Three-dimensional tracking of the ciliate Tetrahymena reveals the mechanism of ciliary stroke-driven helical swimming

Helical swimming in free-space is a common behavior among microorganisms, such as ciliates that are covered with thousands hair-like motile cilia, and is thought to be essential for cells to orient directly to an external stimulus. However, a direct quantification of their three-dimensional (3D) helical trajectories has not been reported, in part due to difficulty in tracking 3D swimming behavior of ciliates, especially Tetrahymena with a small, transparent cell body. Here, we conducted 3D tracking of fluorescent microbeads within a cell to directly visualize the helical swimming exhibited by Tetrahymena. Our technique showed that Tetrahymena swims along a right-handed helical path with right-handed rolling of its cell body. Using the Tetrahymena cell permeabilized with detergent treatment, we also observed that influx of Ca2+ into cilia changed the 3D-trajectory patterns of Tetrahymena swimming, indicating that the beating pattern of cilia is the determining factor in its swimming behavior.

Extracellular Vesicles Analysis in the COVID-19 Era: Insights on Serum Inactivation Protocols towards Downstream Isolation and Analysis

Since the outbreak of the COVID-19 crisis, the handling of biological samples from confirmed or suspected SARS-CoV-2-positive individuals demanded the use of inactivation protocols to ensure laboratory operators’ safety. While not standardized, these practices can be roughly divided into two categories, namely heat inactivation and solvent-detergent treatments. These routine procedures should also apply to samples intended for Extracellular Vesicles (EVs) analysis. Assessing the impact of virus-inactivating pre-treatments is therefore of pivotal importance, given the well-known variability introduced by different pre-analytical steps on downstream EVs isolation and analysis. Arguably, shared guidelines on inactivation protocols tailored to best address EVs-specific requirements will be needed among the analytical community, yet deep investigations in this direction have not yet been reported. We here provide insights into SARS-CoV-2 inactivation practices to be adopted prior to serum EVs analysis by comparing solvent/detergent treatment vs. heat inactivation. Our analysis entails the evaluation of EVs recovery and purity along with biochemical, biophysical and biomolecular profiling by means of a set of complementary analytical techniques: Nanoparticle Tracking Analysis, Western Blotting, Atomic Force Microscopy, miRNA content (digital droplet PCR) and tetraspanin assessment by microarrays. Our data suggest an increase in ultracentrifugation (UC) recovery following heat treatment; however, it is accompanied by a marked enrichment in EVs-associated contaminants. On the other hand, solvent/detergent treatment is promising for small EVs (<150 nm range), yet a depletion of larger vesicular entities was detected. This work represents a first step towards the identification of optimal serum inactivation protocols targeted to EVs analysis.

Extracellular Vesicles Analysis in the COVID-19 Era: Insights on Serum Inactivation Protocols Towards Downstream Isolation and Analysis

Since the outbreak of COVID-19 crisis, the handling of biological samples from confirmed or suspected SARS-CoV-2 positive individuals demanded the use of inactivation protocols to ensure laboratory operators safety. While not standardized, these practices can be roughly divided in two categories, namely heat inactivation and solvent-detergent treatments. As such, these routine procedures should also apply to samples intended for Extracellular Vesicles (EVs) analysis. Assessing the impact of virus inactivating pre-treatments is therefore of pivotal importance, given the well-known variability introduced by different pre-analytical steps on downstream EVs isolation and analysis. Arguably, shared guidelines on inactivation protocols tailored to best address EVs-specific requirements will be needed among the EVs community, yet deep investigations in this direction haven’t been reported so far.In the attempt of sparking interest on this highly relevant topic, we here provide preliminary insights on SARS-CoV-2 inactivation practices to be adopted prior serum EVs analysis by comparing solvent/detergent treatment vs. heat inactivation. Our analysis entailed the evaluation of EVs recovery and purity along with biochemical, biophysical and biomolecular profiling by means of Nanoparticle Tracking Analysis, Western Blotting, Atomic Force Microscopy, miRNA content (digital droplet PCR) and tetraspanin assessment by microarrays. Our data suggest an increase in ultracentrifugation (UC) recovery following heat-treatment, however accompanied by a marked enrichment in EVs-associated contaminants. On the contrary, solvent/detergent treatment is promising for small EVs (< 150 nm range), yet a depletion of larger vesicular entities was detected. This work represents a first step towards the identification of optimal serum inactivation protocols targeted to EVs analysis.

Handwashing and Detergent Treatment Greatly Reduce SARS-CoV-2 Viral Load on Halloween Candy Handled by COVID-19 Patients

ABSTRACT Although SARS-CoV-2 is primarily transmitted by respiratory droplets and aerosols, transmission by fomites remains plausible. During Halloween, a major event for children in numerous countries, SARS-CoV-2 transmission risk via candy fomites worries many parents. To address this concern, we enrolled 10 recently diagnosed asymptomatic or mildly/moderately symptomatic COVID-19 patients to handle typical Halloween candy (pieces individually wrapped) under three conditions: normal handling with unwashed hands, deliberate coughing and extensive touching, and normal handling following handwashing. We then used a factorial design to subject the candies to two posthandling treatments: no washing (untreated) and household dishwashing detergent. We measured SARS-CoV-2 load by reverse transcriptase quantitative PCR (RT-qPCR) and loop-mediated isothermal amplification (LAMP). From the candies not washed posthandling, we detected SARS-CoV-2 on 60% of candies that were deliberately coughed on, 60% of candies normally handled with unwashed hands, but only 10% of candies handled after hand washing. We found that treating candy with dishwashing detergent reduced SARS-CoV-2 load by 62.1% in comparison to untreated candy. Taken together, these results suggest that although the risk of transmission of SARS-CoV-2 by fomites is low even from known COVID-19 patients, viral RNA load can be reduced to near zero by the combination of handwashing by the infected patient and ≥1 min detergent treatment after collection. We also found that the inexpensive and fast LAMP protocol was more than 80% concordant with RT-qPCR. IMPORTANCE The COVID-19 pandemic is leading to important tradeoffs between risk of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) transmission and mental health due to deprivation from normal activities, with these impacts being especially profound in children. Due to the ongoing pandemic, Halloween activities will be curtailed as a result of the concern that candy from strangers might act as fomites. Here, we demonstrate that these risks can be mitigated by ensuring that, prior to handling candy, the candy giver washes their hands and, after receipt, by washing candy with household dishwashing detergent. Even in the most extreme case, with candy deliberately coughed on by known COVID-19 patients, viral load was reduced dramatically after washing with household detergent. We conclude that with reasonable precautions, even if followed only by either the candy giver or the candy recipient, the risk of viral transmission by this route is very low.

Characterization of the Novel Mitochondrial Genome Segregation Factor TAP110 in Trypanosoma brucei

Proper mitochondrial genome inheritance is key for eukaryotic cell survival, however little is known about the molecular mechanism controlling this process. Trypanosoma brucei, a protozoan parasite, contains a singular mitochondrial genome aka kinetoplast DNA (kDNA). kDNA segregation requires anchoring of the genome to the basal body via the tripartite attachment complex (TAC). Several components of the TAC as well as their assembly have been described, it however remains elusive how the TAC connects to the kDNA. Here, we characterize the TAC associated protein TAP110 and for the first time use ultrastructure expansion microscopy in trypanosomes to reveal that TAP110 is the currently most proximal kDNA segregation factor. The kDNA proximal positioning is also supported by RNAi depletion of TAC102, which leads to loss of TAP110 at the TAC. Overexpression of TAP110 leads to expression level changes of several mitochondrial proteins and a delay in the separation of the replicated kDNA networks. In contrast to other kDNA segregation factors TAP110 remains only partially attached to the flagellum after DNAse and detergent treatment and can only be solubilized in dyskinetoplastic cells, suggesting that interaction with the kDNA might be important for stability of the TAC association. Furthermore, we demonstrate that the TAC, but not the kDNA, is required for correct TAP110 localization in vivo and suggest that TAP110 might interact with other proteins to form a >669 kDa complex.Summary StatementTAP110 is a novel mitochondrial genome segregation factor in Trypanosoma brucei that associates with the previously described TAC component TAC102. Ultrastructure expansion microscopy reveals its proximal position to the kDNA.

THE EFFECT OF GIVING DETERGENTS TO THE GROWTH OF Azolla microphylla IN BRACKISH WATER

This research conducted on March to April 2019 with the aim of knowing the effect of detergent on the growth of A. microphylla. The results showed wet weight growth ranging from 2-23.81 grams (control) and 1.43-8.35 grams (addition of detergent). Absolute weight growth ranges from 3.63-8.47 grams and 1.77-3.28 grams. Growth in the number of colonies between 39.66-295 colonies and 37.66-98.33 colonies. Growth doubling time day 3-23 and 3.63-7.66 days. The growth of wet weight and the number of colonies of A. microphyla increased in each data collection on the control treatment media, while the addition of detergent treatment increased on the day 5 and day 10 and on the day 15 and day 20 decreased.

Temporary Anosmia in Mice Following Nasal Lavage With Dilute Detergent Solution

Olfactory sensory deprivation induces anosmia and reduces tyrosine hydroxylase and dopamine levels in the olfactory bulb. The behavioral consequences specific to the loss of olfactory bulb dopamine are difficult to determine because sensory deprivation protocols are either confounded by side effects or leave the animal anosmic. A new method to both induce sensory deprivation and to measure the behavioral and circuit consequences is needed. We developed a novel, recoverable anosmia protocol using nasal lavage with a dilute detergent solution. Detergent treatment did not damage the olfactory epithelium as measured by scanning electron microscopy, alcian blue histology, and acetylated tubulin immunohistochemistry. One treatment-induced anosmia that lasted 24 to 48 h. Three treatments over 5 days reduced olfactory bulb tyrosine hydroxylase and dopamine levels indicating that anosmia persists between treatments. Importantly, even with multiple treatments, olfactory ability recovered within 48 h. This is the first report of a sensory deprivation protocol that induces recoverable anosmia and can be paired with biochemical, histological, and behavioral investigations of olfaction.

Functional Correlation between Subcellular Localizations of Japanese Encephalitis Virus Capsid Protein and Virus Production

ABSTRACT The flavivirus capsid protein is considered to be essential for the formation of nucleocapsid complexes with viral genomic RNA at the viral replication organelle that appears on the endoplasmic reticulum (ER), as well as for incorporation into virus particles. However, this protein is also detected at the lipid droplet (LD) and nucleolus, and physiological roles of these off-site localizations are still unclear. In this study, we made a series of alanine substitution mutants of Japanese encephalitis virus (JEV) capsid protein that cover all polar and hydrophobic amino acid residues to identify the molecular surfaces required for virus particle formation and for localization at the LD and nucleolus. Five mutants exhibited a defect in the formation of infectious particles, and two of these mutants failed to be incorporated into the subviral particles (SVP). Three mutants lost the ability to localize to the nucleolus, and only a single mutant, with mutations at α2, was unable to localize to the LD. Unlike the cytoplasmic capsid protein, the nucleolar capsid protein was resistant to detergent treatment, and the α2 mutant was hypersensitive to detergent treatment. To scrutinize the relationship between these localizations and viral particle formation, we made eight additional alanine substitution mutants and found that all the mutants that did not localize at the LD or nucleolus failed to form normal viral particles. These results support the functional correlation between LD or nucleolus localization of the flaviviral capsid protein and the formation of infectious viral particles. IMPORTANCE This study is the first to report the comprehensive mutagenesis of a flavivirus capsid protein. We assessed the requirement of each molecular surface for infectious viral particle formation as well as for LD and nucleolar localization and found functional relationships between the subcellular localization of the virus capsid protein and infectious virus particle formation. We developed a system to independently assess the packaging of viral RNA and that of the capsid protein and found a molecular surface of the capsid protein that is crucial for packaging of viral RNA but not for packaging of the capsid protein itself. We also characterized the biochemical properties of capsid protein mutants and found that the capsid protein localizes at the nucleolus in a different manner than for its localization to the LD. Our comprehensive alanine-scanning mutagenesis study will aid in the development of antiflavivirus small molecules that can target the flavivirus capsid protein.

Assessment of cytocompatibility and mechanical properties of detergent-decellularized ovine pericardial tissue

Background: Autologous pericardium is widely used for the repair of different sized cardiovascular defects. However, its use is limited especially in redo cardiac surgery. We developed an engineered tissue based on decellularized pericardium reseeded with blood-derived endothelial cells. Materials and Methods: Decellularization of ovine pericardium was performed using detergent treatment. Ovine outgrowth blood-derived and green fluorescent protein–labeled endothelial cells were used to reseed the decellularized ovine pericardium on the mesothelial side. The cell adhesion was assessed using fluorescent microscopy up to 15 days of in vitro cultivation. The mechanical properties of the pericardium were evaluated using suturability, burst pressure, and suture retention strength tests. Results: After decellularization the pericardial sheets appeared cell-free and repopulation using ovine blood-derived endothelial cells was successful by forming a robust monolayer. Detergent treatment did not affect the extracellular matrix. The thickness of decellularized tissue was similar to native ovine pericardium (285.3 ± 28.2 µm, respective 276.9 ± 23.8 µm, p = 0.48). Decellularized patch showed similar suturability comparable to the native ovine pericardium. Resulted burst pressure was not significantly different (native/decellularized: 312.5 ± 13.6/304.2 ± 16, p = 0.35). The suture retention strength of native pericardium was 638.33 ± 90.2 gr and comparable to decellularized tissue (622.2 ± 89.9 gr, p = 0.76). No differences were observed concerning elongation of native and decellularized pericardium (8.33 ± 1.5 and 8.5 ± 0.84 mm, respectively; p = 0.82). Conclusion: Mesothelial surface of decellularized ovine pericardium is suitable for reseeding with ovine blood-derived endothelial cells. The mechanical properties of detergent-treated pericardium were comparable to native tissue.