Nitric Oxide in Seed Biology

Nitric oxide (NO) has been recognized as a gasotransmitter in the mainstream of plant research since the beginning of the 21st century. It is produced in plant tissue and the environment. It influences plant physiology during every ontogenetic stage from seed germination to plant senescence. In this review, we demonstrate the increased interest in NO as a regulatory molecule in combination with other signalling molecules and phytohormones in the information network of plant cells. This work is a summary of the current knowledge on NO action in seeds, starting from seed pretreatment techniques applied to increase seed quality. We describe mode of action of NO in the regulation of seed dormancy, germination, and aging. During each stage of seed physiology, NO appears to act as a key agent with a predominantly beneficial effect.     

Preliminary Evidence of the Potent and Selective Adenosine A2B Receptor Antagonist PSB-603 in Reducing Obesity and Some of Its Associated Metabolic Disorders in Mice

The adenosine A_2A and A_2B receptors are promising therapeutic targets in the treatment of obesity and diabetes since the agonists and antagonists of these receptors have the potential to positively affect metabolic disorders. The present study investigated the link between body weight reduction, glucose homeostasis, and anti-inflammatory activity induced by a highly potent and specific adenosine A_2B receptor antagonist, compound PSB-603. Mice were fed a high-fat diet for 14 weeks, and after 12 weeks, they were treated for 14 days intraperitoneally with the test compound. The A₁/A_2A/A_2B receptor antagonist theophylline was used as a reference. Following two weeks of treatment, different biochemical parameters were determined, including total cholesterol, triglycerides, glucose, TNF-α, and IL-6 blood levels, as well as glucose and insulin tolerance. To avoid false positive results, mouse locomotor and spontaneous activities were assessed. Both theophylline and PSB-603 significantly reduced body weight in obese mice. Both compounds had no effects on glucose levels in the obese state; however, PSB-603, contrary to theophylline, significantly reduced triglycerides and total cholesterol blood levels. Thus, our observations showed that selective A_2B adenosine receptor blockade has a more favourable effect on the lipid profile than nonselective inhibition.     

Secondary Structure in Amyloids in Relation to Their Wild Type Forms

The amyloid structures and their wild type forms, available in the PDB database, provide the basis for comparative analyses. Globular proteins are characterised by a 3D spatial structure, while a chain in any amyloid fibril has a 2D structure. Another difference lies in the structuring of the hydrogen bond network. Amyloid forms theoretically engage all the NH and C=O groups of the peptide bonds in a chain with two hydrogen bonds each. In addition, the hydrogen bond network is highly ordered—as perpendicular to the plane of the chain. The β-structure segments provide the hydrogen bond system with an anti-parallel system. The folds appearing in the rectilinear propagation of the segment with the β-structure are caused by just by one of the residues in the sequence—residues with a Rα-helical or Lα-helical conformation. The antiparallel system of the hydrogen bonds in the β-structure sections at the site of the amino acid with a Rα- or Lα-helical conformation changes into a parallel system locally. This system also ensures that the involvement of the C=O and H-N groups in the construction of the interchain hydrogen bond, while maintaining a perpendicular orientation towards the plane of the chain. Conformational analysis at the level of the Phi and Psi angles indicates the presence of the conditions for the structures observed in the amyloids. The specificity of amyloid structures with the dominant conformation expressed as |Psi| = |Phi| reveals the system of organisation present in amyloid fibrils. The Phi, Psi angles, as present in this particular structure, transformed to form |Psi| = |Phi| appear to be ordered co-linearly. Therefore, the calculation of the correlation coefficient may express the distribution around this idealised localisation on the Ramachandran map. Additionally, when the outstanding points are eliminated, the part of amyloid chain can be classified as fulfilling the defined conditions. In addition, the presentation of the chain structure using geometric parameters, V-angle—the angle between the planes of the adjacent peptide bonds (angle versus the virtual axis Cα-Cα) and the radius of the curvature R, depending on the size of the angle V, allows for a quantitative assessment of changes during amyloid transformation.     

Brownian Motion Influence on AFM Exosomes’ Size Measurements

Extracellular vesicles are evaluated by nanoparticle tracking analysis (NTA), providing information on their hydrodynamic diameters, and by atomic force microscopy (AFM) to calculate their geometric diameters. The aim of this study is to explore the influence of Brownian movements in a sample drop and preparation time on imaging-based measurements and to determine the relationship between the geometric and hydrodynamic sizes of the extracellular vesicles measured by the AFM and the NTA, respectively. Exosomes derived from the human prostate cancer cell line PC3 were evaluated by NTA and AFM, and those results were compared with Monte Carlo simulations. The mean size, evaluated by AFM shortly after application on the mica substrate, is less than its real value. It obtains the correct value faster for a thinner sample drop. Fitting the log-normal distribution to the geometric and hydrodynamic diameters leads to the conclusion that the latter could arise from the former by linear scaling by a factor that could be used to characterize the analyzed extracellular vesicles. The size of the vesicles attached to the mica substrate depends on time. The effect of Brownian motion and stretch of the lipid bilayer should be considered in the context of exosome AFM studies.     

Applications of Metabolomics in Calcium Metabolism Disorders in Humans

The pathogenesis of the disorders of calcium metabolism is not fully understood. This review discusses the studies in which metabolomics was applied in this area. Indeed, metabolomics could play an essential role in discovering biomarkers and elucidating pathological mechanisms. Despite the limited bibliography, the present review highlights the potential of metabolomics in identifying the biomarkers of some of the most common endocrine disorders, such as primary hyperparathyroidism (PHPT), secondary hyperparathyroidism (SHPT), calcium deficiency, osteoporosis and vitamin D supplementation. Metabolites related to above-mentioned diseorders were grouped into specific classes and mapped into metabolic pathways. Furthermore, disturbed metabolic pathways can open up new directions for the in-depth exploration of the basic mechanisms of these diseases at the molecular level.     

Boron Clusters as Enhancers of RNase H Activity in the Smart Strategy of Gene Silencing by Antisense Oligonucleotides

Boron cluster-conjugated antisense oligonucleotides (B-ASOs) have already been developed as therapeutic agents with “two faces”, namely as potential antisense inhibitors of gene expression and as boron carriers for boron neutron capture therapy (BNCT). The previously observed high antisense activity of some B-ASOs targeting the epidermal growth factor receptor (EGFR) could not be rationally assigned to the positioning of the boron cluster unit: 1,2-dicarba-closo-dodecaborane (0), [(3,3′-Iron-1,2,1′,2′-dicarbollide) (1-), FESAN], and dodecaborate (2-) in the ASO chain and its structure or charge. For further understanding of this observation, we performed systematic studies on the efficiency of RNase H against a series of B-ASOs models. The results of kinetic analysis showed that pyrimidine-enriched B-ASO oligomers activated RNase H more efficiently than non-modified ASO. The presence of a single FESAN unit at a specific position of the B-ASO increased the kinetics of enzymatic hydrolysis of complementary RNA more than 30-fold compared with unmodified duplex ASO/RNA. Moreover, the rate of RNA hydrolysis enhanced with the increase in the negative charge of the boron cluster in the B-ASO chain. In conclusion, a “smart” strategy using ASOs conjugated with boron clusters is a milestone for the development of more efficient antisense therapeutic nucleic acids as inhibitors of gene expression.     

SARS-CoV-2 Spike Protein (RBD) Subunit Adsorption at Abiotic Surfaces and Corona Formation at Polymer Particles

The adsorption kinetics of the SARS-CoV-2 spike protein subunit with the receptor binding domain at abiotic surfaces was investigated. A combination of sensitive methods was used such as atomic force microscopy yielding a molecular resolution, a quartz microbalance, and optical waveguide lightmode spectroscopy. The two latter methods yielded in situ information about the protein adsorption kinetics under flow conditions. It was established that at pH 3.5–4 the protein adsorbed on mica and silica surfaces in the form of compact quasi-spherical aggregates with an average size of 14 nm. The maximum coverage of the layers was equal to 3 and 1 mg m⁻² at pH 4 and 7.4, respectively. The experimental data were successfully interpreted in terms of theoretical results derived from modeling. The experiments performed for flat substrates were complemented by investigations of the protein corona formation at polymer particles carried out using in situ laser Doppler velocimetry technique. In this way, the zeta potential of the protein layers was acquired as a function of the coverage. Applying the electrokinetic model, these primary data were converted to the dependence of the subunit zeta potential on pH. It was shown that a complete acid-base characteristic of the layer can be acquired only using nanomolar quantities of the protein.     

Computational Pipeline for Reference-Free Comparative Analysis of RNA 3D Structures Applied to SARS-CoV-2 UTR Models

RNA is a unique biomolecule that is involved in a variety of fundamental biological functions, all of which depend solely on its structure and dynamics. Since the experimental determination of crystal RNA structures is laborious, computational 3D structure prediction methods are experiencing an ongoing and thriving development. Such methods can lead to many models; thus, it is necessary to build comparisons and extract common structural motifs for further medical or biological studies. Here, we introduce a computational pipeline dedicated to reference-free high-throughput comparative analysis of 3D RNA structures. We show its application in the RNA-Puzzles challenge, in which five participating groups attempted to predict the three-dimensional structures of 5′- and 3′-untranslated regions (UTRs) of the SARS-CoV-2 genome. We report the results of this puzzle and discuss the structural motifs obtained from the analysis. All simulated models and tools incorporated into the pipeline are open to scientific and academic use.