Single-cell image-based genetic screens systematically identify regulators of Ebola virus subcellular infection dynamics.

Ebola virus (EBOV) is a high-consequence filovirus that gives rise to frequent epidemics with high case fatality rates and few therapeutic options. Here, we applied image-based screening of a genome-wide CRISPR library to systematically identify host cell regulators of Ebola virus infection in 39,085,093 million single cells. Measuring viral RNA and protein levels together with their localization in cells identified over 998 related host factors and provided detailed information about the role of each gene across the virus replication cycle. We trained a deep learning model on single-cell images to associate each host factor with predicted replication steps, and confirmed the predicted relationship for select host factors. Among the findings, we showed that the mitochondrial complex III subunit UQCRB is a post-entry regulator of Ebola virus RNA replication, and demonstrated that UQCRB inhibition with a small molecule reduced overall Ebola virus infection with an IC50 of 5 µM. Using a random forest model, we also identified perturbations that reduced infection by disrupting the equilibrium between viral RNA and protein. One such protein, STRAP, is a spliceosome-associated factor that was found to be closely associated with VP35, a viral protein required for RNA processing. Loss of STRAP expression resulted in a reduction in full-length viral genome production and subsequent production of non-infectious virus particles. Overall, the data produced in this genome-wide high-content single-cell screen and secondary screens in additional cell lines and related filoviruses (MARV and SUDV) revealed new insights about the role of host factors in virus replication and potential new targets for therapeutic intervention.

A protein-proximity screen reveals Ebola virus co-opts the mRNA decapping complex through the scaffold protein EDC4.

The interaction of host and Ebola virus (EBOV) proteins is required for establishing infection of the cell. To identify protein binding partners, a proximity-dependent protein interaction screen was performed for six EBOV proteins. Hits were computationally mapped onto a human protein-protein interactome and then annotated with viral proteins to reveal known and previously undescribed EBOV-host protein interactions and processes. Importantly, this approach efficiently arranged proteins into functional complexes associated with single viral proteins. Focused characterization of interactions between EBOV VP35 and the mRNA decapping complex demonstrated that VP35 binds the scaffold protein EDC4 through the C-terminal subdomain, with each protein found associated in EBOV-infected cells. Mechanistically, depletion of three components of the complex each similarly inhibited viral replication by reducing early viral RNA synthesis. Overall, we demonstrate successful identification of EBOV protein interaction with entire cellular machines, providing a deeper understanding of replication mechanism for therapeutic intervention.

Membrane mimetic-dependence of GPCR energy landscapes.

We leveraged variable-temperature 19F-NMR spectroscopy to compare the conformational equilibria of the human A2A adenosine receptor (A2AAR), a class A G protein-coupled receptor (GPCR), across a range of temperatures ranging from lower temperatures typically employed in 19F-NMR experiments to physiological temperature. A2AAR complexes with partial agonists and full agonists showed large increases in the population of a fully active conformation with increasing temperature. NMR data measured at physiological temperature were more in line with functional data. This was pronounced for complexes with partial agonists, where the population of active A2AAR was nearly undetectable at lower temperature but became evident at physiological temperature. Temperature-dependent behavior of complexes with either full or partial agonists exhibited a pronounced sensitivity to the specific membrane mimetic employed. Cellular signaling experiments correlated with the temperature-dependent conformational equilibria of A2AAR in lipid nanodiscs but not in some detergents, underscoring the importance of the membrane environment in studies of GPCR function.

A critical review on the recent trends of photocatalytic, antibacterial, antioxidant and nanohybrid applications of anatase and rutile TiO2 nanoparticles.

Titanium dioxide nanoparticles (TiO2 NPs) have become a focal point of research due to their widespread daily use and diverse synthesis methods, including physical, chemical, and environmentally sustainable approaches. These nanoparticles possess unique attributes such as size, shape, and surface functionality, making them particularly intriguing for applications in the biomedical field. The continuous exploration of TiO2 NPs is driven by the quest to enhance their multifunctionality, aiming to create next-generation products with superior performance. Recent research efforts have specifically focused on understanding the anatase and rutile phases of TiO2 NPs and evaluating their potential in various domains, including photocatalytic processes, antibacterial properties, antioxidant effects, and nanohybrid applications. The hypothesis guiding this research is that by exploring different synthesis methods, particularly chemical and environmentally friendly approaches, and incorporating doping and co-doping techniques, the properties of TiO2 NPs can be significantly improved for diverse applications. The study employs a comprehensive approach, investigating the effects of nanoparticle size, shape, dose, and exposure time on performance. The synthesis methods considered encompass both conventional chemical processes and environmentally friendly alternatives, with a focus on how doping and co-doping can enhance the properties of TiO2 NPs. The research unveils valuable insights into the distinct phases of TiO2 NPs and their potential across various applications. It sheds light on the improved properties achieved through doping and co-doping, showcasing advancements in photocatalytic processes, antibacterial efficacy, antioxidant capabilities, and nanohybrid applications. The study concludes by emphasizing regulatory aspects and offering suggestions for product enhancement. It provides recommendations for the reliable application of TiO2 NPs, addressing a comprehensive spectrum of critical aspects in TiO2 NP research and application. Overall, this research contributes to the evolving landscape of TiO2 NP utilization, offering valuable insights for the development of innovative and high-performance products.

Single-molecule visualization of human A2A adenosine receptor activation by a G protein and constitutively activating mutations.

Mutations that constitutively activate G protein-coupled receptors (GPCRs), known as constitutively activating mutations (CAMs), modify cell signaling and interfere with drugs, resulting in diseases with limited treatment options. We utilize fluorescence imaging at the single-molecule level to visualize the dynamic process of CAM-mediated activation of the human A2A adenosine receptor (A2AAR) in real time. We observe an active-state population for all CAMs without agonist stimulation. Importantly, activating mutations significantly increase the population of an intermediate state crucial for receptor activation, notably distinct from the addition of a partner G protein. Activation kinetics show that while CAMs increase the frequency of transitions to the intermediate state, mutations altering sodium sensitivity increase transitions away from it. These findings indicate changes in GPCR function caused by mutations may be predicted based on whether they favor or disfavor formation of an intermediate state, providing a framework for designing receptors with altered functions or therapies that target intermediate states.

Biomass waste-derived carbon materials for sustainable remediation of polluted environment: A comprehensive review.

In response to the growing global concern over environmental pollution, the exploration of sustainable and eco-friendly materials derived from biomass waste has gained significant traction. This comprehensive review seeks to provide a holistic perspective on the utilization of biomass waste as a renewable carbon source, offering insights into the production of environmentally benign and cost-effective carbon-based materials. These materials, including biochar, carbon nanotubes, and graphene, have shown immense promise in the remediation of polluted soils, industrial wastewater, and contaminated groundwater. The review commences by elucidating the intricate processes involved in the synthesis and functionalization of biomass-derived carbon materials, emphasizing their scalability and economic viability. With their distinctive structural attributes, such as high surface areas, porous architectures, and tunable surface functionalities, these materials emerge as versatile tools in addressing environmental challenges. One of the central themes explored in this review is the pivotal role that carbon materials play in adsorption processes, which represent a green and sustainable technology for the removal of a diverse array of pollutants. These encompass noxious organic compounds, heavy metals, and organic matter, encompassing pollutants found in soils, groundwater, and industrial wastewater. The discussion extends to the underlying mechanisms governing adsorption, shedding light on the efficacy and selectivity of carbon-based materials in different environmental contexts. Furthermore, this review delves into multifaceted considerations, spanning the spectrum from biomass and biowaste resources to the properties and applications of carbon materials. This holistic approach aims to equip researchers and practitioners with a comprehensive understanding of the synergistic utilization of these materials, ultimately facilitating effective and affordable strategies for combatting industrial wastewater pollution, soil contamination, and groundwater impurities.

Membrane Mimetic-Dependence of GPCR Energy Landscapes.

Protein function strongly depends on temperature, which is related to temperature-dependent changes in the equilibria of protein conformational states. We leveraged variable-temperature 19F-NMR spectroscopy to interrogate the temperature dependence of the conformational landscape of the human A2A adenosine receptor (A2AAR), a class A GPCR. Temperature-induced changes in the conformational equilibria of A2AAR in lipid nanodiscs were markedly dependent on the efficacy of bound drugs. While antagonist complexes displayed only modest changes as the temperature rose, both full and partial agonist complexes exhibited substantial increases in the active state population. Importantly, the temperature-dependent response of complexes with both full and partial agonists exhibited a pronounced sensitivity to the specific membrane mimetic employed. In striking contrast to observations within lipid nanodiscs, in detergent micelles the active state population exhibited different behavior for A2AAR complexes with both full and partial agonists. This underscores the importance of the protein environment in understanding the thermodynamics of GPCR activation.

Illuminating GPCR signaling mechanisms by NMR spectroscopy with stable-isotope labeled receptors.

G protein-coupled receptors (GPCRs) exhibit remarkable structural plasticity, which underlies their capacity to recognize a wide range of extracellular molecules and interact with intracellular partner proteins. Nuclear magnetic resonance (NMR) spectroscopy is uniquely well-suited to investigate GPCR structural plasticity, enabled by stable-isotope "probes" incorporated into receptors that inform on structure and dynamics. Progress with stable-isotope labeling methods in Eukaryotic expression systems has enabled production of native or nearly-native human receptors with varied and complementary distributions of NMR probes. These advances have opened up new avenues for investigating the roles of conformational dynamics in signaling processes, including by mapping allosteric communication networks, understanding the specificity of GPCR interactions with partner proteins and exploring the impact of membrane environments on GPCR function.

Removal of organic dyes and free radical assay by encapsulating polyvinylpyrrolidone and Tinospora Cordifolia in dual (Co-Cu) doped TiO2 nanoparticles.

Aquatic pollution refers to any water that has been used and discarded in different water bodies by industrial and commercial activities which contains a wide range of toxic substances and required treatment so that water can be safely reused for various purposes. In present paper, polymer polyvinylpyrrolidone (PVP) and plant Tinospora Cordifolia (T. Cordifolia) encapsulated dual doped cobalt-copper titanium dioxide nanoparticles (Co-Cu TNPs) has been synthesized via microwave-assisted method for the degradation aquatic pollutant dyes: Methyl Orange (MO) & Methylene Blue (MB). Using the encapsulated dual doped Co-Cu TNPs, free radical assays (2,2-diphenyl-1-picrylhydrazyl: DPPH; Hydrogen peroxide: HP & Nitric oxide: NO) were also performed. Several physicochemical properties of encapsulated TNPs were examined using a variety of characterization techniques that helps in photocatalytic and antioxidant activity. The encapsulated TNPs exhibit tetragonal crystal lattice having average particles size between 25 and 38 nm with spherical shape morphology. The bandgap of encapsulated dual doped Co-Cu TNPs was found in the range of 3.25-3.29 eV. The binding of encapsulated dual doped Co-Cu TNPs were also calculated by using XPS which confirms the presence of dopants. The photocatalytic activity was performed with using control experiment and using encapsulated dual doped Co-Cu TNPs against MO and MB dyes. The results revealed that the degradation was observed up to 100% for the both MO and MB dyes. Also, antioxidant activity of encapsulated dual doped Co-Cu TNPs was observed against the DPPH, HO and NO assays.

Ebolavirus Species-Specific Interferon Antagonism Mediated by VP24.

Members of the Ebolavirus genus demonstrate a marked differences in pathogenicity in humans with Ebola (EBOV) being the most pathogenic, Bundibugyo (BDBV) less pathogenic, and Reston (RESTV) is not known to cause a disease in humans. The VP24 protein encoded by members of the Ebolavirus genus blocks type I interferon (IFN-I) signaling through interaction with host karyopherin alpha nuclear transporters, potentially contributing to virulence. Previously, we demonstrated that BDBV VP24 (bVP24) binds with lower affinities to karyopherin alpha proteins relative to EBOV VP24 (eVP24), and this correlated with a reduced inhibition in IFN-I signaling. We hypothesized that modification of eVP24-karyopherin alpha interface to make it similar to bVP24 would attenuate the ability to antagonize IFN-I response. We generated a panel of recombinant EBOVs containing single or combinations of point mutations in the eVP24-karyopherin alpha interface. Most of the viruses appeared to be attenuated in both IFN-I-competent 769-P and IFN-I-deficient Vero-E6 cells in the presence of IFNs. However, the R140A mutant grew at reduced levels even in the absence of IFNs in both cell lines, as well as in U3A STAT1 knockout cells. Both the R140A mutation and its combination with the N135A mutation greatly reduced the amounts of viral genomic RNA and mRNA suggesting that these mutations attenuate the virus in an IFN-I-independent attenuation. Additionally, we found that unlike eVP24, bVP24 does not inhibit interferon lambda 1 (IFN-λ1), interferon beta (IFN-ß), and ISG15, which potentially explains the lower pathogenicity of BDBV relative to EBOV. Thus, the VP24 residues binding karyopherin alpha attenuates the virus by IFN-I-dependent and independent mechanisms.

Dual mechanisms of cholesterol-GPCR interactions that depend on membrane phospholipid composition.

Cholesterol is a critical component of mammalian cell membranes and an allosteric modulator of G protein-coupled receptors (GPCRs), but divergent views exist on the mechanisms by which cholesterol influences receptor functions. Leveraging the benefits of lipid nanodiscs, i.e., quantitative control of lipid composition, we observe distinct impacts of cholesterol in the presence and absence of anionic phospholipids on the function-related conformational dynamics of the human A2A adenosine receptor (A2AAR). Direct receptor-cholesterol interactions drive activation of agonist-bound A2AAR in membranes containing zwitterionic phospholipids. Intriguingly, the presence of anionic lipids attenuates cholesterol's impact through direct interactions with the receptor, highlighting a more complex role for cholesterol that depends on membrane phospholipid composition. Targeted amino acid replacements at two frequently predicted cholesterol interaction sites showed distinct impacts of cholesterol at different receptor locations, demonstrating the ability to delineate different roles of cholesterol in modulating receptor signaling and maintaining receptor structural integrity.

Anionic phospholipids control mechanisms of GPCR-G protein recognition.

G protein-coupled receptors (GPCRs) are embedded in phospholipids that strongly influence drug-stimulated signaling. Anionic lipids are particularly important for GPCR signaling complex formation, but a mechanism for this role is not understood. Using NMR spectroscopy, we explore the impact of anionic lipids on the function-related conformational equilibria of the human A2A adenosine receptor (A2AAR) in bilayers containing defined mixtures of zwitterionic and anionic phospholipids. Anionic lipids prime the receptor to form complexes with G proteins through a conformational selection process. Without anionic lipids, signaling complex formation proceeds through a less favorable induced fit mechanism. In computational models, anionic lipids mimic interactions between a G protein and positively charged residues in A2AAR at the receptor intracellular surface, stabilizing a pre-activated receptor conformation. Replacing these residues strikingly alters the receptor response to anionic lipids in experiments. High sequence conservation of the same residues among all GPCRs supports a general role for lipid-receptor charge complementarity in signaling.

Anionic Phospholipids Control Mechanisms of GPCR-G Protein Recognition.

G protein-coupled receptors (GPCRs) are embedded in phospholipids that strongly influence drug-stimulated signaling. Anionic lipids are particularly important for GPCR signaling complex formation, but a mechanism for this role is not understood. Using NMR spectroscopy, we visualized the impact of anionic lipids on the function-related conformational equilibria of the human A 2A adenosine receptor (A 2A AR) in bilayers containing defined mixtures of zwitterionic and anionic phospholipids. Anionic lipids primed the receptor to form complexes with G proteins through a conformational selection process. Without anionic lipids, signaling complex formation proceeded through a less favorable induced fit mechanism. In computational models, anionic lipids mimicked interactions between a G protein and positively charged residues in A 2A AR at the receptor intracellular surface, stabilizing a pre-activated receptor conformation. Replacing these residues strikingly altered the receptor response to anionic lipids in experiments. High sequence conservation of the same residues among all GPCRs supports a general role for lipid-receptor charge complementarity in signaling.

Global insights into the fine tuning of human A2AAR conformational dynamics in a ternary complex with an engineered G protein viewed by NMR.

G protein-coupled receptor (GPCR) conformational plasticity enables formation of ternary signaling complexes with intracellular proteins in response to binding extracellular ligands. We investigate the dynamic process of GPCR complex formation in solution with the human A2A adenosine receptor (A2AAR) and an engineered Gs protein, mini-Gs. 2D nuclear magnetic resonance (NMR) data with uniform stable isotope-labeled A2AAR enabled a global comparison of A2AAR conformations between complexes with an agonist and mini-Gs and with an agonist alone. The two conformations are similar and show subtle differences at the receptor intracellular surface, supporting a model whereby agonist binding alone is sufficient to populate a conformation resembling the active state. However, an A2AAR "hot spot" connecting the extracellular ligand-binding pocket to the intracellular surface is observed to be highly dynamic in the ternary complex, suggesting a mechanism for allosteric connection between the bound G protein and the drug-binding pocket involving structural plasticity of the "toggle switch" tryptophan.

A comparative study of interfacial environments in lipid nanodiscs and vesicles.

Membrane protein conformations and dynamics are driven by the protein-lipid interactions occurring within the local environment of the membrane. These environments remain challenging to accurately capture in structural and biophysical experiments using bilayers. Consequently, there is an increasing need for realistic cell-membrane mimetics for in vitro studies. Lipid nanodiscs provide certain advantages over vesicles for membrane protein studies. Nanodiscs are increasingly used for structural and spectroscopic characterization of membrane proteins. Despite the common use of nanodiscs, the interfacial environments of lipids confined to a ~10-nm diameter area have remained relatively underexplored. Here, we use ultrafast two-dimensional infrared spectroscopy and temperature-dependent infrared absorption measurements of the ester carbonyls to compare the interfacial hydrogen bond structure and dynamics in lipid nanodiscs of varying lipid compositions and sizes with ~100-nm vesicles. We examine the effects of lipid composition and nanodisc size. We found that nanodiscs and vesicles share largely similar lipid-water H-bond environments and interfacial dynamics. Differences in measured enthalpies of H-bonding suggest that H-bond dynamics in nanodiscs are modulated by the interaction between the annular lipids and the scaffold protein.

Production of human A2AAR in lipid nanodiscs for 19F-NMR and single-molecule fluorescence spectroscopy.

We describe production of the human A2A adenosine receptor (A2AAR), a class A G protein-coupled receptor (GPCR) for 19F-NMR and single-molecule fluorescence (SMF) spectroscopy. We explain in detail steps shared between the two sample preparation strategies, including expression and isolation of A2AAR and assembly of A2AAR in lipid nanodiscs and procedures for incorporation of either 19F-NMR or fluorescence probes. Protocols for SMF experiments include sample setup, data acquisition, data processing, and error analysis. For complete details on the use and execution of this protocol, please refer to Wei et al. (2022) and Susac et al. (2018).

Effect of natural radionuclide's in the environment along the Jwalamukhi thrust of Himachal Pradesh, North West Himalayas, India.

This research paper is devoted to measure the activity contents of natural radionuclide, like, radium (226Ra), thorium (232Th) and potassium (40K) in the soil gathered along the Jwalamukhi thrust of Himachal Pradesh, North Western Himalayas, India. NaI(Tl) Scintillator detector was utilized for the estimation of activity content. The activity concentration of 226Ra, 232Th and 40K for some of the soil samples have been observed to be above the global normal mean values. The outcomes acquired for indoor and outdoor effective dosage are well below the normal international and national proposed results. The determined values of external hazard (Hex) for studied locations are less than unity, therefore; samples assembled from these regions are safe from a health hazard point of view and can be utilized as a construction purposes without producing any radio-logical hazard to human beings. The average estimations of radium equivalent activity were found to be within the limits suggested by Organization for Economic Cooperation and Development (OECD). Radon (222Rn) and thoron (220Rn) exhalation rates have also been calculated and discussed.

Slow conformational dynamics of the human A2A adenosine receptor are temporally ordered.

A more complete depiction of protein energy landscapes includes the identification of different function-related conformational states and the determination of the pathways connecting them. We used total internal reflection fluorescence (TIRF) imaging to investigate the conformational dynamics of the human A2A adenosine receptor (A2AAR), a class A G protein-coupled receptor (GPCR), at the single-molecule level. Slow, reversible conformational exchange was observed among three different fluorescence emission states populated for agonist-bound A2AAR. Transitions among these states predominantly occurred in a specific order, and exchange between inactive and active-like conformations proceeded through an intermediate state. Models derived from molecular dynamics simulations with available A2AAR structures rationalized the relative fluorescence emission intensities for the highest and lowest emission states but not the transition state. This suggests that the functionally critical intermediate state required to achieve activation is not currently visualized among available A2AAR structures.

Phytoantioxidant Functionalized Nanoparticles: A Green Approach to Combat Nanoparticle-Induced Oxidative Stress.

Nanotechnology is gaining significant attention, with numerous biomedical applications. Silver in wound dressings, copper oxide and silver in antibacterial preparations, and zinc oxide nanoparticles as a food and cosmetic ingredient are common examples. However, adverse effects of nanoparticles in humans and the environment from extended exposure at varied concentrations have yet to be established. One of the drawbacks of employing nanoparticles is their tendency to cause oxidative stress, a significant public health concern with life-threatening consequences. Cardiovascular, renal, and respiratory problems and diabetes are among the oxidative stress-related disorders. In this context, phytoantioxidant functionalized nanoparticles could be a novel and effective alternative. In addition to performing their intended function, they can protect against oxidative damage. This review was designed by searching through various websites, books, and articles found in PubMed, Science Direct, and Google Scholar. To begin with, oxidative stress, its related diseases, and the mechanistic basis of oxidative damage caused by nanoparticles are discussed. One of the main mechanisms of action of nanoparticles was unearthed to be oxidative stress, which limits their use in humans. Secondly, the role of phytoantioxidant functionalized nanoparticles in oxidative damage prevention is critically discussed. The parameters for the characterization of nanoparticles were also discussed. The majority of silver, gold, iron, zinc oxide, and copper nanoparticles produced utilizing various plant extracts were active free radical scavengers. This potential is linked to several surface fabricated phytoconstituents, such as flavonoids and phenols. These phytoantioxidant functionalized nanoparticles could be a better alternative to nanoparticles prepared by other existing approaches.

Nanotechnology Interventions in the Management of COVID-19: Prevention, Diagnosis and Virus-Like Particle Vaccines.

SARS-CoV-2 claimed numerous lives and put nations on high alert. The lack of antiviral medications and the small number of approved vaccines, as well as the recurrence of adverse effects, necessitates the development of novel treatment ways to combat COVID-19. In this context, using databases such as PubMed, Google Scholar, and Science Direct, we gathered information about nanotechnology's involvement in the prevention, diagnosis and virus-like particle vaccine development. This review revealed that various nanomaterials like gold, polymeric, graphene and poly amino ester with carboxyl group coated magnetic nanoparticles have been explored for the fast detection of SARS-CoV-2. Personal protective equipment fabricated with nanoparticles, such as gloves, masks, clothes, surfactants, and Ag, TiO2 based disinfectants played an essential role in halting COVID-19 transmission. Nanoparticles are used not only in vaccine delivery, such as lipid nanoparticles mediated transport of mRNA-based Pfizer and Moderna vaccines, but also in the development of vaccine as the virus-like particles elicit an immune response. There are now 18 virus-like particle vaccines in pre-clinical development, with one of them, developed by Novavax, reported being in phase 3 trials. Due to the probability of upcoming COVID-19 waves, and the rise of new diseases, the future relevance of virus-like particles is imperative. Furthermore, psychosocial variables linked to vaccine reluctance constitute a critical problem that must be addressed immediately to avert pandemic.