Assessment of Benchmark Dose in BEAS-2B Cells by Evaluating the Cell Relative Viability with Particulates in Motorcycle Exhaust via the Air-liquid Interface Exposure

Objective This study aimed to use an air-liquid interface(ALI)exposure system to simulate the inhalation exposure of motorcycle exhaust particulates(MEPs)and then investigate the benchmark dose(BMD)of MEPs by evaluating cell relative viability(CRV)in lung epithelial BEAS-2B cells.Methods The MEPs dose was characterized by measuring the number concentration(NC),surface area concentration(SAC),and mass concentration(MC).BEAS-2B cells were exposed to MEPs at different concentrations via ALI and CRV was determined using Cell Counting Kit(CCK-8)assay.BMD software was applied to calculate BMD and the lower limit of benchmark dose(BMDL)according to Akaike Information Coefficient(AIC),with P-value based on Hill,Linear,Polynomial,and Power model.Results Our results reveal that BMD of NC and SAC were estimated by the best-fitting Hill model,while MC was estimated by Polynomial model.The BMDL for CRV following ALI exposure to MEPs were as follows:364.2#/cm^(3)for NC;0.662×10^(7)nm^(2)/cm^(3)for SAC;and 0.278μg/m^(3)for MC.Conclusion These results indicate that MEPs exposure via ALI system induces a dose-dependent decrease of CRV and provides the potential exposure threshold of MEPs in a lung cell model.

Assessing the Adverse Impacts of PM_(2.5)on Olfactory System Using an Air-liquid Interface Culture Model of Primary Olfactory Epithelial Cells

The air-liquid interface(ALI)culture is a kind of recently developed system,which has proved its availability in simulating the biology of respiratory tract epithelial tissues.In this study,an ALI-based mouse primary olfactory epithelial cell(OEC)model was established to perform the exposure of PM_(2.5)(PM=particulate matter)collected from Dianshan Lake(Shanghai)and Wangdu(Hebei).The results showed that PM_(2.5)in both regions caused a decrease in cell viability in a dose-dependent manner.The 0.5 and 5µg/cm^(2)(around ambient concentrations)of PM_(2.5)disrupted OEC membrane integrity and produced oxidative stress with elevated indicators of malondialdehyde(MDA)and reactive oxygen species(ROS).In transcriptomic sequencing,the terms concerning inflammatory cytokines and second messenger cyclic adenosine-3′,5′-monophoshate(cAMP)were enriched in two treatments.The cytokine array showed the levels of some cytokines were altered,although inflammatory responses may not remarkably occur.Meanwhile,PM_(2.5)disturbed cAMP contents and key genes in the cAMP signaling pathway.The effects of PM_(2.5)of both regions were largely consistent,while Wangdu samples caused more ROS and Dianshan Lake samples tended to induce inflammatory injury.Thus,with the application of a novel ALI-based in vitro OEC model,our study demonstrated that ambient PM_(2.5)has the ability to threaten the physiologies and functions of the olfactory system.

Organoid co-culture models of the tumor microenvironment promote precision medicine

In recent years,the three-dimensional(3D)culture system has emerged as a promising preclinical model for tumor research owing to its ability to replicate the tissue structure and molecular characteristics of solid tumors in vivo.This system offers several advantages,including high throughput,efficiency,and retention of tumor heterogeneity.Traditional Matrigel-submerged organoid cultures primarily support the long-term proliferation of epithelial cells.One solution for the exploration of the tumor microenvironment is a reconstitution approach involving the introduction of exogenous cell types,either in dual,triple or even multiple combinations.Another solution is a holistic approach including patient-derived tumor fragments,air-liquid interface,suspension 3D culture,and microfluidic tumor-on-chip models.Organoid co-culture models have also gained popularity for studying the tumor microenvironment,evaluating tumor immunotherapy,identifying predictive biomarkers,screening for effective drugs,and modeling infections.By leveraging these 3D culture systems,it is hoped to advance the clinical application of therapeutic approaches and improve patient outcomes.

Intensified inactivation of model and environmental bacteria by an atmospheric-pressure air-liquid discharge plasma compared with chlorination

Water-borne pathogenic bacteria are always the top priority to be removed through disinfection process in water treatment due to their threat to human health. It was necessary to develop novel disinfection methods since the conventional chlorine disinfection was inefficient in inactivating chlorine-resistant bacteria, inducing the viable but non-culturable(VBNC) bacteria and forming disinfection by-products(DBPs). In this study, the inactivation of four model strains including Gram-negative(G), Gram-positive(G) and environmental samples by atmospheric-pressure air-liquid discharge plasma(ALDP) was assessed systematically. The results showed that ALDP was superior in inactivating all of the samples compared with chlorination. During 10 min ALDP treatment, the Gbacteria were completely inactivated, and the Gone was inactivated by more than 4.61 logs. The inactivation of bacteria from a campus lake and a wastewater treatment plant effluent exceeded 99.82% and 97.78%, respectively. For G-bacteria, ALDP resulted in a much lower(10~2~10~3 times) levels of VBNC cells than chlorination. ALDP could effectively remove the chlorine-resistant bacteria. More than 96.41% of the intracellular DNA and 99.99% of the extracellular DNA were removed, whereas it was only 56.35% and 12.82% for chlorination. ALDP had a stronger ability to destroy cell structure than chlorination, presumably due to the existence of ROS( ·OH, ~1Oand O). GC-MS analysis showed that ALDP produced less DBPs than chlorination. These findings provided new insights for the application of discharge plasma in water disinfection, which could be complemental or alternative to the conventional disinfection methods.

气液界面培养肠道类器官的医学研究进展

类器官技术是继2D细胞系、动物、异种移植模型之后崭新的生物学研究体系,2017年被《Nature》杂志评选为年度最佳生物学技术。这种基于基质胶构建的类器官模型是一种能在体外模拟人体器官功能、结构的三维细胞复合体,主要由上皮细胞构成。其中,经由气液界面培养的类器官模型因其能够在体外重现肿瘤免疫微环境而受到国内外学者的关注。本文对气液类器官的诞生、发展及其在以肠道疾病为首的多种疾病中的应用进行总结概述,探讨气液界面培养的类器官体系在药物敏感性实验、中医药研究中的应用价值,并对该模型在目前研究应用中的不足与展望进行分析,旨在为未来疾病研究、药物开发以及实现个体化免疫治疗提供更有力的参考依据。

Composition and solution properties of fluorinated block copolymers and their surface structures in the solid state

A series of diblock copolymers composed of methyl methacrylate and 2-perfluorooctylethyl methacry-late(PMMA144-b-PFMAn) with various PFMA block lengths were prepared by atom transfer radical po-lymerization(ATRP).The surface structures and properties of these polymers in the solid state and in solution were investigated using contact angle measurement,X-ray photoelectron spectroscopy(XPS),sum frequency generation(SFG) vibrational spectroscopy,surface tension and dynamic laser light scattering(DLS).It was found that with increasing PFMA block length,water and oil repellency de-creased,the ratio of F/C increased with increasing film depth,and the degree of ordered packing of the perfluoroalkyl side chains at the surface decreased.When the number of PFMA block units reached 10,PMMA segments were detected at the copolymer surface,which was attributed to the PFMA block length affecting molecular aggregation structure of the copolymer in the solution and the interfacial structure at the air/liquid interface,which in turn affects surface structure formation during solution solidification.The results suggest that copolymer solution properties play an important role in struc-ture formation on the solid surface.

Meteorin-β/Meteorin like/IL-41 attenuates airway inflammation in house dust mite-induced allergic asthma

We sought to examine the regulatory effect of Meteorin-β(Metrnβ)/Meteorin like(Metrnl)/IL-41 on lung inflammation in allergic asthma.We found that Metrnβwas elevated significantly in asthmatic patients and in mice with allergic asthma induced by house dust mite(HDM)extract.Upon exposure to HDM,Metrnβwas secreted predominantly by airway epithelial cells and inflammatory cells,including macrophages and eosinophils.The increased Metrnβeffectively blocked the development of airway hyperreactivity(AHR)and decreased inflammatory cell airway infiltration and type 2 cytokine production,which was associated with downregulated DC-mediated adaptive immune responses.Moreover,Metrnβimpaired the maturation and function of bone marrow-derived dendritic cells in vitro.Asthmatic mice adoptively transferred with dendritic cells isolated from Metrnβ-treated allergic mice displayed decreased AHR,airway inflammation,and lung injury.Metrnβalso displayed anti-inflammatory properties in immunodeficient SCID mice with allergic asthma and in in vitro 3D ALI airway models.Moreover,blockade of Metrnβby anti-Metrnβantibody treatment promoted the development of allergic asthma.These results revealed the unappreciated protective roles of Metrnβin alleviating DC-mediated Th2 inflammation in allergic asthma,providing the novel treatment strategy of therapeutic targeting of Metrnβin allergic asthma.

Pre-treatment of pyridine wastewater by new cathodic–anodic-electrolysis packing

A novel cathodic–anodic-electrolysis packing（CAEP） used in the treatment of pyridine wastewater was researched, which mainly consisted of 4,4′-diamino-2,2′-disulfonic acid（DSD acid） industrial iron sludge. The physical properties and morphology of the packing were studied. The CAEP was used in a column reactor during the pretreatment of pyridine wastewater. The influence of p H, hydraulic retention time（HRT）, the air–liquid ratio（A/L）and the initial concentration of pyridine were investigated by measuring the removal of total organic carbon（TOC） and pyridine. The characterization results showed that the bulk density, grain density, water absorption percentage and specific surface area were 921 kg/m3,1086 kg/m3, 25% and 29.89 m^2/g, respectively; the removal of TOC and pyridine could reach50% and 58% at the optimal experimental conditions（p H = 3, HRT = 8 hr, A/L = 2）. Notably,the surface of the packing was renewed constantly during the running of the filter, and the handling capacity was stable after running for three months.

Patient-derived organoids potentiate precision medicine in advanced clear cell renal cell carcinoma

To investigate the role of patient-derived organoid(PDO)model in the precision medicine of advanced clear cell renal cell carcinoma(ccRCC),we retrospectively analyzed the clinical data of seven cases of ccRCC diagnosed by operation and pathology in Renji Hospital from September 2021 to September 2022.The seven patients were diagnosed with advanced ccRCC with or without remote metastasis.Cytoreductive and radical nephrectomy was performed respectively.To predict the response to immunotherapy and provide personalized medicine recommendation,a PDO model based on air-liquid interface system was established from the surgical resected tumor and subsequent drug screening was performed.Hematoxylin and eosin(H&E)staining and immunohistochemistry revealed that the PDO recapitulated the histological feature of parent tumor.Immunofluorescence staining identified that CD3^(+)T cells,SMA^(+)cancer associated fibroblasts,and CD31^(+)endothelial cells were preserved in PDO models.Fluorescence activated cell sorter(FACS)revealed an evidently increased ratio of CD8^(+)/CD4^(+)T cells and apoptotic tumor cells in PDO treated with toripalimab than those treated with IgG4.The results showed that toripalimab is able to rescue the excessive death of CD8^(+)T cells by critically reversing the immune exhaustion state of ccRCC in PDO model.This research validated that PDO is a promising and faithful preclinical model for prediction of immunotherapy response in patients with ccRCC.

Development of a standardized in vitro approach to evaluate microphysical,chemical,and toxicological properties of combustion-derived fine and ultrafine particles

Ultrafine particles represent a growing concern in the public health community but their precise role in many illnesses is still unknown. This lack of knowledge is related to the experimental difficulty in linking their biological effects to their multiple properties, which are important determinants of toxicity. Our aim is to propose an interdisciplinary approach to study fine(FP) and ultrafine(UFP) particles, generated in a controlled manner using a mini CAST(Combustion Aerosol Standard) soot generator used with two different operating conditions(CAST1 and CAST3). The chemical characterization was performed by an untargeted analysis using ultra-high resolution mass spectrometry. In conjunction with this approach, subsequent analysis by gas chromatography–mass spectrometry(GC–MS) was performed to identify polycyclic aromatic hydrocarbons(PAH). CAST1 enabled the generation of FP with a predominance of small PAH molecules, and CAST3 enabled the generation of UFP, which presented higher numbers of carbon atoms corresponding to larger PAH molecules. Healthy normal human bronchial epithelial(NHBE) cells differentiated at the air-liquid interface(ALI) were directly exposed to these freshly emitted FP and UFP. Expression of MUC5AC, FOXJ1, OCLN and ZOI as well as microscopic observation confirmed the ciliated pseudostratified epithelial phenotype. Study of the mass deposition efficiency revealed a difference between the two operating conditions, probably due to the morphological differences between the two categories of particles. We demonstrated that only NHBE cells exposed to CAST3 particles induced upregulation in the gene expression of IL-8 and NQO1. This approach offers new perspectives to study FP and UFP with stable and controlled properties.

Conditionally Reprogrammed Human Normal Airway Epithelial Cells at ALI: A Physiological Model for Emerging Viruses

Cancer cell lines have been used widely in cancer biology, and as biological or functional cell systems in many biomedical research fields. These cells are usually defective for many normal activities or functions due to significant genetic and epigenetic changes. Normal primary cell yields and viability from any original tissue specimens are usually relatively low or highly variable. These normal cells cease after a few passages or population doublings due to very limited proliferative capacity. Animal models(ferret, mouse, etc.) are often used to study virus-host interaction. However, viruses usually need to be adapted to the animals by several passages due to tropism restrictions including viral receptors and intracellular restrictions. Here we summarize applications of conditionally reprogrammed cells(CRCs), long-term cultures of normal airway epithelial cells from human nose to lung generated by conditional cell reprogramming(CR) technology, as an ex vivo model in studies of emerging viruses. CR allows to robustly propagate cells from non-invasive or minimally invasive specimens, for example, nasal or endobronchial brushing. This process is rapid(2 days) and conditional. The CRCs maintain their differentiation potential and lineage functions, and have been used for studies of adenovirus, rhinovirus, respiratory syncytial virus, influenza viruses, parvovirus, and SARS-CoV. The CRCs can be easily used for airliquid interface(ALI) polarized 3 D cultures, and these coupled CRC/ALI cultures mimic physiological conditions and are suitable for studies of viral entry including receptor binding and internalization, innate immune responses, viral replications, and drug discovery as an ex vivo model for emerging viruses.