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Abstract Background The organism-wide effects of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) viral infection are well studied, but little is known about the dynamics of how the infection spreads in time among or within cells due to the scarcity of suitable high-resolution experimental systems. It has been reported that SARS-CoV-2 infection pathways converge at calcium influx and subcellular calcium distribution changes. Imaging combined with a proper staining technique is an effective tool for studying subcellular calcium-related infection and replication mechanisms at such resolutions. Methods Using two-photon (2P) fluorescence imaging with our novel Ca-selective dye, automated image analysis and clustering analysis were applied to reveal titer and variant effects on SARS-CoV-2-infected Vero E6 cells. Results The application of a new calcium sensor molecule is shown, combined with a high-end 2P technique for imaging and identifying the patterns associated with cellular infection damage within cells. Vero E6 cells infected with SARS-CoV-2 variants, D614G or B.1.1.7, exhibit elevated cytosolic calcium levels, allowing infection monitoring by tracking the cellular changes in calcium level by the internalized calcium sensor. The imaging provides valuable information on how the level and intracellular distribution of calcium are perturbed during the infection. Moreover, two-photon calcium sensing allowed the distinction of infections by two studied viral variants via cluster analysis of the image parameters. This approach will facilitate the study of cellular correlates of infection and their quantification depending on viral variants and viral load. Conclusions We propose a new two-photon microscopy-based method combined with a cell-internalized sensor to quantify the level of SARS-CoV-2 infection. We optimized the applied dye concentrations to not interfere with viral fusion and viral replication events. The presented method ensured the proper monitoring of viral infection, replication, and cell fate. It also enabled distinguishing intracellular details of cell damage, such as vacuole and apoptotic body formation. Using clustering analysis, 2P microscopy calcium fluorescence images were suitable to distinguish two different viral variants in cell cultures. Cellular harm levels read out by calcium imaging were quantitatively related to the initial viral multiplicity of infection numbers. Thus, 2P quantitative calcium imaging might be used as a correlate of infection or a correlate of activity in cellular antiviral studies. Graphical Abstract

Investigating the kinetics of polymerization reactions is a powerful tool to obtain information for the engineering design of such processes. It provides insights into how the relative rates of elementary reactions which influence both the kinetics of the reactions and thus characteristics of the products as well, particularly when the reaction parameters of these reactions are unknown. Among polymerization processes, ring-opening metathesis polymerization (ROMP) has gained broad academic and industrial interest, due to its mild conditions to obtain a wide range of polymer products. In this study, kinetic Monte Carlo simulation was applied to reveal the effect of the elementary reactions of ROMP on the kinetics and product distribution. Both the propagation and the cross-metathesis reactions were considered, with the latter leading to the formation of various types of macrospecies whose population levels were also monitored. Relevant tendencies were observed regarding how the variations in the reaction parameters of the elementary reactions of this polymerization process, such as reaction probabilities, monomer addition method, catalyst-to-monomer ratio, and reaction time, affect the kinetics of the polymerization process and the product distributions. These variations also influence the key macromolecular parameters of the resulting polymeric materials, including chain length distribution (CLD), population levels, and polymer functionalities. The results of this study indicate that this simulation technique is a valuable tool for mapping the tailoring possibilities of modifying the reaction parameters of ROMP to achieve desired properties. These properties include number average molecular weight, polydispersity, chain end functionality, and macrocycle-free products.

Not all amide bonds are created equally. The purpose of the present paper is the reinterpretation of the amide group by means of two concepts: amidicity and carbonylicity. These concepts are meant to provide a new viewpoint in defining the stability and reactivity of amides. With the help of simple quantum-chemical calculations, practicing chemists can easily predict the outcome of a desired process. The main benefit of the concepts is their simplicity. They provide intuitive, but quasi-thermodynamic data, making them a practical rule of thumb for routine use. In the current paper we demonstrate the performance of our methods to describe the chemical character of an amide bond strength and the way of its activation methods. Examples include transamidation, acyl transfer and amide reductions. Also, the method is highly capable for simple interpretation of mechanisms for biological processes, such as protein splicing and drug mechanisms. Finally, we demonstrate how these methods can provide information about photo-activation of amides, through the examples of two caged neurotransmitter derivatives.

Sex pheromones are commonly used in traps to monitor populations and movements of male click beetles, but to date few attractants have been identified for females. Notable exceptions are plant-derived kairomones for female Agriotes brevis and A. ustulatus, allowing the monitoring of both males and females of these species with lures containing both pheromones and plant volatiles. The attractiveness of these plant volatiles for two congeners, A. obscurus and A. lineatus, which are agricultural pests in Europe and North America, was evaluated in the current study. Both the four-component MINIM plant-derived lure for A. brevis, and the blend of (E)-anethol and (E)-cinnamaldehyde for A. ustulatus, were not attractive to A. obscurus and A. lineatus, and instead appeared to reduce captures—both when compared to blank controls, and when blended with and compared to the sex pheromones of these species. This was most pronounced in A. obscurus, where (E)-anethol and (E)-cinnamaldehyde reduced male captures by 43 and 37%, respectively. Combining the pheromones of A. obscurus and A. lineatus reduced captures of these species by 77 and 19%, respectively, compared to these pheromones singly. This suggests that attractants for female click beetles can be highly species-specific, and that the blending of pheromones of congeneric species with each other, or with plant volatiles, can reduce captures. Further research into developing such attractants for economic species is urgently needed.