Earlier In Vitro Viral Production With SARS-CoV-2 Alpha Than With Beta, Gamma, B, or A.27 Variants

Since its emergence in China at the end of 2019, SARS-CoV-2 has rapidly spread across the world to become a global public health emergency. Since then, the pandemic has evolved with the large worldwide emergence of new variants, such as the Alpha (B.1.1.7 variant), Beta (B.1.351 variant), and Gamma (P.1 variant), and some other under investigation such as the A.27 in France. Many studies are focusing on antibody neutralisation changes according to the spike mutations, but to date, little is known regarding their respective replication capacities. In this work, we demonstrate that the Alpha variant provides an earlier replication in vitro, on Vero E6 and A549 cells, than Beta, Gamma, A.27, and historical lineages. This earlier replication was associated with higher infectious titres in cell-culture supernatants, in line with the higher viral loads observed among Alpha-infected patients. Interestingly, Beta and Gamma variants presented similar kinetic and viral load than the other non-Alpha-tested variants.

Proteolytic cleavage of Arabidopsis thaliana phosphoenolpyruvate carboxykinase-1 modifies its allosteric regulation

Phosphoenolpyruvate carboxykinase (PEPCK) plays a crucial role in gluconeogenesis. In this work, we analyze the proteolysis of Arabidopsis thaliana PEPCK1 (AthPEPCK1) in germinating seedlings. We found that the amount of AthPEPCK1 protein peaks at 24-48 hours post-imbibition. Concomitantly, we observed shorter versions of AthPEPCK1, putatively generated by metacaspase-9 (AthMC9). To study the impact of AthMC9 cleavage on the kinetic and regulatory properties of AthPEPCK1, we produced truncated mutants based on the reported AthMC9 cleavage sites. The Δ19 and Δ101 truncated mutants of AthPEPCK1 showed similar kinetic parameters and the same quaternary structure than the WT. However, activation by malate and inhibition by glucose 6-phosphate were abolished in the Δ101 mutant. We propose that proteolysis of AthPEPCK1 in germinating seedlings operates as a mechanism to adapt the sensitivity to allosteric regulation during the sink-to-source transition.

Proteolytic cleavage of Arabidopsis thaliana phosphoenolpyruvate carboxykinase-1 modifies its allosteric regulation

Phosphoenolpyruvate carboxykinase (PEPCK) plays a crucial role in gluconeogenesis. In this work, we analyze the proteolysis of Arabidopsis thaliana PEPCK1 (AthPEPCK1) in germinating seedlings. We found that expression of AthPEPCK1 peaks at 24-48 hours post-imbibition. Concomitantly, we observed shorter versions of AthPEPCK1, putatively generated by metacaspase-9 (AthMC9). To study the impact of AthMC9 cleavage on the kinetic and regulatory properties of AthPEPCK1, we produced truncated mutants based on the reported AthMC9 cleavage sites. The Δ19 and Δ101 truncated mutants of AthPEPCK1 showed similar kinetic parameters and the same quaternary structure than the WT. However, activation by malate and inhibition by glucose 6-phosphate were abolished in the Δ101 mutant. We propose that proteolysis of AthPEPCK1 in germinating seedlings operates as a mechanism to adapt the sensitivity to allosteric regulation during the sink-to-source transition.HighlightThis paper describes the effects of the N-terminal proteolytic cleavage on the kinetic and regulatory properties of Arabidopsis thaliana phosphoenolpyruvate carboxykinase-1.

Several geranylgeranyl diphosphate synthase isoforms supply metabolic substrates for carotenoid biosynthesis in tomato

ABSTRACTGeranylgeranyl diphosphate (GGPP) produced by GGPP synthase (GGPPS) serves as a precursor for many plastidial isoprenoids, including carotenoids. Here we show that five different GGPPS isoforms exist in tomato (Solanum lycopersicum). From these, SlGGPPS1, 2 and 3 (or SlG1-3 in short) produce GGPP in plastids and exhibit similar kinetic parameters. Phytoene synthase (PSY) converts GGPP into phytoene, the first committed intermediate of the carotenoid pathway. Gene expression and co-expression network analyses showed a preferential association of individual GGPPS and PSY isoforms in processes linked to carotenoid biosynthesis such as root mycorrhization, seedling deetiolation and fruit ripening. Co-immunoprecipitation experiments showed that SlG2, but not SlG3, physically interacts with PSY proteins. By contrast, CRISPR-Cas9 mutants defective in SlG3 showed a stronger impact on carotenoid levels and derived metabolic, physiological and developmental phenotypes that those impaired in SlG2. Double mutants with a simultaneous knockout of both genes could not be found. Our work demonstrates that the bulk of GGPP production in tomato chloroplasts and chromoplasts relies on two cooperating GGPPS paralogs, unlike other plant species such as Arabidopsis thaliana, rice or pepper, which produce their essential plastidial isoprenoids using a single GGPPS isoform.

Ancestral Resurrection and Directed Evolution of Fungal Mesozoic Laccases

ABSTRACT Ancestral sequence reconstruction and resurrection provides useful information for protein engineering, yet its alliance with directed evolution has been little explored. In this study, we have resurrected several ancestral nodes of fungal laccases dating back ∼500 to 250 million years. Unlike modern laccases, the resurrected Mesozoic laccases were readily secreted by yeast, with similar kinetic parameters, a broader stability, and distinct pH activity profiles. The resurrected Agaricomycetes laccase carried 136 ancestral mutations, a molecular testimony to its origin, and it was subjected to directed evolution in order to improve the rate of 1,3-cyclopentanedione oxidation, a β–diketone initiator commonly used in vinyl polymerization reactions. IMPORTANCE The broad variety of biotechnological uses of fungal laccases is beyond doubt (food, textiles, pulp and paper, pharma, biofuels, cosmetics, and bioremediation), and protein engineering (in particular, directed evolution) has become the key driver for adaptation of these enzymes to harsh industrial conditions. Usually, the first requirement for directed laccase evolution is heterologous expression, which presents an important hurdle and often a time-consuming process. In this work, we resurrected a fungal Mesozoic laccase node which showed strikingly high heterologous expression and pH stability. As a proof of concept that the ancestral laccase is a suitable blueprint for engineering, we performed a quick directed evolution campaign geared to the oxidation of the β-diketone 1,3-cyclopentanedione, a poor laccase substrate that is used in the polymerization of vinyl monomers.

A subpopulation of human Adark spermatogonia behaves as the reserve stem cell

Human spermatogonial stem cells (SSCs) are an essential source to maintain spermatogenesis as an efficient process for daily sperm production with high self-renewal capacity along adulthood. However, the phenotype and the subpopulation that represent the real reserve SSC for the human testis remain unknown. Moreover, although SSC markers have been described for undifferentiated spermatogonia (Adark and Apale), the existence of a specific subtype that could be identified as the actual/true SSC has not yet been fully determined. Herein we evaluated spermatogonial morphology, kinetics, positioning regarding blood vasculature in relation to protein expression (UTF1, GFRA1, and KIT) as well as proliferative activity (MCM7) and identified a small subpopulation of Adark with nuclear rarefaction zone (AdVac) that behaves as the human reserve SSC. We show that AdVac is the smallest human spermatogonial population (10%), staying quiescent (89%) and positioned close to blood vessels throughout most of the stages of the seminiferous epithelium cycle (SEC) and divides only at stages I and II. Within this AdVac population, we found a smaller pool (2% of A undifferentiated spermatogonia) of entirely quiescent cells exhibiting a high expression of UTF1 and lacking GFRA1. This finding suggests them as the real human reserve SSC (AdVac UTF1+/GFRA1-/MCM7-). Additionally, Adark without nuclear vacuole (AdNoVac) and Apale have similar kinetic and high proliferative capacity throughout the SEC (47%), indicating that they are actively dividing undifferentiated spermatogonia. Identification of human stem cells with evident reserve SSC functionality may help further studies intending to sort SSCs to treat male diseases and infertility.

Mechanism of Action of the Tumor Vessel Targeting Agent NGR-hTNF: Role of Both NGR Peptide and hTNF in Cell Binding and Signaling

NGR-hTNF is a therapeutic agent for a solid tumor that specifically targets angiogenic tumor blood vessels, through the NGR motif. Its activity has been assessed in several clinical studies encompassing tumors of different histological types. The drug’s activity is based on an improved permeabilization of newly formed tumor vasculature, which favors intratumor penetration of chemotherapeutic agents and leukocyte trafficking. This work investigated the binding and the signaling properties of the NGR-hTNF, to elucidate its mechanism of action. The crystal structure of NGR-hTNF and modeling of its interaction with TNFR suggested that the NGR region is available for binding to a specific receptor. Using 2D TR-NOESY experiments, this study confirmed that the NGR-peptides binds to a specific CD13 isoform, whose expression is restricted to tumor vasculature cells, and to some tumor cell lines. The interaction between hTNF or NGR-hTNF with immobilized TNFRs showed similar kinetic parameters, whereas the competition experiments performed on the cells expressing both TNFR and CD13 showed that NGR-hTNF had a higher binding affinity than hTNF. The analysis of the NGR-hTNF-triggered signal transduction events showed a specific impairment in the activation of pro-survival pathways (Ras, Erk and Akt), compared to hTNF. Since a signaling pattern identical to NGR-hTNF was obtained with hTNF and NGR-sequence given as distinct molecules, the inhibition observed on the survival pathways was presumably due to a direct effect of the NGR-CD13 engagement on the TNFR signaling pathway. The reduced activation of the pro survival pathways induced by NGR-hTNF correlated with the increased caspases activation and reduced cell survival. This study demonstrates that the binding of the NGR-motif to CD13 determines not only the homing of NGR-hTNF to tumor vessels, but also the increase in its antiangiogenic activity.

The Modern Image, I

The argument in this chapter and the next is that the aesthetic field during the modern period is defined by an elastic and differential regime of motion. These two chapters marshal support for this thesis by looking closely at major arts of the modern age: steel, photographic image, and the novel in Chapter 13, and meter, the action arts, and molecular arts in the next chapter. Although empirically quite different and distributed over hundreds of years, each follows a similar kinetic pattern or regime. This chapter looks at the way in which steel architecture, photography, and the novel move elastically by expanding and contracting and open series of frames (steel frames, photographic frames, and print frames).

Pilot Scale Extraction of Orthosiphon stamineus Raw Material as Rosmarinic Acid Extract

A scale-up protocol for rosmarinic acid extraction from Orthosiphon stamineus was developed using the concept of constant power dissipation for a similar kinetic profile. Rosmarinic acid is the bioactive compound in the herb and therefore, large scale extraction for that compound is important for commercial application. The first-order kinetic equation which was established in the preparative scale extraction was used to describe the extraction of rosmarinic acid from the herb. The agitation speed of the pilot large-scale extractor (451 rpm) was determined based on the constant power dissipation to meet the minimal required speed, namely just-suspended speed (Njs), 450.84 ± 0.88 rpm. The experimental data was fitted well to the proposed kinetic equation with a high correlation coefficient, R2 0.88, and a low root mean square error, RMSE 3.85. This indicates the good performance of the scale-up approach based on the dynamic criterion. Therefore, the use of constant power dissipation to obtain a similar kinetic profile could be applied for phytochemical extraction from herbal plants.

Writing I

This chapter argues that writing did not emerge out of nowhere. It took thousands of years for it to rise to its place of kinetic dominance, particularly toward the end of the ancient period. Writing emerged, like the concept of eternity, through four similar kinetic operations, resulting in a reproducible pattern of centrifugal motion. These operations were not evolutionary or developmental. Three of the four sections in this chapter look at one of the four major kinographic operations as it emerged during a certain time: the introduction of the first tokens of the ancient Near East, dating from around 8500 BCE, around the time of the invention of agriculture; their storage in clay spheres around 3700–3500 BCE; and the first abstract inscriptions as numbers (3400 BCE) and pictographs on tablets (3300 BCE). The fourth of these operations, the creation of written letters in alphabetic systems around 1850 BCE, will be addressed in Chapter 22.