Search

Papain-like protease PLpro, a domain within a large polyfunctional protein, nsp3, plays key roles in the life cycle of SARS-CoV-2, being responsible for the first events of cleavage of a polyprotein into individual proteins (nsp1–4) as well as for the suppression of cellular immunity. Here, we developed a new genetically encoded fluorescent sensor, named PLpro-ERNuc, for detection of PLpro activity in living cells using a translocation-based readout. The sensor was designed as follows. A fragment of nsp3 protein was used to direct the sensor on the cytoplasmic surface of the endoplasmic reticulum (ER) membrane, thus closely mimicking the natural target of PLpro. The fluorescent part included two bright fluorescent proteins—red mScarlet I and green mNeonGreen—separated by a linker with the PLpro cleavage site. A nuclear localization signal (NLS) was attached to ensure accumulation of mNeonGreen into the nucleus upon cleavage. We tested PLpro-ERNuc in a model of recombinant PLpro expressed in HeLa cells. The sensor demonstrated the expected cytoplasmic reticular network in the red and green channels in the absence of protease, and efficient translocation of the green signal into nuclei in the PLpro-expressing cells (14-fold increase in the nucleus/cytoplasm ratio). Then, we used PLpro-ERNuc in a model of Huh7.5 cells infected with the SARS-CoV-2 virus, where it showed robust ER-to-nucleus translocation of the green signal in the infected cells 24 h post infection. We believe that PLpro-ERNuc represents a useful tool for screening PLpro inhibitors as well as for monitoring virus spread in a culture. [ABSTRACT FROM AUTHOR]

The computation of the real part of the nucleus-nucleus optical potential based on the microscopic double-folding model was implemented within both the MPI and OpenMP parallelising techniques. Test calculations of the total cross section of the 6He + 28Si scattering at the energy 50 A MeV show that both techniques provide significant comparable speedup of the calculations.

Fitness landscapes (1,2) depict how genotypes manifest at the phenotypic level and form the basis of our understanding of many areas of biology (2-7), yet their properties remain elusive. Previous [...]

The data on the 12,14Be + p elastic scattering cross sections at 700 Mev are compared with those obtained by solving the relativistic wave equation with the microscopic optical potentials calculated as folding of the NN amplitude of scattering with densities of these nuclei in the form of the symmetrized Fermi function with the fitted radius and diffuseness parameters, and also with the densities obtained in two microscopic models, based on the Generator Coordinate Method (GCM) and the other one – on the Variational Method of Calculations (VMC). For 12Be, above models turn out to be in a small disagreement with the data at "large" angles of scattering θ ≥ 9°, while for the 14Be one sees some inconsistence at smaller angles, too.

Live-cell super-resolution of proteins labeled with genetically encoded fluorescent tags is a challenging task because of the imperfect labeling and the inevitable deterioration of the signal in the course of the experiment. Incomplete maturation of the covalently attached fluorescent tags, inefficient photoconversion, and photobleaching further complicate prolonged live-cell nanoscopy. We have implemented two strategies for lowering the photodamage: ensuring the dynamic replacement of damaged molecules and establishing conditions for the robust intrinsic blinking of the tags at lower illumination powers.

Epigenome contains a lot of information about cell state. Epigenetic analysis includes primarily sequence-based methods, which provide detailed data on distribution of modifications along the genome, but are poorly applicable for screenings. Specific fluorescence labeling and imaging of epigenetic modifications is an attractive complementary approach. It is currently based mainly on histone modifications study. We expect that inclusion of DNA modifications into imaging-based study would empower the method. In this review we discuss methods for fluorescence imaging of DNA modifications (mainly 5-methylcytosine). It opens an easy way to single cell analysis and high-throughput screening. Moreover, tracking epigenome changes in live cells becomes possible with genetically encoded probes. • Genomic DNA modification profile reflects a state of the cell. • Specific probes enable fluorescence imaging of DNA methylation. • Genetically encoded probes can be used to track epigenome changes in live cells. [ABSTRACT FROM AUTHOR]

Elastic scattering cross sections are calculated and compared to the data of π± + 28Si and 40Ca at energies 130, 180, and 230 MeV by using the both microscopic optical potentials (OP), the high-energy folding and the local Kisslinger type potentials. In the folding OP, we use the known nuclear density distributions while parameters of the elementary πN-scattering amplitude are fitted to the data with the aim to estimate the inmedium effect on pions scattered on bound nucleons. As to the local modified Kisslinger OP its parameters are known from the earlier studies. The cross sections for both OPs are calculated by solving the Klein-Gordon wave equation, and thus the relativistic and distortion effects on the process are accounted for exactly. A fairly well agreement with experimental data was obtained and the decisive role of a surface region of potentials was established where both OPs occur in close coincidence.