Luteoloside inhibits Aß1-42 fibrillogenesis, disintegrates preformed fibrils, and alleviates amyloid-induced cytotoxicity.

Abnormal aggregation and fibrillogenesis of amyloid-ß protein (Aß) can cause Alzheimer's disease (AD). Thus, the discovery of effective drugs that inhibit Aß fibrillogenesis in the brain is crucial for the treatment of AD. Luteoloside, as one of the polyphenolic compounds, is found to have a certain therapeutic effect on nervous system diseases. However, it remains unknown whether luteoloside is a potential drug for treating AD by modulating Aß aggregation pathway. In this study, we performed diverse biophysical and biochemical methods to explore the inhibition of luteoloside on Aß1-42 which is linked to AD. The results demonstrated that luteoloside efficiently prevented amyloid oligomerization and cross-ß-sheet formation, reduced the rate of amyloid growth and the length of amyloid fibrils in a dose-dependent manner. Moreover, luteoloside was able to influence aggregation and conformation of Aß1-42 during different fiber-forming phases, and it could disintegrate already preformed fibrils of Aß1-42 and convert them into nontoxic aggregates. Furthermore, luteoloside protected cells from amyloid-induced cytotoxicity and hemolysis, and attenuated the level of reactive oxygen species (ROS). The molecular docking study showed that luteoloside interacted with Aß1-42 mainly via Conventional Hydrogen Bond, Carbon Hydrogen Bond, Pi-Pi T-shaped, Pi-Alkyl and Pi-Anion, thereby possibly preventing it from forming the aggregates. These observations indicate that luteoloside, a natural anti-oxidant molecule, may be applicable as an effective inhibitor of Aß, and promote further exploration of the therapeutic strategy against AD.

Ethanol extract of Verbena officinalis alleviates MCAO-induced ischaemic stroke by inhibiting IL17A pathway-regulated neuroinflammation.

BACKGROUND: The prevention and treatment of ischaemic stroke is a worldwide challenge, and effective clinical treatment strategies are lacking. Studies have demonstrated the efficacy of Verbena officinalis in managing cerebrovascular disorders. However, the neuroprotective bioactive components and mechanisms remain unclear. PURPOSE: To investigate the pharmacological combinatorial components and mechanism underlying the anti-ischemic stroke effect of the ethanol extract of Verbena officinalis (VO Ex). STUDY DESIGN AND METHODS: The components of VO Ex were identified by HPLC. A middle cerebral artery occlusion (MCAO) induced brain injury model was used to assess the therapeutic effect of VO Ex. The activity of the chemical components of VO Ex was evaluated using a primary astrocyte injury model induced by oxygen-glucose deprivation/reperfusion (OGD/R). RNA sequencing was used to reveal the potential targets of VO Ex against cerebral ischemia-reperfusion injury (CIRI), and the results were verified by qRT-PCR and western blotting. The key components and target binding ability were predicted by molecular docking. Finally, the mechanism of combinatorial components was verified by experiments. RESULTS: The HPLC results indicated that the main ingredients of VO Ex were hastatoside, verbenalin, acteoside, luteolin, apigenin and hispidulin. In vivo experiments showed that VO Ex improved MCAO-induced acute cerebral ischemic injury. Transcriptomic data and biological experiments suggested that VO Ex exerted therapeutic effects through IL17A signalling pathways. The in vitro experiments indicated that verbenalin, acteoside, luteolin, apigenin and hispidulin exhibited neuroprotective activities. The novel formula of VALAH, derived from the aforementioned active ingredients, exhibited superior efficacy compared to each individual component. Molecular docking and mechanistic studies have confirmed that VALAH functions in the treatment of ischaemic stroke by suppressing the activation of the IL17A signalling pathway. CONCLUSION: This work is the first to reveal that VO Ex effectively inhibits the IL17A signaling pathway and mitigates neuroinflammation following ischemic stroke. Moreover, we identified the novel formula VALAH as the bioactive combinatorial components for VO Ex. Further research suggests that the activity of VALAH is associated with IL17A-mediated regulation of neuroinflammation. This finding provides new insights into the efficacious components and mechanisms of traditional Chinese medicine.

Decreased S100A9 expression alleviates Clostridium perfringens beta2 toxin-induced inflammatory injury in IPEC-J2 cells.

Background: S100 calcium-binding protein A9 (S100A9) is a commonly known pro-inflammatory factor involved in various inflammatory responses. Clostridium perfringens (C. perfringens ) type C is known to cause diarrhea in piglets. However, the role of S100A9 in C. perfringens type C-induced infectious diarrhea is unclear. Methods: Here, the S100A9 gene was overexpressed and knocked down in the IPEC-J2 cells, which were treated with C. perfringens beta2 (CPB2) toxin. The role of S100A9 in CPB2 toxin-induced injury in IPEC-J2 cells was assessed by measuring the levels of inflammatory cytokines, reactive oxygen species (ROS), lactate dehydrogenase (LDH), cell proliferation, and tight junction-related proteins. Results: The results showed elevated expression of S100A9 in diarrhea-affected piglet tissues, and the elevation of S100A9 expression after CPB2 toxin treatment of IPEC-J2 was time-dependent. In CPB2 toxin-induced IPEC-J2 cells, overexpression of S100A9 had the following effects: the relative expression of inflammatory factors IL-6, IL8, TNF-α, and IL-1ß was increased; the ROS levels and LDH viability were significantly increased; cell viability and proliferation were inhibited; the G0/G1 phase cell ratio was significantly increased. Furthermore, overexpression of S100A9 reduced the expression of tight junction proteins in CPB2-induced IPEC-J2 cells. The knockdown of S100A9 had an inverse effect. In conclusion, our results confirmed that S100A9 exacerbated inflammatory injury in CPB2 toxin-induced IPEC-J2 cells, inhibited cell viability and cell proliferation, and disrupted the tight junctions between cells. Thus, decreased S100A9 expression alleviates CPB2 toxin-induced inflammatory injury in IPEC-J2 cells.

S100A9 plays a key role in Clostridium perfringens beta2 toxin-induced inflammatory damage in porcine IPEC-J2 intestinal epithelial cells.

BACKGROUND: As an important regulator of autoimmune responses and inflammation, S100A9 may serve as a therapeutic target in inflammatory diseases. However, the role of S100A9 in Clostridium perfringens type C infectious diarrhea is poorly studied. The aim of our study was to screen downstream target genes regulated by S100A9 in Clostridium perfringens beta2 (CPB2) toxin-induced IPEC-J2 cell injury. We constructed IPEC-J2 cells with S100A9 knockdown and a CPB2-induced cell injury model, screened downstream genes regulated by S100A9 using RNA-Seq technique, and performed functional enrichment analysis. The function of S100A9 was verified using molecular biology techniques. RESULTS: We identified 316 differentially expressed genes (DEGs), of which 221 were upregulated and 95 were downregulated. Functional enrichment analysis revealed that the DEGs were significantly enriched in cilium movement, negative regulation of cell differentiation, immune response, protein digestion and absorption, and complement and coagulation cascades. The key genes of immune response were TNF, CCL1, CCR7, CSF2, and CXCL9. When CPB2 toxin-induced IPEC-J2 cells overexpressed S100A9, Bax expression increased, Bcl-2 expression and mitochondrial membrane potential decreased, and SOD activity was inhibited. CONCLUSION: In conclusion, S100A9 was involved in CPB2-induced inflammatory response in IPEC-J2 cells by regulating the expression of downstream target genes, namely, TNF, CCL1, CCR7, CSF2, and CXCL9; promoting apoptosis; and aggravating oxidative cell damage. This study laid the foundation for further study on the regulatory mechanism underlying piglet diarrhea.

Development and validation of a three-dimensional deep learning-based system for assessing bowel preparation on colonoscopy video.

Background: The performance of existing image-based training models in evaluating bowel preparation on colonoscopy videos was relatively low, and only a few models used external data to prove their generalization. Therefore, this study attempted to develop a more precise and stable AI system for assessing bowel preparation of colonoscopy video. Methods: We proposed a system named ViENDO to assess the bowel preparation quality, including two CNNs. First, Information-Net was used to identify and filter out colonoscopy video frames unsuitable for Boston bowel preparation scale (BBPS) scoring. Second, BBPS-Net was trained and tested with 5,566 suitable short video clips through three-dimensional (3D) convolutional neural network (CNN) technology to detect BBPS-based insufficient bowel preparation. Then, ViENDO was applied to complete withdrawal colonoscopy videos from multiple centers to predict BBPS segment scores in clinical settings. We also conducted a human-machine contest to compare its performance with endoscopists. Results: In video clips, BBPS-Net for determining inadequate bowel preparation generated an area under the curve of up to 0.98 and accuracy of 95.2%. When applied to full-length withdrawal colonoscopy videos, ViENDO assessed bowel cleanliness with an accuracy of 93.8% in the internal test set and 91.7% in the external dataset. The human-machine contest demonstrated that the accuracy of ViENDO was slightly superior compared to most endoscopists, though no statistical significance was found. Conclusion: The 3D-CNN-based AI model showed good performance in evaluating full-length bowel preparation on colonoscopy video. It has the potential as a substitute for endoscopists to provide BBPS-based assessments during daily clinical practice.

METTL3 Regulates the Inflammatory Response in CPB2 Toxin-Exposed IPEC-J2 Cells through the TLR2/NF-κB Signaling Pathway.

Clostridium perfringens beta2 (CPB2) toxin is one of the main pathogenic toxins produced by Clostridium perfringens, which causes intestinal diseases in animals and humans. The N6-methyladenosine (m6A) modification is the most common reversible modification in eukaryotic disease processes. Methyltransferase-like 3 (METTL3) regulates immunity and inflammatory responses induced by the bacterial infections in animals. However, METTL3's involvement in CPB2-treated intestinal porcine epithelial cell line-J2 (IPEC-J2) remains unclear. In the current study, we used methylated RNA immunoprecipitation-quantitative polymerase chain reaction, Western blotting and immunofluorescence assay to determine the role of METTL3 in CPB2-exposed IPEC-J2 cells. The findings revealed that m6A and METTL3 levels were increased in CPB2 treated IPEC-J2 cells. Functionally, METTL3 overexpression promoted the release of inflammatory factors, increased cytotoxicity, decreased cell viability and disrupted tight junctions between cells, while the knockdown of METTL3 reversed these results. Furthermore, METTL3 was involved in the inflammatory response of IPEC-J2 cells by activating the TLR2/NF-κB signaling pathway through regulating TLR2 m6A levels. In conclusion, METTL3 overexpression triggered the TLR2/NF-κB signaling pathway and promoted CPB2-induced inflammatory responses in IPEC-J2 cells. These findings may provide a new strategy for the prevention and treatment of diarrhea caused by Clostridium perfringens.

Higher serum sST2 is associated with increased left atrial low-voltage areas and atrial fibrillation recurrence in patients undergoing radiofrequency ablation.

OBJECTIVE: To conduct a comprehensive analysis of prospectively measuring the concentration of soluble suppression of tumorigenicity 2 (sST2) to predict left atrial (LA) low-voltage areas (LVAs) and atrial fibrillation (AF) recurrence after radiofrequency catheter ablation (RFA). METHODS: This was a prospective cohort study. A total of 84 patients, including 54 paroxysmal AF cases and 30 persistent AF cases who underwent RFA, were recruited. Electroanatomical voltage mapping determined the extent of LVAs. The serum level of sST2 was measured by enzyme-linked immunosorbent assay. All patients were followed for 12 months after the RFA procedure to verify AF recurrence. RESULTS: The concentration of sST2 measured in the sample was 17.90-198.77 pg/mL, and the range of LA LVAs was 0-85.6%. The sST2 level positively correlated with LVAs (r = 0.40; P = 0.005). When comparing the top and bottom quartile, sST2 is significantly associated with LA LVAs (OR = 1.833, 95% CI: 1.582-2.011, P = 0.004). When compared with the 1st quartile group, the multivariable adjusted hazard ratios for AF recurrence after RFA were 1.57 (95% CI: 1.182-1.795) for the 4th quartile group, 1.44 (95% CI: 1.085-1.598) for the 3rd quartile group, and 1.27 (95% CI: 0.954-1.318) for the 2nd quartile group. The AF-free survival rates of patients with 1st quartile and 4th quartile sST2 levels after ablation were 95% and 59.6%, respectively (Log Rank test, P = 0.027). CONCLUSION: Elevated sST2 levels of AF patients were associated with higher LA LVAs and a significantly increased risk of recurrence. The circulating sST2 concentration might be a pre-diagnostic marker of AF recurrence after RFA.

ssc-microRNA-132 targets DACH1 to exert anti-inflammatory and anti-apoptotic effects in Clostridium perfringens beta2 toxin-treated porcine intestinal epithelial cells.

Clostridium perfringens (C. perfringens) type C (CPC) is one of the chief pathogens that causes diarrhea in piglets, and C. perfringens beta2 (CPB2) toxin is the main virulence factor of CPC. Our previous research demonstrated that ssc-microR-132 was differentially expressed in ileal tissues of CPC-mediated diarrheic piglets and healthy piglets, which implied a potential role of ssc-microR-132 in this process. Here, we found that ssc-microR-132 was notably down-regulated in CPB2-exposed intestinal porcine epithelial cells (IPEC-J2), which was consistent with the ileal tissue expression. Moreover, ssc-microR-132 upregulation alleviated CPB2-induced inflammatory damage and apoptosis in IPEC-J2, whereas ssc-microR-132 knockdown presented the opposite effects. Furthermore, the dual-luciferase reporter assay indicated that ssc-microR-132 directly targeted Dachshund homolog 1 (DACH1). Moreover, DACH1 overexpression intensified CPB2-induced inflammatory injury and apoptosis in IPEC-J2. Remarkably, the introduction of DACH1 weakened the anti-inflammatory and anti-apoptotic effects of ssc-microR-132 in CPB2-exposed IPEC-J2. Overall, the results reveal that ssc-microR-132 targeted DACH1 to alleviate CPB2-mediated inflammation and apoptosis in IPEC-J2.

Component-target network and mechanism of Qufeng Zhitong capsule in the treatment of neuropathic pain.

ETHNOPHARMACOLOGICAL RELEVANCE: Qufeng Zhitong capsule (QFZTC) is a traditional Chinese medicine (TCM) clinically used for treating pain. However, the active ingredients of QFZTC and its pharmacological mechanism in the treatment of neuropathic pain (NP) remain unclear. AIM OF THE STUDY: We aimed to identify the active ingredients of QFZTC and reveal its target genes and underlying mechanism of action in NP. MATERIALS AND METHODS: High-performance liquid chromatography (HPLC) was used to identify the active ingredients of QFZTC. Network pharmacology analysis was conducted to determine the core targets and pathway enrichment of QFZTC. An NP mice model was established through chronic compression injury (CCI) surgery of the sciatic nerve, while von Frey instrumentation and a thermal stimulator were employed to measure the sensitivity of mice to mechanical and thermal stimuli. Immunofluorescence was used to observe the expression of TLR4 and p-P65 in microglia. Western blotting was used to detect the levels of protein expression of Iba-1, TLR4, MyD88, P65, p-P65, and c-Fos, while ELISA kits were used to detect the release of TNF-α, IL-6, and IL-1ß. RESULTS: Seven active ingredients were identified in QFZTC: gallic acid, loganylic acid, syringin, corilagin, loganin, ellagic acid, and osthole. Network analysis identified TLR4, TNF, IL6, IL1ß, and c-Fos as core targets, and Toll-like receptors and NF-κB as core signaling pathways. Treatment with QFZTC significantly relieved mechanical allodynia and thermal hyperalgesia in CCI mice models. CCI induced an increase in the expression of TLR4 and p-P65 in microglia, whereas QFZTC dose-dependently reduced the expression of Iba-1, TLR4, MyD88, and p-P65 in the spinal cord. QFZTC inhibited the expression of the c-Fos pain marker and reduced the expression of the TNF-α, IL-6, and IL-1ß inflammatory factors. CONCLUSION: We combined the active ingredients of QFZTC with network pharmacology research to clarify its biological mechanism in the treatment of NP. We demonstrated that QFZTC reduced NP in mice probably through regulating the spinal microglia via the TLR4/MyD88/NF-κB signaling pathway. Hence, QFZTC could be regarded as a potential drug for relieving NP.

N6-Methyladenosine Methylation Analysis of Long Noncoding RNAs and mRNAs in IPEC-J2 Cells Treated With Clostridium perfringens beta2 Toxin.

Background: The n6-methyladenosine (m6A) modification is present widely in mRNAs and long non-coding RNAs (lncRNAs), and is related to the occurrence and development of certain diseases. However, the role of m6A methylation in Clostridium perfringens type C infectious diarrhea remains unclear. Methods: Here, we treated intestinal porcine jejunum epithelial cells (IPEC-J2 cells) with Clostridium perfringens beta2 (CPB2) toxin to construct an in vitro model of Clostridium perfringens type C (C. perfringens type C) infectious diarrhea, and then used methylated RNA immunoprecipitation sequencing (MeRIP-seq) and RNA sequencing (RNA-seq) to identify the methylation profiles of mRNAs and lncRNAs in IPEC-J2 cells. Results: We identified 6,413 peaks, representing 5,825 m6A-modified mRNAs and 433 modified lncRNAs, of which 4,356 m6A modified mRNAs and 221 m6A modified lncRNAs were significantly differential expressed between the control group and CPB2 group. The motif GGACU was enriched significantly in both the control group and the CPB2 group. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) annotation analysis showed that the differentially methylated modified mRNAs were mainly enriched in Hippo signaling pathway and Wnt signaling pathway. In addition, the target genes of the differentially m6A modified lncRNAs were related to defense response to virus and immune response. For example, ENSSSCG00000042575, ENSSSCG00000048701 and ENSSSCG00000048785 might regulate the defense response to virus, immune and inflammatory response to resist the harmful effects of viruses on cells. Conclusion: In summary, this study established the m6A transcription profile of mRNAs and lncRNAs in IPEC-J2 cells treated by CPB2 toxin. Further analysis showed that m6A-modified RNAs were related to defense against viruses and immune response after CPB2 toxin treatment of the cells. Threem6A-modified lncRNAs, ENSSSCG00000042575, ENSSSCG00000048785 and ENSSSCG00000048701, were most likely to play a key role in CPB2 toxin-treated IPEC-J2 cells. The results provide a theoretical basis for further research on the role of m6A modification in piglet diarrhea.

Comprehensive Analysis of Transcriptome-wide m6A Methylome Upon Clostridium perfringens Beta2 Toxin Exposure in Porcine Intestinal Epithelial Cells by m6A Sequencing.

Piglet diarrhea is a swine disease responsible for serious economic impacts in the pig industry. Clostridium perfringens beta2 toxin (CPB2), which is a major toxin of C. perfringens type C, may cause intestinal diseases in many domestic animals. N6-methyladenosine (m6A) RNA methylation plays critical roles in many immune and inflammatory diseases in livestock and other animals. However, the role of m6A methylation in porcine intestinal epithelial (IPEC-J2) cells exposed to CPB2 has not been studied. To address this issue, we treated IPEC-J2 cells with CPB2 toxin and then quantified methylation-related enzyme expression by RT-qPCR and assessed the m6A methylation status of the samples by colorimetric N6-methyladenosine quantification. The results showed that the methylation enzymes changed to varying degrees while the m6A methylation level increased (p < 0.01). On this basis, we performed N6-methyladenosine sequencing (m6A-seq) and RNA sequencing (RNA-seq) to examine the detailed m6A modifications and gene expression of the IPEC-J2 cells following CPB2 toxin exposure. Our results indicated that 1,448 m6A modification sites, including 437 up-regulated and 1,011 down-regulated, differed significantly between CPB2 toxin exposed cells and non-exposed cells (p < 0.05). KEGG pathway analysis results showed that m6A peaks up-regulated genes (n = 394) were mainly enriched in cancer, Cushing syndrome and Wnt signaling pathways, while m6A peaks down-regulated genes (n = 920) were mainly associated with apoptosis, small cell lung cancer, and the herpes simplex virus 1 infection signaling pathway. Furthermore, gene expression (RNA-seq data) analysis identified 1,636 differentially expressed genes (DEGs), of which 1,094 were up-regulated and 542 were down-regulated in the toxin exposed group compared with the control group. In addition, the down-regulated genes were involved in the Hippo and Wnt signaling pathways. Interestingly, the combined results of m6A-seq and RNA-seq identified genes with up-regulated m6A peaks but with down-regulated expression, here referred to as "hyper-down" genes (n = 18), which were mainly enriched in the Wnt signaling pathway. Therefore, we speculate that the genes in the Wnt signaling pathway may be modified by m6A methylation in CPB2-induced IPEC-J2 cells. These findings provide new insights enabling further exploration of the mechanisms underlying piglet diarrhea caused by CPB2 toxin.

Effects of miR-204 on apoptosis and inflammatory response of Clostridium perfringens beta2 toxin induced IPEC-J2 cells via targeting BCL2L2.

Clostridium perfringens beta2 (CPB2) toxin can cause intestinal damage and inflammatory responses in a variety of animals, which seriously endanger the healthy development of animal husbandry. Increasing evidence has demonstrated that microRNAs (miRNAs) can play an important regulatory role in the process of pathogenic infection. In our previous study, we found that miR-204 was highly expressed in the ileum tissues of the susceptible group diarrhea piglets after infection with Clostridium perfringens (C. perfringens) type C. In this study, we found that miR-204 was also up-regulated in different time points after CPB2 toxin treatment. Overexpression of miR-204 promoted apoptosis and inflammatory response of intestinal porcine epithelial cells (IPEC-J2), whereas the opposite results were displayed after transfected with miR-204 inhibitor. Furthermore, the luciferase reporter assays confirmed that BCL2L2 was a direct target gene of miR-204. Interestingly, we found that overexpression BCL2L2 attenuated the apoptosis and inflammatory response of CPB2 toxin induced IPEC-J2 cells. In conclusion, these results find that miR-204 promotes the apoptosis and intensify inflammatory response of CPB2 toxin induced IPEC-J2 cells via targeting BCL2L2. These data provide a valuable reference for the piglets resistance diarrhea at the molecular level.

Tectorigenin attenuates the OGD/R-induced HT-22 cell damage through regulation of the PI3K/AKT and the PPARγ/NF-κB pathways.

Tectorigenin (TEC) is an effective compound that derived from many plants, such as Iris unguicularis, Belamcanda chinensis and Pueraria thunbergiana Benth. Evidence suggested that TEC has anti-tumor, anti-oxidant activity, anti-bacterial and anti-inflammatory effects. In addition, there has some evidence indicated that TEC is a potential anti-stroke compound; however, its specific roles and associated mechanism have not yet been elucidated. In the present study, we aimed to investigate the anti-inflammatory, anti-oxidant activity and anti-apoptosis effects of TEC on oxygen-glucose deprivation/reperfusion (OGD/R)-induced HT-22 cells, and clarified the relevant mechanisms. Here, we observed that TEC significantly promoted cell survival, impeded cell apoptosis, inhibited ROS and inflammatory cytokines IL-1ß, IL-6, TNF-α production in OGD/R-induced HT-22 cells. Moreover, TEC activated PI3K/AKT signal pathway, increased PPARγ expression and inhibited NF-κB pathway activation in OGD/R-induced HT-22 cells. Further studies indicated that PPARγ inhibitor GW9662 activated NF-κB pathway after TEC treatment in OGD/R-induced HT-22 cells. Also, PI3K/AKT inhibitor LY294002, PPARγ inhibitor GW9662 and NF-κB activator LPS both reversed the effects of TEC on OGD/R-induced HT-22 cell biology. Taken together, this research confirmed that TEC benefit to HT-22 cell survival and against OGD/R damage through the PI3K/AKT and PPARγ/NF-κB pathways. These results indicated that TEC might be an effective compound in the treatment for ischemic brain injury.

Construction of a reference material panel for detecting KRAS/NRAS/EGFR/BRAF/MET mutations in plasma ctDNA.

BACKGROUND: The absence of high-quality next-generation sequencing (NGS) reference material (RM) has impeded the clinical use of liquid biopsies with plasma cell-free DNA (cfDNA) in China. OBJECTIVE: This study aimed to develop a national RM panel for external quality assessment and performance evaluation during kit registration of non-small-cell lung cancer (NSCLC)-related Kirsten rat sarcoma viral oncogene (KRAS)/neuroblastoma ras oncogene (NRAS)/epidermal growth factor receptor (EGFR)/B-type Raf kinase (BRAF)/mesenchymal-epithelial transition factor (MET) genetic assays using plasma circulating tumor DNA (ctDNA). METHODS: Mutation cell lines detected by NGS and validated by Sanger sequencing were selected to establish the RM. Cell line genomic DNA was sheared and used to spike basal plasma cfDNA at 10% concentration. Then, the calibration accuracy was determined by four sequencing platforms. Average values were adopted and diluted to 0.1%, 0.3%, 1% and 3% concentrations with basal plasma as the RM panel. Then, five manufacturers were invited to evaluate the performance of the RM panel. RESULTS: 20 cell lines with 23 clinically important mutations were selected, including six mutations in KRAS, two mutations in NRAS, three in BRAF, four in phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha (PIK3CA), six in EGFR, one EGFR Gain (4-5 copy) and one MET Gain (2-5 copy). The RM panel consisted of 87 samples, including these 21 mutations at four concentrations (0.1%, 0.3%, 1% and 3%), one MET gain, one EGFR gain and one wild type. The detection rate was 100% for the 3%, 1% and 0.3% samples at all five companies. For the 0.1% concentration, 15 samples had inconsistent results, but at least three companies had correct results for each mutation. CONCLUSION: RM for a KRAS/NRAS/EGFR/BRAF/MET mutation panel for plasma ctDNA was developed, which will be essential for quality control of the performance of independent laboratories.

Echinacoside reverses myocardial remodeling and improves heart function via regulating SIRT1/FOXO3a/MnSOD axis in HF rats induced by isoproterenol.

Myocardial remodelling is important pathological basis of HF, mitochondrial oxidative stress is a promoter to myocardial hypertrophy, fibrosis and apoptosis. ECH is the major active component of a traditional Chinese medicine Cistanches Herba, plenty of studies indicate it possesses a strong antioxidant capacity in nerve cells and tumour, it inhibits mitochondrial oxidative stress, protects mitochondrial function, but the specific mechanism is unclear. SIRT1/FOXO3a/MnSOD is an important antioxidant axis, study finds that ECH binds covalently to SIRT1 as a ligand and up-regulates the expression of SIRT1 in brain cells. We hypothesizes that ECH may reverse myocardial remodelling and improve heart function of HF via regulating SIRT1/FOXO3a/MnSOD signalling axis and inhibit mitochondrial oxidative stress in cardiomyocytes. Here, we firstly induce cellular model of oxidative stress by ISO with AC-16 cells and pre-treat with ECH, the level of mitochondrial ROS, mtDNA oxidative injury, MMP, carbonylated protein, lipid peroxidation, intracellular ROS and apoptosis are detected, confirm the effect of ECH in mitochondrial oxidative stress and function in vitro. Then, we establish a HF rat model induced by ISO and pre-treat with ECH. Indexes of heart function, myocardial remodelling, mitochondrial oxidative stress and function, expression of SIRT1/FOXO3a/MnSOD signalling axis are measured, the data indicate that ECH improves heart function, inhibits myocardial hypertrophy, fibrosis and apoptosis, increases the expression of SIRT1/FOXO3a/MnSOD signalling axis, reduces the mitochondrial oxidative damages, protects mitochondrial function. We conclude that ECH reverses myocardial remodelling and improves cardiac function via up-regulating SIRT1/FOXO3a/MnSOD axis and inhibiting mitochondrial oxidative stress in HF rats.

The synthesized transporter K16APoE enabled the therapeutic HAYED peptide to cross the blood-brain barrier and remove excess iron and radicals in the brain, thus easing Alzheimer's disease.

Alzheimer's disease (AD) is currently incurable and places a large burden on the caregivers of AD patients. In the AD brain, iron is abundant, catalyzing free radicals and impairing neurons. The blood-brain barrier hampers antidementia drug delivery via circulation to the brain, which limits the therapeutic effects of drugs. Here, according to the method described by Gobinda, we synthesized a 16 lysine (K) residue-linked low-density lipoprotein receptor-related protein (LRP)-binding amino acid segment of apolipoprotein E (K16APoE). By mixing this protein with our designed therapeutic peptide HAYED, we successfully transported HAYED into an AD model mouse brain, and the peptide scavenged excess iron and radicals and decreased the necrosis of neurons, thus easing AD.