SARS-CoV-2 viral budding and entry can be modeled using BSL-2 level virus-like particles.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was first discovered in December 2019 in Wuhan, China, and expeditiously spread across the globe causing a global pandemic. Research on SARS-CoV-2, as well as the closely related SARS-CoV-1 and MERS coronaviruses, is restricted to BSL-3 facilities. Such BSL-3 classification makes SARS-CoV-2 research inaccessible to the majority of functioning research laboratories in the United States; this becomes problematic when the collective scientific effort needs to be focused on such in the face of a pandemic. However, a minimal system capable of recapitulating different steps of the viral life cycle without using the virus' genetic material could increase accessibility. In this work, we assessed the four structural proteins from SARS-CoV-2 for their ability to form virus-like particles (VLPs) from human cells to form a competent system for BSL-2 studies of SARS-CoV-2. Herein, we provide methods and resources of producing, purifying, fluorescently and APEX2-labeling of SARS-CoV-2 VLPs for the evaluation of mechanisms of viral budding and entry as well as assessment of drug inhibitors under BSL-2 conditions. These systems should be useful to those looking to circumvent BSL-3 work with SARS-CoV-2 yet study the mechanisms by which SARS-CoV-2 enters and exits human cells.

CryoAPEX - an electron tomography tool for subcellular localization of membrane proteins.

We describe a method, termed cryoAPEX, which couples chemical fixation and high-pressure freezing of cells with peroxidase tagging (APEX) to allow precise localization of membrane proteins in the context of a well-preserved subcellular membrane architecture. Further, cryoAPEX is compatible with electron tomography. As an example, we apply cryoAPEX to obtain a high-resolution three-dimensional contextual map of the human FIC (filamentation induced by cAMP) protein, HYPE (also known as FICD). HYPE is a single-pass membrane protein that localizes to the endoplasmic reticulum (ER) lumen and regulates the unfolded protein response. Alternate cellular locations for HYPE have been suggested. CryoAPEX analysis shows that, under normal and/or resting conditions, HYPE localizes robustly within the subdomains of the ER and is not detected in the secretory pathway or other organelles. CryoAPEX is broadly applicable for assessing both lumenal and cytosol-facing membrane proteins.

N-terminal autoprocessing and acetylation of multifunctional-autoprocessing repeats-in-toxins (MARTX) Makes Caterpillars Floppy-like effector is stimulated by adenosine diphosphate (ADP)-Ribosylation Factor 1 in advance of Golgi fragmentation.

Studies have successfully elucidated the mechanism of action of several effector domains that comprise the multifunctional-autoprocessing repeats-in-toxins (MARTX) toxins of Vibrio vulnificus. However, the biochemical linkage between the cysteine proteolytic activity of Makes Caterpillars Floppy (MCF)-like effector and its cellular effects remains unknown. In this study, we identify the host cell factors that activate in vivo and in vitro MCF autoprocessing as adenosine diphosphate (ADP)-Ribosylation Factor 1 (ARF1) and ADP-Ribosylation Factor 3 (ARF3). Autoprocessing activity is enhanced when ARF1 is in its active [guanosine triphosphate (GTP)-bound] form compared to the inactive [guanosine diphosphate (GDP)-bound] form. Subsequent to auto-cleavage, MCF is acetylated on its exposed N-terminal glycine residue. Acetylation apparently does not dictate subcellular localization as MCF is found localized throughout the cell. However, the cleaved form of MCF gains the ability to bind to the specialized lipid phosphatidylinositol 5-phosphate enriched in Golgi and other membranes necessary for endocytic trafficking, suggesting that a fraction of MCF may be subcellularly localized. Traditional thin-section electron microscopy, high-resolution cryoAPEX localization, and fluorescent microscopy show that MCF causes Golgi dispersal resulting in extensive vesiculation. In addition, host mitochondria are disrupted and fragmented. Mass spectrometry analysis found no reproducible modifications of ARF1 suggesting that ARF1 is not post-translationally modified by MCF. Further, catalytically active MCF does not stably associate with ARF1. Our data indicate not only that ARF1 is a cross-kingdom activator of MCF, but also that MCF may mediate cytotoxicity by directly targeting another yet to be identified protein. This study begins to elucidate the biochemical activity of this important domain and gives insight into how it may promote disease progression.

A physical interaction network of dengue virus and human proteins.

Dengue virus (DENV), an emerging mosquito-transmitted pathogen capable of causing severe disease in humans, interacts with host cell factors to create a more favorable environment for replication. However, few interactions between DENV and human proteins have been reported to date. To identify DENV-human protein interactions, we used high-throughput yeast two-hybrid assays to screen the 10 DENV proteins against a human liver activation domain library. From 45 DNA-binding domain clones containing either full-length viral genes or partially overlapping gene fragments, we identified 139 interactions between DENV and human proteins, the vast majority of which are novel. These interactions involved 105 human proteins, including six previously implicated in DENV infection and 45 linked to the replication of other viruses. Human proteins with functions related to the complement and coagulation cascade, the centrosome, and the cytoskeleton were enriched among the DENV interaction partners. To determine if the cellular proteins were required for DENV infection, we used small interfering RNAs to inhibit their expression. Six of 12 proteins targeted (CALR, DDX3X, ERC1, GOLGA2, TRIP11, and UBE2I) caused a significant decrease in the replication of a DENV replicon. We further showed that calreticulin colocalized with viral dsRNA and with the viral NS3 and NS5 proteins in DENV-infected cells, consistent with a direct role for calreticulin in DENV replication. Human proteins that interacted with DENV had significantly higher average degree and betweenness than expected by chance, which provides additional support for the hypothesis that viruses preferentially target cellular proteins that occupy central position in the human protein interaction network. This study provides a valuable starting point for additional investigations into the roles of human proteins in DENV infection.

Contribution of the Golgi apparatus in morphogenesis of a virus-induced cytopathic vacuolar system.

The Golgi apparatus (GA) in mammalian cells is pericentrosomally anchored and exhibits a stacked architecture. During infections by members of the alphavirus genus, the host cell GA is thought to give rise to distinct mobile pleomorphic vacuoles known as CPV-II (cytopathic vesicle-II) via unknown morphological steps. To dissect this, we adopted a phased electron tomography approach to image multiple overlapping volumes of a cell infected with Venezuelan equine encephalitis virus (VEEV) and complemented it with localization of a peroxidase-tagged Golgi marker. Analysis of the tomograms revealed a pattern of progressive cisternal bending into double-lamellar vesicles as a central process underpinning the biogenesis and the morphological complexity of this vacuolar system. Here, we propose a model for the conversion of the GA to CPV-II that reveals a unique pathway of intracellular virus envelopment. Our results have implications for alphavirus-induced displacement of Golgi cisternae to the plasma membrane to aid viral egress operating late in the infection cycle.

A comparative assessment of pomegranate extract, sodium hypochlorite, chlorhexidine, Myrrh (Commiphora molmol), tulsi extract against Enterococcus faecalis, Fusobacterium nucleatum and Staphylococci epidermidis.

BACKGROUND: The presence of pathogenic bacteria, toxins and byproducts in the root canal system show a decisive part in success of endodontic therapy. Thus, the complete removal of this bacterium is highly desirable. Several intracanal medicaments were tried to disinfect the root canal before obturation. AIM: The present study was conducted to compare Pomegranate, sodium hypochlorite, Chlorhexidine, Myrrh (Commiphora molmol), tulsi extract against Enterococcus faecalis, Fusobacterium nucleatum and Staphylococci epidermidis. SETTINGS AND DESIGN: Cross-sectional observational prospective study. MATERIALS AND METHODOLOGY: Aqueous extract of 20% pomegranate peel, 20% pomegranate peel, 0.2% CHX, 2.5% sodium hypochlorite, Tulsi extract and Myrrh (Commiphora molmol) was used as agent against E. faecalis, F. nucleatum and Staphylococci Epidermidis. Zone of inhibition and minimum inhibitory concentration (MIC) was calculated and compared using analysis of variance and Mann-Whitney test. The information was statistically evaluated with SPSS software version 20 with P < 0.05. RESULTS AND CONCLUSION: The mean zone of inhibition against E. faecalis, F. nucleatum and S. Epidermidis was highest in chlorhexidine and sodium hypochlorite groups compared to herbal groups. MIC was least with group III followed by group II against all bacterial species (P < 0.05). Sodium hypochlorite found comparatively better followed by chlorhexidine and other agents against E. faecalis, F nucleatum and S. epidermidis.

Brain response to color stimuli: an EEG study with nonlinear approach.

Color perception is a major guiding factor in the evolutionary process of human civilization, but most of the neurological background of the same are yet unknown. This work attempts to address this area with an EEG based neuro-cognitive study on response of brain to different color stimuli. With respect to a Grey baseline seven colors of the VIBGYOR were shown to 16 participants with normal color vision and corresponding EEG signals from different lobes (Frontal, Occipital & Parietal) were recorded. In an attempt to quantify the brain response while watching these colors, the corresponding EEG signals were analysed using two of the latest state of the art non-linear techniques (MFDFA and MFDXA) of dealing complex time series. MFDFA revealed that for all the participants the spectral width, and hence the complexity of the EEG signals, reaches a maximum while viewing color Blue, followed by colors Red and Green in all the brain lobes. MFDXA, on the other hand, suggests a lower degree of inter and intra lobe correlation while watching the VIBGYOR colors compared to baseline Grey, hinting towards a post processing of visual information. We hope that along with the novelty of methodologies, the unique outcomes of this study may leave a long term impact in the domain of color perception research.

Assessment of expression of podoplanin in odontogenic tumors and cysts-An immunohistochemical study.

AIM: The present study was conducted to determine the podoplanin expression in odontogenic tumors and cysts. MATERIALS AND METHODS: It consisted of 12 cases of the keratocystic odontogenic tumor (KCOT), 10 cases of ameloblastoma, 8 cases of dentigerous cysts (DC), 8 cases of radicular cysts (RC) and 8 dental follicles (DF) as controls which were immunohistochemically evaluated using an antibody against podoplanin. RESULTS: Immunostaining intensity, % of PPC and total score of ameloblastoma was higher than DC and DF but less than KCOT (t- 1.48). When DC was compared with KCOT and RC, the podoplanin expression was significantly higher with DC (P < 0.05). The podoplanin expression was comparatively higher with KCOT as compared to RC and DC. OT (t-4.40) revealed higher podoplanin expression as compared to OC and DF (t-5.54). CONCLUSION: There was significantly higher expression of podoplanin in cases of ameloblastoma and KCOT as compared to the RC, DC and DF. Podoplanin may be considered as a useful marker to delineate the aggressiveness of ameloblastoma and KCOT.

The Cytosolic Phospholipase A2α N-terminal C2 Domain Binds and Oligomerizes on Membranes with Positive Curvature.

Group IV phospholipase A2α (cPLA2α) regulates the production of prostaglandins and leukotrienes via the formation of arachidonic acid from membrane phospholipids. The targeting and membrane binding of cPLA2α to the Golgi involves the N-terminal C2 domain, whereas the catalytic domain produces arachidonic acid. Although most studies of cPLA2α concern its catalytic activity, it is also linked to homeostatic processes involving the generation of vesicles that traffic material from the Golgi to the plasma membrane. Here we investigated how membrane curvature influences the homeostatic role of cPLA2α in vesicular trafficking. The cPLA2α C2 domain is known to induce changes in positive membrane curvature, a process which is dependent on cPLA2α membrane penetration. We showed that cPLA2α undergoes C2 domain-dependent oligomerization on membranes in vitro and in cells. We found that the association of the cPLA2α C2 domain with membranes is limited to membranes with positive curvature, and enhanced C2 domain oligomerization was observed on vesicles ~50 nm in diameter. We demonstrated that the cPLA2α C2 domain localizes to cholesterol enriched Golgi-derived vesicles independently of cPLA2α catalytic activity. Moreover, we demonstrate the C2 domain selectively localizes to lipid droplets whereas the full-length enzyme to a much lesser extent. Our results therefore provide novel insight into the molecular forces that mediate C2 domain-dependent membrane localization in vitro and in cells.

SARS-CoV-2 viral budding and entry can be modeled using virus-like particles.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was first discovered in December 2019 in Wuhan, China and expeditiously spread across the globe causing a global pandemic. While a select agent designation has not been made for SARS-CoV-2, closely related SARS-CoV-1 and MERS coronaviruses are classified as Risk Group 3 select agents, which restricts use of the live viruses to BSL-3 facilities. Such BSL-3 classification make SARS-CoV-2 research inaccessible to the majority of functioning research laboratories in the US; this becomes problematic when the collective scientific effort needs to be focused on such in the face of a pandemic. In this work, we assessed the four structural proteins from SARS-CoV-2 for their ability to form viruslike particles (VLPs) from human cells to form a competent system for BSL-2 studies of SARS-CoV-2. Herein, we provide methods and resources of producing, purifying, fluorescently and APEX2-labeling of SARS-CoV-2 VLPs for the evaluation of mechanisms of viral budding and entry as well as assessment of drug inhibitors under BSL-2 conditions.

Cryofixation of Inactivated Hantavirus-Infected Cells as a Method for Obtaining High-Quality Ultrastructural Preservation for Electron Microscopic Studies.

Hantaviruses rewire the host cell and induce extensive membrane rearrangements for their replication and the morphogenesis of the virion. Transmission electron microscopy (TEM) is a powerful technique for imaging these pathological membrane changes especially when combined with large volume electron tomography. Excellent preservation of membrane structure can be obtained when chemical fixation is combined with cryofixation via high pressure freezing making the samples amenable to serial-section tomographic reconstruction. Taking advantage of this, we have optimized a hybrid method that employs aldehyde fixation, a step that is essential for virus inactivation, followed by high-pressure freezing for ultrastructural study of Hantaan (HTN) and Andes (AND) virus infected Vero E6 cells. HTNV and ANDV are two species of the Orthohantavirus, from the Old and New World, respectively, and the causative agents of hemorrhagic fever with renal syndrome and hantavirus pulmonary syndrome in humans. We applied the method for the qualitative assessment of the perturbation of the endomembrane system induced by HTNV and ANDV in infected vs. mock-infected cells. Screening of serial-sections revealed consistency of membrane preservation across large volumes indicating potential of these samples for tomographic studies. Images revealed large-scale perturbations of the endomembrane system following HTNV-infection that included the dilation of the rough endoplasmic reticulum and fragmentation of the Golgi apparatus. Infected cells exhibited a tendency to accumulate large numbers of vacuoles that were especially apparent in ANDV. In summary, our hybrid method provides a path for the study of BSL-3 pathogens using cutting edge 3D-imaging technologies.

Music of brain and music on brain: a novel EEG sonification approach.

Can we hear the sound of our brain? Is there any technique which can enable us to hear the neuro-electrical impulses originating from the different lobes of brain? The answer to all these questions is YES. In this paper we present a novel method with which we can sonify the electroencephalogram (EEG) data recorded in "control" state as well as under the influence of a simple acoustical stimuli-a tanpura drone. The tanpura has a very simple construction yet the tanpura drone exhibits very complex acoustic features, which is generally used for creation of an ambience during a musical performance. Hence, for this pilot project we chose to study the nonlinear correlations between musical stimulus (tanpura drone as well as music clips) and sonified EEG data. Till date, there have been no study which deals with the direct correlation between a bio-signal and its acoustic counterpart and also tries to see how that correlation varies under the influence of different types of stimuli. This study tries to bridge this gap and looks for a direct correlation between music signal and EEG data using a robust mathematical microscope called Multifractal Detrended Cross Correlation Analysis (MFDXA). For this, we took EEG data of 10 participants in 2 min "control condition" (i.e. with white noise) and in 2 min 'tanpura drone' (musical stimulus) listening condition. The same experimental paradigm was repeated for two emotional music, "Chayanat" and "Darbari Kanada". These are well known Hindustani classical ragas which conventionally portray contrast emotional attributes, also verified from human response data. Next, the EEG signals from different electrodes were sonified and MFDXA technique was used to assess the degree of correlation (or the cross correlation coefficient γx) between the EEG signals and the music clips. The variation of γx for different lobes of brain during the course of the experiment provides interesting new information regarding the extraordinary ability of music stimuli to engage several areas of the brain significantly unlike any other stimuli (which engages specific domains only).

Alpha-Synuclein Is a Target of Fic-Mediated Adenylylation/AMPylation: Possible Implications for Parkinson's Disease.

During disease, cells experience various stresses that manifest as an accumulation of misfolded proteins and eventually lead to cell death. To combat this stress, cells activate a pathway called unfolded protein response that functions to maintain endoplasmic reticulum (ER) homeostasis and determines cell fate. We recently reported a hitherto unknown mechanism of regulating ER stress via a novel post-translational modification called Fic-mediatedadenylylation/AMPylation. Specifically, we showed that the human Fic (filamentation induced by cAMP) protein, HYPE/FicD, catalyzes the addition of an adenosine monophosphate (AMP) to the ER chaperone, BiP, to alter the cell's unfolded protein response-mediated response to misfolded proteins. Here, we report that we have now identified a second target for HYPE-alpha-synuclein (αSyn), a presynaptic protein involved in Parkinson's disease. Aggregated αSyn has been shown to induce ER stress and elicit neurotoxicity in Parkinson's disease models. We show that HYPE adenylylates αSyn and reduces phenotypes associated with αSyn aggregation invitro, suggesting a possible mechanism by which cells cope with αSyn toxicity.