A phosphorylated guanidine chitosan and UiO-66-NH2 modified magnetic nanoparticle platform for enrichment and detection of multiple bacteria.

Wastewater-based epidemiology (WBE) is a powerful tool for early warning of infectious disease outbreaks. Hence, a rapid and portable pathogen monitoring system is urgent needed for on-site detection. In this work, we first reported synthesis of an artificial modulated wide-spectrum bacteria capture nanoparticle (Arg-CSP@UiO@Fe3O4). Arginine-modified phosphorylated chitosan (Arg-CSP) coating could provide strongly positive charged guanidinium group for pathogen interaction by electrostatic attraction, and UiO-66-NH2 layer could help Arg-CSP graft onto Fe3O4 magnetic particles. The capture efficiency of Arg-CSP@UiO@Fe3O4 reached 92.2 % and 97.3 % for Escherichia coli (E.coli) and Staphylococcus epidermidis (S.epidermidis)within 40 min, in 10 mL sample. To prevent pathogen degradation in sewage, a portable nucleic acid extraction-free method was also developed. UiO-66-NH2 could disintegrate in buffer with high concentration of PO43- for bacterium desorption, and then nucleic acid of the bacteria was released by heating. The DNA template concentration obtained by this method was 779.28 times higher than that of the direct thermal lysis product and 8.63 times higher than that of the commercial kit. Afterwards, multiple detection of bacteria was realized by loop-mediated isothermal amplification (LAMP). Artificial regulated pathogen desorption could prevent non-specific adsorption of nucleic acid by nanoparticles. The detection limit of Arg-CSP@UiO@Fe3O4-LAMP method was 80 cfu/mL for E.coli and 300 cfu/mL for S.epidermidis. The accuracy and reliability of the method was validated by spiked sewage samples. In conclusion, this bio-monitoring system was able to detect multiple bacteria in environment conveniently and have good potential to become an alternative solution for rapid on-site pathogen detection.

Air-Liquid Interface Microfluidic Monitoring Sensor Platform for Studying Autophagy Regulation after PM2.5 Exposure.

Undoubtedly, a deep understanding of PM2.5-induced tumor metastasis at the molecular level can contribute to improving the therapeutic effects of related diseases. However, the underlying molecular mechanism of fine particle exposure through long noncoding RNA (lncRNA) regulation in autophagy and, ultimately, lung cancer (LC) metastasis remains elusive; on the other hand, the related monitoring sensor platform used to investigate autophagy and cell migration is lacking. Herein, this study performed an air-liquid interface microfluidic monitoring sensor (AIMMS) platform to analyze human bronchial epithelial cells after PM2.5 stimulation. The multiomics analysis [RNA sequencing (RNA-seq) on lncRNA and mRNA expressions separately] showed that MALAT1 was highly expressed in the PM2.5 treatment group. Furthermore, RNA-seq analysis demonstrated that autophagy-related pathways were activated. Notably, the main mRNAs associated with autophagy regulation, including ATG4D, ATG12, ATG7, and ATG3, were upregulated. Inhibition or downregulation of MALAT1 inhibited autophagy via the ATG4D/ATG12/ATG7/ATG3 pathway after PM2.5 exposure and ultimately suppressed LC metastasis. Thus, based on the AIMMS platform, we found that MALAT1 might become a promising therapeutic target. Furthermore, this low-cost AIMMS system as a fluorescence sensor integrated with the cell-monitor module could be employed to study LC migration after PM2.5 exposure. With the fluorescence cell-monitoring module, the platform could be used to observe the migration of LC cells and construct the tumor metastasis model. In the future, several fluorescence probes, including nanoprobes, could be used in the AIMMS platform to investigate many other biological processes, especially cell interaction and migration, in the fields of toxicology and pharmacology.

Proteinase K-pretreated ConA-based ELISA assay: a novel urine LAM detection strategy for TB diagnosis.

Objectives: Lipoarabinomannan (LAM), an abundant cell wall glycolipid of mycobacteria including Mycobacterium tuberculosis (Mtb), is a promising TB diagnostic marker. The current commercially available urine LAM assays are not sufficiently sensitive, and more novel detection strategies are urgently needed to fill the current diagnostic gap. Methods: A proteinase K-pretreated Concanavalin A (ConA)-based ELISA assay was developed. Diagnostic performance was assessed by several bacterial strains and clinical urine samples. Results: The limit of detection (LoD) of the assay against ManLAM was 6 ng/ml. The assay reacted strongly to Mtb H37Rv and M. bovis BCG, intermediately to M. smegmatis mc2155, and weakly to four non-mycobacteria pathogens. This method could distinguish TB patients from healthy controls (HCs) and close contacts (CCs) in 71 urine samples treated with proteinase K, which increases urine LAM antibody reactiveness. In TB+HIV+ and TB+HIV- patients, the sensitivity was 43.8 and 37.5%, respectively, while the specificity was 100.0%. The areas under ROC curves (AUCs) were 0.74 and 0.82, respectively. Conclusion: This study implies that ConA can be paired with antibodies to detect LAM. Proteinase K treatment could effectively enhance the sensitivity by restoring the reactiveness of antibodies to LAM.

Development and evaluation of a centrifugal disk system for the rapid detection of multiple pathogens and their antibiotic resistance genes in urinary tract infection.

Background: Urinary tract infections (UTIs) are some of the most common bacterial infections in the world. Nevertheless, as uncomplicated UTIs are treated empirically without culturing the urine, adequate knowledge of the resistance pattern of uropathogens is essential. Conventional urine culture and identification take at least 2 days. Here, we developed a platform based on LAMP and centrifugal disk system (LCD) to simultaneously detect the main pathogens and antibiotic resistant genes (ARGs) of urgent concern multidrug-resistant among UTIs. Methods: We designed specific primers to detect the target genes above and evaluated their sensitivity and specificity. We also assessed the result of our preload LCD platform on 645 urine specimens with a conventional culturing method and Sanger sequencing. Results: The results obtained with the 645 clinical samples indicated that the platform has high specificity (0.988-1) and sensitivity (0.904-1) for the studied pathogens and ARGs. Moreover, the kappa value of all pathogens was more than 0.75, revealing an excellent agreement between the LCD and culture method. Compared to phenotypic tests, the LCD platform is a practical and fast detection approach for methicillin-resistant Staphylococcus aureus, vancomycin-resistant Enterococci, carbapenem-resistant Enterobacteriaceae, carbapenem-resistant Acinetobacter, carbapenem-resistant Pseudomonas aeruginosa (kappa value of all >0.75), and non-extended-spectrum ß-lactamase producers. Conclusion: We developed a detection platform that has high accuracy and that meets the need for rapid diagnosis, which can be completed within 1.5 h from specimen collection. It may be a powerful tool for evidence-based UTIs diagnosis, which has essential support for the rational use of antibiotics. More high-quality clinical studies are required to prove the effectiveness of our platform.

Assessment of cancer-related signaling pathways in responses to polystyrene nanoplastics via a kidney-testis microfluidic platform (KTP).

As a new type of environmental pollutants, micro/nano plastics (MPs/NPs) derived from plastic products are commonly contact in daily life and lead to some serious health issues. The toxicity effects of MPs/NPs on the human body have aroused wide concerns. Although MPs/NPs have been reported to be transmitted into the kidney and reproductive organs, the molecular mechanisms of MPs/NPs toxicity remain unclear due to the lack of a physiologically relevant organ-organ linking platform in vitro. Here, we present a kidney-testis microfluidic platform (KTP) with NPs exposure that enables the communication of kidney and testis chambers and reproduces endothelium-linked chambers to simulate the state in vivo. The function of KTP was assessed by cell counting kit (CCK-8), tight junction protein claudin-2 and glucose consumption. Results revealed that MPs/NPs entered the kidney and testis via endocytosis. Immunofluorescence and ELISA analysis were performed on KTP at 200 µg/mL PS-NP to identify the dysregulated proteins on cancer-related signaling pathways, including the MAPK signaling pathway (RTK, RAS, ERK, JNK, P38, NRF2, TNF-α, and TNF-α-R) and the PI3K-AKT signaling pathway (PI3K, AKT, MDM2, P53, and ΒΑD). This multi-organ platform (KTP) contributes to clarifying cancer pathways triggered by MPs/NPs exposure and provides a promising method for assessing diseases induced by environmental pollutants.

Primary exploration of host-microorganism interaction and enteritis treatment with an embedded membrane microfluidic chip of the human intestinal-vascular microsystem.

Intestinal flora plays a crucial role in the host's intestinal health. Imbalances in the intestinal flora, when accompanied by inflammation, affect the host's intestinal barrier function. Understanding it requires studying how living cells and tissues work in the context of living organs, but it is difficult to form the three-dimensional microstructure intestinal-vascular system by monolayer cell or co-culture cell models, and animal models are costly and slow. The use of microfluidic-based organ chips is a fast, simple, and high-throughput method that not only solves the affinity problem of animal models but the lack of microstructure problem of monolayer cells. In this study, we designed an embedded membrane chip to generate an in vitro gut-on-a-chip model. Human umbilical vein endothelial cells and Caco-2 were cultured in the upper and lower layers of the culture chambers in the microfluidic chip, respectively. The human peripheral blood mononuclear cells were infused into the capillary side at a constant rate using an external pump to simulate the in vitro immune system and the shear stress of blood in vivo. The model exhibited intestine morphology and function after only 5 days of culture, which is significantly less than the 21 days required for static culture in the Transwell® chamber. Furthermore, it was observed that drug-resistant bacteria triggered barrier function impairment and inflammation, resulting in enteritis, whereas probiotics (Lactobacillus rhamnosus GG) improved only partially. The use of Amikacin for enteritis is effective, whereas other antibiotic therapies do not work, which are consistent with clinical test results. This model may be used to explore intestinal ecology, host and intestinal flora interactions, and medication assessment.

Fully Automated CRISPR-LAMP Platform for SARS-CoV-2 Delta and Omicron Variants.

Integrated clustered regularly interspaced short palindromic repeat (CRISPR)-loop-mediated amplification (LAMP) technology is of great importance in CRISPR-based diagnostic systems, which urgently needs to be developed to improve diagnostic accuracy. A labor-free, contamination-free, and fully automated droplet manipulation platform for the CRISPR-LAMP technology has not been developed before. Herein, we propose a fully automated CRISPR-LAMP platform, which can precisely manipulate the CRISPR-LAMP droplet and perform combined reactions with high sensitivity and specificity. SARS-CoV-2 Spike T478K, D614G, P681R, and P681H mutations, typical point mutations of B.1.617.2 (Delta) and Omicron variants, are monitored with this platform with a detection limit of 102 copies/µL. Allele discrimination between the mutants and wild type is significant with the designed one/two-mismatch CRISPR RNA (crRNA) at a limit of 102 copies/µL. Chemically synthesized and modified crRNAs greatly increase the CRISPR-LAMP signal, which advance the wide application. Combined with the previously developed RdRp CRISPR-LAMP assay, clinical results showed that Spike T478K and P681H can discriminate the mutant type form the wild type with 70% (49.66-85.50%, 95% confidence interval) and 78% (57.27-90.62%, 95% confidence interval) sensitivity, respectively, and 100% specificity (51.68-100%, 95% confidence interval), and the RdRp target can detect SARS-CoV-2 strains with 85% sensitivity (65.39-95.14%, 95% confidence interval) and 100% specificity (51.68-100%, 95% confidence interval). We believe that this automatic digital microfluid (DMF) system can advance the integrated CRISPR-LAMP technology with higher stability, sensitivity, and practicability, also for other CRISPR-associated diagnostic platforms.

A rapid multiplex nucleic acid detection system of airborne fungi by an integrated DNA release device and microfluidic chip.

Occupational health problems, such as asthma, in specific work environments arise from the presence of airborne fungi. Rapid detection of pathogenic airborne fungi is therefore important to reduce or avoid any adverse effects on staff health. Herein, we established a new integrated rapid Lyticase-Motor-Chemical reagent nucleic acid releasing (LMC) method for the release of fungal DNA. Aspergillus fumigatus, Aspergillus flavus, and Cryptococcus neoformans were chosen to evaluate the LMC method. The results of Loop-Mediated Isothermal Amplification (LAMP) analyses showed that this method could release the nucleic acid of 4 × 104 fungal spores, equaling to 400 copies per microliter. This rapid multiplex nucleic acid detection system of airborne fungi included an integrated DNA release device and a portable microfluidic chip. The integrated DNA release device combined mechanical lysing and biochemical reagent treatment to automate DNA release. The microfluidic chip was capable of multiplex nucleic acid detection. The detection limit of this system was 4 × 104 spores per test, meeting the requirement of early warnings. The whole analysis from the sample input to readout could be completed within 90 min, including 30 min for fungal DNA release and 45 min for LAMP analysis. The integrated DNA release device and microfluidic chip were portable, showing tremendous potential in point-of-care tests of airborne fungi.

Recent developments and trends of automatic nucleic acid detection systems.

Nucleic acid detection, widely used in clinical diagnosis, biological analysis, and environmental monitoring, is of great significance for disease diagnosis and basic research. With the outbreak of COVID-19, the demand for fast and high-throughput nucleic acid detection from large numbers of samples has increased sharply. Automated nucleic acid detection systems can meet these needs, and also play important roles in disease screening and infectious disease prevention and control. In this review, we introduce and compare the current mainstream nucleic acid automatic detection instruments and equipment, then discuss the future demands of nucleic acid detection.

Biosafety of human environments can be supported by effective use of renewable biomass.

Preventing pathogenic viral and bacterial transmission in the human environment is critical, especially in potential outbreaks that may be caused by the release of ancient bacteria currently trapped in the permafrost. Existing commercial disinfectants present issues such as a high carbon footprint. This study proposes a sustainable alternative, a bioliquid derived from biomass prepared by hydrothermal liquefaction. Results indicate a high inactivation rate of pathogenic virus and bacteria by the as-prepared bioliquid, such as up to 99.99% for H1N1, H5N1, H7N9 influenza A virus, and Bacillus subtilis var. niger spores and 99.49% for Bacillus anthracis Inactivation of Escherichia coli and Staphylococcus epidermidis confirmed that low-molecular-weight and low-polarity compounds in bioliquid are potential antibacterial components. High temperatures promoted the production of antibacterial substances via depolymerization and dehydration reactions. Moreover, bioliquid was innoxious as confirmed by the rabbit skin test, and the cost per kilogram of the bioliquid was $0.04427, which is notably lower than that of commercial disinfectants. This study demonstrates the potential of biomass to support our biosafety with greater environmental sustainability.

A point-of-care test device for MRSA rapid detection.

Staphylococcus aureus (SA) is one of the most common pathogenic bacteria, and methicillin-resistant SA (MRSA) is an equally common drug-resistant bacteria. MRSA detection is of great significance for clinical diagnosis, medication guidance, and prevention of antibiotic abuse. Traditional MRSA detection using the culture method is time-consuming, laborious, and difficult to conduct rapid on-site detection. In this research, we developed a device for rapid MRSA detection, which can detect the nuc gene in SA and mecA gene in MRSA simultaneously for 30-40 min. After simple sample processing, the mixture can be directly loaded onto the chip device. The detection results can be directly determined by a color change, with a limitation of approximately 102 copies. This isothermal amplification chip device can be widely applied in many fields, with simple operation and low contamination.

A LAMP-based microfluidic module for rapid detection of pathogen in cryptococcal meningitis.

Cryptococcal meningitis (CM) is a global threat with significant attributable morbidity and mortality. Information on microfluidic detection for CM diagnosis is still limited. We developed a multifunctional microfluidic module that integrated the pathogen enrichment and on-chip nucleic acid extraction. The module adopted a simple filtration membrane to effectively capture Cryptococcus cells and simplify the process, and combined lyticase digestion, alkaline lysis and heating methods to optimize the strategy to achieve nucleic acid extraction. The entire process was operated in the module, which reduced the exposure risk of directly processing cryptococcal samples. A portable one-pot lyophilized LAMP reagent bead with no temperature limit was developed, which improved the flexibility of operation. This module did not require any additional instrument, and is promising to develop a simple, rapid, and efficient approach to realize the "sample in and answer out" detection of real CSF samples. This microfluidic module had practical prospects and is expected to replace LFA for efficacy evaluation and follow-up in the middle and late stages of CM treatment, and could be used as an auxiliary method to confirm cases with questionable LFA results in the early diagnosis of CM.

Universally Stable and Precise CRISPR-LAMP Detection Platform for Precise Multiple Respiratory Tract Virus Diagnosis Including Mutant SARS-CoV-2 Spike N501Y.

Nowadays, rapid and accurate diagnosis of respiratory tract viruses is an urgent need to prevent another epidemic outbreak. To overcome this problem, we have developed a clustered, regularly interspaced short palindromic repeats (CRISPR) loop mediated amplification (LAMP) technology to detect influenza A virus, influenza B virus, respiratory syncytial A virus, respiratory syncytial B virus, and severe acute respiratory syndrome coronavirus 2, including variants of concern (B.1.1.7), which utilized CRISPR-associated protein 12a (Cas12a) to advance LAMP technology with the sensitivity increased 10 times. To reduce aerosol contamination in CRISPR-LAMP technology, an uracil-DNA-glycosylase-reverse transcription-LAMP system was also developed which can effectively remove dUTP-incorporated LAMP amplicons. In vitro Cas12a cleavage reaction with 28 crRNAs showed that there were no position constraints for Cas12a/CRISPR RNA (crRNA) recognition and cleavage in LAMP amplicons, and even the looped position of LAMP amplicons could be effectively recognized and cleaved. Wild-type or spike N501Y can be detected with a limit of detection of 10 copies/µL (wild-type) even at a 1% ratio level on the background (spike N501Y). Combining UDG-RT-LAMP technology, CRISPR-LAMP design, and mutation detection design, we developed a CRISPR-LAMP detection platform that can precisely diagnose pathogens with better stability and significantly improved point mutation detection efficiency.

A LAMP-based system for rapid detection of eight common pathogens causing lower respiratory tract infections.

Lower respiratory tract infections (LRTIs) are a leading cause of morbidity and mortality worldwide and lack a rapid diagnostic method. To improve the diagnosis of LRTIs, we established an available loop-mediated isothermal amplification (LAMP) assay for the detection of eight common lower respiratory pathogens, including Klebsiella pneumoniae, Pseudomonas aeruginosa, Acinetobacter baumannii, Staphylococcus aureus, Escherichia coli, Haemophilus influenzae, Streptococcus pneumoniae, and Moraxella catarrhalis. The whole process can be achieved within 1 h (sample to results read out). We established an extraction free isothermal system. 528 sputum samples collected from patients suspected to have LRTIs were analyzed by the system (8 tests in each sample, a total of 4224 tests) and compared with the standard culture method (SCM). The samples with inconsistent results were further verified by Sanger sequencing and High-throughput sequencing (NGS). The detection limits of the LAMP assay for the 8 pathogens ranged from 103 to 104 CFU/mL. Upon testing 528 samples, the Kappa coefficients of all pathogens ranged between 0.5 and 0.7 indicated a moderate agreement between the LAMP assay and the SCM. All inconsistent samples were further verified by Sanger sequencing, we found that the developed LAMP assay had a higher consistency level with Sanger sequencing than the SCM for all pathogens. Additionally, when the NGS was set to a diagnostic gold standard, the specificity and sensitivity of the LAMP assay for LRTIs were 94.49% and 75.00%. The present study demonstrated that the developed LAMP has high consistency with the sequencing methods. Meanwhile, the LAMP assay has a higher detection rate compared to the SCM. It may be a powerful tool for rapid and reliable clinical diagnosis of LRTIs in primary hospitals.

Cytotoxicity analysis of biomass combustion particles in human pulmonary alveolar epithelial cells on an air-liquid interface/dynamic culture platform.

BACKGROUND: Exposure to indoor air pollution from solid fuel combustion is associated with lung diseases and cancer. This study investigated the cytotoxicity and molecular mechanisms of biomass combustion-derived particles in human pulmonary alveolar epithelial cells (HPAEpiC) using a platform that combines air-liquid interface (ALI) and dynamic culture (DC) systems. METHODS: HPAEpiC were cultured on the surface of polycarbonate (PC) membranes on the ALI-DC platform. The cells were sprayed with an aerosolized solution of biomass combustion soluble constituents (BCSCs) and simultaneously nourished with culture medium flowing beneath the permeable PC membranes. The ALI-DC method was compared with the traditional submerged culture approach. BCSC particle morphology and dosages deposited on the chip were determined for particle characterization. Flow cytometry, scanning electron microscopy, and transmission electron microscopy were used to investigate the apoptosis rate of HPAEpiC and changes in the cell ultrastructure induced by BCSCs. Additionally, the underlying apoptotic pathway was examined by determining the protein expression levels by western blotting. RESULTS: Scanning electron microscope images demonstrated that the sample processing and delivering approach of the ALI-DC platform were suitable for pollutant exposure. Compared with the submerged culture method, a significant decline in cell viability and increase in apoptosis rate was observed after BCSC exposure on the ALI-DC platform, indicating that the ALI-DC platform is a more sensitive system for investigating cytotoxicity of indoor air pollutants in lung cells. The morphology and ultrastructure of the cells were damaged after exposure to BCSCs, and the p53 pathway was activated. The Bcl-2/Bax ratio was reduced, upregulating caspase-9 and caspase-3 expression and subsequently inducing apoptosis of HPAEpiC. The addition of N-acetyl cysteine antioxidant significantly alleviated the cytotoxicity induced by BCSCs. CONCLUSION: A novel ALI-DC platform was developed to study the cytotoxicity of air pollutants on lung cells. Using the platform, we demonstrated that BCSCs could damage the mitochondria, produce reactive oxygen species, and activate p53 in HPAEpiC, ultimately inducing apoptosis.

A Three-Dimensional Micromixer Using Oblique Embedded Ridges.

A micromixer is one of the most significant components in a microfluidic system. A three-dimensional micromixer was developed with advantages of high efficiency, simple fabrication, easy integration, and ease of mass production. The designed principle is based on the concepts of splitting-recombination and chaotic advection. A numerical model of this micromixer was established to characterize the mixing performance for different parameters. A critical Reynolds number (Re) was obtained from the simulation results. When the Re number is smaller than the critical value, the fluid mixing is mainly dependent on the mechanism of splitting-recombination, therefore, the length of the channel capable of complete mixing (complete mixing length) increases as the Re number increases. When the Re number is larger than the critical value, the fluid mixing is dominated by chaotic advection, and the complete mixing length decreases as the Re number increases. For normal fluids, a complete mixing length of 500 µm can be achieved at a very small Re number of 0.007 and increases to 2400 µm as the Re number increases to the critical value of 4.7. As the Re number keep increasing and passes the critical Re number, the complete mixing length continues to descend to 650 µm at the Re number of 66.7. For hard-to-mix fluids (generally referring to fluids with high viscosity and low diffusion coefficient, which are difficult to mix), even though no evidence of strong chaotic advection is presented in the simulation, the micromixer can still achieve a complete mixing length of 2550 µm. The mixing performance of the micromixer was also verified by experiments. The experimental results showed a consistent trend with the numerical simulation results, which both climb upward when the Re number is around 0.007 (flow rate of 0.03 µm/min) to around 10 (flow rate of 50 µm/min), then descend when the Re number is around 13.3 (flow rate of 60 µm/min).

Sorption of organochlorine pesticides on polyethylene microplastics in soil suspension.

As a new type of environmental pollutant, microplastics (MPs) can adsorb residual organochlorine pesticides (OCPs) in the soil and pose a severe threat to the soil ecosystems. To understand the interaction between soil MPs and OCPs, the sorption of two kinds of OCPs, including hexachlorocyclohexanes (HCHs) and dichlorodiphenyltrichloroethanes (DDTs), on polyethylene (PE) microplastics in soil suspension was studied through sorption kinetics and isotherm models. The effects of solution/soil ratio and MPs diameter on sorption were examined. The kinetic experiment results show that the sorption equilibrium was 12 h， and the sorption process of OCPs on MPs can be well described by a pseudo-second-order model. The Freundlich model (R2 = 0.942-0.997) provides a better fit to the sorption isotherm data than the Langmuir model (R2 = 0.062-0.634), indicating that the sorption process takes place on the nonuniform surface of MPs. The MPs had a good sorption effect on OCPs when the solution/soil ratio was from 75:1 to 100:1. As the diameter of MPs increases, the sorption capacity decreases. These results provide support for further research on microplastic pollution in soil.

The decay of airborne bacteria and fungi in a constant temperature and humidity test chamber.

Despite substantial research to profile the microbial characteristics in the atmosphere, the changing metabolism underpinning microbial successional dynamics remains ambiguous. Herein, we applied qPCR, high-throughput sequencing of the genes encoding 16S and ITS rRNA to render the bacterial/fungal dynamics of ambient PM2.5 filters maintained at constant conditions of temperature (20 ± 2 °C) and humidity (50 ± 5%). The incubation experiments which lasted for 50 days aim to simulate a metabolic process of microbe in two types PM2.5 (polluted and non-polluted). The results show that microbial community species in polluted PM2.5 had faster decay rates, more bacterial diversity and less fungal community compared to the non-polluted ones. For bacteria, the proportion of anaerobic species is higher than aerobic ones, and their performance of contain mobile elements, form-biofilms, and pathogenic risks declined rapidly as times went by. Whereas for fungi, saprotroph species occupied about 70% of the population, resulting in a specified peak of abundance due to the adequacy nutrients supplied by the apoptosis cells. Combining the classified microbial species, we found stable community structure and the volatile ones related to the various metabolic survival strategies during different time. Without the input of peripheral environment, the health risks of airborne microbe descend to a healthy level after 20 days, implying their biologic effectiveness was about 20 days no matter the air is polluted or not. This study provided new insights into the different metabolic survival of airborne microorganisms in ideal and stable conditions.

Association of PM2.5 with Insulin Resistance Signaling Pathways on a Microfluidic Liver-Kidney Microphysiological System (LK-MPS) Device.

Insulin resistance (IR) is a typical sign of metabolic dysregulation caused by fine particulate matter (PM2.5), but the underlying signaling has not been clearly determined. Herein, a microfluidic liver-kidney microphysiological system (LK-MPS) is presented to assess the signaling pathways of IR generated by PM2.5 at 200 µg/mL for 24 h. The LK-MPS device consisted of a biomimetic liver-kidney architecture and reconstructed two circulation paths: the liver metabolism-kidney excretion (LM-KE) and kidney excretion-liver metabolism (KE-LM), by which PM2.5 is feasibly distributed in the two organs. Transmission electron microscopy (TEM) analysis revealed that PM2.5 can embed in the cytoplasm and nuclei, undergo transport by vesicles, and lead to the destruction of mitochondria. Further comprehensive immunofluorescence, enzyme-linked immunosorbent assays (ELISAs) and untargeted metabolomic analyses confirmed that PM2.5 disturbed the classic IRS-1/AKT signaling pathway (INSR, IRS-1, PI3K, AKT, GLUT2, GLUT4, and FOXO1 downregulated) and IR-related metabolic pathways: UDP-hexosamine (UDP-GlcNAc), gluconeogenesis (ß-d-glucose 6-phosphate), and lipid biosynthesis (ceramide (Cer) and triacylglycerol (TG)) pathways, leading to the disorder of glucose levels. Collectively, these disorders aggravate hepatic and renal IR. Pearson's correlation coefficient test showed that elemental carbon (EC), polycyclic aromatic hydrocarbons (PAHs), and metals (Ca, Co, and V) were negatively correlated to the dysregulated proteins (INSR, IRS-1, AKT, FOXO1, GLUT2, and GLUT4). These findings may partially explain IR-related signaling pathways triggered by PM2.5.