High-Throughput Fluorescent Assay for Inhibitor Screening of Proteases from RNA Viruses

Spanish flu, polio epidemics, and the ongoing COVID-19 pandemic are the most profound examples of severe widespread diseases caused by RNA viruses. The coronavirus pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) demands affordable and reliable assays for testing antivirals. To test inhibitors of viral proteases, we have developed an inexpensive high-throughput assay based on fluorescent energy transfer (FRET). We assayed an array of inhibitors for papain-like protease from SARS-CoV-2 and validated it on protease from the tick-borne encephalitis virus to emphasize its versatility. The reaction progress is monitored as loss of FRET signal of the substrate. This robust and reproducible assay can be used for testing the inhibitors in 96- or 384-well plates.     

Isolation of metallothionein from cells derived from aggressive form of high-grade prostate carcinoma using paramagnetic antibody-modified microbeads off-line coupled with electrochemical and electrophoretic analysis

Prostate cancer with altered zinc(II) cell metabolism is the second most frequently diagnosed cancer in developed countries. The alterations of zinc(II) metabolism can influence metabolism of other metal ions and can also be associated with the expression and translation of metal-binding proteins including metallothioneins. The aim of this article was to optimize immunoseparation protocol based on paramagnetic beads conjugated with protein G for the isolation of metallothionein. Isolated metallothionein was determined by differential pulse voltammetry Brdicka reaction and SDS-PAGE. Optimal conditions: antigen-binding time - 60 min, temperature - 70°C, and buffer composition and pH - acetate buffer, pH 4.3, were determined. Under the optimized conditions, lysates from 22Rv1 prostate cancer cells treated with various concentrations of cadmium(II) and copper(II) ions were analyzed. We observed strong correlation in all experimental groups and all lysate types (r>0.83 at p<0.041) between metallothionein concentration related to viability and concentration of copper(II) ions and cadmium(II) ions in medium. Moreover, the results were compared with standard sample preparation as heat treatment and SDS-PAGE analysis.     

Silver baits for the "miraculous draught" of amphiphilic lanthanide helicates

The axial connection of flexible thioalkyls chains of variable length (n=1–12) within the segmental bis‐tridentate 2‐benzimidazole‐8‐hydroxyquinoline ligands [ L12 ^Cn?2 H]^2? provides amphiphilic receptors designed for the synthesis of neutral dinuclear lanthanides helicates. However, the stoichiometric mixing of metals and ligands in basic media only yields intricate mixtures of poorly soluble aggregates. The addition of Ag^I in solution restores classical helicate architectures for n=3, with the quantitative formation of the discrete D₃‐symmetrical [Ln₂Ag2( L12 ^C3?2 H)₃]²⁺ complexes at millimolar concentration (Ln=La, Eu, Lu). The X‐ray crystal structure supports the formation of [La₂Ag₂( L12 ^C3?2 H)₃][OTf]₂, which exists in the solid state as infinite linear polymers bridged by S‐Ag‐S bonds. In contrast, molecular dynamics (MD) simulations in the gas phase and in solution confirm the experimental diffusion measurements, which imply the formation of discrete molecular entities in these media, in which the sulfur atoms of each lipophilic ligand are rapidly exchanged within the Ag^I coordination sphere. Turned as a predictive tool, MD suggests that this Ag^I templating effect is efficient only for n=1–3, while for n>3 very loose interactions occur between Ag^I and the thioalkyl residues. The subsequent experimental demonstration that only 25 % of the total ligand speciation contributes to the formation of [Ln₂Ag₂( L12 ^C12?2 H)₃]²⁺ in solution puts the bases for a rational approach for the design of amphiphilic helical complexes with predetermined molecular interfaces.     

Structural phenomena in glassy carbon induced by cobalt ion implantation

Glassy carbon (GC) was implanted by 150 keV Co⁺ ions to the doses of 1×10¹⁶ (low dose) and 1×10¹⁷ ions/cm² (high dose). The low dose implantation results in GC structure disordering with formation of amorphous carbon (a-C). Analysis of Rutherford backscattering (RBS) and Raman spectra has revealed 15 at.% of sp³-bonded C atoms in the a-C structure. The in-pane size of sp² clusters was estimated to be 1.1 nm. On the contrary, the high dose ion implantation results in ordering of the a-C structure. Content of the sp³ atoms in a-C was reduced to about 5% and, respectively, the in-plane sp² cluster size was increased up to 2.8 nm. Together with the a-C structure ordering the Raman spectra identifies formation of transpolyacetylene (TPA)-like chains after the high-dose Co⁺ implantation. In parallel, RBS suggests an enhanced diffusion of the implanted cobalt within the modified carbon layer. Correlation of the RBS and Raman results argues a driving role of cobalt diffusion in the TPA-like chains formation and a-C ordering. Great surface roughening observed after the high dose Co⁺ implantation suggests also the pronounced cobalt clustering causing large flux of “free volume” to the surface.     

Different evolutionary trails in the related genomes Cricetus cricetus and Peromyscus eremicus (Rodentia, Cricetidae) uncovered by orthologous satellite DNA repositioning

Constitutive heterochromatin comprises a substantial fraction of the eukaryotic genomes and is mainly composed of tandemly arrayed satellite DNAs (satDNA). These repetitive sequences represent a very dynamic and fast evolving component of genomes. In the present work we report the isolation of Cricetus cricetus (CCR, Cricetidae, Rodentia) centromeric repetitive sequences from chromosome 4 (CCR4/10sat), using the laser microdissection and laser pressure catapulting procedure, followed by DOP-PCR amplification and labelling. Physical mapping by fluorescent in situ hybridisation of these sequences onto C. cricetus and another member of Cricetidae, Peromyscus eremicus, displayed quite interesting patterns. Namely, the centromeric sequences showed to be present in another C. cricetus chromosome (CCR10) besides CCR4. Moreover, these almost chromosome-specific sequences revealed to be present in the P. eremicus genome, and most interestingly, displaying a ubiquitous scattered distribution throughout this karyotype. Finally and in both species, a co-localisation of CCR4/10sat with constitutive heterochromatin was found, either by classical C-banding or C-banding sequential to in situ endonuclease restriction.The presence of these orthologous sequences in both genomes is suggestive of a phylogenetic proximity. Furthermore, the existence of common repetitive DNA sequences with a different chromosomal location foresees the occurrence of an extensive process of karyotype restructuring somehow related with intragenomic movements of these repetitive sequences during the evolutionary process of C. cricetus and P. eremicus species.     

Accelerated Electrophotocatalytic C(sp3)−H Heteroarylation Enabled by an Efficient Continuous-Flow Reactor**

Electrophotocatalytic transformations are garnering attention in organic synthesis, particularly for accessing reactive intermediates under mild conditions. Moving these methodologies to continuous-flow systems, or flow ElectroPhotoCatalysis (f-EPC), showcases potential for scalable processes due to enhanced irradiation, increased electrode surface, and improved mixing of the reaction mixture. Traditional methods sequentially link photochemical and electrochemical reactions, using flow reactors connected in series, yet struggle to accommodate reactive transient species. In this study, we introduce a new flow reactor concept for electrophotocatalysis (EPC) that simultaneously utilizes photons and electrons. The reactor is designed with a transparent electrode and employs cost-effective materials. We used this technology to develop an efficient process for electrophotocatalytic heteroarylation of C(sp³)−H bonds. Importantly, the same setup can also facilitate purely electrochemical and photochemical transformations. This reactor represents a significant advancement in electrophotocatalysis, providing a framework for its application in flow for complex synthetic transformations.     

Effect of SBS on Rheological Properties of Different Base Asphalts

In this note rheological properties of two different polymer modified asphalts (PMA) are compared. PMAs were prepared from two different base asphalts, a soft one (200/300 Pen grade) and a hard one (70/100 Pen grade), by addition of 4% by weight of radial SBS as modifier. Dynamic mechanical properties were determined in a wide temperature range for bases and modified asphalts both before and after artificial aging. As expected, the moduli of the soft base are more sensitive to polymer addition and the two PMAs attain similar maximum values after aging. However a significant difference in the frequency at which the maxima of the loss modulus are reached is maintained within the two materials irrespectively of aging or polymer addition.     

Blends and composites based on fluoropolymers

Fluoropolymers represent a rather unique group of polymeric materials. Essentially, current most widely used commercial fluoropolymers are derivatives of ethylene and propylene, also known as fluorocarbon polymers. Other, more complex fluorinated polymers are also important technically, but these are used in considerably smaller amounts. Because of the unique chemistry and properties, fluorocarbon polymers rarely form good blends. The only exceptions are homopolymers and copolymers of vinylidene fluoride, which form blends based on thermodynamic compatibility with certain polymers, such as acrylates and methacrylates. However, most known fluoropolymers can be used to produce fiber and fabric reinforced composites as well as composite films and coatings.