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1.
Nat Commun ; 13(1): 1758, 2022 04 01.
Article in English | MEDLINE | ID: covidwho-1886207

ABSTRACT

Fluorescence techniques dominate the field of live-cell microscopy, but bleaching and motion blur from too long integration times limit dynamic investigations of small objects. High contrast, label-free life-cell imaging of thousands of acquisitions at 160 nm resolution and 100 Hz is possible by Rotating Coherent Scattering (ROCS) microscopy, where intensity speckle patterns from all azimuthal illumination directions are added up within 10 ms. In combination with fluorescence, we demonstrate the performance of improved Total Internal Reflection (TIR)-ROCS with variable illumination including timescale decomposition and activity mapping at five different examples: millisecond reorganization of macrophage actin cortex structures, fast degranulation and pore opening in mast cells, nanotube dynamics between cardiomyocytes and fibroblasts, thermal noise driven binding behavior of virus-sized particles at cells, and, bacterial lectin dynamics at the cortex of lung cells. Using analysis methods we present here, we decipher how motion blur hides cellular structures and how slow structure motions cover decisive fast motions.


Subject(s)
Actins , Lighting , Fibroblasts , Microscopy, Fluorescence/methods
2.
Nat Biotechnol ; 40(7): 1132-1142, 2022 07.
Article in English | MEDLINE | ID: covidwho-1805637

ABSTRACT

The low photostability of fluorescent proteins is a limiting factor in many applications of fluorescence microscopy. Here we present StayGold, a green fluorescent protein (GFP) derived from the jellyfish Cytaeis uchidae. StayGold is over one order of magnitude more photostable than any currently available fluorescent protein and has a cellular brightness similar to mNeonGreen. We used StayGold to image the dynamics of the endoplasmic reticulum (ER) with high spatiotemporal resolution over several minutes using structured illumination microscopy (SIM) and observed substantially less photobleaching than with a GFP variant optimized for stability in the ER. Using StayGold fusions and SIM, we also imaged the dynamics of mitochondrial fusion and fission and mapped the viral spike proteins in fixed cells infected with severe acute respiratory syndrome coronavirus 2. As StayGold is a dimer, we created a tandem dimer version that allowed us to observe the dynamics of microtubules and the excitatory post-synaptic density in neurons. StayGold will substantially reduce the limitations imposed by photobleaching, especially in live cell or volumetric imaging.


Subject(s)
COVID-19 , Endoplasmic Reticulum , Green Fluorescent Proteins/genetics , Humans , Microscopy, Fluorescence/methods
3.
Commun Biol ; 5(1): 212, 2022 03 08.
Article in English | MEDLINE | ID: covidwho-1735294

ABSTRACT

Internalization of membrane proteins plays a key role in many physiological functions; however, highly sensitive and versatile technologies are lacking to study such processes in real-time living systems. Here we describe an assay based on bioluminescence able to quantify membrane receptor trafficking for a wide variety of internalization mechanisms such as GPCR internalization/recycling, antibody-mediated internalization, and SARS-CoV2 viral infection. This study represents an alternative drug discovery tool to accelerate the drug development for a wide range of physiological processes, such as cancer, neurological, cardiopulmonary, metabolic, and infectious diseases including COVID-19.


Subject(s)
Drug Discovery/methods , Membrane Proteins , Protein Transport/physiology , Spectrometry, Fluorescence/methods , COVID-19 , Drug Development/methods , HEK293 Cells , Humans , Luciferases/genetics , Luciferases/metabolism , Membrane Proteins/chemistry , Membrane Proteins/metabolism , Microscopy, Fluorescence , Nanotechnology , Receptors, G-Protein-Coupled , SARS-CoV-2/chemistry , SARS-CoV-2/metabolism , Virus Internalization
4.
Toxicol Appl Pharmacol ; 440: 115913, 2022 04 01.
Article in English | MEDLINE | ID: covidwho-1671180

ABSTRACT

The COVID-19 pandemic raises significance for a potential influenza therapeutic compound, cetylpyridinium chloride (CPC), which has been extensively used in personal care products as a positively-charged quaternary ammonium antibacterial agent. CPC is currently in clinical trials to assess its effects on severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) morbidity. Two published studies have provided mouse and human data indicating that CPC may alleviate influenza infection, and here we show that CPC (0.1 µM, 1 h) reduces zebrafish mortality and viral load following influenza infection. However, CPC mechanisms of action upon viral-host cell interaction are currently unknown. We have utilized super-resolution fluorescence photoactivation localization microscopy to probe the mode of CPC action. Reduction in density of influenza viral protein hemagglutinin (HA) clusters is known to reduce influenza infectivity: here, we show that CPC (at non-cytotoxic doses, 5-10 µM) reduces HA density and number of HA molecules per cluster within the plasma membrane of NIH-3T3 mouse fibroblasts. HA is known to colocalize with the negatively-charged mammalian lipid phosphatidylinositol 4,5-bisphosphate (PIP2); here, we show that nanoscale co-localization of HA with the PIP2-binding Pleckstrin homology (PH) reporter in the plasma membrane is diminished by CPC. CPC also dramatically displaces the PIP2-binding protein myristoylated alanine-rich C-kinase substrate (MARCKS) from the plasma membrane of rat RBL-2H3 mast cells; this disruption of PIP2 is correlated with inhibition of mast cell degranulation. Together, these findings offer a PIP2-focused mechanism underlying CPC disruption of influenza and suggest potential pharmacological use of this drug as an influenza therapeutic to reduce global deaths from viral disease.


Subject(s)
COVID-19 , Influenza, Human , Animals , Cell Communication , Cetylpyridinium/chemistry , Cetylpyridinium/pharmacology , Dinucleoside Phosphates , Humans , Immunity , Mammals , Mice , Microscopy, Fluorescence , Pandemics , Phosphatidylinositols , Rats , SARS-CoV-2 , Zebrafish
5.
Biochim Biophys Acta Mol Basis Dis ; 1868(4): 166347, 2022 04 01.
Article in English | MEDLINE | ID: covidwho-1636951

ABSTRACT

As epitomised by the COVID-19 pandemic, diseases caused by viruses are one of the greatest health and economic burdens to human society. Viruses are 'nanostructures', and their small size (typically less than 200 nm in diameter) can make it challenging to obtain images of their morphology and structure. Recent advances in fluorescence microscopy have given rise to super-resolution techniques, which have enabled the structure of viruses to be visualised directly at a resolution in the order of 20 nm. This mini-review discusses how recent state-of-the-art super-resolution imaging technologies are providing new nanoscale insights into virus structure.


Subject(s)
Microscopy, Fluorescence , Viruses/chemistry , Humans , Imaging, Three-Dimensional , Virion/chemistry
6.
Cells ; 11(1)2022 01 03.
Article in English | MEDLINE | ID: covidwho-1580990

ABSTRACT

Extracellular vesicles (EVs) and viruses share common features: size, structure, biogenesis and uptake. In order to generate EVs expressing the SARS-CoV-2 spike protein on their surface (S-EVs), we collected EVs from SARS-CoV-2 spike expressing human embryonic kidney (HEK-293T) cells by stable transfection with a vector coding for the S1 and S2 subunits. S-EVs were characterized using nanoparticle tracking analysis, ExoView and super-resolution microscopy. We obtained a population of EVs of 50 to 200 nm in size. Spike expressing EVs represented around 40% of the total EV population and co-expressed spike protein with tetraspanins on the surfaces of EVs. We subsequently used ACE2-positive endothelial and bronchial epithelial cells for assessing the internalization of labeled S-EVs using a cytofluorimetric analysis. Internalization of S-EVs was higher than that of control EVs from non-transfected cells. Moreover, S-EV uptake was significantly decreased by anti-ACE2 antibody pre-treatment. Furthermore, colchicine, a drug currently used in clinical trials, significantly reduced S-EV entry into the cells. S-EVs represent a simple, safe, and scalable model to study host-virus interactions and the mechanisms of novel therapeutic drugs.


Subject(s)
COVID-19/metabolism , Extracellular Vesicles/metabolism , SARS-CoV-2/metabolism , Spike Glycoprotein, Coronavirus/metabolism , Angiotensin-Converting Enzyme 2/immunology , Antibodies, Blocking/pharmacology , COVID-19/virology , Cell Line , Cells, Cultured , Colchicine/pharmacology , Flow Cytometry/methods , HEK293 Cells , Host Microbial Interactions/drug effects , Human Umbilical Vein Endothelial Cells/metabolism , Human Umbilical Vein Endothelial Cells/virology , Humans , Microscopy, Fluorescence/methods , Protein Binding/drug effects , SARS-CoV-2/physiology
7.
AAPS J ; 24(1): 8, 2021 12 06.
Article in English | MEDLINE | ID: covidwho-1555615

ABSTRACT

Lipidoid nanoparticles (LNPs) are the delivery platform in Onpattro, the first FDA-approved siRNA drug. LNPs are also the carriers in the Pfizer-BioNTech and Moderna COVID-19 mRNA vaccines. While these applications have demonstrated that LNPs effectively deliver nucleic acids to hepatic and muscle cells, it is unclear if LNPs could be used for delivery of siRNA to neural cells, which are notoriously challenging delivery targets. Therefore, the purpose of this study was to determine if LNPs could efficiently deliver siRNA to neurons. Because of their potential delivery utility in either applications for the central nervous system and the peripheral nervous system, we used both cortical neurons and sensory neurons. We prepared siRNA-LNPs using C12-200, a benchmark ionizable cationic lipidoid along with helper lipids. We demonstrated using dynamic light scattering that the inclusion of both siRNA and PEG-lipid provided a stabilizing effect to the LNP particle diameters and polydispersity indices by minimizing aggregation. We found that siRNA-LNPs were safely tolerated by primary dorsal root ganglion neurons. Flow cytometry analysis revealed that Cy5 siRNA delivered via LNPs into rat primary cortical neurons showed uptake levels similar to Lipofectamine RNAiMAX-the gold standard commercial transfection agent. However, LNPs demonstrated a superior safety profile, whereas the Lipofectamine-mediated uptake was concomitant with significant toxicity. Fluorescence microscopy demonstrated a time-dependent increase in the uptake of LNP-delivered Cy5 siRNA in a human cortical neuron cell line. Overall, our results suggest that LNPs are a viable platform that can be optimized for delivery of therapeutic siRNAs to neural cells.


Subject(s)
Ganglia, Spinal/metabolism , Lipids/chemistry , Nanoparticles , Neurons/metabolism , RNA, Small Interfering/administration & dosage , RNAi Therapeutics , Transfection , Animals , Carbocyanines/metabolism , Fluorescent Dyes/metabolism , Ganglia, Spinal/cytology , Humans , MCF-7 Cells , Microscopy, Fluorescence , Nanotechnology , Primary Cell Culture , RNA, Small Interfering/genetics , RNA, Small Interfering/metabolism , Rats , Time Factors
8.
Cell ; 184(24): 5932-5949.e15, 2021 11 24.
Article in English | MEDLINE | ID: covidwho-1549679

ABSTRACT

Anosmia, the loss of smell, is a common and often the sole symptom of COVID-19. The onset of the sequence of pathobiological events leading to olfactory dysfunction remains obscure. Here, we have developed a postmortem bedside surgical procedure to harvest endoscopically samples of respiratory and olfactory mucosae and whole olfactory bulbs. Our cohort of 85 cases included COVID-19 patients who died a few days after infection with SARS-CoV-2, enabling us to catch the virus while it was still replicating. We found that sustentacular cells are the major target cell type in the olfactory mucosa. We failed to find evidence for infection of olfactory sensory neurons, and the parenchyma of the olfactory bulb is spared as well. Thus, SARS-CoV-2 does not appear to be a neurotropic virus. We postulate that transient insufficient support from sustentacular cells triggers transient olfactory dysfunction in COVID-19. Olfactory sensory neurons would become affected without getting infected.


Subject(s)
Autopsy/methods , COVID-19/mortality , COVID-19/virology , Olfactory Bulb/virology , Olfactory Mucosa/virology , Respiratory Mucosa/virology , Aged , Anosmia , COVID-19/physiopathology , Endoscopy/methods , Female , Glucuronosyltransferase/biosynthesis , Humans , Immunohistochemistry , In Situ Hybridization , Male , Microscopy, Fluorescence , Middle Aged , Olfaction Disorders , Olfactory Receptor Neurons/metabolism , Respiratory System , SARS-CoV-2 , Smell
9.
J Am Soc Nephrol ; 32(1): 99-114, 2021 01.
Article in English | MEDLINE | ID: covidwho-1496673

ABSTRACT

BACKGROUND: C3 glomerulopathy (C3G) is characterized by the alternative-pathway (AP) hyperactivation induced by nephritic factors or complement gene mutations. Mice deficient in complement factor H (CFH) are a classic C3G model, with kidney disease that requires several months to progress to renal failure. Novel C3G models can further contribute to understanding the mechanism behind this disease and developing therapeutic approaches. METHODS: A novel, rapidly progressing, severe, murine model of C3G was developed by replacing the mouse C3 gene with the human C3 homolog using VelociGene technology. Functional, histologic, molecular, and pharmacologic assays characterize the presentation of renal disease and enable useful pharmacologic interventions in the humanized C3 (C3hu/hu) mice. RESULTS: The C3hu/hu mice exhibit increased morbidity early in life and die by about 5-6 months of age. The C3hu/hu mice display elevated biomarkers of kidney dysfunction, glomerulosclerosis, C3/C5b-9 deposition, and reduced circulating C3 compared with wild-type mice. Administration of a C5-blocking mAb improved survival rate and offered functional and histopathologic benefits. Blockade of AP activation by anti-C3b or CFB mAbs also extended survival and preserved kidney function. CONCLUSIONS: The C3hu/hu mice are a useful model for C3G because they share many pathologic features consistent with the human disease. The C3G phenotype in C3hu/hu mice may originate from a dysregulated interaction of human C3 protein with multiple mouse complement proteins, leading to unregulated C3 activation via AP. The accelerated disease course in C3hu/hu mice may further enable preclinical studies to assess and validate new therapeutics for C3G.


Subject(s)
Complement C3/genetics , Disease Models, Animal , Glomerulonephritis, Membranoproliferative/genetics , Kidney Diseases/genetics , Animals , Complement C3/metabolism , Complement Pathway, Alternative/genetics , Exons , Gene Expression Regulation , Glomerulonephritis, Membranoproliferative/metabolism , Humans , Kidney Diseases/metabolism , Liver/metabolism , Male , Mice , Mice, Knockout , Microscopy, Fluorescence , Phenotype , Polymorphism, Single Nucleotide , Renal Insufficiency/genetics , Renal Insufficiency/metabolism
10.
Biol Cell ; 113(7): 311-328, 2021 Jul.
Article in English | MEDLINE | ID: covidwho-1294968

ABSTRACT

BACKGROUND INFORMATION: Comprehensive libraries of plasmids for SARS-CoV-2 proteins with various tags (e.g., Strep, HA, Turbo) are now available. They enable the identification of numerous potential protein-protein interactions between the SARS-CoV-2 virus and host proteins. RESULTS: We present here a large library of SARS CoV-2 protein constructs fused with green and red fluorescent proteins and their initial characterisation in various human cell lines including lung epithelial cell models (A549, BEAS-2B), as well as in budding yeast. The localisation of a few SARS-CoV-2 proteins matches their proposed interactions with host proteins. These include the localisation of Nsp13 to the centrosome, Orf3a to late endosomes and Orf9b to mitochondria. CONCLUSIONS AND SIGNIFICANCE: This library should facilitate further cellular investigations, notably by imaging techniques.


Subject(s)
COVID-19/virology , Peptide Library , SARS-CoV-2/metabolism , Viral Proteins/metabolism , A549 Cells , Cell Line , Green Fluorescent Proteins/genetics , Green Fluorescent Proteins/metabolism , Host Microbial Interactions/physiology , Humans , Luminescent Proteins/genetics , Luminescent Proteins/metabolism , Microscopy, Fluorescence , Protein Interaction Domains and Motifs , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , SARS-CoV-2/genetics , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/metabolism , Time-Lapse Imaging , Viral Proteins/genetics
11.
Invest Ophthalmol Vis Sci ; 62(7): 25, 2021 06 01.
Article in English | MEDLINE | ID: covidwho-1280514

ABSTRACT

Purpose: The ocular surface is considered an important route for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) transmission. The expression level of the SARS-CoV-2 receptor angiotensin-converting enzyme 2 (ACE2) is vital for viral infection. However, the regulation of ACE2 expression on the ocular surface is still unknown. We aimed to determine the change in ACE2 expression in inflamed corneal epithelium and explore potential drugs to reduce the expression of ACE2 on the ocular surface. Methods: The expression of the SARS-CoV-2 receptors ACE2 and TMPRSS2 in human corneal epithelial cells (HCECs) was examined by qPCR and Western blotting. The altered expression of ACE2 in inflammatory corneal epithelium was evaluated in TNFα- and IL-1ß-stimulated HCECs and inflamed mouse corneal epithelium, and the effect of resveratrol on ACE2 expression in HCECs was detected by immunofluorescence and Western blot analysis. Results: ACE2 and TMPRSS2 are expressed on the human corneal epithelial cells. ACE2 expression is upregulated in HCECs by stimulation with TNFα and IL-1ß and inflamed mouse corneas, including dry eye and alkali-burned corneas. In addition, resveratrol attenuates the increased expression of ACE2 induced by TNFα in HCECs. Conclusions: This study demonstrates that ACE2 is highly expressed in HCECs and can be upregulated by stimulation with inflammatory cytokines and inflamed mouse corneal epithelium. Resveratrol may be able to reduce the increased expression of ACE2 on the inflammatory ocular surface. Our work suggests that patients with an inflammatory ocular surface may display higher ACE2 expression, which increases the risk of SARS-CoV-2 infection.


Subject(s)
Angiotensin-Converting Enzyme 2/genetics , Enzyme Inhibitors/pharmacology , Epithelium, Corneal/enzymology , Gene Expression Regulation, Enzymologic/physiology , Keratitis/enzymology , Resveratrol/pharmacology , SARS-CoV-2/physiology , Adult , Angiotensin-Converting Enzyme 2/metabolism , Animals , Blotting, Western , Cells, Cultured , Epithelium, Corneal/drug effects , Gene Expression Regulation, Enzymologic/drug effects , Humans , Inflammation/drug therapy , Inflammation/enzymology , Interleukin-1beta/pharmacology , Keratitis/drug therapy , Male , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Microscopy, Fluorescence , RNA, Messenger/genetics , Real-Time Polymerase Chain Reaction , Receptors, Virus/metabolism , Serine Endopeptidases/genetics , Serine Endopeptidases/metabolism , Tumor Necrosis Factor-alpha/pharmacology , Up-Regulation
12.
Int J Mol Sci ; 22(12)2021 Jun 18.
Article in English | MEDLINE | ID: covidwho-1273462

ABSTRACT

The COVID-19 pandemic caused by SARS-CoV-2 coronavirus deeply affected the world community. It gave a strong impetus to the development of not only approaches to diagnostics and therapy, but also fundamental research of the molecular biology of this virus. Fluorescence microscopy is a powerful technology enabling detailed investigation of virus-cell interactions in fixed and live samples with high specificity. While spatial resolution of conventional fluorescence microscopy is not sufficient to resolve all virus-related structures, super-resolution fluorescence microscopy can solve this problem. In this paper, we review the use of fluorescence microscopy to study SARS-CoV-2 and related viruses. The prospects for the application of the recently developed advanced methods of fluorescence labeling and microscopy-which in our opinion can provide important information about the molecular biology of SARS-CoV-2-are discussed.


Subject(s)
Microscopy, Fluorescence , SARS-CoV-2/physiology , COVID-19/pathology , COVID-19/virology , Endocytosis , Fluorescent Dyes/chemistry , Genes, Reporter , Humans , RNA, Viral/chemistry , RNA, Viral/metabolism , SARS-CoV-2/genetics , SARS-CoV-2/isolation & purification , Viral Envelope Proteins/chemistry , Viral Envelope Proteins/metabolism , Virus Internalization
15.
Cornea ; 39(12): 1556-1562, 2020 Dec.
Article in English | MEDLINE | ID: covidwho-1109355

ABSTRACT

PURPOSE: To confirm the ocular tropism of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) by evaluating the expression of viral entry factors in human ocular tissues using immunohistochemistry. METHODS: Fresh donor corneas and primary explant cultures of corneal, limbal, and conjunctival epithelial cells were evaluated for the expression of viral entry factors. Using immunohistochemistry, the samples were tested for the expression of angiotension-converting enzyme 2 (ACE2), dendritic cell-specific intracellular adhesion molecule 3-grabbing nonintegrin (DC-SIGN), DC-SIGN-related protein (DC-SIGNR), and transmembrane serine protease 2 (TMPRSS2). RESULTS: In total, 5 donor corneas were evaluated for the expression of viral entry factors. In all specimens, both ACE2 and TMPRSS2 were expressed throughout the surface epithelium (corneal, limbal, and conjunctival) and corneal endothelium. In corneal stromal cells, ACE2 was sporadically expressed, whereas TMPRSS2 was absent. DC-SIGN/DC-SIGNR expression varied between donor specimens. Four specimens expressed DC-SIGN/DC-SIGNR in a similar distribution to ACE2, but 1 specimen from a young donor showed no expression of DC-SIGN/DC-SIGNR. ACE2, TMPRSS2, and DC-SIGN/DC-SIGNR were all expressed in the cultured corneal, limbal, and conjunctival epithelial cells. CONCLUSIONS: Both corneal and conjunctival epithelia express ACE2, DC-SIGN/DC-SIGNR, and TMPRSS2, suggesting that the ocular surface is a potential route for the transmission of SARS-CoV-2. The risk of viral transmission with corneal transplantation cannot be ruled out, given the presence of ACE2 in corneal epithelium and endothelium. Cultured corneal, limbal, and conjunctival epithelial cells mimic the expression of viral entry factors in fresh donor tissue and may be useful for future in vitro SARS-CoV-2 infection studies.


Subject(s)
Betacoronavirus/physiology , Cell Adhesion Molecules/metabolism , Conjunctiva/metabolism , Epithelium, Corneal/metabolism , Lectins, C-Type/metabolism , Peptidyl-Dipeptidase A/metabolism , Receptors, Cell Surface/metabolism , Serine Endopeptidases/metabolism , Adult , Aged , Aged, 80 and over , Angiotensin-Converting Enzyme 2 , COVID-19 , Cells, Cultured , Conjunctiva/cytology , Coronavirus Infections/immunology , Epithelial Cells/metabolism , Female , Fluorescent Antibody Technique, Indirect , Humans , Limbus Corneae/cytology , Male , Microscopy, Fluorescence , Middle Aged , Pandemics , Pneumonia, Viral/immunology , SARS-CoV-2 , Tissue Donors , Viral Tropism/physiology , Virus Internalization , Young Adult
16.
ACS Appl Mater Interfaces ; 13(8): 10321-10327, 2021 Mar 03.
Article in English | MEDLINE | ID: covidwho-1087402

ABSTRACT

Early diagnosis of SARS-CoV-2 infection is critical for facilitating proper containment procedures, and a rapid, sensitive antigen assay is a critical step in curbing the pandemic. In this work, we report the use of a high-purity semiconducting (sc) single-walled carbon nanotube (SWCNT)-based field-effect transistor (FET) decorated with specific binding chemistry to assess the presence of SARS-CoV-2 antigens in clinical nasopharyngeal samples. Our SWCNT FET sensors, with functionalization of the anti-SARS-CoV-2 spike protein antibody (SAb) and anti-nucleocapsid protein antibody, detected the S antigen (SAg) and N antigen (NAg), reaching a limit of detection of 0.55 fg/mL for SAg and 0.016 fg/mL for NAg in calibration samples. SAb-functionalized FET sensors also exhibited good sensing performance in discriminating positive and negative clinical samples, indicating a proof of principle for use as a rapid COVID-19 antigen diagnostic tool with high analytical sensitivity and specificity at low cost.


Subject(s)
Antigens, Viral/analysis , Biosensing Techniques , COVID-19 Testing/instrumentation , Nanotubes, Carbon/chemistry , Semiconductors , Transistors, Electronic , COVID-19 Testing/methods , Calibration , Electrodes , Gold , Humans , Limit of Detection , Materials Testing , Microscopy, Atomic Force , Microscopy, Fluorescence , Nanotechnology , SARS-CoV-2 , Sensitivity and Specificity , Spectrophotometry, Ultraviolet , Spectroscopy, Near-Infrared , Spectrum Analysis, Raman , Spike Glycoprotein, Coronavirus/analysis
17.
ACS Appl Bio Mater ; 4(2): 1307-1318, 2021 02 15.
Article in English | MEDLINE | ID: covidwho-1069089

ABSTRACT

Recent evidence suggests that proinflammatory cytokines, such as tumor necrosis factor α (TNF-α), play a pivotal role in the development of inflammatory-related pathologies (covid-19, depressive disorders, sepsis, cancer, etc.,). More importantly, the development of TNF-α biosensors applied to biological fluids (e.g. sweat) could offer non-invasive solutions for the continuous monitoring of these disorders, in particular, polydimethylsiloxane (PDMS)-based biosensors. We have therefore investigated the biofunctionalization of PDMS surfaces using a silanization reaction with 3-aminopropyltriethoxysilane, for the development of a human TNF-α biosensor. The silanization conditions for 50 µm PDMS surfaces were extensively studied by using water contact angle measurements, electron dispersive X-ray and Fourier transform infrared spectroscopies, and fluorescamine detection. Evaluation of the wettability of the silanized surfaces and the Si/C ratio pointed out to the optimal silanization conditions supporting the formation of a stable and reproducible aminosilane layer, necessary for further bioconjugation. An ELISA-type immunoassay was then successfully performed for the detection and quantification of human TNF-α through fluorescent microscopy, reaching a limit of detection of 0.55 µg/mL (31.6 nM). Finally, this study reports for the first time a promising method for the development of PDMS-based biosensors for the detection of TNF-α, using a quick, stable, and simple biofunctionalization process.


Subject(s)
Dimethylpolysiloxanes/chemistry , Immunoassay/methods , Tumor Necrosis Factor-alpha/analysis , Antibodies, Immobilized/chemistry , Antibodies, Immobilized/immunology , COVID-19/metabolism , COVID-19/pathology , COVID-19/virology , Carbon/chemistry , Humans , Immunoassay/instrumentation , Limit of Detection , Microfluidics , Microscopy, Fluorescence , SARS-CoV-2/isolation & purification , Silicon/chemistry , Tumor Necrosis Factor-alpha/immunology , Wettability
18.
Sci Rep ; 11(1): 1822, 2021 01 19.
Article in English | MEDLINE | ID: covidwho-1065937

ABSTRACT

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) responsible for the COVID-19 global pandemic has infected over 25 million people worldwide and resulted in the death of millions. The COVID-19 pandemic has also resulted in a shortage of personal protective equipment (PPE) in many regions around the world, particularly in middle- and low-income countries. The shortages of PPE, such as N95 respirators, is something that will persist until an effective vaccine is made available. Thus, devices that while being easy to operate can also be rapidly deployed in health centers, and long-term residences without the need for major structural overhaul are instrumental to sustainably use N95 respirators. In this report, we present the design and validation of a decontamination device that combines UV-C & B irradiation with mild-temperature treatment. The device can decontaminate up to 20 masks in a cycle of < 30 min. The decontamination process did not damage or reduce the filtering capacity of the masks. Further, the efficacy of the device to eliminate microbes and viruses from the masks was also evaluated. The photothermal treatment of our device was capable of eradicating > 99.9999% of the bacteria and > 99.99% of the virus tested.


Subject(s)
Bacteria/radiation effects , Decontamination/methods , Ultraviolet Rays , Viruses/radiation effects , COVID-19/pathology , COVID-19/virology , Equipment Reuse , HEK293 Cells , Humans , Microscopy, Fluorescence , N95 Respirators/virology , SARS-CoV-2/isolation & purification , SARS-CoV-2/radiation effects , Temperature , Viruses/metabolism
19.
EBioMedicine ; 63: 103182, 2021 Jan.
Article in English | MEDLINE | ID: covidwho-1014448

ABSTRACT

BACKGROUND: SARS-CoV-2 infection (COVID-19 disease) can induce systemic vascular involvement contributing to morbidity and mortality. SARS-CoV-2 targets epithelial and endothelial cells through the ACE2 receptor. The anatomical involvement of the coronary tree is not explored yet. METHODS: Cardiac autopsy tissue of the entire coronary tree (main coronary arteries, epicardial arterioles/venules, epicardial capillaries) and epicardial nerves were analyzed in COVID-19 patients (n = 6). All anatomical regions were immunohistochemically tested for ACE2, TMPRSS2, CD147, CD45, CD3, CD4, CD8, CD68 and IL-6. COVID-19 negative patients with cardiovascular disease (n = 3) and influenza A (n = 6) served as controls. FINDINGS: COVID-19 positive patients showed strong ACE2 / TMPRSS2 expression in capillaries and less in arterioles/venules. The main coronary arteries were virtually devoid of ACE2 receptor and had only mild intimal inflammation. Epicardial capillaries had a prominent lympho-monocytic endotheliitis, which was less pronounced in arterioles/venules. The lymphocytic-monocytic infiltrate strongly expressed CD4, CD45, CD68. Peri/epicardial nerves had strong ACE2 expression and lympho-monocytic inflammation. COVID-19 negative patients showed minimal vascular ACE2 expression and lacked endotheliitis or inflammatory reaction. INTERPRETATION: ACE2 / TMPRSS2 expression and lymphomonocytic inflammation in COVID-19 disease increases crescentically towards the small vessels suggesting that COVID-19-induced endotheliitis is a small vessel vasculitis not involving the main coronaries. The inflammatory neuropathy of epicardial nerves in COVID-19 disease provides further evidence of an angio- and neurotrophic affinity of SARS-COV2 and might potentially contribute to the understanding of the high prevalence of cardiac complications such as myocardial injury and arrhythmias in COVID-19. FUNDING: No external funding was necessary for this study.


Subject(s)
Capillaries/pathology , Coronary Vessels/pathology , SARS-CoV-2/metabolism , Aged , Aged, 80 and over , Angiotensin-Converting Enzyme 2/genetics , Angiotensin-Converting Enzyme 2/metabolism , COVID-19/pathology , COVID-19/virology , Capillaries/metabolism , Coronary Vessels/metabolism , Female , Humans , Inflammation/pathology , Male , Microscopy, Fluorescence , Middle Aged , RNA, Viral/metabolism , SARS-CoV-2/genetics , SARS-CoV-2/isolation & purification , Serine Endopeptidases/genetics , Serine Endopeptidases/metabolism , Spike Glycoprotein, Coronavirus/metabolism
20.
Nat Commun ; 11(1): 6041, 2020 11 27.
Article in English | MEDLINE | ID: covidwho-947535

ABSTRACT

The etiologic agent of the Covid-19 pandemic is the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The viral membrane of SARS-CoV-2 surrounds a helical nucleocapsid in which the viral genome is encapsulated by the nucleocapsid protein. The nucleocapsid protein of SARS-CoV-2 is produced at high levels within infected cells, enhances the efficiency of viral RNA transcription, and is essential for viral replication. Here, we show that RNA induces cooperative liquid-liquid phase separation of the SARS-CoV-2 nucleocapsid protein. In agreement with its ability to phase separate in vitro, we show that the protein associates in cells with stress granules, cytoplasmic RNA/protein granules that form through liquid-liquid phase separation and are modulated by viruses to maximize replication efficiency. Liquid-liquid phase separation generates high-density protein/RNA condensates that recruit the RNA-dependent RNA polymerase complex of SARS-CoV-2 providing a mechanism for efficient transcription of viral RNA. Inhibition of RNA-induced phase separation of the nucleocapsid protein by small molecules or biologics thus can interfere with a key step in the SARS-CoV-2 replication cycle.


Subject(s)
COVID-19/virology , Coronavirus Nucleocapsid Proteins/metabolism , RNA, Viral/metabolism , SARS-CoV-2/physiology , Animals , Antiviral Agents/pharmacology , Antiviral Agents/therapeutic use , COVID-19/epidemiology , Coronavirus Nucleocapsid Proteins/antagonists & inhibitors , Coronavirus RNA-Dependent RNA Polymerase/metabolism , HeLa Cells , Humans , Insecta , Intravital Microscopy , Microscopy, Fluorescence , Molecular Dynamics Simulation , Pandemics/prevention & control , Phosphoproteins/antagonists & inhibitors , Phosphoproteins/metabolism , RNA, Viral/antagonists & inhibitors , SARS-CoV-2/drug effects , Viral Transcription/drug effects , Viral Transcription/physiology , Virus Replication/drug effects , Virus Replication/genetics
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