Thermodynamic Swings: How Ideal Complex of Cas9–RNA/DNA Forms

Most processes of the recognition and formation of specific complexes in living systems begin with collisions in solutions or quasi-solutions. Then, the thermodynamic regulation of complex formation and fine tuning of complexes come into play. Precise regulation is very important in all cellular processes, including genome editing using the CRISPR–Cas9 tool. The Cas9 endonuclease is an essential component of the CRISPR–Cas-based genome editing systems. The attainment of high-specificity and -efficiency Cas9 during targeted DNA cleavage is the main problem that limits the practical application of the CRISPR–Cas9 system. In this study, we analyzed the thermodynamics of interaction of a complex’s components of Cas9–RNA/DNA through experimental and computer simulation methods. We found that there is a small energetic preference during Cas9–RNA/DNA formation from the Cas9–RNA and DNA/DNA duplex. The small difference in binding energy is relevant for biological interactions and could be part of the sequence-specific recognition of double-stranded DNA by the CRISPR–Cas9 system.     

Antiviral Activity of N1,N3-Disubstituted Uracil Derivatives against SARS-CoV-2 Variants of Concern

Despite the widespread use of the COVID-19 vaccines, the search for effective antiviral drugs for the treatment of patients infected with SARS-CoV-2 is still relevant. Genetic variability leads to the continued circulation of new variants of concern (VOC). There is a significant decrease in the effectiveness of antibody-based therapy, which raises concerns about the development of new antiviral drugs with a high spectrum of activity against VOCs. We synthesized new analogs of uracil derivatives where uracil was substituted at the N₁ and N₃ positions. Antiviral activity was studied in Vero E6 cells against VOC, including currently widely circulating SARS-CoV-2 Omicron. All synthesized compounds of the panel showed a wide antiviral effect. In addition, we determined that these compounds inhibit the activity of recombinant SARS-CoV-2 RdRp. Our study suggests that these non-nucleoside uracil-based analogs may be of future use as a treatment for patients infected with circulating SARS-CoV-2 variants.     

Case Report: Compound Heterozygous Variants of the MAN1B1 Gene in a Russian Patient with Rafiq Syndrome

Rafiq syndrome (RAFQS) is a congenital disorder of glycosylation (CDG) that is caused by mutations in the MAN1B1 gene and characterized by impaired protein and lipid glycosylation. RAFQS is characterized by a delay in intellectual and motor development, facial and other dysmorphism, truncal obesity, behavior problems, and hypotonia. We describe a Russian patient with delayed intellectual and motor development, a lack of speech, disorientation in space and time, impaired attention and memory, and episodes of aggression. Screening for lysosomal, amino acid, organic acid, and mitochondrial disorders was normal. The patient was referred for the targeted sequencing of the “Hereditary Metabolic Disorders” panel. The genetic testing revealed two heterozygous pathogenic variants in the MAN1B1 gene: the previously reported c.1000C > T (p.Arg334Cys) and the novel c.1065 + 1 G > C. Thus, the patient’s clinical picture and genetic analysis confirmed RAFQS in the patient.     

Identification of a Novel de Novo Variant in the CASZ1 Causing a Rare Type of Dilated Cardiomyopathy

A new de novo frameshift variant has been identified in the CASZ1 gene leading to severe dilated cardiomyopathy. Methods: The proband was analyzed with WES NGS, post-mortem, using dried blood spots on filters. The variant was verified with Sanger sequencing for the proband and her parents. Results: We reported a proband with a new de novo frameshift mutation, c.3781del (p.(Trp1261GlyfsTer29)), in the CASZ1 gene. The clinical presentation was similar to the severe phenotype described in previous studies. Conclusions: In this study, we described a new case with a frameshift mutation in CASZ1 causing a severe phenotype of dilated cardiomyopathy.     

Microarray Profiling of Vaccination-Induced Antibody Responses to SARS-CoV-2 Variants of Interest and Concern

Mutations in surface proteins enable emerging variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) to escape a substantial fraction of neutralizing antibodies and may thus weaken vaccine-driven immunity. To compare available vaccines and justify revaccination, rapid evaluation of antibody (Ab) responses to currently circulating SARS-CoV-2 variants of interest (VOI) and concern (VOC) is needed. Here, we developed a multiplex protein microarray-based system for rapid profiling of anti-SARS-CoV-2 Ab levels in human sera. The microarray system was validated using sera samples from SARS-CoV-2-free donors and those diagnosed with COVID-19 based on PCR and enzyme immunoassays. Microarray-based profiling of vaccinated donors revealed a substantial difference in anti-VOC Ab levels elicited by the replication-deficient adenovirus vector-base (Sputnik V) and whole-virion (CoviVac Russia COVID-19) vaccines. Whole-virion vaccine-induced Abs showed minor but statistically significant cross-reactivity with the human blood coagulation factor 1 (fibrinogen) and thrombin. However, their effects on blood clotting were negligible, according to thrombin time tests, providing evidence against the concept of pronounced cross-reactivity-related side effects of the vaccine. Importantly, all samples were collected in the pre-Omicron period but showed noticeable responses to the receptor-binding domain (RBD) of the Omicron spike protein. Thus, using the new express Ab-profiling system, we confirmed the inter-variant cross-reactivity of the anti-SARS-CoV-2 Abs and demonstrated the relative potency of the vaccines against new VOCs.     

Evaluation of Auranofin Loading within Ferritin Nanocages

Auranofin (AF), a gold(I) compound that is currently used for the treatment of rheumatoid arthritis and is in clinical trials for its promising anticancer activity, was encapsulated within the human H-chain and the horse spleen ferritin nanocages using the alkaline disassembly/reassembly protocol. The aim of the work was to highlight possible differences in their drug loading capacity and efficacy. The drug-loaded ferritins were characterized via UV-vis absorption spectroscopy and inductively coupled plasma-atomic emission spectroscopy to assess AF encapsulation and to define the exact amount of gold atoms trapped in the Ft cavity. The crystal structures allowed us to define the nature of AF interaction with both ferritins and to identify the gold binding sites. Moreover, the biological characterization let us to obtain preliminary information on the cytotoxic effect of AF when bound to the human H-chain.     

Properties of Protein Isolates from Marine Hydrobionts Obtained by Isoelectric Solubilisation/Precipitation: Influence of Temperature and Processing Time

Protein isolates were obtained from marine hydrobionts by the method of isoelectric precipitation with a preliminary stage of protein alkaline solubilisation. Northern blue whiting was chosen as the raw material. Various technological modes of the solubilisation stage were used: the temperature of the reaction mixture was 4 or 20 °C, and the duration was 4 or 16 h. The yield of the product was 44–45% with a high content of the main component (protein) equal to about 95%. It has been shown that a decrease in the temperature and duration of the alkaline solubilisation stage provides the production of protein isolates with good technological properties, a low solubility, high swelling and high emulsifying ability, necessary for its use in the production of functional food products, including therapeutic and prophylactic effects. These technological properties are explained by a change in the composition and structure of the protein, the change being an increase in the content of essential amino acids and the proportion of α-helices in the polypeptide chain. The main patterns obtained will be used to obtain protein isolates from marine molluscs.     

Structure of pectic polysaccharides isolated from onion Allium cepa L. using a simulated gastric medium and their effect on intestinal absorption

The polysaccharide fraction extracted with simulated gastric juice from onion bulbs contained a mixture of galactan with short-length sugar chains, pectic polysaccharides and evident content of proteinaceous material. Galacturonan and rhamnogalacturonan were the main constituents of the linear regions of the sugar chains of the pectic polysaccharides. The ramified regions included rhamnogalacturonan-I. NMR data revealed that the side chains of the ramified region contained mainly 1,4-linked β-d-galactopyranose residues and lesser content of 1,3-linked β-d-galactopyranose and 1,5-linked α-l-arabinofuranose residues. Furthermore, the proteinaceous material was determined to be partly linked to the sugar chains.     

Dependence of thermoelectric behaviour on severe plastic deformation parameters: A case study on p-type skutterudite DD0.60Fe3CoSb12

High-pressure torsion (HPT), a severe plastic deformation technique, can effectively improve the thermoelectric performance of skutterudites, resulting in ZT values higher than for ball-milled and hot-pressed (BMHP) samples. In this paper the influence of the HPT parameters, i.e. the number of revolutions (equivalent to the applied strain), the processing temperature and the hydrostatic pressure on the microstructural and thermoelectric properties of the skutterudite DD_0.60Fe₃CoSb₁₂ are evaluated and compared with the BMHP samples before HPT processing. Whilst the three parameters have specific effects on (i) the crystallite size, (ii) the density of lattice defects and (ii) the density of cracks, a suitable combination thereof allows for an increase of the figure of merit by at least 20%.