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Background: Acute lung injury (ALI) is the main cause of death in clinical respiratory virus infection. Liang-Ge-San (LGS), a famous traditional Chinese formula, has been proved to be effective in treating ALI caused by lipopolysaccharide. However, the effects of LGS on ALI induced by viral infections remain uncertain., Purpose: To investigate the effect and mechanism of action of LGS on viral infection-induced ALI., Methods: The inhibitory effects of LGS on virus-induced inflammation in vitro were evaluated by qRT-PCR and ELISA. The protein expression of α7nAChR was examined by Western blotting. α7nAChR was inhibited by the transfection of siRNA or methyllycaconitine citrate (MLA, an α7nAChR inhibitor) to investigate the role of α7nAChR in the anti-inflammatory effect of LGS. Adoptive culture and co-culture systems of macrophages RAW264.7 and alveolar epithelial cells MLE-12 were established to mimic their interaction. Western blotting, immunofluorescence, flow cytometry and transmission electron microscopy were used to examine the effects of LGS on mitophagy inhibition. In vivo, ALI mouse models induced by SARS-CoV-2, H1N1 or Poly(I:C) infection were established to explore the therapeutic effect and mechanism of LGS., Results: LGS reduced the release of IL-6, TNF-α and IL-1β and increased the expression of α7nAChR in virus-infected RAW264.7 cells. The blockage of α7nAChR counteracted the anti-inflammatory effect of LGS. Moreover, LGS significantly inhibited autophagy in MLE-12 cells induced by Poly(I:C) in adoptive culture and co-culture systems of RAW264.7 and MLE-12 cells, which could be attenuated after the inhibition of α7nAChR in RAW264.7 cells by decreasing the secretion of IL-6, TNF-α and IL-1β. Further study showed that LGS suppressed TNF-α-induced mitochondrial damage and mitophagy by inhibiting the generation of ROS in MLE-12 cells. In vivo, LGS significantly prolonged the survival time, alleviated pathological injury and acute inflammation of ALI mice induced by SARS-CoV-2, H1N1 or Poly(I:C) infection which were associated with the inhibition of α7nAChR-mediated mitophagy., Conclusion: This study first demonstrates that LGS inhibits virus infection-induced inflammation in RAW246.7 cells by increasing the expression of α7nAChR, thereby inhibiting mitophagy induction in MLE-12 cells to alleviate ALI. This work indicates that LGS may serve as a candidate drug for treating ALI/ARDS caused by viral infection., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier GmbH.)

Broccoli ( Brassica oleracea L.) is a vegetable with numerous nutritional properties, with sulforaphane (SFN) being the most abundant and unique bioactive ingredient. SFN has anti-inflammatory, antioxidant, and anti-cancer activities. In this study, a series of SFN derivatives were synthesized and screened for improved antidepressant effects. Among these, the SFN derivatives SLL-III-9 and SLL-III-120 were the best candidates, and the potential antidepressant mechanism of SFN, SLL-III-9, and SLL-III-120 associated with their effects in a chronic unpredictable mild stress (CUMS) mouse model was explored based on the microbiota-gut-brain axis. All three compounds were able to relieve depression-like behaviors in CUMS mice and regulate the composition of the gut bacteria Firmicutes, Actinobacteria, Parabasalia, and Tenericutes at the phylum level and Bacteroidales bacterium , Lachnospiraceae bacterium A4 , Muribaculum intestinale , Muribaculaceae bacterium , and Prevotella sp. MGM1 at the species level, possibly altering their function associated with the anti-inflammatory effect. Additionally, SFN and its derivatives upregulated the expression of the tight junction proteins ZO-1, occludin, and claudin and increased the concentration of IL-10, dopamine (DA), 5-hydroxytryptamine (5-HT) and the brain-derived neurotrophic factor (BDNF), while downregulating the expressions of proteins related to the NF-κB/NLRP3 pathway and reducing the concentration of TNF-α. Further in vitro studies revealed significant inhibition of the production of inflammatory factors IL-1β, IL-18, IL-6, and TNF-α in LPS-activated BV2 cells via the NF-κB/NLRP3 pathway when these cells were treated with SFN or its two derivatives. Taken together, the results suggested that SFN and its two derivatives, SLL-III-9 and SLL-III-120, could be considered potential compounds for the development of a promising and safe agent for combating depression.

Background: Hepatocellular carcinoma (HCC) is one of the most common and deadly cancers worldwide. POC1 centriolar protein A ( POC1A ) is a gene encoding a protein that plays a key role in the centrosome, and is one of the two isoforms of POC1. To date, the expression of POC1A in HCC and its potential as a biomarker and tumor therapeutic target have not been examined. This study aimed to explore the effect of POC1A on patients with HCC and its potential mechanism., Methods: This study investigated the role of POC1A in the occurrence and development of HCC. It analyzed the expression of POC1A in various types of HCC patients and its effect on survival using HCC patient information from the Gene Expression Omnibus (GEO), The Cancer Genome Atlas (TCGA), the Human Protein Atlas (HPA), and the Hepatocellular Carcinoma Cell DataBase (HCCDB). It then explored the major enrichment pathways and gene functions of POC1A in HCC using the gene set enrichment analysis (GSEA) method and examined its protein-protein interactions (PPIs). Finally, it predicted the potential transcription factors (TFs) and target microRNAs (miRNAs) of POC1A , and analyzed the single nucleotide variation (SNV) and copy number variation (CNV) mutations of POC1A and the related genes in HCC, as well as their effects on immune cells., Results: The results showed that POC1A was significantly overexpressed in HCC and was significantly associated with a poor prognosis. The Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses indicated that POC1A was mainly involved in the regulation of cell-cycle pathways and chromosome segregation functions. POC1A showed significant interactions with NUDC and PPARG, and they both had different numbers of SNV and CNV mutations in the HCC samples. In relation to immunity, the high expression of POC1A and its reciprocal genes may play an important role in B cells and macrophages., Conclusions: In general, our findings suggest that POC1A overexpression could have an important effect on the development of HCC by regulating cell-cycle pathways, and that it could serve as a novel prognostic biomarker and a potential therapeutic target for HCC., Competing Interests: Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://tcr.amegroups.com/article/view/10.21037/tcr-23-2398/coif). The authors have no conflicts of interest to declare., (2024 AME Publishing Company. All rights reserved.)

Competing Interests: Declaration of competing interest The authors declared that they have no competing financial interests reported in this paper.

Background: Tanreqing injection (TRQ) has been employed in clinical practice as a treatment for dengue fever (DF). Nevertheless, the precise pharmacological mechanism underlying its efficacy remains elusive., Method: Network pharmacology, molecular docking, transcriptome sequencing, and experimental evaluation were employed to analyze and study the inhibitory potential of TRQ against dengue virus (DENV)., Result: We found that TRQ inhibited the replication of DENV in human umbilical vein endothelial cells, Huh-7 cells, and Hep3B cells. In addition, TRQ prolonged the survival duration of AG129 mice infected with DF, decreased the viral load in serum and organs, and alleviated organ damage. Subsequently, ultra-high-performance liquid chromatography-tandem mass spectrometry analysis of TRQ was performed to identify 314 targets associated with 36 active compounds present in TRQ. Integration of multiple databases yielded 47 DF-related genes. Then, 15 hub targets of TRQ in DF were determined by calculating the network topology parameters (Degree). Gene Ontology and Kyoto Encyclopedia of Genes and Genomes analyses revealed that these pathways were primarily enriched in the processes of cytokine activation and leukocyte cross-endothelial migration, with significant enrichment of cell adhesion molecules. Molecular docking revealed favorable binding affinity between TRQ's key active compounds and the predicted hub targets. Transcriptome sequencing results showed TRQ's ability to restore the expression of vascular cell adhesion molecule-1 (VCAM-1) post-DENV infection. Finally, TRQ was found to modulate the immune status by regulating the nuclear factor kappa-B (NF-κB)- intercellular cell adhesion molecule-1 (ICAM-1)/VCAM-1 axis, as well as reduce immune cell alterations, inflammatory factor secretion, vascular permeability, and bleeding tendencies induced by DENV infection., Conclusion: Our research suggests that TRQ exerts therapeutic effects on DF by regulating the NF-κB-ICAM-1/VCAM-1 axis., Competing Interests: Declaration of competing interest The authors declared no conflict of interest., (Copyright © 2024. Published by Elsevier GmbH.)

The widespread use of silver nanoparticles (AgNPs) in commercial and industrial applications has led to their increased presence in the environment, raising concerns about their ecological and health impacts. This study pioneers an investigation into the chronic versus short-term acute toxicological impacts of differently coated AgNPs on zebrafish, with a novel focus on the thyroid-disrupting effects previously unexplored. The results showed that acute toxicity ranked from highest to lowest as AgNO 3 (0.128 mg/L), PVP-AgNPs (1.294 mg/L), Citrate-AgNPs (6.984 mg/L), Uncoated-AgNPs (8.269 mg/L). For bioaccumulation, initial peaks were observed at 2 days, followed by fluctuations over time, with the eventual highest enrichment seen in Uncoated-AgNPs and Citrate-AgNPs at concentrations of 13 and 130 μg/L. Additionally, the four exposure groups showed a significant increase in T3 levels, which was 1.28-2.11 times higher than controls, and significant changes in thyroid peroxidase (TPO) and thyroglobulin (TG) content, indicating thyroid disruption. Gene expression analysis revealed distinct changes in the HPT axis-related genes, providing potential mechanisms underlying the thyroid toxicity induced by different AgNPs. The higher the Ag concentration in zebrafish, the stronger the thyroid disrupting effects, which in turn affected growth and development, in the order of Citrate-AgNPs, Uncoated-AgNPs > AgNO 3 , PVP-AgNPs. This research underscores the importance of considering nanoparticle coatings in risk assessments and offers insights into the mechanisms by which AgNPs affect aquatic organisms' endocrine systems, highlighting the need for careful nanotechnology use and the relevance of these findings for understanding environmental pollutants' role in thyroid disease., Competing Interests: Declaration of competing interest The authors enjoy no competing financial interests to declare., (Copyright © 2024. Published by Elsevier Ltd.)

Microplastics (MPs), which are prevalent and increasingly accumulating in aquatic environments. Other pollutants coexist with MPs in the water, such as pesticides, and may be carried or transferred to aquatic organisms, posing unpredictable ecological risks. This study sought to assess the adsorption of lambda-cyhalothrin (LCT) by virgin and aged polyethylene MPs (VPE and APE, respectively), and to examine their influence on LCT's toxicity in zebrafish, specifically regarding acute toxicity, oxidative stress, gut microbiota and immunity. The adsorption results showed that VPE and APE could adsorb LCT, with adsorption capacities of 34.4 mg∙g -1 and 39.0 mg∙g -1 , respectively. Compared with LCT exposure alone, VPE and APE increased the acute toxicity of LCT to zebrafish. Additionally, exposure to LCT and PE-MPs alone can induce oxidative stress in the zebrafish gut, while combined exposure can exacerbate the oxidative stress response and intensify intestinal lipid peroxidation. Moreover, exposure to LCT or PE-MPs alone promotes inflammation, and combined exposure leads to downregulation of the myd88-nf-κb related gene expression, thus impacting intestinal immunity. Furthermore, exposure to APE increased LCT toxicity to zebrafish more than VPE. Meanwhile, exposure to PE-MPs and LCT alone or in combination has the potential to affect gut microbiota function and alter the abundance and diversity of the zebrafish gut flora. Collectively, the presence of PE-MPs may affect the toxicity of pesticides in zebrafish. The findings emphasize the importance of studying the interaction between MPs and pesticides in the aquatic environment., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

The interaction between pesticides and microplastics (MPs) can lead to changes in their mode of action and biological toxicity, creating substantial uncertainty in risk assessments. Succinate dehydrogenase inhibitor (SDHI) fungicides, a common fungicide type, are widely used. However, little is known about how penthiopyrad (PTH), a member of the SDHI fungicide group, interacts with polyethylene microplastics (PE-MPs). This study primarily investigates the individual and combined effects of virgin or aged PE-MPs and penthiopyrad on zebrafish (Danio rerio), including acute toxicity, bioaccumulation, tissue pathology, enzyme activities, gut microbiota, and gene expression. Short-term exposure revealed that PE-MPs enhance the acute toxicity of penthiopyrad. Long-term exposure demonstrated that PE-MPs, to some extent, enhance the accumulation of penthiopyrad in zebrafish, leading to increased oxidative stress injury in their intestines by the 7th day. Furthermore, exposure to penthiopyrad and/or PE-MPs did not result in histopathological damage to intestinal tissue but altered the gut flora at the phylum level. Regarding gene transcription, penthiopyrad exposure significantly modified the expression of pro-inflammatory genes in the zebrafish gut, with these effects being mitigated when VPE or APE was introduced. These findings offer a novel perspective on environmental behavior and underscore the importance of assessing the combined toxicity of PE-MPs and fungicides on organisms., Competing Interests: Declaration of competing interest The authors enjoy no competing financial interests to declare., (Copyright © 2024 Elsevier B.V. All rights reserved.)

This study evaluated the impact of pulsed electric fields (PEFs) combined with three-phase partitioning (TPP) extraction methods on the physicochemical properties, functional properties, and structural characterization of the soluble dietary fiber (SDF) derived from peanut shells (PS). The findings of this study indicated that the application of a PEF-TPP treatment leads to a notable improvement in both the extraction yield and purity of SDF. Consequently, the PEF-TPP treatment resulted in the formation of more intricate and permeable structures, a decrease in molecular weight, and an increase in thermal stability compared to SDFs without TPP treatment. An analysis revealed that the PEF-TPP method resulted in an increase in the levels of arabinose and galacturonic acid, leading to enhanced antioxidant capacities. Specifically, the IC 50 values were lower in SDFs which underwent PEF-TPP (4.42 for DPPH and 5.07 mg/mL for ABTS) compared to those precipitated with 40% alcohol (5.54 mg/mL for DPPH, 5.56 mg/mL for ABTS) and PEF75 (6.60 mg/mL for DPPH, 7.61 mg/mL for ABTS), respectively. Notably, the SDFs which underwent PEF-TPP demonstrated the highest water- and oil-holding capacity, swelling capacity, emulsifying activity, emulsion stability, glucose adsorption, pancreatic lipase inhibition, cholesterol adsorption, nitric ion adsorption capacity, and the least gelation concentration. Based on the synthesis scores obtained through PCA (0.536 > -0.030 > -0.33), which indicated that SDFs which underwent PEF-TPP exhibited the highest level of quality, the findings indicate that PEF-TPP exhibits potential and promise as a method for preparing SDFs.

Background: Encephalitis caused by dengue virus (DENV) is considered a manifestation of severe dengue. Tanreqing injection (TRQ) is a well-known Chinese patented medicine, which has been used to treat brain-related disorders by inhibiting inflammation. Nevertheless, the effects of TRQ on DENV encephalitis have not been studied. The aim of this study was to evaluate the effects of TRQ on DENV encephalitis and to explore its potential mechanisms., Methods: The cytotoxicity of TRQ was examined by MTT assay, and the anti-DENV activities of TRQ in BHK-21 baby hamster kidney fibroblast were evaluated through CCK-8 and plaque assays. The expression levels of NO, IL1B/IL-1β, TNFα and IL6 were measured by qRT‒PCR and ELISA in the BV2 murine microglial cell line. The inhibitory effects of TRQ on NLRP3 inflammasome activation in BV2 cells were examined by Western blotting, qRT‒PCR and ELISA. The effects of TRQ on HT22 mouse hippocampal neuronal cells were examined by CCK-8 assay, morphology observation and flow cytometry. Moreover, a DENV-infected ICR suckling mouse model was developed to investigate the protective role of TRQ in vivo., Results: TRQ decreased the release of NO, IL6, TNFα and IL1B from BV2 cells and inhibited the activation of NLRP3. The presence of the NLRP3 agonist nigericin reversed the anti-inflammatory activities of TRQ. Furthermore, TRQ inhibited the death of HT22 cells by decreasing IL1B in DENV-infected BV2 cells. In addition, TRQ significantly attenuated weight loss, reduced clinical scores and extended the survival in DENV-infected ICR suckling mice. Critically, TRQ ameliorated pathological changes in ICR suckling mice brain by inhibiting microglia and NLRP3 activation and decreasing the production of inflammatory factors and the number of dead neurons., Conclusion: TRQ exerts potent inhibitory effects on dengue encephalitis in vitro and in vivo by reducing DENV-2-induced microglial activation and subsequently decreasing the inflammatory response, thereby protecting neurons. These findings demonstrate the potential of TRQ in the treatment of dengue encephalitis., (© 2024. The Author(s).)

The application of chemotherapy drugs in tumor treatment has a long history, but the lack of selectivity of drugs often leads to serious side effects during chemotherapy. The natural anti-tumor ingredients derived from Chinese herbal medicine are attracting increased attention due to their diverse anti-tumor effects, abundant resources, and minimal side effects. An effective anti-tumor strategy may lie in the combination of these naturally derived anti-tumor ingredients with conventional chemotherapy drugs. This approach could potentially inhibit tumor growth and the development of drug resistance in tumor cells while reducing the adverse effects of chemotherapy drugs. This review provides a comprehensive overview of the combined therapy strategies integrating natural anti-tumor components from Chinese herbal medicine with chemotherapy drugs in current research. We primarily summarize various compounds in Chinese herbal medicine exhibiting natural anti-tumor activities and the relevant mechanisms in synergistic anti-tumor combination therapy. The focus of this paper is on underlining that this integrative approach, combining natural anti-tumor components of Chinese herbal medicine with chemotherapy drugs, presents a novel cancer treatment methodology, thereby providing new insights for future oncological research.

The co-exposure of microplastics (MPs) and other contaminants has aroused extensive attention, but the combined impacts of MPs and pesticides remain poorly understood. Acetochlor (ACT), a widely used chloroacetamide herbicide, has raised concerns for its potential bio-adverse effects. This study evaluated the influences of polyethylene microplastics (PE-MPs) for acute toxicity, bioaccumulation, and intestinal toxicity in zebrafish to ACT. We found that PE-MPs significantly enhanced ACT acute toxicity. Also, PE-MPs increased the accumulation of ACT in zebrafish and aggravate the oxidative stress damage of ACT in intestines. Exposure to PE-MPs or/and ACT causes mild damage to the gut tissue of zebrafish and altered gut microbial composition. In terms of gene transcription, ACT exposure triggered a significant increase in inflammatory response-related gene expressions in the intestines, while some pro-inflammatory factors were found to be inhibited by PE-MPs. This study provides a new perspective on the fate of MPs in the environment and on the assessment of the combined effects of MPs and pesticides on organisms., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Degradable plastics are gradually regarded as alternatives of conventional, synthetic organic polymers to reduce the plastics or microplastics (MPs) pollution; however, the reports upon environmental risk of degradable plastics are still limited. In order to evaluate the potential vector effect of biodegradable MPs on coexisting contaminants, sorption of atrazine onto pristine and ultraviolet-aged (UV) polybutylene adipate co-terephthalate (PBAT) MPs and polybutylene succinate co-terephthalate (PBST) MPs were investigated. The results showed that, UV aging led to more wrinkles and cracks on the surface, increased homogeneous chains proportion, enhanced hydrophobicity, and enlarged crystallinity of both MPs. The sorption kinetics of atrazine to MPs fitted well into pseudo-first-order (R 2  = 0.809-0.996) and pseudo-second-order (R 2  = 0.889-0.994) models. In the concentration range of 0.5-25 mg L -1 , the sorption isotherm fitted into linear (R 2  = 0.967-0.996) and Freundlich model (R 2  = 0.972-0.997), indicating that the absorption partitioning was the dominant sorption mechanism. The partition coefficient (K d ) of atrazine to PBAT- MPs (40.11-66.01 L kg -1 ) was higher than that of PBST- MPs (34.34-57.96 L kg -1 ), and the K d values of both MPs declined for aged MPs. The specific surface area, hydrophobicity, polarity and crystallinity of MPs jointly interpreted the changing sorption capacity of the MPs. In the present study, both aged PBAT- and aged PBST- MPs exhibited lower vector potential to atrazine than pristine MPs, suggesting reduced risk of being a pollutant carrier, which is of great significance for the development of biodegradable plastics., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Ethnopharmacological Relevance: Dengue virus (DENV) infection is a global public health issue without effective therapeutic interventions. Chinese medicine with heat-clearing and detoxifying properties has been frequently used in the treatment of viral infection. Ampelopsis Radix (AR) is a traditional Chinese medicine for clearing heat and detoxification that has been widely used in the prevention and treatment of infectious diseases. However, no studies on the effects of AR against viral infection have been reported, thus far., Aim of the Study: To explore the anti-DENV activities of the fraction (AR-1) obtained from AR both in vitro and in vivo., Materials and Methods: The chemical composition of AR-1 was identified by liquid chromatography-tandem MS (LC‒MS/MS). The antiviral activities of AR-1 were studied in baby hamster kidney fibroblast BHK-21 cells, ICR suckling mice and induction of interferon α/β (IFN-α/β) and IFN-γ R -/- (AG129) mice., Results: Based on LC‒MS/MS analysis, 60 compounds (including flavonoids, phenols, anthraquinones, alkaloids and other types) were tentatively characterized from AR-1. AR-1 inhibited the cytopathic effect, the production of progeny virus and the synthesis of viral RNA and proteins by blocking DENV-2 binding to BHK-21 cells. Moreover, AR-1 significantly attenuated weight loss, decreased clinical scores and prolonged the survival of DENV-infected ICR suckling mice. Critically, the viral load in blood, brain and kidney tissues and the pathological changes in brain were remarkably alleviated after AR-1 treatment. Further study on AG129 mice showed that AR-1 obviously improved the clinical manifestations and survival rate, reduced viremia, attenuated gastric distension and relieved the pathological lesions caused by DENV., Conclusions: In summary, this is the first report that AR-1 exhibits anti-DENV effects both in vitro and in vivo, which suggests that AR-1 may be developed as a therapeutic candidate against DENV infection., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Objective: Mid- to late-Holocene large-scale population migration profoundly impacted the interaction of ethnic groups and cultures across Eurasia, notably in Central Asia. However, due to a lack of thorough historical documents, distinctive burial items, and human remains, the process of population migration during this historical era in the area is still unclear. Using an interdisciplinary approach at the Lafuqueke (LFQK) cemetery, this study investigates the spatiotemporal processes and explores the factors that influenced human migration in the eastern Tianshan Mountains between the 7th and 12th centuries., Materials and Methods: In this study, tooth enamel from 56 human remains found in the LFQK cemetery in Hami Basin, eastern Tianshan Mountains, is examined for strontium and lead isotopes., Results: The early, middle, and late phases of migration might potentially be represented by a three-phase migration model, according to the isotopic study. The highest proportion of the early phase (ca. 7th-mid 7th century) comprised non-locals (54.55%), although this percentage decreased in the middle phase (mid 7th-mid 8th centuries, 30.77%). After the 10th century, the proportion of non-locals again fell (16.13%)., Conclusion: In this study, the interdisciplinary approach was employed to propose a new model for the diachronic changes that accompanied human migration and cultural interaction in the eastern Tianshan Mountains and identified geopolitics as a significant factor influencing the migratory behavior of LFQK population in this region between the 7th and 12th centuries., (© 2023 Wiley Periodicals LLC.)

Background: Licorice (Glycyrrhiza uralensis Fisch.), a well-known traditional medicine, is traditionally used for the treatment of respiratory disorders, such as cough, sore throat, asthma and bronchitis. We aim to investigate the effects of liquiritin (LQ), the main bioactive compound in licorice against acute lung injury (ALI) and explore the potential mechanism., Methods: Lipopolysaccharide (LPS) was used to induce inflammation in RAW264.7 cells and zebrafish. Intratracheal instillation of 3 mg/kg of LPS was used for induction an ALI mice model. The concentrations of IL-6 and TNF-α were tested using the enzyme linked immunosorbent assay. Western blot analysis was used to detect the expression of JNK/Nur77/c-Jun related proteins. Protein levels in bronchoalveolar lavage fluid (BALF) was measured by BCA protein assay. The effect of JNK on Nur77 transcriptional activity was determined by luciferase reporter assay, while electrophoretic mobility shift assay was used to examine the c-Jun DNA binding activity., Results: LQ has significant anti-inflammatory effects in zebrafish and RAW264.7 cells. LQ inhibited the expression levels of p-JNK (Thr183/Tyr185), p-Nur77 (Ser351) and p-c-Jun (Ser63), while elevated the Nur77 expression level. Inhibition of JNK by a specific inhibitor or small interfering RNA enhanced the regulatory effect of LQ on Nur77/c-Jun, while JNK agonist abrogated LQ-mediated effects. Moreover, Nur77-luciferase reporter activity was suppressed after JNK overexpression. The effects of LQ on the expression level of c-Jun and the binding activity of c-Jun with DNA were attenuated after Nur77 siRNA treatment. LQ significantly ameliorated LPS-induced ALI with the reduction of lung water content and BALF protein content, the downregulation of TNF-α and IL-6 levels in lung BALF and the suppression of JNK/Nur77/c-Jun signaling, which can be reversed by a specific JNK agonist., Conclusion: Our results indicated that LQ exerts significant protective effects against LPS-induced inflammation both in vivo and in vitro via suppressing the activation of JNK, and consequently inhibiting the Nur77/c-Jun signaling pathway. Our study suggests that LQ may be a potential therapeutic candidate for ALI and inflammatory disorders., (© 2023. The Author(s).)

Lauraceae is a large family with significant economic and medicinal value. Bioactive ingredients from Lauraceae plants have contributed greatly to medicines, food nutrients and fine chemical products. In recent years, quite a few sesquiterpenes and diterpenes with unique structures have been achieved from Lauraceae and their potential benefits are embodied in a wide range of health areas. To our knowledge, there is no review to summarizes these constituents and their biological effects systematically. This current work aims to classify and ascribe the structural types and bioactivities of the identified sesquiterpenes and diterpenes. Herein, a total of 362 sesquiterpenes and 69 diterpenes were comprehensively complied. The various bioactivities could be recognized as cytotoxicity, anti-proliferation and/or anti-apoptosis, anti-inflammation, anti-oxidation, anti-bacterium, etc. This updated data could serve as a catalysis of these sesquiterpenes and diterpenes for the future medical and industrial applications., Competing Interests: The authors declare no competing interests., (© 2022 Published by Elsevier Ltd.)

Objective: A set of cardiac MRI short-axis image dataset is constructed, and an automatic segmentation based on an improved SegNet model is developed to evaluate its performance based on deep learning techniques., Methods: The Affiliated Hospital of Qingdao University collected 1354 cardiac MRI between 2019 and 2022, and the dataset was divided into four categories: for the diagnosis of cardiac hypertrophy and myocardial infraction and normal control group by manual annotation to establish a cardiac MRI library. On the basis, the training set, validation set and test set were separated. SegNet is a classical deep learning segmentation network, which borrows part of the classical convolutional neural network, that pixelates the region of an object in an image division of levels. Its implementation consists of a convolutional neural network. Aiming at the problems of low accuracy and poor generalization ability of current deep learning frameworks in medical image segmentation, this paper proposes a semantic segmentation method based on deep separable convolutional network to improve the SegNet model, and trains the data set. Tensorflow framework was used to train the model and the experiment detection achieves good results., Results: In the validation experiment, the sensitivity and specificity of the improved SegNet model in the segmentation of left ventricular MRI were 0.889, 0.965, Dice coefficient was 0.878, Jaccard coefficient was 0.955, and Hausdorff distance was 10.163 mm, showing good segmentation effect., Conclusion: The segmentation accuracy of the deep learning model developed in this paper can meet the requirements of most clinical medicine applications, and provides technical support for left ventricular identification in cardiac MRI., Competing Interests: Declaration of Competing Interest The authors declare no conflict of interest for this paper., (Copyright © 2022. Published by Elsevier B.V.)

Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous soil contaminants, and their bioaccessibility determines their environmental risks in contaminated land. In the present study, the residual concentrations of PAHs in the soils of two industrial sites were determined, and their bioaccessibility was estimated by the hydroxypropyl-β-cyclodextrin extraction (HPCD) extraction method. The results showed heavy PAH contamination at both site S1 (0.38-3342.5 mg kg -1 ) and site S2 (0.2-138.18 mg kg -1 ), of which high molecular weight (HMW) PAHs (4-, 5-, and 6-ring compounds) accounted for approximately 80%. The average bioaccessibility of PAHs at sites S1 and S2 was 52.02% and 29.28%, respectively. The bioaccessibility of certain PAH compounds decreased with increasing ring number of the molecule. Lower PAH bioaccessibility was detected in loamy and silty soil textures than in sandy soil. Moreover, among the soil properties, the dissolved organic matter, total organic carbon, total potassium, and total manganese concentrations had significant effects on the bioaccessibility of PAHs. The toxicity analysis showed that the composition and bioaccessibility of PAHs could affect their potential toxicity in soil. We suggest that bioaccessibility should be taken into consideration when assessing the toxicity of PAHs in soil, and more attention should be given to low-ring PAHs with high bioaccessibility., (© 2022. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Lactate can play an immunosuppressive role in the tumor microenvironment and promote tumor development by recruiting and inducing the activity of immunosuppressive cells and molecules. High lactate concentrations are important for tumor cell metastasis, angiogenesis, and treatment resistance. With the in-depth studies on tumor metabolism, lactate, one of the key factors involved in glycolysis, has been increasing emerged its characteristic clinical value in colorectal cancer (CRC). In this study, lactate genes were screened based on lactate metabolism pathways. Subsequently, the lactate subtypes were determined by clustering and analysis of the subtypes at all levels, including immune checkpoints, immune infiltration, and clinical characteristics, which revealed the biological significance of lactate metabolism in CRC. Subtype-based differential gene analysis resulted in a lactate score, which stratifies the prognosis of CRC. We discovered that 27 lactate genes and 61 lactate-phenotype genes are associated with immune cell infiltration and have a significant prognostic efficacy. The CRC patients were clustered into four subtypes and five clusters, based on lactate genes and lactate-phenotype genes, respectively. There are significant differences in survival time and activities of hallmark pathways, namely immune-related signatures and chemokines, among these subtypes and clusters. Particularly, cluster 2 and subtype 1 have significantly higher lactate scores than that of the others. In conclusion, lactate score is an independent prognostic factor for cancer that can be used as a clinical guide for predicting CRC progression and as an evaluation factor for the effect of immunotherapy in CRC., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Zhu, Jiang, Cao, Yang, Shu, Feng, Yang, Sun and Shao.)

Background: Hepatocellular carcinoma (HCC) is characterized by poor diagnosis and high mortality. Novel and efficient therapeutic agents are urgently needed for the treatment. Hedyotis diffusa Willd (HDW) is used to treat cancers, especially HCC in China., Purpose: The study aimed to identify the main anti-HCC extract in HDW and to explore the mechanism of the active extract., Materials and Methods: The high-performance liquid chromatography-quadrupole-time of flight mass spectrometry (HPLC-QTOF-MS) method was used for the simultaneous determination of main compounds in the ethyl acetate fraction of HDW (EHDW). The toxicity test of different HDW fractions was carried out on larvae at 2 day-post-fertilization (dpf) for 72 h. The in vivo anti-HCC effect of different HDW fractions was evaluated on a zebrafish tumor model by immersion administration. The antiproliferative effect of HDW fractions was determined with MTT assay, as well as hematoxylin and eosin (HE) staining assay. Hoechst 33258 staining was used to observe changes in nucleus morphology. Flow cytometry analysis was used to investigate apoptosis induction. Western blot analysis was used to examine apoptosis-related proteins, and key proteins in JNK/Nur77 signaling pathway. SP600125 was served to validate the apoptotic mechanism., Results: EHDW showed the strongest tumor cell growth inhibitory effect on zebrafish tumor model. Further study revealed that EHDW induced apoptosis in zebrafish tumor model and in cultured Hep3B cells. Meanwhile, it has been shown that the levels of BCL2-associated X (Bax), cytochrome c (cyto c), cleaved-caspase 3, and poly-ADP-ribose polymerase (PARP) cells were upregulated. In contrast, the level of antiapoptotic B cell lymphoma-2 (Bcl-2) was downregulated in Hep3B cells. Additionally, EHDW activated JNK/Nur77 pathway by increasing the levels of p-JNK (Thr183/Tyr185) and p-Nur77 (Ser351) . Further study showed that blockage of JNK by SP600125 reversed EHDW-induced JNK/Nur77 pathway and the downstream apoptotic proteins., Conclusion: In conclusion, EHDW exerted the anti-HCC effect, which may be attributed to the activation of JNK/Nur77 pathway. This study supported the rationale of HDW as an HCC therapeutic agent., Competing Interests: The authors claim no conflicts of interest., (Copyright © 2022 Weimin Ning et al.)

Broomcorn and foxtail millet were the most important crops in northern China during the Neolithic period. Although the significance of broomcorn millet in human subsistence exceeded that of foxtail millet during the early Neolithic, this pattern was reversed by the end of Neolithic period. However, the process underlying this shift remains unclear. The recent excavation of the Gedachuan (GDC) in Zhangjiachuan county has revealed an abundance of relics including millet crop remains from relatively continuous strata of the Yangshao and Qijia cultures, and therefore provides a unique opportunity to examine how and when foxtail millet replaced broomcorn millet as the dominant crop in the western Loess Plateau during the Neolithic period. In this study, we identify 1,738 and 2,686 broomcorn and foxtail millet remains, respectively, from 74 flotation samples, accounting for 38.81% and 59.98% of total plant remains, respectively. Compared with 23 direct dates of carbonized crop grains in GDC, we propose that the weight of foxtail millet in plant subsistence of GDC first exceeded that of broomcorn millet as early as ∼5,500 BP, filling an important gap in the archaeobotanical record from the western Loess Plateau. Further comparative analysis of multidisciplinary data suggests the shift in significance of these two millet crops during the late Neolithic may have been triggered by variations in human settlement intensity and climate change in the western Loess Plateau. The results of this study also suggest that the Banpo Phase of Yangshao Culture survived in the western Loess Plateau as late as ∼5,600 BP., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Yang, Wang, Li, Dong, Cao, Ma, Chen and Dong.)

Background: Type A aortic dissection (TAAD) has a rapid onset and high mortality. Currently, aortic diameter is the major criterion for evaluating the risk of TAAD. We attempted to find other aortic morphological indicators to further analyze their relationships with the risk of type A dissection., Methods: We included the imaging and clinical data of 112 patients. The patients were divided into three groups, of which Group 1 had 49 patients with normal aortic diameter, Group 2 had 22 patients with ascending aortic aneurysm, and Group 3 had 41 patients with TAAD. We used AW Server software, version 3.2, to measure aorta-related morphological indicators., Results: First, in Group 1, the univariate analysis results showed that ascending aortic diameter was correlated with patient age ( r 2 = 0.35) and ascending aortic length (AAL) ( r 2 = 0.43). AAL was correlated with age ( r 2 = 0.12) and height ( r 2 = 0.11). Further analysis of the aortic morphological indicators among the three groups found that the median aortic diameter was 36.20 mm in Group 1 (Q1-Q3: 33.40-37.70 mm), 42.5 mm in Group 2 (Q1-Q3: 41.52-44.17 mm) and 48.6 mm in Group 3 (Q1-Q3: 42.4-55.3 mm). There was no significant difference between Groups 2 and 3 ( P > 0.05). Group 3 had the longest AAL (median: 109.4 mm, Q1-Q3: 118.3-105.3 mm), followed by Group 2 (median: 91.0 mm, Q1-Q3: 95.97-84.12 mm) and Group 1 (81.20 mm, Q1-Q3: 76.90-86.20 mm), and there were statistically significant differences among the three groups ( P < 0.05). The Aortic Bending Index (ABI) was 14.95 mm/cm in Group 3 (Q1-Q3: 14.42-15.78 mm/cm), 13.80 mm/cm in Group 2 (Q1-Q3: 13.42-14.42 mm/cm), and 13.29 mm/cm in Group 1 (Q1-Q3: 12.71-13.78 mm/cm), and the difference was statistically significant in comparisons between any two groups ( P < 0.05). Regression analysis showed that aortic diameter + AAL + ABI differentiated Group 2 and Group 3 with statistical significance (area under the curve (AUC) = 0.834), which was better than aortic diameter alone (AUC = 0.657; P < 0.05)., Conclusions: We introduced the new concept of ABI, which has certain clinical significance in distinguishing patients with aortic dissection and aneurysm. Perhaps the ascending aortic diameter combined with AAL and ABI could be helpful in predicting the occurrence of TAAD., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Sun, Li, Wang, Sun, Zhang, Chi, Cao, Ma, Yan and Liu.)

Toad venom ( Chansu ) is used in the treatment of infectious and inflammatory diseases in China and East/Southeast Asian countries. However, the anti-inflammatory components of toad venom have not yet been systematically evaluated and clearly defined. To investigate the anti-inflammatory effects of toad venom and identify new anti-inflammatory ingredients, we used zebrafish, an alternative drug screening model, to evaluate the anti-inflammatory effects of 14 bufadienolides previously isolated from toad venom. Most of the bufadienolides were found to exert significant anti-inflammatory effects on lipopolysaccharide-, CuSO 4 -, or tail transection-induced zebrafish inflammatory models. Moreover, gammabufotalin ( 6: ) inhibits lipopolysaccharide-induced inflammation by suppressing the myeloid differentiation primary response 88/nuclear factor-kappa B and STAT3 signal pathways. This study confirms the potential of zebrafish in drug screening, clarifies the anti-inflammatory effects of bufadienolides from toad venom, and indicates that gammabufotalin may be developed as a novel therapeutic agent for inflammatory diseases in the future., Competing Interests: The authors declare that they have no conflict of interest., (Thieme. All rights reserved.)

Column experiments were conducted to investigate the effects of ion type, ion strength, humic acid (HA), and nanometer alumina (NA) particles on the transport of hexavalent chromium (HC) in saturated porous media. A one-dimensional model is developed to simulate the migration of HC affected by NA particles. The results show that nano-alumina particles would enhance the mobility of HC in saturated porous media. However, the influence of NA on the migration of HC in porous media is complex. When the concentration of NA reaches 30 mg/L, HC has minimum retention parameter and best mobility. The transport of HC also is affected by ion strength and ion type. Higher ionic strength would decrease the retention of HC and enhance its mobility. Compared with sodium ion, calcium ion has larger effects on the transport of HC. Moreover, HA can improve the mobility of HC in saturated porous media, but the corresponding promoting effect decreases with the increase of HA concentration. As nanometer contaminants and HC come into the subsurface environment, findings from this study elucidate the key factors and processes controlling the transport of HC in porous media, which can promote the prediction and assessment of HC in the groundwater system., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Flower and fruit colors are important agronomic traits. To date, there is no forward genetic evidence that the glutathione S-transferase (GST) gene is responsible for the white flower color in peach (Prunus persica). In this study, genetic analysis indicated that the white-flower trait is monogenetic, is recessive to the non-white allele, and shows pleiotropic effects with non-white-flowered types. The genetic locus underpinning this trait was mapped onto chromosome 3 between 0.421951 and 3.227115 Mb by using bulked segregant analysis in conjunction with whole-genome sequencing, and was further mapped between 0 and 1.178149 Mb by using the backcross 1 (BC 1 ) population. Finally, the locus was fine-mapped within 535.974- and 552.027-kb intervals by using 151 F 2 individuals and 75 individuals from a BC 1 self-pollinated (BC 1 S 1 ) population, respectively. Pp3G013600, encoding a GST that is known to transport anthocyanin, was identified within the mapping interval. The analysis of genome sequence data showed Pp3G013600 in white flowers has a 2-bp insertion or a 5-bp deletion in the third exon. These variants likely render the GST non-functional because of early stop codons that reduce the protein length from 215 amino acids to 167 and 175 amino acids, respectively. Genetic markers based on these variants validated a complete correlation between the GST loss-of-function alleles and white flower in 128 peach accessions. This correlation was further confirmed by silencing of Pp3G013600 using virus-induced gene silencing technology, which reduced anthocyanin accumulation in peach fruit. The new knowledge from this study is useful for designing peach breeding programs to generate cultivars with white flower and fruit skin., (© 2021 Society for Experimental Biology and John Wiley & Sons Ltd.)

The sorption behavior of phthalate additives in plastic and microplastic litter is an important process controlling the exposure, net health risk and ecotoxicity of these co-occurring pollutants. Plastic crystallinity and particle morphology are hypothesized to be important variables for microplastics sorption behavior, but to date there have been few direct studies to explicitly test for the influence of these parameters. To address this, in this study we explored the sorption of dibutyl phthalate (DBP) as a probe molecule to diverse polyethylene microplastics including irregularly-shaped pure polyethylene microplastics (IPPM), black plastic film microplastics (BPFM), white plastic film microplastics (WPFM), and commercial microspheres (CM), which had crystallinities ranging from 17 to 99%. Sorption kinetics for all materials could be well represented with both a pseudo-first-order (R 2  = 0.87-0.93) and pseudo-second-order model (R 2  = 0.87-0.93). Further, sorption was highly linear in the concentration range of 0.5-10 mg L -1 , with no greater performance from a linear sorption model (R 2  = 0.96-0.99) than the non-linear Freundlich or Temkin sorption models. The partition coefficient (K d ) of DBP sorption onto IPPM, BPFM, WPFM and CMs were 1974.55 L kg -1 , 1483.85 L kg -1 , 1477.45 L kg -1 and 509.37 L kg -1 , respectively, showing a significant decrease with increasing crystallinity (r 2  = 0.98). The particle size of microplastics (27-1000 μm) is, however, an indecisive factor affecting their sorption behavior for DBP in this study. This study provides new insight that crystallinity plays a governing role on the sorption of phthalate from microplastic. This should be considered in future exposure studies and assessments of phthalates from plastics and microplastics., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Although accelerated cellular senescence is closely related to the progression of chronic kidney disease (CKD) and renal fibrosis, the underlying mechanisms remain largely unknown. Here, we reported that tubular aberrant expression of Brahma-related gene 1 (BRG1), an enzymatic subunit of the SWItch/Sucrose Non-Fermentable complex, is critically involved in tubular senescence and renal fibrosis. BRG1 was significantly up-regulated in the kidneys, predominantly in tubular epithelial cells, of both CKD patients and unilateral ureteral obstruction (UUO) mice. In vivo, shRNA-mediated knockdown of BRG1 significantly ameliorated renal fibrosis, improved tubular senescence, and inhibited UUO-induced activation of Wnt/β-catenin pathway. In mouse renal tubular epithelial cells (mTECs) and primary renal tubular cells, inhibition of BRG1 diminished transforming growth factor-β1 (TGF-β1)-induced cellular senescence and fibrotic responses. Correspondingly, ectopic expression of BRG1 in mTECs or normal kidneys increased p16INK4a, p19ARF, and p21 expression and senescence-associated β-galactosidase (SA-β-gal) activity, indicating accelerated tubular senescence. Additionally, BRG1-mediated pro-fibrotic responses were largely abolished by small interfering RNA (siRNA)-mediated p16INK4a silencing in vitro or continuous senolytic treatment with ABT-263 in vivo. Moreover, BRG1 activated the Wnt/β-catenin pathway, which further inhibited autophagy. Pharmacologic inhibition of the Wnt/β-catenin pathway (ICG-001) or rapamycin (RAPA)-mediated activation of autophagy effectively blocked BRG1-induced tubular senescence and fibrotic responses, while bafilomycin A1 (Baf A1)-mediated inhibition of autophagy abolished the effects of ICG-001. Further, BRG1 altered the secretome of senescent tubular cells, which promoted proliferation and activation of fibroblasts. Taken together, our results indicate that BRG1 induces tubular senescence by inhibiting autophagy via the Wnt/β-catenin pathway, which ultimately contributes to the development of renal fibrosis., (© 2021 The Author(s).)

Raw milk is a nature media of microbiota that access milk from various sources, which constitutes a challenge in dairy production. This study characterizes the relationship between the raw milk quality and the bacteria diversity at different sampling sites in dairy farms, aiming to provide a strong scientific basis for good hygienic practices and optimized procedure in milk production. High-throughput sequencing of 16S rRNA V3-V4 region was used to analyze the components, abundance and diversity of 48 bacterial population sampled from 8 different sites in dairy farm: pre-sterilized cow's teats (C1), post-sterilized cow's teats (C2), milking cluster (E), milk in storage tank (M1), transport vehicle (M2), storage equipment (E2), cow's dung samples (F) and drinking water (W). Firmicutes account for predominantly 32.36% (C1), 44.62% (C2), 44.71% (E), 41.10% (M1), 45.08% (M2), 53.38% (F) of all annotated phyla. Proteobacteria accounts for 81.79% in W group and Actinobacteria 56.43% in E2 group. At the genus level, Acinetobacter was the most abundant genus that causes bovine mastitis, Acinetobacter and Arthrobacter were dominant in C1, C2, and E groups, Kocuria in E2 group and Arcobacter in W group. E, C1, and C2 group have very similar bacterial composition, and M1 and M2 demonstrated similar composition, indicating that the milking cluster was polluted by the environment or contact with cow udders. Bacterial population composition in different sampling sites identified by NGS reveals a correlation between the bacteria communities of raw milk production chain and the quality of raw milk., Competing Interests: The authors declare no potential conflicts of interest., (© Korean Society for Food Science of Animal Resources.)

Ethnopharmacological Relevance: Liang-Ge-San (LGS), a traditional Chinese medicine (TCM) formula, is usually used in acute inflammatory diseases in China., Aim of the Study: This study aims to detect the optimal combination of anti-inflammatory components from LGS., Materials and Methods: Four mainly representative components (phillyrin, emodin, baicalin, and liquiritin) from LGS were chosen. The optimal combination was investigated by orthogonal design study. Zebrafish inflammation model was established by lipopolysaccharide (LPS)-yolk microinjection, and then the anti-inflammatory activities of different combinations were determined by survival analysis, changes on inflammatory cells infiltration, the MyD88/NF-κB and MAPK pathways and inflammatory cytokines production., Results: The different combinations of bioactive ingredients from LGS significantly protected zebrafish from LPS-induced inflammation, as evidenced by decreased recruitment of macrophages and neutrophils, inhibition of the MyD88/NF-κB and MAPK pathways and down-regulation of TNF-α and IL-6. Among them, the combination group 8 most significantly protected against LPS. The combination of group 8 is: 0.1 μM of emodin, 2 μM of baicalin, 20 μM of phillyrin and 12.5 μM of liquiritin., Conclusion: The optimized combination group 8 exerts the most significant anti-inflammatory activity by inhibiting the recruitment of inflammatory cells, activation of the MyD88/NF-κB and MAPK pathways and the secretion of pro-inflammatory cytokines. This present study provides pharmacological evidences for the further development of new modern Chinese drug from LGS to treat acute inflammatory diseases, but indicated the use of zebrafish in the screening of components from formulas., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Ethnopharmacological Relevance: Sheng-Mai Yin (SMY), a famous traditional Chinese medicine formula, has been commonly used in China for centuries to treat various diseases, such as inflammation-related diseases. However, the anti-inflammatory activity of SMY and its potential mechanisms still have not yet been clearly understood., Aim of the Study: In this study, we aimed to determine the anti-inflammatory effect of SMY and explore its underlying mechanisms both on RAW 264.7 cells and zebrafish., Materials and Methods: The levels of pro-inflammatory cytokines IL-6 and TNF-α secreted by RAW 264.7 cells were measured by ELISA. The protein expressions of IκBα, p-IκBα (Ser32), STAT3 and p-STAT3 (Tyr705) were determined by Western blotting. And the nuclear translocation of NF-κB p65 in LPS-induced RAW 264.7 macrophage cells was detected by confocal microscopy. Moreover, the in vivo anti-inflammatory effect of SMY and its potential mechanisms were further investigated by survival analysis, hematoxylin-eosin staining (H&E), observation of neutrophil migration and quantitative real-time PCR (qRT-PCR) analysis in zebrafish inflammatory models., Results: SMY reduced the release of IL-6 and TNF-α, inhibited the phosphorylation of IκBα and STAT3 as well as the nuclear translocation of NF-κB p65 in LPS-induced RAW 264.7 cells. Furthermore, the increased survival, decreased infiltration of inflammatory cells and the attenuated migration of neutrophils together suggested the in vivo anti-inflammatory effects of SMY. More importantly, SMY reduced the gene expressions of pro-inflammatory cytokines and suppressed LPS-induced up-regulation of NF-κB, IκBα and STAT3 in zebrafish inflammatory models., Conclusion: SMY exerts significant anti-inflammatory effects with a potential mechanism of inhibiting the NF-κB and STAT3 signal pathways. Our findings suggest a scientific rationale of SMY to treat inflammatory diseases in clinic., (Copyright © 2020. Published by Elsevier B.V.)

Ethnopharmacological Relevance: Qingwen Baidu Decoction (QBD), a famous traditional Chinese medicine prescription with heat-clearing and detoxifying efficacies, is widely used in the treatment of inflammatory diseases. However, due to lack of holistic quality evaluation research, the further study on the detailed molecular mechanisms of action are still insufficient., Aim of the Study: This study aimed to evaluate the overall quality of QBD and to explore the anti-inflammatory effects and associated intracellular signaling pathways., Materials and Methods: a comprehensive method of chemical fingerprint analysis and simultaneous multi-component quantification was firstly developed by high performance liquid chromatography with diode array detector (HPLC-DAD). Similarity analysis, principal component analysis and hierarchical cluster analysis with heatmap were also applied to screen out the markers components in QBD samples. Moreover, its anti-inflammatory effects and mechanisms were further investigated by survival analysis, hematoxylin-eosin staining (H&E), neutrophil observation, quantitative real-time PCR analysis (qRT-PCR), Western blotting and confocal microscopy., Results: Twenty-one characteristic peaks from 11 herbs were chemically identified in the chromatographic fingerprint. Fifteen quantitative markers from 11 herbs, such as baicalin, wogonoside, geniposidic acid, oxypaeoniflora and so on, were screened out with the aid of chemometrics to further quantitatively assess the quality of QBD. The results of survival analysis, H&E and neutrophil observation in zebrafish inflammatory models consistently showed that QBD exerts potent anti-inflammatory effects in a dose-dependent manner. Additionally, QBD inhibited the activation of NF-κB and STAT3 signal pathways in LPS-induced zebrafish and RAW 264.7 macrophage cells., Conclusion: Collectively, our investigations firstly described the chemical profile of QBD and its possible mechanism of anti-inflammation, which provides a preferred strategy for monitoring the overall quality of QBD and supports its clinical application in treating inflammation-related diseases., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Acute lung injury (ALI) is a life-threatening disease without effective pharmacotherapies, so far. Forsythia suspensa is frequently used in the treatment of lung infection in traditional Chinese medicine. In search for natural anti-inflammatory components, the activity and the underlying mechanism of Forsythoside A (FA) from Forsythia suspensa were explored. In the present paper, BALB/c mice and murine RAW 264.7 cells were stimulated by LPS to establish inflammation models. Data showed that FA inhibited the production of TNF-α and IL-6 and the activation of STAT3 in LPS-stimulated RAW 264.7 cells. Additionally, FA increased the expression level of microRNA-124 (miR-124). Furthermore, the inhibitory effect of FA on STAT3 was counteracted by the treatment of miR-124 inhibitor. Critically, FA ameliorated LPS-induced ALI pathological damage, the increase in lung water content and inflammatory cytokine, cells infiltration and activation of the STAT3 signaling pathway in BALB/c mice. Meanwhile, FA up-regulated the expression of miR-124 in lungs, while administration with miR-124 inhibitor attenuated the protective effects of FA. Our results indicated that FA alleviates LPS-induced inflammation through up-regulating miR-124 in vitro and in vivo. These findings indicate the potential of FA and miR-124 in the treatment of ALI., (© 2020 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.)

ShengMai Formula (SMF), a famous traditional Chinese medicine (TCM) formula, has been extensively used for treating the diseases caused by Qi-Yin deficiency for almost 1000 years. However, few studies are elucidated about its batch-to-batch quality control system and the quality control markers remain largely unrevealed, which have hindered the development and utilization of SMF. In this study, we aimed to screen the optimal quality control markers to evaluate the overall quality consistency of SMF. High-performance liquid chromatography (HPLC) fingerprint coupled with similarity analysis (SA), principal components analysis (PCA) and hierarchical cluster analysis (HCA) was firstly established to hunt for the discriminant components that resulting in the chemical inconsistence among different batches of SMF. Subsequently, different batches of samples were selected to explore their immunomodulatory activities by neutral red method, Cell Counting Kit-8 (CCK-8) assay and enzyme-linked immunosorbent assay (ELISA). Finally, the fingerprint-efficacy relationships were further illuminated to discover the major bioactive compositions using grey relational analysis (GRA), partial least squares regression (PLSR) analysis and artificial neural network (ANN) analysis. As a result, schisandrol A, schisandrol B, methylophiopogonanone A, schisandrin B, ginsenoside Rf, ginsenoside Rb1, ginsenoside Rg2 and ginsenoside Rb2 were selected as the quality control markers and thus their simultaneous quantification was performed to both evaluate the batch-to-batch chemical and bioactive consistency among different batches of SMF. Our investigation not only stresses the necessity of consistency in efficacy besides chemical consistency, but also provides a comprehensive and powerful quality assessment approach, which is promising to monitor the overall quality consistency of SMF., Competing Interests: Declaration of Competing Interest The researchers claim no conflicts of interests., (Copyright © 2020. Published by Elsevier B.V.)

Purpose: To investigate commuter exposures to particulate matter (PM) in urban public transportation buses and subways, PM concentrations were simultaneously monitored for these two modes, over the same routes, in Xi'an, China., Methods: The microenvironment variabilities in each stage of the total trip were analyzed. Exposure doses for the different commute processes were estimated based on the heart rates of volunteers. Experimental measurements were taken during peak traffic hours in July and October (summer and autumn) on two typical commute routes, for a total of 36 trips. One-way ANOVA was used to analyze the effects of different variables on commuter exposures., Results: On the same route, the average PM exposure concentration of bus commuters was higher than those of subway commuters. For example, on Route 1 in the case study, the average PM10, PM2.5, and PM1 exposure concentrations of bus commuters were 71.6%, 19%, and 10.4% higher, respectively, than those of subway commuters. In the ground transportation mode, the exposure concentration of bus commuters was affected by the type of vehicle. Particle concentrations were significantly higher inside compressed natural gas (CNG) buses, than in pure electric (PE) buses, and in summer, the PM10 concentration in a CNG bus was 4.3 times higher than that in a PE bus. In a CNG bus, commuters in the back door area suffered the highest PM10 exposure concentration (179.6 μg/m 3 ), followed by those in the rear of the carriage (142.8 μg/m 3 ), and then those in the front door area (105.4 μg/m 3 )., Conclusion: Commuters' avoidance of ground traffic sources, effective ventilation systems in buses, and the use of screens in subway systems can all help to lower the PM exposure of commuters. For all the modes of transportation in our study, the hottest spots for PM exposure appeared in the period when commuters were waiting for transit vehicles to arrive., Competing Interests: Conflict of interestThe study was carried according to a protocol approved by Chang’an University, the approval number is 20180517. The objectives of the study were explained to the study participants and all participants signed volunteer consent form.Conflict of interestThe authors declare that they have no conflict of interest., (© Springer Nature Switzerland AG 2020.)

Ethnopharmacological Relevance: Liang-Ge-San (LGS) is a traditional Chinese medicine formula that commonly used in acute inflammatory diseases. However, the anti-inflammatory effects and the underlying mechanisms of LGS are not fully studied., Aim of the Study: This study aims to investigate the anti-inflammatory activity and explore the underlying mechanisms of LGS in zebrafish and cell inflammation models., Materials and Methods: LPS-induced zebrafish inflammation model was established by LPS-yolk microinjection. The protective effect of LGS on zebrafish injected with LPS was observed using survival analysis. Infiltration of inflammatory cells was determined by H&E staining assay. Expression levels of key inflammatory cytokines TNF-α and IL-6 were measured by q-PCR assay. Recruitment of neutrophils and macrophages were observed by fluorescence microscopy, SB staining and NR staining. In vitro anti-inflammatory effects of LGS were evaluated on LPS-stimulated RAW 264.7 cells. The generation of IL-6 and TNF-α was detected by ELISA. The protein expression levels of JNK, p-JNK (Thr183/Tyr185), Nur77 and p-Nur77 (Ser351) were determined by Western blotting. Finally, two additional inflammatory models in zebrafish, which were induced by CuSO 4 or tail fin injury, were also established and the recruitment of neutrophils and macrophages were observed for the determination of the anti-inflammatory activity of LGS., Results: LGS protected zebrafish against LPS-induced death and dose-dependently inhibited LPS-induced acute inflammatory response in zebrafish, as indicated by increased survival rate, reduced infiltration of inflammatory cells, decreased recruitment of macrophages and neutrophils, and downregulated expression levels of TNF-α and IL-6. Additionally, LGS inhibited the secretion of TNF-α and IL-6, increased the expression of Nur77, and reduced the expression of p-Nur77 (Ser351) and p-JNK (Thr183/Tyr185) in LPS-stimulated RAW 264.7 cells. The anti-inflammatory action of LGS was also observed in another two zebrafish inflammation models, which was supported by the inhibition on neutrophils and macrophages recruitment., Conclusion: The present study demonstrates that LGS possesses anti-inflammatory activity in zebrafish inflammation models and LPS-stimulated RAW 264.7 cells, which is related to the inhibition on p-JNK and p-Nur77. This finding provides a pharmacological basis for LGS in the control of inflammatory disorder., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Phytochemical investigation of the roots of Arnebia euchroma led to the isolation of a new dimeric naphthoquinone, shikometabolin H (1), a new meroterpeniod, epoxyarnebinol (5) and a new ring A-seco hopane triterpenoid, 2, 3-secodiplopterol dioic acid (7), together with 18 known compounds. The structures of the new compounds were elucidated by extensive spectroscopic analyses, including UV, IR, HRESIMS, 1D and 2D NMR. The absolute configuration of 1 was assigned by comparison of the experimental and calculated ECD data, whereas that of 5 was achieved by the comparison between experimental and calculated OR values. Compound 7 was found to be the first ring A-seco hopane triterpenoid ever reported. Data from MTT assays showed that compounds 1, 2, 4, 8, 11-14 and 19 possessed promising anti-proliferative activities in human melanoma cells A375 and A2058, and human colorectal adenocarcinoma cells HCT116 and SW620. In addition, molecular docking study was carried out to predict the possible binding modes of these active compounds with STAT3. Immunoblotting results further confirmed the inhibitory effects of compounds 1, 4, 8, 11, 14 and 19 on STAT3, which was indicated by the downregulated protein levels of phosphorylated and/or total STAT3 in A2058 and HCT116 cells., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Background: Acute lung injury (ALI) is a life-threatening disease without effective chemotherapy at present. Liang-Ge-San (LGS) is a famous traditional Chinese medicine formula, which is used to treat ALI in China. However, only a few studies have addressed the mechanisms of LGS in ALI. Purpose: To evaluate the anti-inflammatory effects of LGS on lipopolysaccharide (LPS)-induced ALI, and to explore its underlying molecular mechanism. Methods: Murine RAW264.7 cells were treated with LGS and LPS (1 μg/ml). The generation of IL-6, TNF-α, IL-1β was detected by ELISA. The protein expressions of STAT3 and P-STAT3 (Tyr705) were determined by Western blotting and fluorescence confocal microscopy. STAT3 transcriptional activity was investigated by luciferase reporter gene assay. qPCR was used to detect the expressions of microRNA-21 (miR-21), STAT3, and IL-6. DSS cross-linking assay was used to assess the change of STAT3 dimer. In vivo anti-inflammatory effects of LGS were evaluated in an ALI mouse model induced by tracheal instillation of LPS (3 mg/kg). The anti-ALI effects were evaluated by ELISA, qPCR, Western blotting, BCA, and H&E assays. Results: LGS suppressed LPS-stimulated IL-6, TNF-α, and IL-1β generation in murine macrophages RAW264.7. Moreover, LGS down-regulated protein levels of P-STAT3 (Tyr705) and STAT3, inhibited STAT3 transcriptional activity, and up-regulated miR-21. Furthermore, blockage of miR-21 antagonized the inhibitory effects of LGS on the production of IL-6 and the expressions of P-STAT3 (Tyr705) and STAT3 as well as the formation of STAT3 dimer. Critically, LGS up-regulated the expression of miR-21 and inhibited the protein expressions of STAT3 and P-STAT3 (Tyr705) to reduce the release of IL-6 and inflammatory cell infiltration as well as the degree of edema in LPS-induced ALI mice. Conclusion: LGS inhibited LPS-induced ALI through up-regulating miR-21 and subsequently inhibiting the STAT3 signaling pathway, thereby decreasing the release of IL-6., (Copyright © 2019 Yang, Lu, Huo, Chen, Cao, Xie, Zhou, Liu, Liu and Yu.)

Exosome, a membranous vesicle with biological activity, not only transmits active substances between cells but also transfers information between cells to participate in cell communication. Epithelial-mesenchymal transition (EMT) is a process by which epithelial cells acquire migratory and invasive properties to become mesenchymal stem cells. EMT is essential for the development of a spectrum of diseases. Studies have shown that exosomes have dual effects on EMT to, on the one hand, promote EMT and tumor cell invasion and metastasis and accelerate angiogenesis and tumor growth; on the other hand, exosomes can suppress tumor cell invasion, inhibit fibrosis of the heart, liver and kidney, and improve myocardial infarction by inhibiting EMT. Exosomes modulate EMT-related signaling pathways by carrying EMT-related proteins or miRNA to exert their bi-directional regulatory effects.

Since the first description of canine circovirus (CanineCV)-associated infection, there have been several reports on the distribution of the disease in worldwide. To investigate the prevalence and genetic diversity of CanineCV in China, we conducted PCR screening of 1226 dog serum samples collected from different regions in mainland China between 2014 and 2016. CanineCV DNA was found in 81/926 serum samples from Guangxi Province. Furthermore, 25 full-length genomes of CanineCV from positive samples were sequenced and compared with CanineCV sequences in the GenBank database. Pairwise analysis showed that the determined genome sequences shared 84.9%-100% identity among themselves and 81.4%-90.5% with the other 28 sequences. Phylogenetic analysis revealed that the 52 viral genome sequences could be divided into two genotypes (CanineCV-1 and CanineCV-2). Analysis of the amino acid sequences of the capsid protein revealed the existence of 9 major regions of variation. The present work contributes to the understanding of CanineCV molecular epidemiology., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Acute lung injury (ALI) is a deadly disease without effective chemotherapy, so far. Traditional Chinese medicine andrographis herba is frequently used in the treatment of respiratory diseases. In searching for natural anti-ALI components from andrographis herba, the activities of 3-dehydroandrographolide (3-DA), a new natural andrographolide product from andrographis herba were evaluated. In this study, murine macrophage RAW 264.7 cells and BALB/c mice were treated with LPS (lipopolysaccharide, 100 ng/ml in vitro; 3 mg/kg, intratracheal) to establish inflammation models. 3-DA attenuated the release of pro-inflammatory cytokines IL-6 and TNF-α, inhibited the degradation and phosphorylation of IκBα, and suppressed the nuclear translocation of NF-κB p65 as well as the phosphorylation of Akt at Ser473 in LPS-stimulated RAW 264.7 macrophage cells. Furthermore, 3-DA increased α7nAchR expression level and bound with α7nAchR. More importantly, the anti-inflammatory effects of 3-DA were counteracted in the presence of α7nAchR siRNA or methyllycaconitine (MLA, a α7nAchR specific inhibitor), suggesting that α7nAchR is a potential target in the anti-inflammatory effects of 3-DA. Besides, 3-DA significantly inhibited inflammation in LPS-induced ALI mice, which was associated with the decrease of lung water content and inflammatory cytokines, the inhibition of neutrophil and macrophage infiltration, and activation of the NF-κB/Akt signaling pathway. Moreover, these protective effects were attenuated by the treatment of MLA. Taken together, 3-DA alleviates LPS-induced inflammation via the cholinergic anti-inflammatory pathway in vitro and in vivo. These findings provide a rationale for the role of the cholinergic anti-inflammatory pathway in inflammation and the promising clinical application of 3-DA to treat ALI., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Manipulating the degradation rate of biomedical Mg alloys has always been a challenge. In this study, a two-step treatment including sliding friction treatment (SFT) and micro-arc oxidation (MAO) was adopted to acquire a unique Mg-based architecture containing three typical layers comprising a MAO coating/nanocrystalline (NC) layer/coarse-grained (CG) matrix. It was found that the modified topmost MAO coating possessed enhanced corrosion resistance, cytocompatibility and hemocompatibility. The intermediate NC layer sandwiched between the coating and CG matrix was an ideal transition layer capable of avoiding degradation rate upsurge caused by coating breakdown; meanwhile, it provided an effective reinforcing effect on the overall mechanical strength. More importantly, the corrosion resistance of these layers was ranked in the order: MAO coating > NC layer > CG matrix. This kind of gradually increasing corrosion rate of the three layers with depth renders the two-step treatment a promising approach to design Mg-based implants possessing controllable degradation rates.

For the purpose of detecting the influence of grain structure of a Mg matrix on the microstructure and corrosion resistance of micro-arc oxidation (MAO) coating, prior to MAO processing, sliding friction treatment (SFT) was adopted to generate a fine-grained (FG) layer on coarse-grained (CG) pure Mg surface. It showed that the FG layer had superior corrosion resistance, as compared to the CG matrix, owing to the grain refinement; furthermore, it successfully survived after MAO treatment. Thus, an excellent FG-MAO coating was gained by combining SFT and MAO. The surface morphology and element composition of FG-MAO and CG-MAO samples did not show significant changes. However, the FG layer favorably facilitated the formation of an excellent MAO coating, which possessed a superior bonding property and greater thickness. Consequently, the modified FG-MAO sample possessed enhanced corrosion resistance, since a lower hydrogen evolution rate, a larger impedance modulus and a lower corrosion current were observed on the FG-MAO sample.

Background: Physical exercise has been long recognized for its therapeutic effects on depressive disorders, but the underlying mechanisms remain largely unknown. In the study, we investigated whether the physical exercise by voluntary wheel running (VWR) alters depression-like behaviors and its impact on brain blood oxygen level-dependent (BOLD) signals in mice., Methods: Adult male C57BL/6 mice were assigned to one of the following groups; (1) no exercise control (noEx), housed in a standard cage; (2) exercise (Ex), 2h/day in a running wheel apparatus; (3) chronic unpredictable mild stress (CUMS), which was imitating adult stress; and (4) CUMS+Ex. The differences in functional brain changes were determined by BOLD functional magnetic resonance imaging (fMRI)., Results: The results showed that VWR exercise significantly reversed the CUMS-induced behavioral abnormalities. Base on the fMRI amplitude of low-frequency fluctuation (ALFF) analysis, we found that VWR exercise could restore the CUMS-induced excessive BOLD activation in parts of limbic system, such as cortex, hippocampus and corpus callosum. Furthermore, CUMS-induced BOLD suppressive regions were also partially attenuated by VWR exercise, such as amygdala, cerebellum anterior lobe, thalamus, midbrain, and pontine. Most of these regions are involved in mood-regulating circuit, suggesting dysfunction of the circuit in CUMS model of depression, and VWR exercise could adjust the mood-regulating circuit., Conclusions: These results suggested that VWR exercise ameliorated depression-like behaviors and brain BOLD signals in CUMS induced depression mice., (Copyright © 2017 Elsevier B.V. All rights reserved.)

In this study, YN26/2013, a novel recombinant duck circovirus (DuCV), was isolated from a Muscovy duck in Yunnan Province, southern China. The whole genome of YN26/2013 consists of 1,987 nucleotides (nt), the same genomic size as that of the DuCV-2 genotype. However, YN26/2013 shares 91.5 to 94.3% nucleotide identity similarity with previously reported type I (DuCV-1) viruses. Importantly, a novel putative recombinant event between DuCV-1 and DuCV-2 was identified as occurring within the 987- to 1111-nt region of the YN26/2013 genome., (Copyright © 2016 Sun et al.)

Icaritin (IT) is a flavonoid isolated from Herba Epimedii. In this study, we evaluated the anti-melanoma activities of IT, and determined its cytotoxic mechanism. We found that IT exerted cytotoxicity to melanoma cells. Furthermore, IT induced melanoma cell apoptosis, which was accompanied with PARP cleavage. Mechanistically, IT suppressed p-STAT3 (tyr705) level in parallel with increases of p-STAT3 (ser727), p-ERK and p-AKT. IT significantly inhibited STAT3 nuclear translocation and reduced the levels of STAT3 -targeted genes. IT also inhibited IGF-1-induced STAT3 activation through down-regulation of total IGF-1R level. No dramatic changes in IGF-1R mRNA levels were observed in IT-treated cells, suggesting that IT acted primarily at a post-transcriptional level. Using molecular docking analysis, IT was identified as a novel fatty acid synthase (FASN) inhibitor. We found that IT reduced the level of total IGF-1R via FASN inhibition. In summary, we reported that IT exerted anti-melanoma activities, and these effects were partially due to inhibition of FASN/IGF-1R/STAT3 signaling., Competing Interests: The authors declare no conflicts of interest.

Dog circovirus (DogCV) is a newly discovered mammalian circovirus. To investigate the genomic characteristics and genetic diversity of DogCV spreads in China, the first genome sequence of Chinese isolate, designated as JZ98/2014,was obtained by overlap PCR using the DNA extracted from dog serum as template for amplification. The nucleotide content and genome organization were subsequently analyzed. The results showed that the full-length genome of JZ98/2014 is 2063nt,and contains three major open reading frame: ORF V1 (encodes the 303 amino acid Rep protein),ORF C1(encodes the 270 amino acid Cap protein),and ORF C2(encodes 106 amino acids).JZ98/2014 shared 82.1%-89.5% homology with the complete genome sequences of DogCV isolates from America and Europe. The Rep gene and Cap gene of JZ98/2014 shared 82.1%-89.5%and 84.6%-89.1% homology, respectively, with the same genes from other DogCVs. Phylogenetic tree analysis indicated that there were several different genetic clades of DogCV spread in the world, and JZ98/2014 formed a clade by itself.

Proline-rich antimicrobial peptides (PR-AMPs) are a group of cationic host defense peptides that are characterized by a high content of proline residues. Up to now, they have been reported in some insects, vertebrate and invertebrate animals, but are not found in plants. In this study, we performed an in silico screening of antimicrobial peptides, which led to discovery of a Brassica napus gene encoding a novel PR-AMP. This gene encodes a 35-amino acid peptide with 13 proline residues, designated BnPRP1. BnPRP1 has 40.5% identity with a known proline-rich antimicrobial peptide SP-B from the pig. BnPRP1 was artificially synthetized and cloned into the prokaryotic expression vector pET30a/His-EDDIE-GFP. Recombinant BnPRP1 was produced in Escherichia coli and has a predicted molecular mass of 3.8 kDa. Analysis of its activity demonstrated that BnPRP1 exhibited strong antimicrobial activity against Gram-positive bacterium, Gram-negative bacterium, yeast and also had strong antifungal activity against several pathogenic fungi, such as Sclerotinia sclerotiorum, Mucor sp., Magnaporthe oryzae and Botrytis cinerea. Circular dichroism (CD) revealed the main secondary structure of BnPRP1 was the random coil. BnPRP1 gene expression detected by qRT-PCR is responsive to pathogen inoculation. At 48 hours after S. sclerotiorum inoculation, the expression of BnPRP1 increased significantly in the susceptible lines while slight decrease occurred in resistant lines. These suggested that BnPRP1 might play a role in the plant defense response against S. sclerotiorum. BnPRP1 isolated from B. napus was the first PR-AMP member that was characterized in plants, and its homology sequences were found in some other Brassicaceae plants by the genome sequences analysis. Compared with the known PR-AMPs, BnPRP1 has the different primary sequences and antimicrobial activity. Above all, this study gives a chance to cast a new light on further understanding about the AMPs' mechanism and application.

Background: Brassica napus is the third leading source of vegetable oil in the world after soybean and oil palm. The accumulation of gene sequences, especially expressed sequence tags (ESTs) from plant cDNA libraries, has provided a rich resource for genes discovery including potential antimicrobial peptides (AMPs). In this study, we used ESTs including those generated from B. napus cDNA libraries of seeds, pathogen-challenged leaves and deposited in the public databases, as a model, to perform in silico identification and consequently in vitro confirmation of putative AMP activities through a highly efficient system of recombinant AMP prokaryotic expression., Results: In total, 35,788 were generated from cDNA libraries of pathogen-challenged leaves and 187,272 ESTs from seeds of B. napus, and the 644,998 ESTs of B. napus were downloaded from the EST database of PlantGDB. They formed 201,200 unigenes. First, all the known AMPs from the AMP databank (APD2 database) were individually queried against all the unigenes using the BLASTX program. A total of 972 unigenes that matched the 27 known AMP sequences in APD2 database were extracted and annotated using Blast2GO program. Among these unigenes, 237 unigenes from B. napus pathogen-challenged leaves had the highest ratio (1.15 %) in this unigene dataset, which is 13 times that of the unigene datasets of B. napus seeds (0.09 %) and 2.3 times that of the public EST dataset. About 87 % of each EST library was lipid-transfer protein (LTP) (32 % of total unigenes), defensin, histone, endochitinase, and gibberellin-regulated proteins. The most abundant unigenes in the leaf library were endochitinase and defensin, and LTP and histone in the pub EST library. After masking of the repeat sequence, 606 peptides that were orthologous matched to different AMP families were found. The phylogeny and conserved structural motifs of seven AMPs families were also analysed. To investigate the antimicrobial activities of the predicted peptides, 31 potential AMP genes belonging to different AMP families were selected to test their antimicrobial activities after bioinformatics identification. The AMP genes were all optimized according to Escherichia coli codon usage and synthetized through one-step polymerase chain reaction method. The results showed that 28 recombinant AMPs displayed expected antimicrobial activities against E. coli and Micrococcus luteus and Sclerotinia sclerotiorum strains., Conclusion: The study not only significantly expanded the number of known/predicted peptides, but also contributed to long-term plant genetic improvement for increased resistance to diverse pathogens of B.napus. These results proved that the high-throughput method developed that combined an in silico procedure with a recombinant AMP prokaryotic expression system is considerably efficient for identification of new AMPs from genome or EST sequence databases.