Intracellular Particles Research Articles

Novel Bioengineered Antibacterial and Anticancer ZnO Nanoparticles.

Transition metal oxide NPs have delivered wide applications in various fields. Therefore, in this study, a novel fungus, Alternaria sp. (NCBI Accession No: MT982648) was isolated and characterized from the vicinity of medicinal plants. Eventually, in this method extracted proteins from isolated fungus were utilized to synthesize highly biocompatible zinc nanoparticles (ZnO NPs). The various physical techniques including UV-visible spectroscopy, TEM, HR-TEM, XRD, DLS, zeta potential, and FTIR were used to characterize particles. The UV-visible absorption (λMax) and binding energy for the as-synthesized particles were found to be 329 nm and 3.91 eV, respectively. Further, the polydispersed particles were revealed to have regular crystallinity with hexagonal wurtzite phase of ZnO with the spacing of ~2.46 Å under XRD and HR-TEM. The average size of a particle under TEM was found to be ~18 nm. The evaluation of various surface functional groups of particles was done by FTIR. The average hydrodynamic diameter of particles was found to be ~57 d. nm with 0.44 particle distribution index whereas the nanoemulsion stability was explained by Zeta potential (-9.47 mV). These particles were found to exhibit potential antibacterial and anticancer activities. They were found to be bactericidal against S. abony (MIC 5.73 μg/mL); B. pumilis (MIC 6.64 μg/mL); K. pneumonia (MIC 14.4 μg/mL); E. coli (MIC 8.7 μg/mL); B. subtilis (MIC 5.63 μg/mL) and S. aureus (MIC 12.04 μg/mL). Further, they are also found to be concentration-dependent anticancer and inhibited the growth of A549 cells (IC50-65.3 μg/mL) whereas they were found to demonstrate no any cytotoxicity against NRK normal kidney cell line. The internalization of particles into the nucleus (i.e., nuclear fragmentation and DNA damage) was confirmed by DAPI staining. The intracellular particles were found to generate excessive ROS. Further, the anticancer potential was also estimated by noticing a hike in oxidative stress parameters, cell viability, cell morphology, and change in mitochondrial membrane potential. We effectively synthesized potentially potent antibacterial and anticancer novel bioengineered ZnO NPs.

Modeling intracellular transport and traffic jam in 3D neurons using PDE-constrained optimization

Abstract The intracellular transport process plays an important role in delivering essential materials throughout branched geometries of neurons for their survival and function. Many neurodegenerative diseases have been associated with the disruption of transport. Therefore, it is essential to study how neurons control the transport process to localize materials to necessary locations. Here, we develop a novel optimization model to simulate the traffic regulation mechanism of material transport in three-dimensional complex geometries of neurons. The transport is controlled to avoid traffic jams of materials by minimizing a predefined objective function. The optimization subjects to a set of partial differential equation (PDE) constraints that describe the material transport process based on a macroscopic molecular-motor-assisted transport model of intracellular particles. The proposed PDE-constrained optimization model is solved in complex tree structures by using the isogeometric analysis. Different simulation parameters are used to introduce traffic jams and study how neurons handle the transport issue. Specifically, we successfully model and explain the traffic jam caused by the reduced number of microtubules (MTs) and MT swirls. In summary, our model effectively simulates the material transport process in healthy neurons and also explains the formation of a traffic jam in abnormal neurons. Our results demonstrate that both geometry and MT structure play important roles in achieving an optimal transport process in neurons.

Modeling material transport regulation and traffic jam in neurons using PDE-constrained optimization

The intracellular transport process plays an important role in delivering essential materials throughout branched geometries of neurons for their survival and function. Many neurodegenerative diseases have been associated with the disruption of transport. Therefore, it is essential to study how neurons control the transport process to localize materials to necessary locations. Here, we develop a novel optimization model to simulate the traffic regulation mechanism of material transport in complex geometries of neurons. The transport is controlled to avoid traffic jam of materials by minimizing a pre-defined objective function. The optimization subjects to a set of partial differential equation (PDE) constraints that describe the material transport process based on a macroscopic molecular-motor-assisted transport model of intracellular particles. The proposed PDE-constrained optimization model is solved in complex tree structures by using isogeometric analysis (IGA). Different simulation parameters are used to introduce traffic jams and study how neurons handle the transport issue. Specifically, we successfully model and explain the traffic jam caused by reduced number of microtubules (MTs) and MT swirls. In summary, our model effectively simulates the material transport process in healthy neurons and also explains the formation of a traffic jam in abnormal neurons. Our results demonstrate that both geometry and MT structure play important roles in achieving an optimal transport process in neuron.

Faster Crystallization during Coral Skeleton Formation Correlates with Resilience to Ocean Acidification.

The mature skeletons of hard corals, termed stony or scleractinian corals, are made of aragonite (CaCO3). During their formation, particles attaching to the skeleton’s growing surface are calcium carbonate, transiently amorphous. Here we show that amorphous particles are observed frequently and reproducibly just outside the skeleton, where a calicoblastic cell layer envelops and deposits the forming skeleton. The observation of particles in these locations, therefore, is consistent with nucleation and growth of particles in intracellular vesicles. The observed extraskeletal particles range in size between 0.2 and 1.0 μm and contain more of the amorphous precursor phases than the skeleton surface or bulk, where they gradually crystallize to aragonite. This observation was repeated in three diverse genera of corals, Acropora sp., Stylophora pistillata—differently sensitive to ocean acidification (OA)—and Turbinaria peltata, demonstrating that intracellular particles are a major source of material during the additive manufacturing of coral skeletons. Thus, particles are formed away from seawater, in a presumed intracellular calcifying fluid (ICF) in closed vesicles and not, as previously assumed, in the extracellular calcifying fluid (ECF), which, unlike ICF, is partly open to seawater. After particle attachment, the growing skeleton surface remains exposed to ECF, and, remarkably, its crystallization rate varies significantly across genera. The skeleton surface layers containing amorphous pixels vary in thickness across genera: ∼2.1 μm in Acropora, 1.1 μm in Stylophora, and 0.9 μm in Turbinaria. Thus, the slow-crystallizing Acropora skeleton surface remains amorphous and soluble longer, including overnight, when the pH in the ECF drops. Increased skeleton surface solubility is consistent with Acropora’s vulnerability to OA, whereas the Stylophora skeleton surface layer crystallizes faster, consistent with Stylophora’s resilience to OA. Turbinaria, whose response to OA has not yet been tested, is expected to be even more resilient than Stylophora, based on the present data.

PM1-loaded recombinant human H-ferritin nanocages: A novel pH-responsive sensing platform for the identification of cancer cells

The aggregation-induced emission (AIE) material has been widely used in biological detection due to their unique property of fluorescing in aggregation state. However, the poor dispersion and biocompatibility limit its application in in vivo real-time imaging. Here, a novel strategy is designed to obtain pH-responsive AIE nanomaterials, working through 4-Undecoxy Tetraphenyl Ethylene Methacrylate (PM1) block, with excellent features (dispersion, biocompatibility, self-reconstruction and cancer specific recognition). The recombinant human H-ferritin (rHuHF) was used to prepare rHuHF-PM1 nanocomposites which effectively supported the dispersion and transfer of PM1 in the biological environment, even making it target tumor cells due to the overexpression of ferritin receptors on tumor cells. To simulate the changes of rHuHF in intracellular lysosomes, particle size and fluorescence of rHuHF-PM1 were analyzed, which reflected the loose structural changes of rHuHF nanocages in weak acid system that facilitated the degradation of macromolecular rHuHF in intracellular lysosomes and following release of PM1. The released PM1 molecules aggregated and emitted brilliant blue fluorescence. Several cell lines, Hela, HT-29, HepG2, L-O2 and HUVEC have all been sensitively detected and distinguished. Accordingly, this nanocage has a potential to be applied to disease diagnosis and provides a novel sensing platform for the identification of cancer.

Application of three-dimensional Raman imaging to determination of the relationship between cellular localization of diesel exhaust particles and the toxicity

A diesel exhaust particle (DEP) is a type of particulate matter that is easily produced from combustion in a diesel power engine. It has been reported that DEPs can cause short- and long-term health problems. This is because DEPs are complex mixtures that are highly inhalable through the airways due to their small particle size. However, the relationship between intracellular localization of DEPs after their deposition in the lungs and the subsequent biological responses remains to be clarified. This is due to difficulties in distinguishing particles that are inside the cells from those that are outside. In this study, A549 human lung epithelial cells were exposed to DEPs at concentrations of 0, 25, 75, or 200 µg/mL for different periods, after that particles in the A549 cells were analyzed by three-dimensional (3D) images obtained from a Raman microscope. The cytotoxic effects of DEPs on the A549 cells were investigated by measuring cell viability, the levels of intracellular reactive oxygen species (ROS) and cell death. The Raman microscopy revealed that the particles invaded the A549 cells, and at a concentration of 200 µg/mL, they markedly decreased cell viability, increased intracellular ROS production, triggered late apoptosis/necrosis and induced nuclear damage. These results suggest that intracellular DEPs exposed at a high concentration may be highly toxic and can impair the viability of A549 cells. Furthermore, the 3D images from the Raman microscopy can be used to evaluate intracellular particle dynamics.

Vertical Transmission of Diverse Cultivation-Recalcitrant Endophytic Bacteria Elucidated Using Watermelon Seed Embryos.

Seed transmission of endophytic microorganisms is a growing research area in plant biology and microbiology. We employed cultivation versus cultivation-independent approaches on excised embryos from watermelon seeds (6–12 months in storage) and on embryo-derived in vitro seedlings (EIVS) to assess the vertical transmission of endophytic bacteria. Surface-disinfected watermelon seeds bore abundant residual bacteria in the testa and perisperm tissues, predominantly Bacillus spp. propounding the essentiality of excluding all non-embryonic tissues for vertical transmission studies. Tissue homogenates from re-disinfected seed embryos displayed no cultivable bacteria during the 1-week monitoring. Bright-field live microscopy revealed abundant bacteria in tissue homogenates and in embryo sections as intracellular motile particles. Confocal imaging on embryo sections after SYTO-9 staining and eubacterial fluorescent in situ hybridization (FISH) endorsed enormous bacterial colonization. Quantitative Insights Into Microbial Ecology (QIIME)-based 16S rRNA V3–V4 taxonomic profiling excluding the preponderant chloroplast and mitochondrial sequences revealed a high bacterial diversity in watermelon seed embryos mainly Firmicutes barring spore formers followed by Proteobacteria, Bacteroidetes, and Actinobacteria, and other minor phyla. Embryo-base (comprising the radicle plus plumule parts) and embryo-cotyledon parts differed in bacterial profiles with the abundance of Firmicutes in the former and Proteobacteria dominance in the latter. EIVS displayed a higher bacterial diversity over seed embryos indicating the activation from the dormant stage of more organisms in seedlings or their better amenability to DNA techniques. It also indicated embryo-to-seedling bacterial transmission, varying taxonomic abundances for seed embryos and seedlings, and differing phylogenic profiles for root, hypocotyl, and cotyledon/shoot-tip tissues. Investigations on different watermelon cultivars confirmed the embryo transmission of diverse cultivation recalcitrant endophytic bacteria. Firmicutes, Proteobacteria, Actinobacteria, and Bacteroidetes formed the core phyla across different cultivars with 80–90% similarity at genus to phylum levels. Conversely, freshly harvested seeds displayed a dominance of Proteobacteria. The findings revealed that dicot seeds such as in different watermelon cultivars come packaged with abundant and diverse vertical and seedling-transmissible cultivation recalcitrant endophytic bacteria with significant implications for plant biology.

How the spleen reshapes and retains young and old red blood cells: A computational investigation.

The spleen, the largest secondary lymphoid organ in humans, not only fulfils a broad range of immune functions, but also plays an important role in red blood cell’s (RBC) life cycle. Although much progress has been made to elucidate the critical biological processes involved in the maturation of young RBCs (reticulocytes) as well as removal of senescent RBCs in the spleen, the underlying mechanisms driving these processes are still obscure. Herein, we perform a computational study to simulate the passage of RBCs through interendothelial slits (IES) in the spleen at different stages of their lifespan and investigate the role of the spleen in facilitating the maturation of reticulocytes and in clearing the senescent RBCs. Our simulations reveal that at the beginning of the RBC life cycle, intracellular non-deformable particles in reticulocytes can be biomechanically expelled from the cell upon passage through IES, an insightful explanation of why this peculiar “pitting” process is spleen-specific. Our results also show that immature RBCs shed surface area by releasing vesicles after crossing IES and progressively acquire the biconcave shape of mature RBCs. These findings likely explain why RBCs from splenectomized patients are significantly larger than those from nonsplenectomized subjects. Finally, we show that at the end of their life span, senescent RBCs are not only retained by IES due to reduced deformability but also become susceptible to mechanical lysis under shear stress. This finding supports the recent hypothesis that transformation into a hemolyzed ghost is a prerequisite for phagocytosis of senescent RBCs. Altogether, our computational investigation illustrates critical biological processes in the spleen that cannot be observed in vivo or in vitro and offer insights into the role of the spleen in the RBC physiology.

Data-Driven Modeling of the Cellular Pharmacokinetics of Degradable Chitosan-Based Nanoparticles.

Nanoparticle drug delivery vehicles introduce multiple pharmacokinetic processes, with the delivery, accumulation, and stability of the therapeutic molecule influenced by nanoscale processes. Therefore, considering the complexity of the multiple interactions, the use of data-driven models has critical importance in understanding the interplay between controlling processes. We demonstrate data simulation techniques to reproduce the time-dependent dose of trimethyl chitosan nanoparticles in an ND7/23 neuronal cell line, used as an in vitro model of native peripheral sensory neurons. Derived analytical expressions of the mean dose per cell accurately capture the pharmacokinetics by including a declining delivery rate and an intracellular particle degradation process. Comparison with experiment indicates a supply time constant, τ = 2 h. and a degradation rate constant, b = 0.71 h−1. Modeling the dose heterogeneity uses simulated data distributions, with time dependence incorporated by transforming data-bin values. The simulations mimic the dynamic nature of cell-to-cell dose variation and explain the observed trend of increasing numbers of high-dose cells at early time points, followed by a shift in distribution peak to lower dose between 4 to 8 h and a static dose profile beyond 8 h.

Fabrication of iron nanoparticles using Leptolyngbya valderiana and investigation of its Cr (VI) removal potential in the free and biomass associated forms

This study reported the ability of L. valderiana to fabricate iron nano particles of definite shape and size, as well as the enhanced effectiveness of the synthesized nanoparticle loaded biomass in removal of hexavalent chromium [Cr (VI)] from aqueous solution compared to free iron nano particles. Spindle-shaped nano scale zerovalent irons were synthesized by live biomass at intracellular and extracellular levels with the dimensions of 47.42 ± 3.86 × 7.7 ± 2.01 nm and 66.37 ± 3.09 × 12.49 ± 4.01 nm, respectively. The nanosorbents were prepared separately from intracellular particles (NI), extracellular particles (NE) and nano‑iron loaded biomass (BN). All the adsorbents were tested for chromium removal from Cr (VI) aqueous solutions with concentrations in the range of 5–20 mg L−1. The adsorption parameters were optimized. The removal rate and adsorption capacity, i.e., Langmuir constant (KL) of nano‑iron loaded biomass (BN) toward Cr (VI) reached the peak at 0.0439 L mg−1 of adsorbent, followed by intracellular particles (NI) (0.0162 L mg−1 of adsorbent) and extracellular particles (NE) (0.009 L mg−1 of adsorbent). The adsorption kinetics data of nano‑iron loaded biomass (BN) fitted well with the pseudo-second-order kinetic model and Freundlich isotherm model whereas, the other two nanosorbent-assisted removal processes followed the pseudo-first-order kinetic model and Langmuir isotherm model. Moreover, nano‑iron loaded biomass (BN) was the easiest to handle and showed maximum adsorption capacity; therefore, portraying it as a low-cost, environmentally benign potential nanosorbent for the removal of Cr (VI) from industrial waste.

Dynamic enzymatic synthesis of γ-cyclodextrin using a photoremovable hydrazone template

Dynamic enzymatic synthesis of γ-cyclodextrin using a photoremovable hydrazone template

Spectral Analysis of ATP-Dependent Mechanical Vibrations in T Cells.

Mechanical vibrations affect multiple cell properties, including its diffusivity, entropy, internal content organization, and thus—function. Here, we used Differential Interference Contrast (DIC), confocal, and Total Internal Reflection Fluorescence (TIRF) microscopies to study mechanical vibrations in live (Jurkat) T cells. Vibrations were measured via the motion of intracellular particles and plasma membrane. These vibrations depend on adenosine triphosphate (ATP) consumption and on Myosin II activity. We then used spectral analysis of these vibrations to distinguish the effects of thermal agitation, ATP-dependent mechanical work and cytoskeletal visco-elasticity. Parameters of spectral analyses could be related to mean square displacement (MSD) analyses with specific advantages in characterizing intracellular mechanical work. We identified two spectral ranges where mechanical work dominated vibrations of intracellular components: 0–3 Hz for intracellular particles and the plasma-membrane, and 100–150 Hz for the plasma-membrane. The 0–3 Hz vibrations of the cell membrane that we measured in an experimental model of immune synapse (IS) are expected to affect the IS formation and function in effector cells. It may also facilitate immunological escape of extensively vibrating malignant cells.

Seasonal Variation in Viral Infection Rates and Cell Sizes of Infected Prokaryotes in a Large and Deep Freshwater Lake (Lake Biwa, Japan).

As viruses regulate prokaryotic abundance and the carbon cycle by infecting and lysing their prokaryotic hosts, the volume of infected prokaryotes is an important parameter for understanding the impact of viruses on aquatic environments. However, literature regarding the seasonal and spatial variations in the cell volume of infected prokaryotes is limited, despite the volume of the prokaryotic community varying dynamically with season and water column depth. Here, we conducted a field survey for two annual cycles in a large and deep freshwater lake (Lake Biwa, Japan), where large prokaryotes inhabit the deeper layer during the stratified period. We used transmission electron microscopy to reveal the seasonal and spatial variation in the frequency of viral infection and cell volume of infected prokaryotes. We found that the viral infection rate in the surface layer increased when estimated contact rates increased during the middle of the stratified period, whereas the infection rate in the deeper layer increased despite low estimated contact rates during the end of the stratified period. In addition, in the deeper layer, the fraction of large prokaryotes in the total and infected prokaryotic communities increased progressively while the number of intracellular viral particles increased. We suggest different ways in which the viral abundance is maintained in the two water layers. In the surface layer, it is speculated that viral abundance is supported by the high viral infection rate because of the high activity of prokaryotes, whereas in the deeper layer, it might be supported by the larger number of intracellular viral particles released from large prokaryotes. Moreover, large prokaryotes could contribute as important sources of organic substrates via viral lysis in the deeper layer, where labile dissolved organic matter is depleted.

Impact of Differentiated Macrophage-Like Cells on the Transcriptional Toxicity Profile of CuO Nanoparticles in Co-Cultured Lung Epithelial Cells.

To mimic more realistic lung tissue conditions, co-cultures of epithelial and immune cells are one comparatively easy-to-use option. To reveal the impact of immune cells on the mode of action (MoA) of CuO nanoparticles (NP) on epithelial cells, A549 cells as a model for epithelial cells have been cultured with or without differentiated THP-1 cells, as a model for macrophages. After 24 h of submerged incubation, cytotoxicity and transcriptional toxicity profiles were obtained and compared between the cell culture systems. Dose-dependent cytotoxicity was apparent starting from 8.0 µg/cm2 CuO NP. With regard to gene expression profiles, no differences between the cell models were observed concerning metal homeostasis, oxidative stress, and DNA damage, confirming the known MoA of CuO NP, i.e., endocytotic particle uptake, intracellular particle dissolution within lysosomes with subsequent metal ion deliberation, increased oxidative stress, and genotoxicity. However, applying a co-culture of epithelial and macrophage-like cells, CuO NP additionally provoked a pro-inflammatory response involving NLRP3 inflammasome and pro-inflammatory transcription factor activation. This study demonstrates that the application of this easy-to-use advanced in vitro model is able to extend the detection of cellular effects provoked by nanomaterials by an immunological response and emphasizes the use of such models to address a more comprehensive MoA.

Visualizing Diffusional Dynamics of Gold Nanorods on Cell Membrane using Single Nanoparticle Darkfield Microscopy.

Analyzing the diffusional dynamics of nanoparticles on cell membrane plays a significant role in better understanding the cellular uptake process and provides a theoretical basis for the rational design of nano-medicine delivery. Single particle tracking (SPT) analysis could probe the position and orientation of individual nanoparticles on cell membrane, and reveal their translational and rotational states. Here, we show how to use traditional dark-field microscopy to monitor the dynamics of gold nanorods (AuNRs) on live cell membrane. We also show how to extract the location and orientation of AuNRs using ImageJ and MATLAB, and how to characterize the diffusive states of AuNRs. Statistical analysis of hundreds of particles show that single AuNRs perform Brownian motion on the surface of U87 MG cell membrane. However, individual long trajectory analysis shows that AuNRs have two distinctly different types of motion states on the membrane, namely long-range transport and limited-area confinement. Our SPT methods can be potentially used to study the surface or intracellular particle diffusion in different biological cells and can become a powerful tool for investigations of complex cellular mechanisms.

Role of Damage-Associated Molecular Patterns in Septic Acute Kidney Injury, From Injury to Recovery.

Damage-associated molecular patterns (DAMPs) are a group of immunostimulatory molecules, which take part in inflammatory response after tissue injury. Kidney-specific DAMPs include Tamm-Horsfall glycoprotein, crystals, and uromodulin, released by tubular damage for example. Non-kidney-specific DAMPs include intracellular particles such as nucleus [histones, high-mobility group box 1 protein (HMGB1)] and cytosol parts. DAMPs trigger innate immunity by activating the NRLP3 inflammasome, G-protein coupled class receptors or the Toll-like receptor. Tubular necrosis leads to acute kidney injury (AKI) in either septic, ischemic or toxic conditions. Tubular necrosis releases DAMPs such as histones and HMGB1 and increases vascular permeability, which perpetuates shock and hypoperfusion via Toll Like Receptors. In acute tubular necrosis, intracellular abundance of NADPH may explain a chain reaction where necrosis spreads from cell to cell. The nature AKI in intensive care units does not have preclinical models that meet a variation of blood perfusion or a variation of glomerular filtration within hours before catecholamine infusion. However, the dampening of several DAMPs in AKI could provide organ protection. Research should be focused on the numerous pathophysiological pathways to identify the relative contribution to renal dysfunction. The therapeutic perspectives could be strategies to suppress side effect of DAMPs and to promote renal function regeneration.

Severe Acute Respiratory Syndrome Coronavirus 2, Olfactory Mucosa, and the Entrance in the Central Nervous System: Expanding the Evidence?

Meinhardt, J, Radke, J, Dittmayer, C, et al. Olfactory transmucosal SARS-CoV-2 invasion as a port of central nervous system entry in individuals with COVID-19. Nat Neurosci 2021; 24( 2): 168– 175. https://doi.org/10.1038/s41593-020-00758-5. The world faces a unique and historic challenge as a result of the pandemic associated with the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. Beyond the respiratory compromise, SARS-CoV-2 infection produces a myriad of neurological manifestations that have been progressively characterized.1 These neurological manifestations can potentially be (1) a consequence of the pulmonary and systemic disease, (2) attributed to a postinfectious (immune-mediated) mechanism, or (3) related to the direct viral central nervous system (CNS) invasion (either via trans-synaptic spreading or through the disruption of the blood–brain barrier). The lack of detection of SARS-CoV-2 in the cerebrospinal fluid in most patients with coronavirus disease 2019 (COVID-19)–related encephalitis, despite evidence of brain inflammation, suggests an immune-mediated mechanism rather than a direct viral invasion.1, 2 The neurotropism of coronaviruses is known and the recent detection of viral RNA in the brains of a subset of patients with COVID-19 led to the hypothesis of SARS-CoV-2 to reach the CNS. Because of the high number of patients with COVID-19 experiencing hyposmia, the trans-synaptic olfactory pathway has been pointed to as the route for entry into the CNS.3 A ramification of this theory affecting us, movement disorders experts, is the hypothetical link between SARS-CoV-2 infection and Parkinson's disease development.4 The featured article by Meinhardt and colleagues5 represents the most extensive postmortem study published so far. The authors assessed the olfactory mucosa and several defined CNS regions to address the important question of whether the olfactory mucosa acts as an entrance for SARS-CoV-2 into the CNS.5 The rather comprehensive evaluation of the tissue samples from 33 patients who died in the context of severe COVID-19 infection included detailed histological and genetic assessments. As a first step, the authors mapped the regional presence of SARS-CoV-2 RNA by real-time quantitative polymerase chain reaction and subgenomic RNA. Interestingly, the olfactory mucosa revealed the highest viral load compared with other tissue samples such as the oral mucosa or trigeminal ganglion. Only active viral replication was identified in the olfactory and oral mucosa. In additional analyses, the SARS-CoV-2 RNA in situ hybridization technique showed intense signaling in the mucous layer and cells of the olfactory epithelium, whereas it was absent in the olfactory sensory neurons. In line with this result, the ultrastructural imaging using electron microscopy showed both extracellular and intracellular viral particles within different cellular compartments of the olfactory epithelium. To further characterize which cells within the olfactory mucosa were invaded by the virus, they performed immunofluorescence colocalization studies. Remarkably, in 3 of 33 patients, the authors found colocalization of the SARS-CoV-2 spike protein with Olig2, an oligodendrocyte marker. This finding provided evidence of the presence of the viral protein in the olfactory sensory neurons. Finally, SARS-CoV-2 was not found in the neuronal or glial cells of the CNS parenchyma of the studied patients. Overall, these data suggested that in the absence of angiotensin-converting enzyme 2 receptors, such as in the olfactory neuronal cells, SARS-CoV-2 infection could be mediated through a transcytosis mechanism: from the olfactory mucosa cells to the olfactory neuronal cells. However, it cannot be discarded that other receptors may facilitate cell entry and infectivity.6 Furthermore, the virus could have been transported through the blood vessels into the brain, as the virus was also found in blood vessel endothelium. The extent to which the virus might have spread into the CNS in those 3 cases remains speculative, as neither viral RNA within the brain parenchyma or neuron-to-neuron propagation were shown. In summary, these results provide evidence that SARS-CoV-2 neuroinvasion can occur at the olfactory neural–mucosal interface. This critical aspect may help to characterize further the extension and mechanisms underlying the entry and action of SARS-CoV-2 in the brain. Broader CNS sampling studies also elucidating the molecular details of SARS-CoV-2 infection and its repercussions in the human brain are needed. Ethical Compliance Statement: Informed patient consent was not necessary for this work. The approval of an institutional review board was not required. We confirm that we have read the Journal's position on issues involved in ethical publication and affirm that this work is consistent with those guidelines. Funding Sources and Conflicts of Interest: No specific funding was received for this work. There are no conflicts of interest relevant to this work. Financial Disclosures for the Previous 12 Months: M.H.G.M. is an employee of the Department of Neurology, Northwestern University Feinberg School of Medicine and has received honoraria from the Autonomous University of Madrid and HM-Puerta del Sur University Hospital and speaker honoraria from Novartis Pharmaceutical.

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.

Exosome as potential biomarkers for gastrointestinal tumors.

Introduction:Exosomes are polyvesicles that are formed by invagination of intracellular lysosomal particles, and are released into the extracellular matrix after the fusion of polyvesicular outer membrane and cell membrane. In the body, immune response, antigen presentation, cell migration, cell differentiation and tumor invasion are closely related to tumorigenesis and tumor progression. This study aimed to conduct a meta-analysis for evaluating the clinicopathological, diagnostic and prognostic significance of exosomal expression in gastrointestinal tumors.Methods:The original English articles were systematically searched in the online databases. The diagnostic accuracy, prognostic utility and clinicopathological correlation of gastrointestinal tumors were investigated. The quality assessment for studies of diagnostic accuracy II and Newcastle-Ottawa scale were used for quality evaluation, and the data was strictly extracted to judge the deviation of the study.Results:A total of 14 studies with 1837 gastrointestinal tumor patients were included. The change in exosomal expression showed significant correlation with poor clinicopathological parameters (tumor diameter: combined P = .00024394; differentiation: combined P = 2.796e-08; lymphatic metastasis: P = 9.610e-07; distant metastasis: combined P = .00017326; pathological classification: combined P = .00875213; invasion depth: combined P = 3.504e-08) carcinoembryonic antigen (combined P = . 04458857) and tumor location (combined P = .00145983). The difference in the area under the curve between gastrointestinal tumor patients and healthy people showed an area under the curve of 0.89 (95%Cl 0.85–0.91) and heterogeneity of 0.59, 95% CI=[0.55–0.68]. The sensitivity was 0.88 (95%Cl 0.83 mi 0.91), the specificity was 0.72 (95%Cl 0.63 mi 0.80), and the diagnostic odds ratio was 18 (10–33). The results of survival analysis revealed that the abnormally expressed exosomes were significantly correlated with poor overall survival (hazard ratio =2.81, 95% CI: 2.02–3.93, P=0.013∗ 62.7%∗).Conclusion:The abnormally expressed exosomes might act as auxiliary biomarkers in diagnosing gastrointestinal tumors and demonstrated good prognostic significance in predicting the survival of patients with gastrointestinal tumors.

Quantifying Intracellular Particle Flows by DIC Object Tracking

Label-free imaging techniques such as differential interference contrast (DIC) allow the observation of cells and large subcellular structures in their native, unperturbed states with minimal exposure to light. The development of robust computational image-analysis routines is vital to quantitative label-free imaging. The reliability of quantitative analysis of time-series microscopy data based on single-particle tracking relies on accurately detecting objects as distinct from the background, i.e., segmentation. Typical approaches to segmenting DIC images either involve converting images to those resembling phase contrast, mimicking the optics of DIC object formation, or using the morphological properties of objects. Here, we describe MATLAB based, single-particle tracking tool with a GUI for mobility analysis of objects from in vitro and in vivo DIC time-series microscopy. The tool integrates contrast enhancement with multiple modified Gaussian filters, automated threshold detection for segmentation and minimal distance-based two-dimensional single-particle tracking. We compare the relative performance of multiple filters and demonstrate the utility of the tool for DIC object tracking (DICOT). We quantify subcellular dynamics of a time series of Caenorhabditis elegans embryos in the one-celled stage by detecting birefringent yolk granules in the cytoplasm with high precision. The resulting two-dimensional map of oscillatory dynamics of granules quantifies the cytoplasmic flows driven by anaphasic spindle oscillations. The frequency of oscillations across the anterior-posterior (A-P) and transverse axes of the embryo correspond well with the reported frequency of spindle oscillations. We validate the quantitative accuracy of our method by tracking the in vitro diffusive mobility of micron-sized beads in glycerol solutions. Estimates of the diffusion coefficients of the granules are used to measure the viscosity of a dilution series of glycerol. Thus, our computational method is likely to be useful for both intracellular mobility and in vitro microrheology.