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1.
Sci Rep ; 11(1): 20877, 2021 10 22.
Article in English | MEDLINE | ID: covidwho-1479811

ABSTRACT

Adenovirus vectors offer a platform technology for vaccine development. The value of the platform has been proven during the COVID-19 pandemic. Although good stability at 2-8 °C is an advantage of the platform, non-cold-chain distribution would have substantial advantages, in particular in low-income countries. We have previously reported a novel, potentially less expensive thermostabilisation approach using a combination of simple sugars and glass micro-fibrous matrix, achieving excellent recovery of adenovirus-vectored vaccines after storage at temperatures as high as 45 °C. This matrix is, however, prone to fragmentation and so not suitable for clinical translation. Here, we report an investigation of alternative fibrous matrices which might be suitable for clinical use. A number of commercially-available matrices permitted good protein recovery, quality of sugar glass and moisture content of the dried product but did not achieve the thermostabilisation performance of the original glass fibre matrix. We therefore further investigated physical and chemical characteristics of the glass fibre matrix and its components, finding that the polyvinyl alcohol present in the glass fibre matrix assists vaccine stability. This finding enabled us to identify a potentially biocompatible matrix with encouraging performance. We discuss remaining challenges for transfer of the technology into clinical use, including reliability of process performance.


Subject(s)
Adenoviridae/genetics , Adenovirus Vaccines/chemistry , COVID-19 Vaccines/therapeutic use , COVID-19/prevention & control , Vaccine Potency , Adenoviruses, Simian , Biocompatible Materials , Calorimetry, Differential Scanning , Glass , HEK293 Cells , Humans , Light , Magnetic Resonance Spectroscopy , Materials Testing , Microscopy, Confocal , Microscopy, Electron, Scanning , Polyvinyl Alcohol , Rabies Vaccines , Scattering, Radiation , Spectroscopy, Fourier Transform Infrared , Sugars/chemistry , Temperature , Thermogravimetry , Trehalose/chemistry
2.
Eur Rev Med Pharmacol Sci ; 25(19): 5904-5912, 2021 Oct.
Article in English | MEDLINE | ID: covidwho-1478932

ABSTRACT

OBJECTIVE: Liver injury has been reported in patients with COVID-19. This condition is characterized by severe outcome and could be related with the ability of SARS-CoV-2 to activate cytotoxic T cells. The purpose of this study is to show the histological and scanning electron microscopy features of liver involvement in COVID-19 to characterize the liver changes caused by the activation of multiple molecular pathways following this infection. PATIENTS AND METHODS: Liver biopsies from 4 patients (3 post-mortems and 1 in vivo) with COVID-19 were analyzed with histology and by scanning electron microscopy. RESULTS: The liver changes showed significant heterogeneity. The first case showed ground glass hepatocytes and scattered fibrin aggregates in the sinusoidal lumen. The second evidenced intra-sinusoidal thrombi. The third was characterized by sinusoidal dilatation, atrophy of hepatocytes, Disse's spaces dilatation and intra-sinusoidal aggregates of fibrin and red blood cells. The fourth case exhibited diffuse fibrin aggregates in the dilated Disse spaces and microthrombi in the sinusoidal lumen. CONCLUSIONS: In COVID-19-related liver injury, a large spectrum of pathological changes was observed. The most peculiar features were very mild inflammation, intra-sinusoidal changes, including sinusoidal dilatation, thrombotic sinusoiditis and diffuse intra-sinusoidal fibrin deposition. These findings suggested that a thrombotic sinusoiditis followed by a local diffuse intra-vascular (intra-sinusoidal) coagulation could be the typical features of the SARS-CoV-2-related liver injury.


Subject(s)
Blood Coagulation Disorders/pathology , COVID-19/pathology , Liver Diseases/pathology , Liver/pathology , Thrombosis/pathology , Aged , Autopsy , Biopsy , Erythrocytes/pathology , Fibrin , Hepatocytes/pathology , Humans , Male , Microscopy, Electron, Scanning , Middle Aged , Thrombosis/complications , Young Adult
3.
Curr Top Med Chem ; 21(14): 1235-1250, 2021 Oct 05.
Article in English | MEDLINE | ID: covidwho-1441869

ABSTRACT

BACKGROUND: Virus-like Particles (VLPs) are non-genetic multimeric nanoparticles synthesized through in vitro or in vivo self-assembly of one or more viral structural proteins. Immunogenicity and safety of VLPs make them ideal candidates for vaccine development and efficient nanocarriers for foreign antigens or adjuvants to activate the immune system. AIMS: The present study aimed to design and synthesize a chimeric VLP vaccine of the phage Qbeta (Qß) coat protein presenting the universal epitope of the coronavirus. METHODS: The RNA phage Qß coat protein was designed and synthesized, denoted as Qbeta. The CoV epitope, a universal epitope of coronavirus, was inserted into the C-terminal of Qbeta using genetic recombination, designated as Qbeta-CoV. The N-terminal of Qbeta-CoV was successively inserted into the TEV restriction site using mCherry red fluorescent label and modified affinity purified histidine label 6xHE, which was denoted as HE-Qbeta-CoV. Isopropyl ß-D-1-thiogalactopyranoside (IPTG) assessment revealed the expression of Qbeta, Qbeta-CoV, and HE-Qbeta-CoV in the BL21 (DE3) cells. The fusion protein was purified by salting out using ammonium sulfate and affinity chromatography. The morphology of particles was observed using electron microscopy. The female BALB/C mice were immunized intraperitoneally with the Qbeta-CoV and HE-Qbeta-- CoV chimeric VLPs vaccines and their sera were collected for the detection of antibody level and antibody titer using ELISA. The serum is used for the neutralization test of the three viruses of MHV, PEDV, and PDCoV. RESULTS: The results revealed that the fusion proteins Qbeta, Qbeta-CoV, and HE-Qbeta-CoV could all obtain successful expression. Particles with high purity were obtained after purification; the chimeric particles of Qbeta-CoV and HE-Qbeta-CoV were found to be similar to Qbeta particles in morphology and formed chimeric VLPs. In addition, two chimeric VLP vaccines induced specific antibody responses in mice and the antibodies showed certain neutralizing activity. CONCLUSION: The successful construction of the chimeric VLPs of the phage Qß coat protein presenting the universal epitope of coronavirus provides a vaccine form with potential clinical applications for the treatment of coronavirus disease.


Subject(s)
Antibodies, Neutralizing/immunology , Capsid Proteins/immunology , Coronavirus/immunology , Vaccines, Virus-Like Particle/immunology , Animals , Antigens, Viral/genetics , Antigens, Viral/immunology , Enzyme-Linked Immunosorbent Assay , Female , Mice, Inbred BALB C , Microscopy, Electron, Scanning , Phylogeny , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/isolation & purification , Recombinant Fusion Proteins/metabolism , Vaccines, Virus-Like Particle/genetics , Viral Proteins/genetics
4.
ACS Appl Mater Interfaces ; 13(40): 47996-48008, 2021 Oct 13.
Article in English | MEDLINE | ID: covidwho-1440455

ABSTRACT

Use of masks is a primary tool to prevent the spread of the novel COVID-19 virus resulting from unintentional close contact with infected individuals. However, detailed characterization of the chemical properties and physical structure of common mask materials is lacking in the current literature. In this study, a series of commercial masks and potential mask materials, including 3M Particulate Respirator 8210 N95, a material provided by Oak Ridge National Laboratory Carbon Fiber Technology Facility (ORNL/CFTF), and a Filti Face Mask Material, were characterized by a suite of techniques, including scanning electron microscopy, X-ray diffraction, Raman spectroscopy, and X-ray photoelectron spectroscopy. Wetting properties of the mask materials were quantified by measurements of contact angle with a saliva substitute. Mask pass-through experiments were performed using a dispersed metal oxide nanoparticle suspension to model the SARS-CoV-2 virus, with quantification via spatially resolved X-ray fluorescence mapping. Notably, all mask materials tested provided a strong barrier against respiratory droplet breakthrough. The comparisons and characterizations provided in this study provide useful information when evaluating mask materials for respiratory protection.


Subject(s)
Filtration , Masks , Materials Testing/methods , N95 Respirators , COVID-19/prevention & control , Metal Nanoparticles/chemistry , Microscopy, Electron, Scanning , Photoelectron Spectroscopy , Polyesters/chemistry , Polypropylenes/chemistry , Porosity , SARS-CoV-2 , Spectrum Analysis, Raman , Wettability , X-Ray Diffraction
7.
Microsc Microanal ; 27(4): 815-827, 2021 08.
Article in English | MEDLINE | ID: covidwho-1345523

ABSTRACT

Manual selection of targets in experimental or diagnostic samples by transmission electron microscopy (TEM), based on single overview and detail micrographs, has been time-consuming and susceptible to bias. Substantial information and throughput gain may now be achieved by the automated acquisition of virtually all structures in a given EM section. Resulting datasets allow the convenient pan-and-zoom examination of tissue ultrastructure with preserved microanatomical orientation. The technique is, however, critically sensitive to artifacts in sample preparation. We, therefore, established a methodology to prepare large-scale digitization samples (LDS) designed to acquire entire sections free of obscuring flaws. For evaluation, we highlight the supreme performance of scanning EM in transmission mode compared with other EM technology. The use of LDS will substantially facilitate access to EM data for a broad range of applications.


Subject(s)
Microscopy, Electron, Scanning , Specimen Handling , Cells , Microscopy, Electron, Transmission
8.
Molecules ; 26(12)2021 Jun 10.
Article in English | MEDLINE | ID: covidwho-1282534

ABSTRACT

Multi-drug resistant pathogens are a rising danger for the future of mankind. Iodine (I2) is a centuries-old microbicide, but leads to skin discoloration, irritation, and uncontrolled iodine release. Plants rich in phytochemicals have a long history in basic health care. Aloe Vera Barbadensis Miller (AV) and Salvia officinalis L. (Sage) are effectively utilized against different ailments. Previously, we investigated the antimicrobial activities of smart triiodides and iodinated AV hybrids. In this work, we combined iodine with Sage extracts and pure AV gel with polyvinylpyrrolidone (PVP) as an encapsulating and stabilizing agent. Fourier transform infrared spectroscopy (FT-IR), Ultraviolet-visible spectroscopy (UV-Vis), Surface-Enhanced Raman Spectroscopy (SERS), microstructural analysis by scanning electron microscopy (SEM), energy dispersive spectroscopy (EDS), and X-Ray-Diffraction (XRD) analysis verified the composition of AV-PVP-Sage-I2. Antimicrobial properties were investigated by disc diffusion method against 10 reference microbial strains in comparison to gentamicin and nystatin. We impregnated surgical sutures with our biohybrid and tested their inhibitory effects. AV-PVP-Sage-I2 showed excellent to intermediate antimicrobial activity in discs and sutures. The iodine within the polymeric biomaterial AV-PVP-Sage-I2 and the synergistic action of the two plant extracts enhanced the microbial inhibition. Our compound has potential for use as an antifungal agent, disinfectant and coating material on sutures to prevent surgical site infections.


Subject(s)
Anti-Bacterial Agents/chemistry , Anti-Bacterial Agents/chemical synthesis , Aloe/chemistry , Antifungal Agents/chemistry , Gentamicins/chemistry , Microbial Sensitivity Tests , Microscopy, Electron, Scanning/methods , Nystatin/chemistry , Plant Extracts/chemistry , Povidone/chemistry , Salvia/chemistry , Salvia officinalis/chemistry , Spectrometry, X-Ray Emission/methods , Spectroscopy, Fourier Transform Infrared/methods , X-Ray Diffraction/methods
9.
Curr Top Med Chem ; 21(14): 1235-1250, 2021 Oct 05.
Article in English | MEDLINE | ID: covidwho-1274595

ABSTRACT

BACKGROUND: Virus-like Particles (VLPs) are non-genetic multimeric nanoparticles synthesized through in vitro or in vivo self-assembly of one or more viral structural proteins. Immunogenicity and safety of VLPs make them ideal candidates for vaccine development and efficient nanocarriers for foreign antigens or adjuvants to activate the immune system. AIMS: The present study aimed to design and synthesize a chimeric VLP vaccine of the phage Qbeta (Qß) coat protein presenting the universal epitope of the coronavirus. METHODS: The RNA phage Qß coat protein was designed and synthesized, denoted as Qbeta. The CoV epitope, a universal epitope of coronavirus, was inserted into the C-terminal of Qbeta using genetic recombination, designated as Qbeta-CoV. The N-terminal of Qbeta-CoV was successively inserted into the TEV restriction site using mCherry red fluorescent label and modified affinity purified histidine label 6xHE, which was denoted as HE-Qbeta-CoV. Isopropyl ß-D-1-thiogalactopyranoside (IPTG) assessment revealed the expression of Qbeta, Qbeta-CoV, and HE-Qbeta-CoV in the BL21 (DE3) cells. The fusion protein was purified by salting out using ammonium sulfate and affinity chromatography. The morphology of particles was observed using electron microscopy. The female BALB/C mice were immunized intraperitoneally with the Qbeta-CoV and HE-Qbeta-- CoV chimeric VLPs vaccines and their sera were collected for the detection of antibody level and antibody titer using ELISA. The serum is used for the neutralization test of the three viruses of MHV, PEDV, and PDCoV. RESULTS: The results revealed that the fusion proteins Qbeta, Qbeta-CoV, and HE-Qbeta-CoV could all obtain successful expression. Particles with high purity were obtained after purification; the chimeric particles of Qbeta-CoV and HE-Qbeta-CoV were found to be similar to Qbeta particles in morphology and formed chimeric VLPs. In addition, two chimeric VLP vaccines induced specific antibody responses in mice and the antibodies showed certain neutralizing activity. CONCLUSION: The successful construction of the chimeric VLPs of the phage Qß coat protein presenting the universal epitope of coronavirus provides a vaccine form with potential clinical applications for the treatment of coronavirus disease.


Subject(s)
Antibodies, Neutralizing/immunology , Capsid Proteins/immunology , Coronavirus/immunology , Vaccines, Virus-Like Particle/immunology , Animals , Antigens, Viral/genetics , Antigens, Viral/immunology , Enzyme-Linked Immunosorbent Assay , Female , Mice, Inbred BALB C , Microscopy, Electron, Scanning , Phylogeny , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/isolation & purification , Recombinant Fusion Proteins/metabolism , Vaccines, Virus-Like Particle/genetics , Viral Proteins/genetics
10.
Viruses ; 13(2)2021 01 28.
Article in English | MEDLINE | ID: covidwho-1058918

ABSTRACT

During infection with positive-strand RNA viruses, viral RNA synthesis associates with modified intracellular membranes that form unique and captivating structures in the cytoplasm of the infected cell. These viral replication organelles (ROs) play a key role in the replicative cycle of important human pathogens like coronaviruses, enteroviruses, or flaviviruses. From their discovery to date, progress in our understanding of viral ROs has closely followed new developments in electron microscopy (EM). This review gives a chronological account of this progress and an introduction to the different EM techniques that enabled it. With an ample repertoire of imaging modalities, EM is nowadays a versatile technique that provides structural and functional information at a wide range of scales. Together with well-established approaches like electron tomography or labeling methods, we examine more recent developments, such as volume scanning electron microscopy (SEM) and in situ cryotomography, which are only beginning to be applied to the study of viral ROs. We also highlight the first cryotomography analyses of viral ROs, which have led to the discovery of macromolecular complexes that may serve as RO channels that control the export of newly-made viral RNA. These studies are key first steps towards elucidating the macromolecular complexity of viral ROs.


Subject(s)
Microscopy, Electron , RNA Viruses/physiology , Viral Replication Compartments/ultrastructure , Virus Replication , Cryoelectron Microscopy , Electron Microscope Tomography , Image Processing, Computer-Assisted , Intracellular Membranes/ultrastructure , Microscopy, Electron, Scanning , Microscopy, Electron, Transmission , Microscopy, Immunoelectron , RNA, Viral/biosynthesis , Viral Nonstructural Proteins/analysis , Viral Nonstructural Proteins/metabolism , Viral Replication Compartments/chemistry
11.
Carbohydr Polym ; 264: 118011, 2021 Jul 15.
Article in English | MEDLINE | ID: covidwho-1172080

ABSTRACT

Veklury™ by Gilead Sciences, Inc., containing antiviral drug, remdesivir (REM) has received emergency authorization in the USA and in Europe for COVID-19 therapy. Here, for the first time, we describe details of the non-covalent, host-guest type interaction between REM and the solubilizing excipient, sulfobutylether-beta-cyclodextrin (SBECD) that results in significant solubility enhancement. Complete amorphousness of the cyclodextrin-enabled REM formulation was demonstrated by X-ray diffraction, thermal analysis, Raman chemical mapping and electron microscopy/energy dispersive spectroscopy. The use of solubilizing carbohydrate resulted in a 300-fold improvement of the aqueous solubility of REM, and enhanced dissolution rate of the drug enabling the preparation of stable infusion solutions for therapy. 2D ROESY NMR spectroscopy provided information on the nature of REM-excipient interaction and indicated the presence of inclusion phenomenon and the electrostatic attraction between anionic SBECD and nitrogen-containing REM in aqueous solution.


Subject(s)
Adenosine Monophosphate/analogs & derivatives , Alanine/analogs & derivatives , Excipients/chemistry , beta-Cyclodextrins/chemistry , Adenosine Monophosphate/chemistry , Alanine/chemistry , Antiviral Agents/chemistry , COVID-19/drug therapy , Calorimetry, Differential Scanning , Freeze Drying/methods , Magnetic Resonance Spectroscopy , Microscopy, Electron, Scanning , Molecular Docking Simulation , Nanofibers/chemistry , Powders , Solubility , Spectrum Analysis, Raman , X-Ray Diffraction
12.
Viruses ; 13(4)2021 04 02.
Article in English | MEDLINE | ID: covidwho-1167763

ABSTRACT

The visualization of cellular ultrastructure over a wide range of volumes is becoming possible by increasingly powerful techniques grouped under the rubric "volume electron microscopy" or volume EM (vEM). Focused ion beam scanning electron microscopy (FIB-SEM) occupies a "Goldilocks zone" in vEM: iterative and automated cycles of milling and imaging allow the interrogation of microns-thick specimens in 3-D at resolutions of tens of nanometers or less. This bestows on FIB-SEM the unique ability to aid the accurate and precise study of architectures of virus-cell interactions. Here we give the virologist or cell biologist a primer on FIB-SEM imaging in the context of vEM and discuss practical aspects of a room temperature FIB-SEM experiment. In an in vitro study of SARS-CoV-2 infection, we show that accurate quantitation of viral densities and surface curvatures enabled by FIB-SEM imaging reveals SARS-CoV-2 viruses preferentially located at areas of plasma membrane that have positive mean curvatures.


Subject(s)
COVID-19/pathology , Host Microbial Interactions , Image Interpretation, Computer-Assisted/methods , Imaging, Three-Dimensional/methods , Microscopy, Electron, Scanning/methods , SARS-CoV-2 , Animals , Cell Communication , Cell Membrane , Chlorocebus aethiops , Epithelial Cells/virology , Humans , Lung , Vero Cells
13.
Mol Pharm ; 18(5): 1970-1984, 2021 05 03.
Article in English | MEDLINE | ID: covidwho-1164785

ABSTRACT

Physicochemical properties, in particular solubility and the associated bioavailability, are key factors in determining efficacy of poorly water-soluble drugs, which constitute 40% of new drugs in the market, and improving them is an important challenge for modern pharmacy. A recent strategy to achieve this goal is formation of stable co-amorphous solid dispersions with co-formers of low molecular weight. Here, the amorphization strategy was applied for low-soluble anti-hypertensive valsartan (VAL), an angiotensin II receptor blocker, and nicotinamide, which exhibits lung- and cardio-protective effects. Through interactions with the renin-angiotensin-aldosteron system, VAL may be used to treat both hypertension and the current pandemic coronavirus SARS-CoV-2 infection. Using mechanochemical and liquid- and solid-state approaches, solvated co-amorphous solid dispersions of VAL with nicotinamide were obtained. They were characterized by spectroscopic, thermal, and X-ray analyses. The density functional theory, quantum theory of atoms in molecules, and non-covalent interaction index calculations revealed the presence of two types of hydrogen bonds between VAL and NIC (i.e., N-H···O and O-H···O). One of them had a partially covalent character, which caused conformational changes in the flexible VAL molecule, restricting contribution of the tetrazolyl N-H donor and thus limiting the possibility of co-crystal formation. The recognized VAL/NIC1- and VAL/NIC2-type heterodimeric interactions were responsible for the excellent durability of the solid compositions and up to 24-fold better solubility than VAL alone. The synthesized dispersions constitute a new class of dually acting drugs, containing an active pharmaceutical ingredient (VAL) and supporting nutraceutical (nicotinamide).


Subject(s)
Angiotensin II Type 1 Receptor Blockers/chemistry , Antihypertensive Agents/chemistry , COVID-19/drug therapy , Chemistry, Pharmaceutical/methods , Drug Carriers/chemistry , Niacinamide/chemistry , Valsartan/chemistry , Antihypertensive Agents/chemical synthesis , Biological Availability , Calorimetry, Differential Scanning , Drug Compounding , Humans , Hydrogen Bonding , Magnetic Resonance Spectroscopy , Microscopy, Electron, Scanning , Quantum Theory , Solubility , Spectroscopy, Fourier Transform Infrared , X-Ray Diffraction
14.
J Pharm Biomed Anal ; 196: 113924, 2021 Mar 20.
Article in English | MEDLINE | ID: covidwho-1051793

ABSTRACT

Owing to its simplicity and low cost, the lateral flow assay (LFA) is one of the most commonly used point-of-care diagnostic techniques, despite its low sensitivity and poor quantification. Here, we report a newly developed LFA-NanoSuit method (LNSM) combined with a desktop scanning electron microscope (SEM) for the direct observation of immunocomplexes labeled with a colloidal metal instead of signal enhancement strategies, such as using color, electrochemical signals, silver enhancement, magnetic properties, luminescent, and surface-enhanced Raman spectroscopy (SERS). The proposed LNSM suppresses cellulose deformity, thereby allowing the acquisition of high-resolution images of gold/platinum-labeled immunocomplexed pathogens such as influenza A, without conductive treatment as in conventional SEM. Electron microscopy-based diagnosis of influenza A exhibited 94 % clinical sensitivity (29/31; 95 % confidence interval [CI]: 79.3-98.2 %) and 100 % clinical specificity (95 % CI: 98.1-100 %), which was more sensitive (71.4 %) than visual detection (14.3 %), especially in the lower influenza A-RNA copy number group. The detection ability of our method was nearly comparable to that of real-time reverse transcription-PCR. This is the first report on the diagnosis of clinical diseases using LFA equipped with a desktop SEM. This simple and highly sensitive quantitative analysis method involving LFA can be used to diagnose various diseases in humans and livestock, including highly infectious diseases such as COVID-19.


Subject(s)
Biological Assay/methods , Gold/chemistry , Metal Nanoparticles/chemistry , Microscopy, Electron, Scanning/methods , Platinum/chemistry , Animals , Evaluation Studies as Topic , Humans , Limit of Detection , Livestock , Point-of-Care Testing , Spectrum Analysis, Raman/methods
15.
Nano Lett ; 21(3): 1508-1516, 2021 02 10.
Article in English | MEDLINE | ID: covidwho-1049951

ABSTRACT

Following the COVID-19 outbreak, swabs for biological specimen collection were thrust to the forefront of healthcare materials. Swab sample collection and recovery are vital for reducing false negative diagnostic tests, early detection of pathogens, and harvesting DNA from limited biological samples. In this study, we report a new class of nanofiber swabs tipped with hierarchical 3D nanofiber objects produced by expanding electrospun membranes with a solids-of-revolution-inspired gas foaming technique. Nanofiber swabs significantly improve absorption and release of proteins, cells, bacteria, DNA, and viruses from solutions and surfaces. Implementation of nanofiber swabs in SARS-CoV-2 detection reduces the false negative rates at two viral concentrations and identifies SARS-CoV-2 at a 10× lower viral concentration compared to flocked and cotton swabs. The nanofiber swabs show great promise in improving test sensitivity, potentially leading to timely and accurate diagnosis of many diseases.


Subject(s)
COVID-19 Testing/instrumentation , COVID-19/diagnosis , Nanofibers , SARS-CoV-2 , COVID-19/virology , COVID-19 Testing/methods , COVID-19 Testing/statistics & numerical data , False Negative Reactions , Humans , Materials Testing , Microscopy, Electron, Scanning , Nanofibers/ultrastructure , Nanotechnology , SARS-CoV-2/isolation & purification , Specimen Handling/instrumentation , Specimen Handling/methods , Specimen Handling/statistics & numerical data
16.
Int J Mol Sci ; 22(1)2020 Dec 24.
Article in English | MEDLINE | ID: covidwho-1041240

ABSTRACT

Thymosin α1 (Tα1) is an immunostimulatory peptide for the treatment of hepatitis B virus (HBV) and hepatitis C virus (HCV) infections and used as an immune enhancer, which also offers prospects in the context of COVID-19 infections and cancer. Manufacturing of this N-terminally acetylated 28-residue peptide is demanding, and its short plasma half-life limits in vivo efficacy and requires frequent dosing. Here, we combined the PASylation technology with enzymatic in situ N-acetylation by RimJ to produce a long-acting version of Tα1 in Escherichia coli at high yield. ESI-MS analysis of the purified fusion protein indicated the expected composition without any signs of proteolysis. SEC analysis revealed a 10-fold expanded hydrodynamic volume resulting from the fusion with a conformationally disordered Pro/Ala/Ser (PAS) polypeptide of 600 residues. This size effect led to a plasma half-life in rats extended by more than a factor 8 compared to the original synthetic peptide due to retarded kidney filtration. Our study provides the basis for therapeutic development of a next generation thymosin α1 with prolonged circulation. Generally, the strategy of producing an N-terminally protected PASylated peptide solves three major problems of peptide drugs: (i) instability in the expression host, (ii) rapid degradation by serum exopeptidases, and (iii) low bioactivity because of fast renal clearance.


Subject(s)
Adjuvants, Immunologic/pharmacokinetics , Thymalfasin/pharmacokinetics , Acetylation , Acetyltransferases/metabolism , Adjuvants, Immunologic/genetics , Adjuvants, Immunologic/pharmacology , Animals , COVID-19/drug therapy , Escherichia coli/metabolism , Escherichia coli Proteins/metabolism , Female , Half-Life , Mass Spectrometry , Microscopy, Electron, Scanning , Neoplasms/drug therapy , Peptides/chemistry , Proteolysis , Rats , Rats, Wistar , Recombinant Fusion Proteins/blood , Recombinant Fusion Proteins/isolation & purification , Recombinant Fusion Proteins/pharmacokinetics , Recombinant Fusion Proteins/ultrastructure , Ribosomal Proteins/metabolism , Thymalfasin/blood , Thymalfasin/chemistry , Thymalfasin/genetics , Virus Diseases/drug therapy
17.
Talanta ; 225: 122064, 2021 Apr 01.
Article in English | MEDLINE | ID: covidwho-1003087

ABSTRACT

Quantum dots (QDs) based fluorescent nanobeads are considered as promising materials for next generation point-of-care diagnosis systems. In this study, we carried out, for the first time, the synthesis of QDs nanobeads using polystyrene (PS) nanobead as the template. QDs loading on PS nanobead surface in this method can be readily achieved by the use of polyelectrolyte, avoiding the time-consuming and uncontrollable silane reagents-involved functionalization procedure that conventional synthesis of silica-based QDs nanobeads often suffer from. Notably, the application of QDs nanobeads in suspension microarray for H5N1 virus detection leads to a sensitivity lower than 25 PFU/mL. In addition, QDs nanobead was also incorporated into lateral flow assay for SARS-CoV-2 antibody detection, leading to more than one order of magnitude detection sensitivity as compared to that of commercial one based on colloid gold.


Subject(s)
Biosensing Techniques/methods , COVID-19/diagnosis , Influenza, Human/diagnosis , Microspheres , Nanostructures/chemistry , Polystyrenes/chemistry , Quantum Dots , Antibodies, Viral/immunology , COVID-19/virology , Fluorescent Dyes/chemistry , Humans , Influenza A Virus, H5N1 Subtype/physiology , Influenza, Human/virology , Microscopy, Electron, Scanning , Microscopy, Electron, Transmission , Nanostructures/ultrastructure , SARS-CoV-2/immunology , SARS-CoV-2/physiology , Sensitivity and Specificity , Silicon Dioxide/chemistry
18.
Nano Lett ; 21(1): 651-657, 2021 01 13.
Article in English | MEDLINE | ID: covidwho-962235

ABSTRACT

The global COVID-19 pandemic has changed many aspects of daily lives. Wearing personal protective equipment, especially respirators (face masks), has become common for both the public and medical professionals, proving to be effective in preventing spread of the virus. Nevertheless, a detailed understanding of respirator filtration-layer internal structures and their physical configurations is lacking. Here, we report three-dimensional (3D) internal analysis of N95 filtration layers via X-ray tomography. Using deep learning methods, we uncover how the distribution and diameters of fibers within these layers directly affect contaminant particle filtration. The average porosity of the filter layers is found to be 89.1%. Contaminants are more efficiently captured by denser fiber regions, with fibers <1.8 µm in diameter being particularly effective, presumably because of the stronger electric field gradient on smaller diameter fibers. This study provides critical information for further development of N95-type respirators that combine high efficiency with good breathability.


Subject(s)
COVID-19/prevention & control , N95 Respirators/virology , Pandemics , SARS-CoV-2/ultrastructure , Air Microbiology , COVID-19/transmission , COVID-19/virology , Deep Learning , Filtration/statistics & numerical data , Humans , Imaging, Three-Dimensional , Microscopy, Electron, Scanning , N95 Respirators/standards , N95 Respirators/statistics & numerical data , Nanoparticles/ultrastructure , Pandemics/prevention & control , Particle Size , Polypropylenes , Porosity , Textiles/virology , Tomography, X-Ray
19.
Sci Rep ; 10(1): 16099, 2020 09 30.
Article in English | MEDLINE | ID: covidwho-809222

ABSTRACT

SARS-CoV-2 is the cause of the ongoing COVID-19 pandemic. Here, we investigated the interaction of this new coronavirus with Vero cells using high resolution scanning electron microscopy. Surface morphology, the interior of infected cells and the distribution of viral particles in both environments were observed 2 and 48 h after infection. We showed areas of viral processing, details of vacuole contents, and viral interactions with the cell surface. Intercellular connections were also approached, and viral particles were adhered to these extensions suggesting direct cell-to-cell transmission of SARS-CoV-2.


Subject(s)
Betacoronavirus/ultrastructure , Coronavirus Infections/transmission , Host-Pathogen Interactions/physiology , Pneumonia, Viral/transmission , Animals , COVID-19 , Cell Line , Chlorocebus aethiops , Coronavirus Infections/pathology , Humans , Microscopy, Electron, Scanning , Pandemics , Pneumonia, Viral/pathology , SARS-CoV-2 , Vero Cells
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