Special Isue: Virtual anatomy, histology and embryology in research and education. (Special Isue: Virtual anatomy, histology and embryology in research and education.)

This issue contains 13 articles on the use of virtual anatomy, histology and embryology in research and education;digital histological morphometry of the human pineal gland in a postmortem study, with endocrine and neurological clinical implications;an international collaborative approach to learning histology using a virtual microscope;delivery anatomy kits to help keep practical veterinary classes during the COVID-19 pandemic;how virtual animal anatomy facilitated a successful transition to online instruction and supported student learning during the coronavirus pandemic;using videos in active learning in veterinary anatomy;dissection videos as a virtual veterinary anatomy peer learning tool at the University of Tehran during the COVID-19 pandemic;a new virtual platform for teaching comparative animal neuroanatomy based on metameric slices of the central nervous system;application of student remote and distance research in neuroanatomy by mapping Dscaml1 expression with a LacZ gene trap in mouse brain;implementing a multi-colour genetic marker analysis technique for embryology education;impact of COVID-19 on student attainment and pedagogical needs when undertaking independent scientific research;extended reality veterinary medicine case studies for diagnostic veterinary imaging instruction and assessing student perceptions and examination performance and students' performance in teaching neuroanatomy using traditional and technology-based methods. 16 proceedings from the Trans-European Pedagogic Anatomy Research Group (TEPARG) Hybrid Meeting entitled "Hybrid Anatomy Education: Barriers and Enablers for Students and Educators" held in Barcelona, Spain, during 5 March 2022, are also included.

Identification of selected earthworm species of Northeast India using DNA barcoding

Aim: This study aimed at the species identification of selected indigenous earthworms of Manipur and Assam, Northeast India along with an exotic species using morpho-anatomical study and DNA barcoding. Methodology: Indigenous species of earthworms were collected from Imphal and Jorhat, North-eastern part of India. The exotic species of earthworm were collected from Indian Council of Agricultural Research Complex, Manipur. The samples were collected by digging and hand sorting method. Identification of samples was done by both conventional and molecular methods. Molecular characterization was accomplished through PCR amplification of the mitochondrial cytochrome oxidase I (COI) genes. Automatic sequencing reactions were performed for the amplified PCR products on ABI3100 Genetic Analyser (Applied Biosystems). Result(s): Out of five specimens (EM1, EM2, EM4, EG5 and EM6) examined through morpho-anatomical studies, three were identified to species level while the other two were identified to their genus level only. Out of EM1 and EM2 specimens in the genus Perionyx as per the morpho-anatomical studies, DNA barcoding could deduce the EM2 specimen up to the species level as P. excavatus. The exotic EM6 specimen morphologically identified as Eisenia fetida showed 99% COI gene sequence similarity with both E. fetida and E. andrei but its sequence divergence with E. andrei was less than 1%, so, it belonged to E. andrei. Interpretation(s): This study shows the reliability of clubbing DNA barcoding experiments with classical taxonomy in supplementing and strengthening the traditional taxonomy for accurate identification of earthworms.Copyright © Triveni Enterprises, Lucknow (India)

Toxicity effects of disinfection byproduct chloroacetic acid to Microcystis aeruginosa: Cytotoxicity and mechanisms.

Chlorine-based disinfectants are widely used for disinfection in wastewater treatment. The mechanism of the effects of chlorinated disinfection by-products on cyanobacteria was unclear. Herein, the physiological effects of chloroacetic acid (CAA) on Microcystis aeruginosa (M. aeruginosa), including acute toxicity, oxidative stress, apoptosis, production of microcystin-LR (MC-LR), and the microcystin transportation-related gene mcyH transcript abundance have been investigated. CAA exposure resulted in a significant change in the cell ultrastructure, including thylakoid damage, disappearance of nucleoid, production of gas vacuoles, increase in starch granule, accumulation of lipid droplets, and disruption of cytoplasm membranes. Meanwhile, the apoptosis rate of M. aeruginosa increased with CAA concentration. The production of MC-LR was affected by CAA, and the transcript abundance of mcyH decreased. Our results suggested that CAA poses acute toxicity to M. aeruginosa, and it could cause oxidative damage, stimulate MC-LR production, and damage cell ultrastructure. This study may provide information about the minimum concentration of CAA in the water environment, which is safe for aquatic organisms, especially during the global coronavirus disease 2019 pandemic period.

Visualization of SARS-CoV-2 particles in naso/oropharyngeal swabs by thin section electron microscopy.

BACKGROUND: SARS-CoV-2 replicates efficiently in the upper airways of humans and produces high loads of virus RNA and, at least in the initial phase after infection, many infectious virus particles. Studying virus ultrastructure, such as particle integrity or presence of spike proteins, and effects on their host cells in patient samples is important to understand the pathogenicity of SARS-CoV-2. METHODS: Suspensions from swab samples with a high load of virus RNA (Ct < 20) were sedimented by desktop ultracentrifugation and prepared for thin section electron microscopy using a novel method which is described in detail. Embedding was performed in Epon or in LR White resin using standard or rapid protocols. Thin sections were examined using transmission electron microscopy. RESULTS: Virus particles could be regularly detected in the extracellular space, embedded in a background of heterogenous material (e.g. vesicles and needle-like crystals), and within ciliated cells. Morphology (i.e. shape, size, spike density) of virus particles in the swab samples was very similar to particle morphology in cell culture. However, in some of the samples the virus particles hardly revealed spikes. Infected ciliated cells occasionally showed replication organelles, such as double-membrane vesicles. The most common cells in all samples were keratinocytes from the mucosa and bacteria. CONCLUSIONS: The new method allows the ultrastructural visualization and analysis of coronavirus particles and of infected host cells from easy to collect naso/oropharyngeal patient swab samples.

Ultrastructural analysis and three-dimensional reconstruction of cellular structures involved in SARS-CoV-2 spread.

The cytoskeleton not only deals with numerous interaction and communication mechanisms at the cellular level but also has a crucial role in the viral infection cycle. Although numerous aspects of SARS-CoV-2 virus interaction at the cellular level have been widely studied, little has been reported about the structural and functional response of the cytoskeleton. This work aims to characterize, at the ultrastructural level, the modifications in the cytoskeleton of infected cells, namely, its participation in filopodia formation, the junction of these nanostructures forming bridges, the viral surfing, and the generation of tunnel effect nanotubes (TNT) as probable structures of intracellular viral dissemination. The three-dimensional reconstruction from the obtained micrographs allowed observing viral propagation events between cells in detail for the first time. More profound knowledge about these cell-cell interaction models in the viral spread mechanisms could lead to a better understanding of the clinical manifestations of COVID-19 disease and to find new therapeutic strategies.

Observations of nemaline bodies in muscle biopsies of critically ill patients infected with SARS-CoV-2.

Patients infected with SARS-CoV-2 who have been admitted to the intensive care unit (ICU) often face months of physical disability after discharge. To optimize recovery, it is important to understand the role of musculoskeletal alterations in critically ill patients infected with SARS-CoV-2. The main aim of the present study was to describe the presence and morphology of nemaline bodies found in skeletal muscle tissue from critically ill patients infected with SARS-CoV-2. In n=7 patients infected with SARS-CoV-2, ultrastructural characteristics of vastus lateralis muscle obtained on days 1-3 (T0) and days 5-8 (T1) following ICU admission were investigated in more detail with electron microscopy. Those muscle biopsies consistently showed variable degrees of myofiber necrosis and myofibrillar disorganisation. In 4/7 (57%) patients at T1, the Z-line material accumulated into nemaline bodies with a typical lattice-like appearance at higher magnification, similar to that found in nemaline myopathy. This study is the first to describe the disintegration of myofibrils and accumulation of Z-line material into nemaline bodies in skeletal muscle tissue obtained from critically ill COVID-19 patients following ICU admission, which should be interpreted primarily as a non-specific pathological response of extreme myofibrillar disintegration associated with myofiber necrosis. Mini abstract This study describes the presence and morphology of typical nemaline bodies in skeletal muscle ti0ssue from critically ill patients infected with SARS-CoV-2, which should be interpreted primarily as a non-specific pathological response of extreme myofibrillar disintegration associated with myofiber necrosis.

An ultrastructural and genomic study on the SARS-CoV-2 variant B.1.210 circulating during the first wave of COVID-19 pandemic in India.

PURPOSE: The study aims to isolate and understand cytopathogenesis, ultrastructure, genomic characteristics and phylogenetic analysis of SARS-CoV-2 virus of B.1.210 lineage, that circulated in India during first wave of the pandemic. METHODS: Clinical specimen from an interstate traveller from Maharashtra to Karnataka, in May 2020, who was positive by RT PCR for SARS-CoV-2 infection was subjected to virus isolation and Whole Genome Sequencing. Vero cells were used to study cytopathogenesis and ultrastructural features by Transmission Electron Microscopy (TEM). Phylogenetic analysis of the whole genome sequences of several SARS-CoV-2 variants downloaded from GISAID was performed in comparison with the B.1.210 variant identified in this study. RESULTS: The virus was isolated in Vero cells and identified by immunofluorescence assay and RT PCR. The growth kinetics in infected Vero cells revealed a peak viral titre at 24 â€‹h post-infection. Ultrastructural studies revealed distinct morphological changes with accumulation of membrane-bound vesicles containing pleomorphic virions in the cytoplasm, with single or multiple intranuclear filamentous inclusions and dilated rough endoplasmic reticulum with viral particles. Whole genome sequence of the clinical specimen as well as the isolated virus revealed the virus to be of lineage B.1.210 with the D614G mutation in the spike protein. Phylogenetic analysis of the whole genome sequence in comparison with other variants reported globally revealed that the isolated SARS-CoV-2 virus of lineage B.1.210 is closely related to the original Wuhan virus reference sequence. CONCLUSIONS: The SARS-CoV-2 variant B.1.210 virus isolated here showed ultrastructural features and cytopathogenesis similar to that of the virus reported during early phase of pandemic. Phylogenetic analysis showed that the isolated virus is closely related to the original Wuhan virus, thereby suggesting that the SARS-CoV-2 lineage B.1.210 that was circulating in India during the early phase of pandemic is likely to have evolved from the original Wuhan strain.

Pathological involvement of placenta in COVID-19: a systematic review.

The mammalian placenta, which is responsible for bonding between the mother and the fetus, is one of the first organs to develop. Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) infection has caused a great threat to public health and affected almost all the organs including the placenta. Owing to limited available data on vertical transmission and pathological changes in the placenta of SARS-CoV-2 positive patients, we aim to review and summarize histopathological and ultrastructural changes in the placental tissue following SARS-CoV-2 infection. Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) 2009 guidelines were used for review writing. Multiple studies have reported significant pathological changes in the placental tissue of SARS-CoV-2 positive mothers. On the other hand, some studies have demonstrated either no or very little involvement of the placental tissue. The most common pathological changes reported are fetal and maternal vascular malformation, villitis of unknown etiology, thrombus formation in the intervillous space and sub-chorionic space, and chorangiosis. Reports on vertical transmission are less in number. The observations of this review present a strong base for the pathological involvement of the placenta in SARS-CoV-2 infected mothers. However, a smaller number of original studies have been done until now, and most of them have small sample sizes and lack matched control groups, which are the big limitations for drawing an effective conclusion at this stage. Antenatal care can be improved by a better understanding of the correlation between maternal SARS-CoV-2 infection and placental pathology in COVID-19.

RED BLOOD CELL COLOUR VARIATION AS A MARKER OF CALCIUM DISTRIBUTION: WHAT ROLE DOES IT HAVE IN HEART FAILURE PATIENTS WITH HYPERTENSION AND LONG COVID?

Objective: Red blood cell (RBC) role is both passive action, oxygen delivery to the tissues as well as carbon dioxide to the lungs and active action involvement in the regulation of vascular tone. The aim was to investigate pathophysiological and ultrastructural changes of RBC in heart failure (HF) patients with hypertension (HT) and long Covid. Design and method: In total 12 patients with HF of Coronary Artery Disease origin, HT, and long Covid were examined. Mean age of patients was 62 ± 5.8 years. The control group consisted of 10 apparently healthy people. The functional state and ultrastructure of RBC were studied using electron microscopy. Results: During ultrastructure examination, structural pathologies of RBC in HF patients with HT and long Covid were revealed. RBC anisocytosis and poikilocytosis as structural damage variations in size and shape were found respectively. Reticulocytes were found much more often in HF patients with HT and long Covid than in the control group. In healthy control group, RBC had a typical discoid shape. In the presence of long Covid, both calcification as a marker of RBC apoptosis and destruction was also detected (Fig.1). Neutrophil extracellular traps (NETs) were found in RBC surrounding (Fig.1). Conclusions: Altered RBC function has important implications for HF patients with HT and long Covid. RBC has been shown to induce endothelial cell dysfunction and to increase cardiac injury as well as increased inflammatory processes in long Covid. The presence of HF, HT and long Covid leads to RBC calcification and activation of blood cell apoptosis. Prognostic role of RBC calcium distribution in combination with other important prognostic measures, such as biomarkers like Thrombospondin - 1, NT-proBNP and ST2 is subject of interest and requires further research.

Detection and identification of coronaviruses in human tissues using electron microscopy.

The identification of viral particles within a tissue specimen requires specific knowledge of viral ultrastructure and replication, as well as a thorough familiarity with normal subcellular organelles. The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic has underscored how challenging the task of identifying coronavirus by electron microscopy (EM) can be. Numerous articles have been published mischaracterizing common subcellular structures, including clathrin- or coatomer- coated vesicles, multivesicular bodies, and rough endoplasmic reticulum, as coronavirus particles in SARS-CoV-2 positive patient tissue specimens. To counter these misinterpretations, we describe the morphological features of coronaviruses that should be used to differentiate coronavirus particles from subcellular structures. Further, as many of the misidentifications of coronavirus particles have stemmed from attempts to attribute tissue damage to direct infection by SARS-CoV-2, we review articles describing ultrastructural changes observed in specimens from SARS-CoV-2-infected individuals that do not necessarily provide EM evidence of direct viral infection. Ultrastructural changes have been observed in respiratory, cardiac, kidney, and intestinal tissues, highlighting the widespread effects that SARS-CoV-2 infection may have on the body, whether through direct viral infection or mediated by SARS-CoV-2 infection-induced inflammatory and immune processes. HIGHLIGHTS: The identification of coronavirus particles in SARS-CoV-2 positive tissues continues to be a challenging task. This review provides examples of coronavirus ultrastructure to aid in the differentiation of the virus from common cellular structures.

SARS-CoV-2 Immunohistochemistry In Placenta.

Compared to the parental SARS-CoV-2 virus, infections by the now dominant Delta variant of SARS-CoV-2 appear to be more common and more severe in pregnant women. The need for a robust, cheap, and quick method for diagnosing placental infection by SARS-CoV-2 has thus become more acute. Here, we describe a highly sensitive and specific immunohistochemical assay for SARS-CoV-2 nucleocapsid protein for routine use in placental pathology practice.

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.

Neuropathological analysis of the brains of fifty-two patients with COVID-19.

Coronavirus disease 2019 (COVID-19) poses a global challenge to healthcare and society in the early 21st century. We report neuropathological changes in 52 patients aged between 22 years and 88 years (median 58 years) who were infected with the CoV-2 coronavirus. Patients died under various circumstances and had various pre-existing diseases. The inclusion criteria for this study were: positive result for the nasopharyngeal swab for SARS-CoV-2 RNA, diagnosis of pneumonia of SARS-CoV-2 or nucleoproteins of SARS-CoV-2 in pulmonary tissue confirmed by immunohistochemical methods (IHC). Samples from all brain structures and lung specimens were taken for histopathological examinations. Brain and pulmonary samples were stained typically with histological and immunohistochemical methods and small tissue fragments were examined with the transmission electron microscope (TEM). The light and electron microscopy examination confirmed the numerous neuropathological changes in the brains of the patients infected with the CoV-2. Many of these changes were caused by pre-existing diseases of patients and/or by necessary treatment. However, vascular lesions and the inflammatory process seem to be characteristic of the CoV-2 infection. In all of the structures of 52 brains of patients, damage of the vessel walls and morphological feature of the damage to the blood-brain barrier were observed. Lymphocytic and microglial infiltrates, both perivascular and diffuse, were also observed. Hence, the brain changes due to COVID-19 infection, could be called COVID-19 cerebral angiopathy with diffuse inflammation.

Cellular tropism of SARS-CoV-2 in the respiratory tract of Syrian hamsters and B6.Cg-Tg(K18-ACE2)2Prlmn/J transgenic mice.

Several animal models have been developed to study the pathophysiology of SARS-CoV-2 infection and to evaluate vaccines and therapeutic agents for this emerging disease. Similar to infection with SARS-CoV-1, infection of Syrian hamsters with SARS-CoV-2 results in moderate respiratory disease involving the airways and lung parenchyma but does not lead to increased mortality. Using a combination of immunohistochemistry and transmission electron microscopy, we showed that the epithelium of the conducting airways of hamsters was the primary target for viral infection within the first 5 days of infection, with little evidence of productive infection of pneumocytes. At 6 days postinfection, antigen was cleared but parenchymal damage persisted, and the major pathological changes resolved by day 14. These findings are similar to those previously reported for hamsters with SARS-CoV-1 infection. In contrast, infection of K18-hACE2 transgenic mice resulted in pneumocyte damage, with viral particles and replication complexes in both type I and type II pneumocytes together with the presence of convoluted or cubic membranes; however, there was no evidence of virus replication in the conducting airways. The Syrian hamster is a useful model for the study of SARS-CoV-2 transmission and vaccination strategies, whereas infection of the K18-hCE2 transgenic mouse results in lethal disease with fatal neuroinvasion but with sparing of conducting airways.

Difficulties in Differentiating Coronaviruses from Subcellular Structures in Human Tissues by Electron Microscopy.

Efforts to combat the coronavirus disease (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have placed a renewed focus on the use of transmission electron microscopy for identifying coronavirus in tissues. In attempts to attribute pathology of COVID-19 patients directly to tissue damage caused by SARS-CoV-2, investigators have inaccurately reported subcellular structures, including coated vesicles, multivesicular bodies, and vesiculating rough endoplasmic reticulum, as coronavirus particles. We describe morphologic features of coronavirus that distinguish it from subcellular structures, including particle size range (60-140 nm), intracellular particle location within membrane-bound vacuoles, and a nucleocapsid appearing in cross section as dense dots (6-12 nm) within the particles. In addition, although the characteristic spikes of coronaviruses may be visible on the virus surface, especially on extracellular particles, they are less evident in thin sections than in negative stain preparations.

Best practices for correctly identifying coronavirus by transmission electron microscopy.

This guidance provides clear, concise strategies for identifying coronaviruses by transmission electron microscopy of ultrathin sections of tissues or infected tissue cultures. These include a description of virus morphology as well as cell organelles that can resemble viruses. Biochemical testing and caveats are discussed. Numerous references provide information for documentation and further study.

Hunting coronavirus by transmission electron microscopy - a guide to SARS-CoV-2-associated ultrastructural pathology in COVID-19 tissues.

Transmission electron microscopy has become a valuable tool to investigate tissues of COVID-19 patients because it allows visualisation of SARS-CoV-2, but the 'virus-like particles' described in several organs have been highly contested. Because most electron microscopists in pathology are not accustomed to analysing viral particles and subcellular structures, our review aims to discuss the ultrastructural changes associated with SARS-CoV-2 infection and COVID-19 with respect to pathology, virology and electron microscopy. Using micrographs from infected cell cultures and autopsy tissues, we show how coronavirus replication affects ultrastructure and put the morphological findings in the context of viral replication, which induces extensive remodelling of the intracellular membrane systems. Virions assemble by budding into the endoplasmic reticulum-Golgi intermediate complex and are characterised by electron-dense dots of cross-sections of the nucleocapsid inside the viral particles. Physiological mimickers such as multivesicular bodies or coated vesicles serve as perfect decoys. Compared to other in-situ techniques, transmission electron microscopy is the only method to visualise assembled virions in tissues, and will be required to prove SARS-CoV-2 replication outside the respiratory tract. In practice, documenting in tissues the characteristic features seen in infected cell cultures seems to be much more difficult than anticipated. In our view, the hunt for coronavirus by transmission electron microscopy is still on.

Ultrastructure of cell trafficking pathways and coronavirus: how to recognise the wolf amongst the sheep.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic has resulted in an urgent need to understand the pathophysiology of SARS-CoV-2 infection, to assist in the identification of treatment strategies. Viral tissue tropism is an active area of investigation, one approach to which is identification of virus within tissues by electron microscopy of post-mortem and surgical specimens. Most diagnostic histopathologists have limited understanding of the ultrastructural features of normal cell trafficking pathways, which can resemble intra- and extracellular coronavirus; in addition, viral replication pathways make use of these trafficking pathways. Herein, we review these pathways and their ultrastructural appearances, with emphasis on structures which may be confused with coronavirus. In particular, we draw attention to the fact that, when using routine fixation and processing, the typical 'crown' that characterises a coronavirus is not readily identified on intracellular virions, which are located in membrane-bound vacuoles. In addition, the viral nucleocapsid is seen as black dots within the virion and is more discriminatory in differentiating virions from other cellular structures. The identification of the viral replication organelle, a collection of membranous structures (convoluted membranes) seen at a relatively low scanning power, may help to draw attention to infected cells, which can be sparse. © 2020 The Authors. The Journal of Pathology published by John Wiley & Sons, Ltd. on behalf of The Pathological Society of Great Britain and Ireland.

What can we learn from a COVID-19 lung biopsy?

We report the case of a patient diagnosed with severe pneumonia due to coronavirus disease 2019 (COVID-19). A percutaneous lung biopsy was performed under ultrasound guidance. Morphological and ultrastructural characteristics of the patient's lungs are presented, along with details of some important changes in inflammatory biological markers, in order to help better understand the disease and provide clues to allow members of the multidisciplinary team to save more people.