Mycobacterium abscessus extracellular vesicles increase mycobacterial resistance to clarithromycin in vitro.

Nontuberculous Mycobacteria (NTM) are a group of emerging bacterial pathogens that have been identified in cystic fibrosis (CF) patients with microbial lung infections. The treatment of NTM infection in CF patients is challenging due to the natural resistance of NTM species to many antibiotics. Mycobacterium abscessus is one of the most common NTM species found in the airways of CF patients. In this study, we characterized the extracellular vesicles (EVs) released by drug-sensitive M. abscessus untreated or treated with clarithromycin (CLR), one of the frontline anti-NTM drugs. Our data show that exposure to CLR increases mycobacterial protein trafficking into EVs as well as the secretion of EVs in culture. Additionally, EVs released by CLR-treated M. abscessus increase M. abscessus resistance to CLR when compared to EVs from untreated M. abscessus. Proteomic analysis further indicates that EVs released by CLR-treated M. abscessus carry an increased level of 50S ribosomal subunits, the target of CLR. Taken together, our results suggest that EVs play an important role in M. abscessus resistance to CLR treatment.

The use of human iPSC-derived alveolar organoids to explore SARS-CoV-2 variant infections and host responses.

Severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2) primarily targets the respiratory system. Physiologically relevant human lung models are indispensable to investigate virus-induced host response and disease pathogenesis. In this study, we generated human induced pluripotent stem cell (iPSC)-derived alveolar organoids (AOs) using an established protocol that recapitulates the sequential steps of in vivo lung development. AOs express alveolar epithelial type II cell protein markers including pro-surfactant protein C and ATP binding cassette subfamily A member 3. Compared to primary human alveolar type II cells, AOs expressed higher mRNA levels of SARS-CoV-2 entry factors, angiotensin-converting enzyme 2 (ACE2), asialoglycoprotein receptor 1 (ASGR1) and basigin (CD147). Considering the localization of ACE2 on the apical side in AOs, we used three AO models, apical-in, sheared and apical-out for SARS-CoV-2 infection. All three models of AOs were robustly infected with the SARS-CoV-2 irrespective of ACE2 accessibility. Antibody blocking experiment revealed that ASGR1 was the main receptor for SARS-CoV2 entry from the basolateral in apical-in AOs. AOs supported the replication of SARS-CoV-2 variants WA1, Alpha, Beta, Delta, and Zeta and Omicron to a variable degree with WA1 being the highest and Omicron being the least. Transcriptomic profiling of infected AOs revealed the induction of inflammatory and interferon-related pathways with NF-κB signaling being the predominant host response. In summary, iPSC-derived AOs can serve as excellent human lung models to investigate infection of SARS-CoV-2 variants and host responses from both apical and basolateral sides.

Compact auto-aligning interferometers with picometer precision.

This research introduces a compact, auto-aligning interferometer engineered for measuring translations with a wide angular working range and picometer precision above 1H z. It presents a design ensuring automatic beam alignment during movement through secondary reflection from a corner reflector. The sensor head, a 20×10×10m m 3 all-glass quasi-monolithic structure, exhibits a displacement sensitivity below 1p m/H z 1/2 above 1H z and a wide angular working range of ±200m r a d. This versatile optical design holds promise to improve the sensitivity in applications such as laser ranging, optical seismometers, precision manufacturing, and metrology.

Cellular microRNAs target SARS-CoV-2 spike protein and restrict viral replication.

MicroRNAs (miRNAs) regulate gene expression posttranscriptionally and are implicated in viral replication and host tropism. miRNAs can impact the viruses either by directly interacting with the viral genome or modulating host factors. Although many miRNAs have predicted binding sites in the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) viral RNA genome, little experimental validation has been done. We first identified 492 miRNAs that have binding site(s) on the spike (S) viral RNA by a bioinformatics prediction. We then validated the selected 39 miRNAs by examining S-protein levels after coexpressing the S-protein and a miRNA into the cells. Seven miRNAs were found to reduce the S-protein levels by more than 50%. Among them, miR-15a, miR-153, miR-298, miR-508, miR-1909, and miR-3130 also significantly reduced SARS-CoV-2 viral replication. SARS-CoV-2 infection decreased the expression levels of miR-298, miR-497, miR-508, miR-1909, and miR-3130, but had no significant effects on miR-15a and miR-153 levels. Intriguingly, the targeting sequences of these miRNAs on the S viral RNA showed sequence conservation among the variants of concern. Our results suggest that these miRNAs elicit effective antiviral defense against SARS-CoV-2 by modulating S-protein expression and are likely targeting all the variants. Thus, the data signify the therapeutic potential of miRNA-based therapy for SARS-CoV-2 infections.NEW & NOTEWORTHY MicroRNAs can impact viruses either by directly interacting with the virus genome or by modulating host factors. We identified that cellular miRNAs regulate effective antiviral defense against SARS-CoV-2 via modulating spike protein expression, which may offer a potential candidate for antiviral therapy.

Axin1: A novel scaffold protein joins the antiviral network of interferon.

Acute respiratory infection by influenza virus is a persistent and pervasive public health problem. Antiviral innate immunity initiated by type I interferon (IFN) is the first responder to pathogen invasion and provides the first line of defense. We discovered that Axin1, a scaffold protein, was reduced during influenza virus infection. We also found that overexpression of Axin1 and the chemical stabilizer of Axin1, XAV939, reduced influenza virus replication in lung epithelial cells. This effect was also observed with respiratory syncytial virus and vesicular stomatitis virus. Axin1 boosted type I IFN response to influenza virus infection and activated JNK/c-Jun and Smad3 signaling. XAV939 protected mice from influenza virus infection. Thus, our studies provide new mechanistic insights into the regulation of the type I IFN response and present a new potential therapeutic of targeting Axin1 against influenza virus infection.

High-precision laser beam lateral displacement measurement based on differential wavefront sensing.

Accurately lateral displacement measurement is essential for a vast of non-contact sensing technologies. Here, we introduce a high-precision lateral displacement measurement method based on differential wavefront sensing (DWS). Compared to the conventional differential power sensing (DPS) method, the DWS method based on phase readout has the potential to achieve a higher resolution. The beam lateral displacement can be obtained by the curvature distribution of the wavefront on the surface of the detector. According to the theoretical model of the DWS method, the sensitivity of the lateral displacement can be greatly improved by increasing the wavefront curvature of the measured laser beam by means of lenses. An optical system for measuring the lateral displacement of the laser beam is built and calibrated by a high-precision hexapod. The experimental results show that the DWS-based lateral displacement measurement achieves a resolution of 40 pm/Hz1/2 (at 1-10 Hz) with a linear range of about 40 µm, which is consistent with the theoretical model. This technique can be applied to high-precision multi-degree-of-freedom interferometers.

SNHG15 aids SARS-CoV-2 entry via RABL2A.

Angiotensin-converting enzyme 2 (ACE2) and several proteins have been identified as entry factors for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). However, whether long noncoding RNAs are involved in SARS-CoV-2 entry remains unknown. In this study, we investigated the role of small nucleolar RNA host gene 15 (SNHG15) in SARS-CoV-2 entry using a SARS-CoV-2 spike pseudotyped lentivirus with a luciferase reporter. Overexpression of SNHG15 promoted but SNHG15 knockdown limited SARS-CoV-2 entry in a dose- and time-dependent manner. SNHG15 interacted with Rab-like protein 2A (RABL2A). Overexpression and knockdown of RABL2A produced similar effects on SARS-CoV-2 entry as those of SNHG15. Furthermore, RABL2A knockdown abolished the SNHG15-mediated increase in SARS-CoV-2 entry. In conclusion, SNHG15 is a critical regulatory factor that aids SARS-CoV-2 entry through RABL2A.

Regulation of influenza A virus infection by Lnc-PINK1-2:5.

Influenza virus causes approximately 291,000 to 646,000 human deaths worldwide annually. It is also a disease of zoonotic importance, affecting animals such as pigs, horses, and birds. Even though vaccination is being used to prevent influenza virus infection, there are limited options available to treat the disease. Long noncoding RNAs (lncRNAs) are RNA molecules with more than 200 nucleotides that do not translate into proteins. They play important roles in the physiological and pathological processes. In this study, we identified a novel transcript, Lnc-PINK1-2:5 that was upregulated by influenza virus. This lncRNA was predominantly located in the nucleus and was not affected by type I interferons. Overexpression of Lnc-PINK1-2:5 reduced the influenza viral mRNA and protein levels in cells as well as titres in culture media. Knockdown of Lnc-PINK1-2:5 using CRISPR interference enhanced the virus replication. Antiviral activity of Lnc-PINK1-2:5 was independent of influenza virus strains. RNA sequencing analysis revealed that Lnc-PINK1-2:5 upregulated thioredoxin interacting protein (TXNIP) during influenza virus infection. Overexpression of TXNIP reduced influenza virus infection, suggesting that TXNIP is an antiviral gene. Knockdown of TXNIP abolished the Lnc-PINK1-2:5-mediated increase in influenza virus infection. In conclusion, the newly identified Lnc-PINK1-2:5 isoform is an anti-influenza lncRNA acting through the upregulation of TXNIP gene expression.

MicroRNA-206 inhibits influenza A virus replication by targeting tankyrase 2.

Due to the frequent mutations, influenza A virus (IAV) becomes resistant to anti-viral drugs targeting influenza viral proteins. There are increasing interests in anti-viral agents that target host cellular proteins required for virus replication. Tankyrase (TNKS) has poly (ADP-ribose) polymerase activity and is a negative regulator of many host proteins. The objectives of this study are to study the role of TNKS2 in IAV infection, identify the microRNAs targeting TNKS2, and to understand the mechanisms involved. We found that TNKS2 expression was elevated in human lung epithelial cells and mouse lungs during IAV infection. Knock-down of TNKS2 by RNA interference reduced viral replication. Using a computation approach and 3'-untranslation regions (3'-UTR) reporter assay, we identified miR-206 as the microRNA that targeted TNKS2. Overexpression of miR-206 reduced viral protein levels and virus production in cell culture. The effect of miR-206 on IAV replication was strain-independent. miR-206 activated JNK/c-Jun signalling, induced type I interferon expression and enhanced Stat signalling. Finally, the delivery of an adenovirus expressing miR-206 into the lung of mice challenged with IAV increased type I interferon response, suppressed viral load in the lungs and increased survival. Our results indicate that miR-206 has anti-influenza activity by targeting TNKS2 and subsequently activating the anti-viral state.

All-fiber heterodyne velocity and displacement interferometer based on DPLL Doppler tracking with sub-nanometer per second and picometer sensitivity.

Velocity and displacement measurements play an important role not only in the process of industrial production and metrology on the ground but also in satellite gravity measurement in space. A high-precision all-fiber heterodyne velocity and displacement interferometer based on digital phase-locked loop (DPLL) Doppler tracking is proposed in this paper. The target velocity is measured by tracking the heterodyne frequency changes of the beat-note signal, and the displacement is obtained by the integrated phase of the Doppler frequency change. A dual-signal differential optical-path scheme combined with DPLL signal tracking technology enables high-precision and high-linearity measurement of velocity and displacement simultaneously. For integration and compactness, the interferometer uses all-fiber optics that are packaged in a small box with dimensions of 150×150×70m m 3, except for an externally fiber-connected collimator as the sensor head. The experimental results show a velocity sensitivity below 30p m/s/H z 1/2 in the 0.03-2 Hz band and a displacement sensitivity below 10p m/H z 1/2 above 0.4 Hz.

Weak-Light Phase-Locking Time Delay Interferometry with Optical Frequency Combs.

In the future space-borne gravitational wave (GW) detector, the optical transponder scheme, i.e., the phase-locking scheme, will be utilized so as to maintain the signal-to-noise ratio (SNR). In this case, the whole constellation will share one common laser equivalently, which enables the considerable simplification of time delay interferometry (TDI) combinations. Recently, and remarkably, the unique combination of TDI and optical frequency comb (OFC) has shown a bright prospect for the future space-borne missions. When the laser frequency noise and the clock noise are synchronized using OFC as the bridge, the data streams will be reasonably simplified. However, in the optical transponder scheme, the weak-light phase-locking (WLPL) loops could bring additional noises. In this work, we analyze the phase-locking scheme with OFC and transfer characteristics of the noises including the WLPL noise. We show that the WLPL noise can be efficiently reduced by using the specific TDI combination, and the cooperation of phase-locking and frequency combs can greatly simplify the post-processing.

Shot-noise-limit performance of a weak-light phase readout system for intersatellite heterodyne interferometry.

A laser interferometer will be used in the spaceborne gravitational-wave detection missions to measure the inter-satellite optical pathlength variations. The phase readout system of the interferometer needs to be carefully designed and tested to accomplish a shot-noise-limited detection performance under the situation of pico-Watt level received lights. In this work, a scheme based on dual-tone acousto-optic diffraction is presented to verify the performance of the weak-light phase readout system. By optimizing the parameters of the photoreceiver and the local strong-light power, the signal-to-noise ratio of the beat-note signal is enhanced. Thanks to the scheme's common-mode noise rejections for the laser frequency noise, and the optical-path noise, etc., the differential phase noise has achieved a performance of 2×10-4 rad/Hz1/2, which is dominated by the weak-light (∼13 pW) shot noise above the frequencies of 2 mHz.

miR-29a is a negative regulator of influenza virus infection through targeting of the frizzled 5 receptor.

Influenza A virus (IAV) infections result in a large number of deaths and substantial economic losses each year. MicroRNAs repress gene expression and are involved in virus-host interactions. miR-29a is known to have anti-tumor and anti-fibrotic effects. However, the role of miR-29a in IAV infection is unclear. In the present study, we investigated the effect of miR-29a on IAV infection and the mechanisms by which it functions. IAV infection was found to cause decreased miR-29a expression in lung epithelial A549 cells and mouse lungs. Overexpression of miR-29a reduced IAV mRNA and protein levels and progeny virus production in HEK293 and A549 cells. Inhibition of IAV infection by miR-29a was observed with different strains of IAV, including A/PR/8/34, A/WSN/1933, and clinical isolates A/OK/3052/09 and A/OK/309/06 H3N2. Knockout of miR-29a using CRISPR/Cas9 resulted in an increase in viral mRNA and protein levels, confirming that miR-29a suppresses IAV infection. A 3' untranslated region (3'-UTR) reporter assay showed that miR-29a had binding sites in the 3'-UTR of the Wnt-Ca2+ signaling receptor frizzled 5 gene, and overexpression of miR-29a reduced the level of the endogenous frizzled 5 protein. Wnt5a treatment of HEK293 and A549 cells enhanced IAV infection. Our results suggest that miR-29a inhibits IAV infection, probably via the frizzled 5 receptor.

Long Noncoding RNA FENDRR Inhibits Lung Fibroblast Proliferation via a Reduction of ß-Catenin.

Idiopathic Pulmonary Fibrosis (IPF) is a chronic, progressive, and usually lethal lung disease and it has been widely accepted that fibroblast proliferation is one of the key characteristics of IPF. Long noncoding RNAs (lncRNAs) play vital roles in the pathogenesis of many diseases. In this study, we investigated the role of lncRNA FENDRR on fibroblast proliferation. Human lung fibroblasts stably overexpressing FENDRR showed a reduced cell proliferation compared to those expressing the control vector. On the other hand, FENDRR silencing increased fibroblast proliferation. FENDRR bound serine-arginine rich splicing factor 9 (SRSF9) and inhibited the phosphorylation of p70 ribosomal S6 kinase 1 (PS6K), a downstream protein of the mammalian target of rapamycin (mTOR) signaling. Silencing SRSF9 reduced fibroblast proliferation. FENDRR reduced ß-catenin protein, but not mRNA levels. The reduction of ß-catenin protein levels in lung fibroblasts by gene silencing or chemical inhibitor decreased fibroblast proliferation. Adenovirus-mediated FENDRR transfer to the lungs of mice reduced asbestos-induced fibrotic lesions and collagen deposition. RNA sequencing of lung tissues identified 7 cell proliferation-related genes that were up-regulated by asbestos but reversed by FENDRR. In conclusion, FENDRR inhibits fibroblast proliferation and functions as an anti-fibrotic lncRNA.

Long Noncoding RNA FENDRR Exhibits Antifibrotic Activity in Pulmonary Fibrosis.

Abnormal activation of lung fibroblasts contributes to the initiation and progression of idiopathic pulmonary fibrosis (IPF). The objective of the present study was to investigate the role of fetal-lethal noncoding developmental regulatory RNA (FENDRR) in the activation of lung fibroblasts. Dysregulated long noncoding RNAs in IPF lungs were identified by next-generation sequencing analysis from the two online datasets. FENDRR expression in lung tissues from patients with IPF and mice with bleomycin-induced pulmonary fibrosis was determined by quantitative real-time PCR. IRP1 (iron-responsive element-binding protein 1), a protein partner of FENDRR, was identified by RNA pulldown-coupled mass spectrometric analysis and confirmed by RNA immunoprecipitation. The interaction region between FENDRR and IRP1 was determined by cross-linking immunoprecipitation. The in vivo role of FENDRR in pulmonary fibrosis was studied using adenovirus-mediated gene transfer in mice. The expression of FENDRR was downregulated in fibrotic human and mouse lungs as well as in primary lung fibroblasts isolated from bleomycin-treated mice. TGF-ß1 (transforming growth factor-ß1)-SMAD3 signaling inhibited FENDRR expression in lung fibroblasts. FENDRR was preferentially localized in the cytoplasm of adult lung fibroblasts and bound IRP1, suggesting its role in iron metabolism. FENDRR reduced pulmonary fibrosis by inhibiting fibroblast activation by reducing iron concentration and acting as a competing endogenous RNA of the profibrotic microRNA-214. Adenovirus-mediated FENDRR gene transfer in the mouse lung attenuated bleomycin-induced lung fibrosis and improved lung function. Our data suggest that FENDRR is an antifibrotic long noncoding RNA and a potential therapeutic target for pulmonary fibrosis.

Repeated Exposure to Streptococcus pneumoniae Exacerbates Chronic Obstructive Pulmonary Disease.

Streptococcus pneumoniae is commonly found in patients with chronic obstructive pulmonary disease (COPD) and is linked to acute exacerbation of COPD. However, current clinical therapy neglects asymptomatic insidious S. pneumoniae colonization. We studied the roles of repeated exposure to S. pneumoniae in COPD progression using a mouse model. C57BL/6J mice were intranasally inoculated with S. pneumoniae ST262 every 4 weeks with or without cigarette smoke (CS) exposure up to 20 weeks to maintain persistent S. pneumoniae presence in the lower airways. Streptococcus pneumoniae enhanced CS-induced inflammatory cell infiltration at 12 to 20 weeks of exposure. Streptococcus pneumoniae also increased CS-induced release of inflammatory cytokines, including IL-1ß, tumor necrosis factor-α, IL-12 (p70), and IL-5 at 20 weeks of exposure. Moreover, a combination of CS and S. pneumoniae caused alveolar epithelial injury, a decline in lung function, and an increased expression of platelet-activating factor receptor and bacterial load. Our results suggest that repeated exposure to S. pneumoniae in lower airways exacerbates CS-induced COPD.

miR-193b represses influenza A virus infection by inhibiting Wnt/ß-catenin signalling.

Due to an increasing emergence of new and drug-resistant strains of the influenza A virus (IAV), developing novel measures to combat influenza is necessary. We have previously shown that inhibiting Wnt/ß-catenin pathway reduces IAV infection. In this study, we aimed to identify antiviral human microRNAs (miRNAs) that target the Wnt/ß-catenin signalling pathway. Using a miRNA expression library, we identified 85 miRNAs that up-regulated and 20 miRNAs that down-regulated the Wnt/ß-catenin signalling pathway. Fifteen miRNAs were validated to up-regulate and five miRNAs to down-regulate the pathway. Overexpression of four selected miRNAs (miR-193b, miR-548f-1, miR-1-1, and miR-509-1) that down-regulated the Wnt/ß-catenin signalling pathway reduced viral mRNA, protein levels in A/PR/8/34-infected HEK293 cells, and progeny virus production. Overexpression of miR-193b in lung epithelial A549 cells also resulted in decreases of A/PR/8/34 infection. Furthermore, miR-193b inhibited the replication of various strains, including H1N1 (A/PR/8/34, A/WSN/33, A/Oklahoma/3052/09) and H3N2 (A/Oklahoma/309/2006), as determined by a viral reporter luciferase assay. Further studies revealed that ß-catenin was a target of miR-193b, and ß-catenin rescued miR-193b-mediated suppression of IAV infection. miR-193b induced G0/G1 cell cycle arrest and delayed vRNP nuclear import. Finally, adenovirus-mediated gene transfer of miR-193b to the lung reduced viral load in mice challenged by a sublethal dose of A/PR/8/34. Collectively, our findings suggest that miR-193b represses IAV infection by inhibiting Wnt/ß-catenin signalling.

Serum amyloid A promotes LPS clearance and suppresses LPS-induced inflammation and tissue injury.

Lipopolysaccharide (LPS) is a major microbial mediator for tissue injury and sepsis resulting from Gram-negative bacterial infection. LPS is an external factor that induces robust expression of serum amyloid A (SAA), a major constituent of the acute-phase proteins, but the relationship between SAA expression and LPS-induced tissue injury remains unclear. Here, we report that mice with inducible transgenic expression of human SAA1 are partially protected against inflammatory response and lung injury caused by LPS and cecal ligation and puncture (CLP). In comparison, transgenic SAA1 does not attenuate TNFα-induced lung inflammation and injury. The SAA1 expression level correlates inversely with the endotoxin concentrations in serum and lung tissues since SAA1 binds directly to LPS to form a complex that promotes LPS uptake by macrophages. Disruption of the SAA1-LPS interaction with a SAA1-derived peptide partially reduces the protective effect and exacerbates inflammation. These findings demonstrate that acute-phase SAA provides innate feedback protection against LPS-induced inflammation and tissue injury.

Long non-coding RNA PSMB8-AS1 regulates influenza virus replication.

Long non-coding RNAs (lncRNAs) are a new arm of gene regulatory mechanism as discovered by sequencing techniques and follow-up functional studies. There are only few studies on lncRNAs as related to gene expression regulation and anti-viral activity during influenza virus infection. We sought to identify and characterize lncRNAs involved in influenza virus replication. Using RNA sequencing analysis, we found that 1,912 lncRNAs were significantly changed in human lung epithelial A549 cells infected with influenza A/Puerto Rico/8/34. Gene ontology analysis on neighboring genes of these lncRNAs revealed that the genes involved in type I interferon signaling and cellular response were highly enriched. Seven selected up-regulated lncRNAs (AC015849.2, RP-1-7H24.1, PSMB8-AS1, CTD-2639E6.9, PSOR1C3, AC007283.5 and RP11-670E13.5) were verified by real-time PCR. These lncRNAs were also induced by other two influenza H1N1 virus strains (A/WSN/1933 and A/Oklahoma/3052/09) and interferon ß1. Repression of PSMB8 antisense RNA 1 (PSMB8-AS1) using CRISPR interference reduced viral mRNA and protein levels as well as the release of progeny influenza virus particles. Our study suggests that lncRNA PSMB8-AS1 could be a new host factor target for developing antiviral therapy against influenza virus infection.

MicroRNA-101 attenuates pulmonary fibrosis by inhibiting fibroblast proliferation and activation.

Aberrant proliferation and activation of lung fibroblasts contribute to the initiation and progression of idiopathic pulmonary fibrosis (IPF). However, the mechanisms responsible for the proliferation and activation of fibroblasts are not fully understood. The objective of this study was to investigate the role of miR-101 in the proliferation and activation of lung fibroblasts. miR-101 expression was determined in lung tissues from patients with IPF and mice with bleomycin-induced pulmonary fibrosis. The regulation of miR-101 and cellular signaling was investigated in pulmonary fibroblasts in vitro The role of miR-101 in pulmonary fibrosis in vivo was studied using adenovirus-mediated gene transfer in mice. The expression of miR-101 was down-regulated in fibrotic lungs from patients with IPF and bleomycin-treated mice. The down-regulation of miR-101 occurred via the E26 transformation-specific (ETS) transcription factor. miR-101 suppressed the WNT5a-induced proliferation of lung fibroblasts by inhibiting NFATc2 signaling via targeting Frizzled receptor 4/6 and the TGF-ß-induced activation of lung fibroblasts by inhibition of SMAD2/3 signaling via targeting the TGF-ß receptor 1. Adenovirus-mediated miR-101 gene transfer in the mouse lung attenuated bleomycin-induced lung fibrosis and improved lung function. Our data suggest that miR-101 is an anti-fibrotic microRNA and a potential therapeutic target for pulmonary fibrosis.