Dry powder inhalation containing muco-inert ciprofloxacin and colistin co-loaded liposomes for pulmonary P. Aeruginosa biofilm eradication.

Pseudomonas aeruginosa (PA), a predominant pathogen in lung infections, poses significant challenges due to its biofilm formation, which is the primary cause of chronic and recalcitrant pulmonary infections. Bacteria within these biofilms exhibit heightened resistance to antibiotics compared to their planktonic counterparts, and their secreted toxins exacerbate lung infections. Diverging from traditional antibacterial therapy for biofilm eradication, this study introduces a novel dry powder inhalation containing muco-inert ciprofloxacin and colistin co-encapsulated liposomes (Cipro-Col-Lips) prepared using ultrasonic spray freeze drying (USFD) technique. This USFD dry powder is designed to efficiently deliver muco-inert Cipro-Col-Lips to the lungs. Once deposited, the liposomes rapidly diffuse into the airway mucus, reaching the biofilm sites. The muco-inert Cipro-Col-Lips neutralize the biofilm-secreted toxins and simultaneously trigger the release of their therapeutic payload, exerting a synergistic antibiofilm effect. Our results demonstrated that the optimal USFD liposomal dry powder formulation exhibited satisfactory in vitro aerosol performance in terms of fine particle fraction (FPF) of 44.44 ± 0.78 %, mass median aerodynamic diameter (MMAD) of 4.27 ± 0.21 µm, and emitted dose (ED) of 99.31 ± 3.31 %. The muco-inert Cipro-Col-Lips effectively penetrate the airway mucus and accumulate at the biofilm site, neutralizing toxins and safeguarding lung cells. The triggered release of ciprofloxacin and colistin works synergistically to reduce the biofilm's antibiotic resistance, impede the development of antibiotic resistance, and eliminate 99.99 % of biofilm-embedded bacteria, including persister bacteria. Using a PA-beads induced biofilm-associated lung infection mouse model, the in vivo efficacy of this liposomal dry powder aerosol was tested, and the results demonstrated that this liposomal dry powder aerosol achieved a 99.7 % reduction in bacterial colonization, and significantly mitigated inflammation and pulmonary fibrosis. The USFD dry powder inhalation containing muco-inert Cipro-Col-Lips emerges as a promising therapeutic strategy for treating PA biofilm-associated lung infections.

Bufei Huoxue capsule attenuates COPD-related inflammation and regulates intestinal microflora, metabolites.

Background: Bufei Huoxue capsule (BFHX) is widely used for the clinical treatment of chronic obstructive pulmonary disease (COPD) in China. Objectives: The aim of this study is to explore the effects on COPD and the underlying mechanism of BFHX. The process and methods: In this study, we established a COPD mouse model through cigarette smoke (CS) exposure in combination with lipopolysaccharide (LPS) intratracheal instillation. Subsequently, BFHX was orally administrated to COPD mice, and their pulmonary function, lung pathology, and lung inflammation, including bronchoalveolar lavage fluid (BALF) cell count and classification and cytokines, were analyzed. In addition, the anti-oxidative stress ability of BFHX was detected by Western blotting, and the bacterial diversity, abundance, and fecal microbiome were examined using 16S rRNA sequencing technology. Outcome: BFHX was shown to improve pulmonary function, suppress lung inflammation, decrease emphysema, and increase anti-oxidative stress, whereas 16S rRNA sequencing indicated that BFHX can dynamically regulate the diversity, composition, and distribution of the intestinal flora microbiome and regulate the lysine degradation and phenylalanine metabolism of COPD mice. These results highlight another treatment option for COPD and provide insights into the mechanism of BFHX.

Quanzhen Yiqi decoction attenuates inflammation in mice with smoking-induced COPD by activating the Nrf2/HO-1 pathway and inhibiting the NLRP3 inflammasome.

OBJECTIVE: Quanzhen Yiqi decoction (QZYQ) is a traditional Chinese medicine for treating chronic obstructive pulmonary disease. METHODS: Mice were exposed to cigarette smoke (CS) 6 days/week (40 cigarettes/day) for 24 weeks and then intragastrically administered QZYQ (4.72, 9.45, or 18.89 g/kg) or dexamethasone (DEX, 0.6 mg/kg) for 6 weeks. We examined the lung function and collected bronchoalveolar lavage fluid for inflammatory cell and cytokine quantification. The pathological lung changes, ROS and oxidative biomarkers were measured. We used immunohistochemistry and western blotting to evaluate the levels of Nrf2/HO-1, NLRP3/ASC/Caspase1/IL-1ß/IL-18. RESULTS: The CS group showed significant increases in the forced vital capacity, lung resistance, and chord compliance and a lower FEV50/FVC compared with the control, and QZYQ improved these changes. In addition, QZYQ effectively reduced emphysema, immune cell infiltration, and airway remodeling. QZYQ stimulated HO-1 expression and reduced oxidative stress through the Nrf2 pathway. QZYQ inhibited the production of NLRP3/ASC/Caspase-1 to inhibit IL-1ß and IL-18. CONCLUSION: Our study suggested that QZYQ can improve the function and histology of the lungs and reduce inflammatory cell recruitment. QZYQ inhibits ROS production and NLRP3 inflammasome activation by upregulating Nrf2 to reduce lung injury. The anti-inflammatory effects of QZYQ are similar to those of DEX.

Therapeutic effect of phycocyanin on chronic obstructive pulmonary disease in mice.

INTRODUCTION: The prevention and treatment of chronic obstructive pulmonary disease (COPD) is closely tied to antioxidation and anti-inflammation. Phycocyanin (PC) has numerous pharmacological effects, such as antioxidation and anti-inflammation. However, it remains unclear whether PC can play a therapeutic role in COPD. OBJECTIVE: As inflammation and oxidative stress can aggravate COPD, this study is to explore the effect of PC on COPD mice and its mechanisms. METHODS: The COPD mice model was established by exposing them to lipopolysaccharide (LPS) and cigarette smoke (CS); PC was administrated in a concentration of 50 mg/kg for 30 days. On the last day, lung function was measured, and bronchoalveolar lavage fluid (BALF) was obtained and classified for cells. Lung tissue pathological change was analyzed, and organ indices statistics were measured. Based on molecular docking, the mechanism was explored with Western blotting, immunohistochemical, and immunofluorescence in vivo and in vitro. RESULTS: PC significantly ameliorated the pulmonary function of COPD mice and reduced inflammation of the lung (p < 0.05), and hematoxylin and eosin (H&E) staining showed PC depressed lung inflammatory cell accumulation and emphysema. Periodic acid Schiff (PAS) and Masson staining revealed that PC retarded goblet cells metaplasia and collagen deposition (p < 0.05). In addition, in vivo PC regulated Heme oxygenase 1 (HO-1) (p < 0.05) and NAD(P)H dehydrogenase quinone 1 (NQO1) level (p < 0.01) in the lung, as well as NOX2 level in pulmonary macrophages. Molecular docking results indicate that phycocyanobilin (PCB) in PC had a good binding site in Keap1 and NOX2 proteins; the phycocyanobilin-bound phycocyanin peptide (PCB-PC-peptide) was obtained for further studies. In vitro, PCB-PC-peptide could depress the phospho-NF-E2-related factor 2 (p-Nrf2) and NQO1 protein expression in RAW264.7 cells induced by cigarette smoke extract (CSE) (p < 0.05). CONCLUSION: PC exerts beneficial effects on COPD via anti-inflammatory and antioxidative stress, which may be achieved through PCB.

[18 F] -FAPI-42 PET/CT assessment of Progressive right ventricle fibrosis under pressure overload.

BACKGROUND: Right heart failure (RHF) is a complication of pulmonary hypertension (PH) and increases the mortality independently of the underlying disease. However, the process of RHF development and progression is not fully understood. We aimed to develop effective approaches for early diagnosis and precise evaluation of RHF. METHODS: Right ventricle (RV) pressure overload was performed via pulmonary artery banding (PAB) surgery in Sprague-Dawley (SD) rats to induce RHF. Echocardiography, right heart catheterization, histological staining, fibroblast activation protein (FAP) immunofluorescence and 18 F-labelled FAP inhibitor-42 ([18 F] -FAPI-42) positron emission tomography/computed tomography (PET/CT) were performed at day 3, week 1, 2, 4 and 8 after PAB. RNA sequencing was performed to explore molecular alterations between PAB and sham group at week 2 and week 4 after PAB respectively. RESULTS: RV hemodynamic disorders were aggravated, and RV function was declined based on right heart catheterization and echocardiography at week 2, 4 and 8 after PAB. Progressive cardiac hypertrophy, fibrosis and capillary rarefaction could be observed in RV from 2 to 8 weeks after PAB. RNA sequencing indicated 80 upregulated genes and 43 downregulated genes in the RV at both week 2 and week 4 after PAB; Gene Ontology (GO) analysis revealed that fibrosis as the most significant biological process in the RV under pressure overload. Immunofluorescence indicated that FAP was upregulated in the RV from week 2 to week 8 after PAB; and [18 F] -FAPI-42 PET/CT revealed FAPI uptake was significantly higher in RV at week 2 and further increased at week 4 and 8 after PAB. CONCLUSION: RV function is progressively declined with fibrosis as the most prominent molecular change after pressure overload, and [18 F] -FAPI-42 PET/CT is as sensitive and accurate as histopathology in RV fibrosis evaluation.

Echocardiographic evaluation of right heart failure which might be associated with DNA damage response in SU5416-hypoxia induced pulmonary hypertension rat model.

Right heart failure is the leading cause of death in pulmonary hypertension (PH), and echocardiography is a commonly used tool for evaluating the risk hierarchy of PH. However, few studies have explored the dynamic changes in the structural and functional changes of the right heart during the process of PH. Previous studies have found that pulmonary circulation coupling right ventricular adaptation depends on the degree of pressure overload and other factors. In this study, we performed a time-dependent evaluation of right heart functional changes using transthoracic echocardiography in a SU5416 plus hypoxia (SuHx)-induced PH rat model. Rats were examined in 1-, 2-, 4-, and 6-week using right-heart catheterization, cardiac echocardiography, and harvested heart tissue. Our study found that echocardiographic measures of the right ventricle (RV) gradually worsened with the increase of right ventricular systolic pressure, and right heart hypofunction occurred at an earlier stage than pulmonary artery thickening during the development of PH. Furthermore, sarco-endoplasmic reticulum calcium ATPase 2 (SERCA2), a marker of myocardial damage, was highly expressed in week 2 of SuHx-induced PH and had higher levels of expression of γ-H2AX at all timepoints, as well as higher levels of DDR-related proteins p-ATM and p53/p-p53 and p21 in week 4 and week 6. Our study demonstrates that the structure and function of the RV begin to deteriorate with DNA damage and cellular senescence during the early stages of PH development.

SARS-CoV-2 spike protein receptor-binding domain perturbates intracellular calcium homeostasis and impairs pulmonary vascular endothelial cells.

Exposure to the spike protein or receptor-binding domain (S-RBD) of SARS-CoV-2 significantly influences endothelial cells and induces pulmonary vascular endotheliopathy. In this study, angiotensin-converting enzyme 2 humanized inbred (hACE2 Tg) mice and cultured pulmonary vascular endothelial cells were used to investigate how spike protein/S-RBD impacts pulmonary vascular endothelium. Results show that S-RBD leads to acute-to-prolonged induction of the intracellular free calcium concentration ([Ca2+]i) via acute activation of TRPV4, and prolonged upregulation of mechanosensitive channel Piezo1 and store-operated calcium channel (SOCC) key component Orai1 in cultured human pulmonary arterial endothelial cells (PAECs). In mechanism, S-RBD interacts with ACE2 to induce formation of clusters involving Orai1, Piezo1 and TRPC1, facilitate the channel activation of Piezo1 and SOCC, and lead to elevated apoptosis. These effects are blocked by Kobophenol A, which inhibits the binding between S-RBD and ACE2, or intracellular calcium chelator, BAPTA-AM. Blockade of Piezo1 and SOCC by GsMTx4 effectively protects the S-RBD-induced pulmonary microvascular endothelial damage in hACE2 Tg mice via normalizing the elevated [Ca2+]i. Comparing to prototypic strain, Omicron variants (BA.5.2 and XBB) of S-RBD induces significantly less severe cell apoptosis. Transcriptomic analysis indicates that prototypic S-RBD confers more severe acute impacts than Delta or Lambda S-RBD. In summary, this study provides compelling evidence that S-RBD could induce persistent pulmonary vascular endothelial damage by binding to ACE2 and triggering [Ca2+]i through upregulation of Piezo1 and Orai1. Targeted inhibition of ACE2-Piezo1/SOCC-[Ca2+]i axis proves a powerful strategy to treat S-RBD-induced pulmonary vascular diseases.

Bufei huoxue capsule alleviates bleomycin-induced pulmonary fibrosis in mice via TGF-ß1/Smad2/3 signaling.

ETHNOPHARMACOLOGICAL RELEVANCE: Bufei huoxue (BFHX) is a Traditional Chinese Medicine formulation that consists of Astragalus Exscapus L, Paeonia Lactiflora Pall, and Psoralea Aphylla L. It can ameliorate collagen deposition and inhibit EMT. However, it remains unknown whether and how BFHX alleviates IPF. AIM OF THE STUDY: Our work aimed to explore the therapeutic efficacy of BFHX on IPF and dissect the mechanisms involved. MATERIALS AND METHODS: A mouse model of IPF was induced by bleomycin. BFHX was administered on the first day of modeling and maintained for 21 days. Pulmonary fibrosis and inflammation were evaluated by micro-CT, lung histopathology, pulmonary function assessment, and cytokines in BALF. In addition, we examined the signaling molecules involved in EMT and ECM by immunofluorescence, western Blot, EdU, and MMP (Δψm) assays. RESULTS: BFHX alleviated lung parenchyma fibrosis as evidenced by Hematoxylin-eosin (H&E), Masson's trichrome staining, and micro-CT, and it improved lung function. In addition, BFHX treatment not only decreased the levels of interleukin (IL)-6 and tumor necrosis factor-α (TNF-α), but also upregulated E-cadherin (E-Cad) and downregulated α-smooth muscle actin (α-SMA), collagen Ӏ (Col Ӏ), vimentin, and fibronectin (FN). Mechanistically, BFHX repressed TGF-ß1-driven Smad2/3 phosphorylation, which, in turn, suppressed EMT and transition of fibroblasts to myofibroblasts in vivo and in vitro. CONCLUSION: BFHX effectively reduces the occurrence of EMT and inhibits the production of ECM by inhibiting the TGF-ß1/Smad2/3 signaling pathway, which provides a potential novel therapeutic strategy for IPF.

Fracture Characteristics of Commercial PEEK Dental Crowns: Combining the Effects of Aging Time and TiO2 Content.

Polyetheretherketone (PEEK) is an emerging thermoplastic polymer with good mechanical properties and an elastic modulus similar to that of alveolar bone. PEEK dental prostheses for computer-aided design/computer-aided manufacturing (CAD/CAM) systems on the market often have additives of titanium dioxide (TiO2) to strengthen their mechanical properties. However, the effects of combining aging, simulating a long-term intraoral environment, and TiO2 content on the fracture characteristics of PEEK dental prostheses have rarely been investigated. In this study, two types of commercially available PEEK blocks, containing 20% and 30% TiO2, were used to fabricate dental crowns by CAD/CAM systems and were aged for 5 and 10 h based on the ISO 13356 specifications. The compressive fracture load values of PEEK dental crowns were measured using a universal test machine. The morphology and crystallinity of the fracture surface were analyzed by scanning electron microscopy and an X-ray diffractometer, respectively. Statistical analysis was performed using the paired t-test (α = 0.05). Results showed no significant difference in the fracture load value of the test PEEK crowns with 20% and 30% TiO2 after 5 or 10 h of aging treatment; all test PEEK crowns have satisfactory fracture properties for clinical applications. Fracture surface analysis revealed that all test crowns fractured from the lingual side of the occlusal surface, with the fracture extending along the lingual sulcus to the lingual edge, showing a feather shape at the middle part of the fracture extension path and a coral shape at the end of the fracture. Crystalline analysis showed that PEEK crowns, regardless of aging time and TiO2 content, remained predominantly PEEK matrix and rutile phase TiO2. We would conclude that adding 20% or 30% TiO2 to PEEK crowns may have been sufficient to improve the fracture properties of PEEK crowns after 5 or 10 h of aging. Aging times below 10 h may still be safe for reducing the fracture properties of TiO2-containing PEEK crowns.

Airway delivery of Streptococcus salivarius is sufficient to induce experimental pulmonary hypertension in rats.

BACKGROUND AND PURPOSE: The causal relationship between altered host microbiome composition, especially the respiratory tract microbiome, and the occurrence of pulmonary hypertension (PH) has not yet been studied. An increased abundance of airway streptococci is seen in patients with PH compared with healthy individuals. This study aimed to determine the causal link between elevated airway exposure to Streptococcus and PH. EXPERIMENTAL APPROACH: The dose-, time- and bacterium-specific effects of Streptococcus salivarius (S. salivarius), a selective streptococci, on PH pathogenesis were investigated in a rat model established by intratracheal instillation. KEY RESULTS: Exposure to S. salivarius successfully induced typical PH characteristics, such as elevated right ventricular systolic pressure (RVSP), right ventricular hypertrophy (Fulton's index) and pulmonary vascular remodelling, in a dose- and time-dependent manner. Moreover, the S. salivarius-induced characteristics were absent in either the inactivated S. salivarius (inactivated bacteria control) treatment group or the Bacillus subtilis (active bacteria control) treatment group. Notably, S. salivarius-induced PH is characterized by elevated inflammatory infiltration in the lungs, in a pattern different from the classic hypoxia-induced PH model. Moreover, in comparison with the SU5416/hypoxia-induced PH model (SuHx-PH), S. salivarius-induced PH causes similar histological changes (pulmonary vascular remodelling) but less severe haemodynamic changes (RVSP, Fulton's index). S. salivarius-induced PH is also associated with altered gut microbiome composition, suggesting potential communication of the lung-gut axis. CONCLUSION AND IMPLICATIONS: This study provides the first evidence that the delivery of S. salivarius in the respiratory tract could cause experimental PH in rats.

Proteomics and metabolomics profiling reveal panels of circulating diagnostic biomarkers and molecular subtypes in stable COPD.

BACKGROUND: Chronic obstructive pulmonary disease (COPD) is a complex and heterogeneous disease with high morbidity and mortality, especially in advanced patients. We aimed to develop multi-omics panels of biomarkers for the diagnosis and explore its molecular subtypes. METHODS: A total of 40 stable patients with advanced COPD and 40 controls were enrolled in the study. Proteomics and metabolomics techniques were applied to identify potential biomarkers. An additional 29 COPD and 31 controls were enrolled for validation of the obtained proteomic signatures. Information on demographic, clinical manifestation, and blood test were collected. The ROC analyses were carried out to evaluate the diagnostic performance, and experimentally validated the final biomarkers on mild-to-moderate COPD. Next, molecular subtyping was performed using proteomics data. RESULTS: Theophylline, palmitoylethanolamide, hypoxanthine, and cadherin 5 (CDH5) could effectively diagnose advanced COPD with high accuracy (auROC = 0.98, sensitivity of 0.94, and specificity of 0.95). The performance of the diagnostic panel was superior to that of other single/combined results and blood tests. Proteome based stratification of COPD revealed three subtypes (I-III) related to different clinical outcomes and molecular feature: simplex COPD, COPD co-existing with bronchiectasis, and COPD largely co-existing with metabolic syndrome, respectively. Two discriminant models were established using the auROC of 0.96 (Principal Component Analysis, PCA) and 0.95 (the combination of RRM1 + SUPV3L1 + KRT78) in differentiating COPD and COPD with co-morbidities. Theophylline and CDH5 were exclusively elevated in advanced COPD but not in its mild form. CONCLUSIONS: This integrative multi-omics analysis provides a more comprehensive understanding of the molecular landscape of advanced COPD, which may suggest molecular targets for specialized therapy.

Prediction and prognosis of adverse maternal and foetal/neonatal outcomes in pulmonary hypertension: an observational study and nomogram construction.

BACKGROUND: Pregnant women with pulmonary hypertension (PH) have higher mortality rates and poor foetal/neonatal outcomes. Tools to assess these risk factors are not well established. METHODS: Predictive and prognostic nomograms were constructed using data from a "Development" cohort of 420 pregnant patients with PH, recorded between January 2009 and December 2018. Logistic regression analysis established models to predict the probability of adverse maternal and foetal/neonatal events and overall survival by Cox analysis. An independent "Validation" cohort comprised data of 273 consecutive patients assessed from January 2019 until May 2022. Nomogram performance was evaluated internally and implemented with online software to increase the ease of use. RESULTS: Type I respiratory failure, New York Heart Association functional class, N-terminal pro-brain natriuretic peptide [Formula: see text] 1400 ng/L, arrhythmia, and eclampsia with pre-existing hypertension were independent risk factors for maternal mortality or heart failure. Type I respiratory failure, arrhythmia, general anaesthesia for caesarean section, New York Heart Association functional class, and N-terminal pro-brain natriuretic peptide [Formula: see text] 1400 ng/L were independent predictors of pulmonary hypertension survival during pregnancy. For foetal/neonatal adverse clinical events, type I respiratory failure, arrhythmia, general anaesthesia for caesarean section, parity, platelet count, fibrinogen, and left ventricular systolic diameter were important predictors. Nomogram application for the Development and Validation cohorts showed good discrimination and calibration; decision curve analysis demonstrated their clinical utility. CONCLUSIONS: The nomogram and its online software can be used to analyse individual mortality, heart failure risk, overall survival prediction, and adverse foetal/neonatal clinical events, which may be useful to facilitate early intervention and better survival rates.

Influence of Aging on the Fracture Characteristics of Polyetheretherketone Dental Crowns: A Preliminary Study.

Although polyetheretherketone (PEEK) is becoming more widely used in dentistry applications, little is known about how aging will affect this material. Therefore, this study aimed to investigate the influence of an aging treatment on fracture characteristics of PEEK dental crowns. Additionally, the impact of the addition of titanium dioxide (TiO2) into PEEK was examined. Two types of commercial PEEK discs were used in this study, including TiO2-free and 20% TiO2-containing PEEK. The PEEK dental crowns were fabricated and aging-treated at 134 °C and 0.2 MPa for 5 h in accordance with the ISO 13356 specification before being cemented on artificial tooth abutments. The fracture loads of all crown samples were measured under compression tests. Results demonstrated that adding TiO2 enhanced the fracture load of PEEK crowns compared to TiO2-free PEEK crowns before the aging treatment. However, the aging treatment decreased the fracture load of TiO2-containing PEEK crowns while increasing the fracture load of TiO2-free PEEK crowns. The fracture morphology of TiO2-containing PEEK crowns revealed finer feather shapes than that of the TiO2-free PEEK crowns. We concluded that adding TiO2 increased the fracture load of PEEK crowns without aging treatment. Still, the aging treatment influenced the fracture load and microscopic fracture morphology of PEEK crowns, depending on the addition of TiO2.

Rural environment reduces allergic inflammation by modulating the gut microbiota.

Rural environments and microbiota are linked to a reduction in the prevalence of allergies. However, the mechanism underlying the reduced allergies modulated by rural residency is unclear. Here, we assessed gut bacterial composition and metagenomics in urban and rural children in the EuroPrevall-INCO cohort. Airborne dusts, including mattress and rural henhouse dusts, were profiled for bacterial and fungal composition by amplicon sequencing. Mice were repeatedly exposed to intranasal dust extracts and evaluated for their effects on ovalbumin (OVA)-induced allergic airway inflammation, and gut microbiota restoration was validated by fecal microbiota transplant (FMT) from dust-exposed donor mice. We found that rural children had fewer allergies and unique gut microbiota with fewer Bacteroides and more Prevotella. Indoor dusts in rural environments harbored higher endotoxin level and diversity of bacteria and fungi, whereas indoor urban dusts were enriched with Aspergillus and contained elevated pathogenic bacteria. Intranasal administration of rural dusts before OVA sensitization reduced respiratory eosinophils and blood IgE level in mice and also led to a recovery of gut bacterial diversity and Ruminiclostridium in the mouse model. FMT restored the protective effect by reducing OVA-induced lung eosinophils in recipient mice. Together, these results support a cause-effect relationship between exposure to dust microbiota and allergy susceptibility in children and mice. Specifically, rural environmental exposure modulated the gut microbiota, which was essential in reducing allergy in children from Southern China. Our findings support the notion that the modulation of gut microbiota by exposure to rural indoor dust may improve allergy prevention.

Morpho-histology, endogenous hormone dynamics, and transcriptome profiling in Dacrydium pectinatum during female cone development.

Dacrydium pectinatum de Laubenfels is a perennial dioeciously gymnosperm species dominant in tropical montane rain forests. Due to deforestation, natural disasters, long infancy, and poor natural regeneration ability, the population of this species has been significantly reduced and listed as an endangered protected plant. To better understand the female cone development in D. pectinatum, we examined the morphological and anatomical changes, analyzed the endogenous hormone dynamics, and profiled gene expression. The female reproductive structures were first observed in January. The morpho-histological observations suggest that the development of the D. pectinatum megaspore can be largely divided into six stages: early flower bud differentiation, bract primordium differentiation, ovule primordium differentiation, dormancy, ovule maturity, and seed maturity. The levels of gibberellins (GA), auxin (IAA), abscisic acid (ABA), and cytokinin (CTK) fluctuate during the process of female cone development. The female cones of D. pectinatum need to maintain a low level of GA3-IAA-ABA steady state to promote seed germination. The first transcriptome database for female D. pectinatum was generated, revealing 310,621 unigenes. Differential expression analyses revealed several floral (MADS2, AGL62, and LFY) and hormone biosynthesis and signal transduction (CKX, KO, KAO, ABA4, ACO, etc.) genes that could be critical for female cone development. Our study provides new insights into the cone development in D. pectinatum and the foundation for female cone induction with hormones.

Gene Expression Trajectories from Normal Nonsmokers to COPD Smokers and Disease Progression Discriminant Modeling in Response to Cigarette Smoking.

Background: Cigarette smoking (CS) is considered to the predominant risk factor contributing to the etiopathogenesis of chronic obstructive pulmonary disease (COPD); meanwhile, genetic predisposition likely plays a role in determining disease susceptibility. Objectives: We aimed to investigate gene expression trajectories from normal nonsmokers to COPD smokers and disease progression discriminant modeling in response to cigarette smoking. Methods: Small airway epithelial samples of human with different smoking status using fiberoptic bronchoscopy and corresponding rat lung tissues following 0, 3, and 6 months of CS exposure were obtained. The expression of the significant overlapping genes between human and rats was confirmed in 16HBE cells, rat lung tissues, and human peripheral PBMC using qRT-PCR. Binary logistic regression analysis was carried out to establish discrimination models. Results: The integrated bioinformatic analysis of 8 human GEO datasets (293 individuals) and 9 rat transcriptome databases revealed 13 overlapping genes between humans and rats in response to smoking exposure during COPD progression. Of these, 5 genes (AKR1C3/Akr1c3, ERP27/Erp27, AHRR/Ahrr, KCNMB2/Kcnmb2, and MRC1/Mrc1) were consistently identified in both the human and rat and validated by qRT-PCR. Among them, ERP27/Erp27, KCNMB2/Kcnmb2, and MRC1/Mrc1 were newly identified. On the basis of the overlapping gene panel, discriminant models were established with the receiver operating characteristic curve (AUC) of 0.98 (AKR1C3/Akr1c3 + ERP27/Erp27) and 0.99 (AHRR/Ahrr + KCNMB2/Kcnmb2) in differentiating progressive COPD from normal nonsmokers. In addition, we also found that DEG obtained from each expression profile dataset was better than combined analysis as more genes could be identified. Conclusion: This study identified 5 DEG candidates of COPD progression in response to smoking and developed effective and convenient discriminant models that can accurately predict the disease progression.

Effects of bone morphogenetic protein 4 on TGF-ß1-induced cell proliferation, apoptosis, activation and differentiation in mouse lung fibroblasts via ERK/p38 MAPK signaling pathway.

Fibroblasts, in particular myofibroblasts, are the critical effector cells in idiopathic pulmonary fibrosis (IPF), a deadly lung disease characterized by abnormal lung remodeling and the formation of "fibroblastic foci". Aberrant activation of TGF-ß1 is frequently encountered and promotes fibroblast proliferation, activation, and differentiation in pulmonary fibrosis. Hence, the inhibition of TGF-ß1-induced lung fibroblast activation holds promise as a therapeutic strategy for IPF. The present study aimed to investigate the potential effect and underlying mechanisms of bone morphogenetic protein 4 (BMP4) on TGF-ß1-induced proliferation, apoptosis, activation and myofibroblast differentiation of adult lung fibroblasts. Here, we demonstrated that BMP4 expression was significantly decreased in TGF-ß1-stimulated mouse primary lung fibroblasts (PLFs). BMP4 inhibited proliferation and apoptosis resistance of TGF-ß1-stimulated mouse PLFs. BMP4 suppressed TGF-ß1-induced fibroblast activation and differentiation in mouse PLFs. We also found that BMP4 inhibited TGF-ß1-induced ERK and p38 MAPK phosphorylation. Our findings indicate that BMP4 exerts its anti-fibrotic effects by regulating fibroblast proliferation, apoptosis, activation and differentiation via the inhibition of the ERK/p38 MAPK signaling pathway, and thus has a potential for the treatment of pulmonary fibrosis.

Bone morphogenetic protein 4 inhibits pulmonary fibrosis by modulating cellular senescence and mitophagy in lung fibroblasts.

BACKGROUND: Accumulation of myofibroblasts is critical to fibrogenesis in idiopathic pulmonary fibrosis (IPF). Senescence and insufficient mitophagy in fibroblasts contribute to their differentiation into myofibroblasts, thereby promoting the development of lung fibrosis. Bone morphogenetic protein 4 (BMP4), a multifunctional growth factor, is essential for the early stage of lung development; however, the role of BMP4 in modulating lung fibrosis remains unknown. METHODS: The aim of this study was to evaluate the role of BMP4 in lung fibrosis using BMP4-haplodeleted mice, BMP4-overexpressed mice, primary lung fibroblasts and lung samples from patients with IPF. RESULTS: BMP4 expression was downregulated in IPF lungs and fibroblasts compared to control individuals, negatively correlated with fibrotic genes, and BMP4 decreased with transforming growth factor (TGF)-ß1 stimulation in lung fibroblasts in a time- and dose-dependent manner. In mice challenged with bleomycin, BMP4 haploinsufficiency perpetuated activation of lung myofibroblasts and caused accelerated lung function decline, severe fibrosis and mortality. BMP4 overexpression using adeno-associated virus 9 vectors showed preventative and therapeutic efficacy against lung fibrosis. In vitro, BMP4 attenuated TGF-ß1-induced fibroblast-to-myofibroblast differentiation and extracellular matrix (ECM) production by reducing impaired mitophagy and cellular senescence in lung fibroblasts. Pink1 silencing by short-hairpin RNA transfection abolished the ability of BMP4 to reverse the TGF-ß1-induced myofibroblast differentiation and ECM production, indicating dependence on Pink1-mediated mitophagy. Moreover, the inhibitory effect of BMP4 on fibroblast activation and differentiation was accompanied with an activation of Smad1/5/9 signalling and suppression of TGF-ß1-mediated Smad2/3 signalling in vivo and in vitro. CONCLUSION: Strategies for enhancing BMP4 signalling may represent an effective treatment for pulmonary fibrosis.

Identification of genetic variants of the IL-22 gene in association with an altered risk of COPD susceptibility.

The incidence of chronic obstructive pulmonary disease (COPD) is related to the interaction between environmental exposure and genetic factors. Far more than 15% of smokers eventually develop COPD. In addition to smoking, genetic susceptibility may be another factor in the development of COPD. IL-22 and its receptors are increased in human and experimental COPD and contribute to pathogenesis. Here, we conducted a case-control study to evaluate the association between IL-22 tag-single nucleotide polymorphisms (SNPs) and COPD risk. Four tag-SNPs (rs2227478, rs2227481, rs2227484 and rs2227485) were identified according to linkage disequilibrium (LD) analysis in 30 healthy controls. A total of 513 COPD cases and 504 controls were recruited to perform an association study between these four tag-SNPs and COPD risk. We found that the "C" allele of rs2227478T>C and the "T" allele of rs2227481C>T were obviously related to decreased COPD susceptibility. Genetic model analysis showed that rs2227478T>C and rs2227481C>T were significantly associated with a decreased risk of COPD under dominant models after adjusting for the above factors. In the recessive model, rs2227485T>C was obviously associated with decreased COPD risk. Our data showed that only rs2227485T>C was associated with a decreased COPD risk after Bonferroni correction. The eQTL analysis showed that rs2227485T>C was significantly associated with IL-22 expression. The pGL4-rs2227485-C gene reporter had a higher promoter activity than pGL4-rs2227485-T. In our study, rs2227485T>C, located in the promoter region of IL-22, was associated with a decreased risk of COPD and increased IL-22 promoter activity, suggesting that this variant might modulate COPD susceptibility.