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.

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.

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.

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.

Quantitative Detection and Viral Load Analysis of SARS-CoV-2 in Infected Patients.

BACKGROUND: Coronavirus disease 2019 (COVID-19) has become a public health emergency. The widely used reverse transcription-polymerase chain reaction (RT-PCR) method has limitations for clinical diagnosis and treatment. METHODS: A total of 323 samples from 76 COVID-19-confirmed patients were analyzed by droplet digital PCR (ddPCR) and RT-PCR based 2 target genes (ORF1ab and N). Nasal swabs, throat swabs, sputum, blood, and urine were collected. Clinical and imaging data were obtained for clinical staging. RESULTS: In 95 samples that tested positive by both methods, the cycle threshold (Ct) of RT-PCR was highly correlated with the copy number of ddPCR (ORF1ab gene, R2 = 0.83; N gene, R2 = 0.87). Four (4/161) negative and 41 (41/67) single-gene positive samples tested by RT-PCR were positive according to ddPCR with viral loads ranging from 11.1 to 123.2 copies/test. The viral load of respiratory samples was then compared and the average viral load in sputum (17 429 ±â€…6920 copies/test) was found to be significantly higher than in throat swabs (2552 ±â€…1965 copies/test, P < .001) and nasal swabs (651 ±â€…501 copies/test, P < .001). Furthermore, the viral loads in the early and progressive stages were significantly higher than that in the recovery stage (46 800 ±â€…17 272 vs 1252 ±â€…1027, P < .001) analyzed by sputum samples. CONCLUSIONS: Quantitative monitoring of viral load in lower respiratory tract samples helps to evaluate disease progression, especially in cases of low viral load.

"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.

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.

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.

Alternaria induces airway epithelial cytokine expression independent of protease-activated receptor.

BACKGROUND AND OBJECTIVE: A novel fungal allergen, Alternaria (Alt), has been previously shown to associate with the pathogenesis of allergic rhinitis and bronchial asthma, particularly in arid and semi-arid regions. Airway epithelial cells are among the first to encounter Alt, and epithelial cytokine production and subsequent airway inflammation are early events in the response to Alt exposure. However, the underlying mechanism is unclear. As protease-activated receptor 2 (PAR2) has been implicated in most of the Alt-induced biological events, we investigated the regulation of airway inflammation and epithelial cytokine expression by PAR2. METHODS: Wild-type (WT) and Par2 knockout (Par2-KO) mice were used to evaluate the in vivo role of PAR2. Primary human and mouse airway epithelial cells were used to examine the mechanistic basis of epithelial cytokine regulation in vitro. RESULTS: Surprisingly, Par2 deficiency had no negative impact on the change of lung function, inflammation and cytokine production in the mouse model of Alt-induced asthma. Alt-induced cytokine production in murine airway epithelial cells from Par2-KO mice was not significantly different from the WT cells. Consistently, PAR2 knockdown in human cells also had no effect on cytokine expression. In contrast, the cytokine expressions induced by synthetic PAR2 agonist or other asthma-related allergens (e.g. cockroach extracts) were indeed mediated via a PAR2-dependent mechanism. Finally, we found that EGFR pathway was responsible for Alt-induced epithelial cytokine expression. CONCLUSION: The activation of EGFR, but not PAR2, was likely to drive the airway inflammation and epithelial cytokine production induced by Alt.

The Club Cell Marker SCGB1A1 Downstream of FOXA2 is Reduced in Asthma.

Human SCGB1A1 protein has been shown to be significantly reduced in BAL, sputum, and serum from humans with asthma as compared with healthy individuals. However, the mechanism of this reduction and its functional impact have not been entirely elucidated. By mining online datasets, we found that the mRNA of SCGB1A1 was significantly repressed in brushed human airway epithelial cells from individuals with asthma, and this repression appeared to be associated with reduced expression of FOXA2. Consistently, both Scgb1A1 and FoxA2 were downregulated in an ovalbumin-induced mouse model of asthma. Furthermore, compared with wild-type mice, Scgb1a1 knockout mice had increased airway hyperreactivity and inflammation when they were exposed to ovalbumin, confirming the antiinflammatory role of Scgb1a1 in protection against asthma phenotypes. To search for potential asthma-related stimuli of SCGB1A1 repression, we tested T-helper cell type 2 cytokines. Both IL-4 and IL-13 repressed epithelial expression of SCGB1A1 and FOXA2. Importantly, infection of epithelial cells with human rhinovirus similarly reduced expression of these two genes, which suggests that FOXA2 may be the common regulator of SCGB1A1. To establish the causal role of reduced FOXA2 in SCGB1A1 repression, we demonstrated that FOXA2 was required for SCGB1A1 expression at baseline. FOXA2 overexpression was sufficient to drive promoter activity and expression of SCGB1A1 and was also able to restore the repressed SCGB1A1 expression in IL-13-treated or rhinovirus-infected cells. Taken together, these findings suggest that low levels of epithelial SCGB1A1 in asthma are caused by reduced FOXA2 expression.

Exploring the roles of substrate-binding surface of the chaperone site in the chaperone activity of trigger factor.

Trigger factor (TF) is a key component of the prokaryotic chaperone network, which is involved in many basic cellular processes, such as protein folding, protein trafficking, and ribosome assembly. The major chaperone site of TF has a cradle-like structure in which protein substrate may fold without interference from other proteins. Here, we investigated in vivo and in vitro the roles of hydrophobic and charged patches on the edge and interior of cradle during TF-assisted protein folding. Our results showed that most of the surface of the cradle was involved in TF-assisted protein folding, which was larger than found in early studies. Although the inner surface of cradle was mostly hydrophobic, both hydrophobic and electrostatic patches were indispensable for TF to facilitate correct protein folding. However, hydrophobic patches were more important for the antiaggregation activity of TF. Furthermore, it was found that the patches on the surface of cradle were involved in TF-assisted protein folding in a spatial and temporal order. These results suggest that the folding-favorable interface between the cradle and substrate was dynamic during TF-assisted protein folding, which enabled TF to be involved in the folding of substrate in an aggressive manner rather than acting as a classic holdase.-Fan, D., Cao, S., Zhou, Q., Zhang, Y., Yue, L., Han, C., Yang, B., Wang, Y., Ma, Z., Zhu, L., Liu, C. Exploring the roles of substrate-binding surface of chaperone site in the chaperone activity of trigger factor.

A multiplex droplet digital PCR assay for non-invasive prenatal testing of fetal aneuploidies.

Higher multiplexing in droplet digital PCR (ddPCR) can simplify the detection process of ddPCR-based non-invasive prenatal testing (NIPT) and improve its reliability, making it a practical approach in clinical practice. However, a high level of multiplex ddPCR-based NIPT has rarely been reported. In this study, we developed a multiplex ddPCR assay using universal locked nucleic acid (LNA) probes to reliably identify fetal aneuploidies. We first performed statistical analysis based on the Poisson distribution to evaluate the required number of target DNA molecules and the total number of droplets for a ddPCR assay. Next, we designed two sets of primers and probes to quantify cfDNA from chromosomes 21 and 18 and then determined the disease status of a sample. Finally, we evaluated our multiplex ddPCR assay with 60 clinical plasma samples. All of the 60 clinical samples were correctly identified. The accessibility and cost-effectiveness of our multiplex ddPCR-based NIPT make it a competitive prenatal testing method in clinical use.

A density-watershed algorithm (DWA) method for robust, accurate and automatic classification of dual-fluorescence and four-cluster droplet digital PCR data.

Droplet digital PCR (ddPCR) is a single-molecule amplification technology with broad applications in precision medicine and clinical diagnosis. Dual-fluorescence and four-cluster ddPCR (two/four-ddPCR) assay is an effective way to quantify copy numbers. Currently, two/four-ddPCR data are usually classified with manual thresholds. For clinical applications, automatic and accurate methods are required to avoid subjectivity in diagnosis. Although there are some automatic classification algorithms, their accuracy and robustness still need to be improved to meet the needs of clinical diagnosis. Therefore, a new method is in high demand to automatically classify two/four-ddPCR data in an accurate and robust way. Here, a novel density-watershed algorithm (DWA) method was developed for the accurate, automatic and unsupervised classification of two/four-ddPCR data. First, data gridding was applied to a scatter plot of the fluorescence signal intensity to calculate data densities. Based on the data densities, the watershed algorithm was used to divide the gridded scatter plot into isolated regions automatically. Next, an optimal cluster pattern was determined based on these isolated regions, and excess regions were merged. Finally, the two/four-ddPCR data were classified based on the merged regions, and DNA template copy numbers were calculated accordingly. Using the DWA method for the quantification of both wild types and mutants of epidermal growth factor receptor (EGFR) L858R and T790M, the classification results were highly consistent with expectations, and significantly better than commonly-used automatic algorithms for now. The computed template copy numbers scaled proportionally to the relative concentration of input templates (r2 > 0.998) in four orders of magnitude with a good reproducibility, and achieved a limit of detection over 40 times lower than the commonly-used automatic algorithms. Furthermore, the DWA method was validated on 254 clinical DNA samples derived from frozen tissues, formalin-fixed paraffin-embedded tissues and peripheral blood. In most cases, the DWA method derived accurate and valid classification results. This highly effective DWA method may be widely used for automatically classifying two/four-ddPCR data, and it will greatly promote the application of ddPCR in clinical diagnosis.

Noninvasive and Accurate Detection of Hereditary Hearing Loss Mutations with Buccal Swab Based on Droplet Digital PCR.

Hereditary hearing loss is a common clinical neurosensory disorder in humans and has a high demand for genetic screening. Current screening techniques using peripheral blood or dried blood spots (DBSs) are invasive. Therefore, this study aims to develop a noninvasive and accurate detection method for eight hotspot deafness-associated mutations based on buccal swab and droplet digital PCR (ddPCR). First, this method was evaluated for analytic performance including specificity, detection limit, dynamic range using plasmid DNA. The specificity was 100% and the detection limit was 5 copies. The dynamic range of this ddPCR-based method was from 10 to 105 copies/µL. Next, the method was found to accurately quantify mitochondrial gene heteroplasmy rate as low as 1% for both m.1494C > T and m.1555A > G sites. Then, we demonstrated that buccal swab was a reliable sample. DNA can be extracted and accurately quantified after a buccal swab had been stored for 90 days at either room temperature or -20 °C. Finally, clinical samples (23 DBSs and 42 buccal swabs) were tested to further evaluate the accuracy and clinical applicability of this method. All clinical samples were accurately quantified and genotyped. This noninvasive and accurate method is highly promising as a genetic screening method for deafness-associated mutations due to its high sensitivity and accuracy.

Precision methylome characterization of Mycobacterium tuberculosis complex (MTBC) using PacBio single-molecule real-time (SMRT) technology.

Tuberculosis (TB) remains one of the most common infectious diseases caused by Mycobacterium tuberculosis complex (MTBC). To panoramically analyze MTBC's genomic methylation, we completed the genomes of 12 MTBC strains (Mycobacterium bovis; M. bovis BCG; M. microti; M. africanum; M. tuberculosis H37Rv; H37Ra; and 6 M. tuberculosis clinical isolates) belonging to different lineages and characterized their methylomes using single-molecule real-time (SMRT) technology. We identified three (m6)A sequence motifs and their corresponding methyltransferase (MTase) genes, including the reported mamA, hsdM and a newly discovered mamB. We also experimentally verified the methylated motifs and functions of HsdM and MamB. Our analysis indicated the MTase activities varied between 12 strains due to mutations/deletions. Furthermore, through measuring 'the methylated-motif-site ratio' and 'the methylated-read ratio', we explored the methylation status of each modified site and sequence-read to obtain the 'precision methylome' of the MTBC strains, which enabled intricate analysis of MTase activity at whole-genome scale. Most unmodified sites overlapped with transcription-factor binding-regions, which might protect these sites from methylation. Overall, our findings show enormous potential for the SMRT platform to investigate the precise character of methylome, and significantly enhance our understanding of the function of DNA MTase.

Human Airway Epithelial Cells Direct Significant Rhinovirus Replication in Monocytic Cells by Enhancing ICAM1 Expression.

Human rhinovirus (RV) is the major cause of common cold, and it also plays a significant role in asthma and asthma exacerbation. The airway epithelium is the primary site of RV infection and production. In contrast, monocytic cells (e.g., monocytes and macrophages) are believed to be nonpermissive for RV replication. Instead, RV has been shown to modulate inflammatory gene expressions in these cells via a replication-independent mechanism. In the study presented here, replication of RV16 (a major-group RV) was found to be significantly enhanced in monocytes when it was cocultivated with airway epithelial cells. This effect appeared to be mediated by secretory components from epithelial cells, which stimulated RV16 replication and significantly elevated the expression of a number of proinflammatory cytokines. The lack of such an effect on RV1A, a minor-group RV that enters the cell by a different receptor, suggests that intercellular adhesion molecule 1 (ICAM1), the receptor for major-group RVs, may be involved. Indeed, conditioned media from epithelial cells significantly increased ICAM1 expression in monocytes. Consistently, ICAM1 overexpression and ICAM1 knockdown enhanced and blocked RV production, respectively, confirming the role of ICAM1 in this process. Thus, this is the first report demonstrating that airway epithelial cells direct significant RV16 replication in monocytic cells via an ICAM1-dependent mechanism. This finding will open a new avenue for the study of RV infection in airway disease and its exacerbation.

Global Analysis of the Impact of Deleting Trigger Factor on the Transcriptome Profile of Escherichia coli.

Trigger factor (TF) is a key component of prokaryotic chaperone network, which is involved various basic cellular processes such as nascent peptide folding, protein trafficking, ribosome assembly. To better understanding the physiological roles of TF, global transcriptome profiles of a variety of TF deletion mutant strains of Escherichia coli were determined. We found that deletion of the tig gene, encoding TF, led to a dramatic alteration of transcriptome profile, not only affecting the gene expression of members of the chaperone network, but also changing the levels of quite a few RNAs related to metabolism and other cellular processes. Further studies showed that this alteration was only partially recovered by knockin of TF domain-deletion mutants into the endogenous tig locus, indicating that structural integrity is crucial for the biological function of TF. Finally, by combining the transcriptome and phenotype results, a physiological mechanism underlying the impact of TF deletion on the transcriptome profile was proposed. J. Cell. Biochem. 118: 141-153, 2017. © 2016 Wiley Periodicals, Inc.

Distribution patterns of haplotypes for symbionts from Umbilicaria esculenta and U. muehlenbergii reflect the importance of reproductive strategy in shaping population genetic structure.

BACKGROUND: The diversity of lichen fungal components and their photosynthetic partners reflects both ecological and evolutionary factors. In present study, molecular investigations of the internal transcribed spacer of the nuclear ribosomal DNA (ITS nrDNA) region were conducted to analyze the genetic diversity of Umbilicaria esculenta and U. muehlenbergii together with their associated green algae. RESULT: It was here demonstrated that the reproductive strategy is a principal reason for fungal selectivity to algae. U. muehlenbergii, which disperses via sexual spores, exhibits lower selectivity to its photosynthetic partners than U. esculenta, which has a vegetative reproductive strategy. The difference of genotypic diversity (both fungal and algal) between these two Umbilicaria species is low, although their nucleotide diversity can vary greatly. CONCLUSIONS: The present study illustrates that lichen-forming fungi with sexual reproductive strategies are less selective with respect to their photobionts; and reveals that both sexual and vegetative reproduction allow lichens to generate similar amounts of diversity to adapt to the environments. The current study will be helpful for elucidating how lichens with different reproductive strategies adapt to changing environments.

Modulation of airway epithelial antiviral immunity by fungal exposure.

Multiple pathogens, such as bacteria, fungi, and viruses, have been frequently found in asthmatic airways and are associated with the pathogenesis and exacerbation of asthma. Among these pathogens, Alternaria alternata (Alt), a universally present fungus, and human rhinovirus have been extensively studied. However, their interactions have not been investigated. In the present study, we tested the effect of Alt exposure on virus-induced airway epithelial immunity using live virus and a synthetic viral mimicker, double-stranded RNA (dsRNA). Alt treatment was found to significantly enhance the production of proinflammatory cytokines (e.g., IL-6 and IL-8) induced by virus infection or dsRNA treatment. In contrast to this synergistic effect, Alt significantly repressed type I and type III IFN production, and this impairment led to elevated viral replication. Mechanistic studies suggested the positive role of NF-κB and mitogen-activated protein kinase pathways in the synergism and the attenuation of the TBK1-IRF3 pathway in the inhibition of IFN production. These opposite effects are caused by separate fungal components. Protease-dependent and -independent mechanisms appear to be involved. Thus, Alt exposure alters the airway epithelial immunity to viral infection by shifting toward more inflammatory but less antiviral responses.

Universal Probe Library based real-time PCR for rapid detection of bacterial pathogens from positive blood culture bottles.

A set of real-time PCR based assays using the locked nucleic acid probes from Roche Universal ProbeLibrary were developed for rapid detection of eight bacterial species from positive blood culture bottles. Four duplex real-time PCR reactions targeting to one Gram-positive bacterium and one Gram-negative bacterium were optimized for species identification according to Gram stain results. We also included mecA-specific primers and probes in the assays to indicate the presence of methicillin resistance in the bacterial species. The analytical sensitivity was in the range of 1-10 CFU per PCR reaction mixture. The specificity and cross reactivity of the assay was validated by 28 ATCC reference strains and 77 negative blood culture specimens. No cross-reactivity was observed in these samples thus demonstrating 100 % specificity. 72 previously characterized clinical isolates were tested by the real-time PCR assay and validated the accuracy and feasibility of the real-time PCR assay. Furthermore, 55 positive blood culture samples were tested using real-time PCR and 50 (90.9 %) of them were identified as the same species as judged by biochemical analysis. In total, real-time PCR showed 98.2 % consistent to that of traditional methods. Real-time PCR can be used as a supplement for early detection of the frequently-occurred pathogens from the positive blood cultures.