Protective Effect of the Total Alkaloid Extract from Bulbus Fritillariae pallidiflorae in a Mouse Model of Cigarette Smoke-Induced Chronic Obstructive Pulmonary Disease.

Purpose: In recent years, the incidence of chronic obstructive pulmonary disease (COPD) has been increasing year by year, but therapeutic drugs has no breakthrough. The total alkaloid extract from Bulbus Fritillariae pallidiflorae (BFP-TA) is widely used in treating lung diseases. Therefore, this study aimed to investigate the protective effect and mechanism of BFP-TA in COPD mice. Methods: BFP-TA was prepared by macroporous adsorbent resin, and the material basis of BFP-TA was analyzed by HPLC-ELSD and UHPLC-MS/MS. Then, the COPD mouse model was induced by cigarette smoke (CS) for 12 weeks, administered at weeks 9-12. Subsequently, the body weight, lung-body ratio, pulmonary function, histopathology, and the levels of pro-inflammatory cytokines, matrix metalloproteinases (MMPs) and oxidative stress markers in the serum of mice were determined. The expressions of related protein of EMT and MAPK signaling pathways in the lung tissues of mice were detected by Western blot. Results: The alkaloid relative content of BFP-TA is 64.28%, and nine alkaloids in BFP-TA were identified and quantified by UHPLC-MS/MS. Subsequently, the animal experiment showed that BFP-TA could improve pulmonary function, and alleviate inflammatory cell infiltration, pulmonary emphysema, and collagen fiber deposition in the lung of COPD mice. Furthermore, BFP-TA could decrease the levels of pro-inflammatory cytokines (TNF-α, IL-6 and IL-1ß), MMPs (MMP-9 and MMP-12) and MDA, while increase the levels of TIMP-1 and SOD. Moreover, BFP-TA could decrease the protein expressions of collagen I, vimentin, α-SMA, MMP-9, MMP-9/TIMP-1, Bax, p-JNK/JNK, p-P38/P38, and p-ERK/ERK, while increase the level of E-cadherin. Conclusion: This study is the first to demonstrate the protective effect of BFP-TA in CS-induced COPD mouse model. Furthermore, BFP-TA may improve airway remodeling by inhibiting the EMT process and potentially exert anti-inflammatory effect by inhibiting the MAPK signaling pathway.

Experimental congenital diaphragmatic hernia features an alteration of DNA sensing targets cGAS and STING.

BACKGROUND: The pathogenesis of congenital diaphragmatic hernia (CDH) depends on multiple factors. Activation of the DNA-sensing cyclic-GMP-AMP-synthase (cGAS) and Stimulator-of-Interferon-Genes (STING) pathway by double-stranded DNA (dsDNA) links environmental stimuli and inflammation. We hypothesized that nitrofen exposure alters cGAS and STING in human bronchial epithelial cells and fetal rat lungs. METHODS: We used the Quant-IT™-PicoGreen™ assay to assess dsDNA concentration in BEAS-2B cells after 24 h of nitrofen-exposure and performed immunofluorescence of cGAS/STING. We used nitrofen to induce CDH and harvested control and CDH lungs at embryonic day E15, E18 and E21 for cGAS/STING immunofluorescence, RT-qPCR and RNA-Scope™ in-situ-hybridization (E18, E21). RESULTS: We found a higher concentration of dsDNA following nitrofen treatment. Nitrofen-exposure to BEAS-2B cells increased cGAS and STING protein abundance. cGAS abundance was higher in nitrofen lungs at E15, E18 and E21. RNA-Scope in-situ-hybridization showed higher cGAS and STING expression in E18 and E21 lungs. RT-qPCR revealed higher mRNA expression levels of STING in E21 nitrofen-induced lungs. CONCLUSION: Our data suggest that nitrofen-exposure increases dsDNA content which leads to stimulation of the cGAS/STING pathway in human BEAS-2B cells and the nitrofen rat model of CDH. Consequently, DNA sensing and the cGAS-STING-pathway potentially contribute to abnormal lung development in CDH. IMPACT STATEMENT: We found an alteration of DNA sensing targets cGAS and STING in human BEAS-2B cells and experimental congenital diaphragmatic hernia with higher protein abundance and mRNA expression in cells and lung sections of nitrofen-treated rat pups. This is the first study to investigate DNA sensing, a potential link between environmental stimuli and inflammation, in experimental CDH. Our study extends the knowledge on the pathogenesis of experimental CDH.

Cevanine-type steroidal alkaloids from the bulbs of Fritillaria cirrhosa D. Don.

Eight previously undescribed cevanine-type steroidal alkaloids, cirrhosinones I-N and cirrhosinols A-B, along with five known analogs, were isolated from the bulbs of Fritillaria cirrhosa D. Don. Their structures were elucidated on the basis of comprehensive analysis of HRESIMS, 1D and 2D NMR spectroscopic data, and single-crystal X-ray diffraction analyses. All compounds revealed weak NO inhibitory activities in the LPS-stimulated NR8383 cells at the concentration of 20 µM, with inhibition ratios ranging from 5.1% to 14.3%.

Patient-Reported Outcome Measures in High-Risk Medical Device Registries: A Scoping Review.

Little is known about the methods and outcomes of patient-reported outcome measure (PROM) use among high-risk medical device registries. The objective of this scoping review was to assess the utility and predictive ability of PROMs in high-risk medical device registries. We searched Ovid Medline, Embase, APA PsychINFO, Cochrane Library, and Scopus databases for published literature. After searching, 4323 titles and abstracts were screened, and 262 full texts were assessed for their eligibility. Seventy-six papers from across orthopedic (n = 64), cardiac (n = 10), penile (n = 1), and hernia mesh (n = 1) device registries were identified. Studies predominantly used PROMs as an outcome measure when comparing cohorts or surgical approaches (n = 45) or to compare time points (n = 13) including pre- and postintervention. Fifteen papers considered the predictive ability of PROMs. Of these, 8 treated PROMs as an outcome, 5 treated PROMs as a risk factor through regression analysis, and 2 papers treated PROMs as both a risk factor and as an outcome. One paper described PROMs to study implant survival. To advance methods of PROM integration into clinical decision-making for medical devices, an understanding of their use in high-risk device registries is needed. This scoping review found that there is a paucity of studies using PROMs to predict long-term patient and clinical outcomes in high-risk medical device registries. Determination as to why PROMs are rarely used for predictive purposes in long-term data collection is needed if PROM data are to be considered suitable as real-world evidence for high-risk device regulatory purposes, as well as to support clinical decision-making.

The RNA-binding protein quaking is upregulated in nitrofen-induced congenital diaphragmatic hernia lungs at the end of gestation.

BACKGROUND: The RNA-binding protein Quaking (QKI) increases during epithelial-to-mesenchymal transition and its expression is controlled by microRNA-200 family members. Here, we aimed to describe the expression of QKI in the developing lungs of control and nitrofen-induced congenital diaphragmatic hernia lungs (CDH). METHODS: To investigate the expression of QKI, we dissected lungs from control and nitrofen-induced CDH rats on embryonic day 15, 18, 21 (E15, E18, E21). We performed immunofluorescence (IF) and quantitative reverse transcription PCR (RT-qPCR) for QKI expression. Additionally, we assessed Interleukin-6 (IL-6) abundance using IF. RESULTS: On E21, IF showed that the abundance of all three QKI isoforms and IL-6 protein was higher in CDH lungs compared to control lungs (QKI5: p = 0.023, QKI6: p = 0.006, QKI7: p = 0.014, IL-6: p = 0.045, respectively). Furthermore, RT-qPCR data showed increased expression of QKI5, QKI6, and QKI7 mRNA in E21 nitrofen lungs by 1.63 fold (p = 0.001), 1.63 fold (p = 0.010), and 1.48 fold (p = 0.018), respectively. CONCLUSIONS: Our data show an increase in the abundance and expression of QKI at the end of gestation in nitrofen-induced CDH lungs. Therefore, a disruption in the regulation of QKI during the late stage of pregnancy could be associated with the pathogenesis of abnormal lung development in CDH.

Estimating the cost of visiting hospital outpatient.

Objectives: This study aims to investigate the cost incurred by people travelling to the neurology outpatient clinic of a large metropolitan hospital. As outpatients are a substantial portion of a hospital's demographic, we aimed to understand the patient experience of various commuters. Methods: We conducted an observational study collecting demographic details and travel information for how people attended the neurology clinic of Monash Medical Centre. Statistical analysis was performed using R. 165 participants were randomly selected and interviewed in-person. Data were collected via an anonymous questionnaire. The study was approved by the Monash Health Human Ethics Research Committee. Results: 155 responses were included in the analysis. Patients paid an average of $A16.64 to travel to Monash Medical Centre. Drivers paid on average $A16.70 and those taking public transport paid on average $A9.64, with the maximum cost overall being $A120.00. For patients driving to hospital, parking accounted for 60% of their travel costs. The average to Monash Medical centre was 20.82 km with the maximum being 190.88 km. Distance from hospital was correlated with a higher cost of travel (p<0.001, Spearman's rank correlation coefficient=0.48). There was also an inverse association between distance from hospital and socioeconomic status (p<0.001, Spearman's rank correlation coefficient=-0.26). Conclusion: Travelling to hospital can be a costly endeavour. Driving is the most popular form of transport, but a large portion of the cost involved is hospital parking. Further research should be conducted at other tertiary centres with larger samples.

Ataxia-Telangiectasia Mutated Loss-of-Function Displays Variant and Tissue-Specific Differences across Tumor Types.

PURPOSE: Mutations in the ATM gene are common in multiple cancers, but clinical studies of therapies targeting ATM-aberrant cancers have yielded mixed results. Refinement of ATM loss of function (LOF) as a predictive biomarker of response is urgently needed. EXPERIMENTAL DESIGN: We present the first disclosure and preclinical development of a novel, selective ATR inhibitor, ART0380, and test its antitumor activity in multiple preclinical cancer models. To refine ATM LOF as a predictive biomarker, we performed a comprehensive pan-cancer analysis of ATM variants in patient tumors and then assessed the ATM variant-to-protein relationship. Finally, we assessed a novel ATM LOF biomarker approach in retrospective clinical data sets of patients treated with platinum-based chemotherapy or ATR inhibition. RESULTS: ART0380 had potent, selective antitumor activity in a range of preclinical cancer models with differing degrees of ATM LOF. Pan-cancer analysis identified 10,609 ATM variants in 8,587 patient tumors. Cancer lineage-specific differences were seen in the prevalence of deleterious (Tier 1) versus unknown/benign (Tier 2) variants, selective pressure for loss of heterozygosity, and concordance between a deleterious variant and ATM loss of protein (LOP). A novel ATM LOF biomarker approach that accounts for variant classification, relationship to ATM LOP, and tissue-specific penetrance significantly enriched for patients who benefited from platinum-based chemotherapy or ATR inhibition. CONCLUSIONS: These data help to better define ATM LOF across tumor types in order to optimize patient selection and improve molecularly targeted therapeutic approaches for patients with ATM LOF cancers.

Microinjection With Nanoparticles to Deliver Drugs in Prenatal Lung Explants - A Pilot Study for Prenatal Therapy in Congenital Diaphragmatic Hernia.

BACKGROUND: Fetoscopic endoluminal tracheal occlusion (FETO) improves the survival rate in fetuses with severe congenital diaphragmatic hernia (CDH). We hypothesize that prenatal therapies into the trachea during FETO can further improve outcomes. Here, we present an ex vivo microinjection technique with rat lung explants to study prenatal therapy with nanoparticles. METHODS: We used microsurgery to isolate lungs from rats on embryonic day 18. We injected chitosan nanoparticles loaded with fluorescein (FITC) into the trachea of the lung explants. We compared the difference in biodistribution of two types of nanoparticles, functionalized IgG-conjugated nanoparticles (IgG-nanoparticles) and bare nanoparticles after 24 h culture with immunofluorescence (IF). We used IF to mark lung epithelial cells with E-cadherin and to investigate an apoptosis (Active-caspase 3) and inflammatory marker (Interleukin, IL-6) and compared its abundance between the two experimental groups and control lung explants. RESULTS: We detected the presence of nanoparticles in the lung explants, and the relative number of nanoparticles to cells was 2.49 fold higher in IgG-nanoparticles than bare nanoparticles (p < 0.001). Active caspase-3 protein abundance was similar in the control, bare nanoparticles (1.20 fold higher), and IgG-nanoparticles (1.34 fold higher) groups (p = 0.34). Similarly, IL-6 protein abundance was not different in the control, bare nanoparticles (1.13 fold higher), and IgG-nanoparticles (1.12 fold higher) groups (p = 0.33). CONCLUSIONS: Functionalized nanoparticles had a higher presence in lung cells and this did not result in more apoptosis or inflammation. Our proof-of-principle study will guide future research with therapies to improve lung development prenatally. LEVELS OF EVIDENCE: N/A TYPE OF STUDY: Animal and laboratory study.

How environmental impact is considered in economic evaluations of critical care: a scoping review.

PURPOSE: Health care is a major contributor to climate change, and critical care is one of the sector's highest carbon emitters. Health economic evaluations form an important component of critical care and may be useful in identifying economically efficient and environmentally sustainable strategies. The purpose of this scoping review was to synthesise available literature on whether and how environmental impact is considered in health economic evaluations of critical care. METHODS: A robust scoping review methodology was used to identify studies reporting on environmental impact in health economic evaluations of critical care. We searched six academic databases to locate health economic evaluations, costing studies and life cycle assessments of critical care from 1993 to present. RESULTS: Four studies met the review's inclusion criteria. Of the 278 health economic evaluations of critical care identified, none incorporated environmental impact into their assessments. Most included studies (n = 3/4) were life cycle assessments, and the remaining study was a prospective observational study. Life cycle assessments used a combination of process-based data collection and modelling to incorporate environmental impact into their economic assessments. CONCLUSIONS: Health economic evaluations of critical care have not yet incorporated environmental impact into their assessments, and few life cycle assessments exist that are specific to critical care therapies and treatments. Guidelines and standardisation regarding environmental data collection and reporting in health care are needed to support further research in the field. In the meantime, those planning health economic evaluations should include a process-based life cycle assessment to establish key environmental impacts specific to critical care.

An external validation of the Kidney Donor Risk Index in the UK transplant population in the presence of semi-competing events.

BACKGROUND: Transplantation represents the optimal treatment for many patients with end-stage kidney disease. When a donor kidney is available to a waitlisted patient, clinicians responsible for the care of the potential recipient must make the decision to accept or decline the offer based upon complex and variable information about the donor, the recipient and the transplant process. A clinical prediction model may be able to support clinicians in their decision-making. The Kidney Donor Risk Index (KDRI) was developed in the United States to predict graft failure following kidney transplantation. The survival process following transplantation consists of semi-competing events where death precludes graft failure, but not vice-versa. METHODS: We externally validated the KDRI in the UK kidney transplant population and assessed whether validation under a semi-competing risks framework impacted predictive performance. Additionally, we explored whether the KDRI requires updating. We included 20,035 adult recipients of first, deceased donor, single, kidney-only transplants between January 1, 2004, and December 31, 2018, collected by the UK Transplant Registry and held by NHS Blood and Transplant. The outcomes of interest were 1- and 5-year graft failure following transplantation. In light of the semi-competing events, recipient death was handled in two ways: censoring patients at the time of death and modelling death as a competing event. Cox proportional hazard models were used to validate the KDRI when censoring graft failure by death, and cause-specific Cox models were used to account for death as a competing event. RESULTS: The KDRI underestimated event probabilities for those at higher risk of graft failure. For 5-year graft failure, discrimination was poorer in the semi-competing risks model (0.625, 95% CI 0.611 to 0.640;0.611, 95% CI 0.597 to 0.625), but predictions were more accurate (Brier score 0.117, 95% CI 0.112 to 0.121; 0.114, 95% CI 0.109 to 0.118). Calibration plots were similar regardless of whether the death was modelled as a competing event or not. Updating the KDRI worsened calibration, but marginally improved discrimination. CONCLUSIONS: Predictive performance for 1-year graft failure was similar between death-censored and competing event graft failure, but differences appeared when predicting 5-year graft failure. The updated index did not have superior performance and we conclude that updating the KDRI in the present form is not required.

Management of adult-onset Still's disease: evidence- and consensus-based recommendations by experts.

BACKGROUND: Adult-onset Still's disease (AOSD) is a rare condition characterized by fevers, rash, and arthralgia/arthritis. Most doctors treating AOSD in the Netherlands treat <5 patients per year. Currently, there is no internationally accepted treatment guideline for AOSD. OBJECTIVES: To conduct a Delphi panel aimed at reaching consensus about diagnostic and treatment strategies for patients with AOSD and to use the outcomes as a basis for a treatment algorithm. METHODS: The Delphi panel brought together 18 AOSD experts: rheumatologists, internists and paediatricians. The Delphi process consisted of 3 rounds. In the first two rounds, online list of questions and statements were completed. In the third round, final statements were discussed during a virtual meeting and a final vote took place. Consensus threshold was set at 80%. Two targeted literature searches were performed identifying the level of evidence of the consensus-based statements. RESULTS: Consensus was reached on 29 statements, including statements related to diagnosis and diagnostic tests, definition of response and remission, the therapy, the use of methotrexate, and tapering of treatment. The panel consented on reduction of the use of glucocorticoids to avoid side-effect, and preferred the use of biologics over conventional treatment. The role of interleukin-1 and interleukin-6 blocking agents was considered important in the treatment of AOSD. CONCLUSIONS: In this Delphi panel, a high level of consensus was achieved on recommendations for diagnosis and therapy of AOSD that can serve as a basis for a treatment guideline.

Overactivated Epithelial NF-κB Disrupts Lung Development in Congenital Diaphragmatic Hernia.

Abnormal lung development is the main cause of morbidity and mortality in neonates with congenital diaphragmatic hernia (CDH), a common birth defect (1:2,500) of largely unknown pathobiology. Recent studies discovered that inflammatory processes, and specifically NF-κB-associated pathways, are enriched in human and experimental CDH. However, the molecular signaling of NF-κB in abnormal CDH lung development and its potential as a therapeutic target require further investigation. Using sections and hypoplastic lung explant cultures from the nitrofen rat model of CDH and human fetal CDH lungs, we demonstrate that NF-κB and its downstream transcriptional targets are hyperactive during abnormal lung formation in CDH. NF-κB activity was especially elevated in the airway epithelium of nitrofen and human CDH lungs at different developmental stages. Fetal rat lung explants had impaired pseudoglandular airway branching after exposure to nitrofen, together with increased phosphorylation and transcriptional activity of NF-κB. Dexamethasone, the broad and clinically applicable antiinflammatory NF-κB antagonist, rescued lung branching and normalized NF-κB signaling in hypoplastic lung explants. Moreover, specific NF-κB inhibition with curcumenol similarly rescued ex vivo lung hypoplasia and restored NF-κB signaling. Last, we showed that prenatal intraperitoneal dexamethasone administration to pregnant rat dams carrying fetuses with hypoplastic lungs significantly improves lung branching and normalizes NF-κB in vivo. Our results indicate that NF-κB is aberrantly activated in human and nitrofen CDH lungs. Antiinflammatory treatment with dexamethasone and/or specific NF-κB inhibition should be investigated further as a therapeutic avenue to target lung hypoplasia in CDH.

I know what you did last summer: a cross-sectional study of personal COVID-19 risk reduction strategies used by Victorian adults, December 2021-January 2022.

OBJECTIVE: We describe COVID-19 risk reduction strategies adopted by Victorian adults during December 2021-January 2022, a period of high COVID-19 infection and limited government mandated public health measures. METHODS: In February 2022, participants of a Victorian-based cohort study (Optimise) completed a cross-sectional survey on risk reduction behaviours during December 2021-January 2022. Regression modelling estimated the association between risk reduction and demographics. RESULTS: A total of 556 participants were included (median age 47 years; 75% women; 82% in metropolitan Melbourne). Two-thirds (61%) adopted at least one risk reduction behaviour, with uptake highest among younger participants (18-34 years; adjusted relative risk (aRR): 1.20, 95% confidence interval [CI]: 1.01, 1.41) and those with a chronic health condition (aRR: 1.17, 95% CI: 1.02, 1.35). CONCLUSIONS: Participants adopted their own COVID-19 risk reduction strategies in a setting of limited government restrictions, with young people more likely to adopt a risk reduction strategy that did not limit social mobility. IMPLICATION FOR PUBLIC HEALTH: A public health response to COVID-19 that focusses on promoting personal risk reduction behaviours, as opposed to mandated restrictions, could be enhanced by disseminating information on and increasing availability of effective risk reduction strategies tailored to segments of the population.

Engaging with communities to encourage adoption of a harm reduction approach to COVID-19.

Since the emergence of the Omicron variant in Australia in late 2021 there have been over 5.47 million cases and 4993 deaths, disproportionately impacting on people with social and structural disadvantage. However there has been increasing reluctance by governments to intervene to reduce the impact of COVID-19 transmission and its consequences. This commentary article provides a perspective on the support and guidance required to mitigate individuals and communities risk by using a harm reduction approach to highlight strategies that can reduce COVID-19 transmission and infection.

Microtubule-associated proteins MAP7 and MAP7D1 promote DNA double-strand break repair in the G1 cell cycle phase.

The DNA-damage response is a complex signaling network that guards genomic integrity. The microtubule cytoskeleton is involved in the repair of DNA double-strand breaks; however, little is known about which cytoskeleton-related proteins are involved in DNA repair and how. Using quantitative proteomics, we discovered that microtubule associated proteins MAP7 and MAP7D1 interact with several DNA repair proteins including DNA double-strand break repair proteins RAD50, BRCA1 and 53BP1. We observed that downregulation of MAP7 and MAP7D1 leads to increased phosphorylation of p53 after γ-irradiation. Moreover, we determined that the downregulation of MAP7D1 leads to a strong G1 arrest and that the downregulation of MAP7 and MAP7D1 in G1 arrested cells negatively affects DNA repair, recruitment of RAD50 to chromatin and localization of 53BP1 to the sites of damage. These findings describe for the first time a novel function of MAP7 and MAP7D1 in cell cycle regulation and repair of DNA double-strand breaks.

Research on 90-day subchronic toxicities of the ethanol extract from the cultivated Fritillaria Cirrhosa bulbs by oral administration in Sprague-Dawley rats.

Fritillaria Cirrhosa bulbus (BFC) is a Chinese herbal medicine. In the present study, subchronic toxicities of the ethanol extract from cultivated Fritillaria Cirrhosa bulbus (ECBFC) were performed by oral daily administration in Sprague-Dawley rats. The subchronic toxicity test of ECBFC was conducted at doses of 0.34, 0.68, and 2.04 g/kg/day for 90 days (equivalent to the highest human clinical recommend dosage of 25, 50, and 150-fold) with a 4-week satellite group. No mortality or significant changes in behaviors, body weight and food consumption were observed during the experimental and recovery periods. According to the data from ematological analysis, biochemistry, organ coefficient and the results of histopathology, the ECBFC have toxicity to the spleen and liver at the highest (2.04 g/kg), medium (0.68 g/kg) dose and nephrotoxicity at the highest dose. Subchronic oral toxicity of ECBFC in SD rats (90 days) with NOAEL was 0.34 g/kg and LOAEL was 0.68 g/kg. In addition, the toxicity is gender neutral and reversible. The NOAEL value (0.34 g/kg) is 25-fold of the highest human clinical recommend dosage thus the ECBFC could be long-term used as Chinese patent medicine or functional food.

A distinction between Fritillaria Cirrhosa Bulbus and Fritillaria Pallidiflora Bulbus via LC-MS/MS in conjunction with principal component analysis and hierarchical cluster analysis.

Fritillaria Cirrhosa Bulbus (known as chuanbeimu in Chinese, FCB) is one of the most used Chinese medicines for lung disease. However, a variety of substitutes have entered the market, with Fritillaria Pallidiflora Bulbus (FPB) being the most common. Due to their similarity in appearance, morphology, and chemical composition but a large price difference, the FCB has frequently been adulterated with the FPB, posing a serious challenge to the distinction and quality of the FCB. Therefore, we aimed to distinguish FCB and FPB based on their main nine isosteroidal alkaloid contents and test the potential of chemometrics as a discrimination approach for evaluating quality. The nine major isosteroidal alkaloids were measured using a liquid chromatography with tandem mass spectrometry (LC-MS/MS) approach in 41 batches of FCB and 17 batches of FPB. Additionally, they were categorized and distinguished using the methods of hierarchical cluster analysis (HCA) and principal component analysis (PCA). Quantitative analysis revealed that the nine alkaloids were present in different amounts in the two types of Fritillariae bulbus. In FCB, the highest amount was peimisine (17.92-123.53 µg/g) and the lowest was delavine (0.42-29.18 µg/g), while in FPB, imperialine was higher (78.05-344.09 µg/g), but verticinone and verticine were less than the other seven alkaloids. The FCB and FPB were successfully classified and distinguished by the HCA and PCA. Taken together, the method has a good linear relationship (R2 > 0.9975). The LOD and LOQ of the nine alkaloids were in the range of 0.0651-0.6510 and 0.1953-1.9531 ng/mL, respectively. The intra- and inter-day precision were shown to be excellent, with relative standard deviations (RSDs) below 1.63% and 2.39%, respectively. The LC-MS/MS method in conjunction with HCA and PCA can effectively differentiate FCB and FPB. It may be a promising strategy for quality evaluation and control at the FCB.

A Tracheal Aspirate-derived Airway Basal Cell Model Reveals a Proinflammatory Epithelial Defect in Congenital Diaphragmatic Hernia.

Rationale: Congenital diaphragmatic hernia (CDH) is characterized by incomplete closure of the diaphragm and lung hypoplasia. The pathophysiology of lung defects in CDH is poorly understood. Objectives: To establish a translational model of human airway epithelium in CDH for pathogenic investigation and therapeutic testing. Methods: We developed a robust methodology of epithelial progenitor derivation from tracheal aspirates of newborns. Basal stem cells (BSCs) from patients with CDH and preterm and term non-CDH control subjects were derived and analyzed by bulk RNA sequencing, assay for transposase accessible chromatin with sequencing, and air-liquid interface differentiation. Lung sections from fetal human CDH samples and the nitrofen rat model of CDH were subjected to histological assessment of epithelial defects. Therapeutics to restore epithelial differentiation were evaluated in human epithelial cell culture and the nitrofen rat model of CDH. Measurements and Main Results: Transcriptomic and epigenetic profiling of CDH and control BSCs reveals a proinflammatory signature that is manifested by hyperactive nuclear factor kappa B and independent of severity and hernia size. In addition, CDH BSCs exhibit defective epithelial differentiation in vitro that recapitulates epithelial phenotypes found in fetal human CDH lung samples and fetal tracheas of the nitrofen rat model of CDH. Furthermore, blockade of nuclear factor kappa B hyperactivity normalizes epithelial differentiation phenotypes of human CDH BSCs in vitro and in nitrofen rat tracheas in vivo. Conclusions: Our findings have identified an underlying proinflammatory signature and BSC differentiation defects as a potential therapeutic target for airway epithelial defects in CDH.

Proteomic Profiling of Hypoplastic Lungs Suggests an Underlying Inflammatory Response in the Pathogenesis of Abnormal Lung Development in Congenital Diaphragmatic Hernia.

The pathogenesis of lung hypoplasia in congenital diaphragmatic hernia (CDH), a common birth defect, is poorly understood. The diaphragmatic defect can be repaired surgically, but the abnormal lung development contributes to a high mortality in these patients. To understand the underlying pathobiology, we compared the proteomic profiles of fetal rat lungs at the alveolar stage (E21) that were either exposed to nitrofen in utero (CDH lungs, n=5) or exposed to vehicle only (non-CDH control lungs, n=5). Pathway analysis of proteomic datasets showed significant enrichment in inflammatory response proteins associated with cytokine signaling and Epstein Barr Virus in nitrofen CDH lungs. Among the 218 significantly altered proteins between CDH and non-CDH control lungs were Tenascin C, CREBBP, LYN, and STAT3. We showed that Tenascin C was decreased around the distal airway branches in nitrofen rat lungs and human CDH lungs, obtained from stillborn fetuses that did not receive pre- or postnatal treatment. In contrast, STAT3 was significantly increased in the airway epithelium of nitrofen lungs at E21. STAT3 inhibition after direct nitrofen exposure to fetal rat lung explants (E14.5) partially rescued the hypoplastic lung phenotype ex vivo by increasing peripheral lung budding. Moreover, we demonstrated that several STAT3-associated cytokines (IL-15, IL-9, andIL-2) are increased in fetal tracheal aspirates of CDH survivors compared with nonsurvivors after fetoscopic endoluminal tracheal occlusion. With our unbiased proteomics approach, we showed for the first time that downstream inflammatory processes are likely involved in the pathogenesis of abnormal lung development in CDH.

Total alkaloids of bulbus of Fritillaria cirrhosa alleviate bleomycin-induced inflammation and pulmonary fibrosis in rats by inhibiting TGF-ß and NF-κB signaling pathway.

Background: Bulbus of Fritillaria cirrhosa is a medicinal and edible plant that has the functions of clearing away heat and moisturizing the lungs, resolving phlegm, and relieving coughs. Its ethanol extract has been proven to have a therapeutic effect on lung diseases. Pulmonary fibrosis is a respiratory disease that forms scars in lung tissue, leading to severe respiratory problems. However, the therapeutic effect of total alkaloids of bulbus of Fritillaria cirrhosa (BFC-TA) on pulmonary fibrosis has not been confirmed. Objective: This study aimed to investigate the therapeutic effect of total alkaloids of Fritillaria cirrhosa on pulmonary fibrosis rat model and explore its potential mechanism. Design: The total alkaloids in the bulbus of Fritillaria cirrhosa were purified using cation exchange resin. The alkaloids contained in the BFC-TA were identified, and the concentration of alkaloids was determined by High Performance Liquid Chromatography-Diode Array Detector-Evaporative Light Scattering Detector (HPLC-DAD-ELSD). Bleomycin (BLM) (5.0 mg/kg) was instilled into the trachea of 60 rats to establish a pulmonary fibrosis model. After 7 days, BFC-TA (34.2, 68.4, and 136.8 mg/kg) was administered continuously for 21 days. During this period, the body weight changes of the rats were measured, the levels of hydroxyproline (HYP) and inflammatory factors were measured in the collected serum, and the histological analysis of the lung tissue was performed by staining technology. Western blotting and quantitative Polymerase Chain Reaction (qPCR) were used to assess the protein and gene composition of inflammation and transforming growth factor-ß (TGF-ß) signaling pathways. Results: Nine main components (Peimisine, Imperialine-3-ß-D-glucoside, Yibeinoside A, Imperialine, Peiminine, Isopeimine, Hupehenine, Delavinone, Ebeiedinone) were determined by HPLC-DAD-ELSD, and the contents of Peimisine, Imperialine-3-ß-D-glucoside and Imperialine were determined. BFC-TA (34.2, 68.4, and 136.8 mg/kg) reduced the levels of pro-inflammatory factors, increased the levels of anti-inflammatory factors, dose-dependently improved the morphology of lung tissue. And during epithelial-mesenchymal transition process, BFC-TA dose-dependently reduced the expression of E-cadherin, dose-dependently increased the expression of Fibronectin. In addition, Western blot analysis and qPCR results showed that inhibiting NF-κB and TGF-ß-related signaling pathways effectively slowed down the occurrence of BLM-induced pulmonary fibrosis in rats. And the therapeutic effect of BFC-TA (136.8 mg/kg) is better than that of pirfenidon (PFD) (150 mg/kg). Conclusion: BFC-TA effectively alleviates the progression of the BLM-induced pulmonary fibrosis rat model by regulating the inflammatory response in the lungs and the expression of the TGF-ß signaling pathway.