Concentration of SARS-CoV-2-Infected Cell Culture Supernatants for Detection of Virus-like Particles by Scanning Electron Microscopy.

There is currently a need for new rapid viral diagnostic electron microscopy methods. Although the gold standard remains the transmission electron microscopy (TEM) negative staining method for electron microscopic examination of samples containing a virus, difficulties can arise when the virus particle content of the sample that has to be examined is poor. Such samples include supernatants of virus-infected cells that can be difficult to examine, as sometimes only a few virus particles are released in the culture medium upon infection. In addition to TEM, scanning electron microscopy (SEM) can also be used for visualizing virus particles. One advantage of SEM over TEM is its ability to rapidly screen several large specimens, such as microscopy slides. In this study, we investigated this possibility and tested different coating molecules as well as the effect of centrifugation for analyzing SARS-CoV-2-virus-infected cell culture supernatants deposited on microscopy glass slides by SEM. We found that centrifugation of 25XConcanavalinA-coated microscopy glass slides in shell vials provided an improved method for concentrating SARS-CoV-2-virus-infected cell supernatants for virus-like particle detection by SEM.

Effectiveness of Different Methods in Removing Dentin Caries of Primary Teeth: Micro-CT and SEM Evaluation.

OBJECTIVES: With the recent improvements in technology, the expectation of minimal invasion and maximal comfort in caries removal techniques is increasing. This study aims to examine the effectiveness of six caries removal methods in primary teeth. STUDY DESIGN: Sixty primary molars (10 teeth in each groups) were used. The groups were: Group I (Tungsten Carbide Bur), Group II (Sono abrasion), Group III (Air abrasion), Group IV (Carisolv), Group V (Er:YAG Laser), Group VI (ART). In micro-CT scanning, mineral density at the cavity floor was examined before and after caries removal. After caries removal, the patency of the dentinal tubules was examined in two teeth from each group on SEM images. Statistical analyses were performed using Kruskal-Wallis, Wilcoxon tests. RESULTS: For six different caries removal methods, tooth mineral (inorganic, total) densities at cavity floors were compared among the groups after the procedures, and no statistically significant difference was found (p>0.05). On the SEM images, it was seen that the dentinal tubules were exposed and no smear layer was formed in the Carisolv group. Significant rough surfaces were exposed in the laser group. CONCLUSION: It was observed that alternative caries removal methods are at least as effective as the traditional method in primary teeth for clinical applications.

Determination of Alteration in Micromeritic Properties of a Solid Dispersion: Brunauer-Emmett-Teller Based Adsorption and Other Structured Approaches.

The present study is focused on the use of solid dispersion technology to triumph over the solubility-related problems of bexarotene which is currently used for treating various types of cancer and has shown potential inhibitory action on COVID-19 main protease and human ACE2 receptors. It is based on comparison of green locust bean gum and synthetic poloxamer as polymers using extensive mechanistic methods to explore the mechanism behind solubility enhancement and to find suitable concentration of drug to polymer ratio to prepare porous 3rd generation solid dispersion. The prepared solid dispersions were characterized using different studies like X-ray diffraction (XRD), thermal gravimetric analysis (TGA), scanning electron microscopy (SEM), Brunauer-Emmett-Teller (BET), differential scanning calorimetry (DSC), and particle size analysis in order to determine the exact changes occurred in the product which are responsible for enhancing solubility profiles of an insoluble drug. The results showed different profiles for particle size, solubility, dissolution rate, porosity, BET, and Langmuir specific surface area of prepared solid dispersions by using different polymers. In addition to the comparison of polymers, the BET analysis deeply explored the changes occurred in all dispersions when the concentration of polymer was increased. The optimized solid dispersion prepared with MLBG using lyophilization technique showed reduced particle size of 745.7±4.4 nm, utmost solubility of 63.97%, pore size of 211.597 Å, BET and Langmuir specific surface area of 5.6413 m2/g and 8.2757 m2/g, respectively.

Drug delivery of amoxicillin molecule as a suggested treatment for covid-19 implementing functionalized mesoporous SBA-15 with aminopropyl groups.

SARS-CoV-2 is a novel coronavirus that was isolated and identified for the first time in Wuhan, China in 2019. Nowadays, it is a worldwide danger and the WHO named it a pandemic. In this investigation, a functionalization post-synthesis method was used to assess the ability of an adapted SBA-15 surface as a sorbent to load the drug from an aqueous medium. Different characterization approaches were used to determine the characterization of the substance before and after functionalization such as X-ray diffraction (XRD), Fourier transform infrared (FTIR) spectroscopy, scanning electron microscopy (SEM), nitrogen adsorption-desorption porosimetry (Brunauer-Emmett-Teller) BET surface area analysis, and thermal gravimetric analysis (TGA). Batch adsorption testing was carried out in a single adsorption device to find the impact of multiple variables on the drug amoxicillin charge output. The following parameters were studied: 0-72 hr. contact time, 20-120 mg/l initial concentration, and 20-250 mg of NH2-SBA-15 dose. The outcomes from such experiments revealed the strong influence and behavior of the amino-functional group to increase the drug's load. Drug delivery outcomes studies found that amoxicillin loading was directly related to NH2-SBA-15 contact time and dose, but indirectly related to primary concentration. It was observed that 80% of amoxicillin was loaded while the best release test results were 1 hour and 51%.

Thermometric lateral flow immunoassay with colored latex beads as reporters for COVID-19 testing.

Temperature sensing is a promising method of enhancing the detection sensitivity of lateral flow immunoassay (LFIA) for point-of-care testing. A temperature increase of more than 100 °C can be readily achieved by photoexcitation of reporters like gold nanoparticles (GNPs) or colored latex beads (CLBs) on LFIA strips with a laser power below 100 mW. Despite its promise, processes involved in the photothermal detection have not yet been well-characterized. Here, we provide a fundamental understanding of this thermometric assay using non-fluorescent CLBs as the reporters deposited on nitrocellulose membrane. From a measurement for the dependence of temperature rises on the number density of membrane-bound CLBs, we found a 1.3-fold (and 3.2-fold) enhancement of the light absorption by red (and black) latex beads at 520 nm. The enhancement was attributed to the multiple scattering of light in this highly porous medium, a mechanism that could make a significant impact on the sensitivity improvement of LFIA. The limit of detection was measured to be 1 × 105 particles/mm2. In line with previous studies using GNPs as the reporters, the CLB-based thermometric assay provides a 10× higher sensitivity than color visualization. We demonstrated a practical use of this thermometric immunoassay with rapid antigen tests for COVID-19.

Ultrastructural findings of lung injury due to Vaccine-induced Immune Thrombotic Thrombo- cytopenia (VITT) following COVID-19 vaccination: a scanning electron microscopic study.

Vaccine-induced immune thrombotic thrombocytopenia (VITT) is a rare new syndrome occurring after the ChAdOx1 nCoV-19 vaccine immunization. Patients with VITT are characterized by a variable clinical presentation, likewise also the outcome of these patients is very variable. Here we report the lung ultrastructural findings in the course of VITT of a 58-year-old male patient. Alveoli were mainly dilated, irregular in shape, and occupied by a reticular network of fibrin, while interalveolar septa appeared thickened. The proliferation of small capillaries gave rise to plexiform structures and pulmonary capillary hemangiomatosis-like features. Near the alveoli occupied by a dense fibrin network, the medium-sized arteries showed a modified wall and an intraluminal thrombus. This scenario looks quite similar to that found during COVID-19, where the lungs suffer from the attack of the antigen-antibodies complexes and the virus respectively. In both diseases, the final outcome is a severe inflammation, activation of the haemostatic system and fibrinolysis.

Imperative role of electron microscopy in toxicity assessment: A review.

Electron microscope (EM) was developed in 1931 and since then microscopical examination of both the biological and non-biological samples has been revolutionized. Modifications in electron microscopy techniques, such as scanning EM and transmission EM, have widened their applicability in the various sectors such as understanding of drug toxicity, development of mechanism, criminal site investigation, and characterization of the nano-molecule. The present review summarizes its role in important aspects such as toxicity assessment and disease diagnosis in special reference to SARS-COV2. In the biological system, EM studies have elucidated the impact of toxicants at the ultra-structural level in various tissue in conformity to physiological alterations. Thus, EM can be concluded as an important tool in toxicity assessment and disease prognosis.

Ultrasensitive Detection of COVID-19 Causative Virus (SARS-CoV-2) Spike Protein Using Laser Induced Graphene Field-Effect Transistor.

COVID-19 is a highly contagious human infectious disease caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), and the war with the virus is still underway. Since no specific drugs have been made available yet and there is an imbalance between supply and demand for vaccines, early diagnosis and isolation are essential to control the outbreak. Current nucleic acid testing methods require high sample quality and laboratory conditions, which cannot meet flexible applications. Here, we report a laser-induced graphene field-effect transistor (LIG-FET) for detecting SARS-CoV-2. The FET was manufactured by different reduction degree LIG, with an oyster reef-like porous graphene channel to enrich the binding point between the virus protein and sensing area. After immobilizing specific antibodies in the channel, the FET can detect the SARS-CoV-2 spike protein in 15 min at a concentration of 1 pg/mL in phosphate-buffered saline (PBS) and 1 ng/mL in human serum. In addition, the sensor shows great specificity to the spike protein of SARS-CoV-2. Our sensors can realize fast production for COVID-19 rapid testing, as each LIG-FET can be fabricated by a laser platform in seconds. It is the first time that LIG has realized a virus sensing FET without any sample pretreatment or labeling, which paves the way for low-cost and rapid detection of COVID-19.

Bio-Performance of Hydrothermally and Plasma-Treated Titanium: The New Generation of Vascular Stents.

The research presented herein follows an urgent global need for the development of novel surface engineering techniques that would allow the fabrication of next-generation cardiovascular stents, which would drastically reduce cardiovascular diseases (CVD). The combination of hydrothermal treatment (HT) and treatment with highly reactive oxygen plasma (P) allowed for the formation of an oxygen-rich nanostructured surface. The morphology, surface roughness, chemical composition and wettability of the newly prepared oxide layer on the Ti substrate were characterized by scanning electron microscopy (SEM) with energy-dispersive X-ray analysis (EDX), atomic force microscopy (AFM), X-ray photoelectron spectroscopy (XPS) and water contact angle (WCA) analysis. The alteration of surface characteristics influenced the material's bio-performance; platelet aggregation and activation was reduced on surfaces treated by hydrothermal treatment, as well as after plasma treatment. Moreover, it was shown that surfaces treated by both treatment procedures (HT and P) promoted the adhesion and proliferation of vascular endothelial cells, while at the same time inhibiting the adhesion and proliferation of vascular smooth muscle cells. The combination of both techniques presents a novel approach for the fabrication of vascular implants, with superior characteristics.

A rapid lateral flow immunoassay strip for detection of SARS-CoV-2 antigen using latex microspheres.

BACKGROUND: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a highly infectious and concealed virus that causes pneumonia, severe acute respiratory syndrome, and even death. Although the epidemic has been controlled since the development of vaccines and quarantine measures, many people are still infected, particularly in third-world countries. Several methods have been developed for detection of SARS-CoV-2, but owing to its price and efficiency, the immune strip could be a better method for the third-world countries. METHODS: In this study, two antibodies were linked to latex microspheres, using 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide, as the bridge to decrease the cost further and improve the detection performance. The specificity of the lateral flow immunoassay strip (LFIA) was tested by several common viruses and respiratory bacterial infections. Besides, the reproducibility and stability of the LFIAs were tested on the same batch of test strips. Under optimal conditions, the sensitivity of LFIA was determined by testing different dilutions of the positive specimens. RESULTS: The proposed LFIAs were highly specific, and the limit of detection was as low as 25 ng/mL for SARS-CoV-2 antigens. The clinical applicability was evaluated with 659 samples (230 positive and 429 negative samples) by using both LFIA and rRT-PCR. Youden's index (J) was used to assess the performance of these diagnostic tests. The sensitivity and specificity were 98.22% and 97.93%, respectively, and J is 0.9615. The sensitivity and specificity were 98.22% and 97.93%, respectively, and J is 0.9615. In addition, the consistency of our proposed LFIA was analyzed using Cohen's kappa coefficient (κ = 0.9620). CONCLUSION: We found disease stage, age, gender, and clinical manifestations have only a slight influence on the diagnosis. Therefore, the lateral flow immunoassay SARS-CoV-2 antigen test strip is suitable for point-of-care detection and provides a great application for SARS-CoV-2 epidemic control in the third-world countries.

Characterisation of factors contributing to the performance of nonwoven fibrous matrices as substrates for adenovirus vectored vaccine stabilisation.

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.

Thrombotic sinusoiditis and local diffuse intrasinusoidal coagulation in the liver of subjects affected by COVID-19: the evidence from histology and scanning electron microscopy.

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.

Chimeric Virus-like Particles of Universal Antigen Epitopes of Coronavirus and Phage Qß Coat Protein Trigger the Production of Neutralizing Antibodies.

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.

Characterization of Materials Used as Face Coverings for Respiratory Protection.

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.

Impact of olokizumab on platelets, leukocytes and erythrocytes during mild COVID-19.

No abstract present.

Mild COVID-19 and Impaired Blood Cell-Endothelial Crosstalk: Considering Long-Term Use of Antithrombotics?

BACKGROUND: Current coronavirus disease 2019 (COVID-19) pandemic reveals thrombotic, vascular, and endothelial dysfunctions at peak disease. However, the duration, degree of damage, and appropriate long-term use of antithrombotic strategies are unclear. Most COVID data are yielded from random clinical observations or autopsy of postmortem samples, while precise blood cellular data in survivors are insufficient. METHODS: We analyzed erythrocytes, circulating endothelial cells, and echinocytes by electron microscopy and flow cytometry in patients with confirmed COVID-19 (n = 31) and matched healthy controls (n = 32) on admission and at hospital discharge. RESULTS: All patients experienced mild disease, none required pulmonary support, and all survived. Admission number of circulating endothelial cells was significantly (40-100 times) higher in COVID-19 patients. Cells were massively damaged by multiple fenestrae in membranes with diameter comparable to the size of supercapsid in SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) virus. COVID-19 also provoked formation of stacked aggregated erythrocytes capable of clogging microvascular bed and of diminishing oxygen supply. In some patients, such abnormalities persisted at hospital discharge revealing remaining intracellular penetration of SARS-CoV-2 where it may be replicated and returned to circulation. CONCLUSION: These observational and descriptive data suggest that persistent viral cell injury may cause blood vessel damage; their increased permeability resulted in tissue edema, inflammation, platelet activation, and augmented thrombosis. There is a residual blood cell damage following the acute phase in some COVID-19 survivors. Controlled outcome-driven trials are urgently needed for exploring optimal use of long-term antithrombotics and vascular protection strategies even after mild COVID-19.

Preparation of Samples for Large-Scale Automated Electron Microscopy of Tissue and Cell Ultrastructure.

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.

Facile Synthesis of Bio-Antimicrobials with "Smart" Triiodides.

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.

Multiscale Electron Microscopy for the Study of Viral Replication Organelles.

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.