Growing attention on the toxicity of Chinese herbal medicine: a bibliometric analysis from 2013 to 2022.

Introduction: Despite the clinical value of Chinese herbal medicine (CHM), restricted comprehension of its toxicity limits the secure and efficacious application. Previous studies primarily focused on exploring specific toxicities within CHM, without providing an overview of CHM's toxicity. The absence of a quantitative assessment of focal points renders the future research trajectory ambiguous. Therefore, this study aimed to reveal research trends and areas of concern for the past decade. Methods: A cross-sectional study was conducted on publications related to CHM and toxicity over the past decade from Web of Science Core Collection database. The characteristics of the publication included publication year, journal, institution, funding, keywords, and citation counts were recorded. Co-occurrence analysis and trend topic analysis based on bibliometric analysis were conducted on keywords and citations. Results: A total of 3,225 publications were analyzed. Number of annal publications increased over the years, with the highest number observed in 2022 (n = 475). The Journal of Ethnopharmacology published the most publications (n = 425). The most frequently used toxicity classifications in keywords were hepatotoxicity (n = 119) or drug-induced liver injury (n = 48), and nephrotoxicity (n = 40). Co-occurrence analysis revealed relatively loose connections between CHM and toxicity, and their derivatives. Keywords emerging from trend topic analysis for the past 3 years (2019-2022) included ferroptosis, NLRP3 inflammasome, machine learning, network pharmacology, traditional uses, and pharmacology. Conclusion: Concerns about the toxicity of CHM have increased in the past decade. However, there remains insufficient studies that directly explore the intersection of CHM and toxicity. Hepatotoxicity and nephrotoxicity, as the most concerned toxicity classifications associated with CHM, warrant more in-depth investigations. Apoptosis was the most concerned toxicological mechanism. As a recent increase in attention, exploring the mechanisms of ferroptosis in nephrotoxicity and NLRP3 inflammasome in hepatotoxicity could provide valuable insights. Machine learning and network pharmacology are potential methods for future studies.

Panax notoginseng transcription factor WRKY15 modulates resistance to Fusarium solani by up-regulating osmotin-like protein expression and inducing JA/SA signaling pathways.

BACKGROUND: Panax notoginseng (Burk) F. H. Chen is a valuable traditional Chinese medicinal plant, but its commercial production is seriously affected by root rot caused by some pathogenic fungi, including Fusarium solani. Nevertheless, the genetic breeding for disease resistance of P. notoginseng remains limited. The WRKY transcription factors have been revealed to play important roles in plant defense responses, which might provide an inspiration for resistance improvement in P. notoginseng. RESULTS: In this study, the regulatory mechanism of transcription factor PnWRKY15 on P. notoginseng resistance to F. solani infection was revealed. The suppressed expression of PnWRKY15 via RNA interference increased the sensitivity of P. notoginseng to F. solani and decreased the expression levels of some defense-related genes, including PnOLP1, which encodes an osmotin-like protein that confers resistance to F. solani. Ectopic expression of PnWRKY15 in the model plant tobacco significantly enhanced the resistance to F. solani. Moreover, the transcriptome sequencing analysis discovered that some pathogenesis-related genes were expressed at higher levels in the PnWRKY15-overexpressing tobacco than that in the wild-type tobacco. In addition, the jasmonic acid (JA) and salicylic acid (SA) signaling pathways were evidently induced by PnWRKY15-overexpression, that was evidenced by that the JA and SA contents were significantly higher in the PnWRKY15-overexpressing tobacco than that in the wild-type. Furthermore, PnWRKY15, which was localized in the nucleus, can trans-activate and up-regulate PnOLP1 expression according to the EMSA, yeast one-hybrid and co-expression assays. CONCLUSIONS: PnWRKY15 contributes to P. notoginseng resistance to F. solani by up-regulating the expression of resistance-related gene PnOLP1 and activating JA/SA signaling pathways. These findings will help to further elucidate the transcriptional regulatory mechanism associated with the P. notoginseng defense response to F. solani.

An MYB Transcription Factor Modulates Panax notoginseng Resistance Against the Root Rot Pathogen Fusarium solani by Regulating the Jasmonate Acid Signaling Pathway and Photosynthesis.

Root rot of Panax notoginseng, a precious Chinese medicinal plant, seriously impacts its sustainable production. However, the molecular regulatory mechanisms employed by P. notoginseng against root rot pathogens, including Fusarium solani, are still unclear. In this study, the PnMYB2 gene was isolated, and its expression was affected by independent treatments with four signaling molecules (methyl jasmonate, ethephon, salicylic acid, and hydrogen peroxide) as assessed by quantitative real-time PCR. Moreover, the PnMYB2 expression level was induced by F. solani infection. The PnMYB2 protein localized to the nucleus and may function as a transcription factor. When overexpressed in transgenic tobacco, the PnMYB2 gene conferred resistance to F. solani. Jasmonic acid (JA) metabolism and disease resistance-related genes were induced in the transgenic tobacco, and the JA content significantly increased compared with in the wild type. Additionally, transcriptome sequencing, Kyoto Encyclopedia of Genes and Genomes annotation enrichment, and metabolic pathway analyses of the differentially expressed genes in the transgenic tobacco revealed that JA metabolic, photosynthetic, and defense response-related pathways were activated. In summary, PnMYB2 is an important transcription factor in the defense responses of P. notoginseng against root rot pathogens that acts by regulating JA signaling, photosynthesis, and disease-resistance genes.

Serratia symbiotica Enhances Fatty Acid Metabolism of Pea Aphid to Promote Host Development.

Bacterial symbionts associated with insects are often involved in host development and ecological adaptation. Serratia symbiotica, a common facultative endosymbiont harbored in pea aphids, improves host fitness and heat tolerance, but studies concerning the nutritional metabolism and impact on the aphid host associated with carrying Serratia are limited. In the current study, we showed that Serratia-infected aphids had a shorter nymphal developmental time and higher body weight than Serratia-free aphids when fed on detached leaves. Genes connecting to fatty acid biosynthesis and elongation were up-regulated in Serratia-infected aphids. Specifically, elevated expression of fatty acid synthase 1 (FASN1) and diacylglycerol-o-acyltransferase 2 (DGAT2) could result in accumulation of myristic acid, palmitic acid, linoleic acid, and arachidic acid in fat bodies. Impairing fatty acid synthesis in Serratia-infected pea aphids either by a pharmacological inhibitor or through silencing FASN1 and DGAT2 expression prolonged the nymphal growth period and decreased the aphid body weight. Conversely, supplementation of myristic acid (C14:0) to these aphids restored their normal development and weight gain. Our results indicated that Serratia promoted development and growth of its aphid host through enhancing fatty acid biosynthesis. Our discovery has shed more light on nutritional effects underlying the symbiosis between aphids and facultative endosymbionts.

Ophiocordyceps lanpingensis polysaccharides alleviate chronic kidney disease through MAPK/NF-κB pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: Ophiocordyceps lanpingensis (O. lanpingensis) is a traditional ethno-medicine distributed in Eastern Himalayas, which has been used by local minorities to prevent and treat urinary diseases for hundreds of years. However, the corresponding active components and related pharmacological mechanism of such medication are not clear yet. AIMS OF THE STUDY: This study was performed to investigate the effects and potential mechanisms of O. lanpingensis polysaccharides (OLP) in the treatment of chronic kidney disease (CKD) based on our previous research results. MATERIALS AND METHODS: Methylation analysis was used to investigate the monosaccharide composition and glycosidic linkages in OLP. The animals were divided into the control group, CKD model group, losartan group and three different doses of OLP groups. The CKD mouse model was established by the adenine gavage. The histological changes of renal tissue were observed by Hematoxylin-eosin and Masson staining. Biochemical indicators, including blood urea nitrogen (BUN), serum creatinine (Scr), serum phosphorus (P), plasma calcium (Ca), reactive oxygen species (ROS), superoxide dismutase (SOD), glutathione peroxidase (GSH-PX) and malondialdehyde (MDA) were measured to evaluate the alleviation of CKD by OLP. Moreover, the expression levels of a series of cytokines related to the inflammation, apoptosis and fibrosis were analyzed to explore the possible mechanisms of OLP to treat CKD. RESULTS: OLP is composed of three kinds of monosaccharides. There are eight kinds of glycosidic linkages in OLP, among which →4)-Glcp-(1→ is the main linkage. OLP could significantly attenuate CKD in mice and the tubulointerstitial damage was recovered to almost normal after the treatment of OLP. Compared with the CKD model group, the levels of Scr, BUN, MDA, P in OLP treatment groups were significantly decreased; and the levels of SOD and Ca were increased after OLP treatment. Furthermore, OLP could reduce the oxidative stress of the renal tissues, decrease the expression levels of pro-inflammatory factors through TLR4-mediated MAPK and NF-κB pathway, inhibit the apoptosis of renal cells by MAPK pathway, and relieve the renal fibrosis by down-regulating the expression of TGF-ß1. CONCLUSIONS: OLP is composed of three kinds of monosaccharides and →4)-Glcp-(1→ is the main glycosidic linkage in the polysaccharide. OLP could ameliorate CKD in mice by declining the oxidative stress, inflammation, apoptosis and fibrosis in the kidneys. The study provided some evidences for the potential application of OLP in alleviating CKD.

[Synergistic effect on biosynthesis of Panax notoginseng saponins by overexpressing a transcription factor PnbHLH and RNA interference of cycloartenol synthase gene].

This study cloned the transcription factor gene PnbHLH which held an open reading frame of 966 bp encoding 321 amino acids. This study constructed the overexpression vector of transcription factor PnbHLH of Panax notoginseng. The combination of PnbHLH overexpression and RNAi of the key enzyme gene PnCAS involved in the phytosterol biosynthesis was achieved in P. notoginseng cells, thus exploring the biosynthetic regulation of P. notoginseng saponins(PNS) by the synergistic effect of PnbHLH overexpression and PnCAS RNAi. The results showed that the PnbHLH transcription factor interacted with the promoters of key enzyme genes PnDS, PnSS and PnSE in the biosynthetic pathway of PNS, and then regulated the expression levels of key enzyme genes and affected the biosynthesis of saponins indirectly. Further study indicated that the synergistic effect of PnbHLH overexpression and PnCAS RNAi was a more effective approach to regulate the biosynthesis of saponins. Compared with the wild type and PnCAS RNAi cells of P. notoginseng, the contents of total saponins and monomeric saponins(Rd, Rb_1, Re, Rg_1 and R_1) were increased to some extent in the cell lines of PnbHLH overexpression and PnCAS RNAi. This indicated that the two ways of forward regulation and reverse regulation of saponin biosynthesis showed superposition effect. This study explored a more rational and efficient regulation strategy of PNS biosynthesis based on the advantages of multi-point regulation of transcription factors as well as the down-regulation of by-product synthesis of saponins.

Is combined medication with natural medicine a promising therapy for bacterial biofilm infection?

Bacterial biofilms widely exist in nature and seriously threaten global public health. Biofilms always cause persistent infection and seriously aggravate the occurrence of antibiotic resistance, which makes the treatment of bacterial infection difficult. Current conventional therapies, such as antibiotics, bacteriophages and quorum sensing inhibitors, are widely used to combat biofilms. However, these therapies are inadequate for the safe and effective treatment of biofilms. Antibiotics often produce resistance in treated bacteria, and antibacterial peptides are easily decomposed by proteases, so their efficacy is reduced. These results indicate that the treatment of biofilms needs further improvement. Increasing evidence has shown that natural medicine therapies have significant inhibitory effects on biofilms. This review summarized and analyzed the efficacy characteristics and corresponding mechanisms of conventional and natural medicine therapies combatting biofilms. By comparison, the advantages and disadvantages of those therapies have been classified and interpreted, so we have inferred that combined medication with natural medicines will be a more effective strategy against biofilms. This review lays a promising foundation for the development of antibiofilm agents and provides novel thinking for the treatment of bacterial biofilm infections.

Antioxidant-related catalase CTA1 regulates development, aflatoxin biosynthesis, and virulence in pathogenic fungus Aspergillus flavus.

Reactive oxygen species (ROS) induce the synthesis of a myriad of secondary metabolites, including aflatoxins. It raises significant concern as it is a potent environmental contaminant. In Aspergillus flavus., antioxidant enzymes link ROS stress response with coordinated gene regulation of aflatoxin biosynthesis. In this study, we characterized the function of a core component of the antioxidant enzyme catalase (CTA1) of A. flavus. Firstly, we verified the presence of cta1 corresponding protein (CTA1) by Western blot analysis and mass-spectrometry based analysis. Then, the functional study revealed that the growth, sporulation and sclerotia formation significantly increased, while aflatoxins production and virulence were decreased in the cta1 deletion mutant as compared with the WT and complementary strains. Furthermore, the absence of the cta1 gene resulted in a significant rise in the intracellular ROS level, which in turn added to the oxidative stress level of cells. A further quantitative proteomics investigation hinted that in vivo, CTA1 might maintain the ROS level to facilitate the aflatoxin synthesis. All in all, the pleiotropic phenotype of A. flavus CTA1 deletion mutant revealed that the antioxidant system plays a crucial role in fungal development, aflatoxins biosynthesis and virulence.

A Pathogenesis-Related Protein-Like Gene Is Involved in the Panax notoginseng Defense Response to the Root Rot Pathogen.

Pathogenesis-related proteins (PRs) are a class of proteins that accumulate in response to biotic and abiotic stresses to protect plants from damage. In this study, a gene encoding a PR-like protein (PnPR-like) was isolated from Panax notoginseng, which is used in traditional Chinese herbal medicines. An analysis of gene expression in P. notoginseng indicated that PnPR-like was responsive to an infection by the root rot pathogen Fusarium solani. The expression of this gene was induced by several signaling molecules, including methyl jasmonate, ethephon, hydrogen peroxide, and salicylic acid. The PnPR-like-GFP fusion gene was transiently expressed in onion (Allium cepa) epidermal cells, which revealed that PnPR-like is a cytoplasmic protein. The purified recombinant PnPR-like protein expressed in Escherichia coli had antifungal effects on F. solani and Colletotrichum gloeosporioides as well as inhibited the spore germination of F. solani. Additionally, the in vitro ribonuclease (RNase) activity of the recombinant PnPR-like protein was revealed. The PnPR-like gene was inserted into tobacco (Nicotiana tabacum) to verify its function. The gene was stably expressed in T2 transgenic tobacco plants, which exhibited more RNase activity and greater disease resistance than the wild-type tobacco. Moreover, the transient expression of hairpin RNA targeting PnPR-like in P. notoginseng leaves increased the susceptibility to F. solani and decreased the PnPR-like expression level. In conclusion, the cytoplasmic protein PnPR-like, which has RNase activity, is involved in the P. notoginseng defense response to F. solani.

A transcriptome analysis uncovers Panax notoginseng resistance to Fusarium solani induced by methyl jasmonate.

BACKGROUND: Panax notoginseng is a famous Chinese herbal medicine, but the root rot disease mainly caused by Fusarium solani severely reduces the yield and quality of its medicinal materials. OBJECTIVE: The defense priming in P. notoginseng through exogenous application of signaling molecule will supply theoretical support for the exogenous regulation of disease resistance in P. notoginseng. METHODS: In this study, the exogenous application of methyl jasmonate (MeJA) increased P. notoginseng's resistance to F. solani. Furthermore, the P. notoginseng transcriptome during F. solani infection was investigated through next-generation sequencing to uncover the resistance mechanism of P. notogingseng induced by MeJA. RESULTS: The de novo assembly of transcriptome sequences produced 80,551 unigenes, and 36,771 of these unigenes were annotated by at least one database. A differentially expressed gene analysis revealed that a large number of genes related to terpenoid backbone biosynthesis, phenylalanine metabolism, and plant-pathogen interactions were predominantly up-regulated by MeJA. Moreover, jasmonic acid (JA) biosynthesis-related genes and the JA signaling pathway genes, such as linoleate 13S-lipoxygenase, allene oxide cyclase, allene oxide synthase, TIFY, defensin, and pathogenesis-related proteins, showed increased transcriptional levels after inoculation with F. solani. Notably, according to the gene expression analysis, JA and ethylene signaling pathways may act synergistically to positively regulate the defense responses of P. notoginseng to F. solani. CONCLUSION: JA signaling appears to play a vital role in P. notoginseng responses to F. solani infection, which will be helpful in improving the disease resistance of P. notoginseng cultivars as well as in developing an environmentally friendly biological control method for root rot disease.

Oleanane-Type Saponins Biosynthesis in Panax notoginseng via Transformation of ß-Amyrin Synthase Gene from Panax japonicus.

Oleanane-type saponins considered as the main medicinal ingredients in Panax japonicus are not found in Panax notoginseng. ß-Amyrin synthase (ßAS) was recognized as the first key enzyme in the biosynthetic branch of oleanane-type saponins. In this study, ßAS gene from P. japonicus ( PjßAS) was transferred into P. notoginseng cells. Along with PjßAS expression in the transgenic cells, the expression levels of several key enzyme genes related to triterpenoid saponins biosynthesis and the content of P. notoginseng saponins were also increased. Two oleanane-type saponins, chikusetsusaponin IV and chikusetsusaponin IVa, contained in P. japonicus were first discovered in transgenic P. notoginseng cells. This study successfully constructed a biosynthetic pathway of oleanane-type saponins in P. notoginseng by introducing just one gene into the species. On the basis of this discovery and previous studies, the common biosynthetic pathway of triterpenoid saponins in Panax genus may be unified to some extent.

Protective Effects of Ophiocordyceps lanpingensis on Glycerol-Induced Acute Renal Failure in Mice.

OBJECTIVE: Oxidative stress and immune response are associated with acute renal failure (ARF). Ophiocordyceps lanpingensis (OL) might be an antioxidant and immunopotentiator. In this study, we explored the protective effects of OL on glycerol-induced ARF. METHODS: Male mice were randomly divided into four groups, specifically, glycerol-induced ARF model group, low-dose OL-treated group (1.0 g/kg/d), high-dose OL-treated group (2.0 g/kg/d), and control group. Renal conditions were evaluated using kidney index, serum creatinine (Cr), blood urea nitrogen (BUN), and histological analysis. Rhabdomyolysis was monitored using creatine kinase (CK) level. Oxidative stress was determined using kidney tissue glutathione (GSH), malondialdehyde (MDA), and superoxide dismutase (SOD) levels. Immune status was evaluated using immune organ indices and immunoglobulin G (IgG) level. RESULTS: OL could relieve renal pathological injury and decrease the abnormal levels of kidney index, serum Cr, CK, BUN, and MDA, as well as increase the immune organ indices and the levels of IgG, GSH, and SOD. Treatment with a high dose of OL had more positive therapeutic effects on ARF than using a low dose of OL. CONCLUSION: OL could ameliorate renal dysfunction in glycerol-induced ARF in mice by inhibiting oxidative stress and enhancing immune response.

Molecular Cloning and Characterization of PnbHLH1 Transcription Factor in Panax notoginseng.

Panax notoginseng has been extensively used as a traditional Chinese medicine. In the current study, molecular cloning and characterization of PnbHLH1 transcription factor were explored in Panax notoginseng. The full length of the PnbHLH1 gene obtained by splicing was 1430 bp, encoding 321 amino acids. Prokaryotic expression vector pET-28a-PnbHLH1 was constructed and transferred into the BL21 prokaryotic expression strain. An electrophoretic mobility shift assay of PnbHLH1 protein binding to E-box cis-acting elements verified that PnbHLH1 belonged to the bHLH class transcription factor which could interact with the promoter region of the E-box core sequence. The expression levels of key genes involved in the biosynthesis of triterpenoid saponins in PnbHLH1 transgenic cells were higher than those in the wild cells. Similarly, the total saponin contents were increased in the PnbHLH1 transgenic cell lines compared with the wild cell lines. Such results suggest that the PnbHLH1 transcription factor is a positive regulator in the biosynthesis of triterpenoid saponins in Panax notoginseng.

Proteogenomic analysis and global discovery of posttranslational modifications in prokaryotes.

We describe an integrated workflow for proteogenomic analysis and global profiling of posttranslational modifications (PTMs) in prokaryotes and use the model cyanobacterium Synechococcus sp. PCC 7002 (hereafter Synechococcus 7002) as a test case. We found more than 20 different kinds of PTMs, and a holistic view of PTM events in this organism grown under different conditions was obtained without specific enrichment strategies. Among 3,186 predicted protein-coding genes, 2,938 gene products (>92%) were identified. We also identified 118 previously unidentified proteins and corrected 38 predicted gene-coding regions in the Synechococcus 7002 genome. This systematic analysis not only provides comprehensive information on protein profiles and the diversity of PTMs in Synechococcus 7002 but also provides some insights into photosynthetic pathways in cyanobacteria. The entire proteogenomics pipeline is applicable to any sequenced prokaryotic organism, and we suggest that it should become a standard part of genome annotation projects.

A preliminary metagenomic study of puer tea during pile fermentation.

BACKGROUND: Up to now, there has been no report on the taxonomic and functional analysis of the microbial community in fermenting puer tea by pyrosequencing. In this study, metagenomic pyrosequencing was first used in fermenting puer tea to delineate a relatively comprehensive overview of the microbial taxonomy while also preliminarily characterising the functional ontologies of microbial genes present in puer tea pile fermentation. RESULTS: A total of 251 738 pyrosequencing reads (9197 contigs and 145 402 singletons) were generated by pyrosequencing. Taxonomic analysis revealed three dominant bacterial phyla, Actinobacteria (30.08%), Proteobacteria (24.47%) and Firmicutes (20.23%), and one dominant eukaryotic phylum, Ascomycota (15.21%) [corrected]. A total of 58 664 hits were categorised into 28 functional subsystems based on the SEED database. Moreover, two categories, 'metabolism of terpenoids and polyketides' and 'biosynthesis of other secondary metabolites', were selectively analysed and 69 enzyme genes were presented in 16 pathways. CONCLUSION: The dominant microbes of puer tea fermentation were bacteria in the present study, and yeasts rather than moulds accounted for the overwhelming majority of Eukaryota. The analysis of functional genes and metabolic pathways will be helpful for further study of the mechanism of puer tea fermentation at molecular level.

[The construction of over-expression vector for Panax notoginseng SS gene and its transformation].

PNS (Panax notoginseng saponins) is the main medical bioactive component in Panax notoginseng. The medical value of PNS cannot be extended because of its low production. With the deep study of saponins biosynthetic pathway, the control of PNS biosynthesis through metabolic engineering has gradually become possible. In this study, the Squalene synthase (SS) over-expression vector was established. By the way of agrobacterium-mediated method, the vector was transfered and integrated into the Panax notoginseng genome. The result of the PCR detection and the saponin content detection shows that over-expression SS is able to produce high level of Panax notoginseng saponins, and confirms the regulatory function of SS in the biosynthesis of ginsenosides in Panax notoginseng. It provides a theoretical basis and technical basis for the construction of PNS homologous or heterologous efficient expression system in the future.

Study on the mechanism of acupoint sticking therapy with Chuan fuling for treatment of asthma model rats / 中国针灸

<p><b>OBJECTIVE</b>To investigate the mechanism of the acupoint sticking therapy with Chuanfuling for preventing and treating asthma.</p><p><b>METHODS</b>Thirty male SD rats were randomly divided into a control group (normal saline, p.i. +no acupoint sticking+ normal saline, spray inhalation), model group (normal saline with ovalbumin, p.i. +no acupoint sticking+ normal saline with ovalbumin, spray inhalation), and acupoint sticking group (normal saline with ovalbumin, p.i. +acupoint sticking with Chuan fuling+normal saline with ovalbumin, spray inhalation), 10 rats in each group. The incubation period of nodding breath, symptom of asthmatic attack, expression level of interleukin-4 mRNA (IL-4 mRNA) and interferon-gamma mRNA (IF-gamma mRNA), as well as pathological changes on the middle leaf of right lung, were observed in each group.</p><p><b>RESULTS</b>(1) Comparing with the control group, the model group was showed that the expression level of IL-4 mRNA in the peripheral blood cells (PBMC) was increased, while hyperemia, edema and eosinocyte (EOS) invasion of lung tissue was more serious (P < 0.01). (2) Comparing with the model group, the acupoint sticking group was showed that the expression level of IL-4 mRNA in PBMC was decreased, the incubation period of nodding breath was prolonged for induced asthma on the fifth and seventh time with lower frequency, while in the lung tissue EOS invasion was reduced (P < 0.05), but there were no significant changes on the hyperemia and edema (P > 0.05).</p><p><b>CONCLUSION</b>Acupoint sticking for treating asthma of model rats with Chuanfuling can inhibit the expression level of IL-4 mRNA in PBMC, and the release of the inflammatory mediator and cytokine from the EOS to the air passage, in order to reduce the injury of epithelial layer and high reaction on the air passage.</p>

High-throughput assay using a GFP-expressing replicon for SARS-CoV drug discovery.

The causative agent of severe acute respiratory syndrome (SARS) has been identified as a novel coronavirus, SARS-CoV. The development of rapid screening assays is essential for antiviral drug discovery. By using a cell line expressing a SARS-CoV subgenomic replicon, we developed a high-throughput assay and used it to screen small molecule compounds for inhibitors of SARS-CoV replication in the absence of live virus. The assay system involves minimal manipulation after assay set-up, facilitates automated read-out and minimizes risks associated with hazardous viruses. Based on this assay system, we screened 7035 small molecule compounds from which we identified 7 compounds with anti-SARS-CoV activity. We demonstrate that the compounds inhibited SARS-CoV replication-dependent GFP expression in the replicon cells and reduced SARS-CoV viral protein accumulation and viral RNA copy number in the replicon cells. In a SARS-CoV plaque reduction assay, these compounds were confirmed to have antiviral activity. The target of one of the hit compounds, C12344, was validated by the generation of resistant replicon cells and the identification of the mutations conferring the resistant phenotype. These compounds should be valuable for developing anti-SARS therapeutic drugs as well as research tools to study the mechanism of SARS-CoV replication.

[Lysosome of Eisenia fetida as biomarker of soil pollution].

The lysosome of Eisenia fetida can accumulate neutral red dye rapidly. When exposed to environment pollution, the membrane of the lysosome is easily to be cracked, and the dye shall leak to the cytoplasm gradually, making the cell red. Therefore, neutral red retention time (NRRT) can reflect the damaged degree of lysosome. In this paper, the NRRT of the lysosome in coelomocytes of E. fetida was determined after exposed to different concentrations of acetochlor and selenium. The results showed that with increasing exposure time and increasing concentration of acetochlor and Se, the NRRT was decreased significantly, and had significant correlations with the concentrations of acetochlor in soil and of accumulated Se in E. fetida's body. NRRT could be served as a biomarker of soil pollution, and a promising tool for the early warning of potential danger.

[Factors influencing the activity of fibrinolytic enzymes from earthworm, Eeisenia foetida].

OBJECTIVE: To study the factors influencing the activity of fibrinolytic enzymes from earthworm and to obtain the better way to extract fibrinolytic enzymes as well as keep its optimum activity. METHOD: 75% alcohol, 0.9% NaCl and 10% saccharose was used to extract the crude fibrinolytic enzymes from earthworm, the method of urokinase gelose-fibrin plate was used to measure the activity of fibrinolytic enzymes from earthworm. and the method of 3,3'-diaminobezidine tetrahydrochloride colorimetry to was used measure the content of selenium. The method use ts of measuring the content of arsenic was silver diethyldithiocarbamate colorimetry. RESULT: The fibrinolytin of earthworms reared with cattle soils had higher activity than that reared with garbage. The arsenic in the earthworm's body could improve the activity of earthworm's fibrinolytin. However, the selenium had litter influence on it. Among the three methods of extraction, the 75% alcohol one was the most efficient, the 0.9% NaCl was next, and the 10% saccharose was the lowest. The influence of dialysis on the activity of fibrinolytin was less than that of ultrafiltration, when the earthworm's fibrinolytin enzyme was further sublimated. CONCLUSION: The activity of the earthworm's fibrinolytin will be increased earthworm is reared with the fitting baits and when appropriate methods, of extraction and purification are used.