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2.
Sci Rep ; 12(1): 3794, 2022 03 08.
Article in English | MEDLINE | ID: covidwho-1735281

ABSTRACT

SARS-CoV-2 virions enter the host cells by docking their spike glycoproteins to the membrane-bound Angiotensin Converting Enzyme 2. After intracellular assembly, the newly formed virions are released from the infected cells to propagate the infection, using the extra-cytoplasmic ACE2 docking mechanism. However, the molecular events underpinning SARS-CoV-2 transmission between host cells are not fully understood. Here, we report the findings of a scanning Helium-ion microscopy study performed on Vero E6 cells infected with mNeonGreen-expressing SARS-CoV-2. Our data reveal, with unprecedented resolution, the presence of: (1) long tunneling nanotubes that connect two or more host cells over submillimeter distances; (2) large scale multiple cell fusion events (syncytia); and (3) abundant extracellular vesicles of various sizes. Taken together, these ultrastructural features describe a novel intra-cytoplasmic connection among SARS-CoV-2 infected cells that may act as an alternative route of viral transmission, disengaged from the well-known extra-cytoplasmic ACE2 docking mechanism. Such route may explain the elusiveness of SARS-CoV-2 to survive from the immune surveillance of the infected host.


Subject(s)
Microscopy/methods , SARS-CoV-2/physiology , Virus Internalization , Angiotensin-Converting Enzyme 2/metabolism , Animals , COVID-19/transmission , COVID-19/virology , Chlorocebus aethiops , Cytoplasm/chemistry , Cytoplasm/ultrastructure , Cytoplasm/virology , Extracellular Vesicles/chemistry , Extracellular Vesicles/ultrastructure , Giant Cells/chemistry , Giant Cells/physiology , Helium/chemistry , Humans , Ions/chemistry , SARS-CoV-2/isolation & purification , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/metabolism , Vero Cells
3.
Nat Methods ; 18(12): 1496-1498, 2021 12.
Article in English | MEDLINE | ID: covidwho-1612200

ABSTRACT

The rapid pace of innovation in biological imaging and the diversity of its applications have prevented the establishment of a community-agreed standardized data format. We propose that complementing established open formats such as OME-TIFF and HDF5 with a next-generation file format such as Zarr will satisfy the majority of use cases in bioimaging. Critically, a common metadata format used in all these vessels can deliver truly findable, accessible, interoperable and reusable bioimaging data.


Subject(s)
Computational Biology/instrumentation , Computational Biology/standards , Metadata , Microscopy/instrumentation , Microscopy/standards , Software , Benchmarking , Computational Biology/methods , Data Compression , Databases, Factual , Information Storage and Retrieval , Internet , Microscopy/methods , Programming Languages , SARS-CoV-2
4.
J Am Chem Soc ; 144(4): 1498-1502, 2022 02 02.
Article in English | MEDLINE | ID: covidwho-1586041

ABSTRACT

Several applications in health diagnostics, food, safety, and environmental monitoring require rapid, simple, selective, and quantitatively accurate viral load monitoring. Here, we introduce the first label-free biosensing method that rapidly detects and quantifies intact virus in human saliva with single-virion resolution. Using pseudotype SARS-CoV-2 as a representative target, we immobilize aptamers with the ability to differentiate active from inactive virions on a photonic crystal, where the virions are captured through affinity with the spike protein displayed on the outer surface. Once captured, the intrinsic scattering of the virions is amplified and detected through interferometric imaging. Our approach analyzes the motion trajectory of each captured virion, enabling highly selective recognition against nontarget virions, while providing a limit of detection of 1 × 103 copies/mL at room temperature. The approach offers an alternative to enzymatic amplification assays for point-of-collection diagnostics.


Subject(s)
Aptamers, Nucleotide/chemistry , Biosensing Techniques/methods , DNA/chemistry , Immobilized Nucleic Acids/chemistry , SARS-CoV-2/isolation & purification , Biosensing Techniques/instrumentation , Humans , Limit of Detection , Microscopy/methods , Optics and Photonics/instrumentation , Optics and Photonics/methods , SARS-CoV-2/chemistry , Saliva/virology , Spike Glycoprotein, Coronavirus/chemistry
5.
J Med Internet Res ; 23(2): e24266, 2021 02 22.
Article in English | MEDLINE | ID: covidwho-1574391

ABSTRACT

BACKGROUND: Transition to digital pathology usually takes months or years to be completed. We were familiarizing ourselves with digital pathology solutions at the time when the COVID-19 outbreak forced us to embark on an abrupt transition to digital pathology. OBJECTIVE: The aim of this study was to quantitatively describe how the abrupt transition to digital pathology might affect the quality of diagnoses, model possible causes by probabilistic modeling, and qualitatively gauge the perception of this abrupt transition. METHODS: A total of 17 pathologists and residents participated in this study; these participants reviewed 25 additional test cases from the archives and completed a final psychologic survey. For each case, participants performed several different diagnostic tasks, and their results were recorded and compared with the original diagnoses performed using the gold standard method (ie, conventional microscopy). We performed Bayesian data analysis with probabilistic modeling. RESULTS: The overall analysis, comprising 1345 different items, resulted in a 9% (117/1345) error rate in using digital slides. The task of differentiating a neoplastic process from a nonneoplastic one accounted for an error rate of 10.7% (42/392), whereas the distinction of a malignant process from a benign one accounted for an error rate of 4.2% (11/258). Apart from residents, senior pathologists generated most discrepancies (7.9%, 13/164). Our model showed that these differences among career levels persisted even after adjusting for other factors. CONCLUSIONS: Our findings are in line with previous findings, emphasizing that the duration of transition (ie, lengthy or abrupt) might not influence the diagnostic performance. Moreover, our findings highlight that senior pathologists may be limited by a digital gap, which may negatively affect their performance with digital pathology. These results can guide the process of digital transition in the field of pathology.


Subject(s)
COVID-19/epidemiology , Clinical Competence , Diagnostic Imaging/methods , Diagnostic Imaging/standards , Image Processing, Computer-Assisted/methods , Image Processing, Computer-Assisted/standards , Pathology, Clinical/methods , Pathology, Clinical/standards , Bayes Theorem , Disease Outbreaks , Humans , Internship and Residency/methods , Internship and Residency/standards , Italy/epidemiology , Microscopy , Surveys and Questionnaires
6.
Nat Commun ; 12(1): 7135, 2021 12 09.
Article in English | MEDLINE | ID: covidwho-1565715

ABSTRACT

A characteristic clinical feature of COVID-19 is the frequent incidence of microvascular thrombosis. In fact, COVID-19 autopsy reports have shown widespread thrombotic microangiopathy characterized by extensive diffuse microthrombi within peripheral capillaries and arterioles in lungs, hearts, and other organs, resulting in multiorgan failure. However, the underlying process of COVID-19-associated microvascular thrombosis remains elusive due to the lack of tools to statistically examine platelet aggregation (i.e., the initiation of microthrombus formation) in detail. Here we report the landscape of circulating platelet aggregates in COVID-19 obtained by massive single-cell image-based profiling and temporal monitoring of the blood of COVID-19 patients (n = 110). Surprisingly, our analysis of the big image data shows the anomalous presence of excessive platelet aggregates in nearly 90% of all COVID-19 patients. Furthermore, results indicate strong links between the concentration of platelet aggregates and the severity, mortality, respiratory condition, and vascular endothelial dysfunction level of COVID-19 patients.


Subject(s)
COVID-19/diagnosis , Platelet Aggregation , Single-Cell Analysis , Thrombosis/virology , COVID-19/blood , Female , Humans , Male , Microscopy , Sex Factors
7.
CRISPR J ; 4(6): 854-871, 2021 12.
Article in English | MEDLINE | ID: covidwho-1545880

ABSTRACT

The lack of efficient tools to label multiple endogenous targets in cell lines without staining or fixation has limited our ability to track physiological and pathological changes in cells over time via live-cell studies. Here, we outline the FAST-HDR vector system to be used in combination with CRISPR-Cas9 to allow visual live-cell studies of up to three endogenous proteins within the same cell line. Our approach utilizes a novel set of advanced donor plasmids for homology-directed repair and a streamlined workflow optimized for microscopy-based cell screening to create genetically modified cell lines that do not require staining or fixation to accommodate microscopy-based studies. We validated this new methodology by developing two advanced cell lines with three fluorescent-labeled endogenous proteins that support high-content imaging without using antibodies or exogenous staining. We applied this technology to study seven severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2/COVID-19) viral proteins to understand better their effects on autophagy, mitochondrial dynamics, and cell growth. Using these two cell lines, we were able to identify the protein ORF3a successfully as a potent inhibitor of autophagy, inducer of mitochondrial relocalization, and a growth inhibitor, which highlights the effectiveness of live-cell studies using this technology.


Subject(s)
Autophagy , COVID-19 , CRISPR-Cas Systems , Gene Targeting , Mitochondrial Dynamics , SARS-CoV-2 , Viroporin Proteins , COVID-19/genetics , COVID-19/metabolism , HCT116 Cells , HEK293 Cells , HeLa Cells , Humans , Microscopy , SARS-CoV-2/genetics , SARS-CoV-2/metabolism , Viroporin Proteins/genetics , Viroporin Proteins/metabolism
8.
Pathologe ; 42(Suppl 2): 142-148, 2021 Dec.
Article in German | MEDLINE | ID: covidwho-1527330

ABSTRACT

BACKGROUND: Due to the corona pandemic, digital teaching has become especially important in education and has led to a restructuring of teaching, not only in the subject of surgical pathology. OBJECTIVES: In this article, different forms of e­learning are presented and illustrated using the example of teaching surgical pathology and neuropathology at the University Medical Center Mainz. RESULTS: Before the onset of the corona pandemic in spring 2020, digitization had already assumed great importance for teaching in the technology- and method-oriented subject of surgical pathology. In particular, the possibility of virtual microscopy via scanned slides with a digital slide server has been used in many pathology institutes. Virtual microscopy often partially or completely replaced conventional microscopy of histologic slide collections. Complementary virtual learning offers are becoming more and more important. These include asynchronously provided lectures or macroscopy videos, video conferences, scripts and communication via learning platforms. In addition, electronic exams have become an indispensable part of teaching. Nevertheless, the corona pandemic revealed how important personal contact with students is to achieve optimal learning success; learning forms with a combination of face-to-face teaching and e­learning in the sense of blended learning are of particular importance. CONCLUSIONS: As part of blended learning, digital teaching is an ideal complement to face-to-face teaching and is changing teaching in the longer term, not only in the field of surgical pathology. Digital learning formats will remain in the future and will at least partially replace classroom formats such as lectures.


Subject(s)
Learning , Pathology, Surgical , Humans , Microscopy , Pandemics
9.
Virol J ; 18(1): 149, 2021 07 18.
Article in English | MEDLINE | ID: covidwho-1496197

ABSTRACT

BACKGROUND: The novel coronavirus SARS-CoV-2 is the etiological agent of COVID-19. This virus has become one of the most dangerous in recent times with a very high rate of transmission. At present, several publications show the typical crown-shape of the novel coronavirus grown in cell cultures. However, an integral ultramicroscopy study done directly from clinical specimens has not been published. METHODS: Nasopharyngeal swabs were collected from 12 Cuban individuals, six asymptomatic and RT-PCR negative (negative control) and six others from a COVID-19 symptomatic and RT-PCR positive for SARS CoV-2. Samples were treated with an aldehyde solution and processed by scanning electron microscopy (SEM), confocal microscopy (CM) and, atomic force microscopy. Improvement and segmentation of coronavirus images were performed by a novel mathematical image enhancement algorithm. RESULTS: The images of the negative control sample showed the characteristic healthy microvilli morphology at the apical region of the nasal epithelial cells. As expected, they do not display virus-like structures. The images of the positive sample showed characteristic coronavirus-like particles and evident destruction of microvilli. In some regions, virions budding through the cell membrane were observed. Microvilli destruction could explain the anosmia reported by some patients. Virus-particles emerging from the cell-surface with a variable size ranging from 80 to 400 nm were observed by SEM. Viral antigen was identified in the apical cells zone by CM. CONCLUSIONS: The integral microscopy study showed that SARS-CoV-2 has a similar image to SARS-CoV. The application of several high-resolution microscopy techniques to nasopharyngeal samples awaits future use.


Subject(s)
COVID-19/pathology , Nasopharynx/ultrastructure , SARS-CoV-2/ultrastructure , Antigens, Viral/metabolism , COVID-19/diagnosis , COVID-19/virology , Epithelial Cells/ultrastructure , Epithelial Cells/virology , Humans , Image Enhancement , Microscopy , Microvilli/ultrastructure , Nasal Mucosa/ultrastructure , Nasal Mucosa/virology , Nasopharynx/virology , SARS-CoV-2/isolation & purification , Virion/ultrastructure
10.
EMBO Rep ; 22(11): e54024, 2021 11 04.
Article in English | MEDLINE | ID: covidwho-1451019

ABSTRACT

Many people have discovered new hobbies and pastimes during the COVID crisis. David Smith describes how he developed an obsession with rescuing old microscopes.


Subject(s)
Hobbies , Microscopy , COVID-19 , Pandemics , SARS-CoV-2
12.
Blood ; 138(22): 2256-2268, 2021 12 02.
Article in English | MEDLINE | ID: covidwho-1443788

ABSTRACT

SARS-CoV-2 vaccine ChAdOx1 nCoV-19 (AstraZeneca) causes a thromboembolic complication termed vaccine-induced immune thrombotic thrombocytopenia (VITT). Using biophysical techniques, mouse models, and analysis of VITT patient samples, we identified determinants of this vaccine-induced adverse reaction. Super-resolution microscopy visualized vaccine components forming antigenic complexes with platelet factor 4 (PF4) on platelet surfaces to which anti-PF4 antibodies obtained from VITT patients bound. PF4/vaccine complex formation was charge-driven and increased by addition of DNA. Proteomics identified substantial amounts of virus production-derived T-REx HEK293 proteins in the ethylenediaminetetraacetic acid (EDTA)-containing vaccine. Injected vaccine increased vascular leakage in mice, leading to systemic dissemination of vaccine components known to stimulate immune responses. Together, PF4/vaccine complex formation and the vaccine-stimulated proinflammatory milieu trigger a pronounced B-cell response that results in the formation of high-avidity anti-PF4 antibodies in VITT patients. The resulting high-titer anti-PF4 antibodies potently activated platelets in the presence of PF4 or DNA and polyphosphate polyanions. Anti-PF4 VITT patient antibodies also stimulated neutrophils to release neutrophil extracellular traps (NETs) in a platelet PF4-dependent manner. Biomarkers of procoagulant NETs were elevated in VITT patient serum, and NETs were visualized in abundance by immunohistochemistry in cerebral vein thrombi obtained from VITT patients. Together, vaccine-induced PF4/adenovirus aggregates and proinflammatory reactions stimulate pathologic anti-PF4 antibody production that drives thrombosis in VITT. The data support a 2-step mechanism underlying VITT that resembles the pathogenesis of (autoimmune) heparin-induced thrombocytopenia.


Subject(s)
Antigen-Antibody Complex/immunology , Autoantibodies/immunology , COVID-19/prevention & control , Capsid Proteins/adverse effects , Drug Contamination , Genetic Vectors/adverse effects , HEK293 Cells/immunology , Immunoglobulin G/immunology , Platelet Factor 4/immunology , Purpura, Thrombocytopenic, Idiopathic/etiology , SARS-CoV-2 , Spike Glycoprotein, Coronavirus/adverse effects , Adenoviridae/immunology , Animals , Antigen-Antibody Complex/ultrastructure , Autoantibodies/biosynthesis , Capillary Leak Syndrome/etiology , Capsid Proteins/immunology , Cell Line, Transformed , /immunology , Dynamic Light Scattering , Epitopes/chemistry , Epitopes/immunology , Extracellular Traps/immunology , Extravasation of Diagnostic and Therapeutic Materials/etiology , Genetic Vectors/immunology , HEK293 Cells/chemistry , Humans , Imaging, Three-Dimensional , Immunoglobulin G/biosynthesis , Inflammation , Mice , Microscopy/methods , Platelet Activation , Proteomics , Purpura, Thrombocytopenic, Idiopathic/blood , Purpura, Thrombocytopenic, Idiopathic/immunology , Sinus Thrombosis, Intracranial/diagnostic imaging , Sinus Thrombosis, Intracranial/immunology , Spike Glycoprotein, Coronavirus/immunology , Virus Cultivation
13.
Indian J Ophthalmol ; 69(10): 2844-2845, 2021 10.
Article in English | MEDLINE | ID: covidwho-1441275

ABSTRACT

In this manuscript, we report a rapid intra-operative detection test for visualisation of mucor, with the use of the Smartphone-based intraocular lens microscope (IOLSCOPE). IOLSCOPE helps not only in detecting tissue affected by mucor, but also makes sure that the surgeon does not leave any fungus behind; similar to the principles employed by a 'Frozen section' in conventional general surgery. In the technique proposed by us, we have used an undigested tissue sample, to quickly diagnose mucor, intra-operatively. Thus, in view of tackling the COVID-19 and mucor duplet, IOLSCOPE can be considered by ophthalmologists and otorhinolaryngologists for its rapid screening property, convenience of ease, good quality images and cost effectiveness.


Subject(s)
COVID-19 , Mucor , Frozen Sections , Humans , Microscopy , SARS-CoV-2
14.
Cytometry A ; 97(9): 882-886, 2020 09.
Article in English | MEDLINE | ID: covidwho-1384154

ABSTRACT

Operating shared resource laboratories (SRLs) in times of pandemic is a challenge for research institutions. In a multiuser, high-turnover working space, the transmission of infectious agents is difficult to control. To address this challenge, imaging core facility managers being members of German BioImaging discussed how shared microscopes could be operated with minimal risk of spreading SARS-CoV-2 between users and staff. Here, we describe the resulting guidelines and explain their rationale, with a focus on separating users in space and time, protective face masks, and keeping surfaces virus-free. These recommendations may prove useful for other types of SRLs. © 2020 The Authors. Cytometry Part A published by Wiley Periodicals LLC. on behalf of International Society for Advancement of Cytometry.


Subject(s)
Betacoronavirus/pathogenicity , Biomedical Research/organization & administration , Coronavirus Infections/prevention & control , Infection Control , Laboratories/organization & administration , Microscopy , Occupational Health , Pandemics/prevention & control , Pneumonia, Viral/prevention & control , COVID-19 , Cooperative Behavior , Coronavirus Infections/transmission , Coronavirus Infections/virology , Decontamination , Equipment Contamination/prevention & control , Germany , Humans , Occupational Exposure/prevention & control , Personal Protective Equipment , Pneumonia, Viral/transmission , Pneumonia, Viral/virology , Protective Factors , Research Personnel/organization & administration , Risk Assessment , Risk Factors , SARS-CoV-2 , Workflow
15.
Nat Rev Genet ; 22(2): 69, 2021 02.
Article in English | MEDLINE | ID: covidwho-1387375
16.
J Laryngol Otol ; 135(8): 710-717, 2021 Aug.
Article in English | MEDLINE | ID: covidwho-1340961

ABSTRACT

OBJECTIVE: The application of a 4K display resolution three-dimensional exoscope system (Vitom 3D) was evaluated to determine the feasibility of adopting the system in ENT surgery in the coronavirus disease 2019 era and beyond. METHODS: Eighteen ENT surgeons performed structured otological tasks on fresh-frozen sheep heads using the Vitom 3D. Structured feedback of the participants' experience was analysed. RESULTS: Seventy-four per cent and 94 per cent of participants reported that the Vitom 3D was ergonomic and comfortable to use respectively. Whilst colour fidelity and image quality were very good, 50 per cent of participants reported image distortion and pixilation at the highest magnification. All participants agreed that there was an increased educational value to exoscope technology. Half the participants preferred the microscope over the Vitom 3D for fine otological work, which may reflect the learning curve. CONCLUSION: The Vitom 3D exoscope is a promising and viable alternative for performing otological surgery when using full personal protective equipment in the coronavirus disease 2019 era.


Subject(s)
COVID-19/epidemiology , Microscopy/instrumentation , Otologic Surgical Procedures/methods , Animals , Disease Models, Animal , Feasibility Studies , Head and Neck Neoplasms/pathology , Head and Neck Neoplasms/surgery , Humans , Microscopy/methods , Otologic Surgical Procedures/education , Otologic Surgical Procedures/instrumentation , Sheep
18.
GMS J Med Educ ; 38(5): Doc87, 2021.
Article in English | MEDLINE | ID: covidwho-1295609

ABSTRACT

At the start of the COVID-pandemic in March 2020, the Institutes of Veterinary Anatomy of the five German educational institutions were confronted with the challenge of digitalising all lectures for the second and fourth semesters of veterinary students. After an online kick-off event and a preliminary status quo meeting, available digital teaching material was exchanged for students to stream from learning platforms. Lectures were either synchronized or made available as audio recordings and connotated slides on the learning platforms. Fortunately, digital microscopic slides had already been in use, which made it easy for students to access them. Dissection exercises mostly consisted of self-study, using instructive videos and interactive exercises. In the second half of the semester, four of the educational institutions were able to offer a restricted number of in-person gross anatomy classes under reinforced conditions. Success monitoring took place online through different formats, and partially on a voluntary basis, via the learning platforms. Although the past two semesters had to almost exclusively take place online due to the unprecedented circumstances, and joint efforts of the five veterinary institutions, there is a general consensus that the practical education in anatomy, histology and embryology is essential to veterinary students. In fact, it is the only way they can obtain the necessary skills to successfully complete the rest of their degree.


Subject(s)
Anatomy/education , COVID-19 , Education, Distance , Education, Veterinary/methods , Pandemics , Schools, Veterinary , Teaching , Audiovisual Aids , Digital Technology , Dissection , Germany , Humans , Internet , Microscopy , Physical Distancing , Professional Competence , Students , Universities , Veterinary Medicine
20.
Opt Lett ; 46(10): 2344-2347, 2021 May 15.
Article in English | MEDLINE | ID: covidwho-1229026

ABSTRACT

Rapid screening of red blood cells for active infection of COVID-19 is presented using a compact and field-portable, 3D-printed shearing digital holographic microscope. Video holograms of thin blood smears are recorded, individual red blood cells are segmented for feature extraction, then a bi-directional long short-term memory network is used to classify between healthy and COVID positive red blood cells based on their spatiotemporal behavior. Individuals are then classified based on the simple majority of their cells' classifications. The proposed system may be beneficial for under-resourced healthcare systems. To the best of our knowledge, this is the first report of digital holographic microscopy for rapid screening of COVID-19.


Subject(s)
COVID-19 Testing/methods , COVID-19/blood , Deep Learning , Erythrocytes/pathology , Holography/instrumentation , SARS-CoV-2 , COVID-19/classification , Humans , Image Enhancement/instrumentation , Microscopy/instrumentation , Reproducibility of Results , Sensitivity and Specificity
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