A new chapter of healthy indoor air: antiviral air treatments

Aerosol transmission remains a major challenge for the control of respiratory viruses. To date, prevention strategies include masks, vaccinations, physical distancing, travel restrictions, and lockdowns. Such measures are effective but come with heavy societal burdens and rely on public compliance. Additionally, most are simply not suitable as long-term measures. Other strategies evolve around the concept of improved indoor air quality and involve ventilation, relative humidity (RH) control, and air filtration. Unfortunately, natural ventilation increases exposure to airborne pollutants and vector-borne diseases, and incurs substantial energy losses in colder months. Mechanical ventilation concepts, including regular air changes and filtration, are effective but costly, and often require expensive engineering solutions and widespread renovations. Alternative options to reduce the spread of emerging and seasonal infections are sorely needed. In this issue of EMBO Molecular Medicine, Styles et al (2023) describe the use of propylene glycol (PG) to inactivate infectious bioaerosols and virus-containing droplets deposited on surfaces

SARS-CoV-2 variants reveal features critical for replication in primary human cells

Since entering the human population, Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2; the causative agent of Coronavirus Disease 2019 [COVID-19]) has spread worldwide, causing >100 million infections and >2 million deaths. While large-scale sequencing efforts have identified numerous genetic variants in SARS-CoV-2 during its circulation, it remains largely unclear whether many of these changes impact adaptation, replication, or transmission of the virus. Here, we characterized 14 different low-passage replication-competent human SARS-CoV-2 isolates representing all major European clades observed during the first pandemic wave in early 2020. By integrating viral sequencing data from patient material, virus stocks, and passaging experiments, together with kinetic virus replication data from nonhuman Vero-CCL81 cells and primary differentiated human bronchial epithelial cells (BEpCs), we observed several SARS-CoV-2 features that associate with distinct phenotypes. Notably, naturally occurring variants in Orf3a (Q57H) and nsp2 (T85I) were associated with poor replication in Vero-CCL81 cells but not in BEpCs, while SARS-CoV-2 isolates expressing the Spike D614G variant generally exhibited enhanced replication abilities in BEpCs. Strikingly, low-passage Vero-derived stock preparation of 3 SARS-CoV-2 isolates selected for substitutions at positions 5/6 of E and were highly attenuated in BEpCs, revealing a key cell-specific function to this region. Rare isolate-specific deletions were also observed in the Spike furin cleavage site during Vero-CCL81 passage, but these were rapidly selected against in BEpCs, underscoring the importance of this site for SARS-CoV-2 replication in primary human cells. Overall, our study uncovers sequence features in SARS-CoV-2 variants that determine cell-specific replication and highlights the need to monitor SARS-CoV-2 stocks carefully when phenotyping newly emerging variants or potential variants of concern

Expiratory Aerosol pH: The Overlooked Driver of Airborne Virus Inactivation

Respiratory viruses, including influenza virus and SARS-CoV-2, are transmitted by the airborne route. Air filtration and ventilation mechanically reduce the concentration of airborne viruses and are necessary tools for disease mitigation. However, they ignore the potential impact of the chemical environment surrounding aerosolized viruses, which determines the aerosol pH. Atmospheric aerosol gravitates toward acidic pH, and enveloped viruses are prone to inactivation at strong acidity levels. Yet, the acidity of expiratory aerosol particles and its effect on airborne virus persistence have not been examined. Here, we combine pH-dependent inactivation rates of influenza A virus (IAV) and SARS-CoV-2 with microphysical properties of respiratory fluids using a biophysical aerosol model. We find that particles exhaled into indoor air (with relative humidity ≥ 50%) become mildly acidic (pH ∼ 4), rapidly inactivating IAV within minutes, whereas SARS-CoV-2 requires days. If indoor air is enriched with nonhazardous levels of nitric acid, aerosol pH drops by up to 2 units, decreasing 99%-inactivation times for both viruses in small aerosol particles to below 30 s. Conversely, unintentional removal of volatile acids from indoor air may elevate pH and prolong airborne virus persistence. The overlooked role of aerosol acidity has profound implications for virus transmission and mitigation strategies

Inactivation mechanisms of influenza A virus under pH conditions encountered in aerosol particles as revealed by whole-virus HDX-MS

Multiple respiratory viruses, including influenza A virus (IAV), can be transmitted via expiratory aerosol particles, and aerosol pH was recently identified as a major factor influencing airborne virus infectivity. Indoors, small exhaled aerosols undergo rapid acidification to pH ~4. IAV is known to be sensitive to mildly acidic conditions encountered within host endosomes; however, it is unknown whether the same mechanisms could mediate viral inactivation within the more acidic aerosol micro-environment. Here, we identified that transient exposure to pH 4 caused IAV inactivation by a two-stage process, with an initial sharp decline in infectious titers mainly attributed to premature attainment of the post-fusion conformation of viral protein haemagglutinin (HA). Protein changes were observed by hydrogen-deuterium exchange coupled to mass spectrometry (HDX-MS) as early as 10 s post-exposure to acidic conditions. Our HDX-MS data are in agreement with other more labor-intensive structural analysis techniques, such as X-ray crystallography, highlighting the ease and usefulness of whole-virus HDX-MS for multiplexed protein analyses, even within enveloped viruses such as IAV. Additionally, virion integrity was partially but irreversibly affected by acidic conditions, with a progressive unfolding of the internal matrix protein 1 (M1) that aligned with a more gradual decline in viral infectivity with time. In contrast, no acid-mediated changes to the genome or lipid envelope were detected. Improved understanding of respiratory virus fate within exhaled aerosols constitutes a global public health priority, and information gained here could aid the development of novel strategies to control the airborne persistence of seasonal and/or pandemic influenza in the future. IMPORTANCE: It is well established that COVID-19, influenza, and many other respiratory diseases can be transmitted by the inhalation of aerosolized viruses. Many studies have shown that the survival time of these airborne viruses is limited, but it remains an open question as to what drives their infectivity loss. Here, we address this question for influenza A virus by investigating structural protein changes incurred by the virus under conditions relevant to respiratory aerosol particles. From prior work, we know that expelled aerosols can become highly acidic due to equilibration with indoor room air, and our results indicate that two viral proteins are affected by these acidic conditions at multiple sites, leading to virus inactivation. Our findings suggest that the development of air treatments to quicken the speed of aerosol acidification would be a major strategy to control infectious bioburdens in the air

Phosphoproteomic profiling of influenza virus entry reveals infection-triggered filopodia induction counteracted by dynamic cortactin phosphorylation

Binding of influenza virus to its receptor triggers signaling cascades that reprogram the cell for infection. To elucidate global virus-induced changes to the cellular signaling landscape, we conducted a quantitative phosphoproteomic screen with human and avian influenza viruses. Proteins with functions in cell adhesion and cytoskeletal remodeling are overrepresented among the hits, and the majority of factors undergoing phosphorylation changes have a significant impact on infection efficiency. We show that influenza virus induces the formation of filopodia through Cdc42 signaling, which results in enhanced virus endocytosis. The host cell counteracts this mechanism with cortactin, a regulator of actin polymerization that becomes phosphorylated in response to virus binding and translocates to the cell cortex, where it limits filopodia formation and virus uptake. Overall, our study reveals the signaling cascades induced by influenza virus receptor engagement and uncovers virus-induced filopodia formation that is counteracted by the host cell.ISSN:2666-3864ISSN:2211-124

Adaptive Feedback from Artificial Neural Networks Facilitates Pre-Service Teachers’ Diagnostic Reasoning in Simulation-based Learning

In simulations, pre-service teachers need sophisticated feedback to develop complex skills such as diagnostic reasoning. In an experimental study with N = 178 pre-service teachers about simulated pupils with learning difficulties, we investigated the effects of automatic adaptive feedback, which is based on artificial neural networks, on pre-service teachers’ diagnostic reasoning. Diagnostic reasoning was operationalised as diagnostic accuracy and the quality of justifications. We compared automatic adaptive feedback with static feedback, which we provided in form of an expert solution. Further, we experimentally manipulated whether the learners worked individually or in dyads on the computer lab-based simulations. Results show that adaptive feedback facilitates pre-service teachers’ quality of justifications in written assignments, but not their diagnostic accuracy. Further, static feedback even had detrimental effects on the learning process in dyads. Automatic adaptive feedback in simulations offers scalable, elaborate, process-oriented feedback in real-time to high numbers of students in higher education.This research was supported by a grant of the German Federal Ministry of Research and Education (Grant No.: 16DHL1040) and by the Elite Network of Bavaria (K-GS-2012-209)

Combined computational and cellular screening identifies synergistic inhibition of SARS-CoV-2 by lenvatinib and remdesivir

Rapid repurposing of existing drugs as new therapeutics for COVID-19 has been an important strategy in the management of disease severity during the ongoing SARS-CoV-2 pandemic. Here, we used high-throughput docking to screen 6000 compounds within the DrugBank library for their potential to bind and inhibit the SARS-CoV-2 3 CL main protease, a chymotrypsin-like enzyme that is essential for viral replication. For 19 candidate hits, parallel in vitro fluorescence-based protease-inhibition assays and Vero-CCL81 cell-based SARS-CoV-2 replication-inhibition assays were performed. One hit, diclazuril (an investigational anti-protozoal compound), was validated as a SARS-CoV-2 3 CL main protease inhibitor in vitro (IC$_{50}$ value of 29 µM) and modestly inhibited SARS-CoV-2 replication in Vero-CCL81 cells. Another hit, lenvatinib (approved for use in humans as an anti-cancer treatment), could not be validated as a SARS-CoV-2 3 CL main protease inhibitor in vitro, but serendipitously exhibited a striking functional synergy with the approved nucleoside analogue remdesivir to inhibit SARS-CoV-2 replication, albeit this was specific to Vero-CCL81 cells. Lenvatinib is a broadly-acting host receptor tyrosine kinase (RTK) inhibitor, but the synergistic effect with remdesivir was not observed with other approved RTK inhibitors (such as pazopanib or sunitinib), suggesting that the mechanism-of-action is independent of host RTKs. Furthermore, time-of-addition studies revealed that lenvatinib/remdesivir synergy probably targets SARS-CoV-2 replication subsequent to host-cell entry. Our work shows that combining computational and cellular screening is a means to identify existing drugs with repurposing potential as antiviral compounds. Future studies could be aimed at understanding and optimizing the lenvatinib/remdesivir synergistic mechanism as a therapeutic option

From the Well to the Bottle: Identifying Sources of Microplastics in Mineral Water

Microplastics (MP) have been detected in bottled mineral water across the world. Because only few MP particles have been reported in ground water-sourced drinking water, it is suspected that MP enter the water during bottle cleaning and filling. However, until today, MP entry paths were not revealed. For the first time, this study provides findings of MP from the well to the bottle including the bottle washing process. At four mineral water bottlers, five sample types were taken along the process: raw and deferrized water samples were filtered in situ; clean bottles were sampled right after they left the bottle washer and after filling and capping. Caustic cleaning solutions were sampled from bottle washers and MP particles isolated through enzymatic and chemical treatments. The samples were analyzed for eleven synthetic and natural polymer particles ≥11 µm with Fourier-transform infrared imaging and random decision forests. MP were present in all steps of mineral water bottling, with a sharp increase from <1 MP L−1 to 317 ± 257 MP L−1 attributed to bottle capping. As 81% of MP resembled the PE-based cap sealing material, abrasion from the sealings was identified as the main entry path for MP into bottled mineral water

Synthetic Epigenetic Reprogramming of Mesenchymal to Epithelial States Using the CRISPR/dCas9 Platform in Triple Negative Breast Cancer

Altres ajuts: National Health and Medical Research Council (NHMRC) grant APP1147528; NHMRC grants APP1165208 and APP1187328; National Breast Cancer Foundation IIRS-22-044; Cancer Council New South Wales APP2013068; Cancer Council Western Australia APP2004608Epithelial-mesenchymal transition (EMT) is a reversible transcriptional program invoked by cancer cells to drive cancer progression. Transcription factor ZEB1 is a master regulator of EMT, driving disease recurrence in poor-outcome triple negative breast cancers (TNBCs). Here, this work silences ZEB1 in TNBC models by CRISPR/dCas9-mediated epigenetic editing, resulting in highly-specific and nearly complete suppression of ZEB1 in vivo, accompanied by long-lasting tumor inhibition. Integrated "omic" changes promoted by dCas9 linked to the KRAB domain (dCas9-KRAB) enabled the discovery of a ZEB1-dependent-signature of 26 genes differentially-expressed and -methylated, including the reactivation and enhanced chromatin accessibility in cell adhesion loci, outlining epigenetic reprogramming toward a more epithelial state. In the ZEB1 locus transcriptional silencing is associated with induction of locally-spread heterochromatin, significant changes in DNA methylation at specific CpGs, gain of H3K9me3, and a near complete erasure of H3K4me3 in the ZEB1 promoter. Epigenetic shifts induced by ZEB1-silencing are enriched in a subset of human breast tumors, illuminating a clinically-relevant hybrid-like state. Thus, the synthetic epi-silencing of ZEB1 induces stable "lock-in" epigenetic reprogramming of mesenchymal tumors associated with a distinct and stable epigenetic landscape. This work outlines epigenome-engineering approaches for reversing EMT and customizable precision molecular oncology approaches for targeting poor outcome breast cancers