Autonomous enzymatic synthesis of functional nucleic acids for sensitive measurement of long noncoding RNA in human lung tissues.

Aberrant long noncoding RNA (lncRNA) expression is linked to varied pathological processes and malignant tumors, and lncRNA can serve as potential disease biomarkers. Herein, we demonstrate the autonomous enzymatic synthesis of functional nucleic acids for sensitive measurement of lncRNA in human lung tissues on the basis of multiple primer generation-mediated rolling circle amplification (mPG-RCA). This assay involves two padlock probes that act as both a detection probe for recognizing target lncRNA and a domain for producing complementary DNAzyme. Two padlock probes can hybridize with target lncRNA at different sites, followed by ligation to form a circular template with the aid of RNA ligase. The circular template can initiate mPG-RCA to generate abundant Mg2+-dependent DNAzymes that can specifically cleave signal probes to induce the recovery of Cy3 fluorescence. The inherent characteristics of ligase-based ligation reaction and DNAzymes endow this assay with excellent specificity, and the introduction of multiple padlock probes endows this assay with high sensitivity. This strategy can rapidly and sensitively measure lncRNA with a wide linear range of 1 fM - 1 nM and a detection limit of 678 aM within 1.5 h, and it shows distinct advantages of simplicity and immobilization-free without the need of precise temperature control and tedious procedures of nanomaterial preparation. Moreover, it enables accurate measurement of lncRNA level in normal cells and malignant tumor cells as well as differentiation of lncRNA expressions in tissues of non-small cell lung cancer (NSCLC) patients and normal individuals, with promising applications in biomedical studies and disease diagnosis.

Intracellular Pseudomonas aeruginosa within the Airway Epithelium of Cystic Fibrosis Lung Tissues.

Rationale: Pseudomonas aeruginosa is the major bacterial pathogen colonizing the airways of adult patients with cystic fibrosis (CF) and causes chronic infections that persist despite antibiotic therapy. Intracellular bacteria may represent an unrecognized reservoir of bacteria that evade the immune system and antibiotic therapy. Although the ability of P. aeruginosa to invade and survive within epithelial cells has been described in vitro in different epithelial cell models, evidence of this intracellular lifestyle in human lung tissues is currently lacking. Objectives: To detect and characterize intracellular P. aeruginosa in CF airway epithelium from human lung explant tissues. Methods: We sampled lung explant tissues from patients with CF undergoing lung transplantation and non-CF lung donor control tissue. We analyzed lung tissue sections for the presence of intracellular P. aeruginosa using quantitative culture and microscopy, in parallel to histopathology and airway morphometry. Measurements and Main Results: P. aeruginosa was isolated from the lungs of seven patients with CF undergoing lung transplantation. Microscopic assessment revealed the presence of intracellular P. aeruginosa within airway epithelial cells in three of the seven patients analyzed at a varying but low frequency. We observed those events occurring in lung regions with high bacterial burden. Conclusions: This is the first study describing the presence of intracellular P. aeruginosa in CF lung tissues. Although intracellular P. aeruginosa in airway epithelial cells is likely relatively rare, our findings highlight the plausible occurrence of this intracellular bacterial reservoir in chronic CF infections.

Microplastics exposure causes the senescence of human lung epithelial cells and mouse lungs by inducing ROS signaling.

Microplastics (MPs) are environmental pollutants and can be inhaled by humans to threaten health. The lung tissue, responsible for the gas exchange between the body and the environment, is vulnerable to MPs exposure. However, from the perspective of cellular senescence, the effect of MPs on lung cells and tissues has not yet been deeply dissected. In this study, we reported that all the four typical MPs exhibited the significant biological effects in term of inducing senescence of human lung derived cells A549 and BEAS-2B in vitro. We further found that polyvinyl chloride (PVC) increased the reactive oxygen species (ROS) level in A549 cells and that PVC-induced senescent characteristics could be largely reversed by antioxidant treatment. Importantly, intratracheal instillation of PVC MPs in mice could effectively impair their physical function, induce the increased systemic inflammation level, cause the accumulation of senescent cells. Our study demonstrates that MPs induce senescence in human lung epithelial cells and mouse lungs by activating ROS signaling, and provides new insight into the potential pathogenesis of MPs on lung diseases.

Type-II IFN inhibits SARS-CoV-2 replication in human lung epithelial cells and ex vivo human lung tissues through indoleamine 2,3-dioxygenase-mediated pathways.

Interferons (IFNs) are critical for immune defense against pathogens. While type-I and -III IFNs have been reported to inhibit SARS-CoV-2 replication, the antiviral effect and mechanism of type-II IFN against SARS-CoV-2 remain largely unknown. Here, we evaluate the antiviral activity of type-II IFN (IFNγ) using human lung epithelial cells (Calu3) and ex vivo human lung tissues. In this study, we found that IFNγ suppresses SARS-CoV-2 replication in both Calu3 cells and ex vivo human lung tissues. Moreover, IFNγ treatment does not significantly modulate the expression of SARS-CoV-2 entry-related factors and induces a similar level of pro-inflammatory response in human lung tissues when compared with IFNß treatment. Mechanistically, we show that overexpression of indoleamine 2,3-dioxygenase 1 (IDO1), which is most profoundly induced by IFNγ, substantially restricts the replication of ancestral SARS-CoV-2 and the Alpha and Delta variants. Meanwhile, loss-of-function study reveals that IDO1 knockdown restores SARS-CoV-2 replication restricted by IFNγ in Calu3 cells. We further found that the treatment of l-tryptophan, a substrate of IDO1, partially rescues the IFNγ-mediated inhibitory effect on SARS-CoV-2 replication in both Calu3 cells and ex vivo human lung tissues. Collectively, these results suggest that type-II IFN potently inhibits SARS-CoV-2 replication through IDO1-mediated antiviral response.

SARS-CoV-2 infection dysregulates the expression of clinically relevant drug metabolizing enzymes in Vero E6 cells and membrane transporters in human lung tissues.

SARS-CoV-2-mediated interactions with drug metabolizing enzymes and membrane transporters (DMETs) in different tissues, especially lung, the main affected organ may limit the clinical efficacy and safety profile of promising COVID-19 drugs. Herein, we investigated whether SARS-CoV-2 infection could dysregulate the expression of 25 clinically relevant DMETs in Vero E6 cells and postmortem lung tissues from COVID-19 patients. Also, we assessed the role of 2 inflammatory and 4 regulatory proteins in modulating the dysregulation of DMETs in human lung tissues. We showed for the first time that SARS-CoV-2 infection dysregulates CYP3A4 and UGT1A1 at the mRNA level, as well as P-gp and MRP1 at the protein level, in Vero E6 cells and postmortem human lung tissues, respectively. We observed that at the cellular level, DMETs could potentially be dysregulated by SARS-CoV-2-associated inflammatory response and lung injury. We uncovered the pulmonary cellular localization of CYP1A2, CYP2C8, CYP2C9, and CYP2D6, as well as ENT1 and ENT2 in human lung tissues, and observed that the presence of inflammatory cells is the major driving force for the discrepancy in the localization of DMETs between COVID-19 and control human lung tissues. Because alveolar epithelial cells and lymphocytes are both sites of SARS-CoV-2 infection and localization of DMETs, we recommend further investigation of the pulmonary pharmacokinetic profile of current COVID-19 drug dosing regimen to improve clinical outcomes.

Tonifying-Qi-and-Detoxification Decoction attenuated injuries of colon and lung tissues in ulcerative colitis rat model via regulating NF-κB and p38MAPK pathway.

Background: Tonifying-Qi-and-Detoxification Decoction (TQDD) is a Chinese medicine compound. This research probed the possible protective effects of TQDD on injuries of the colon and lung tissues in ulcerative colitis (UC) rat model. Methods: UC rat model was established by colon mucosal tissue sensitization combined with TNBS-ethanol. Ninety-six rats were randomly divided into normal control (NC), model, sulfasalazine (SASP), and TQDD (low, middle, and high dosages) groups. After 4 weeks intervention, all rats were sacrificed. The microstructure of lung tissue was observed using hematoxylin-eosin (HE) staining. Transmission electron microscope (TEM) was utilized to assess the ultrastructure change of alveolar epithelial type II cells (AEC-II). The mRNA expressions of Bax, Caspase 3, nuclear factor kappa B (NF-κB) and NF-κB inhibitor α (IKBα) in tissues were measured via quantitative reverse transcription PCR (qRT-PCR) assay. Western blotting and immunohistochemistry (IHC) were used to test p38MAPK, activating transcription factor 2 (ATF2), c-jun and c-fos expressions in tissues. Results: TQDD alleviated microstructure change of lung tissues, lung cell apoptosis and ultrastructure alterations of AEC-II in UC rat model. Moreover, TQDD suppressed activation of NF-κB pathway in colon and lung tissues. Besides, TQDD inhibited p38MAPK pathway in colon and lung tissues, as well as reduced ATF2, c-jun, and c-fos expressions in colon and lung tissues. Conclusions: This research confirmed the beneficial effect of TQDD on injuries of colon and lung tissues in UC rat model. TQDD attenuated injuries of lung and colon tissues in colon mucosal tissue sensitization combined with TNBS-ethanol-caused UC model via regulating NF-κB and p38MAPK pathways.

A 3D in vitro comparison of two undiluted e-cigarette aerosol generating systems.

In vitro studies play an important role in supporting the toxicological assessment of e-cigarettes, with many current methods reliant on sophisticated in vitro exposure systems designed for conventional cigarette testing. In this study, we have compared two distinct systems; the modified Vitrocell VC10 and Borgwaldt LM4E designed to deliver undiluted e-cigarette aerosol. We assessed the cytotoxicity response of 3D reconstituted lung tissue (MucilAir) exposed to undiluted aerosol from ePen3 (closed modular e-cigarette) using these two exposure systems. As the induced cytotoxicity profiles were comparable, we then compared these responses against historical eBox (open modular e-cigarette) and 3R4F reference cigarette data to show evolution of product technology. This latter approach was deemed possible by monitoring intrinsic donor-to-donor control variability over a three-year period, bridging between exposure systems and observed biological responses. Despite the differences in the technology, on a puff-by-puff basis these machines gave remarkably similar cytotoxicity profiles for ePen3, as determined by MTT, and consistency of pre-cytotoxicity markers: transepithelial electrical resistance (TEER), cilia beat frequency and cilia active area. When responses are compared as a function of exposed nicotine concentration, we see differences due to the dynamics of the exposure systems. The parity of responses between the systems in generated undiluted aerosol has allowed us to compare back to previously published eBox data, irrespective of aerosol generating system and MucilAir donor, showing how evolution from open systems to podmod e-cigarette design can make a step change in the cytotoxicity profile of the product.

Essential Genes and MiRNA-mRNA Network Contributing to the Pathogenesis of Idiopathic Pulmonary Arterial Hypertension.

Background: Idiopathic pulmonary arterial hypertension (IPAH) is a life-threatening disease. Owing to its high fatality rate and narrow therapeutic options, identification of the pathogenic mechanisms of IPAH is becoming increasingly important. Methods: In our research, we utilized the robust rank aggregation (RRA) method to integrate four eligible pulmonary arterial hypertension (PAH) microarray datasets and identified the significant differentially expressed genes (DEGs) between IPAH and normal samples. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways were performed to analyze their functions. The interaction network of protein-protein interaction (PPI) was constructed to explore the correlation between these DEGs. The functional modules and hub genes were further identified by the weighted gene coexpression network analysis (WGCNA). Moreover, a miRNA microarray dataset was involved and analyzed to filter differentially expressed miRNAs (DE-miRNAs). Potential target genes of screened DE-miRNAs were predicted and merged with DEGs to explore a miRNA-mRNA network in IPAH. Some hub genes were selected and validated by RT-PCR in lung tissues from the PAH animal model. Results: A total of 260 DEGs, consisting of 183 upregulated and 77 downregulated significant DEGs, were identified, and some of those genes were novel. Their molecular roles in the etiology of IPAH remained vague. The most crucial functional module involved in IPAH is mainly enriched in biological processes, including leukocyte migration, cell chemotaxis, and myeloid leukocyte migration. Construction and analysis of the PPI network showed that CXCL10, CXCL9, CCR1, CX3CR1, CX3CL1, CXCR2, CXCR1, PF4, CCL4L1, and ADORA3 were recognized as top 10 hub genes with high connectivity degrees. WGCNA further identified five main functional modules involved in the pathogenesis of IPAH. Twelve upregulated DE-miRNAs and nine downregulated DE-miRNAs were identified. Among them, four downregulated DEGs and eight upregulated DEGs were supposed to be negatively regulated by three upregulated DE-miRNAs and three downregulated DE-miRNAs, respectively. Conclusions: This study identifies some key and functional coexpression modules involved in IPAH, as well as a potential IPAH-related miRNA-mRNA regulated network. It provides deepening insights into the molecular mechanisms and provides vital clues in seeking novel therapeutic targets for IPAH.

Suppression of SARS-CoV-2 infection in ex-vivo human lung tissues by targeting class III phosphoinositide 3-kinase.

The novel betacoronavirus severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerged at the end of 2019 and caused the coronavirus disease 19 (COVID-19) pandemic due to its high transmissibility and early immunosuppression. Previous studies on other betacoronaviruses suggested that betacoronavirus infection is associated with the host autophagy pathway. However, it is unclear whether any components of autophagy or virophagy can be therapeutic targets for COVID-19 treatment. In this report, we examined the antiviral effect of four well-characterized small molecule inhibitors that target the key cellular factors involved in key steps of the autophagy pathway. They include small molecules targeting the ULK1/Atg1 complex involved in the induction stage of autophagy (ULK1 inhibitor SBI0206965), the ATG14/Beclin1/VPS34 complex involved in the nucleation step of autophagy (class III PI3-kinase inhibitor VPS34-IN1), and a widely-used autophagy inhibitor that persistently inhibits class I and temporary inhibits class III PI3-kinase (3-MA) and a clinically approved autophagy inhibitor that suppresses autophagy by inhibiting lysosomal acidification and prevents the formation of autophagolysosome (HCQ). Surprisingly, not all the tested autophagy inhibitors suppressed SARS-CoV-2 infection. We showed that inhibition of class III PI3-kinase involved in the initiation step of both canonical and noncanonical autophagy potently suppressed SARS-CoV-2 at a nano-molar level. In addition, this specific kinase inhibitor VPS34-IN1, and its bioavailable analogue VVPS34-IN1, potently inhibited SARS-CoV-2 infection in ex vivo human lung tissues. Taken together, class III PI3-kinase may be a possible target for COVID-19 therapeutic development.

Comparison of MS2, synchronous precursor selection MS3, and real-time search MS3 methodologies for lung proteomes of hydrogen sulfide treated swine.

Tandem mass tags (TMTs) have increasingly become an attractive technique for global proteomics. However, its effectiveness for multiplexed quantitation by traditional tandem mass spectrometry (MS2) suffers from ratio distortion. Synchronous precursor selection (SPS) MS3 has been widely accepted for improved quantitation accuracy, but concurrently decreased proteome coverage. Recently, a Real-Time Search algorithm has been integrated with the SPS MS3 pipeline (RTS MS3) to provide accurate quantitation and improved depth of coverage. In this mechanistic study of the impact of exposure to hydrogen sulfide (H2S) on the respiration of swine, we used TMT-based comparative proteomics of lung tissues from control and H2S-treated subjects as a test case to evaluate traditional MS2, SPS MS3, and RTS MS3 acquisition methods on both the Orbitrap Fusion and Orbitrap Eclipse platforms. Comparison of the results obtained by the MS2 with those of SPS MS3 and RTS MS3 methods suggests that the MS3-driven quantitative strategies provided a more accurate global-scale quantitation; however, only RTS MS3 provided proteomic coverage that rivaled that of traditional MS2 analysis. RTS MS3 not only yields more productive MS3 spectra than SPS MS3 but also appears to focus the analysis more effectively on unique peptides. Furthermore, pathway enrichment analyses of the H2S-altered proteins demonstrated that an additional apoptosis pathway was discovered exclusively by RTS MS3. This finding was verified by RT-qPCR, western blotting, and TUNEL staining experiments. We conclude that RTS MS3 workflow enables simultaneous improvement of quantitative accuracy and proteome coverage over alternative approaches (MS2 and SPS MS3). Graphical abstract.

Proteomic analysis of sex differences in hyperoxic lung injury in neonatal mice.

Sex-specific differences in the severity of bronchopulmonary dysplasia (BPD) are due to different susceptibility to hyperoxic lung injury, but the mechanism is unclear. In this study, neonatal male and female mouse pups (C57BL/6J) were exposed to hyperoxia and lung tissues were excised on postnatal day 7 for histological analysis and tandem mass tags proteomic analysis. We found that the lung sections from the male mice following postnatal hyperoxia exposure had increased alveolar simplification, significant aberrant pulmonary vascularization and arrest in angiogenesis compared with females. Comparison of differentially expressed proteins revealed 377 proteins unique to female and 425 unique to male as well as 750 proteins in both male and female. Bioinformatics analysis suggested that several differentially expressed proteins could contribute to the differences in sex-specific susceptibility to hyperoxic lung injury. Our results may help identify sex-specific biomarkers and therapeutic targets of BPD.

Glucosamine protects against radiation-induced lung injury via inhibition of epithelial-mesenchymal transition.

Radiotherapy is one of the most important treatments for chest tumours. Although there are plenty of strategies to prevent damage to normal lung tissues, it cannot be avoided with the emergence of radiation-induced lung injury. The purpose of this study was to investigate the potential radioprotective effects of glucosamine, which exerted anti-inflammatory activity in joint inflammation. In this study, we found glucosamine relieved inflammatory response and structural damages in lung tissues after radiation via HE staining. Then, we detected the level of epithelial-mesenchymal transition marker in vitro and in vivo, which we could clearly observe that glucosamine treatment inhibited epithelial-mesenchymal transition. Besides, we found glucosamine could inhibit apoptosis and promote proliferation of normal lung epithelial cells in vitro caused by radiation. In conclusion, our data showed that glucosamine alleviated radiation-induced lung injury via inhibiting epithelial-mesenchymal transition, which indicated glucosamine could be a novel potential radioprotector for radiation-induced lung injury.

Systematic analysis of concentrations of 52 elements in tumor and counterpart normal tissues of patients with non-small cell lung cancer.

BACKGROUND: Many studies have documented the abnormal concentrations of major/trace elements in serum or malignant tissues of patients, but very few works systematically tested the concentrations of elements in tumor tissues in comparison with paired adjacent normal tissues from the same patients. METHODS: Tumor and adjacent normal lung tissues were obtained from 93 patients with previously untreated NSCLC, and 43 patients whose tumor and paired normal lung tissues reached 200 mg or more were selected for measurement of the elements' concentrations using an inductively coupled plasma-atomic emission spectrometer. RESULTS: We found that the concentrations of the 52 elements varied from 0.4 ng/g tissue (Lu, Pd, and Tm) to 1 658 000 ng/g (Na), 1 951 000 ng/g (P), and 2 495 000 ng/g (K). Thirty eight of the 52 (73.1%) elements showed approximately equal concentrations in tumor and adjacent normal lung tissues of the patients. The concentrations of nine elements (K, P, Mg, Zn, Rb, Cu, Se, Cs, and Tl) in tumor samples were significantly higher than their paired normal lung tissues, and five elements (Na, Fe, Cr, Cd, and Ge) exhibited decreased concentrations in cancer samples compared to counterpart normal lung tissues. Low Fe in tumor samples was associated with smoking history, whereas low Cr was associated with histology (squamous cell carcinoma) of the patients. CONCLUSIONS: Our results demonstrate that measurement of elements' concentrations in both cancer and paired normal tissues is important to get insights into the roles of these elements in carcinogenesis, and therapeutic approaches to normalize the elements are warranted to treat NSCLCs.

Raman spectroscopic discrimination of normal and cancerous lung tissues.

Raman spectroscopy is non-destructive method that allows monitoring of biological tissues with minimal intervention. FT-Raman (λex 1064 nm) and NIR-Vis-Raman (λex 785 nm) spectroscopic measurements were used in ex vivo analysis of normal, non-cancerous abnormal and cancerous lung tissues. Spectroscopic discrimination of the lung tissue samples was made by the use of the ratio of characteristic bands and multivariate statistical methods (PCA, LDA). The combination of Raman spectroscopy and multivariate statistics may have a diagnostic potential for recognizing of cancer lesions in lung.

An approach to testing undiluted e-cigarette aerosol in vitro using 3D reconstituted human airway epithelium.

The data presented here show that to provide an estimate of the relative cytotoxicity and therefore potency of e-cigarettes, undiluted aerosol techniques can be used. With the emergence of electronic nicotine delivery systems, fit-for-purpose in vitro screening methods are required. Reconstituted 3D human airway epithelium, was exposed to undiluted aerosols at the air-liquid interface, using a Vitrocell VC 10. TEER, cilia beat frequency and cytotoxic responses were assessed. Using two smoking regimes (ISO and HCI) a 3R4F reference cigarette, produced IC50s of 5.2 and 2.1 min, 1458 ng/mL and 1640 ng/mL nicotine respectively. Using an open tank e-cigarette device, a full cytotoxicity dose-response curve was obtained giving an IC50 of 30 min with corresponding nicotine of 10,957 ng/mL, 6-14 times less cytotoxic than cigarette smoke. A commonly used e-liquid flavourant cinnamaldehyde and known skin sensitizer was added to the standard e-liquid formulation and used as an aerosolised positive control, at 0.1, 0.025, 0.01 and 0%, demonstrating a full dose response. The delivery of undiluted aerosols in vitro has resulted in increased method sensitivity, throughput and quantitative e-cigarette comparisons. A positive control aerosol generated from a 'safe' e-liquid benchmark can inform risk assessments on supportable levels of flavour ingredients.

Protective effect of ginsenoside Rg3 on lung injury in diabetic rats.

Ginsenoside has been used to treat diabetes, while ginsenoside Rg3 is the main active ingredient component of ginseng and is used to study its effects on lung tissue damage in diabetic rats. In this paper, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay and flow cytometry were applied to detect the proliferation and apoptosis of BEAS-2B cells treated with different concentrations of Rg3. The inflammatory response and pathological change in the lung tissue of diabetic rats treated with Rg3 were evaluated by enzyme-linked immunosorbent assay, quantative real-time polymerase chain reaction, and hematoxylin and eosin staining immunohistochemistry. Meanwhile, PI3K and MAPK signaling pathway proteins in lung tissue were determined by Western blot analysis. The results showed that ginsenoside Rg3 had no significant influence on the proliferation and apoptosis of BEAS-2B cells. Ginsenoside Rg3 can inhibit inflammatory response and promote the activation of PI3K and MAPK signaling pathways to prevent damages of lung tissues induced by hyperglycemia. The protective effect provided by ginsenoside Rg3 indicates that ginsenoside Rg3 is a potential drug for the treatment of diabetes.

Peptidome analysis of lung tissues from a hyperoxia-induced bronchopulmonary dysplasia mouse model: Insights into the pathophysiological process of bronchopulmonary dysplasia.

The aim of this study was to identify and compare the peptidomic profiles of lung tissues from neonatal mice with and without bronchopulmonary dysplasia (BPD). Hyperoxia was used to establish the BPD mouse model. Lung tissues obtained on postnatal day (PND) 9 were processed for analysis via histological staining and label-free liquid chromatography-mass spectrometry (LC-MS/MS). Histological analysis of the lung sections from the BPD group showed significant alveolar simplification and aberrant pulmonary vascularization. We identified 3,704 total peptides, of which 63 were differentially expressed in the lung tissues from the BPD group compared with those from the control group. Within this subset, 31 peptides were downregulated, and 32 peptides were upregulated. Bioinformatics analysis suggested several potential roles of the differentially expressed peptides in the pathophysiological process of BPD. In summary, this study highlights novel peptide candidates, and provides new insights for further understanding the molecular mechanism of BPD development.

TRPA1 channels: expression in non-neuronal murine lung tissues and dispensability for hyperoxia-induced alveolar epithelial hyperplasia.

Transient receptor potential A1 (TRPA1) channels were originally characterized in neuronal tissues but also identified in lung epithelium by staining with fluorescently coupled TRPA1 antibodies. Its exact function in non-neuronal tissues, however, is elusive. TRPA1 is activated in vitro by hypoxia and hyperoxia and is therefore a promising TRP candidate for sensing hyperoxia in pulmonary epithelial cells and for inducing alveolar epithelial hyperplasia. Here, we isolated tracheal, bronchial, and alveolar epithelial cells and show low but detectable TRPA1 mRNA levels in all these cells as well as TRPA1 protein by Western blotting in alveolar type II (AT II) cells. We quantified changes in intracellular Ca2+ ([Ca2+]i) levels induced by application of hyperoxic solutions in primary tracheal epithelial, bronchial epithelial, and AT II cells isolated from wild-type (WT) and TRPA1-deficient (TRPA1-/-) mouse lungs. In all cell types, we detected hyperoxia-induced rises in [Ca2+]i levels, which were not significantly different in TRPA1-deficient cells compared to WT cells. We also tested TRPA1 function in a mouse model for hyperoxia-induced alveolar epithelial hyperplasia. A characteristic significant increase in thickening of alveolar tissues was detected in mouse lungs after exposure to hyperoxia, but not in normoxic WT and TRPA1-/- controls. Quantification of changes in lung morphology in hyperoxic WT and TRPA1-/- mice, however, again revealed no significant changes. Therefore, TRPA1 expression does neither appear to be a key player for hyperoxia-induced changes in [Ca2+]i levels in primary lung epithelial cells, nor being essential for the development of hyperoxia-induced alveolar epithelial hyperplasia.

Investigating the bioavailability of graphene quantum dots in lung tissues via Fourier transform infrared spectroscopy.

Biomolecular fractions affect the fate and behaviour of quantum dots (QDs) in living systems but how the interactions between biomolecules and QDs affect the bioavailability of QDs is a major knowledge gap in risk assessment analysis. The transport of QDs after release into a living organism is a complex process. The majority accumulate in the lungs where they can directly affect the inhalation process and lung architecture. Here, we investigate the bioavailability of graphene quantum dots (GQDs) to the lungs of rats by measuring the alterations in macromolecular fractions via Fourier transform infrared spectroscopy (FTIR). GQDs were intravenously injected into the rats in a dose-dependent manner (low (5 mg kg-1) and high (15 mg kg-1) doses of GQDs per body weight of rat) for 7 days. The lung tissues were isolated, processed and haematoxylin-eosin stained for histological analysis to identify cell death. Key biochemical differences were identified by spectral signatures: pronounced changes in cholesterol were found in two cases of low and high doses; a change in phosphorylation profile of substrate proteins in the tissues was observed in low dose at 24 h. This is the first time biomolecules have been measured in biological tissue using FTIR to investigate the biocompatibility of foreign material. We found that highly accurate toxicological changes can be investigated with FTIR measurements of tissue sections. As a result, FTIR could form the basis of a non-invasive pre-diagnostic tool for predicting the toxicity of GQDs.