Hierarchically porous and single Zn atom-embedded carbon molecular sieves for H2 separations.

Hierarchically porous materials containing sub-nm ultramicropores with molecular sieving abilities and microcavities with high gas diffusivity may realize energy-efficient membranes for gas separations. However, rationally designing and constructing such pores into large-area membranes enabling efficient H2 separations remains challenging. Here, we report the synthesis and utilization of hybrid carbon molecular sieve membranes with well-controlled nano- and micro-pores and single zinc atoms and clusters well-dispersed inside the nanopores via the carbonization of supramolecular mixed matrix materials containing amorphous and crystalline zeolitic imidazolate frameworks. Carbonization temperature is used to fine-tune pore sizes, achieving ultrahigh selectivity for H2/CO2 (130), H2/CH4 (2900), H2/N2 (880), and H2/C2H6 (7900) with stability against water vapor and physical aging during a continuous 120-h test.

Creation of Polymer Datasets with Targeted Backbones for Screening of High-Performance Membranes for Gas Separation.

A simple approach was developed to computationally construct a polymer dataset by combining simplified molecular-input line-entry system (SMILES) strings of a targeted polymer backbone and a variety of molecular fragments. This method was used to create 14 polymer datasets by combining seven polymer backbones and molecules from two large molecular datasets (MOSES and QM9). Polymer backbones that were studied include four polydimethylsiloxane (PDMS) based backbones, poly(ethylene oxide) (PEO), poly(allyl glycidyl ether) (PAGE), and polyphosphazene (PPZ). The generated polymer datasets can be used for various cheminformatics tasks, including high-throughput screening for gas permeability and selectivity. This study utilized machine learning (ML) models to screen the polymers for CO2/CH4 and CO2/N2 gas separation using membranes. Several polymers of interest were identified. The results highlight that employing an ML model fitted to polymer selectivities leads to higher accuracy in predicting polymer selectivity compared to using the ratio of predicted permeabilities.

AG1478 Elicits a Novel Anti-Influenza Function via an EGFR-Independent, GBF1-Dependent Pathway.

Current options for preventing or treating influenza are still limited, and new treatments for influenza viral infection are urgently needed. In the present study, we serendipitously found that a small-molecule inhibitor (AG1478), previously used for epidermal growth factor receptor (EGFR) inhibition, demonstrated a potent activity against influenza both in vitro and in vivo. Surprisingly, the antiviral effect of AG1478 was not mediated by its EGFR inhibitory activity, as influenza virus was insensitive to EGFR blockade by other EGFR inhibitors or by siRNA knockdown of EGFR. Its antiviral activity was also interferon independent as demonstrated by a Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) knockout approach. Instead, AG1478 was found to target the Golgi-specific brefeldin A-resistance guanine nucleotide exchange factor 1 (GBF1)-ADP-ribosylation factor 1 (ARF1) system by reversibly inhibiting GBF1 activity and disrupting its Golgi-cytoplasmic trafficking. Compared to known GBF1 inhibitors, AG1478 demonstrated lower cellular toxicity and better preservation of Golgi structure. Furthermore, GBF1 was found to interact with a specific set of viral proteins including M1, NP, and PA. Additionally, the alternation of GBF1 distribution induced by AG1478 treatment disrupted these interactions. Because targeting host factors, instead of the viral component, imposes a higher barrier for developing resistance, GBF1 modulation may be an effective approach to treat influenza infection.

Single-Tube Multiplex Digital Polymerase Chain Reaction Assay for Molecular Diagnosis and Prediction of Severity of Spinal Muscular Atrophy.

Spinal muscular atrophy (SMA) is an autosomal recessive neuromuscular disease characterized by the degeneration of motor neurons and progressive muscle atrophy. Accurate detection of SMN1 and SMN2 copy numbers is essential for SMA diagnosis, carrier screening, disease severity prediction, therapy, and prognosis. However, a method for SMN1 and SMN2 copy number determination that is simultaneously accurate, simple, rapid, multitargeted, and applicable to various samples has not previously been reported. Here, we developed a single-tube multiplex digital polymerase chain reaction (dPCR) assay for simultaneous determination of the copy numbers of SMN1 exons 7 and 8 and SMN2 exons 7 and 8. A total of 317 clinical samples, including peripheral blood, amniotic fluid, chorionic villus, buccal swabs, and dried blood spots, were collected to evaluate the performance of this dPCR-based assay. The test results were accurate for all the clinical samples. Our assay is accurate, rapid, easy to handle, and applicable to many types of samples and uses a small amount of DNA; it is a powerful tool for SMA molecular diagnosis, large-scale screening, and disease severity assessment.

Applications of digital PCR in COVID-19 pandemic.

The coronavirus disease 2019 (COVID-19) pandemic has led to a public health crisis and global panic. This infectious disease is caused by a novel coronavirus named severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Digital polymerase chain reaction (dPCR), which is an emerging nucleic acid amplification technology that allows absolute quantification of nucleic acids, plays an important role in the detection of SARS-CoV-2. In this review, we introduce the principle and advantages of dPCR, and review the applications of dPCR in the COVID-19 pandemic, including detection of low copy number viruses, measurement of the viral load, preparation of reference materials, monitoring of virus concentration in the environment, detection of viral mutations, and evaluation of anti-SARS-CoV-2 drugs. We also discuss the challenges of dPCR in clinical practice.

Interlaboratory assessment of quantification of SARS-CoV-2 RNA by reverse transcription digital PCR.

The pandemic of the novel coronavirus disease 2019 (COVID-19) has caused severe harm to the health of people all around the world. Molecular detection of the pathogen, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), played a crucial role in the control of the disease. Reverse transcription digital PCR (RT-dPCR) has been developed and used in the detection of SARS-CoV-2 RNA as an absolute quantification method. Here, an interlaboratory assessment of quantification of SARS-CoV-2 RNA was organized by the National Institute of Metrology, China (NIMC), using in vitro transcribed RNA samples, among ten laboratories on six different dPCR platforms. Copy number concentrations of three genes of SARS-CoV-2 were measured by all participants. Consistent results were obtained with dispersion within 2.2-fold and CV% below 23% among different dPCR platforms and laboratories, and Z' scores of all the reported results being satisfactory. Possible reasons for the dispersion included PCR assays, partition volume, and reverse transcription conditions. This study demonstrated the comparability and applicability of RT-dPCR method for quantification of SARS-CoV-2 RNA and showed the capability of the participating laboratories at SARS-CoV-2 test by RT-dPCR platform.

Scalable Polymeric Few-Nanometer Organosilica Membranes with Hydrothermal Stability for Selective Hydrogen Separation.

Nanoporous silica membranes exhibit excellent H2/CO2 separation properties for sustainable H2 production and CO2 capture but are prepared via complicated thermal processes above 400 °C, which prevent their scalable production at a low cost. Here, we demonstrate the rapid fabrication (within 2 min) of ultrathin silica-like membranes (∼3 nm) via an oxygen plasma treatment of polydimethylsiloxane-based thin-film composite membranes at 20 °C. The resulting organosilica membranes unexpectedly exhibit H2 permeance of 280-930 GPU (1 GPU = 3.347 × 10-10 mol m-2 s-1 Pa-1) and H2/CO2 selectivity of 93-32 at 200 °C, far surpassing state-of-the-art membranes and Robeson's upper bound for H2/CO2 separation. When challenged with a 3 d simulated syngas test containing water vapor at 200 °C and a 340 d stability test, the membrane shows durable separation performance and excellent hydrothermal stability. The robust H2/CO2 separation properties coupled with excellent scalability demonstrate the great potential of these organosilica membranes for economic H2 production with minimal carbon emissions.

Establishment of primary reference measurement procedures and reference materials for EGFR variant detection in non-small cell lung cancer.

Circulating tumor DNA (ctDNA)-based mutation detection is promising to change the clinical practice of genotype-directed therapy for cancer. A growing number of non-invasive tests for cancer screening and monitoring that involve the detection of ctDNA have been commercialized. Primary reference measurement procedures (PRMPs) and reference materials (RMs) are urgently needed to assess the non-invasive tests. In this study, a PRMP based on digital PCR (dPCR) and ctDNA RMs for quantification of the frequently occurring variant in epidermal growth factor receptor (EGFR L858R, T790M, and 19Del) in non-small cell lung cancer (NSCLC) were established. The candidate dPCR PRMP showed high specificity (false positive rate 0-0.003%), good repeatability (coefficient of variance (CV), 2-3% for 104 copies/reaction), and high interlaboratory reproducibility (3-10%). A good linearity (0.97 < slope < 1.03, R2 ≥ 0.9999) between the measured mutant (MU) value and prepared value was observed for all assays over the fractional abundance (FA) range, between 25% and 0.05%. The limit of quantification (LoQ) was determined to be 34 L858R, 23 T790M, and 34 19Del copies/reaction, corresponding to a FA of 0.2%. An inter-laboratory study of using the EGFR ctDNA RMs and dPCR assays demonstrated that the participating laboratories produced consistent concentrations of MU and wild-type (WT), as well as FA. This study demonstrates that dPCR can act as a potential PRMP for EGFR mutation for validation of NSCLC genotyping tests and ctDNA quantitative tests. The PRMP and RMs established here could improve interlaboratory repeatability and reproducibility, which supports rapid translation and application of non-invasive tests into clinical practice.

Development of a Droplet Digital Polymerase Chain Reaction for Sensitive Detection of Pneumocystis jirovecii in Respiratory Tract Specimens.

Background: Pneumocystis jirovecii is a human-specific opportunistic fungus that causes Pneumocystis pneumonia (PCP), a life-threatening opportunistic lung infection that affects immunocompromised patients. P. jirovecii colonization may be linked to the transmission of the infection. The detection of P. jirovecii in immunocompromised patients is thus especially important. The low fungal load and the presence of PCR inhibitors limit the usefulness of quantitative PCR (qPCR) for accurate absolute quantification of P. jirovecii in specimens. Droplet digital PCR (ddPCR), however, presents a methodology that allows higher sensitivity and accuracy. Here, we developed a ddPCR method for detecting P. jirovecii DNA in respiratory specimens, and evaluated its sensitivity against qPCR. Materials and Methods: One bronchoalveolar fluid (BALF) sample each was collected from 82 patients with potential PCP to test the presence of P. jirovecii DNA using both ddPCR and qPCR, and samples with inconsistent results between the two methods were further tested by metagenomic next generation sequencing (mNGS). In addition, 37 sputum samples from 16 patients diagnosed with PCP, as well as continuous respiratory tract specimens from nine patients with PCP and treated with sulfonamides, were also collected for P. jirovecii DNA testing using both ddPCR and qPCR. Results: ddPCR and qPCR gave the same results for 95.12% (78/82) of the BALF samples. The remaining four specimens tested positive using ddPCR but negative using qPCR, and they were found to be positive by mNGS. Detection results of 78.37% (29/37) sputum samples were consistent between ddPCR and qPCR, while the other eight samples tested positive using ddPCR but negative using qPCR. The P. jirovecii load of patients with PCP decreased to undetectable levels after treatment according to qPCR, but P. jirovecii was still detectable using ddPCR. Conclusions: ddPCR was more sensitive than qPCR, especially at detecting low-pathogen-load P. jirovecii. Thus, ddPCR represents a useful, viable, and reliable alternative to qPCR in P. jirovecii testing in patients with immunodeficiency.

3D printed MOF-based mixed matrix thin-film composite membranes.

MOF-based mixed-matrix membranes (MMMs) have attracted considerable attention due to their tremendous separation performance and facile processability. In large-scale applications such as CO2 separation from flue gas, it is necessary to have high gas permeance, which can be achieved using thin membranes. However, there are only a handful of MOF MMMs that are fabricated in the form of thin-film composite (TFC) membranes. We propose herein the fabrication of robust thin-film composite mixed-matrix membranes (TFC MMMs) using a three dimensional (3D) printing technique with a thickness of 2-3 µm. We systematically studied the effect of casting concentration and number of electrospray cycles on membrane thickness and CO2 separation performance. Using a low concentration of polymer of intrinsic microporosity (PIM-1) or PIM-1/HKUST-1 solution (0.1 wt%) leads to TFC membranes with a thickness of less than 500 nm, but the fabricated membranes showed poor CO2/N2 selectivity, which could be attributed to microscopic defects. To avoid these microscale defects, we increased the concentration of the casting solution to 0.5 wt% resulting in TFC MMMs with a thickness of 2-3 µm which showed three times higher CO2 permeance than the neat PIM-1 membrane. These membranes represent the first examples of 3D printed TFC MMMs using the electrospray printing technique.

Evaluation of the clinical performance of single-, dual-, and triple-target SARS-CoV-2 RT-qPCR methods.

BACKGROUND: The coronavirus disease 2019 (COVID-19) has become a pandemic. Reverse transcription quantitative PCR (RT-qPCR) has played a vital role in the diagnosis of COVID-19, but the rates of false negatives is not ideal in dealing with this highly infectious virus. It is thus necessary to systematically evaluate the clinical performance of the single-, dual-, triple-target detection kits to guide the clinical diagnosis of this disease. METHODS: A series of reference materials calibrated by droplet digital PCR (ddPCR) and 57 clinical samples were used to evaluate the clinical performance of six single-, dual-, triple-target SARS-CoV-2 nucleic acid detection kits based on RT-qPCR. RESULTS: The dual-target kits, kit B and kit C had the highest and the lowest detection sensitivity, which was 125 copies/mL and 4000 copies/mL, respectively. Among the 57 clinical samples from patients with COVID-19, 47 were tested positive by the kit B, while 35, 29, 28, 30, and 29 were found positive by the kits A, C, D, E, and F, respectively. The number of targets in a detection kit is not a key factor affecting sensitivity, while the amount of sample loading may influence the performance of a detection kit. CONCLUSIONS: This study provides a guide when choosing or developing a nucleic acid detection kit for the diagnosis of COVID-19. Also, the absolute-quantification feature and high-sensitivity performance of ddPCR, suggesting that it can be used to review clinically suspected samples.

Highly Sensitive Droplet Digital PCR Method for Detection of de novo EGFR T790M Mutation in Patients with Non-Small Cell Lung Cancer.

OBJECTIVE: The aim of this study was to investigate the allelic relation between de novo T790M and concomitant sensitizing EGFR mutations in EGFR-TKIs naïve NSCLCs and to explore whether the formalin-fixed and paraffin-embedded (FFPE) materials affect the detection of de novo EGFR T790M mutation. METHODS: Specimens of 300 consecutive EGFR-TKI naïve NSCLCs who received surgical resection between January 2016 and June 2018 were retrospectively investigated. All the snap-frozen tumor tissues from 300 NSCLCs were screened by droplet digital PCR (ddPCR) for the detection of de novo T790M mutation. The allelic relation between de novo T790M mutation and concomitant sensitizing EGFR mutations was also investigated. Furthermore, we assessed de novo T790M mutation in paired FFPE specimens of 50 patients which included tumor tissues and paired normal lung tissues of the pretreatment NSCLCs to investigate whether FFPE materials affect the detection of de novo T790M mutation. RESULTS: The de novo T790M mutation was observed in four patients which included one patient of single de novo T790M mutation and three patients of de novo T790M mutation coexisting with L858R mutation. The incidence of de novo T790M in pretreatment NSCLCs who harboring EGFR mutations was 2.9% (4/139). All the de novo T790M mutations were detected in cis with the concomitant L858R mutations for the three NSCLCs. Our ddPCR method demonstrated that the frequency of de novo T790M mutation was ranging from 0.1% to 0.5% among 90% (45/50) of the FFPE tumor samples and 92% (46/50) of the paired FFPE adjacent normal lung samples. The frequency of de novo T790M mutation in the paired snap-frozen samples was all below 0.1%. CONCLUSION: Our study demonstrated that most de novo T790M mutations were detected in cis with concomitant sensitizing mutations for pretreatment NSCLCs. Analytical cut-off of ddPCR assay for FFPE specimens should be validated carefully considering the possibility of FFPE-derived artificial gene mutations.

Establishment and validation of a novel droplet digital PCR assay for ultrasensitive detection and dynamic monitoring of EGFR mutations in peripheral blood samples of non-small-cell lung cancer patients.

BACKGROUND: Droplet digital PCR (ddPCR)-based blood detection of EGFR mutations plays significant roles in the individualized therapy of non-small-cell lung cancer (NSCLC) patients. However, a standard assay that is approved by health authorities is still lacking. Additionally, the proper application of this method in clinical settings also needs further investigation. METHODS: The performance of a newly established ddPCR assay was first evaluated using reference samples and then validated by comparing this method with the amplification refractory mutation system (ARMS) using cell-free DNA (cfDNA) in patients' peripheral blood. Further, the correlation between dynamic quantification of EGFR mutation in the patients and their clinical outcome of tyrosine kinase inhibitors (TKIs) therapy was investigated. RESULTS: A total of 77 patients were included, with 50 in the test group and 27 in the validation group. According to the results of the reference samples and the blood samples in the test group, the cut-off value for patient detection was proposed as mutation rate ≥ 0.1% (total copy number of cfDNA ≥ 1000) or at least one copy of mutation DNA was detected (total copy number of cfDNA < 1000). With this criterion, superior sensitivity of our assay to that of ARMS was observed (P = 0.002 for Ex19Del & L858R and P < 0.001 for T790M). The dynamic quantification of EGFR mutations during TKI therapy indicated that an increase in mutation abundance was correlated with resistance, while a decline was associated with response. Notably, a rebound in mutation abundance during chemotherapy may indicate a desirable chance for TKI re-treatment. CONCLUSION: The novel ddPCR assay showed superior sensitivity in the detection of EGFR mutation in blood. The dynamic quantification of EGFR mutations by this assay would greatly facilitate the administration of TKI therapy, including the monitoring of resistance and response, as well as cohort screening for retreatment.

Cross-Linked Polyphosphazene Blends as Robust CO2 Separation Membranes.

An effective cross-linking technique allows a viscous and highly gas-permeable hydrophilic polyphosphazene to be cast as solid membrane films. By judicious blending with other polyphosphazenes to improve the mechanical properties, a membrane exhibiting the highest CO2 permeability (610 barrer) among polyphosphazenes combined with a good CO2/N2 selectivity (35) was synthesized and described here. The material demonstrates performance stability after 500 h of exposure to a coal-fired power plant flue gas, making it attractive for use in carbon capture applications. Its CO2/N2 selectivity under conditions up to full humidity is also stable, and although the gas permeability does decline, the performance is fully recovered upon drying. The high molecular weight of these heteropolymers also allows them to be cast as a thin selective layer on an asymmetric porous membrane, yielding a CO2 permeance of 1200 GPU and a CO2/N2 pure gas selectivity of 31, which does not decline over 2000 h. In addition to gas separation membranes, this cross-linked polyphosphazene can potentially be extended to other applications, such as drug delivery or proton exchange membranes, which take advantage of the polyphosphazene's versatile chemistry.

Clinical evaluation of the effectiveness of fusion-induced asymmetric transcription assay-based reverse transcription droplet digital PCR for ALK detection in formalin-fixed paraffin-embedded samples from lung cancer.

BACKGROUND: Accurate detection of anaplastic lymphoma kinase (ALK) rearrangement is the prerequisite for anti-ALK therapy for the patient with non-small cell lung cancer (NSCLC). Fusion-induced asymmetric transcription assay (FIATA)-based reverse transcription droplet digital PCR (RT-ddPCR) was developed and performed for ALK status survey in NSCLC samples. METHODS: A total of 269 cases of formalin-fixed paraffin-embedded (FFPE) specimens from NSCLC, in which ALK status was confirmed by both fluorescence in situ hybridization (FISH) and immunohistochemistry (IHC), were analyzed by FIATA-based RT-ddPCR. RESULTS: In the ALK-positive group, the 3' ALK transcript copies range was 336.6-107 955.4, and the R3 [(the ratio of the 3' ALK transcript copy numbers to the internal reference gene transcript copy numbers) × 100] was 17.23-672.77. In the ALK-negative group, the 3' ALK transcript copies range was 3.7-1370.6, and the R3 range was 0.10-15.57. The lowest R3 level in the ALK-positive group was significantly higher than the highest R3 level in the ALK-negative group. A positive correlation between the proportion of cancer cells in the tissue section and ALK RNA expression level (R3) was found (P < 0.05). There was no relationship between the percentage of FISH positive cells or FISH positive signal patterns and R3 level of the ALK gene. Compared with FISH and IHC, the clinical sensitivity and specificity of FIATA-based RT-ddPCR for ALK detection were 100%, respectively. CONCLUSIONS: An absolute quantitative FIATA-based RT-ddPCR was developed and validated for ALK fusion detection in NSCLC. This method can rapidly, accurately, and objectively classify ALK types and help with individual therapy.

Influence of Different Inactivation Methods on Severe Acute Respiratory Syndrome Coronavirus 2 RNA Copy Number.

The outbreak of coronavirus disease 2019 (COVID-19) has spread across the world and was characterized as a pandemic. To protect medical laboratory personnel from infection, most laboratories inactivate the virus causing COVID-19, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), in clinical samples before testing. However, the effect of inactivation on the detection results remains unknown. Here, we used a digital PCR assay to determine the absolute SARS-CoV-2 RNA copy number in 63 nasopharyngeal swab samples and assess the effect of inactivation methods on viral RNA copy number. Viral inactivation was performed by three different methods: (i) incubation with the TRIzol LS reagent for 10 min at room temperature, (ii) heating in a water bath at 56°C for 30 min, and (iii) high-temperature treatment, including autoclaving at 121°C for 20 min, boiling at 100°C for 20 min, and heating at 80°C for 20 min. Compared to the amount of RNA in the original sample, TRIzol treatment destroyed 47.54% of the nucleocapsid protein (N) gene and 39.85% of open reading frame (ORF) 1ab. For samples treated at 56°C for 30 min, the copy number of the N gene and ORF 1ab was reduced by 48.55% and 56.40%, respectively. The viral RNA copy number dropped by 50 to 66% after heating at 80°C for 20 min. Nearly no viral RNA was detected after autoclaving at 121°C or boiling at 100°C for 20 min. These results indicate that inactivation reduced the quantity of detectable viral RNA and may cause false-negative results, especially in weakly positive cases. Thus, use of the TRIzol reagent rather than heat inactivation is recommended for sample inactivation, as the TRIzol reagent had the least effect on the RNA copy number among the tested methods.

"Sample-to-Answer" Detection of Rare ctDNA Mutation from 2 mL Plasma with a Fully Integrated DNA Extraction and Digital Droplet PCR Microdevice for Liquid Biopsy.

The "sample-to-answer" integration and automation of circulating tumor DNA (ctDNA)-based liquid biopsy using digital PCR (dPCR) has been hampered by the complicated operations of liquids with volumes ranging from milliliter samples to nanoliter droplets. On the basis of a "3D extensible" design paradigm proposed previously, an integrated droplet digital PCR (IddPCR) microdevice was successfully developed to automate the entire process of liquid biopsy, from the extraction of ctDNA in 2 mL of plasma using magnetic beads to the generation, amplification, and screening of over 30 000 droplets for detection. A series of reagent mixing structures, including macro-, meso-, and micromixers, was designed to enable efficient reagent handling and mixing at different volume scales. The volume thresholds of the microscale and macroscale in the IddPCR device were calculated to be 40 and 100 µL, respectively, based on the fluid dynamics and sizes of the device structures, so that different mixers can be selected according to the reagent volumes. The DNA extraction efficiency obtained on the device was determined to be ∼60%, and the on-chip ddPCR demonstrated a high correlation with an R2 of 0.9986 between the readouts and the estimations by a Poisson distribution. Finally, the IddPCR microdevice was able to detect rare tumor mutations (T790M) with an occurring frequency as low as ∼1% from 2 mL of human plasma in a "sample-to-answer" manner. This work offers a feasible solution for the automation of liquid biopsy and paves the way for its broad applications in clinics.

Exposure to mold proteases stimulates mucin production in airway epithelial cells through Ras/Raf1/ERK signal pathway.

Environmental mold (fungus) exposure poses a significant threat to public health by causing illnesses ranging from invasive fungal diseases in immune compromised individuals to allergic hypertensive diseases such as asthma and asthma exacerbation in otherwise healthy people. However, the molecular pathogenesis has not been completely understood, and treatment options are limited. Due to its thermo-tolerance to the normal human body temperature, Aspergillus. fumigatus (A.fumigatus) is one of the most important human pathogens to cause different lung fungal diseases including fungal asthma. Airway obstruction and hyperresponsiveness caused by mucus overproduction are the hallmarks of many A.fumigatus induced lung diseases. To understand the underlying molecular mechanism, we have utilized a well-established A.fumigatus extracts (AFE) model to elucidate downstream signal pathways that mediate A.fumigatus induced mucin production in airway epithelial cells. AFE was found to stimulate time- and dose-dependent increase of major airway mucin gene expression (MUC5AC and MUC5B) partly via the elevation of their promoter activities. We also demonstrated that EGFR was required but not sufficient for AFE-induced mucin expression, filling the paradoxical gap from a previous study using the same model. Furthermore, we showed that fungal proteases in AFE were responsible for mucin induction by activating a Ras/Raf1/ERK signaling pathway. Ca2+ signaling, but ROS, both of which were stimulated by fungal proteases, was an indispensable determinant for ERK activation and mucin induction. The discovery of this novel pathway likely contributes to our understanding of the pathogenesis of fungal sensitization in allergic diseases such as fungal asthma.

Arsenic represses airway epithelial mucin expression by affecting retinoic acid signaling pathway.

Arsenic is a ubiquitous environmental toxicant, found in high concentrations worldwide. Although abundant research has dealt with arsenic-induced cancers, studies on mechanisms of non-malignant lung diseases have not been complete. In addition, decades of research have mostly concentrated on high-dose arsenic exposure, which has very limited use in modeling the biological effects of today's low-dose exposures. Indeed, accumulated evidence has shown that low-dose arsenic exposure (i.e. ≤100 ppb) may also alter lung homeostasis by causing host susceptibility to viral infection. However, the underlying mechanism of this alteration is unknown. In this study, we found that low-dose sodium arsenite (As (III)) repressed major airway mucins-MUC5AC and MUC5B at both mRNA and protein levels. We further demonstrated that this repression was not caused by cellular toxicity or mediated by the reduction of a common mucin-inducing pathway-EGFR. Other established mucin activators- dsRNA, IL1ß or IL17 were not able to override As (III)-induced mucin repression. Interestingly, the suppressing effect of As (III) appeared to be partially reversible, and supplementation of all trans retinoic acid (t-RA) doses dependently restored mucin gene expression. Further analyses indicated that As (III) treatment significantly reduced the protein level of retinoic acid receptors (RARα, γ and RXRα) as well as RARE promoter reporter activity. Therefore, our study fills in an important knowledge gap in the field of low-dose arsenic exposure. The interference of RA signaling, and mucin gene expression may be important pathogenic factors in low-dose arsenic induced lung toxicity.