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Euryale ferox Salisb. is widely grown in China and Southeast Asia as a grain crop and medicinal plant. The composition, morphology, structure, physicochemical properties, thermal properties, and in vitro digestibility of North Euryale ferox seeds starch (NEFS), hybrid Euryale ferox seeds starch (HEFS), and South Euryale ferox seeds starch (SEFS) were studied.Of the varieties that were studied, the amylose content of NEFS (23.03%) was the highest. Starch granules of each variety were smooth, sharp, small, and had an average diameter of 2 μm. All three varieties were A-type crystals with crystallinity ranging from 26.42% to 28.17%. The degree of double helix and the short-range order ranged from 1.9006 to 2.5324 and 1.4294 to 1.6006, respectively. The high proportion of C1 region in NEFS (17.74%) and HEFS (17.66%) were found. Thermodynamic properties in North Euryale ferox seeds included the highest onset temperature (TThe results revealed that the low digestibility of NEFS was attributable to compact granules, high crystallinity, high degree of order, and strong thermal stability. These digestive, physicochemical, and thermodynamic properties provide information for the future application of Euryale ferox seed starch in the food industry. © 2022 Society of Chemical Industry.

Malignant tumors are a major cause of death, and their incidence is increasing worldwide. Although the survival rate for some cancers has improved, treatments for other malignant tumors are limited, and their mortality rate continues to increase. People with type 2 diabetes have a higher risk of malignant tumors and a higher mortality rate than those without diabetes. Metformin is a commonly used hypoglycemic drug. In recent years, a growing number of studies have indicated that metformin has antitumor effects and increases the sensitivity of malignant tumors to chemotherapy. However, the effect of metformin on different tumors is currently controversial, and the mechanism of metformin’s antitumor action is not fully understood. Insights into the effect of metformin on malignant tumors and the possible mechanism may contribute to the development of antitumor drugs., Video abstract Point your SmartPhone at the code above. If you have a QR code reader the video abstract will appear. Or use: https://youtu.be/MWKNn8872gg

Juan Jiang,* Danlan Pu,* Renzhi Hu, Mingyang Hu, Qinan Wu Department of Endocrinology and Nephrology, Chongqing University Cancer Hospital, School of Medicine, Chongqing University, Chongqing, 400030, People’s Republic of China*These authors contributed equally to this workCorrespondence: Qinan WuDepartment of Endocrinology and Nephrology, Chongqing University Cancer Hospital, School of Medicine, Chongqing University, Chongqing, People’s Republic of ChinaEmail wqn11@126.comObjective: To explore the efficacy of the hospital glycemic management system with information integration in patients with malignant tumors and hyperglycemia.Methods: Three hundred ninety-three patients diagnosed with malignant tumors with hyperglycemia and hospitalized in the non-endocrinology department of a specialized cancer hospital from March 2019 to November 2020 were recruited. All the patients were diagnosed and treated according to the clinical department and disease course. In total, 196 patients were divided into the control group, who received the conventional blood glucose management mode, and 197 patients were divided into the intervention group, who received the hospital glycemic management system with information integration. The average daily glucose levels were recorded before and after breakfast, lunch, and dinner, at bedtime and at night. The average glucose level, glucose compliance rate, hypoglycemia rate, hyperglycemia rate, glucose measurements per day, average number of hospitalization days and patient satisfaction were compared between the groups.Results: In the intervention group, the average glucose level was significantly lower than that in the control group (P< 0.05). The hyperglycemia and hypoglycemia rates in the intervention group were lower than those in the control group (P< 0.05). The glucose compliance rate in the intervention group was higher than that in the control group (P< 0.05). The highest blood glucose level in the intervention group was lower than that in the control group (P< 0.05), and the lowest blood glucose level was higher than that in the control group (P< 0.05). The glucose measurements per day in the intervention group were higher than those in the control group, and the average number of hospitalization days in the intervention group was lower than that in the control group (P< 0.05). Patient satisfaction in the intervention group was higher than that in the control group (P< 0.05).Conclusion: The hospital glycemic management system with information integration significantly improved the glycemic management of patients with malignant non-endocrine tumors and hyperglycemia, including their glucose level and glucose compliance rate, as well as patient satisfaction, and reduced the average number of hospitalization days and risk of hyperglycemia/hypoglycemia.Keywords: information integration, malignant tumor with hyperglycemia, hospital glycemic management system

Biosynthetic gene clusters (BGCs) are regions of a genome where genes involved in a biosynthetic pathway are in proximity. The origin and evolution of plant BGCs, and their role in specialised metabolism, remains unclear. We have assembled a chromosome-scale genome of Japanese catnip (Schizonepeta tenuifolia) and discovered a BGC that contains multiple copies of genes involved in four adjacent steps in the biosynthesis of p-menthane monoterpenoids. The BGC has an unprecedented bipartite structure, with mirrored biosynthetic regions separated by 260 kilobases. The bipartite BGC includes identical copies of a gene encoding an old yellow enzyme, a type of flavin-dependent reductase. In vitro assays and virus-induced gene silencing revealed this to encode the missing isopiperitenone reductase enzyme. This gene is from a completely different enzyme family to the isopiperitenone reductases from the closely related Mentha indicating convergent evolution of this pathway step. Phylogenomic analysis revealed that the BGC has emerged uniquely in the S. tenuifolia lineage and through insertion of pathway genes into a region rich in monoterpene synthases. The cluster gained its bipartite structure via an inverted duplication. The S. tenuifolia BGC has similarities to the recently described duplicated p-menthane biosynthesis gene pairs in the M. longifolia genome, providing an example of the convergent evolution of gene order. This work expands our understanding of plant BGCs with respect to both form and evolution, and highlights the power of BGCs for gene discovery in plant biosynthetic pathways.

Inwardly rectifying potassium (Kir) channels make it easier for K+ to enter into a cell and subsequently regulate cellular biological functions. Kir5.1 (encoded by KCNJ16) alone can form a homotetramer and can form heterotetramers with Kir4.1 (encoded by KCNJ10) or Kir4.2 (encoded by KCNJ15). In most cases, homomeric Kir5.1 is non-functional, while heteromeric Kir5.1 on the cell membrane contributes to the inward flow of K+ ions, which can be regulated by intracellular pH and a variety of signaling mechanisms. In the form of a heterotetramer, Kir5.1 regulates Kir4.1/4.2 activity and is involved in the maintenance of nephron function. Actually, homomeric Kir5.1 may also play a very important role in diseases, including in the ventilatory response to hypoxia and hypercapnia, hearing impairment, cardiovascular disease and cancer. With an increase in the number of studies into the roles of Kir channels, researchers are paying more attention to the pathophysiological functions of Kir5.1. This minireview provides an overview regarding these Kir5.1 roles.

We studied the correlation between the soil microbial status and the crop yield or medicinal properties of Angelica s.. Soil from different counties of China were collected and used in this study. Readfq, SOAP denovo, MetaGeneMark, and DIAMOND Resistance Gene Identifier (RGI) software were used in the analysis. A total of 131,345 genes of high quality were obtained, with the number of genes in all samples ranging from 483-42,545 and the average length ranging from 354.88-523.47. The acidobacteria in the non-sterilized group were significantly increased by comparing with the sterilized group, while actinobacteria in the non-sterilized group were significantly reduced by comparing with the sterilized group. There was a strong correlation between the two groups of the planting group and the two groups of the non-planting group, while the correlation between the planting and non-planting group was very weak. There were significant differences in the soil microbial community structure between Min county and Yunnan, as well as significant differences between the dominant genera of Min county and Yunnan. In summary, our study showed that the soil microbial flora in different areas affected the characteristics of Angelica s. roots, while sterilized soil was not conducive to the growth of Angelica s.

Type 2 diabetes mellitus (T2DM) is a global health problem that seriously threatens human health. Vitamin D (VD) has antidiabetic effects. However, the protective mechanism of 1,25-dihydroxyvitamin D3 (1,25D) on T2DM is still unclear.A rat model of T2DM was constructed using a high-fat diet combined with intraperitoneal injection of streptozotocin (STZ). Glucose tolerance was assessed by an oral glucose tolerance test (OGTT). Insulin secretion in blood and cell supernatant was determined by ELISA. Cell viability was analysed by CCK-8 assay. The level of ROS was detected by the DCFH-DA fluorescent probe method. The iron level in pancreatic tissues and cells was detected by an iron assay kit. Immunofluorescence staining was used to detect the expression of the pancreatic β cell marker CD49a. Furthermore, the protein expression levels of ferroptosis pathway-related proteins and vitamin D receptor (VDR) were detected by western blot. Downstream VDR targets were screened by proteomic sequencing.The DM group had increased glucose levels and decreased insulin secretion, while 1,25D treatment decreased glucose levels and increased insulin secretion. 1,25D also suppressed DM-induced ferroptosis in pancreatic tissues in vivo. In addition, 1,25D significantly enhanced the viability of pancreatic β cells and reduced the levels of ROS and iron. 1,25D significantly upregulated the expression of VDR and the ferroptosis-related pathway protein GPX4 and downregulated the expression of ACSL4. Furthermore, knockdown of VDR reversed the effects of 1,25D on cell viability, ROS and iron levels, and ferroptosis-related protein expression in pancreatic β cells. Proteomic sequencing revealed that FOXO1 was the downstream target gene of VDR. Knockdown of FOXO1 reduced pancreatic β cell death, decreased ROS, iron and ACSL4 levels, and increased GPX4 levels.1,25D/VDR inhibited pancreatic β cell ferroptosis in T2DM by downregulating the expression of FOXO1. This study provides a new theoretical basis for basic research on T2DM and is expected to establish a new idea for the treatment of T2DM.

Phenylboronic acid-functionalized nanometer-sized CaCO3 particles (PBA-CaCO3) were designed to determine the carcinoembryonic antigen (CEA) glycoprotein with a portable Ca2+ ion-selective electrode (Ca-ISE) through a typical boronate ester bond. CaCO3 nanospheres were conjugated to 3-aminophenylboronic acid by amine-epoxy reaction, whereas target CEA was captured into the aptasensing interface by the immobilized thiolated aptamer on gold substrate. Upon PBA-CaCO3 introduction, 3-aminophenylboronic acid labeled to CaCO3 microsphere specifically recognized with CEA glycoprotein based on sugar-boronic acid interaction to form a sandwiched complex. The carried CaCO3 was dissolved under acidic conditions to release Ca2+ ion with a portable Ca-ISE readout. Thanks to the specific boronate ester bond between PBA and 1,2-diols, the synthesized PBA-CaCO3 exhibited good conjugation properties for CEA glycoprotein. Under optimum conditions, Ca-ISE-based aptasensing platform exhibited good electrode potential response for evaluation of target CEA, and allowed detection of CEA at a concentration as low as 7.3 pg mL-1. Importantly, Ca-ISE-based aptasensing system is readily extended to detect other disease-related glycoproteins by controlling the corresponding aptamer.

Nepeta tenuifolia (N. tenuifolia) is a common aromatic herb that is widespread in East Asia. The aerial parts and spikes can be used as the traditional phytomedicines for the treatment of cold, fever, respiratory diseases, and skin diseases in the clinic and as a vegetable in salads or cooked foods. These applications have lasted for thousands of years due to their outstanding performance. In this review, with the help of PubMed, Web of Science, Google Scholar, Baidu Scholar, and CNKI, we have summarized the research achievements made in recent years on the various aspects of N. tenuifolia, covering botany, traditional uses, processing, phytochemistry, pharmacology, and pharmaceutics. A total of 102 phytochemicals have been found and identified in N. tenuifolia, which were grouped into terpenoids, flavonoids, organic acids and other components, including many bioactive compounds such as pulegone, hesperidin, and rosmarinic acid. This plant has also exhibited antiviral, immunoregulatory, anti-inflammatory, antioxidant and miscellaneous bioactivities, including antitumour, antidiabetic, antibacterial, and antithrombus activities. The progress to date has provided basic and strong evidence to support the traditional uses of this herbal medicine. However, the present explanations seem insufficient and unsatisfactory, in term of the relationships between the traditional uses and the modern pharmacological actions, the action mechanisms and the material basis. Therefore, in the future, some comprehensive studies should be well designed for further understanding the development and utilization of N. tenuifolia.

We studied the lipid-regulating effect and molecular mechanism of the medical components of Alisma orientalis: alisol A, alisol B, 23-acetyl alisol C (23C) and the 3 (alisol A): 1(alisol B):1(23C) and 2(alisol A):2(alisol B):1(23C) mixtures designed based on the ratio of them in Alisma orientalis from Fujian, Guangxi Province, China. The animal experiment and network pharmacology showed that ACAT was one of its lipid-regulating targets and alisols may reduce the level of TC by inhibiting ACAT activity. The molecular simulation and homologous modeling results suggested that the binding of alisol mixtures with ACAT was stronger than that of monomers because alisol monomers acted on different active regions of ACAT resulting in the superposition effect and caused the synergistic effect. The lipid-regulating effect of Fujian mixture was stronger than that of Guangxi mixture showing that 3:1:1 was a better ratio. The N-terminal lipid-regulating activity of ACAT was stronger than that of transmembrane domain 1.

To assess the potential role of daphnetin, a clinically used anti-inflammatory agent, on the development of the inflammatory and neurodegenerative disease, we investigated its immune regulatory function in a murine model of experimental autoimmune encephalomyelitis (EAE). Significantly, lower levels of pro-inflammatory cytokines including interleukin (IL)-17, interferon-γ, Il6, Il12a, and Il23a were observed in brains of daphnetin-treated EAE mice, compared with those in control littermates. We also confirmed that daphnetin suppressed the production of IL-1β, IL-6, and tumor necrosis factor-α in lipopolysaccharide-stimulated mouse BV2 microglial cells. Mechanistically, heme oxygenase-1 (HO-1), a canonical anti-oxidant and anti-inflammatory factor, was found to be substantially induced by daphnetin treatment in BV2 cells. Also, a significantly higher level of HO-1, accompanied by a decreased level of malondialdehyde, was observed in daphnetin-treated EAE mice. More importantly, the deletion of HO-1 in BV2 microglia largely abrogated daphnetin-mediated inhibition of the inflammatory response. Together, our data demonstrate that daphnetin has an anti-inflammatory and neuroprotective role during the pathogenesis of EAE, which is partially at least, dependent on its regulation of HO-1.

A new electrochemical immunoassay was designed for the sensitive detection of prostate-specific antigen (PSA) via a pyrophosphatase (PPase)-hydrolysed Cu(II)-coordinated pyrophosphate ion (Cu2+-PPi) complex, accompanying the release of Cu(II) ions and capture on a negatively charged electrode. This system involved a sandwiched immunoreaction with nanogold-labeled PPase/detection antibodies on a polystyrene microplate, enzymatic hydrolysis, Cu(II) release and voltammetric measurement. The voltammetric signal is derived from the as-released Cu(II) ions within the applied potential on the electrode. Under optimum conditions, our strategy exhibited a good voltammetric response for the analyte, and allowed determination of PSA at a concentration as low as 5.2 pg mL−1. Good precision, high specificity and acceptable accuracy were achieved for the detection of PSA.

Protostane triterpenes, which are found in Alisma orientale, are tetracyclic triterpenes with distinctive pharmacological activities. The natural distribution of protostane triterpenes is limited mainly to members of the botanical family Alismataceae. Squalene epoxidase (SE) is the key rate-limiting enzyme in triterpene biosynthesis. In this study, we report the characterization of two SEs from A. orientale. AoSE1 and AoSE2 were expressed as fusion proteins in E. coli, and the purified proteins were used in functional research. In vitro enzyme assays showed that AoSE1 and AoSE2 catalyze the formation of oxidosqualene from squalene. Immunoassays revealed that the tubers contain the highest levels of AoSE1 and AoSE2. After MeJA induction, which is the main elicitor of triterpene biosynthesis, the contents of 2,3-oxidosqualene and alisol B 23-acetate increased by 1.96- and 2.53-fold, respectively. In addition, the expression of both AoSE proteins was significantly increased at four days after MeJA treatment. The contents of 2,3-oxidosqualene and alisol B 23-acetate were also positively correlated with AoSEs expression at different times after MeJA treatment. These results suggest that AoSE1 and AoSE2 are the key regulatory points in protostane triterpenes biosynthesis, and that MeJA regulates the biosynthesis of these compounds by increasing the expression of AoSE1 and AoSE2.

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Weiling Leng,1,* Juan Jiang,2,* Bing Chen,1 Qinan Wu3 1Endocrinology Department, The First Affiliated Hospital of the Third Military Medical University (Army Medical University), Chongqing, People’s Republic of China; 2Endocrinology and Nephrology Department, Chongqing University Cancer Hospital and Chongqing Cancer Institute and Chongqing Cancer Hospital, Chongqing, People’s Republic of China; 3Endocrinology Department, Dazu Hospital of Chongqing Medical University, The People’s Hospital of Dazu, Chongqing, People’s Republic of China*These authors contributed equally to this workCorrespondence: Qinan WuEndocrinology Department, Dazu Hospital of Chongqing Medical University, The People’s Hospital of Dazu, Chongqing Email wqn11@126.com View the original paper by Dr Leng and colleagues This is in response to the Letter to the Editor

Euryales Semen (ES) is sought-after for thousand years due to its multiple properties, mainly from the two cultivars (i.e. South Gordon Euryale (SE) and North Gordon Euryale (wild type, WT)). Currently, no effective way was established to recognize the two valuable and analogous kernels. We found high analogies of macroscopical traits and discovered WT seed was affected by ambient temperature, showing ostensible significant relationships with latitude and sea-level pressure. LC-MS based untargeted metabolomics helped us identified 177 putative metabolites. Pathway analysis revealed the underlying and vital roles of flavonoids during seeds development. Our results strongly suggested a strong level of similarity of WT from various regions on the strength of metabolic data. A multivariable model containing 51 chemical markers satisfactorily categorized WT and SE. This study obtained could be used to guide the varietal discrimination of ES.

BackgroundNuclear factor erythroid 2-related factor 2 (Nrf2) is reportedly involved in hepatic lipid metabolism, but the results are contradictory and the underlying mechanism thus remains unclear. Herein we focused on elucidating the effects of Nrf2 on hepatic adipogenesis and on determining the possible underlying mechanism. We established a metabolic associated fatty liver disease (MAFLD) model in high fat diet (HFD) fed Nrf2 knockout (Nrf2 KO) mice; further, a cell model of lipid accumulation was established using mouse primary hepatocytes (MPHs) treated with free fatty acids (FAs). Using these models, we investigated the relationship between Nrf2 and autophagy and its role in the development of MAFLD.ResultsWe observed that Nrf2 expression levels were up-regulated in patients with MAFLD and diet-induced obese mice. Nrf2 deficiency led to hepatic lipid accumulation in vivo and in vitro, in addition to, promoting lipogenesis mainly by increasing SREBP-1 activity. Moreover, Nrf2 deficiency attenuated autophagic flux and inhibited the fusion of autophagosomes and lysosomes in vivo and in vitro. Weakened autophagy caused reduced lipolysis in the liver. Importantly, Chromatin immunoprecipitation-qPCR (ChIP-qPCR) and dual-luciferase assay results proved that Nrf2 bound to LAMP1 promoter and regulated its transcriptional activity. We accordingly report that Nrf2-LAMP1 interaction has an indispensable role in Nrf2-regulated hepatosteatosis. ConclusionsThese data collectively confirm that Nrf2 deficiency promotes hepatosteatosis by enhancing SREBP-1 activity and attenuating autophagy. To conclude, our data reveal a novel multi-pathway effect of Nrf2 on lipid metabolism in the liver, and we believe that multi-target intervention of Nrf2 signaling is a promising new strategy for the prevention and treatment of MAFLD.

Kunitz-like trypsin inhibitors are one of the most noteworthy research objects owing to their significance in pharmacological studies, including anticarcinogenic activity, obesity regulation and anticoagulation. In the current study, a novel Kunitz-like trypsin inhibitor, Kunitzin-AH, was isolated from the skin secretion of Amolops hainanensis. The novel peptide displayed a modest trypsin inhibitory activity with the inhibitor constant (Ki) value of 1.18 ± 0.08 µM without inducing damage to healthy horse erythrocytes. Then, a series of shortened variants of Kunitzin-AH were designed by truncating a peptide loop and site mutation inside the loop to illustrate the structure–activity relationship of the trypsin inhibition function. Among the variants, a significant decrease was observed for the Cys-Cys loop domain, while the extension of an Arg at N-terminus (RCKAAFC) retained the inhibitory activity, indicating that the -RCK-motif is essential in forming the reactive domain for exerting the inhibitory activity. Furthermore, substitutions of Ala by hydrophobic or hydrophilic residues decreased the activity, indicating suitable steric hindrance provides convenience for the combination of trypsin. Additionally, the conformational simulation of the analogues processed with Chimera and Gromacs and further combination simulations between the peptides and trypsin conducted with HDOCK offered a potential opportunity for the natural trypsin inhibitory drug design. The truncated sequence, AH-798, may be a good replacement for the full-length peptide, and can be optimized via cyclization for further study.

Background Angelica sinensis (Oliv.) Diels (A. sinensis) is a Chinese herb grown in different geographical locations. It contains numerous active components with therapeutic value. Rhizosphere microbiomes affect various aspects of plant performance, such as nutrient acquisition, growth and development and plant diseases resistance. So far, few studies have investigated how the microbiome effects level of active components of A. sinensis. This study investigated whether changes in rhizosphere microbial communities and metabolites of A. sinensis vary with the soil microenvironment. Soils from the two main A. sinensis-producing areas, Gansu and Yunnan Province, were used to conduct pot experiments. The soil samples were divided into two parts, one part was sterilized and the other was unsterilized planting with the seedling variety of Gansu danggui 90–01. All seedlings were allowed to grow for 180 days. At the end of the experiment, radix A. sinensis were collected and used to characterize growth targets and chemical compositions. Rhizosphere soils were subjected to microbial analyses. Results Changes in metabolic profiles and rhizosphere microbial communities of A. sinensis grown under different soil microenvironments were similar. The GN (Gansu non-sterilized), YN (Yunnan non-sterilized), GS (Gansu sterilized), and YS (Yunnan sterilized) groups were significantly separated. Notably, antagonistic bacteria such as Sphingomonas, Pseudomonas, Lysobacter, Pseudoxanthomonas, etc. were significantly (p Pseudomonas parafulva; organic acids (including chlorogenic acid, dicaffeoylquinic acid and 5-feruloylquinic acid) and their ester coniferyl ferulate which were enriched in YS Group were positively associated with Gemmatimonadetes bacterium WY71 and Mucilaginibater sp., respectively. Conclusions The soil microenvironment influences growth and level/type of active components in A. sinensis. Further studies should explore the functional features of quality-related bacteria, identify the key response genes and clarify the interactions between genes and soil environments. This will reveal the mechanisms that determine the quality formation of genuine A. sinensis.

We prepared extracts of Alisma orientalis from Sichuan and Fujian Province, China. Based on the ratio of alisol B 23-acetate (23B) to alisol A 24-acetate (24A) in two Alisma orientalis extracts, we prepared two mixtures of 24A and 23B (24A:23B = 1:3 or 1:10). The antitumor molecular mechanism of the monomers 24A and 23B, the two mixtures and the effective components of Alisma orientalis from different habitats were studied. The MTT assay suggested that the difference in the antitumor activity of Alisma orientalis from different habitats was correlated to the ratio of 24A to 23B. The multi-spectroscopic analysis suggested that the effective components, the monomers and mixtures interacted with c-myc DNA in a partial intercalation manner. The binding strength of the alisol acetates to c-myc DNA was consistent with the anticancer activity, indicating that c-myc DNA was the anticancer target. The molecular simulation indicated that the mixtures were all directly bound to different base pairs of c-myc DNA for a superimposed effect, which led to the binding strength of the mixtures to c-myc DNA was stronger than that of the monomers. The molecules in the 1:3 mixture were all bound to different base pairs of c-myc DNA. However, for the 1:10 mixture, seven molecules of 23B bound to the side chain of 24A, resulting in the mixture with a long chain structure which increased the steric hindrance of 24A. As a result, affinity between 24A and c-myc DNA in the 1:10 mixture was weaker than that in the 1:3 mixture. [Formula: see text] The antitumor molecular mechanism of the alisol monomers 24A and 23B, the mixtures with different proportions and the effective components of Alisma orientalis from different habitats were studied. The order of the antitumor activity was as follows: Sichuan Fujian, 24A-23B (1:3)24A-23B (1:10)23B 24A. The antitumor activity of Alisma orientalis from different habitats was consistent with the mixtures which were designed according to the contents of the active ingredients of the medicinal materials, indicating that the antitumor activity of Alisma orientalis from Sichuan is better than that from Fujian which is related to the contents of 24A and 23B and the proportion of 1:3 is better than 1:10. The binding strength of the mixtures to c-myc DNA was consistent with the anticancer activity. The mixtures were all directly bound to different base pairs of c-myc DNA for a superimposed effect, which led to the strength of the interaction of the mixtures to c-myc DNA was stronger than that of the monomers. For the 24A-23B (1:3) mixture, the four small molecules bound to c-myc DNA directly and interacted with different base pairs of c-myc DNA. While for the 24A-23B (1:10) mixture, 24A and three 23B molecules interacted with c-myc DNA, the remaining seven 23B molecules bound to the side chain of 24A, which increased the steric hindrance. The binding of the mixture to c-myc DNA was decreased. Communicated by Ramaswamy H. Sarma.

Comprehensive immune responses are essential for eliminating pathogens but must be tightly controlled to avoid sustained immune activation and potential tissue damage. The engagement of TLR4, a canonical pattern recognition receptor, has been proposed to trigger inflammatory responses with different magnitudes and durations depending on TLR4 cellular compartmentalization. In the present study, we identify an unexpected role of Lamtor5, a newly identified component of the amino acid-sensing machinery, in modulating TLR4 signaling and controlling inflammation. Specifically, Lamtor5 associated with TLR4 via their LZ/TIR domains and facilitated their colocalization at autolysosomes, preventing lysosomal tethering and the activation of mTORC1 upon LPS stimulation and thereby derepressing TFEB to promote autophagic degradation of TLR4. The loss of Lamtor5 was unable to trigger the TFEB-driven autolysosomal pathway and delay degradation of TLR4, leading to sustained inflammation and hence increased mortality among Lamtor5 haploinsufficient mice during endotoxic shock. Intriguingly, nutrient deprivation, particularly leucine deprivation, blunted inflammatory signaling and conferred protection to endotoxic mice. This effect, however, was largely abrogated upon Lamtor5 deletion. We thus propose a homeostatic function of Lamtor5 that couples pathogenic insults and nutrient availability to optimize the inflammatory response; this function may have implications for TLR4-associated inflammatory and metabolic disorders.

The bacterial pneumonia caused by methicillin-resistant Staphylococcus aureus (MRSA) is a potentially fatal disease, featured with extensive infection, inflammation, and airway dysfunction. With the increasing emerging of drug-resistant strains, new therapeutic strategies beyond canonical antibiotic treatment are pressingly needed. Daphnetin (DAPH) is a natural coumarin derivative with anti-inflammation, anti-microorganism and anti-oxidative properties. However, the protective effect of DAPH on S. aureus-caused pneumonia and the mechanism involved are never explored. Here we show that DAPH treatment conferred substantial protection against S. aureus-induced pneumonia, characterized by the reduced inflammatory responses, the augmented bacterial clearance and the alleviated tissue damage. Our study indicates that DAPH significantly enhanced mTOR-dependent autophagic pathway, leading to the boosted microphage bactericidal activity and the suppressed inflammatory responses. Inhibition of autophagic pathway therefore largely abolished DAPH-elicited repression of inflammatory response and macrophage anti-bacterial capability. Together, we herein not only identify a novel, natural agent to combat bacterial pneumonia, but also underscore the significance of autophagic pathway in orchestrating antimicrobial and anti-inflammatory responses, which may have important implication for the treatment of the infectious diseases, particularly that caused by obstinate, antibiotic-resistant pathogens such as MRSA.

6"-O-succinylapigenin [apigenin-7-O-(6′-O-succinyl)-glucoside], a novel compound, is identified in chamomile. Although it is highly produced by Bacillus amyloliquefaciens FJ18, its bioactivity remains unknown. The neuroprotective effects and antioxidative mechanism of 6''-O-succcinylapigenin in the middle cerebral artery occlusion model in male rats was investigated in this study. The structure of this compound was determined by spectroscopic data analysis. After 2 h of occlusion and 24 h of reperfusion, magnetic resonance imaging and assessed neurological scores following middle cerebral artery occlusion (MCAO) in male rats to determine the infarction size and neurological deficits, respectively. In addition, we tested protein levels of the nuclear factor E2-related factor 2 (Nrf2), Kelch-like ECH-associated protein1 (Keap1), heme oxygenase-1 (HO-1), and extracellular-signal-regulated kinase (ERK), to investigate the mechanism of antioxidative action of 6''-O-succcinylapigenin. Finally, we employed immunofluorescence to determine the location of Nrf2 and Keap1 in HT22 cells cultured in vitro. Our results revealed that administration of 6''-O-succcinylapigenin induced a decrease in both infarct volume and neurological scores following MCAO, and significantly increased the activity of HO-1 and nuclear Nrf2 in vivo. Similarly, immunofluorescence assays indicated that Nrf2 is highly expressed in the nucleus following treatment with 6''-O-succcinylapigenin in vitro. Our study suggests that 6''-O-succcinylapigenin exerts an anti-ischemic effect by activating the ERK/Nrf2/HO-1 pathway.

Inflammatory bowel diseases (IBDs) are characterized by chronic pathologies associated with extensive gut dysbiosis and intestinal inflammation. Hence, endeavors to improve the inflammatory pathology by manipulating gut microbiota are ongoing. Daphnetin (DAPH) is a coumarin derivative extracted from Daphne odora var with anti-inflammatory and immune-regulatory properties that has been widely used in treating inflammatory disorders. Herein, we showed that DAPH remarkably alleviated experimental colitis by reducing colonic inflammation, improving colonic integrity, and reestablishing immune and metabolic homeostasis in the inflicted intestines. Our analysis showed that DAPH modified the composition of gut microbiota and altered the metabolic profiles in dextran sulfate sodium-treated mice. In particular, this agent significantly elevated the abundance of short-chain fatty acid (SCFA)-producing gut microbiota, causatively related with the enhanced development of Treg cells and the reduced proinflammatory Th17 cell differentiation. More critically, the protective effect of DAPH was shown to be transmissible among colitic mice through cohousing or fecal microbiota transplantation, further substantiating the importance of SCFA-producing gut microbiota in DAPH action. We thus for the first time reveal the potential of DAPH in resetting the gut microbiome and reestablishing immune homeostasis in colitic mice, which may have clinical implications for treating IBD.-Ji, J., Ge, X., Chen, Y., Zhu, B., Wu, Q., Zhang, J., Shan, J., Cheng, H., Shi, L. Daphnetin ameliorates experimental colitis by modulating microbiota composition and Treg/Th17 balance.

Ethnopharmacological relevance Gardenia jasminoides fruit (GJF) is used as a well-known traditional folk medicine, a food and a natural colorant in Asia with a long history. The herbal medicine has usually been harvested in the autumn from September to November. However, this time span is too long and might result in the quality instability of GJF. Aim of study We aimed to conduct the comprehensive quality evaluation of GJF including the quantitative analysis of the bioactive components and the main bioactivities, and further to determine the most appropriate harvest time of this phytomedicine. Materials and methods In this study, an UFLC-Q-TRAP-MS/MS method was established to quantify 7 iridoid glycosides (geniposide, geniposidic acid, secoxyloganin, gardenoside, genipin 1-gentiobioside, scandoside methyl ester, and shanzhiside), 7 phenylpropanoid acids (chlorogenic acid, cryptochlorogenic acid, neochlorogenic acid, isochlorogenic acid A, isochlorogenic acid B, isochlorogenic acid C, and caffeic acid) and 2 carotenoids (crocin-1 and crocin-2) in GJF. With this method, nine samples of GJF harvested at different times were analyzed and compared. These samples were also investigated and compared in terms of their antioxidant activity (DPPH free radical scavenging, ABTS free radical scavenging, ferric-reducing antioxidation) and anti-influenza activity (neuraminidase inhibition), which are closely related to the GJF efficacies. Then, hierarchical cluster analysis (HCA) was separately performed for the quantitative analysis and bioactivity evaluation in vitro. Results The HCA results demonstrated that three GJF samples (S5, S6, and S7) were clustered into one group for both quantitative analysis and bioactivity evaluation in vitro; these three samples were found to have the highest standardized scores in both the former (12.775, 12.106, 10.817) and the latter (3.406, 3.374, 3.440). Based on the comprehensive results, the optimum harvest period was confirmed to extend from mid-October to early-November. Conclusions This study firstly validated the use of UFLC-Q-TRAP-MS/MS method for the determination of 16 bioactive components in GJF. It was also the first time that a quantitative analysis and a bioactivity assay in vitro were integrated for the determination of the most appropriate harvest period of GJF. We hope this paper may provide some reference to studies of appropriate harvest periods and even the quality control of TCMs.

We first compared long-term clinical outcomes in treating critical limb ischemia (CLI) and foot ulcer in patients with diabetes between autologous bone marrow mesenchymal stem cell (BMMSC) and bone-marrow-derived mononuclear cell (BMMNC) transplants. Forty-one patients were enrolled and followed up for 3 years. They received an 18-day standard treatment before stem cell transplantation. Patients with bilateral CLI and foot ulcer were injected intramuscularly or basally with BMMSC, BMMNC, or normal saline (NS). Cox model analysis showed significant differences in the hazard ratio (HR) for amputation with treatment by BMMSC (HR 0.21 [95% CI (0.05, 0.95)], P = 0.043), infection of foot (HR 5.30 [95% CI (1.89, 14.92)], P = 0.002), and age ≥64 (HR 3.01 [95% CI (1.11, 8.15)], P = 0.030), but no significant differences by BMMNC at 9 months after transplantation. Regarding ulcer healing and recurrence rate, the BMMSC group demonstrated a significant difference from the NS group during the 3–6 months after transplantation or healing, but the BMMNC group did not. This trial suggests that, compared with BMMNC treatment, BMMSC treatment leads to a longer time of limb salvage and blood flow improvement, and, when compared with conventional therapy, it can promote limb blood flow and ulcerative healing, and reduce ulcer recurrence and amputation within 9 months.

The population of Bufo bufo gargarizans Cantor in China has been alarmingly declining due to environmental pollution. Deltamethrin is a pyrethroid pesticide frequently used in agriculture and much of its residues are present in crops, soil and water. Deltamethrin has been shown to have toxicity to toads. Herein, we assumed that deltamethrin contamination might influence the biosynthesis of toxic substances present in toad venom. Bufadienolides present in venom are the toad's chemical defense and highly toxic to predators, and they are important for the survival of toad species against predators. In this study, we determined the contents of bufadienolides in toad venom using a HPLC-triple quadrupole mass spectrometer to evaluate the change in bufadienolide profiles in toad venom before and after cutaneous exposure to deltamethrin. The results indicated that toads exposed to high concentration of deltamethrin survive the least, do not exuviate, and their movements are stiff. Furthermore, it was observed that high level of deltamethrin contamination induces a marked decrease in the levels of toxic bufadienolides in toad venom. These changes in the toxin profiles could lead to the compromised chemical defense of toad, leading to more susceptible to attack by predators. This is the first study to report that environmental contaminants (pesticides) can influence the toad's toxic profiles, suggesting one factor contributing to the decline in the population of B. bufo gargarizans Cantor.

By a pilot trial on investigating immunomodulatory activity and target of ginsenosides, the major bioactive components of ginseng, here we report that structural analogues in herbal medicines hit a shared target to achieve cumulative bioactivity. A ginsenoside analogues combination with definite immunomodulatory activity in vivo was designed by integrating pharmacodynamics, serum pharmacochemistry and pharmacokinetics approaches. The cumulative bioactivity of the ginsenoside analogues was validated on LPS/ATP-induced RAW264.7 macrophages. The potentially shared target NLRP3 involved in this immunomodulatory activity was predicted by systems pharmacology. The steady binding affinity between each ginsenoside and NLRP3 was defined by molecular docking and bio-layer interferometry assay. The activation of NLRP3 inflammasomes in LPS/ATP-induced RAW264.7 was significantly suppressed by the combination, but not by any individual, and the overexpression of NLRP3 counteracted the immunomodulatory activity of the combination. All these results demonstrate that the ginsenoside analogues jointly hit NLRP3 to achieve cumulative immunomodulatory activity., Zhang et al. design ginsenoside structural analogues and demonstrate that their combination shows more potent immunomodulatory activities than individual ginsenosides used alone at the same dosages. They predict that these analogues act on the joint target NLRP3 and consequently suggest that structural analogues hit a shared target to achieve cumulative bioactivity.

Nepeta tenuifolia Briq. (Lamiaceae) is a medicinal plant historically used in the East Asia region to treat cold and fever, and it is currently used as a clinically effective treatment for respiratory diseases. We previously found that monoterpenoids are the dominant volatile secondary metabolites in N. tenuifolia and their biosynthesis occurs in peltate glandular trichomes. To gain an insight into the molecular mechanisms underlying monoterpenoid biosynthesis in N. tenuifolia, we conducted transcriptome sequencing and examined the expression differences in monoterpene molecular pathway-related genes in different tissues and growth stages by qRT-RCR. In total, six p-menthane monoterpene biosynthetic genes in the (+)-menthone pathway were identified and cloned successfully based on transcriptome data. Moreover, the major constituents, including (+)-limonene, (-)-pulegone and (+)-menthone showed greater accumulation in the spikes than in other organs, such as the expression levels of related key enzyme genes. Additionally, the relative expression of pulegone reductase was the highest at 84 days, showing an inverse trend from (-)-pulegone relative content and leading to (+)-menthone accumulation in peltate glandular trichomes. Finished cloning of the gene for limonene 3-hydroxylase in N. tenuifolia (NtL3OH), heterologous expression in yeast, and in vitro assays were performed for functional characterization. Our study provides an important resource for further research of secondary metabolism of monoterpenes in peltate glandular trichomes of N. tenuifolia and other homologous species.

Background Perilla frutescens (L.) Britt is a medicinal and edible plant widely cultivated in Asia. Terpenoids, flavonoids and phenolic acids are the primary source of medicinal ingredients. Glandular trichomes with multicellular structures are known as biochemical cell factories which synthesized specialized metabolites. However, there is currently limited information regarding the site and mechanism of biosynthesis of these constituents in P. frutescens. Herein, we studied morphological features of glandular trichomes, metabolic profiling and transcriptomes through different tissues. Results Observation of light microscopy and scanning electron microscopy indicated the presence of three distinct glandular trichome types based on their morphological features: peltate, capitate, and digitiform glandular trichomes. The oil of peltate glandular trichomes, collected by custom-made micropipettes and analyzed by LC–MS and GC–MS, contained perillaketone, isoegomaketone, and egomaketone as the major constituents which are consistent with the components of leaves. Metabolomics and transcriptomics were applied to explore the bioactive constituent biosynthesis in the leaves, stem, and root of P. frutescens. Transcriptome sequencing profiles revealed differential regulation of genes related to terpenoids, flavonoids, and phenylpropanoid biosynthesis, respectively with most genes expressed highly in leaves. The genes affecting the development of trichomes were preliminarily predicted and discussed. Conclusions The current study established the morphological and chemical characteristics of glandular trichome types of P. frutescens implying the bioactive constituents were mainly synthesized in peltate glandular trichomes. The genes related to bioactive constituents biosynthesis were explored via transcriptomes, which provided the basis for unraveling the biosynthesis of bioactive constituents in this popular medicinal plant.

Background: Angelica sinensis (Oliv.) Diels (A. sinensis), the commonly used famous-region Chinese herbs, grew in different geographical locations which were crucial to active components accumulation in medicinal plants. Rhizosphere microbiome played a critical role in plant performance, including nutrient acquisition, growth and development and plant diseases resistance etc., there were still few studies on how microbiome effecting on accumulation of active components of A. sinensis. This study aim to illustrate whether the variations of rhizosphere microbial communities and metabolites profilings of A. sinensis were dependant on soil microenvironments. Soils from the two main A. sinensis producing areas, Gansu and Yunnan Province, were selected for a pot experiment, and then divided into two parts, one part was sterilized and the other did not, planting with Gansu danggui 90-01, allowed to grow for 180 days. After the trial radix A. sinensis were collected for analysis of growth targets and chemical compositions, rhizosphere soils for microbial analyses. Results: The metabolic profiles and rhizosphere microbial communities of A. sinensis grown under different soil microenvironments showed similar variation. The GN (Gansu non-sterilized), YN (Yunnan non-sterilized), GS (Gansu sterilized), and YS (Yunnan sterilized) group were significantly separated. Specifically, compared with Yunnan soil, antagonistic bacteria such as Sphingomonas, Pseudomonas, Lysobacter, Pseudoxanthomonas, etc. were significantly (p Pseudomonas parafulva; organic acids (including chlorogenic acid, dicaffeoylquinic acid and 5-feruloylquinic acid) and their ester coniferyl ferulate enriched in YS Group were significantly positively relevant with Gemmatimonadetes bacterium WY71 and Mucilaginibater sp., respectively. Conclusions: Given that soil microenvironments may contribute to growth and active components accumulation of A. sinensis. Further study aimes at exploring the functional characteristics of quality-related bacterias, and finding the main response genes and the interactions between genes and the environments, to elucidate the main mechanism of genuine A. sinensis quality formation.

This study was conducted to estimate the protective effect of Cyanidin-3-glucoside (C3G) on myocardial ischemia-reperfusion (IR) injury and to explore its mechanism. The rats were subjected to left anterior descending ligation and perfusion surgery. In vitro experiments were performed on H9c2 cells using the oxygen-glucose deprivation/reoxygenation (OGD/R) model. The results showed the administration of C3G reduced the infarction area, mitigated pathological alterations, inhibited ST segment elevation, and attenuated oxidative stress and ferroptosis-related protein expression. C3G also suppressed the expressions of USP19, Beclin1, NCOA4, and LC3II/LC3I. In addition, treatment with C3G relieved oxidative stress, downregulated LC3II/LC3I, reduced autophagosome number, downregulated TfR1 expression, and upregulated the expressions of FTH1 and GPX4 in OGD/R-induced H9c2 cells. C3G could inhibit the protein levels of USP19 and LC3II. C3G promoted K11-linked ubiquitination of Beclin1. Further evidence that C3G reduced ferroptosis and ameliorated myocardial I/R injury was demonstrated with the ferroptosis promoter RSL3. Taken together, C3G could be a potential agent to protect myocardium from myocardial I/R injury.

Background: Schizonepeta tenuifolia Briq. (ST) has been used as an aromatic exterior-releasing medicine in clinical practice for thousands of years in China. Previous researches have revealed both volatile oil (STVO) and aqueous extract (STAE) from ST showed significant pharmacological activities. However, the influence between each other was still unknown. Methods: This study was designed to compare the pharmacokinetic profiles of three main flavonoids (luteoloside, apigetrin, and hesperidin) in STAE to illustrate the influence of STVO. So, an ultra-flow liquid chromatography-tandem mass spectrometry (UHPLC-MS) method was established to quantitatively analyze the three absorbed ingredients in the plasma of healthy rats. Biological samples were analyzed on an Agilent Eclipse Plus C18 column (3.0 mm × 150 mm, 3.5 μm) with gradient mobile phase (containing 0.2% formic acid and acetonitrile) at a flow rate of 0.8 mL/min. All the analytes and quercitrin (IS) were investigated with an electrospray ionization source (ESI) using multiple-reaction monitoring (MRM) in negative ionization mode. Results: This quantitative method showed good linearities (r ≥0.9995) and the lower limits of quantification were 0.590~1.19 ng/ml. The intra- and inter-day precisions ranged 3.47~10.45% and 4.29~11.28% for the three analytes. The mean extraction recoveries were in the range of 77.41~109.79% and the average matrix effects were within 83.41~112.67%. The validated method has been fully applied to compare the pharmacokinetic parameters of the three flavonoid glycosides in rat plasma after oral administration of STAE and STAE+STVO. In comparison of luteoloside, apigetrin, and hesperidin in STAE group, it was found that different degree of increasing existed for the time to reach the maximum concentration (Tmax), elimination half-life time (T1/2), the area under the concentration curves (AUC0→t and AUC0→∞) and the maximum concentrations (Cmax) in STAE+STVO group. Conclusions: As can be seen from above, STVO could improve the absorption and bioavailability of the three analytes. These findings would provide some active and strong basis of safe clinical application for ST and further exploitation for STVO from the perspective of drug-drug interaction.

A simple and feasible pH meter–based immunoassay is reported for detection of C-reactive protein (CRP) using glucose oxidase (GOD)–conjugated dendrimer loaded with platinum nanozyme. Initially, platinum nanozymes were loaded into the dendrimers through an in situ synthetic method. Then, GOD and monoclonal anti-CRP antibody with a high molar ratio were covalently conjugated onto carboxylated dendrimers via typical carbodiimide coupling. The immunoreaction was carried out with a competitive mode in a CRP-coated microplate. Along with formation of immunocomplex, the added glucose was oxidized into gluconic acid and hydrogen peroxide by GOD, and the latter was further decomposed by platinum nanozyme, thus accelerating chemical reaction in the positive direction. The produced gluconic acid changed the pH of detection solution, which was determined using a handheld pH meter. Under optimum conditions, the pH meter–based immunoassay gave a good signal toward target CRP from 0.01 to 100 ng mL−1. The limit of detection was 5.9 pg mL−1. An intermediate precision ≤ 11.2% was acquired with batch-to-batch identification. No nonspecific adsorption was observed during a series of procedures to detect target CRP, and the cross-reaction against other biomarkers was very low. Importantly, our system gave well-matched results for analysis of human serum samples relative to a referenced ELISA kit. Graphical abstract Supplementary Information The online version contains supplementary material available at 10.1007/s00604-020-04687-9.

Background: Aquatic Euryale ferox Salisb. is an economically important crop in China and India. Unfortunately, low yield limitations seriously hinder market growth. Unveiling the control of seed size is of remarkable importance in improvement of crops. Here, we generated a new hybrid line (HL) with larger seeds by crossing South Gordon Euryale and North Gordon Euryale (WT) which hasn’t been reported before. However, the functional genes and molecular mechanisms controlling the seed size in Euryale ferox Salisb. remain unclear. In this study, we focused on the differentially expressed genes in the auxin signal transduction pathway during fruit development between HL and WT to explore candidate regulatory genes participated in regulating seed size.Results: Both concentration and localization of indole-3-acetic acid (IAA) at two growth stages of fruits of WT and HL were detected by LC-MS and immunofluorescence. Although IAA content between the two lines did not differ, IAA distribution was significantly different. To elucidate the mechanism and to seek the key genes underlying this difference, RNA-seq was performed on young fruits at the two selected growth stages, and differentially expressed genes related to the auxin transduction pathway were selected for further analysis. Conclusion: Hybrid Euryale ferox Salisb. expressed significant heterosis, resulting in non-prickly, thin-coated, large seeds, which accounted for the significantly larger yield of HL than that of WT. Our study indicated that Small Auxin Up RNAs (SAURs) -mediated localization of IAA regulates seed size in Euryale ferox Salisb. We found that some SAURs may act as a positive mediator of the auxin transduction pathway, thereby contributing to the observed heterosis.

Background Cyanidin (CY) is one of the most abundant anthocyanidins found in red-purple diet sources such as grapes, blueberries and purple corns. CY has been proven to exhibit a wide range of biological functions including antioxidant, antiviral, anticarcinogenic and anti-inflammatory properties. Purpose This study investigated the anti-depressive activity of CY and its related mechanism. Methods In the behavioral tests, CY-related effects on depressive symptoms were evaluated. Then the changes in PI3K/AKT/FOXG1/FGF-2 signaling and adult neurogenesis including doublecortin (DCX+) cell number, dendritic length, secondary and third dendrites number in the hippocampus were investigated by Immunofluorescence and Western blot analyses. PI3K antagonist LY 294,002 was used to verify the unique impact of PI3K/AKT/FoxG1/FGF-2 signaling on CY-related antidepressant efficacy. Results CY grossly reversed CUMS-induced behavioral defects, The DCX+ cell number and protein levels increased in CUMS mice receiving CY administration. LY 294,002 successfully blocked CY-induced improvements in depressive behaviors, neurogenesis, and protein levels in CUMS mice. Conclusion The findings demonstrated that CY was efficacious in alleviating depression-like symptoms, which was dependent on PI3K/AKT/FoxG1/FGF-2 signaling-modulated neurogenesis enhancement.

Protostane triterpenes in Alisma orientale (Sam.) Juz., because of their unique structural feature, exhibit distinctive pharmacological activities. However, the biosynthetic pathways for these compounds remain largely unknown. In this study, transcriptome analysis was performed using A. orientale tubers and leaves. We identified 101,107 unigenes, which were annotated against various databases. Importantly, 35 unigenes associated with the terpene biosynthesis pathway were identified, among which 18 encoded key enzymes catalyzing every reaction in the biosynthesis of protostane triterpenes skeleton. In addition, eight candidate transcription factor unigenes and nine candidate cytochrome P450 unigenes related to the terpene biosynthesis pathway were obtained. Moreover, the expression levels of most genes involved in protostane triterpenes biosynthesis in tubers were significantly higher than those in leaves. Furthermore, significant positive correlations were showed between the levels of key enzymes (HMGR2, SS2, SE1, SE2, OSC1, and OSC2), and the contents of the main bioactive compound at different developmental phases of A. orientale. These genes were identified first time in this study. These results provide preliminary evidence that these proteins may play crucially regulatory roles in protostane triterpenes synthesis.

Arsenic sulfide compounds provide nearly all of the world's supply of arsenic. However, the risk of arsenic trisulfide exposure is still not fully investigated. Here, we systemically assessed the toxicology of As4S4 in rats by combining arsenic metabolite detection, routine testing and lipidomic profiling. It was revealed that the oral administration of As4S4 for two months increased the total arsenic content in the liver reaching a saturation level. Further analysis by anion exchange chromatography coupled with inductively coupled plasma mass spectrometry (ICP-MS) technology showed no trace of inorganic arsenic, but there was significant presence of dimethylarsinic acid (DMA), in the livers of rats. This arsenic metabolite was less toxic to rats and did not induce overt liver pathology and functional injury. In contrast, lipidomic profiling provided a comprehensive map of lipids and uncovered a more complex inflammatory response, exhibiting more sensitive change to arsenic exposure. We observed that metabolites of cyclooxygenase, including PGF2α, dhk PGF2α, 15k PGF2α, 8-iso-PGF2a, PGE2, dhk PGE2, PGD2, 15d-PGD2, and PGJ2, were significantly elevated. But mediators from lipoxygenase, cytochrome P450, docosahexaenoic acid, and eicosapentaenoic acid pathways were not markedly affected. In summary, we identified DMA as the predominant arsenic species in the livers of rats, and found cyclooxygenase-derived lipids as the inflammatory mediators before the development of overt liver injury for subchronic As4S4 exposure. These mediators could translate into potential metabolic biomarkers in early arsenic risk assessment and as targets for therapeutic intervention.

Studies on the lipid-regulating effects of alisol compounds are reported that include alisol B, alisol A 24-acetate (24A), alisol A and an alisol B - 24A - alisol A mixture (content ratio = 1:1:1). The effects on the activity of lipoprotein lipase (LPL), a key lipid-modulating enzyme, were studied to investigate the molecular mechanism of lipid-regulating activity of alisols. The effects of alisols on regulating blood lipids and the activities of LPL were determined using a reagent kit method. The structure of LPL was obtained by homology modeling and the interactive mechanism of alisol monomers and the mixture with LPL was investigated by molecular simulation. The alisol monomer and mixture were shown to regulate blood lipids, suggesting that alisols may decrease the level of triglyceride (TG) by improving the activity of LPL. The order of intensity was: mixture > alisol A > alisol B > 24A, indicating that alisols of alismatis rhizoma feature a synergistic effect on LPL. The N- and C-terminus of LPL both represented the catalytic active domains of this lipid-regulating effect. Cys306, Gln129 and Ser166 were the key amino acid residues resulting in the lipid-regulating effect of the alisol monomer while Ser166 and Arg18 were found to be responsible for the lipid-regulating effect of the mixture. The C-terminus of LPL was indirectly involved in the enzymatic process. A folded side chain of alisols or the parent ring was found to bind somewhat weaker to LPL than an open side chain or parent ring. The hydroxyl groups on the C14-, C22-, C28-, C30- and C31-terminus in the side chain, the ring ether structure in C23-position, and the acetyl group in C29-position represented the key sites for the lipid-regulating action of alisols. Meanwhile, the C30-site hydroxyl group played an important role in the synergistic effect of the alisol mixture.

The purpose of the present study was to investigate whether ɑ-tocopherol exhibited neuro-protective effects in chronic unpredictable mild stress (CUMS) mice through the regulation of autophagy. Deficits in behavioural tests, including a sucrose preference test, open field test, forced swim test, and tail suspension test, were ameliorated following ɑ-tocopherol administration. To study the potential mechanism, western blots were performed on both prefrontal cortex and hippocampus samples. Similar to the degree of autophagy, the activities of adenosine monophosphate-activated protein kinase (AMPK) and Unci-51 like autophagy activating kinase-1 (ULK1) were decreased after CUMS stimulation. In addition, we also found increased activity of the mammalian target of rapamycin (mTOR), which was significantly affected following administration of ɑ-tocopherol, as well as its three downstream pathways. Taken together, our study found that ɑ-tocopherol might potentially promote autophagy to induce anti-depressive responses in CUMS mice though the AMPK/mTOR pathway.

Skins of anurans (frogs and toads) are rich sources of bioactive peptides. However, the peptides secreted by the skin glands of Bufo gargarizans, the most common toad in China, remained unexplored to date. Here, a strategy combines LC–MS/MS, RNA sequencing and bioinformation analysis was applied to unravel the peptides in the Bufo gargarizans secretions. Data-dependent LC–MS/MS acquisitions of intact peptides followed by automated chromatographic alignment, De novo analysis, database and homology searches with manual validations showed that the venom is composed by 939 features, with masses ranging from 0.7–4 kDa. These peptides derived from 85 proteins were identified using the PEAKS software with acquired MS and MS/MS spectra of Venenum Bufonis against the house-built protein database using De novo RNA sequencing, while only 23 peptides from 8 proteins were found when searching known amphibian database. Moreover, it was found that many peptides with high abundance in Venenum Bufonis derived from proteolytic processing of a larger precursor protein, named as CL4590. Molecular cloning was applied to validate a short domain of CL4590 to evident the accuracy of these obtained sequences. Although the bioactivities of peptides identified by MS/MS are unknown, the next function annotation showed that they may involve in the cell killing, immune and metabolic process, antioxidant activity and antimicrobial actions. Therefore, the peptidomics analysis on Bufo gargarizans discover abundant novel toad peptides which broaden our horizons on the secretion multiplicity and supplied an assortment of pharmacological candidates.

Background Cancer related inflammation plays a fatal role in the metastatic process, which can foster tumor growth, angiogenesis and dissemination. Sparstolonin B (SsnB), derived from Chinese medicine of the tubers of Scirpus yagara, is a TLR2 and TLR4 antagonists. It has exhibited multiple activities of anti-inflammatory, anti-cancer, anti-obesity and anti-hepatitis. However, whether SsnB is involved in the regulation of inflammation-induced tumor metastasis is not well elucidated. Purpose The aim of this study was to investigate the effectiveness of SsnB as a treatment of inflammation-induced tumor metastasis and identify the underlying mechanisms of its anti-tumor metastatic activity. Method The anti-tumor metastatic activity in vitro was estimated by MTT, wound-healing assay, matrigel invasion analysis and extracellular matrix adhesion assay. Mice lung metastasis and hepatic metastasis experiments were performed to assess the activities in vivo. Lungs or livers were weighed and the number of metastatic nodules was determined after mice were sacrificed. The levels of pro-inflammatory cytokines in the serum, lungs and livers were detected by using enzyme-linked immunosorbent assay (ELISA). Micro-metastasis nodules in lungs or livers were analyzed by histological examination. Immunohistochemistry and western blot analysis were conducted to determine protein expression. Result Herein, SsnB dose-dependently inhibited cell migration and invasion in mouse melanoma B16 cells with or without stimulation of lipopolysaccharide (LPS), Pam3csk4 or molecules from damaged tumor cells (DTC-Ms). The expression of matrix metalloproteinases (MMP)-2 was also significantly abated by SsnB in LPS-modulated B16 cells. And SsnB reduced LPS-activated B16 cells adhesion to extracellular matrix components collagen I and fibronectin in a dose-dependent manner. In vivo, SsnB obviously attenuated LPS-activated pulmonary metastasis in mice by reduction the number of metastatic nodules on the lung surfaces, lung weight and levels of tumor necrosis factor (TNF)-α and interleukin (IL)-6 in serums and lungs. Moreover, in experimental hepatic metastasis model mice, SsnB remarkably repressed LPS-stimulated the number of metastatic nodules along with liver weight; and SsnB significantly suppressed LPS-activated increase levels of TNF-α and IL-6 in livers. Immunohistochemistry analysis indicated that SsnB inhibited the expression of TLR4 in livers. Furthermore, SsnB remarkably blocked p38 and ERK1/2 signaling pathway in LPS-induced B16 cells. P38 and ERK1/2 signaling silencing, using BIRB0796 (small molecular inhibitor of p38 MAPK) and PD184352 (inhibitor of MEK1/2 kinases that activate ERK1/2), significantly abated LPS-induced migration and invasion of B16 cells. Conclusion The present study reports a novel use of SsnB in mitigating TLRs ligands-induced melanoma metastasis by inhibition of p38 and ERK1/2 pathway.

Objective To compare the chemical characters of Sparganii Rhizoma from different areas via chromatographic analysis and to establish a sensitive LC/MS method for quality assessment of Sparganii Rhizoma. Methods Under the optimised HPLC-PDA chromatographic conditions, twenty batches of Sparganii Rhizoma were analyzed by HPLC fingerprints. Principal component analysis (PCA), orthogonal projections to latent structures discriminant analysis (OPLS-DA) and hierarchical cluster analysis (HCA) were performed based on all peak areas of Sparganii Rhizoma fingerprints. Meanwhile, part of common peaks were subsequently quantified by UFLC-QTRAP-MS. Results The similarity values of HPLC fingerprints fluctuated in a wide range of 0.511–0.973, which showed variable differences of chemical characters among Sparganii Rhizoma from twenty habitats. PCA, OPLS-DA and HCA indicated that samples could be divided into five groups with different chemical characters, which generally corresponded with their geographical distributions. A total of 31 peaks in HPLC fingerprints were marked, and eight of them were identified and quantified. The quantitative result was generally in agreement with the classifications based on HPLC fingerprints, which indicated that Sparganii Rhizoma samples from eastern China mostly contained more contents including phenolic acids and flavonoids. Conclusion This study not only proved that there were relationships between geographic distributions and internal chemical compositions of plants, which could provide evidence to the traditional Chinese medicine concept “geo-authentic”, but also supplied a sensitive and rapid simultaneous quantitive method for the quality estimation of Sparganii Rhizoma.

In this work, the effect of phenanthrene and pyrene on biosynthesis and accumulation of bioactive ingredients of Sparganium stoloniferum Buch.-Ham. was evaluated. The analysis of phenanthrene and pyrene was performed using Gas Chromatography-Mass Spectrometry (GC–MS). The bioactive compounds were determined using different analysis technology. In the presence of pyrene and phenanthrene, this plant could grow well. The decreased or increased phenolics content could be observed in response to pyrene and phenanthrene. The low concentration phenanthrene and pyrene could enhance the accumulation of the mannitol and high concentration PAHs inhibit the accumulation. The accumulation of total saponins was not sensitive to pyrene and phenanthrene. To adapt the changes in environmental conditions the polysaccharides accumulation significantly increased. The findings obtained from this study will provide the science basis to investigate the accumulation of bioactive ingredients from plant in the presence of phenanthrene and pyrene.

Long-term exposure to high levels of arsenic (As) will result in damage to organs. Compared with free arsenic, protein-bound arsenic are more difficult to be excreted from the bodies due to their complexation with biological macromolecules. We developed a method of size exclusion chromatography (SEC) and ion exchange chromatography (IEC) combined with inductively coupled plasma-mass spectrometry (ICP-MS) and multiple reaction monitoring (MRM) mode, which was used to determine bound-arsenic species. DMAV was identified as bound arsenic species in rat livers after As4S4 overexposure. Subsequent proteomics analysis showed the potential binding partners included hemoglobin, glutathione S-transferases, superoxide dismutase [Cu-Zn] & [Mn], thiosulfate sulfurtransferase, and metallothionein-2. The method developed here was sensitive, repeatable, and conducive to arsenic analysis, especially for toxicity evaluation of arsenic-containing substances in vivo.

Animal venoms contain many peptides with high specificity and selectivity against their protein targets, a characteristic which makes venoms an invaluable source of potential drugs. High-sensitivity mass spectrometry (MS)- based peptidomic platform has evolved as a predominant method for natural peptide drug discovery due to its strength for direct and rapid identification of peptides and peptide-associated post-translational modifications (PTMs). In this study, we used cell-affinity assays combined with nanoLC-MS/MS based peptidomics to identify cancer cell binding peptides (CBPs) from Bufo Bufo gargarizans. We identified 76 potential cell binding peptides and 237 non-affinity peptides in venom extracts from Asiatic toads, and some were verified with MS-parallel reaction monitoring (PRM) mode. These peptides were further analyzed and internalized within human cells and some demonstrated anti-tumor properties in vitro. These specific peptides might be used as templates for peptide-based drug design or optimization.