CwJAZ4/9 negatively regulates jasmonate-mediated biosynthesis of terpenoids through interacting with CwMYC2 and confers salt tolerance in Curcuma wenyujin.

Plant JASMONATE ZIM-DOMAIN (JAZ) genes play crucial roles in regulating the biosynthesis of specialized metabolites and stressful responses. However, understanding of JAZs controlling these biological processes lags due to numerous JAZ copies. Here, we found that two leaf-specific CwJAZ4/9 genes from Curcuma wenyujin are strongly induced by methyl-jasmonate (MeJA) and negatively correlated with terpenoid biosynthesis. Yeast two-hybrid, luciferase complementation imaging and in vitro pull-down assays confirmed that CwJAZ4/9 proteins interact with CwMYC2 to form the CwJAZ4/9-CwMYC2 regulatory cascade. Furthermore, transgenic hairy roots showed that CwJAZ4/9 acts as repressors of MeJA-induced terpenoid biosynthesis by inhibiting the terpenoid pathway and jasmonate response, thus reducing terpenoid accumulation. In addition, we revealed that CwJAZ4/9 decreases salt sensitivity and sustains the growth of hairy roots under salt stress by suppressing the salt-mediated jasmonate responses. Transcriptome analysis for MeJA-mediated transgenic hairy root lines further confirmed that CwJAZ4/9 negatively regulates the terpenoid pathway genes and massively alters the expression of genes related to salt stress signaling and responses, and crosstalks of multiple phytohormones. Altogether, our results establish a genetic framework to understand how CwJAZ4/9 inhibits terpenoid biosynthesis and confers salt tolerance, which provides a potential strategy for producing high-value pharmaceutical terpenoids and improving resistant C. wenyujin varieties by a genetic approach.

Application of flash GC e-nose and FT-NIR combined with deep learning algorithm in preventing age fraud and quality evaluation of pericarpium citri reticulatae.

Pericarpium citri reticulatae (PCR) is the dried mature fruit peel of Citrus reticulata Blanco and its cultivated varieties in the Brassicaceae family. It can be used as both food and medicine, and has the effect of relieving cough and phlegm, and promoting digestion. The smell and medicinal properties of PCR are aged over the years; only varieties with aging value can be called "Chenpi". That is to say, the storage year of PCR has a great influence on its quality. As the color and smell of PCR of different storage years are similar, some unscrupulous merchants often use PCRs of low years to pretend to be PCRs of high years, and make huge profits. Therefore, we did this study with the aim of establishing a rapid and nondestructive method to identify the counterfeiting of PCR storage year, so as to protect the legitimate rights and interests of consumers. In this study, a classification model of PCR was established by e-eye, flash GC e-nose, and Fourier transform near-infrared (FT-NIR) combined with machine learning algorithms, which can quickly and accurately distinguish PCRs of different storage years. DFA and PLS-DA models were established by flash GC e-nose to distinguish PCRs of different ages, and 8 odor components were identified, among which (+)-limonene and γ-terpinene were the key components to distinguish PCRs of different ages. In addition, the classification and calibration model of PCRs were established by the combination of FT-NIR and machine learning algorithms. The classification models included SVM, KNN, LSTM, and CNN-LSTM, while the calibration models included PLSR, LSTM, and CNN-LSTM. Among them, the CNN-LSTM model built by internal capsule had significantly better classification and calibration performance than the other models. The accuracy of the classification model was 98.21 %. The R2P of age, (+)-limonene and γ-terpinene was 0.9912, 0.9875 and 0.9891, respectively. These results showed that the combination of flash GC e-nose and FT-NIR combined with deep learning algorithm could quickly and accurately distinguish PCRs of different ages. It also provided an effective and reliable method to monitor the quality of PCR in the market.

Research Progress for Probiotics Regulating Intestinal Flora to Improve Functional Dyspepsia: A Review.

Functional dyspepsia (FD) is a common functional gastrointestinal disorder. The pathophysiology remains poorly understood; however, alterations in the small intestinal microbiome have been observed. Current treatments for FD with drugs are limited, and there are certain safety problems. A class of active probiotic bacteria can control gastrointestinal homeostasis, nutritional digestion and absorption, and the energy balance when taken in certain dosages. Probiotics play many roles in maintaining intestinal microecological balance, improving the intestinal barrier function, and regulating the immune response. The presence and composition of intestinal microorganisms play a vital role in the onset and progression of FD and serve as a critical factor for both regulation and potential intervention regarding the management of this condition. Thus, there are potential advantages to alleviating FD by regulating the intestinal flora using probiotics, targeting intestinal microorganisms. This review summarizes the research progress of probiotics regarding improving FD by regulating intestinal flora and provides a reference basis for probiotics to improve FD.

Squalene monooxygenase facilitates bladder cancer development in part by regulating PCNA.

Bladder cancer (BC) is one of the most common cancers worldwide. Although the treatment and survival rate of BC are being improved, the risk factors and the underlying mechanisms causing BC are incompletely understood. Squalene monooxygenase (SQLE) has been associated with the occurrence and development of multiple cancers but whether it contributes to BC development is unclear. In this study, we performed bioinformatics analysis on paired BC and adjacent non-cancerous tissues and found that SQLE expression is significantly upregulated in BC samples. Knockdown of SQLE impairs viability, induces apoptosis, and inhibits the migration and invasion of BC cells. RNA-seq data reveals that SQLE deficiency leads to dysregulated expression of genes regulating proliferation, migration, and apoptosis. Mass spectrometry-directed interactome screening identifies proliferating cell nuclear antigen (PCNA) as an SQLE-interacting protein and overexpression of PCNA partially rescues the impaired viability, migration, and invasion of BC cells caused by SQLE knockdown. In addition, we performed xenograft assays and confirmed that SQLE deficiency inhibits BC growth in vivo. In conclusion, these data suggest that SQLE promotes BC development and SQLE inhibition may be therapeutically useful in BC treatment.

Molecular cloning and functional characterization of peroxinectin from red swamp crayfish, Procambarus clarkii.

Peroxinectin, which has both peroxidase and cell adhesion activities, is crucial for invertebrate innate immune responses. In this study, we first cloned the full-length cDNA of Procambarus clarkii Peroxinectin (denoted as Pc-Px) and evaluated its immune roles. The Pc-Px cDNA had 2460 base pairs (bp) and 819 amino acid residues, including peroxidase domain and a putative integrin-binding motif. Pc-Px tissue expression was found to be ubiquitous in all examined tissues under normal physiological conditions. Pc-Px mRNA levels were highest in hemocytes, followed by gills and heart, and were lowest in the gut. The LPS, PGN, and Poly I:C treatment significantly up-regulated the transcript level of Pc-Px gene, but the expression trends were different after the microbials component treatments. Pc-Px knockdown using double-stranded RNA altered the transcription profiles of various immune-related genes in hepatopancreas of P. clarkii. Taken together, Pc-Px is an important component of immune system that likely to modulate immune function of P. clarkii via regulating immune-associated genes.

A robust method to improve the regression accuracy of LIBS data: determination of heavy metal Cu in Tegillarca granosa.

Tegillarca granosa (T. granosa) is susceptible to contamination by heavy metals, which poses potential health risks for consumers. Laser-induced breakdown spectroscopy (LIBS) combined with the classical partial least squares (PLS) model has shown promise in determining heavy metal concentrations in T. granosa. However, the presence of outliers during calibration can compromise the model's integrity and diminish its predictive capabilities. To address this issue, we propose using a robust method for partial least squares, RSIMPLS, to improve the accuracy of Cu prediction in T. granosa. The RSIMPLS algorithm was employed to analyze and process the high-dimensional LIBS data and utilized diagnostic plots to identify various types of outliers. By selectively eliminating certain outliers, a robust calibration method was achieved. The results showed that LIBS spectroscopy has the potential to predict Cu in T. granosa, with a coefficient of determination (Rp2) of 0.79 and a root mean square error of prediction (RMSEP) of 11.28. RSIMPLS significantly improved the prediction accuracy of Cu concentrations with a 43% decrease in RMSEP compared to the PLS. These findings validated the effectiveness of combining LIBS data with the RSIMPLS algorithm for the prediction of Cu concentrations in T. granosa.

Determinants of switching behavior to wear helmets when riding e-bikes, a two-step SEM-ANFIS approach.

E-bikes have become one of China's most popular travel modes. The authorities have issued helmet-wearing regulations to increase wearing rates to protect e-bike riders' safety, but the effect is unsatisfactory. To reveal the factors influencing the helmet-wearing behavior of e-bike riders, this study constructed a theoretical Push-Pull-Mooring (PPM) model to analyze the factor's relationship from the perspective of travel behavior switching. A two-step SEM-ANFIS method is proposed to test relationships, rank importance and analyze the combined effect of psychological variables. The Partial Least Squares Structural Equation Model (PLS-SEM) was used to obtain the significant influencing factors. The Adaptive Network-based Fuzzy Inference System (ANFIS), a nonlinear approach, was applied to analyze the importance of the significant influencing factors and draw refined conclusions and suggestions from the analysis of the combined effects. The PPM model we constructed has a good model fit and high model predictive validity (GOF = 0.381, R2 = 0.442). We found that three significant factors tested by PLS-SEM, perceived legal norms (ß = 0.234, p < 0.001), perceived inconvenience (ß = -0.117, p < 0.001) and conformity tendency (ß = 0.241, p < 0.05), are the most important factors in the effects of push, mooring and pull. The results also demonstrated that legal norm is the most important factor but has less effect on people with low perceived vulnerability, and low subjective norms will make people with high conformity tendency to follow the crowd blindly. This study could contribute to developing refined interventions to improve the helmet-wearing rate effectively.

Gene coexpression networks allow the discovery of two strictosidine synthases underlying monoterpene indole alkaloid biosynthesis in Uncaria rhynchophylla.

Plant-derived monoterpene indole alkaloids (MIAs) from Uncaria rhynchophylla (UR) have huge medicinal properties in treating Alzheimer's disease, Parkinson's disease, and depression. Although many bioactive UR-MIA products have been isolated as drugs, their biosynthetic pathway remains largely unexplored. In this study, untargeted metabolome identified 79 MIA features in UR tissues (leaf, branch stem, hook stem, and stem), of which 30 MIAs were differentially accumulated among different tissues. Short time series expression analysis captured 58 pathway genes and 12 hub regulators responsible for UR-MIA biosynthesis and regulation, which were strong links with main UR-MIA features. Coexpression networks further pointed to two strictosidine synthases (UrSTR1/5) that were coregulated with multiple MIA-related genes and highly correlated with UR-MIA features (r > 0.7, P < 0.005). Both UrSTR1/5 catalyzed the formation of strictosidine with tryptamine and secologanin as substrates, highlighting the importance of key residues (UrSTR1: Glu309, Tyr155; UrSTR5: Glu295, Tyr141). Further, overexpression of UrSTR1/5 in UR hairy roots constitutively increased the biosynthesis of bioactive UR-MIAs (rhynchophylline, isorhynchophylline, corynoxeine, etc), whereas RNAi of UrSTR1/5 significantly decreased UR-MIA biosynthesis. Collectively, our work not only provides candidates for reconstituting the biosynthesis of bioactive UR-MIAs in heterologous hosts but also highlights a powerful strategy for mining natural product biosynthesis in medicinal plants.

Multiple anti-inflammatory mechanisms of Zedoary Turmeric Oil Injection against lipopolysaccharides-induced acute lung injury in rats elucidated by network pharmacology combined with transcriptomics.

BACKGROUND: Prospects for the drug treatment of acute lung injury (ALI) is unpromising. Managing inflammation can prevent ALI from progressing and minimize further deterioration. Zedoary turmeric oil injection (ZTOI), a patented traditional Chinese medicine (TCM) that has been used against ALI, has shown significant anti-inflammatory effects. However, the mechanisms underlying these effects remain unclear. PURPOSE: Elucidate the anti-inflammatory mechanism by which ZTOI acts against ALI in rats using an ingredients-targets-pathways (I-T-P) interaction network. STUDY DESIGN AND METHODS: The key ingredients of ZTOI were characterized using UPLC-MS/MS combined with literature mining. The target profiles of each ingredient were established using drug-target databases. The anti-inflammatory activity of ZTOI against lipopolysaccharides (LPS)-induced rat ALI was validated using histopathology and inflammatory factor assessments. The therapeutic targets of ZTOI were screened by integrating transcriptomic results of lung tissues with protein-protein interaction (PPI) expansion. Using KEGG pathway enrichment, an I-T-P network was established to determine the essential interactions among ingredients, targets, and pathways of ZTOI against lung inflammation in ALI. Molecular docking and immunofluorescence staining were utilized to confirm the accuracy of the I-T-P network. RESULTS: A total of 11 sesquiterpenes, whose target profiles may characterize the potential function of ZTOI, were identified as key ingredients. In the ALI rat model, ZTOI can alleviate lung inflammation by decreasing the levels of C-reactive protein, interleukin-6, interleukin-1ß, and tumor necrosis factor α both in serum and lung tissues. Based on our biological samples, transcriptomics, PPI network expansion, and KEGG pathway enrichment, 11 ingredients, 174 targets, and 8 signaling pathways were linked in the I-T-P networks. From these results, ZTOI could be inferred to exert multiple anti-inflammatory effects against ALI through Toll-like receptor, NF-kappa B, RIG-I-like receptor, TNF, NOD-like receptor, IL-17, MAPK, and the Toll and Imd signaling pathways. In addition, two significantly regulated targets in the transcriptome, Usp18 and Map3k7, could be the essential anti-inflammatory targets of ZTOI. CONCLUSION: By integrating network pharmacology with ingredient identification and transcriptomics, we show the multiple anti-inflammatory mechanisms by which ZTOI acts against ALI on an I-T-P level. This work also provides a methodological reference for related research into TCM.

LCAT3, a novel m6A-regulated long non-coding RNA, plays an oncogenic role in lung cancer via binding with FUBP1 to activate c-MYC.

BACKGROUND: Long non-coding RNAs (lncRNAs) are important epigenetic regulators, which play critical roles in diverse physiological and pathological processes. However, the regulatory mechanism of lncRNAs in lung carcinogenesis remains elusive. Here, we characterized a novel oncogenic lncRNA, designated as Lung Cancer Associated Transcript 3 (LCAT3). METHODS: We predicted and validated LCAT3 by analyzing RNA-sequencing (RNA-seq) data of lung cancer tissues from TCGA. Methylated RNA immunoprecipitation was performed to assess m6A modification on LCAT3. The LCAT3-FUBP1-MYC axis was assessed by dual-luciferase reporter, RNA immunoprecipitation and Chromatin immunoprecipitation assays. Signaling pathways altered by LCAT3 knockdown were identified using RNA-seq. Furthermore, the mechanism of LCAT3 was investigated using loss-of-function and gain-of-function assays in vivo and in vitro. RESULTS: LCAT3 was found to be up-regulated in lung adenocarcinomas (LUAD), and its over-expression was associated with the poor prognosis of LUAD patients. LCAT3 upregulation is attributable to N6-methyladenosine (m6A) modification mediated by methyltransferase like 3 (METTL3), leading to LCAT3 stabilization. Biologically, loss-of-function assays revealed that LCAT3 knockdown significantly suppressed lung cancer cell proliferation, migration and invasion in vitro, and inhibited tumor growth and metastasis in vivo. LCAT3 knockdown induced cell cycle arrest at the G1 phase. Mechanistically, LCAT3 recruited Far Upstream Element Binding Protein 1 (FUBP1) to the MYC far-upstream element (FUSE) sequence, thereby activating MYC transcription to promote proliferation, survival, invasion and metastasis of lung cancer cells. CONCLUSIONS: Taken together, we identified and characterized LCAT3 as a novel oncogenic lncRNA in the lung, and validated the LCAT3-FUBP1-MYC axis as a potential therapeutic target for LUAD.

Germacrone improves liver fibrosis by regulating the PI3K/AKT/mTOR signalling pathway.

Liver fibrosis is a primary threat to public health, owing to limited therapeutic options. Germacrone (GM) has been shown to exert various curative effects against human diseases, including liver injury. The aim of this study was to investigate the pharmacological effects of GM in the pathophysiology of hepatic fibrosis and determine its potential mechanisms of action. A liver fibrosis rat model was established via carbon tetrachloride (CCl4 ) treatment, and LX-2 cells were stimulated with TGF-ß1. The effects of GM on liver fibrosis and its relationship with the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT)/mammalian target of rapamycin (mTOR) signalling pathway were investigated. In the CCl4 fibrosis-induced rat model, GM improved histological damage, inhibited the activity of hepatic α-smooth muscle actin and improved serum alanine aminotransferase and aspartate aminotransferase levels in a dose-dependent manner. GM potently inhibited hepatic stellate cells (HSCs) growth and epithelial-mesenchymal transition (EMT) progression, as reflected by the altered expression of proliferative (Ki-67, PCNA and cleaved caspase-3) and EMT-related (E-cadherin and vimentin) proteins. In TGF-ß1-stimulated LX-2 cells, GM significantly inhibited the survival and activation of HSCs and induced cell apoptosis. GM also suppressed the migration ability and reversed the EMT process in HSCs. Following GM treatment, the phosphorylation of the PI3K, AKT and mTOR proteins was reduced in the liver of CCl4 -treated rats and TGF-ß1-stimulated LX-2 cells, indicating that GM may attenuate hepatic fibrosis via the PI3K/AKT/mTOR signalling pathway. These outcomes highlight the anti-fibrotic effects of GM and suggest that it is a potential therapeutic agent for the treatment of liver fibrosis.

Co-delivery of miR-29b and germacrone based on cyclic RGD-modified nanoparticles for liver fibrosis therapy.

Hepatic stellate cells (HSCs) were activated and secreted excessive amounts of extracellular matrix (ECM) proteins during pathogenetic progress of liver fibrosis. Germacrone (GMO) and miR-29b can play an important role in inhibiting growth of HSCs and production of type I collagen. GMO and miR-29b were co-encapsulated into nanoparticles (NPs) based on poly(ethylene glycol)-block-poly(lactide-co-glycolide) (PEG-PLGA). Then, NPs were modified with cyclic RGD peptides (cRGDfK). cRGDfK is an effective ligand to bind integrin αvß3 and increase the targeting ability for fibrotic liver. GMO- and miR-29b-loaded NPs exhibited great cytotoxicity to activated HSCs and significantly inhibited production of type I collagen. Liver fibrosis model of mice was induced by administration of carbon tetrachloride. Great targeting ability was achieved in liver fibrotic mice treated with cRGD-modified NPs. Significant ant-fibrotic effects have been presented based on hematoxylin and eosin (H&E), Masson and Sirius Red staining results of liver tissues collected from mice treated with drug-loaded NPs. All these results indicate GMO- and miR-29b-loaded cRGD-modified NPs have the potential for clinical use to treat liver fibrosis.

A Simple and Accurate Method for the Determination of Related Substances in Coenzyme Q10 Soft Capsules.

As a new dosage form, coenzyme Q10 (Co-Q10) soft capsules are easily absorbed and utilized by the human body. Co-Q10 soft capsules can effectively improve the bioavailability and reduce medical costs for patients. A main concern about Co-Q10 as an active pharmaceutical ingredient (API) is how to control the total quantity of related substances. In this article, according to the degradation pattern of the API, the most easily degradable impurity (impurity X) in the sample was prepared and its chemical structure was determined. Furthermore, a simple and accurate method was developed for the determination of related substances and to avert the interference of excipient ingredients in Co-Q10 soft capsules. The approach was validated adequately and the primary impurity X was confirmed accurately. The limit of total quantity of related substances (less than 1%) could be revised to the level of specific impurity X being no more than 0.5%, in this effective quality control method of Co-Q10 soft capsules. The revised level is suggested to be included in the corresponding standard of the supplement taken from the Pharmacopoeia of the People's Republic of China (2015 edition). This can provide a feasible method for the relevant enterprises and regulatory authorities to control the related substances of coenzyme Q10 soft capsules.

Design and optimize N-substituted EF24 as effective and low toxicity NF-κB inhibitor for lung cancer therapy via apoptosis-to-pyroptosis switch.

As NF-κB signaling pathway is constitutively activated in lung cancer, targeting NF-κB has a potential for the treatment. EF24 has been proved to be a NF-κB inhibitor with good antitumor activity, while whose toxicity possibly became one of the obstacles to enter into clinical application. In order to find high efficiency and low toxicity NF-κB inhibitors, EF24 was modified and 13d was screened out. It was proved that 13d possessed an effective combination of inhibiting NF-κB pathway and showing lower cytotoxicity on normal cells as well as less toxicity in acute toxicity experiment compared with the lead compound of EF24. In addition, 13d was found to inhibit cell vitality, arrest cell cycle in G2/M phase, promote cell apoptosis, and suppress the xenograft tumor growth. Furthermore, 13d was elucidated to induce pyroptosis developing from apoptosis, which was associated with the inhibition of NF-κB. Taken together, it was suggested that 13d was a potent antitumor agent.

Association between the use of contact force-sensing catheters and cardiac tamponade in atrial fibrillation ablation.

INTRODUCTION: Cardiac tamponade is a rare but life-threatening complication during atrial fibrillation (AF) catheter ablation. Contact force (CF)-sensing catheters improve ablation effectiveness. However, the impact of the application of CF-sensing catheters on the occurrence of cardiac tamponade remains unclear. The aim of this study is to evaluate the "real-world" impact of CF-sensing catheters on cardiac tamponade during AF ablation in an experienced medical center. METHODS AND RESULTS: This was a retrospective study of consecutive de novo AF ablation procedures at Beijing Anzhen Hospital between 2013 and 2016. The ablation procedure was divided into a CF group and a non-CF group. Logistic regression analysis was used to evaluate the association between the use of CF-sensing catheters and the risk of cardiac tamponade. A total of 5313 patients with AF were involved in this study. The incidence of cardiac tamponade in the CF group was significantly higher than that in the non-CF group (1.07% vs. 0.44%, P = 0.009). Of the cardiac tamponade cases in the non-CF group, 45.45% were delayed compared with 10% in the CF group (P = 0.011). Multivariate logistic regression analysis showed that CF-sensing catheters increased the risk of cardiac tamponade (OR = 2.34, 95% CI = 1.17-4.26, P = 0.015). Stratified analysis revealed patients with a smaller left atrium dimension, lower ejection fraction, longer procedure duration, or longer ablation duration had a greater risk of cardiac tamponade during ablation with CF-sensing catheters. CONCLUSIONS: CF-sensing catheters increase the risk of cardiac tamponade during AF ablation.

A thiopyran derivative with low murine toxicity with therapeutic potential on lung cancer acting through a NF-κB mediated apoptosis-to-pyroptosis switch.

Pyroptosis is a novel manner of cell death that can be mediated by chemotherapy drugs. The awareness of pyroptosis is significantly increasing in the fields of anti-tumor research and chemotherapy drugs. Invoking the occurrence of pyroptosis is an attractive prospect for the treatment of lung cancer. Here, the compound L61H10 was obtained as a thiopyran derivative to compare its activity with curcumin. It was indicated that L61H10 exhibited good anti-tumor activity both in vitro and in vivo via the switch of apoptosis-to-pyroptosis, which was associated with the NF-κB signaling pathway. In addition, L61H10 had no obvious side effects both in vitro and in vivo. In brief, L61H10 is shown to be a potential anti-lung cancer agent and research on its anti-tumor mechanism provides new information for chemotherapy drug research.

Design, synthesis, and evaluation of chalcone analogues incorporate α,ß-Unsaturated ketone functionality as anti-lung cancer agents via evoking ROS to induce pyroptosis.

Chalcone, a natural structure, demonstrates many pharmacological activities including anticancer, and one promising mechanism is to modulate the generation of ROS. It has been known that pyroptosis is associated with anticancer effects, whereas there is fewer researches about ROS-mediated pyroptosis triggered by chemotherapy drugs. Moreover, incorporation of a α,ß-unsaturated ketone unit into chalcone may be an effective strategy for development of chemotherapy drugs. Hence, a number of chalcone analogues bearing a α,ß-unsaturated ketone were synthesized from chalcone analogues 1 with modest anticancer activities as the lead compound. Structure-activity relationship (SAR) studies confirmed the function of α,ß-unsaturated ketone to improve anticancer activity. Notably, compound 8, bearing a α,ß-unsaturated ketone, is the most potent inhibitor of cancer, with IC50 values on NCI-H460, A549 and H1975 cells of 2.3 ±â€¯0.3, 3.2 ±â€¯0.0 and 5.7 ±â€¯1.4 µM, respectively. Besides, 8 showed antiproliferative ability against NCI-H460 cells in a time- and concentration-dependent manner through modulating ROS to induce caspase-3-mediated pyroptosis, and displayed a better safety profile in vivo. Overall, these results demonstrated that compound 8 is a candidate agent and a potential lead compound for development of chemotherapy drugs, and can be used as a probe to further examine the mechanism of ROS-dependent pyroptosis.

Synthesis and anti-tumor activity of EF24 analogues as IKKß inhibitors.

EF24 is an IKKß inhibitor (IC50: 72 µM) containing various anti-tumor activities. In this study, a series of EF24 analogs targeting IKKß were designed and synthesized. Several IKKß inhibitors with better activities than EF24 were screened out and B3 showed best IKKß inhibitory (IC50: 6.6 µM). Molecular docking and dynamic simulation experiments further confirmed this inhibitory effect. B3 obviously suppressed the viability of Hela229, A549, SGC-7901 and MGC-803 cells. Then, in SGC-7901 and MGC-803 cells, B3 blocked the NF-κB signal pathway by inhibiting IKKß phosphorylation, and followed arrested the cell cycle at G2/M phase by suppressing the Cyclin B1 and Cdc2 p34 expression, induced the cell apoptosis by down-regulating Bcl-2 protein and up-regulating cleaved-caspase3. Moreover, B3 significantly reduced tumor growth and suppressed the IKKß-NF-κB signal pathway in SGC-7901 xenograft model. In total, this study present a potential IKKß inhibitor as anti-tumor precursor.

Transcriptome profiling of red swamp crayfish (Procambarus clarkii) hepatopancreas in response to lipopolysaccharide (LPS) infection.

The RNA-sequencing followed by de novo assembly generated 61,912 unigene sequences of P. clarkii hepatopancreas. Comparison of gene expression between LPS challenged and PBS control samples revealed 2552 differentially expressed genes (DEGs). Of these sequences, 1162 DEGs were differentially up-regulated and 1360 DEGs differentially down-regulated. The DEGs were then annotated against gene ontology (GO) database and Kyoto Encyclopedia of Genes and Genomes (KEGG) database. Some immune-related pathways such as PPAR signaling pathway, lysosome, Chemical carcinogenesis, Peroxisome were predicted by canonical pathways analysis. The reliability of transcriptome data was validated by quantitative real time polymerase chain reaction (qRT-PCR) for the selected genes. The data presented here shed light into antibacterial immune responses of crayfish. In addition, these results suggest that transcriptomic data provides valuable sequence resource for immune-related gene identification and helps to understand P. clarkii immune functions.

Molecular structure and functional characterization of the peroxiredoxin 5 in Procambarus clarkii following LPS and Poly I:C challenge.

Peroxiredoxin (Prx) family members play a critical role in host defense against oxidative stress, and are also involved in immune responses following microbial infection. In the present study, we firstly cloned the cDNA of Peroxiredoxin 5 from Procambarus clarkii (denoted as PcPrx5) and investigated its immune functions towards LPS and Poly I:C exposure. The PcPrx5 cDNA was composed of 564 bp and consisting of 187 amino acid residues which included Prx5-like subfamily domain, AHP1 domain and Redoxin domain. The recombinant protein was expressed in Escherichia coli (Transetta DE3), and anti-Prx5 antibodies were prepared. Tissue specific expression analysis showed that PcPrx5 was ubiquitously expressed in all examined tissues. Further, its mRNA transcript was greatest in hepatopancrease, haemocyte followed by gut and stomach, and was weak in muscle. The LPS and Poly I:C exposure could both significantly up-regulate the transcript level of PcPrx5, however the expression trends were different following LPS and Poly I: C treatments. Further, we investigated the antioxidant role of recombinant PcPrx5 protein in vitro by mixed-function oxidase assay; the results demonstrated a dose-dependent inhibition of DNA damage by PcPrx5. Our results implicate PcPrx5 as an important defense against microbial pathogens and oxidants in P. clarkii.