loads from positive-control experiments that establish a detection limit and from dedicated microbiological tests for real food materials. The detection of nonproteolytic C. botulinum employed an optimized protocol that combines selective enrichment culture with multiplex PCR, and the majority of tests on food materials were negative. Posterior beliefs about spore loads center on a concentration range of 1 to 10 spores kg⁻¹. Posterior beliefs for larger spore loads were most significant for dried herbs and spices and were most sensitive to the detailed results from control experiments. Probability distributions for spore loads are represented in a convenient form that can be used for numerical analysis and risk assessments.     

重组人白介素-11蛋白浓度两种测定方法比较

在基因工程蛋白药物开发研究过程中,选定恰当、准确的蛋白浓度测定方法非常重要。本文比较Lowry法和Bradford法测定重组人白介素-11蛋白浓度结果,认为选用Bradford法是可行的,但中国药典三部蛋白浓度测定方法中没有收录Bradford法,建议在中国药典三部修订时收录该方法。     

Identification of Cryptosporidium felis in a Cow by Morphologic and Molecular Methods

Apicomplexan Cryptosporidium parasites infect a wide range of vertebrate hosts. While some species are limited to a single host group, such as Cryptosporidium baileyi, which infects chickens, other species of this genus, such as C. parvum, infect a wide range of mammalian species from mice to humans. During an investigation of Cryptosporidium infection in cattle on a farm in northern Poland, we identified an infection caused by C. felis, in addition to known infections with C. muris and C. parvum. This new infection was identified based on the size of the oocysts (mean size, 4.3 ± 0.4 μm; range, 3.5 to 5.0 μm), as well as by analysis of the molecular sequence of the variable region of the small-subunit rRNA. This finding demonstrates the complex host specificity and circulation in the environment of Cryptosporidium species.     

Comparison of Bacterial Extracellular Polymer Extraction Methods

Five different bacterial extracellular polymer extraction methods and a combination of two of these methods were compared on cultures of activated sludge, synthetic activated sludge, and Klebsiella aerogenes. High-speed centrifugation was the most effective extraction method for the K. aerogenes culture, based on the comparatively small amount of cell disruption and the relatively high extracellular polymer yield. Steaming treatment was the most effective extraction method for the activated sludges, since it released a significant quantity of extracellular polymers from the flocs and caused less cellular disruption than ethylenediaminetetraacetic acid and sodium hydroxide treatments. Sodium hydroxide treatment caused extensive disruption in all cultures. Ultrasonication released low concentrations of extracellular polymers from all cultures. However, it caused no significant cell disruption and therefore may be useful as a preliminary treatment in conjunction with another extraction method.     

Molecular Detection, Quantification, and Diversity Evaluation of Microalgae

This study reviews the available molecular methods and new high-throughput technologies for their practical use in the molecular detection, quantification, and diversity assessment of microalgae. Molecular methods applied to other groups of organisms can be adopted for microalgal studies because they generally detect universal biomolecules, such as nucleic acids or proteins. These methods are primarily related to species detection and discrimination among various microalgae. Among current molecular methods, some molecular tools are highly valuable for small-scale detection [e.g., single-cell polymerase chain reaction (PCR), quantitative real-time PCR (qPCR), and biosensors], whereas others are more useful for large-scale, high-throughput detection [e.g., terminal restriction length polymorphism, isothermal nucleic acid sequence-based amplification, loop-mediated isothermal amplification, microarray, and next generation sequencing (NGS) techniques]. Each molecular technique has its own strengths in detecting microalgae, but they may sometimes have limitations in terms of detection of other organisms. Among current technologies, qPCR may be considered the best method for molecular quantification of microalgae. Metagenomic microalgal diversity can easily be achieved by 454 pyrosequencing rather than by the clone library method. Current NGS, third and fourth generation technologies pave the way for the high-throughput detection and quantification of microalgal diversity, and have significant potential for future use in field monitoring.     

银杏内酯C分子烙印聚合物的制备

以混合银杏内酯为模板分子,它们与单体AA预识别的过程中,利用其在识别性能上的差异,制备了对银杏内酯C有优先选择识别作用的分子烙印聚合物.用红外光谱表征了分子烙印聚合物,并通过紫外光谱和高效液相色谱对其选择吸附性能进行分析.结果表明,所制备的分子烙印聚合物的最大吸附量为15.5 mg/g,混合银杏内酯经MIP选择性吸附后,银杏内酯C的含量提高了12.16%.     

Evaluation of Herpes Simplex Detection in Corneal Scrapings by Three Molecular Methods

Herpes simplex virus (HSV)-1 keratitis (HSK) is a sight-threatening ocular infection with worldwide occurrence. A prompt laboratory diagnosis is often very useful. The purpose of this study was to evaluate molecular methods as rapid diagnostic tools compared with cell culture of HSK. Corneal scrapings from patients with clinically suspected HSK were tested by direct immunofluorescence assay (IFA) for HSV-1 antigen and by polymerase chain reaction (PCR) for HSV-1 DNA, and an attempt for viral isolation was performed on Vero cell line culture. Positive samples by cell culture were 20.8%, whereas PCR was positive in 29.2%, and IFA was positive in 33.3%. IFA had better sensitivity (80%) and negative predictive value (81.8%) than PCR (70% and 76.9%, respectively); however, PCR had better specificity (71.4%) and positive predictive value (63.6%). This indicates that a combination of cell culture, IFA and PCR constitutes the best set of tools for diagnosis of clinically suspected cases of HSK. Documented infection can be further assessed by cell-culture technique or PCR depending laboratory availability.