Interstitial macrophages are a focus of viral takeover and inflammation in COVID-19 initiation in human lung.

Early stages of deadly respiratory diseases including COVID-19 are challenging to elucidate in humans. Here, we define cellular tropism and transcriptomic effects of SARS-CoV-2 virus by productively infecting healthy human lung tissue and using scRNA-seq to reconstruct the transcriptional program in "infection pseudotime" for individual lung cell types. SARS-CoV-2 predominantly infected activated interstitial macrophages (IMs), which can accumulate thousands of viral RNA molecules, taking over 60% of the cell transcriptome and forming dense viral RNA bodies while inducing host profibrotic (TGFB1, SPP1) and inflammatory (early interferon response, CCL2/7/8/13, CXCL10, and IL6/10) programs and destroying host cell architecture. Infected alveolar macrophages (AMs) showed none of these extreme responses. Spike-dependent viral entry into AMs used ACE2 and Sialoadhesin/CD169, whereas IM entry used DC-SIGN/CD209. These results identify activated IMs as a prominent site of viral takeover, the focus of inflammation and fibrosis, and suggest targeting CD209 to prevent early pathology in COVID-19 pneumonia. This approach can be generalized to any human lung infection and to evaluate therapeutics.

Lineage-tracing hematopoietic stem cell origins in vivo to efficiently make human HLF+ HOXA+ hematopoietic progenitors from pluripotent stem cells.

The developmental origin of blood-forming hematopoietic stem cells (HSCs) is a longstanding question. Here, our non-invasive genetic lineage tracing in mouse embryos pinpoints that artery endothelial cells generate HSCs. Arteries are transiently competent to generate HSCs for 2.5 days (∼E8.5-E11) but subsequently cease, delimiting a narrow time frame for HSC formation in vivo. Guided by the arterial origins of blood, we efficiently and rapidly differentiate human pluripotent stem cells (hPSCs) into posterior primitive streak, lateral mesoderm, artery endothelium, hemogenic endothelium, and >90% pure hematopoietic progenitors within 10 days. hPSC-derived hematopoietic progenitors generate T, B, NK, erythroid, and myeloid cells in vitro and, critically, express hallmark HSC transcription factors HLF and HOXA5-HOXA10, which were previously challenging to upregulate. We differentiated hPSCs into highly enriched HLF+ HOXA+ hematopoietic progenitors with near-stoichiometric efficiency by blocking formation of unwanted lineages at each differentiation step. hPSC-derived HLF+ HOXA+ hematopoietic progenitors could avail both basic research and cellular therapies.

Emergent Collective Motion of Self-Propelled Condensate Droplets.

Recently, there is much interest in droplet condensation on soft or liquid or liquidlike substrates. Droplets can deform soft and liquid interfaces resulting in a wealth of phenomena not observed on hard, solid surfaces (e.g., increased nucleation, interdroplet attraction). Here, we describe a unique collective motion of condensate water droplets that emerges spontaneously when a solid substrate is covered with a thin oil film. Droplets move first in a serpentine, self-avoiding fashion before transitioning to circular motions. We show that this self-propulsion (with speeds in the 0.1-1 mm s^{-1} range) is fueled by the interfacial energy release upon merging with newly condensed but much smaller droplets. The resultant collective motion spans multiple length scales from submillimeter to several centimeters, with potentially important heat-transfer and water-harvesting applications.

Visualizing a Nanoscale Lubricant Layer under Blood Flow.

Tethered-liquid perfluorocarbons (TLPs) are a class of liquid-infused surfaces with the ability to reduce blood clot formation (thrombosis) on blood-contacting medical devices. TLP comprises a tethered perfluorocarbon (TP) infused with a liquid perfluorocarbon (LP); this LP must be retained to maintain the antithrombotic properties of the layer. However, the stability of the LP layer remains in question, particularly for medical devices under blood flow. In this study, the lubricant thickness is spatially mapped and quantified in situ through confocal dual-wavelength reflection interference contrast microscopy. TLP coatings prepared on glass substrates are exposed to the flow of 37% glycerol/water mixtures (v/v) or whole blood at a shear strain rate of around 2900 s-1 to mimic physiological conditions (similar to flow conditions found in coronary arteries). Excess lubricant (>2 µm film thickness) is removed upon commencement of flow. For untreated glass, the lubricant is completely depleted after 1 min of shear flow. However, on optimized TLP surfaces, nanoscale films of lubricants (thickness between 100 nm and 2 µm) are retained over many tens of minutes of flow. The nanoscale films conform to the underlying structure of the TP layer and are sufficient to prevent the adhesion of red blood cells and platelets.

Prospective isolation of neural stem and progenitor cells from the developing human brain.

Prospective isolation of defined cell types is critical for the functional study of stem cells, especially in primary human tissues. Here, we present a protocol for purifying 10 transcriptomically and functionally distinct neural stem and progenitor cell types from the developing human brain using fluorescence-activated cell sorting. We describe steps for tissue dissociation, staining, and cell sorting as well as downstream functional experiments for measuring clonogenicity, differentiation, and engraftment potential of purified populations. For complete details on the use and execution of this protocol, please refer to Liu et al. (2023).1.

Droplet detachment force and its relation to Young-Dupre adhesion.

Droplets adhere to surfaces due to their surface tension γ and understanding the vertical force Fd required to detach the droplet is key to many technologies (e.g., inkjet printing, optimal paint formulations). Here, we predicted Fd on different surfaces by numerically solving the Young-Laplace equation. Our numerical results are consistent with previously reported results for a wide range of experimental conditions: droplets subjected to surface vs. body forces with |Fd| ranging from nano- to milli-newtons, droplet radii R ranging from tens of microns to several millimetres, and for various surfaces (micro-/nano-structured superhydrophobic vs. lubricated surfaces). Finally, we derive an analytic solution for Fd on highly hydrophobic surfaces and further show that for receding contact angle θr > 140°, the normalized Fd/πR is equivalent to the Young-Dupre work of adhesion γ(1 + cos θr).

Droplet attraction and coalescence mechanism on textured oil-impregnated surfaces.

Droplets residing on textured oil-impregnated surfaces form a wetting ridge due to the imbalance of interfacial forces at the contact line, leading to a wealth of phenomena not seen on traditional lotus-leaf-inspired non-wetting surfaces. Here, we show that the wetting ridge leads to long-range attraction between millimeter-sized droplets, which coalesce in three distinct stages: droplet attraction, lubricant draining, and droplet merging. Our experiments and model show that the magnitude of the velocity and acceleration at which droplets approach each other horizontally is the same as the vertical oil rise velocity and acceleration in the wetting ridge. Moreover, the droplet coalescence mechanism can be modeled using the classical mass-spring system. The insights gained from this work will inform future fundamental studies on remote droplet interaction on textured oil-impregnated surfaces for optimizing water harvesting and condensation heat transfer.

Purification and characterization of human neural stem and progenitor cells.

The human brain undergoes rapid development at mid-gestation from a pool of neural stem and progenitor cells (NSPCs) that give rise to the neurons, oligodendrocytes, and astrocytes of the mature brain. Functional study of these cell types has been hampered by a lack of precise purification methods. We describe a method for prospectively isolating ten distinct NSPC types from the developing human brain using cell-surface markers. CD24-THY1-/lo cells were enriched for radial glia, which robustly engrafted and differentiated into all three neural lineages in the mouse brain. THY1hi cells marked unipotent oligodendrocyte precursors committed to an oligodendroglial fate, and CD24+THY1-/lo cells marked committed excitatory and inhibitory neuronal lineages. Notably, we identify and functionally characterize a transcriptomically distinct THY1hiEGFRhiPDGFRA- bipotent glial progenitor cell (GPC), which is lineage-restricted to astrocytes and oligodendrocytes, but not to neurons. Our study provides a framework for the functional study of distinct cell types in human neurodevelopment.

Energy Loss for Droplets Bouncing Off Superhydrophobic Surfaces.

A water droplet can bounce off superhydrophobic surfaces multiple times before coming to a stop. The energy loss for such droplet rebounds can be quantified by the ratio of the rebound speed UR and the initial impact speed UI; i.e., its restitution coefficient e = UR/UI. Despite much work in this area, a mechanistic explanation for the energy loss for rebounding droplets is still lacking. Here, we measured e for submillimeter- and millimeter-sized droplets impacting two different superhydrophobic surfaces over a wide range of UI (4-700 cm s-1). We proposed simple scaling laws to explain the observed nonmonotonic dependence of e on UI. In the limit of low UI, energy loss is dominated by contact-line pinning and e is sensitive to the surface wetting properties, in particular to contact angle hysteresis Δ cos θ of the surface. In contrast, e is dominated by inertial-capillary effects and does not depend on Δ cos θ in the limit of high UI.

Presence, origins and effect of stable surface hydration on regenerated cellulose for underwater oil-repellent membranes.

HYPOTHESIS: Underwater oil-repellency of polyelectrolyte brushes has been attributed mainly to electric double-layer repulsion forces based on Derjaguin-Landau-Verwey-Overbeek (DLVO) theory. Many non-polyelectrolyte materials also exhibit oil-repellent behaviour, but it is not clear if there exist similar electric double-layer repulsion and if it is the sole mechanism governing their underwater oil-repellency. EXPERIMENTS/SIMULATIONS: In this article, the oil-repellency of highly amorphous cellulose exhibiting is investigated in detail, through experiments and molecular dynamics simulations (MDS). FINDINGS: It was found that the stable surface hydration on regenerated cellulose was due to a combination of long-range electrostatic repulsions (DLVO theory) and short-range interfacial hydrogen bonding between cellulose and water molecules (as revealed by MDS). The presence of a stable water layer of about 200 nm thick (similar to that of polyelectrolyte brushes) was confirmed. Such stable surface hydration effectively separates cellulose surface from oil droplets, resulting in extremely low adhesion between them. As a demonstration of its practicality, regenerated cellulose membranes were fabricated via electrospinning, and they exhibit high oil/water separation efficiencies (including oil-in-water emulsions) as well as self-cleaning ability.

Mesenchymal stromal cells for the treatment of Alzheimer's disease: Strategies and limitations.

Alzheimer's disease (AD) is a major cause of age-related dementia and is characterized by progressive brain damage that gradually destroys memory and the ability to learn, which ultimately leads to the decline of a patient's ability to perform daily activities. Although some of the pharmacological treatments of AD are available for symptomatic relief, they are not able to limit the progression of AD and have several side effects. Mesenchymal stem/stromal cells (MSCs) could be a potential therapeutic option for treating AD due to their immunomodulatory, anti-inflammatory, regenerative, antioxidant, anti-apoptotic, and neuroprotective effects. MSCs not only secret neuroprotective and anti-inflammatory factors to promote the survival of neurons, but they also transfer functional mitochondria and miRNAs to boost their bioenergetic profile as well as improve microglial clearance of accumulated protein aggregates. This review focuses on different clinical and preclinical studies using MSC as a therapy for treating AD, their outcomes, limitations and the strategies to potentiate their clinical translation.

Wettability-based ultrasensitive detection of amphiphiles through directed concentration at disordered regions in self-assembled monolayers.

Various forms of ecological monitoring and disease diagnosis rely upon the detection of amphiphiles, including lipids, lipopolysaccharides, and lipoproteins, at ultralow concentrations in small droplets. Although assays based on droplets' wettability provide promising options in some cases, their reliance on the measurements of surface and bulk properties of whole droplets (e.g., contact angles, surface tensions) makes it difficult to monitor trace amounts of these amphiphiles within small-volume samples. Here, we report a design principle in which self-assembled monolayer-functionalized microstructured surfaces coated with silicone oil create locally disordered regions within a droplet's contact lines to effectively concentrate amphiphiles within the areas that dominate the droplet static friction. Remarkably, such surfaces enable the ultrasensitive, naked-eye detection of amphiphiles through changes in the droplets' sliding angles, even when the concentration is four to five orders of magnitude below their critical micelle concentration. We develop a thermodynamic model to explain the partitioning of amphiphiles at the contact line by their cooperative association within the disordered, loosely packed regions of the self-assembled monolayer. Based on this local analyte concentrating effect, we showcase laboratory-on-a-chip surfaces with positionally dependent pinning forces capable of both detecting industrially and biologically relevant amphiphiles (e.g., bacterial endotoxins), as well as sorting aqueous droplets into discrete groups based on their amphiphile concentrations. Furthermore, we demonstrate that the sliding behavior of amphiphile-laden aqueous droplets provides insight into the amphiphile's effective length, thereby allowing these surfaces to discriminate between analytes with highly disparate molecular sizes.

AFM Manipulation of EGaIn Microdroplets to Generate Controlled, On-Demand Contacts on Molecular Self-Assembled Monolayers.

Liquid metal droplets, such as eutectic gallium-indium (EGaIn), are important in many research areas, such as soft electronics, catalysis, and energy storage. Droplet contact on solid surfaces is typically achieved without control over the applied force and without optimizing the wetting properties in different environments (e.g., in air or liquid), resulting in poorly defined contact areas. In this work, we demonstrate the direct manipulation of EGaIn microdroplets using an atomic force microscope (AFM) to generate repeated, on-demand making and breaking of contact on self-assembled monolayers (SAMs) of alkanethiols. The nanoscale positional control and feedback loop in an AFM allow us to control the contact force at the nanonewton level and, consequently, tune the droplet contact areas at the micrometer length scale in both air and ethanol. When submerged in ethanol, the droplets are highly nonwetting, resulting in hysteresis-free contact forces and minimal adhesion; as a result, we are able to create reproducible geometric contact areas of 0.8-4.5 µm2 with the alkanethiolate SAMs in ethanol. In contrast, there is a larger hysteresis in the contact forces and larger adhesion for the same EGaIn droplet in air, which reduced the control over the contact area (4-12 µm2). We demonstrate the usefulness of the technique and of the gained insights in EGaIn contact mechanics by making well-defined molecular tunneling junctions based on alkanethiolate SAMs with small geometric contact areas of between 4 and 12 µm2 in air, 1 to 2 orders of magnitude smaller than previously achieved.

LCOR mediates interferon-independent tumor immunogenicity and responsiveness to immune-checkpoint blockade in triple-negative breast cancer.

Ligand-dependent corepressor (LCOR) mediates normal and malignant breast stem cell differentiation. Cancer stem cells (CSCs) generate phenotypic heterogeneity and drive therapy resistance, yet their role in immunotherapy is poorly understood. Here we show that immune-checkpoint blockade (ICB) therapy selects for LCORlow CSCs with reduced antigen processing/presentation machinery (APM) driving immune escape and ICB resistance in triple-negative breast cancer (TNBC). We unveil an unexpected function of LCOR as a master transcriptional activator of APM genes binding to IFN-stimulated response elements (ISREs) in an IFN signaling-independent manner. Through genetic modification of LCOR expression, we demonstrate its central role in modulation of tumor immunogenicity and ICB responsiveness. In TNBC, LCOR associates with ICB clinical response. Importantly, extracellular vesicle (EV) Lcor-messenger RNA therapy in combination with anti-PD-L1 overcame resistance and eradicated breast cancer metastasis in preclinical models. Collectively, these data support LCOR as a promising target for enhancement of ICB efficacy in TNBC, by boosting of tumor APM independently of IFN.

Risk assessment of airborne COVID-19 exposure in social settings.

The COVID-19 pandemic has led to many countries oscillating between various states of lock-down as they seek to balance keeping the economy and essential services running and minimizing the risk of further transmission. Decisions are made about which activities to keep open across a range of social settings and venues guided only by ad hoc heuristics regarding social distancing and personal hygiene. Hence, we propose the dual use of computational fluid dynamic simulations and surrogate aerosol measurements for location-specific assessment of risk of infection across different real-world settings. We propose a 3-tiered risk assessment scheme to facilitate classification of scenarios into risk levels based on simulations and experiments. Threshold values of <54 and >840 viral copies and <5% and >40% of original aerosol concentration are chosen to stratify low, medium, and high risk. This can help prioritize allowable activities and guide implementation of phased lockdowns or re-opening. Using a public bus in Singapore as a case study, we evaluate the relative risk of infection across scenarios such as different activities and passenger positions and demonstrate the effectiveness of our risk assessment methodology as a simple and easily interpretable framework. For example, this study revealed that the bus's air-conditioning greatly influences dispersion and increases the risk of certain seats and that talking can result in similar relative risk to coughing for passengers around an infected person. Both numerical and experimental approaches show similar relative risk levels with a Spearman's correlation coefficient of 0.74 despite differing observables, demonstrating applicability of this risk assessment methodology to other scenarios.

A Clinical PET Imaging Tracer ([18F]DASA-23) to Monitor Pyruvate Kinase M2-Induced Glycolytic Reprogramming in Glioblastoma.

PURPOSE: Pyruvate kinase M2 (PKM2) catalyzes the final step in glycolysis, a key process of cancer metabolism. PKM2 is preferentially expressed by glioblastoma (GBM) cells with minimal expression in healthy brain. We describe the development, validation, and translation of a novel PET tracer to study PKM2 in GBM. We evaluated 1-((2-fluoro-6-[18F]fluorophenyl)sulfonyl)-4-((4-methoxyphenyl)sulfonyl)piperazine ([18F]DASA-23) in cell culture, mouse models of GBM, healthy human volunteers, and patients with GBM. EXPERIMENTAL DESIGN: [18F]DASA-23 was synthesized with a molar activity of 100.47 ± 29.58 GBq/µmol and radiochemical purity >95%. We performed initial testing of [18F]DASA-23 in GBM cell culture and human GBM xenografts implanted orthotopically into mice. Next, we produced [18F]DASA-23 under FDA oversight, and evaluated it in healthy volunteers and a pilot cohort of patients with glioma. RESULTS: In mouse imaging studies, [18F]DASA-23 clearly delineated the U87 GBM from surrounding healthy brain tissue and had a tumor-to-brain ratio of 3.6 ± 0.5. In human volunteers, [18F]DASA-23 crossed the intact blood-brain barrier and was rapidly cleared. In patients with GBM, [18F]DASA-23 successfully outlined tumors visible on contrast-enhanced MRI. The uptake of [18F]DASA-23 was markedly elevated in GBMs compared with normal brain, and it identified a metabolic nonresponder within 1 week of treatment initiation. CONCLUSIONS: We developed and translated [18F]DASA-23 as a new tracer that demonstrated the visualization of aberrantly expressed PKM2 for the first time in human subjects. These results warrant further clinical evaluation of [18F]DASA-23 to assess its utility for imaging therapy-induced normalization of aberrant cancer metabolism.

Visualizing and Quantifying Wettability Alteration by Silica Nanofluids.

An aqueous suspension of silica nanoparticles or nanofluid can alter the wettability of surfaces, specifically by making them hydrophilic and oil-repellent under water. Wettability alteration by nanofluids has important technological applications, including for enhanced oil recovery and heat transfer processes. A common way to characterize the wettability alteration is by measuring the contact angles of an oil droplet with and without nanoparticles. While easy to perform, contact angle measurements do not fully capture the wettability changes to the surface. Here, we employed several complementary techniques, such as cryo-scanning electron microscopy, confocal fluorescence and reflection interference contrast microscopy, and droplet probe atomic force microscopy (AFM), to visualize and quantify the wettability alterations by fumed silica nanoparticles. We found that nanoparticles adsorbed onto glass surfaces to form a porous layer with hierarchical micro- and nanostructures. The porous layer can trap a thin water film, which reduces contact between the oil droplet and the solid substrate. As a result, even a small addition of nanoparticles (0.1 wt %) lowers the adhesion force for a 20 µm sized oil droplet by more than 400 times from 210 ± 10 to 0.5 ± 0.3 nN as measured by using droplet probe AFM. Finally, we show that silica nanofluids can improve oil recovery rates by 8% in a micromodel with glass channels that resemble a physical rock network.

Eye Protection in ENT Practice During the COVID-19 Pandemic.

OBJECTIVES: There is a lack of evidence-based guidelines with regard to eye protection for aerosol-generating procedures in otolaryngology practice. In addition, some recommended personal protective equipment (PPE) is not compatible with commonly used ENT equipment. This study aims to investigate the degree of eye protection that commonly used PPE gives. STUDY DESIGN: Simulation model. SETTING: Simulation laboratory. METHODS: A custom-built setup was utilized to simulate the clinical scenario of a patient cough in proximity of a health care worker. A system that sprays a xanthan-fluorescein mixture was set up and calibrated to simulate a human cough. A mannequin with cellulose paper placed on its forehead, eyes, and mouth was fitted with various PPE combinations and exposed to the simulated cough. The degree of contamination on the cellulose papers was quantified with a fluorescent microscope able to detect aerosols ≥10 µm. RESULTS: When no eye protection was worn, 278 droplets/aerosols reached the eye area. The use of the surgical mask with an attached upward-facing shield alone resulted in only 2 droplets/aerosols reaching the eye area. In this experiment, safety glasses and goggles performed equally, as the addition of either brought the number of droplets/aerosols reaching the eye down to 0. CONCLUSION: When used with an upward-facing face shield, there was no difference in the eye protection rendered by safety goggles or glasses in this study. Safety glasses may be considered a viable alternative to safety goggles in aerosol-generating procedures.

Effective design of barrier enclosure to contain aerosol emissions from COVID-19 patients.

Facing shortages of personal protective equipment, some clinicians have advocated the use of barrier enclosures (typically mounted over the head, with and without suction) to contain aerosol emissions from coronavirus disease 2019 (COVID-19) patients. There is, however, little evidence for its usefulness. To test the effectiveness of such a device, we built a manikin that can expire micron-sized aerosols at flow rates close to physiological conditions. We then placed the manikin inside the enclosure and used a laser sheet to visualize the aerosol leaking out. We show that with sufficient suction, it is possible to effectively contain aerosol from the manikin, reducing aerosol exposure outside the enclosure by 99%. In contrast, a passive barrier without suction only reduces aerosol exposure by 60%.

The Efficacy of Plant-Based Ionizers in Removing Aerosol for COVID-19 Mitigation.

Small-sized droplets/aerosol transmission is one of the factors responsible for the spread of COVID-19, in addition to large droplets and surface contamination (fomites). While large droplets and surface contamination can be relatively easier to deal with (i.e., using mask and proper hygiene measures), aerosol presents a different challenge due to their ability to remain airborne for a long time. This calls for mitigation solutions that can rapidly eliminate the airborne aerosol. Pre-COVID-19, air ionizers have been touted as effective tools to eliminate small particulates. In this work, we sought to evaluate the efficacy of a novel plant-based ionizer in eliminating aerosol. It was found that factors such as the ion concentration, humidity, and ventilation can drastically affect the efficacy of aerosol removal. The aerosol removal rate was quantified in terms of ACH (air changes per hour) and CADR- (clean air delivery rate-) equivalent unit, with ACH as high as 12 and CADR as high as 141 ft3/minute being achieved by a plant-based ionizer in a small isolated room. This work provides an important and timely guidance on the effective deployment of ionizers in minimizing the risk of COVID-19 spread via airborne aerosol, especially in a poorly-ventilated environment.