Synthetic virology approaches to improve the safety and efficacy of oncolytic virus therapies.

The large coding potential of vaccinia virus (VV) vectors is a defining feature. However, limited regulatory switches are available to control viral replication as well as timing and dosing of transgene expression in order to facilitate safe and efficacious payload delivery. Herein, we adapt drug-controlled gene switches to enable control of virally encoded transgene expression, including systems controlled by the FDA-approved rapamycin and doxycycline. Using ribosome profiling to characterize viral promoter strength, we rationally design fusions of the operator element of different drug-inducible systems with VV promoters to produce synthetic promoters yielding robust inducible expression with undetectable baseline levels. We also generate chimeric synthetic promoters facilitating additional regulatory layers for VV-encoded synthetic transgene networks. The switches are applied to enable inducible expression of fusogenic proteins, dose-controlled delivery of toxic cytokines, and chemical regulation of VV replication. This toolbox enables the precise modulation of transgene circuitry in VV-vectored oncolytic virus design.

Substrate profiling of the Arabidopsis Ca2+-dependent protein kinase AtCPK4 and its Ricinus communis ortholog RcCDPK1.

AtCPK4 and AtCPK11 are Arabidopsis thaliana Ca2+-dependent protein kinase (CDPK) paralogs that have been reported to positively regulate abscisic acid (ABA) signal transduction by phosphorylating ABA-responsive transcription factor-4 (AtABF4). By contrast, RcCDPK1, their closest Ricinus communis ortholog, participates in the control of anaplerotic carbon flux in developing castor oil seeds by catalyzing inhibitory phosphorylation of bacterial-type phosphoenolpyruvate carboxylase at Ser451. LC-MS/MS revealed that AtCPK4 and RcCDPK1 transphosphorylated several common, conserved residues of AtABF4 and its castor ortholog, TRANSCRIPTION FACTOR RESPONSIBLE FOR ABA REGULATON. Arabidopsis atcpk4/atcpk11 mutants displayed an ABA-insensitive phenotype that corroborated the involvement of AtCPK4/11 in ABA signaling. A kinase-client assay was employed to identify additional AtCPK4/RcCDPK1 targets. Both CDPKs were separately incubated with a library of 2095 peptides representative of Arabidopsis protein phosphosites; five overlapping targets were identified including PLANT INTRACELLULAR RAS-GROUP-RELATED LEUCINE-RICH REPEAT PROTEIN-9 (AtPIRL9) and the E3-ubiquitin ligase ARABIDOPSIS TOXICOS EN LEVADURA 6 (AtATL6). AtPIRL9 and AtATL6 residues phosphorylated by AtCPK4/RcCDPK1 conformed to a CDPK recognition motif that was conserved amongst their respective orthologs. Collectively, this study provides evidence for novel AtCPK4/RcCDPK1 substrates, which may help to expand regulatory networks linked to Ca2+- and ABA-signaling, immune responses, and central carbon metabolism.

Arabidopsis PAP17 is a dual-localized purple acid phosphatase up-regulated during phosphate deprivation, senescence, and oxidative stress.

A 35 kDa monomeric purple acid phosphatase (APase) was purified from cell wall extracts of Pi starved (-Pi) Arabidopsis thaliana suspension cells and identified as AtPAP17 (At3g17790) by mass spectrometry and N-terminal microsequencing. AtPAP17 was de novo synthesized and dual-localized to the secretome and/or intracellular fraction of -Pi or salt-stressed plants, or senescing leaves. Transiently expressed AtPAP17-green fluorescent protein localized to lytic vacuoles of the Arabidopsis suspension cells. No significant biochemical or phenotypical changes associated with AtPAP17 loss of function were observed in an atpap17 mutant during Pi deprivation, leaf senescence, or salinity stress. Nevertheless, AtPAP17 is hypothesized to contribute to Pi metabolism owing to its marked up-regulation during Pi starvation and leaf senescence, broad APase substrate selectivity and pH activity profile, and rapid repression and turnover following Pi resupply to -Pi plants. While AtPAP17 also catalyzed the peroxidation of luminol, which was optimal at pH 9.2, it exhibited a low Vmax and affinity for hydrogen peroxide relative to horseradish peroxidase. These results, coupled with absence of a phenotype in the salt-stressed or -Pi atpap17 mutant, do not support proposals that the peroxidase activity of AtPAP17 contributes to the detoxification of reactive oxygen species during stresses that trigger AtPAP17 up-regulation.

Aerobic oxidation and oxidative esterification of alcohols through cooperative catalysis under metal-free conditions.

The ABNO@PMO-IL-Br material obtained by anchoring 9-azabicyclo[3.3.1]nonane-3-one N-oxyl (keto-ABNO) within the mesopores of periodic mesoporous organosilica with bridged imidazolium groups is a robust bifunctional catalyst for the metal-free aerobic oxidation of numerous primary and secondary alcohols under oxygen balloon reaction conditions. The catalyst, furthermore, can be successfully employed in the first metal-free self-esterification of primary aliphatic alcohols affording valued esters.

Characterization of Critical Determinants of ACE2-SARS CoV-2 RBD Interaction.

Despite sequence similarity to SARS-CoV-1, SARS-CoV-2 has demonstrated greater widespread virulence and unique challenges to researchers aiming to study its pathogenicity in humans. The interaction of the viral receptor binding domain (RBD) with its main host cell receptor, angiotensin-converting enzyme 2 (ACE2), has emerged as a critical focal point for the development of anti-viral therapeutics and vaccines. In this study, we selectively identify and characterize the impact of mutating certain amino acid residues in the RBD of SARS-CoV-2 and in ACE2, by utilizing our recently developed NanoBiT technology-based biosensor as well as pseudotyped-virus infectivity assays. Specifically, we examine the mutational effects on RBD-ACE2 binding ability, efficacy of competitive inhibitors, as well as neutralizing antibody activity. We also look at the implications the mutations may have on virus transmissibility, host susceptibility, and the virus transmission path to humans. These critical determinants of virus-host interactions may provide more effective targets for ongoing vaccines, drug development, and potentially pave the way for determining the genetic variation underlying disease severity.

Nanoluciferase complementation-based bioreporter reveals the importance of N-linked glycosylation of SARS-CoV-2 S for viral entry.

The ongoing COVID-19 pandemic has highlighted the immediate need for the development of antiviral therapeutics targeting different stages of the SARS-CoV-2 life cycle. We developed a bioluminescence-based bioreporter to interrogate the interaction between the SARS-CoV-2 viral spike (S) protein and its host entry receptor, angiotensin-converting enzyme 2 (ACE2). The bioreporter assay is based on a nanoluciferase complementation reporter, composed of two subunits, large BiT and small BiT, fused to the S receptor-binding domain (RBD) of the SARS-CoV-2 S protein and ACE2 ectodomain, respectively. Using this bioreporter, we uncovered critical host and viral determinants of the interaction, including a role for glycosylation of asparagine residues within the RBD in mediating successful viral entry. We also demonstrate the importance of N-linked glycosylation to the RBD's antigenicity and immunogenicity. Our study demonstrates the versatility of our bioreporter in mapping key residues mediating viral entry as well as screening inhibitors of the ACE2-RBD interaction. Our findings point toward targeting RBD glycosylation for therapeutic and vaccine strategies against SARS-CoV-2.

Luciferase-Based Biosensors in the Era of the COVID-19 Pandemic.

Luciferase-based biosensors have a wide range of applications and assay formats, including their relatively recent use in the study of viruses. Split luciferase, bioluminescence resonance energy transfer, circularly permuted luciferase, cyclic luciferase, and dual luciferase systems have all been used to interrogate the structure and function of prominent viruses infecting humans, animals, and plants. The utility of these assays is demonstrated by numerous studies which have not only successfully characterized interactions between viral and host cell proteins but that have also used these systems to identify viral inhibitors. In the present COVID-19 pandemic, luciferase-based biosensors are already playing a critical role in the study of the culprit virus SARS-CoV-2 as well as in the development of serological assays and drug development via high-throughput screening. In this review paper, we provide a summary of existing luciferase-based biosensors and their applications in virology.

Recent insights into the metabolic adaptations of phosphorus-deprived plants.

Inorganic phosphate (Pi) is an essential macronutrient required for many fundamental processes in plants, including photosynthesis and respiration, as well as nucleic acid, protein, and membrane phospholipid synthesis. The huge use of Pi-containing fertilizers in agriculture demonstrates that the soluble Pi levels of most soils are suboptimal for crop growth. This review explores recent advances concerning the understanding of adaptive metabolic processes that plants have evolved to alleviate the negative impact of nutritional Pi deficiency. Plant Pi starvation responses arise from complex signaling pathways that integrate altered gene expression with post-transcriptional and post-translational mechanisms. The resultant remodeling of the transcriptome, proteome, and metabolome enhances the efficiency of root Pi acquisition from the soil, as well as the use of assimilated Pi throughout the plant. We emphasize how the up-regulation of high-affinity Pi transporters and intra- and extracellular Pi scavenging and recycling enzymes, organic acid anion efflux, membrane remodeling, and the remarkable flexibility of plant metabolism and bioenergetics contribute to the survival of Pi-deficient plants. This research field is enabling the development of a broad range of innovative and promising strategies for engineering phosphorus-efficient crops. Such cultivars are urgently needed to reduce inputs of unsustainable and non-renewable Pi fertilizers for maximum agronomic benefit and long-term global food security and ecosystem preservation.

Home sweet home: how mutualistic microbes modify root development to promote symbiosis.

Post-embryonic organogenesis has uniquely equipped plants to become developmentally responsive to their environment, affording opportunities to remodel organism growth and architecture to an extent not possible in other higher order eukaryotes. It is this developmental plasticity that makes the field of plant-microbe interactions an exceptionally fascinating venue in which to study symbiosis. This review article describes the various ways in which mutualistic microbes alter the growth, development, and architecture of the roots of their plant hosts. We first summarize general knowledge of root development, and then examine how association of plants with beneficial microbes affects these processes. Working our way inwards from the epidermis to the pericycle, this review dissects the cell biology and molecular mechanisms underlying plant-microbe interactions in a tissue-specific manner. We examine the ways in which microbes gain entry into the root, and modify this specialized organ for symbiont accommodation, with a particular emphasis on the colonization of root cortical cells. We present significant advances in our understanding of root-microbe interactions, and conclude our discussion by identifying questions pertinent to root endosymbiosis that at present remain unresolved.

Phosphate and phosphite have a differential impact on the proteome and phosphoproteome of Arabidopsis suspension cell cultures.

Phosphorus absorbed in the form of phosphate (H2 PO4- ) is an essential but limiting macronutrient for plant growth and agricultural productivity. A comprehensive understanding of how plants respond to phosphate starvation is essential for the development of more phosphate-efficient crops. Here we employed label-free proteomics and phosphoproteomics to quantify protein-level responses to 48 h of phosphate versus phosphite (H2 PO3- ) resupply to phosphate-deprived Arabidopsis thaliana suspension cells. Phosphite is similarly sensed, taken up and transported by plant cells as phosphate, but cannot be metabolized or used as a nutrient. Phosphite is thus a useful tool for differentiating between non-specific processes related to phosphate sensing and transport and specific responses to phosphorus nutrition. We found that responses to phosphate versus phosphite resupply occurred mainly at the level of protein phosphorylation, complemented by limited changes in protein abundance, primarily in protein translation, phosphate transport and scavenging, and central metabolism proteins. Altered phosphorylation of proteins involved in core processes such as translation, RNA splicing and kinase signaling was especially important. We also found differential phosphorylation in response to phosphate and phosphite in 69 proteins, including splicing factors, translation factors, the PHT1;4 phosphate transporter and the HAT1 histone acetyltransferase - potential phospho-switches signaling changes in phosphorus nutrition. Our study illuminates several new aspects of the phosphate starvation response and identifies important targets for further investigation and potential crop improvement.

Implications for SARS-CoV-2 Vaccine Design: Fusion of Spike Glycoprotein Transmembrane Domain to Receptor-Binding Domain Induces Trimerization.

The ongoing severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic presents an urgent need for an effective vaccine. Molecular characterization of SARS-CoV-2 is critical to the development of effective vaccine and therapeutic strategies. In the present study, we show that the fusion of the SARS-CoV-2 spike protein receptor-binding domain to its transmembrane domain is sufficient to mediate trimerization. Our findings may have implications for vaccine development and therapeutic drug design strategies targeting spike trimerization. As global efforts for developing SARS-CoV-2 vaccines are rapidly underway, we believe this observation is an important consideration for identifying crucial epitopes of SARS-CoV-2.

The Role of YAP and TAZ in Angiogenesis and Vascular Mimicry.

Angiogenesis, the formation of new blood vessels from pre-existing vasculature, is a physiological process that begins in utero and continues throughout life in both good health and disease. Understanding the underlying mechanism in angiogenesis could uncover a new therapeutic approach in pathological angiogenesis. Since its discovery, the Hippo signaling pathway has emerged as a key player in controlling organ size and tissue homeostasis. Recently, new studies have discovered that Hippo and two of its main effectors, Yes-associated protein (YAP) and its paralog transcription activator with PDZ binding motif (TAZ), play critical roles during angiogenesis. In this review, we summarize the mechanisms by which YAP/TAZ regulate endothelial cell shape, behavior, and function in angiogenesis. We further discuss how YAP/TAZ function as part of developmental and pathological angiogenesis. Finally, we review the role of YAP/TAZ in tumor vascular mimicry and propose directions for future work.

A glycoform of the secreted purple acid phosphatase AtPAP26 co-purifies with a mannose-binding lectin (AtGAL1) upregulated by phosphate-starved Arabidopsis.

The purple acid phosphatase AtPAP26 plays a central role in Pi-scavenging by Pi-starved (-Pi) Arabidopsis. Mass spectrometry (MS) of AtPAP26-S1 and AtPAP26-S2 glycoforms secreted by -Pi suspension cells demonstrated that N-glycans at Asn365 and Asn422 were modified in AtPAP26-S2 to form high-mannose glycans. A 55-kDa protein that co-purified with AtPAP26-S2 was identified as a Galanthus nivalis agglutinin-related and apple domain lectin-1 (AtGAL1; At1g78850). MS revealed that AtGAL1 was bisphosphorylated at Tyr38 and Thr39 and glycosylated at four conserved Asn residues. When AtGAL was incubated in the presence of a thiol-reducing reagent prior to immunoblotting, its cross-reactivity with anti-AtGAL1-IgG was markedly attenuated (consistent with three predicted disulfide bonds in AtGAL1's apple domain). Secreted AtGAL1 polypeptides were upregulated to a far greater extent than AtGAL1 transcripts during Pi deprivation, indicating posttranscriptional control of AtGAL1 expression. Growth of a -Pi atgal1 mutant was unaffected, possibly due to compensation by AtGAL1's closest paralog, AtGAL2 (At1g78860). Nevertheless, AtGAL1's induction by numerous stresses combined with the broad distribution of AtGAL1-like lectins in diverse species implies an important function for AtGAL1 orthologs within the plant kingdom. We hypothesize that binding of AtPAP26-S2's high-mannose glycans by AtGAL1 enhances AtPAP26 function to facilitate Pi-scavenging by -Pi Arabidopsis.

Lectin AtGAL1 interacts with high-mannose glycoform of the purple acid phosphatase AtPAP26 secreted by phosphate-starved Arabidopsis.

Among 29 predicted Arabidopsis purple acid phosphatases (PAPs), AtPAP26 functions as the principle extracellular and intracellular PAP isozyme that is upregulated to recycle and scavenge Pi during Pi-deprivation or leaf senescence. Our companion paper documented the copurification of a secreted, high-mannose AtPAP26-S2 glycoform with AtGAL1 (At1g78850), a Pi starvation-inducible (PSI), and Galanthus nivalis agglutinin-related (mannose-binding) and apple domain lectin. This study tests the hypothesis that AtGAL1 binds AtPAP26-S2 to modify its enzymatic properties. Far-western immunodot blotting established that AtGAL1 readily associates with AtPAP26-S2 but not the low mannose AtPAP26-S1 glycoform nor other secreted PSI PAPs (i.e., AtPAP12 or AtPAP25). Analytical gel filtration indicated that 55-kDa AtGAL1 and AtPAP26-S2 polypeptides associate to form a 112-kDa heterodimer. Microscopic imaging of transiently expressed, fluorescent protein-tagged AtGAL1, and associated bimolecular fluorescence complementation assays demonstrated that (a) like AtPAP26, AtGAL1 also localizes to lytic vacuoles of Pi-deprived Arabidopsis and (b) both proteins interact in vivo. AtGAL1 preincubation significantly enhanced the acid phosphatase activity and thermal stability of AtPAP26-S2 but not AtPAP26-S1. We hypothesize that AtGAL1 plays an important role during Pi deprivation through its interaction with mannose-rich glycans of AtPAP26-S2 and consequent positive impact on AtPAP26-S2 activity and stability.

Electrochemical Alcohol Oxidation Mediated by TEMPO-like Nitroxyl Radicals.

The electrocatalytic oxidation of alcohols mediated by TEMPO-like nitroxyl radicals is an economically and industrially viable method that will shortly find commercial application in the synthesis of valued substances including active pharmaceutical ingredients (APIs), valued natural product derivatives, fine chemicals, and valued nanomaterials.

Extraction and Characterization of Extracellular Proteins and Their Post-Translational Modifications from Arabidopsis thaliana Suspension Cell Cultures and Seedlings: A Critical Review.

Proteins secreted by plant cells into the extracellular space, consisting of the cell wall, apoplastic fluid, and rhizosphere, play crucial roles during development, nutrient acquisition, and stress acclimation. However, isolating the full range of secreted proteins has proven difficult, and new strategies are constantly evolving to increase the number of proteins that can be detected and identified. In addition, the dynamic nature of the extracellular proteome presents the further challenge of identifying and characterizing the post-translational modifications (PTMs) of secreted proteins, particularly glycosylation and phosphorylation. Such PTMs are common and important regulatory modifications of proteins, playing a key role in many biological processes. This review explores the most recent methods in isolating and characterizing the plant extracellular proteome with a focus on the model plant Arabidopsis thaliana, highlighting the current challenges yet to be overcome. Moreover, the crucial role of protein PTMs in cell wall signalling, development, and plant responses to biotic and abiotic stress is discussed.