The Cd resistant mechanism of Proteus mirabilis Ch8 through immobilizing and detoxifying.

Cadmium (Cd) pollution is a serious global environmental problem, which requires a global concern and practical solutions. Microbial remediation has received widespread attention owing to advantages, such as environmental friendliness and soil amelioration. However, Cd toxicity also severely deteriorates the remediation performance of functional microorganisms. Analyzing the mechanism of bacterial resistance to Cd stress will be beneficial for the application of Cd remediation. In this study, the bacteria strain, up to 1400 mg/L Cd resistance, was employed and identified as Proteus mirabilis Ch8 (Ch8) through whole genome sequence analyses. The results indicated that the multiple pathways of immobilizing and detoxifying Cd maintained the growth of Ch8 under Cd stress, which also possessed high Cd extracellular adsorption. Firstly, the changes in surface morphology and functional groups of Ch8 cells were observed under different Cd conditions through SEM-EDS and FTIR analyses. Under 100 mg/L Cd, Ch8 cells exhibited aggregation and less flagella; the Cd biosorption of Ch8 was predominately by secreting exopolysaccharides (EPS) and no significant change of functional groups. Under 500 mg/L Cd, Ch8 were present irregular polymers on the cell surface, some cells with wrapping around; the Cd biosorption capacity exhibited outstanding effects (38.80 mg/g), which was mainly immobilizing Cd by secreting and interacting with EPS. Then, Ch8 also significantly enhanced the antioxidant enzyme activity and the antioxidant substance content under different Cd conditions. The activities of SOD and CAT, GSH content of Ch8 under 500 mg/L Cd were significantly increased by 245.47%, 179.52%, and 241.81%, compared to normal condition. Additionally, Ch8 significantly induced the expression of Acr A and Tol C (the resistance-nodulation-division (RND) efflux pump), and some antioxidant genes (SodB, SodC, and Tpx) to reduce Cd damage. In particular, the markedly higher expression levels of SodB under Cd stress. The mechanism of Ch8 lays a foundation for its application in solving soil remediation.

Wild Cordyceps proteins reinforce intestinal epithelial barrier through MAPK/NF-κB pathway in MRL/lpr mice.

OBJECTIVES: The effects of wild Cordyceps proteins (WCPs) on the gut microbiota and the immune system of MRL/lpr mice were studied. METHODS: The effects of WCP on serum metabolic indexes (total triglyceride, total cholesterol, low-density lipoprotein cholesterol, and high-density lipoprotein cholesterol) content was measured by a biochemical analyzer. CD4+, CD8+ cells, intestinal inflammation, and intestinal barrier function in MRL/lpr mice were measured by flow cytometry, 16S ribosomal RNA, western blot, and quantitative real-time polymerase chain reaction RT-PCR. KEY FINDINGS: The results showed that after the intervention of WCP, the content of CD4+ cells in lupus mice increased, and the levels of proinflammatory cytokines were down-regulated, such as tumor necrosis factor-α and interleukin-6. Secondly, WCP up-regulated the proteins and mRNA levels of ZO-1, Claudin-1, and Occludin. Thirdly, it also increased the Firmicutes/Bacteroidetes ratio and the abundance of Oscillospirales, Lachnospirales, Lachnospiraceae, and Clostridia, as well as negatively regulated the MAPK/NF-кB signaling pathway in lupus nephritis (LN) mice. CONCLUSIONS: These findings suggested that WCP may improve the symptoms of LN by altering immune factors and the intestinal barrier.

Polysaccharides from natural resource: ameliorate type 2 diabetes mellitus via regulation of oxidative stress network.

Diabetes mellitus (DM) is a group of metabolic diseases characterized by hyperglycemia that can occur in children, adults, elderly people, and pregnant women. Oxidative stress is a significant adverse factor in the pathogenesis of DM, especially type 2 diabetes mellitus (T2DM), and metabolic syndrome. Natural polysaccharides are macromolecular compounds widely distributed in nature. Some polysaccharides derived from edible plants and microorganisms were reported as early as 10 years ago. However, the structural characterization of polysaccharides and their therapeutic mechanisms in diabetes are relatively shallow, limiting the application of polysaccharides. With further research, more natural polysaccharides have been reported to have antioxidant activity and therapeutic effects in diabetes, including plant polysaccharides, microbial polysaccharides, and polysaccharides from marine organisms and animals. Therefore, this paper summarizes the natural polysaccharides that have therapeutic potential for diabetes in the past 5 years, elucidating their pharmacological mechanisms and identified primary structures. It is expected to provide some reference for the application of polysaccharides, and provide a valuable resource for the development of new diabetic drugs.

Cordyceps proteins alleviate lupus nephritis through modulation of the STAT3/mTOR/NF-кB signaling pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: Cordyceps is a parasitic edible fungus, which is a unique Chinese medicinal material. It has been reported to have immunomodulatory effects and use in kidney disease. Especially, Cordyceps has been used in the treatment of lupus nephritis (LN). AIM OF STUDY: Cordyceps proteins (CP) have a favorable bidirectional immunomodulatory functions and may have therapeutic potential for LN. However, the underlying molecular mechanism remains unknown. So this study aimed to examine the activities of CP in LN and possible mechanism. MATERIALS AND METHODS: So proteomics was performed to detect proteins components of Cordyceps, and analysis it. In addition, MRL/lpr mice were used to study the progression of LN. The MRL/lpr mice were fed either CP (i.g, 0.5, 1.0, 1.5 g/kg/d), prednisolone acetate (PA, i.g, 6 mg/kg/d), or Bailing capsule (BC, i.g, 0.75 g/kg/d) for 8 weeks. Hematoxylin-eosin (H&E), Periodic Acid Schif (PAS) and Masson's stainings, Immunofluorescence, and Immunohistochemistry were performed to verify the therapeutic effect of CP on MRL/lpr mice. The mechanism by CP alimerated LN was uncovered by Western blotting (WB) and Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) methods. RESULTS: Our results revealed that CP blocked proteinuria production and renal inflammatory infiltratation in MRL/lpr mice to reduce the renal fibrosis. In addition, CP worked better than BC which is artificial Cordyceps fungus powder in regulating proteinuria to urine creatinine ratio and interleukin-4(IL-4) protein amount. Especially, CP modulated the STAT3/mTOR/NF-кB signaling pathway in LN mice and brought a more pronounced lowering effect on the contents of IL-6 and IL-1ß than the PA. CONCLUSION: CP could be a potential anti-inflammatory immune product with strong regulatory effects and potency than BC and PA in nephritis therapeutics.

Regulation of the intestinal flora: A potential mechanism of natural medicines in the treatment of type 2 diabetes mellitus.

Diabetes mellitus comprises a group of heterogeneous disorders, which are usually subdivided into type 1 diabetes mellitus (T1DM) and type 2 diabetes mellitus (T2DM). Both genetic and environmental factors have been implicated in the onset of diabetes. Type 1 diabetes primarily involves autoimmune insulin deficiency. In comparison, type 2 diabetes is contributed by the pathological state of insulin deficiency and insulin resistance. In recent years, significant differences were found in the abundance of microflora, intestinal barrier, and intestinal metabolites in diabetic subjects when compared to normal subjects. To further understand the relationship between diabetes mellitus and intestinal flora, this paper summarizes the interaction mechanism between diabetes mellitus and intestinal flora. Furthermore, the natural compounds found to treat diabetes through intestinal flora were classified and summarized. This review is expected to provide a valuable resource for the development of new diabetic drugs and the applications of natural compounds.

[Comparison of endogenetic microbial community diversity between wild Cordyceps sinensis,artificial C. sinensis and habitat soil].

To obtain the difference of the fungal and bacterial community diversity between wild Cordyceps sinensis, artificial C. sinensis and their habitat soil, Illmina Hiseq high-throughput sequencing technology was applied. The results show that Proteobacteria was the dominant bacterial phylum in C. sinensis, Actinobacteria was the dominant bacterial phylum in soil microhabitat, Ophiocordyceps sinensis was the predominant dominant fungus of C. sinensis. The α diversity analysis showed that the fungal diversity of stroma was lower than other parts, and the fungal diversity of wild C. sinensis was lower than that of artificial C. sinensis. The ß diversity analysis showed that the fungal and bacterial community diversity of soil microhabitat samples was significantly different from that of C. sinensis. The fungal community diversity was less different between wild and artificial C. sinensis, especially in sclerotia. LEfSe analysis showed a lot of species diversity between wild and artificial C. sinensis. Those different species between wild C. sinensis, artificial C. sinensis and their habitat soil provide ideas for further research on breed and components of C. sinensis.

Application for proteomics analysis technology in studying animal-derived traditional Chinese medicine: A review.

Different therapeutically active ingredients, from plants, animals, and mineral sources, are prescribed as traditional Chinese medicines (TCM). TCMs, from animal sources, are rich in proteins and peptides. Different advanced proteomics technologies, such as two-dimensional gel electrophoresis (2-DE), multi-dimensional liquid chromatography (MDLC), matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF-MS), and isobaric tags for relative and absolute quantitation (iTRAQ), have been applied to analyze TCMs, from animal sources. This paper reviews the common proteomic techniques for analyzing animal - derived TCMs. Various scientific studies have reported the application of proteomics for locating drug targets, identifying active components, and elucidating the mechanisms of action of animal - derived TCMs. However, these researches are still at the preliminary stage. This review has also discussed the existing challenges and future directions in this field of research.

Overexpression of ribonucleotide reductase small subunit, RNRM, increases cordycepin biosynthesis in transformed Cordyceps militaris.

Cordycepin was the first adenosine analogue used as an anticancer and antiviral agent, which is extracted from Cordyceps militaris and hasn't been biosynthesized until now. This study was first conducted to verify the role of ribonucleotide reductases (RNRs, the two RNR subunits, RNRL and RNRM) in the biosynthesis of cordycepin by over expressing RNRs genes in transformed C. militaris. Quantitative real-time PCR (qRT-PCR) and western blotting results showed that the mRNA and protein levels of RNR subunit genes were significantly upregulated in transformant C. militaris strains compared to the control strain. The results of the HPLC assay indicated that the cordycepin was significantly higher in the C. militaris transformants carrying RNRM than in the wild-type strain, whereas the RNRML was preferentially downregulated. For the C. militaris transformant carrying RNRL, the content of cordycepin wasn't remarkably changed. Furthermore, we revealed that inhibiting RNRs with Triapine (3-AP) almost abrogated the upregulation of cordycepin. Therefore, our results suggested that RNRM can probably directly participate in cordycepin biosynthesis by hydrolyzing adenosine, which is useful for improving cordycepin synthesis and helps to satisfy the commercial demand of cordycepin in the field of medicine.

[SSR loci information analysis in transcriptome of Cordyceps sinensis].

To analysis the SSR loci information in the transcriptome of Cordyceps sinensis and develop SSR molecular markers,MISA(MicroSatellite) software was used to analyze the microsatellites information from 16 875 unigene sequences and SSR primer designed by Primer 3. 0. In total,5 899 SSRs were detected in 4 252 unigene with the distribution frequency of 34. 99%,which was represented by 74 repeat motifs and SSR loci occurred per 7 952 bp in length. In the SSRs,the mono-nucleotide was the most abundant repeat motif(42. 5%),followed by tri-nucleotide(34. 48%),C/G and CCG/CGG were the dominant repeat motifs,respectively. The number of repetitions of the six SSR repeat types was concentrated on 5 to 12 times,and the length was mostly less than 24 bp. A total of 12 282 pairs of primers were screened and selected 20 pairs of primers for validity detection randomly,10 pairs of primers amplified the expected specific bands,and primer P1 has significant polymorphism. Moreover,it was found that unigene containing SSR loci is mainly related to genetic and environmental functions after GO and KEGG annotation. In conclusion,these SSR loci in the transcriptome of O. sinensis are high in frequency,rich in primitive types,high in polymorphism,and highly available,which will provides abundant candidate molecular markers for its genetic diversity analysis,resource identification protection,and gene function research.

[Development and evaluation of a rapid PCR detection kit for Ophiocordyceps sinensis].

Ophiocordyceps sinensis is a valuable traditional Chinese medicine. Due to resource shortage, expensive price and huge market demand, there are many adulterants of O. sinensis in markets. Therefore, it is necessary to establish a rapid and effective method for distinguishing O. sinensis. Based on the species-specific PCR of O. sinensis, this study developed a detection kit by optimizing the components and evaluated the specificity, detection limit, repeatability and shelf life of the kit. The results showed that when the quality of O. sinensis accounted for more than 1/200 of that mixture, it could be detected successfully. Moreover, only O. sinensis could be amplified and glowed bright green fluorescence under ultraviolet light. The kit was still in effect when it was placed at 37 ℃ for three days, which indicated that it was stable and effective for one year stored in 4 ℃. The kit in the same batch under different operation conditions, and in different batch under the same operation conditions gave the same result and accuracy, which showed good repeatability of the kit. It is simple, rapid and accurate to distinguish O. sinensis from its adulterants using the kit, and lays the foundation for commercialization of traditional Chinese medicine fast detection kit.

[Identification of original species of Mantidis Oötheca (Sangpiaoxiao) based on DNA barcoding].

Both market research and literature reports both found that the ootheca of mantodea was all used as medicine. However, Chinese Pharmacopoeia only records the ootheca of three mantis species. The clinical use of ootheca unrecorded in Chinese Pharmacopoeia, will pose potential risks to drug safety. It's urgent to identify the origin of Mantidis Oötheca. The current researches about original animal in Mantidis Oötheca are based on morphology and unanimous. DNA barcoding fill gaps of the traditional morphological identification, which is widely used in animal classification studies. This study first use DNA barcoding to analyze genetic distance among different Mantidis Oötheca types, align COI sequences between mantis and Mantidis Oötheca and construct the phylogeny tree. The result confirmed that Tenodera sinensis and Hierodula patellifera were the origin insects of Tuanpiaoxiao and Heipiaoxiao, respectively, and Statilia maculate and Mantis religiosa were the origin insects of Changpiaoxiao.

Purification and characterization of a novel type i ribosome inactivating protein, pachyerosin, from Pachyrhizus erosus seeds, and preparation of its immunotoxin against human hepatoma cells.

Pachyrhizus erosus seeds have a high protein content and are used in China due to their cytotoxic effect. Here we report the biological and pharmacological activity of the protein extracts from P. erosus seeds. A novel ribosome-inactivating protein, pachyerosin, from P. erosus seeds was successively purified to homogeneity using ammonium sulfate precipitation, DEAE-sepharose FF, and Sephacryl S-200. Pachyerosin showed to be a type I ribosome-inactivating protein with a molecular mass of 29 kDa and an isoelectric point of 9.19. It strongly inhibited protein synthesis of rabbit reticulocyte lysate with an IC50 of 0.37 ng/mL and showed N-glycosidase activity on rat liver ribosomes with an EC50 of 85.9 pM. The N-terminal 27 amino acids of pachyerosin revealed a 60.71% sequence identity with abrin A from the seeds of Abrus precatorius. With the aim of targeting the delivery of pachyerosin, immunotoxin was prepared by conjugating pachyerosin with anti-human AFP monoclonal antibodies SM0736. The immunotoxin pachyerosin-SM0736 efficiently inhibited the growth of the human hepatoma cell line HuH-7 with an IC50 of 0.050 ± 0.004 nM, 2360 times lower than that of pachyerosin and 430 times lower than that of the immunotoxin against human gastric cancer cell line SGC7901. These results imply that pachyerosin may be used as a new promising anticancer agent.

[Preliminary study on correlation between diversity of soluble proteins and producing area of Cordyceps sinensis].

To analyze the content and type of soluble proteins in Cordyceps sinensis from different producing areas and processed with different methods with bradford method and 2-DE technology, in order to discover significant differences in soluble proteins in C. sinensis processed with different methods and from different producing areas. The preliminary study indicated that the content and diversity of soluble proteins were related to producing areas and processing methods to some extent.

[Corresponding relationship between Mantis and Mantidis Oötheca (Sangpiaoxiao)].

Mantidis Oötheca is commonly used Chinese medicine. Because of the used medicinal part is oötheca and many mantis species can yield ootheca, it is not possible to identify its original animal accurately. There is no unanimous conclusion about the corresponding relationship between Mantis and Mantidis Oötheca (Sangpiaoxiao). This relationship is the basis of the Mantidis Oötheca research. Our study combined the methods of artificial incubation oötheca and capture the living mantis to identify the species of Mantis and Mantidis Oötheca. The results showed that the origin insects of Mantidis Oötheca was Tenodera, Hierodula and Statilia genus insects. This has laid a foundation for further study of Mantidis Oötheca.

[Market survey and identification of hippocampus (Haima)].

OBJECTIVE: To clarify the commercial original species of hippocampus in the market. METHOD: Field survey and interview were applied to the investigation. RESULT: Present study identified the main commercial hippocampus of 13 species, including Pharmacopoeia contained four kinds of hippocampus (without Hippocampus japonicus) listed in Chinese Pharmacopoeia, and the identification method for the 13 kinds of commercially hippocampus was established. CONCLUSION: The further research on hippocampus should be strengthened for the establishment of hippocampal quality control standard.