Iron Overload-Dependent Ferroptosis Aggravates LPS-Induced Acute Lung Injury by Impairing Mitochondrial Function.

Ferroptosis is a newly proposed form of programmed cell death that is iron-dependent and closely linked to oxidative stress. Its specific morphological changes include shrunken mitochondria, increased density of mitochondrial membrane, and rupture or disappearance of mitochondrial cristae. The main mechanism of ferroptosis involves excessive free iron reacting with membrane phospholipids, known as the Fenton reaction, resulting in lipid peroxidation. However, the role of iron in acute lung injury (ALI) remains largely unknown. In this study, LPS was instilled into the airway to induce ALI in mice. We observed a significant increase in iron concentration during ALI, accompanied by elevated levels of lipid peroxidation markers such as malonaldehyde (MDA) and 4-hydroxynonenal (4-HNE). Treatment with the iron chelator deferoxamine (DFO) or ferroptosis inhibitor ferrostatin-1 (Fer-1) reversed lipid peroxidation and significantly attenuates lung injury. Similarly, DFO or Fer-1 treatment improved the cell survival significantly in vitro. These results demonstrated that ferroptosis occurs during ALI and that targeting ferroptosis is an effective treatment strategy. Interestingly, we found that the increased iron was primarily concentrated in mitochondria and DFO treatment effectively restored normal mitochondria morphology. To further confirm the damaging effect of iron on mitochondria, we performed mitochondrial stress tests in vitro, which revealed that iron stimulation led to mitochondrial dysfunction, characterized by impaired basal respiratory capacity, ATP production capacity, and maximum respiratory capacity. MitoTEMPO, an antioxidant targeting mitochondria, exhibited superior efficacy in improving iron-induced mitochondrial dysfunction compared to the broad-spectrum antioxidant NAC. Treatment with MitoTEMPO more effectively alleviated ALI. In conclusion, ferroptosis contributes to the pathogenesis of ALI and aggravates ALI by impairing mitochondrial function.

Regulator of G protein signaling protein 6 alleviates acute lung injury by inhibiting inflammation and promoting cell self-renewal in mice.

BACKGROUND: Acute respiratory distress syndrome (ARDS) is a disease with high mortality and morbidity. Regulator of G protein signaling protein 6 (RGS6), identified as a tumor suppressor gene, has received increasing attention owing to its close relationship with oxidative stress and inflammation. However, the association between ARDS and RGS6 has not been reported. METHODS: Congruously regulated G protein-coupled receptor (GPCR)-related genes and differentially expressed genes (DEGs) in an acute lung injury (ALI) model were identified, and functional enrichment analysis was conducted. In an in vivo study, the effects of RGS6 knockout were studied in a mouse model of ALI induced by lipopolysaccharide (LPS). HE staining, ELISA, and immunohistochemistry were used to evaluate pathological changes and the degree of inflammation. In vitro, qRT‒PCR, immunofluorescence staining, and western blotting were used to determine the dynamic changes in RGS6 expression in cells. The RGS6 overexpression plasmid was constructed for transfection. qRT‒PCR was used to assess proinflammatory factors transcription. Western blotting and flow cytometry were used to evaluate apoptosis and reactive oxygen species (ROS) production. Organoid culture was used to assess the stemness and self-renewal capacity of alveolar epithelial type II cells (AEC2s). RESULTS: A total of 110 congruously regulated genes (61 congruously upregulated and 49 congruously downregulated genes) were identified among GPCR-related genes and DEGs in the ALI model. RGS6 was downregulated in vivo and in vitro in the ALI model. RGS6 was expressed in the cytoplasm and accumulated in the nucleus after LPS stimulation. Compared with the control group, we found higher mortality, more pronounced body weight changes, more serious pulmonary edema and pathological damage, and more neutrophil infiltration in the RGS6 knockout group upon LPS stimulation in vivo. Moreover, AEC2s loss was significantly increased upon RGS6 knockout. Organoid culture assays showed slower alveolar organoid formation, fewer alveolar organoids, and impaired development of new structures after passaging upon RGS6 knockout. In addition, RGS6 overexpression decreased ROS production as well as proinflammatory factor transcription in macrophages and decreased apoptosis in epithelial cells. CONCLUSIONS: RGS6 plays a protective role in ALI not only in early inflammatory responses but also in endogenous lung stem cell regeneration.

Xuebijing alleviates LPS-induced acute lung injury by downregulating pro-inflammatory cytokine production and inhibiting gasdermin-E-mediated pyroptosis of alveolar epithelial cells.

Acute lung injury/acute respiratory distress syndrome (ALI/ARDS) is characterized by diffuse alveolar injury primarily caused by an excessive inflammatory response. Regrettably, the lack of effective pharmacotherapy currently available contributes to the high mortality rate in patients with this condition. Xuebijing (XBJ), a traditional Chinese medicine recognized for its potent anti-inflammatory properties, exhibits promise as a potential therapeutic agent for ALI/ARDS. This study aimed to explore the preventive effects of XBJ on ALI and its underlying mechanism. To this end, we established an LPS-induced ALI model and treated ALI mice with XBJ. Our results demonstrated that pre-treatment with XBJ significantly alleviated lung inflammation and increased the survival rate of ALI mice by 37.5%. Moreover, XBJ substantially suppressed the production of TNF-α, IL-6, and IL-1ß in the lung tissue. Subsequently, we performed a network pharmacology analysis and identified identified 109 potential target genes of XBJ that were mainly involved in multiple signaling pathways related to programmed cell death and anti-inflammatory responses. Furthermore, we found that XBJ exerted its inhibitory effect on gasdermin-E-mediated pyroptosis of lung cells by suppressing TNF-α production. Therefore, this study not only establishes the preventive efficacy of XBJ in ALI but also reveals its role in protecting alveolar epithelial cells against gasdermin-E-mediated pyroptosis by reducing TNF-α release.

Determination of serum CA724 levels using fluorescence immunochromatography.

BACKGROUND: Carbohydrate antigen 724 (CA724) is a sensitive and specific indicator for multiple malignant tumors. The aim of this study was to establish a Eu-time resolved fluorescence immunochromatography (Eu-TRFICO) method for quantitative detection of CA724 in serum. METHODS: Eu-TRFICO strips were optimized and assembled. The sensitivity, specificity and precision were evaluated using CA724 standard dilutions and matrix serum. Meanwhile, the reference interval, comparison, and sensitivity/specificity were performed using clinical negative/positive gastric cancer serum samples. RESULTS: The standard curve equation was y = 9.869 x - 154.12 (R2 = 0.993), and the sensitivity was 0.42 U/mL. The common interferents in serum could not affect the quantitative results with low cross-reactivities (all no more than 1.09%). All average recoveries of the intra- and interbatch ranged from 102.38 to 106.40%, and all CVs were below 10%. The reference interval of the healthy subjects was < 4.68 U/mL and the reference interval of the subjects with grade I/II gastric cancer was > 9.54 U/mL. Additionally, a high Pearson r (0.9503) and sensitivity/specificity (92.86%/94.20%) were obtained. CONCLUSION: This study prepared Eu-TRFICO strips with high sensitivity, specificity, precision and satisfactory clinical testing performance, which provides more options for clinical quantitative and convenient testing of CA724.

Perturbation of arachidonic acid and glycerolipid metabolism promoted particulate matter-induced inflammatory responses in human bronchial epithelial cells.

Particulate matter (PM) has become the main risk factor for public health, being linked with an increased risk of respiratory diseases. However, the potential mechanisms underlying PM-induced lung injury have not been well elucidated. In this study, we systematically integrated the metabolomics, lipidomics, and transcriptomics data obtained from the human bronchial epithelial cells (HBECs) exposed to PM to reveal metabolic disorders in PM-induced lung injury. We identified 170 differentially expressed metabolites (82 upregulated and 88 downregulated metabolites), 218 differentially expressed lipid metabolites (125 upregulated and 93 downregulated lipid metabolites), and 1417 differentially expressed genes (643 upregulated and 774 downregulated genes). Seven key metabolites (prostaglandin E2, inosinic acid, L-arginine, L-citrulline, L-leucine, adenosine, and adenosine monophosphate), and two main lipid subclasses (triglyceride and phosphatidylcholine) were identified in PM-exposed HBECs. The amino acid metabolism, lipid metabolism, and carbohydrate metabolism were the significantly enriched pathways of identified differentially expressed genes. Then, conjoint analysis of these three omics data and further qRT-PCR validation showed that arachidonic acid metabolism, glycerolipid metabolism, and glutathione metabolism were the key metabolic pathways in PM-exposed HBECs. The knockout of AKR1C3 in arachidonic acid metabolism or GPAT3 in glycerolipid metabolism could significantly inhibit PM-induced inflammatory responses in HBECs. These results revealed the potential metabolic pathways in PM-exposed HBECs and provided a new target to protect from PM-induced airway damage.

Tumor mutational burden and efficacy of chemotherapy in lung cancer.

PURPOSE: TMB is one of the potent biomarkers of response to immune checkpoint blockade. The association between TMB and efficacy of chemotherapy in advanced lung cancer has not been comprehensively explored. METHODS: Ninety lung cancer patients receiving first-line chemotherapy with large panel next-generation sequencing data of pre-treatment tumor tissue were identified. The effect of TMB on PFS of chemotherapy were evaluated in univariate and multivariate analyses. RESULTS: The median TMB level of lung cancer patients enrolled in this study was 9.4 mutations/Mb, with TMB levels in smokers significantly higher than those in non-smokers. All patients were divided into high TMB and low TMB groups with the cutoff of the median TMB. The patients with low TMB had longer PFS of first-line chemotherapy (median PFS 9.77 vs 6.33 months, HR = 0.523, 95% CI 0.32-0.852, log-rank P = 0.009). Subgroup analysis showed that PFS of chemotherapy favored low TMB than high TMB among subgroups of male, age < 60, NSCLC, adenocarcinoma, stage IV, ECOG PS 0, driver mutation positive, TP53 wild type and patients not receiving bevacizumab. In multivariate analysis, PFS of chemotherapy remained significantly longer in low TMB group (HR = 0.554, p = 0.036). In those patients received immunotherapy upon unsatisfactory chemotherapy, PFS of immunotherapy was much longer in high TMB group (median PFS 32.88 vs 6.62 months, HR = 0.2426, 95% CI 0.06-0.977, log-rank P = 0.04). CONCLUSIONS: TMB level of tumor tissue is a potent biomarker for efficacy of chemotherapy and immunotherapy in lung cancer. It may provide some clues for the decision of treatment strategy.

Developing and validating a chronic obstructive pulmonary disease quick screening questionnaire using statistical learning models.

BACKGROUND: Active targeted case-finding is a cost-effective way to identify individuals with high-risk for early diagnosis and interventions of chronic obstructive pulmonary disease (COPD). A precise and practical COPD screening instrument is needed in health care settings. METHODS: We created four statistical learning models to predict the risk of COPD using a multi-center randomized cross-sectional survey database (n = 5281). The minimal set of predictors and the best statistical learning model in identifying individuals with airway obstruction were selected to construct a new case-finding questionnaire. We validated its performance in a prospective cohort (n = 958) and compared it with three previously reported case-finding instruments. RESULTS: A set of seven predictors was selected from 643 variables, including age, morning productive cough, wheeze, years of smoking cessation, gender, job, and pack-year of smoking. In four statistical learning models, generalized additive model model had the highest area under curve (AUC) value both on the developing cross-sectional data set (AUC = 0.813) and the prospective validation data set (AUC = 0.880). Our questionnaire outperforms the other three tools on the cross-sectional validation data set. CONCLUSIONS: We developed a COPD case-finding questionnaire, which is an efficient and cost-effective tool for identifying high-risk population of COPD.

A New Method for Early Screening of Gastric Cancer (G17 and CA724 Dual-Labeled Time-Resolved Fluorescence Immunoassay).

Gastric carcinoma (GC) is one of the most common malignancies in the world with the great early screening challenges. The study is aimed at establishing a new detection method for early screening GC using time-resolved fluorescence immunoassay (TRFIA) via quantitative detection of gastrin-17 (G-17) and carbohydrate antigen 724 (CA724) in serum. Time-resolved analyzer measured the fluorescence intensity. The standards of G-17/CA724 were used for drawing the standard curve, which is used to calculate the concentration of G-17 and CA724 in serum sample. The sensitivity for G-17 was 0.54 pg/mL and for CA724 was 0.28 U/mL with a wide-range analyze concentration (0.1-1000) pg/mL or U/mL. The average recoveries ranged from 100.52% to 110.30% for G-17 and 103.02% to 116.00% for CA724. All CVs of the intra- and interassay were below 10% with high specificity. There was a high Pearson coefficient between this TRFIA method and the commercially available kits (Pearson r 0.9117 for G-17 and 0.9449 for CA724). Additionally, the cutoff value was 88.41 pg/mL and 5.47 U/mL for CA724 in health subjects. This study established a TRFIA method for simultaneous detection of the concentrations of G-17 and CA724 in serum, which provide a new method for sensitive, accurate, and specific early screening of gastric cancer.

Weaker Response to XueBiJing Treatment in Severe Community-Acquired Pneumonia Patients With Higher Body Mass Index or Hyperglycemia: A Post Hoc Analysis of a Randomized Controlled Trial.

Background: Overweight and hyperglycemia might result in poor prognosis in patients with severe community-acquired pneumonia (SCAP). XueBiJing treatment could significantly improve the outcomes of patients with SCAP. We investigated the efficacy of XueBiJing injection in patients with SCAP stratified by body mass index (BMI) and fasting blood glucose (FBG). Methods: This is a post hoc analysis of XueBiJing trial, a large prospective, randomized, controlled study conducted in 33 hospitals in China. We compared data from non-overweight (BMI <24 kg/m2, n = 425) vs. overweight (BMI ≥24 kg/m2, n = 250) patients as well as non-hyperglycemia (FBG <7 mmol/L, n = 315) vs. hyperglycemia (FBG ≥7 mmol/L, n = 360) patients with XueBiJing, 100 ml, q12 h, or a visually indistinguishable placebo treatment for 5-7 days. Results: Among patients with BMI <24 kg/m2 (n = 425), 33 (15.3%), XueBiJing recipients and 52 (24.9%) placebo recipients (p = 0.0186) died within 28 days. Among patients with BMI ≥24 kg/m2 (n = 250), XueBiJing recipients still had lower mortality (XueBiJing 16.9% vs. placebo 24.2%; p = 0.2068) but without significantly statistical difference. For the FBG group, patients with FBG <7 mmol/L (n = 315), 18 (11.2%) XueBiJing recipients and 32 (20.8%) placebo recipients (p = 0.030) died within 28 days. Among patients with FBG ≥7 mmol/L (n = 360), XueBiJing recipients still had lower mortality (XueBiJing 20.2% vs. placebo 27.8%; p = 0.120) but without significantly statistical difference. The total duration of the ICU stay and the duration of mechanical ventilation were similar in both groups (p > 0.05). Conclusion: Overweight or hyperglycemia might weaken the efficacy of XueBiJing injection in the treatment of SCAP as indicated by the significant elevated risk of 28-day mortality. Additional studies are needed to validate our findings and to further understand the underlying mechanisms.

Study of mesenchymal stem cells derived from lung-resident, bone marrow and chorion for treatment of LPS-induced acute lung injury.

BACKGROUND: Mesenchymal stem cells (MSCs) have been shown to improve acute lung injury (ALI) and acute respiratory distress syndrome (ARDS). However, the optimal source of MSCs for cell-based therapy remains unknown. To determine which kind of MSCs are more effective, we compared the effects of rat lung resident MSC (LRMSC), human chorion-derived MSC (HMSC-C) and human bone marrow derived MSC (HMSC-BM) in LPS-induced ALI in mice. METHODS: LPS (Pseudomonas aeruginosa) was used to induce ALI model. All three kinds of MSCs were administered via tail vein 4 h after LPS instillation. The mice were sacrificed 48 h after LPS instillation. H&E staining of lung section, wet-to-dry weight ratio of lung tissue, ratio of regulatory T cells (Tregs) and Th17 cells, and total protein concentration, leukocytes counting and cytokines in bronchoalveolar lavage fluid (BALF) were evaluated. RESULTS: The data showed that compared with LRMSC and HMSC-BM, HMSC-C more significantly attenuated lung injury, upregulated the Tregs/Th17 cells ratio, and inhibited release of inflammatory cytokines (IL-1ß, IL-6 and TNF-α) and recruitment of neutrophils and macrophages into alveolus. CONCLUSIONS: Although all three kinds of LRMSC, HMSC-C and HMSC-BM are protective against LPS-induced lung injury, HMSC-C was more effective than LRMSC and HMSC-BM to treat LPS-induced lung injury.

Discovery of efficacy biomarkers for non-small cell lung cancer with first-line anti-PD-1 immunotherapy by data-independent acquisition mass spectrometry.

First-line immune checkpoint inhibitors (ICIs) have greatly ameliorated outcomes in non-small cell lung cancer (NSCLC). However, approximately a quarter of patients receiving ICIs demonstrate long-term clinical benefit, and the true responders have not been fully clarified by the existing biomarkers. To discover potential biomarkers treatment-related outcomes in plasma, mass spectrometry assay for the data-independent acquisition was analyzed plasma samples collected before the anti-PD-1 treatment. From July 2019 to January 2020, 15 patients with EGFR/ALK-negative NSCLC receiving first-line anti-programmed cell death protein 1 (PD-1) inhibitors were enrolled, and six healthy individuals have collected the plasma samples as control. We explored plasma proteome profiles and conducted stratified analyses by anti-PD-1 responders and non-responders. To validate the target proteins by ELISA, we recruited 22 additional independent patients and 15 healthy individuals from April 2021 to August 2021. By identifying biomarkers to predict better efficacy, we performed differential expression analysis in 12 responders and three non-responders. Compared with healthy individuals, hierarchical cluster analysis revealed plasma proteome profiles of NSCLC were markedly changed in 170 differentially expressed proteins. Furthermore, we discovered that SAA1, SAA2, S100A8, and S100A9 were noticeably increased among non-responders than responders, which may serve as predictive biomarkers with unfavorable responses. The validated results from all samples via ELISA have confirmed this observation. Identified a set of plasma-derived protein biomarkers (SAA1, SAA2, S100A8, and S100A9) that could potentially predict the efficacy in cohorts of patients with NSCLC treated with first-line anti-PD-1 inhibitors and deserves further prospective study.

Rational design of small-molecules to recognize G-quadruplexes of c-MYC promoter and telomere and the evaluation of their in vivo antitumor activity against breast cancer.

DNA G4-structures from human c-MYC promoter and telomere are considered as important drug targets; however, the developing of small-molecule-based fluorescent binding ligands that are highly selective in targeting these G4-structures over other types of nucleic acids is challenging. We herein report a new approach of designing small molecules based on a non-selective thiazole orange scaffold to provide two-directional and multi-site interactions with flanking residues and loops of the G4-motif for better selectivity. The ligands are designed to establish multi-site interactions in the G4-binding pocket. This structural feature may render the molecules higher selectivity toward c-MYC G4s than other structures. The ligand-G4 interaction studied with 1H NMR may suggest a stacking interaction with the terminal G-tetrad. Moreover, the intracellular co-localization study with BG4 and cellular competition experiments with BRACO-19 may suggest that the binding targets of the ligands in cells are most probably G4-structures. Furthermore, the ligands that either preferentially bind to c-MYC promoter or telomeric G4s are able to downregulate markedly the c-MYC and hTERT gene expression in MCF-7 cells, and induce senescence and DNA damage to cancer cells. The in vivo antitumor activity of the ligands in MCF-7 tumor-bearing mice is also demonstrated.

The circular RNA circTXNRD1 promoted ambient particulate matter-induced inflammation in human bronchial epithelial cells by regulating miR-892a/COX-2 axis.

Particulate matter (PM)-induced airway inflammation contributes to the development and exacerbation of chronic airway diseases. Circular RNA (circRNA) is a new class of non-coding RNA that participates in gene regulation in various respiratory diseases, but the regulatory role of circRNA in PM-induced airway inflammation has not been fully elucidated. In this study, we performed the human circRNA microarray to reveal differentially expressed circRNAs in PM-induced human bronchial epithelial cells (HBECs). A total of 176 upregulated and 15 downregulated circRNAs were identified. Of these, a new circRNA termed circTXNRD1 was upregulated by PM exposure in a dose- and time-dependent manner. Knockdown of circTXNRD1 significantly attenuated PM-induced expression of proinflammatory cytokine interleukin 6 (IL-6). CircRNA pull-down, dual-luciferase reporter assay and fluorescence in situ hybridization showed that circTXNRD1 acted as an endogenous sponge to decrease miR-892a levels in HBECs. Downregulation of miR-892a could increase cyclooxygenase-2 (COX-2) expression and eventually promote IL-6 secretion in PM-induced HBECs. Taken together, our findings reveal circTXNRD1 as a novel inflammatory mediator in PM-induced inflammation in HBECs via regulating miR-892a/COX-2 axis. These results provide new insight into the biological mechanism underlying PM-induced inflammation in chronic airway diseases.

Lily bulbs' polyphenols extract ameliorates oxidative stress and lipid accumulation in vitro and in vivo.

BACKGROUND: Polyphenols have the potential to reduce the risk of many metabolic disorders. Lily bulbs are rich in polyphenols; however, their effects on lipid metabolism remain unclear. This study aimed to explore the effects of lily bulbs' polyphenols (LBPs) on oxidative stress and lipid metabolism. RESULTS: A total of 14 polyphenolic compounds in LBPs were identified by high-performance liquid chromatography equipped with diode-array detection mass spectrometry. Total phenolic compound in LBPs was 53.76 ± 1.12 g kg-1 dry weight. In cellular experiments, LBPs attenuated the disruption of mitochondrial membrane potential, impeded reactive oxygen species production, alleviated oxidative stress, and reduced lipid accumulation in oleic acid induced HepG2 cells. In in vivo studies, LBPs significantly inhibited body weight gain, reduced lipid levels in serum and liver, and improved oxidative damage in a dose-dependent manner in mice fed a high-fat diet. Moreover, LBPs ameliorated hepatic steatosis and suppressed the expression of hepatic-lipogenesis-related genes (SREBP-1c, FAS, ACC1, and SCD-1) and promoted lipolysis genes (SRB1 and HL) and lipid oxidation genes (PPARα and CPT-1) in mice fed a high-fat diet. CONCLUSION: It was concluded that LBPs are a potential complementary therapeutic alternative in the development of functional foods to curb obesity and obesity-related diseases, such as metabolic syndrome. © 2021 Society of Chemical Industry.

Transcriptomic analysis of key genes and pathways in human bronchial epithelial cells BEAS-2B exposed to urban particulate matter.

Urban particulate matter (PM), a great danger to public health, is associated with increasing risk of pulmonary diseases. However, the involved key genes and signaling pathways mediating the cellular responses to urban PM are largely unknown. In this study, human bronchial epithelial cells BEAS-2B was exposed to Standard reference material (SRM) 1649b, followed by RNA-sequencing (RNA-seq) and a combination of different bioinformatics analysis. A total of 201 genes (111 upregulated and 90 downregulated) were identified as the differentially expressed genes (DEGs). Moreover, Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), and gene set enrichment analysis (GSEA) unveiled several significant genes and pathways involved in PM-induced lung toxicity. Protein-protein interaction (PPI) network was performed with the Search Tool for the Retrieval of Interacting Genes (STRING), and the hub gene modules were recognized by Molecular Complex Detection (MCODE), a plug-in of Cytoscape. Moreover, Connectivity Map (CMap) analysis found six candidate small molecular compounds to reverse PM-altered gene expression, including aminohippuric acid, captopril, cinoxacin, fasudil, pargyline, and altizide. Finally, the expressions of part vital genes related to inflammation (IL-1ß, CXCL2, CXCL5, CXCL8), ferroptosis (HMOX1, GCLM), and autophagy (BECN1, MAPK1LC3B) were in accordance with the RNA-seq data, with a concentration-dependent manner. This study may be helpful in revealing the complex molecular mechanisms underlying PM-induced lung toxicity and provide some new therapeutic targets for PM-related pulmonary diseases.

Identification and mechanism of insecticidal periplocosides from the root bark of Periploca sepium Bunge.

BACKGROUND: The Periploca sepium bark root (PSBR) has been regarded as a potential botanical insecticide because of its significant insecticidal activity of secondary metabolites. Several periplocosides were isolated from it as promising pesticides to control crop pests in agriculture. RESULTS: In our research, two new periplocosides, along with four known periplocosides were isolated from PSBR. The names of new periplocosides were periplocoside T (PST) and periplocoside U (PSU) while another four periplocosides were known as follows: periplocoside A (PSA), periplocoside F (PSF), periplocoside E (PSE) and periplocoside D (PSD). All periplocosides were evalulated for insecticidal activity against 3rd Mythimna separata (Walker) and Plutella xylostella. The biometric data showed that periplocoside T, PSD and PSF had remarkable insecticidal activity against tested insects. Its values of LD50 were 1.31, 3.94 and 3.42 µg·lavare-1 against 3rd M. separata respectively, while the activity of those compounds against 3rd P. xylostella were 5.45, 12.17 and 13.95 µg·lavare-1 , respectively. It was apparent after further study of the mechanism of action against M. separata was conducted that PST possessed the most significant insecticidal activity. The results of enzymatic activity displayed that powerful activation of tryptase, especially weak alkaline tryptase might be a dominant factor causing death of M. separata in vivo. CONCLUSION: We herein report isolation and the mechanisms of action of insecticidal periplocosides, which established the fundamental development of natural agents to prevent pest damage to crops. © 2020 Society of Chemical Industry.

Novel dynein axonemal assembly factor 1 mutations identified using whole­exome sequencing in patients with primary ciliary dyskinesia.

Primary ciliary dyskinesia (PCD) is a rare, genetically heterogeneous disorder caused by dysfunction of the cilia and flagella; however, causative genetic defects have not been detected in all patients with PCD. Seven Chinese Han patients with Kartagener syndrome were enrolled onto the present study. Transmission electron microscopy (TEM) was performed to evaluate the cilial defects and whole­exome sequencing was used to analyze relevant genetic variations in all patients. In two of the seven patients with PCD, four novel dynein axonemal assembly factor 1 (DNAAF1) mutations were identified (NM_178452.6:c.3G>A, c.124+1G>C, c.509delG and c.943A>T) in three alleles. Both of these patients had long­standing infertility. Their chest computed tomography results showed bronchiectasis, lung infections and situs inversus, and paranasal computed tomography revealed sinusitis. Semen analysis of the male patient showed poor sperm motility. TEM showed defects in the inner and outer dynein arms in both patients. The DNAAF1 sequences of family members were then analyzed. Bioinformatics analysis indicated that these mutations may be the cause of the cilial defects in these two probands. Thus, the present study identified novel PCD­causing mutations in DNAAF1 in two patients with PCD. These genetic variations were predicted to alter DNAAF1 amino acid residues and lead to loss of function, thereby inhibiting cilia­mediated motility. Accordingly, the two probands had PCD symptoms, and one of them died due to PCD­associated complications.

Associations of long-term exposure to ambient fine particulate matter and nitrogen dioxide with lung function: A cross-sectional study in China.

BACKGROUND: Few studies have evaluated the effects of ambient air pollution exposure on lung function, especially in areas with high air pollution levels. OBJECTIVES: To investigate the associations of annual concentrations of particulate matter with diameters < 2.5 µm (PM2.5) and nitrogen dioxide (NO2) with adult lung function in Shanghai, China. METHODS: We included 5276 permanent residents aged ≥ 20 years. Annual residential exposure to PM2.5 and NO2 was estimated by validated satellite-based and land use regression models, respectively. The effects of PM2.5 and NO2 on lung function were estimated separately using multivariable linear regression, adjusting for potential confounders. RESULTS: Higher exposure to PM2.5 and NO2 was significantly associated with lower forced vital capacity (FVC), inspiration capacity (IC), and vital capacity (VC). An increase of 10 µg/m3 in the annual average PM2.5 exposure was associated with a 45.83 ml (95% CI: -82.59, -9.07) lower FVC, 1.36 (95% CI: -2.42, -0.29) lower FVC of % predicted (FVC%pred), 121.98 ml (95% CI: -164.38, -79.57) lower IC, and 89.12 ml (95% CI -124.94, -53.3) lower VC. For NO2, an increase of 10 µg/m3 in the annual average concentration was associated with 26.65 ml (95% CI: -46.29, -7.00) lower FVC, 0.70 (95% CI: -1.27, 0.13) lower FVC%pred, 65.26 ml (95% CI: -87.76, -42.76) lower IC, and 45.88 ml (95% CI: -65.03, -26.73) lower VC. The estimated effects on FEV1 were -10.25 ml (95% CI: -40.92, 20.42) and -0.29% (95% CI: -1.40, 0.82) per 10 µg/m3 increase in PM2.5 and -0.74 ml (95% CI: -17.13, 15.65) and 0.01% (95% CI: -0.58, 0.61) per 10 µg/m3 increase in NO2, which were not statistically significant. Stratified analysis showed that the estimated effects of PM2.5 were greater in the healthy subgroup than the COPD patients. Obese individuals were more susceptible to adverse effects of PM2.5 and NO2 on lung function. Education level showed no or only weak evidence of modification of the associations between air pollution and lung function. CONCLUSION: In this study, long-term exposure to ambient air pollutants was significantly associated with impaired lung function, presenting as restrictive ventilatory patterns.

miR-149 regulates the proliferation and apoptosis of human colonic carcinoma cells by targeting FZD5.

OBJECTIVE: To explore the effects of miR-149 on the cell proliferation and apoptosis of colorectal cancer (CRC) and its potential molecular mechanism. METHODS: miR-149 expression patterns were detected in human CRC cell lines by quantitative real-time RT-PCR (Q-PCR). Online prediction software and luciferase reporter assay were performed to screen the functional targets of miR-149. CRC cells were transfected with miR-149 mimics or siRNAs of FZD5 and then divided into NC group (negative control), miR-149 mimics group (cells transfected with miR-149 mimics) and miR-149 mimics + SiFZD5 group (cells transfected by miR-149 mimics and SiFZD5). Moreover, the effects of miR-149 on the proliferation and apoptosis of CRC cells were also analyzed by MTT and flow cytometry assay. In addition, the expression of Wnt/ß-catenin signal pathways related factors were shown by western blot analysis. RESULTS: Q-PCR results demonstrated that the expression of miR-149 was significantly lower in SW480 than that in the FHC cell line. Frizzled class receptor 5 (FZD5) was identified as a functional target of miR-149 through a series of experiments including Q-PCR, western blot analysis, and luciferase assay. Cellular functional experiments demonstrated that the cell viability and proliferation were greatly inhibited after miR-149 overexpression in SW480 cells. Furthermore, the proportion of apoptotic cells increased significantly after introducing miR-149 into SW480 cells. Furthermore, Wnt/ß-catenin signal pathway was activated because of the lower expression of ß-catenin and cyclinD1 in miR-149 mimics group. However, reducing FZD5 expression restored the expression of ß-catenin and cyclin D. CONCLUSIONS: Our data suggested that miR-149 may function as a tumor suppressor in CRC cells lines by targeting FZD5. miR-149/FZD5 may become a new therapeutic target for CRC.

MiR-29b-3p promotes particulate matter-induced inflammatory responses by regulating the C1QTNF6/AMPK pathway.

Inflammatory responses are considered to be the critical mechanism underlying particulate matter (PM)-induced development and exacerbation of chronic respiratory diseases. MiR-29b-3p has been found to participate in various biological processes, but its role in PM-induced inflammatory responses was previously unknown. Here, we constructed a miRNA PCR array to find that miR-29b-3p was the most highly expressed in human bronchial epithelial cells (HBECs) exposed to PM. MiR-29b-3p promoted PM-induced pro-inflammatory cytokines (IL-1ß, IL-6, and IL-8) expression via inhibiting the AMPK signaling pathway in HBECs. RNA sequencing and luciferase reporter assay identified that miR-29b-3p targeted complement C1q tumor necrosis factor-related protein 6 (C1QTNF6), a protein that protected from PM-induced inflammatory responses via activating the AMPK signaling pathway. In vivo, miR-29b-3p antagomirs delivered via the tail vein prior to PM exposure significantly counteracted PM-induced miR-29b-3p upregulation and C1QTNF6 downregulation in lung tissues. Furthermore, miR-29b-3p inhibition alleviated inflammatory cells infiltration and pro-inflammatory cytokines secretion in the lung of PM-exposed mice. These findings firstly revealed that miR-29b-3p acted as a novel modulator of PM-induced inflammatory responses by targeting the C1QTNF6/AMPK signaling pathway, which contributes to a better understanding of the biological mechanisms underlying adverse PM-induced respiratory health effects.