Highly Efficient Red/NIR-Emissive Fluorescent Probe with Polarity-Sensitive Character for Visualizing Cellular Lipid Droplets and Determining Their Polarity.

Lipid droplets (LDs), which are ubiquitous organelles existing in almost all eukaryotic cells, have attracted a lot of attention in the field of cell biology over the last decade. For the biological study of LDs via fluorescence imaging, the superior LD fluorescent probes with environmental polarity-sensitive character are highly desired and powerful but are very scarce. Herein, we have newly developed such a kind of fluorescent probe named LDs-Red which enables us to visualize LDs and to further reveal their polarity information. This fluorescent probe displays the advantages of intense red/near-infrared emission, high LD staining specificity, and good photostability; thus, it would be very useful for LD fluorescence imaging application. As a result, the three-dimensional confocal imaging to visualize spatial distribution of LDs and the multicolor confocal imaging to simultaneously observe LDs and other cellular organelles have been realized using this new LD fluorescent probe. Furthermore, the polarity-sensitive emission character of this probe enables us to quantitatively determine the LD polarity via spectral scan imaging. Consequently, the cancer cells (HepG2, HeLa, and Panc02) displaying lower polarity of LDs than the normal cells (L929, U251, and HT22) have been systematically demonstrated. In addition, this polarity-sensitive probe displaying shorter fluorescence wavelengths in cancer cells than in normal cells has an important and potential ability to distinguish them.

Targeted Delivery of Zinc Pyrithione to Skin Epithelia.

Zinc pyrithione (ZnPT) is an anti-fungal drug delivered as a microparticle to skin epithelia. It is one of the most widely used ingredients worldwide in medicated shampoo for treating dandruff and seborrheic dermatitis (SD), a disorder with symptoms that include skin flaking, erythema and pruritus. SD is a multi-factorial disease driven by microbiol dysbiosis, primarily involving Malassezia yeast. Anti-fungal activity of ZnPT depends on the cutaneous availability of bioactive monomeric molecular species, occurring upon particle dissolution. The success of ZnPT as a topical therapeutic is underscored by the way it balances treatment efficacy with formulation safety. This review demonstrates how ZnPT achieves this balance, by integrating the current understanding of SD pathogenesis with an up-to-date analysis of ZnPT pharmacology, therapeutics and toxicology. ZnPT has anti-fungal activity with an average in vitro minimum inhibitory concentration of 10-15 ppm against the most abundant scalp skin Malassezia species (Malassezia globosa and Malassezia restrica). Efficacy is dependent on the targeted delivery of ZnPT to the skin sites where these yeasts reside, including the scalp surface and hair follicle infundibulum. Imaging and quantitative analysis tools have been fundamental for critically evaluating the therapeutic performance and safety of topical ZnPT formulations. Toxicologic investigations have focused on understanding the risk of local and systemic adverse effects following exposure from percutaneous penetration. Future research is expected to yield further advances in ZnPT formulations for SD and also include re-purposing towards a range of other dermatologic applications, which is likely to have significant clinical impact.

Handheld Multispectral Fluorescence Imaging System to Detect and Disinfect Surface Contamination.

Contamination inspection is an ongoing concern for food distributors, restaurant owners, caterers, and others who handle food. Food contamination must be prevented, and zero tolerance legal requirements and damage to the reputation of institutions or restaurants can be very costly. This paper introduces a new handheld fluorescence-based imaging system that can rapidly detect, disinfect, and document invisible organic residues and biofilms which may host pathogens. The contamination, sanitization inspection, and disinfection (CSI-D) system uses light at two fluorescence excitation wavelengths, ultraviolet C (UVC) at 275 nm and violet at 405 nm, for the detection of organic residues, including saliva and respiratory droplets. The 275 nm light is also utilized to disinfect pathogens commonly found within the contaminated residues. Efficacy testing of the neutralizing effects of the ultraviolet light was conducted for Aspergillus fumigatus, Streptococcus pneumoniae, and the influenza A virus (a fungus, a bacterium, and a virus, respectively, each commonly found in saliva and respiratory droplets). After the exposure to UVC light from the CSI-D, all three pathogens experienced deactivation (> 99.99%) in under ten seconds. Up to five-log reductions have also been shown within 10 s of UVC irradiation from the CSI-D system.

Delivery of Nitric Oxide in the Cardiovascular System: Implications for Clinical Diagnosis and Therapy.

Nitric oxide (NO) is a key molecule in cardiovascular homeostasis and its abnormal delivery is highly associated with the occurrence and development of cardiovascular disease (CVD). The assessment and manipulation of NO delivery is crucial to the diagnosis and therapy of CVD, such as endothelial dysfunction, atherosclerotic progression, pulmonary hypertension, and cardiovascular manifestations of coronavirus (COVID-19). However, due to the low concentration and fast reaction characteristics of NO in the cardiovascular system, clinical applications centered on NO delivery are challenging. In this tutorial review, we first summarized the methods to estimate the in vivo NO delivery process, based on computational modeling and flow-mediated dilation, to assess endothelial function and vulnerability of atherosclerotic plaque. Then, emerging bioimaging technologies that have the potential to experimentally measure arterial NO concentration were discussed, including Raman spectroscopy and electrochemical sensors. In addition to diagnostic methods, therapies aimed at controlling NO delivery to regulate CVD were reviewed, including the NO release platform to treat endothelial dysfunction and atherosclerosis and inhaled NO therapy to treat pulmonary hypertension and COVID-19. Two potential methods to improve the effectiveness of existing NO therapy were also discussed, including the combination of NO release platform and computational modeling, and stem cell therapy, which currently remains at the laboratory stage but has clinical potential for the treatment of CVD.

Renal-Clearable Molecular Probe for Near-Infrared Fluorescence Imaging and Urinalysis of SARS-CoV-2.

Despite the importance of rapid and accurate detection of SARS-CoV-2 in controlling the COVID-19 pandemic, current diagnostic methods are static and unable to distinguish between viable/nonviable virus or directly reflect viral replication activity. Real-time imaging of protease activity specific to SARS-CoV-2 can overcome these issues but remains lacking. Herein, we report a near-infrared fluorescence (NIRF) activatable molecular probe (SARS-CyCD) for detection of SARS-CoV-2 protease in living mice. The probe comprises a hemicyanine fluorophore caged with a protease peptide substrate and a cyclodextrin unit, which function as an NIRF signaling moiety and a renal-clearable enabler, respectively. The peptide substrate of SARS-CyCD can be specifically cleaved by SARS-CoV-2 main protease (Mpro), resulting in NIRF signal activation and liberation of the renal-clearable fluorescent fragment (CyCD). Such a design not only allows sensitive detection of Mpro in the lungs of living mice after intratracheal administration but also permits optical urinalysis of SARS-CoV-2 infection. Thus, this study presents an in vivo sensor that holds potential in preclinical high-throughput drug screening and clinical diagnostics for respiratory viral infections.

Fluorescent glycan fingerprinting of SARS2 spike proteins.

Glycosylation is the most common post-translational modification and has myriad of biological functions. However, glycan analysis has always been a challenge. Here, we would like to present new techniques for glycan fingerprinting based on enzymatic fluorescent labeling and gel electrophoresis. The method is illustrated on SARS2 spike (S) glycoproteins. SARS2, a novel coronavirus and the causative agent of the COVID-19 pandemic, has had significant social and economic impacts since the end of 2019. To obtain the N-glycan fingerprint of an S protein, glycans released from the protein are first labeled through enzymatic incorporation of fluorophore-conjugated sialic acid or fucose, then separated by SDS-PAGE, and finally visualized with a fluorescent imager. To identify the labeled glycans of a fingerprint, glycan standards and glycan ladders are enzymatically generated and run alongside the samples as references. By comparing the mobility of a labeled glycan to that of a glycan standard, the identity of glycans maybe determined. O-glycans can also be fingerprinted. Due to the lack of an enzyme for broad O-glycan release, O-glycans on the S protein can be labeled with fluorescent sialic acid and digested with trypsin to obtain labeled glycan peptides that are then separated by gel electrophoresis. Glycan fingerprinting could serve as a quick method for globally assessing the glycosylation of a specific glycoprotein.

Nanotheranostic Application of Fluorescent Protein-Gold Nanocluster Hybrid Materials: A Mini-review.

The gold nanoclusters (Au NCs) are a special kind of gold nanomaterial containing several gold atoms. Because of their small size and large surface area, Au NCs possess macroscopic quantum tunneling and dielectric domain effects. Furthermore, Au NCs fluorescent materials have longer luminous time and better photobleaching resistance compared with other fluorescent materials. The synthetic process of traditional Au NCs is complicated. Traditional Au NCs are prepared mainly by using polyamide amine type dendrites, and sixteen alkyl trimethylamine bromide or sulfhydryl small molecule as stabilizers. They are consequently synthesized by the reduction of strong reducing agents such as sodium borohydride. Notably, these materials are toxic and environmental-unfriendly. Therefore, there is an urgent need to develop more effective methods for synthesizing Au NCs via a green approach. On the other hand, the self-assembly of protein gold cluster-based materials, and their biomedical applications have become research hotspots in this field. We have been working on the synthesis, assembly and application of protein conjugated gold clusters for a long time. In this review, the synthesis and assembly of protein-gold nanoclusters and their usage in cell imaging and other medical research are discussed.

Cryo-EM structure of SARS-CoV-2 ORF3a in lipid nanodiscs.

SARS-CoV-2 ORF3a is a putative viral ion channel implicated in autophagy inhibition, inflammasome activation and apoptosis. 3a protein and anti-3a antibodies are found in infected patient tissues and plasma. Deletion of 3a in SARS-CoV-1 reduces viral titer and morbidity in mice, suggesting it could be an effective target for vaccines or therapeutics. Here, we present structures of SARS-CoV-2 3a determined by cryo-EM to 2.1-Å resolution. 3a adopts a new fold with a polar cavity that opens to the cytosol and membrane through separate water- and lipid-filled openings. Hydrophilic grooves along outer helices could form ion-conduction paths. Using electrophysiology and fluorescent ion imaging of 3a-reconstituted liposomes, we observe Ca2+-permeable, nonselective cation channel activity, identify mutations that alter ion permeability and discover polycationic inhibitors of 3a activity. 3a-like proteins are found across coronavirus lineages that infect bats and humans, suggesting that 3a-targeted approaches could treat COVID-19 and other coronavirus diseases.

Circulating activated neutrophils in COVID-19: An independent predictor for mechanical ventilation and death.

OBJECTIVES: Critical illness in COVID-19 is attributed to an exaggerated host immune response. Since neutrophils are the major component of innate immunity, we hypothesize that the quantum of activated neutrophils in the blood may predict an adverse outcome. DESIGN: In a retrospective study of 300 adult patients with confirmed COVID-19, we analyzed the impact of neutrophil activation (NEUT-RI), interleukin-6 (IL-6) and the established clinical risk factors of age, diabetes, obesity and hypertension on the clinical outcome. RESULTS: Significant predictors of the need for mechanical ventilation were NEUT-RI (Odds Ratio (OR) = 1.22, P < 0.001), diabetes (OR = 2.56, P = 0.00846) and obesity (OR = 6.55, P < 0.001). For death, the significant predictors were NEUT-RI (OR = 1.14, P = 0.00432), diabetes (OR = 4.11, P = 0.00185) and age (OR = 1.04, P = 0.00896). The optimal cut-off value for NEUT-RI to predict mechanical ventilation and death was 52 fluorescence intensity units (sensitivity 44%, specificity 88%, area under the curve 0.67 and 44%, 86%, 0.64, respectively). CONCLUSION: This finding supports an aberrant neutrophil response in COVID-19, likely due to uncontained viral replication, tissue hypoxia and exacerbated inflammation, introduces a novel biomarker for rapid monitoring and opens new avenues for therapeutic strategies.

Detection of SARS-CoV-2 Neutralizing Antibodies using High-Throughput Fluorescent Imaging of Pseudovirus Infection.

As the COVID-19 pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continues to evolve, it has become evident that the presence of neutralizing antibodies against the virus may provide protection against future infection. Thus, as the creation and translation of effective COVID-19 vaccines continues at an unprecedented speed, the development of fast and effective methods to measure neutralizing antibodies against SARS-CoV-2 will become increasingly important to determine long-term protection against infection for both previously infected and immunized individuals. This paper describes a high-throughput protocol using vesicular stomatitis virus (VSV) pseudotyped with the SARS-CoV-2 spike protein to measure the presence of neutralizing antibodies in convalescent serum from patients who have recently recovered from COVID-19. The use of a replicating pseudotyped virus eliminates the necessity for a containment level 3 facility required for SARS-CoV-2 handling, making this protocol accessible to virtually any containment level 2 lab. The use of a 96-well format allows for many samples to be run at the same time with a short turnaround time of 24 h.

Modeling of aerosol transmission of airborne pathogens in ICU rooms of COVID-19 patients with acute respiratory failure.

The COVID-19 pandemic has generated many concerns about cross-contamination risks, particularly in hospital settings and Intensive Care Units (ICU). Virus-laden aerosols produced by infected patients can propagate throughout ventilated rooms and put medical personnel entering them at risk. Experimental results found with a schlieren optical method have shown that the air flows generated by a cough and normal breathing were modified by the oxygenation technique used, especially when using High Flow Nasal Canulae, increasing the shedding of potentially infectious airborne particles. This study also uses a 3D Computational Fluid Dynamics model based on a Lattice Boltzmann Method to simulate the air flows as well as the movement of numerous airborne particles produced by a patient's cough within an ICU room under negative pressure. The effects of different mitigation scenarii on the amount of aerosols potentially containing SARS-CoV-2 that are extracted through the ventilation system are investigated. Numerical results indicate that adequate bed orientation and additional air treatment unit positioning can increase by 40% the number of particles extracted and decrease by 25% the amount of particles deposited on surfaces 45s after shedding. This approach could help lay the grounds for a more comprehensive way to tackle contamination risks in hospitals, as the model can be seen as a proof of concept and be adapted to any room configuration.

Novel application of indocyanine green fluorescence imaging for real-time detection of thrombus in a membrane oxygenator.

Extracorporeal membrane oxygenation (ECMO) plays an important role in the coronavirus disease 2019 (COVID-19) pandemic. Management of thrombi in ECMO is generally an important issue; especially in ECMO for COVID-19 patients who are prone to thrombus formation, the thrombus formation in oxygenators is an unresolved issue, and it is very difficult to deal with. To prevent thromboembolic complications, it is necessary to develop a method for early thrombus detection. We developed a novel method for detailed real-time observation of thrombi formed in oxygenators using indocyanine green (ICG) fluorescence imaging. The purpose of this study was to verify the efficacy of this novel method through animal experiments. The experiments were performed three times using three pigs equipped with veno-arterial ECMO comprising a centrifugal pump (CAPIOX SL) and an oxygenator (QUADROX). To create thrombogenic conditions, the pump flow rate was set at 1 L/min without anticoagulation. The diluted ICG (0.025 mg/mL) was intravenously administered at a dose of 10 mL once an hour. A single dose of ICG was 0.25mg. The oxygenator was observed with both an optical detector (PDE-neo) and the naked eye every hour after measurement initiation for a total of 8 hours. With this dose of ICG, we could observe it by fluorescence imaging for about 15 minutes. Under ICG imaging, the inside of the oxygenator was observed as a white area. A black dot suspected to be the thrombus appeared 6-8 hours after measurement initiation. The thrombus and the black dot on ICG imaging were finely matched in terms of morphology. Thus, we succeeded in real-time thrombus detection in an oxygenator using ICG imaging. The combined use of ICG imaging and conventional routine screening tests could compensate for each other's weaknesses and significantly improve the safety of ECMO.

Amplification-free detection of SARS-CoV-2 with CRISPR-Cas13a and mobile phone microscopy.

The December 2019 outbreak of a novel respiratory virus, SARS-CoV-2, has become an ongoing global pandemic due in part to the challenge of identifying symptomatic, asymptomatic, and pre-symptomatic carriers of the virus. CRISPR diagnostics can augment gold-standard PCR-based testing if they can be made rapid, portable, and accurate. Here, we report the development of an amplification-free CRISPR-Cas13a assay for direct detection of SARS-CoV-2 from nasal swab RNA that can be read with a mobile phone microscope. The assay achieved ∼100 copies/µL sensitivity in under 30 min of measurement time and accurately detected pre-extracted RNA from a set of positive clinical samples in under 5 min. We combined crRNAs targeting SARS-CoV-2 RNA to improve sensitivity and specificity and directly quantified viral load using enzyme kinetics. Integrated with a reader device based on a mobile phone, this assay has the potential to enable rapid, low-cost, point-of-care screening for SARS-CoV-2.

Quantitative High-speed Assessment of Droplet and Aerosol From an Eye After Impact With an Air-puff Amid COVID-19 Scenario.

PURPOSE: To quantify aerosol and droplets generated during noncontact tonometry (NCT) and assess the spread distance of the same. METHODOLOGY: This was an experimental study on healthy human volunteers (n=8 eyes). In an experimental setup, NCT was performed on eyes (n=8) of human volunteers under normal settings, with a single and 2 drops of lubricant. High-speed shadowgraphy, frontal lighting technique, and fluorescein analysis were used to detect the possible generation of any droplets and aerosols. Mathematical computation of the spread of the droplets was then performed. RESULTS: In a natural setting, there was no droplet or aerosol production. Minimal splatter along with droplet ejection was observed when 1 drop of lubricant was used before NCT. When 2 drops of lubricant were instilled, a significant amount of fluid ejection in the form of a sheet that broke up into multiple droplets was observed. Some of these droplets traversed back to the tonometer. Droplets ranging from 100 to 500 µm in diameter were measured. CONCLUSIONS: There was no droplet generation during NCT performed in a natural setting. However, NCT should be avoided in conditions with high-tear volume (natural or artificial) as it would lead to droplet spread and tactile contamination.

Low-cost measurement of face mask efficacy for filtering expelled droplets during speech.

Mandates for mask use in public during the recent coronavirus disease 2019 (COVID-19) pandemic, worsened by global shortage of commercial supplies, have led to widespread use of homemade masks and mask alternatives. It is assumed that wearing such masks reduces the likelihood for an infected person to spread the disease, but many of these mask designs have not been tested in practice. We have demonstrated a simple optical measurement method to evaluate the efficacy of masks to reduce the transmission of respiratory droplets during regular speech. In proof-of-principle studies, we compared a variety of commonly available mask types and observed that some mask types approach the performance of standard surgical masks, while some mask alternatives, such as neck gaiters or bandanas, offer very little protection. Our measurement setup is inexpensive and can be built and operated by nonexperts, allowing for rapid evaluation of mask performance during speech, sneezing, or coughing.

Hybrid Biopolymer and Lipid Nanoparticles with Improved Transfection Efficacy for mRNA.

Hybrid nanoparticles from lipidic and polymeric components were assembled to serve as vehicles for the transfection of messenger RNA (mRNA) using different portions of the cationic lipid DOTAP (1,2-Dioleoyl-3-trimethylammonium-propane) and the cationic biopolymer protamine as model systems. Two different sequential assembly approaches in comparison with a direct single-step protocol were applied, and molecular organization in correlation with biological activity of the resulting nanoparticle systems was investigated. Differences in the structure of the nanoparticles were revealed by thorough physicochemical characterization including small angle neutron scattering (SANS), small angle X-ray scattering (SAXS), and cryogenic transmission electron microscopy (cryo-TEM). All hybrid systems, combining lipid and polymer, displayed significantly increased transfection in comparison to lipid/mRNA and polymer/mRNA particles alone. For the hybrid nanoparticles, characteristic differences regarding the internal organization, release characteristics, and activity were determined depending on the assembly route. The systems with the highest transfection efficacy were characterized by a heterogenous internal organization, accompanied by facilitated release. Such a system could be best obtained by the single step protocol, starting with a lipid and polymer mixture for nanoparticle formation.