Conserved structures and dynamics in 5'-proximal regions of Betacoronavirus RNA genomes.

Betacoronaviruses are a genus within the Coronaviridae family of RNA viruses. They are capable of infecting vertebrates and causing epidemics as well as global pandemics in humans. Mitigating the threat posed by Betacoronaviruses requires an understanding of their molecular diversity. The development of novel antivirals hinges on understanding the key regulatory elements within the viral RNA genomes, in particular the 5'-proximal region, which is pivotal for viral protein synthesis. Using a combination of cryo-electron microscopy, atomic force microscopy, chemical probing, and computational modeling, we determined the structures of 5'-proximal regions in RNA genomes of Betacoronaviruses from four subgenera: OC43-CoV, SARS-CoV-2, MERS-CoV, and Rousettus bat-CoV. We obtained cryo-electron microscopy maps and determined atomic-resolution models for the stem-loop-5 (SL5) region at the translation start site and found that despite low sequence similarity and variable length of the helical elements it exhibits a remarkable structural conservation. Atomic force microscopy imaging revealed a common domain organization and a dynamic arrangement of structural elements connected with flexible linkers across all four Betacoronavirus subgenera. Together, these results reveal common features of a critical regulatory region shared between different Betacoronavirus RNA genomes, which may allow targeting of these RNAs by broad-spectrum antiviral therapeutics.

Kartogenin-loaded liposomes coated with alkylated chondroitin sulfate for cartilage repair.

Cartilage loss is a common clinical problem, which leads to significant pain, dysfunction, and even disability. As a result, there is growing interest in using small, non-protein molecules to protect or repair cartilage. Kartogenin (KGN), a small hydrophobic molecule, shows chondroprotective and chondrogenic properties. In this study, we embedded KGN in liposomes, and the whole system was stabilized by covering it with n-octadecylated (at two different substitution degrees) chondroitin sulfate (CS) derivatives. We investigated the interactions of empty liposomes and KGN-loaded liposomes with both CS derivatives using various physicochemical techniques, which revealed that hydrophobically modified CSs can interact with both neutral lipid membrane and negatively charged loaded-KGN lipid membrane. The cytotoxicity and chondrogenic properties of the polysaccharides and liposome-CS formulations of KGN were analyzed towards mesenchymal stem cells (MSCs). The results showed that the alkylated CS exhibited cytotoxic properties. The higher substituted CS self-assembles into stable nanoaggregates that can form a corona on the surface of liposomes, eliminating the overall cytotoxicity of this polymer. However, all tested chondrogenic markers' expression levels are enhanced for KGN-loaded liposomes and coated by lower substituted CS. Furthermore, the undesirable hypertrophy effect for this formulation significantly decreased compared to pure polymeric derivative.

Autophagy of Candida albicans cells after the action of earthworm Venetin-1 nanoparticle with protease inhibitor activity.

The present studies show the effect of the Venetin-1 protein-polysaccharide complex obtained from the coelomic fluid of the earthworm Dendrobaena veneta on Candida albicans cells. They are a continuation of research on the mechanisms of action, cellular targets, and modes of cell death. After the action of Venetin-1, a reduced survival rate of the yeast cells was noted. The cells were observed to be enlarged compared to the controls and deformed. In addition, an increase in the number of cells with clearly enlarged vacuoles was noted. The detected autophagy process was confirmed using differential interference contrast, fluorescence microscopy, and transmission electron microscopy. Autophagic vesicles were best visible after incubation of fungus cells with the Venetin-1 complex at a concentration of 50 and 100 µg mL-1. The changes in the vacuoles were accompanied by changes in the size of mitochondria, which is probably related to the previously documented oxidative stress. The aggregation properties of Venetin-1 were characterized. Based on the results of the zeta potential at the Venetin-1/KCl interface, the pHiep = 4 point was determined, i.e. the zeta potential becomes positive above pH = 4 and is negative below this value, which may affect the electrostatic interactions with other particles surrounding Venetin-1.

Studies on Chemical Composition, Structure and Potential Applications of Keratoisis Corals.

The chemical composition and structure of bamboo octocoral Keratoisis spp. skeletons were investigated by using: Scanning Electron Microscopy SEM, Raman Microscopy, X-ray Diffraction XRD, Laser Ablation-Inductively Coupled Plasma LA-ICP, and amino acid analyzers. Elements discovered in the nodes (mainly organic parts of the skeleton) of bamboo corals showed a very interesting arrangement in the growth ring areas, most probably enabling the application of bamboo corals as palaeochronometers and palaeothermometers. LA-ICP results showed that these gorgonian corals had an unusually large content of bromine, larger than any other organism yet studied. The local concentration of bromine in the organic part of the growth rings of one of the studied corals grew up to 29,000 ppm of bromine. That is over 440 times more than is contained in marine water and 35 times more than Murex contains, the species which was used to make Tyrian purple in ancient times. The organic matter of corals is called gorgonin, the specific substance that both from the XRD and Raman studies seem to be very similar to the reptile and bird keratins and less similar to the mammalian keratins. The missing cross-linking by S-S bridges, absence of aromatic rings, and significant participation of ß-turn organization of peptides differs gorgonin from keratins. Perhaps, the gorgonin belongs to the affined but still different substances concerning reptile and bird keratin and in relation to the more advanced version-the mammalian one. Chemical components of bamboo corals seem to have great medical potential, with the internodes as material substituting the hard tissues and the nodes as the components of medicines.

Silmitasertib (CX-4945), a Clinically Used CK2-Kinase Inhibitor with Additional Effects on GSK3ß and DYRK1A Kinases: A Structural Perspective.

A clinical casein kinase 2 inhibitor, CX-4945 (silmitasertib), shows significant affinity toward the DYRK1A and GSK3ß kinases, involved in down syndrome phenotypes, Alzheimer's disease, circadian clock regulation, and diabetes. This off-target activity offers an opportunity for studying the effect of the DYRK1A/GSK3ß kinase system in disease biology and possible line extension. Motivated by the dual inhibition of these kinases, we solved and analyzed the crystal structures of DYRK1A and GSK3ß with CX-4945. We built a quantum-chemistry-based model to rationalize the compound affinity for CK2α, DYRK1A, and GSK3ß kinases. Our calculations identified a key element for CK2α's subnanomolar affinity to CX-4945. The methodology is expandable to other kinase selectivity modeling. We show that the inhibitor limits DYRK1A- and GSK3ß-mediated cyclin D1 phosphorylation and reduces kinase-mediated NFAT signaling in the cell. Given the CX-4945's clinical and pharmacological profile, this inhibitory activity makes it an interesting candidate with potential for application in additional disease areas.

Measurements of Energetic States Resulting from Ion Exchanges in the Isomorphic Crystals of Apatites and Bioapatites.

Developments in the field of nanostructures open new ways for designing and manufacturing innovative materials. Here, we focused on new original ways of calculating energy changes during the substitution of foreign ions into the structure of apatites and bioapatites. Using these tools, the energetic costs of ion exchanges were calculated for the exemplary cases known from the literature. It was established that the most costly were ion exchanges of some cations inside apatites and of anions, and the least costly exchanges in tetrad channel positions. Real energy expenses for bioapatites are much smaller in comparison to mineral apatites due to the limited involvement of magnesium and carbonates in the structure of hard tissues. They are of the order of several electron volts per ion. The rigorous dependences of the energy changes and crystallographic cell volumes on the ionic radii of introduced cations were proved. The differentiation of the positioning of foreign ions in locations of Ca(I) and Ca(II) could be calculated in the case of a Ca-Pb reaction in hydroxyapatite. The energetic effects of tooth aging were indicated. The ability of energy change calculation during the ion exchange for isomorphic substances widens the advantages resulting from X-ray diffraction measurements.

Novel Venetin-1 nanoparticle from earthworm coelomic fluid as a promising agent for the treatment of non-small cell lung cancer.

The present research shows the antitumor activity of a protein-polysaccharide complex Venetin-1 obtained from the coelomic fluid of Dendrobaena veneta earthworms against A549 cancer cells. The investigations are a continuation of experiments on the antitumor activity of coelomic fluid obtained from this species. The Venetin-1 nanoparticle was obtained after thermal treatment of the coelomic fluid, separation from coelomocytes, filtration, and lyophilization. The preparation showed a selective effect on cancer cells, whereas normal cells were unaffected. Venetin-1 was effective against the lung cancer cells at doses of 31.3 and 62.5 µg/ml, and the results were imaged using light microscopy and scanning electron microscopy (SEM). The cells died mainly via the apoptosis pathway. Necrotic cells appeared sporadically in the microscopic view. SEM imaging revealed complete destruction of the A549 cells after the incubation with Venetin-1. The atomic force microscopy (AFM) analyses showed changes in the topography, peak force error images, and Young's modulus (elasticity) of the A549 cells after the incubation with Venetin-1. The transmission electron cryomicroscopy (Cryo-TEM) analysis indicated a polymeric nature of the analyzed preparation. The samples of Venetin-1 showed a very homogeneous size profile with the microparticle size of approximately 58.23 nm. A significant decrease in Venetin-1 binding to sphingomyelin was observed. Venetin-1 lost its pore-forming activity or deactivation of the pore-forming activity occurred. This confirms the absence of hemolytic capacity of Venetin-1 towards red blood cells. The conducted analyses show the suitability of the obtained complex for biomedical research. The next step will consist in analyses of the effect of Venetin-1 on the immune system in mice.

Destabilization of mutated human PUS3 protein causes intellectual disability.

Pseudouridine (Ψ) is an RNA base modification ubiquitously found in many types of RNAs. In humans, the isomerization of uridine is catalyzed by different stand-alone pseudouridine synthases (PUS). Genomic mutations in the human pseudouridine synthase 3 gene (PUS3) have been identified in patients with neurodevelopmental disorders. However, the underlying molecular mechanisms that cause the disease phenotypes remain elusive. Here, we utilize exome sequencing to identify genomic variants that lead to a homozygous amino acid substitution (p.[(Tyr71Cys)];[(Tyr71Cys)]) in human PUS3 of two affected individuals and a compound heterozygous substitution (p.[(Tyr71Cys)];[(Ile299Thr)]) in a third patient. We obtain wild-type and mutated full-length human recombinant PUS3 proteins and characterize the enzymatic activity in vitro. Unexpectedly, we find that the p.Tyr71Cys substitution neither affect tRNA binding nor pseudouridylation activity in vitro, but strongly impair the thermostability profile of PUS3, while the p.Ile299Thr mutation causes protein aggregation. Concomitantly, we observe that the PUS3 protein levels as well as the level of PUS3-dependent Ψ levels are strongly reduced in fibroblasts derived from all three patients. In summary, our results directly illustrate the link between the identified PUS3 variants and reduced Ψ levels in the patient cells, providing a molecular explanation for the observed clinical phenotypes.

Hierarchy of Bioapatites.

Apatites are one of the most intensively studied materials for possible biomedical applications. New perspectives of possible application of apatites correspond with the development of nanomaterials and nanocompounds. Here, an effort to systematize different kinds of human bioapatites forming bones, dentin, and enamel was undertaken. The precursors of bioapatites and hydroxyapatite were also considered. The rigorous consideration of compositions and stoichiometry of bioapatites allowed us to establish an order in their mutual sequence. The chemical reactions describing potential transformations of biomaterials from octacalcium phosphate into hydroxyapatite via all intermediate stages were postulated. Regardless of whether the reactions occur in reality, all apatite biomaterials behave as if they participate in them. To conserve the charge, additional free charges were introduced, with an assumed meaning to be joined with the defects. The distribution of defects was coupled with the values of crystallographic parameters "a" and "c". The energetic balances of bioapatite transformations were calculated. The apatite biomaterials are surprisingly regular structures with non-integer stoichiometric coefficients. The results presented here will be helpful for the further design and development of nanomaterials.

Novel diagnostic and prognostic factors for the advanced melanoma based on the glycosylation-related changes studied by biophysical profiling methods.

Melanoma is a life-threatening disease due to the early onset of metastasis and frequent resistance to the applied treatment. For now, no single histological, immunohistochemical or serological biomarker was able to provide a precise predictive value for the aggressive behavior in melanoma patients. Thus, the search for quantifying methods allowing a simultaneous diagnosis and prognosis of melanoma patients is highly desirable. By investigating specific molecular interactions with some biosensor-based techniques, one can determine novel prognostic factors for this tumor. In our previous study, we have shown the possibility of a qualitative in vitro distinguishing the commercially available melanoma cells at different progression stages based on the measurements of the lectin Concanavalin A interacting with surface glycans present on cells. Here, we present the results of the quantitative diagnostic and prognostic study of both commercial and patient-derived melanoma cells based on the evaluation of two novel factors: lectin affinity and glycan viscoelastic index obtained from the quartz crystal microbalance with dissipation monitoring (QCM-D) measurements. Two approaches to the QCM-D measurements were applied, the first uses the ability of melanoma cells to grow as a monolayer of cells on the sensor (cell-based sensors), and the second shortens the time of the analysis (suspension cell based-sensors). The results were confirmed by the complementary label-free (atomic force microscopy, AFM; and surface plasmon resonance, SPR) and labeling (lectin-ELISA; and microscale thermophoresis, MST) techniques. This new approach provides additional quantitative diagnosis and a personalized prognosis which can be done simultaneously to the traditional histopathological analysis.

Evaluation of Xa inhibitors as potential inhibitors of the SARS-CoV-2 Mpro protease.

Based on previous large-scale in silico screening several factor Xa inhibitors were proposed to potentially inhibit SARS-CoV-2 Mpro. In addition to their known anticoagulants activity this potential inhibition could have an additional therapeutic effect on patients with COVID-19 disease. In this study we examined the binding of the Apixaban, Betrixaban and Rivaroxaban to the SARS-CoV-2 Mpro with the use of the MicroScale Thermophoresis technique. Our results indicate that the experimentally measured binding affinity is weak and the therapeutic effect due to the SARS-CoV-2 Mpro inhibition is rather negligible.

Investigation of Thiocarbamates as Potential Inhibitors of the SARS-CoV-2 Mpro.

In the present study we tested, using the microscale thermophoresis technique, a small library of thionocarbamates, thiolocarbamates, sulfide and disulfide as potential lead compounds for SARS-CoV-2 Mpro drug design. The successfully identified binder is a representative of the thionocarbamates group with a high potential for future modifications aiming for higher affinity and solubility. The experimental analysis was extended by computational studies that show insufficient accuracy of the simplest and widely applied approaches and underline the necessity of applying more advanced methods to properly evaluate the affinity of potential SARS-CoV-2 Mpro binders.

Studies of Peculiar Mg-Containing and Oscillating Bioapatites in Sheep and Horse Teeth.

New types of biological apatites have been discovered in molar sheep and horse teeth and are divided in two types. In the first and more general type, the release of Mg ions is parallel to the changes in composition of apatite leading to a final stoichiometric ratio of Ca to P ions, going from dentin depth towards the boundary of enamel with air. Inside dentin, another apatite sub-types were discovered with alternating layers of Mg-rich and C-rich apatites. The approximate formal stoichiometric relationships for these peculiar types of bioapatites are suggested. We identified two kinds of ion-exchanges responsible for formation of peculiar apatites. Various combinations of main and minor elements lead to new versions of biological apatites.

Opportunities of combinatorial thin film materials design for the sustainable development of magnesium-based alloys.

Magnesium-based lightweight structural materials exhibit potential for energy savings. However, the state-of-the-art quest for novel compositions with improved properties through conventional bulk metallurgy is time, energy, and material intensive. Here, the opportunities provided by combinatorial thin film materials design for the sustainable development of magnesium alloys are evaluated. To characterise the impurity level of (Mg,Ca) solid solution thin films within grains and grain boundaries, scanning transmission electron microscopy and atom probe tomography are correlatively employed. It is demonstrated that control of the microstructure enables impurity levels similar to bulk-processed alloys. In order to substantially reduce time, energy, and material requirements for the sustainable development of magnesium alloys, we propose a three-stage materials design strategy: (1) Efficient and systematic investigation of composition-dependent phase formation by combinatorial film growth. (2) Correlation of microstructural features and mechanical properties for selected composition ranges by rapid alloy prototyping. (3) Establishment of synthesis-microstructure-property relationships by conventional bulk metallurgy.

Novel Approach to Tooth Chemistry. Quantification of the Dental-Enamel Junction.

The dentin-enamel junction (DEJ) is known for its special role in teeth. Several techniques were applied for the investigation of the DEJ in human sound molar teeth. The electron (EPMA) and proton (PIXE) microprobes gave consistent indications about the variability of elemental concentrations on this boundary. The locally increased and oscillating concentrations of Mg and Na were observed in the junction, in the layer adhering to the enamel and covering roughly half of the DEJ width. The chemical results were compared with the optical profiles of the junction. Our chemical and optical results were next compared with the micromechanical results (hardness, elastic modulus, friction coefficient) available in the world literature. A strong correlation of both result sets was proven, which testifies to the self-affinity of the junction structures for different locations and even for different kinds of teeth and techniques applied for studies. Energetic changes in tooth strictly connected with crystallographic transformations were calculated, and the minimum energetic status was discovered for DEJ zone. Modeling of both walls of the DEJ from optical data was demonstrated. Comparing the DEJ in human teeth with the same structure found in dinosaur, shark, and alligator teeth evidences the universality of dentin enamel junction in animal world. The paper makes a contribution to better understanding the joining of the different hard tissues.

Radioactivity of the gas pipeline network in Poland.

The paper presents the study of the radioactivity connected with the transport of natural gas by the gas pipeline network in the selected points in Poland. In the scope of the study the measurements of activity concentration of radon (222Rn) in the gas samples, radiolead (210Pb) in spent filter cartridges and dust samples (black powder) collected from the gas pipeline network were performed. The results show that the 222Rn activity concentration in natural gas varies from the detection limit of the applied method (30 Bq/m3) to around 1400 Bq/m3. The 210Pb activity concentration in black powder samples and spent filter cartridges varies from 500 to 17000 Bq/kg and from 200 to 2900 Bq/kg respectively. The black powder with highest 210Pb concentration should be classified as low-radioactive waste according to nuclear regulations.

Novel Approach to Tooth Chemistry: Quantification of Human Enamel Apatite in Context for New Biomaterials and Nanomaterials Development.

A series of linear profiles of the elements of the enamel in human molar teeth were made with the use of an electron microprobe and a Raman microscope. It is postulated that the enamel can be treated as the superposition of variable "overbuilt" enamel on the stable "core" enamel at the macro-, micro- and nanoscale level. The excessive values characterize the "overbuilt enamel". All the profiles of excessive parameters along the enamel thickness from the enamel surface to the dentin enamel junction (DEJ) can be approximated very precisely with the use of exponential functions, where Ca, P, Cl and F spatial profiles are decaying while Mg, Na, K and CO32- ones are growing distributions. The "overbuilt" apatite formed on the boundary with DEJ, enriched in Na, Mg, OH and carbonates, reacts continuously with Ca, Cl and F, passing into an acid-resistant form of the "overbuilt" enamel. The apparent phases arriving in boundary regions of the "overbuilt enamel" were proposed. Microdiffraction measurements reveal relative variation of energy levels during enamel transformations. Our investigations are the milestones for a further new class of biomaterial and nanomaterial development for biomedical applications.

RNA-binding activity of TRIM25 is mediated by its PRY/SPRY domain and is required for ubiquitination.

BACKGROUND: TRIM25 is a novel RNA-binding protein and a member of the Tripartite Motif (TRIM) family of E3 ubiquitin ligases, which plays a pivotal role in the innate immune response. However, there is scarce knowledge about its RNA-related roles in cell biology. Furthermore, its RNA-binding domain has not been characterized. RESULTS: Here, we reveal that the RNA-binding activity of TRIM25 is mediated by its PRY/SPRY domain, which we postulate to be a novel RNA-binding domain. Using CLIP-seq and SILAC-based co-immunoprecipitation assays, we uncover TRIM25's endogenous RNA targets and protein binding partners. We demonstrate that TRIM25 controls the levels of Zinc Finger Antiviral Protein (ZAP). Finally, we show that the RNA-binding activity of TRIM25 is important for its ubiquitin ligase activity towards itself (autoubiquitination) and its physiologically relevant target ZAP. CONCLUSIONS: Our results suggest that many other proteins with the PRY/SPRY domain could have yet uncharacterized RNA-binding potential. Together, our data reveal new insights into the molecular roles and characteristics of RNA-binding E3 ubiquitin ligases and demonstrate that RNA could be an essential factor in their enzymatic activity.

Radioactivity in wastes generated from shale gas exploration and production - North-Eastern Poland.

In the present study, the K-40, U-238, Ra-226, Pb-210, Ra-228 and Th-228 activity concentrations were measured in 64 samples of wastes generated from shale gas exploration in North-Eastern Poland. The measured samples consist of drill cuttings, solid phase of waste drilling muds, fracking fluids, return fracking fluids and waste proppants. The measured activity concentrations in solid samples vary in a wide range from 116 to around 1100 Bq/kg for K-40, from 14 to 393 Bq/kg for U-238, from 15 to 415 Bq/kg for Ra-226, from 12 to 391 Bq/kg for Pb-210, from a few Bq/kg to 516 Bq/kg for Ra-228 and from a few Bq/kg to 515 Bq/kg for Th-228. Excluding the waste proppants, the measured activity concentrations in solid samples oscillate around their worldwide average values in soil. In the case of the waste proppants, the activity concentrations of radionuclides from uranium and thorium decay series are significantly elevated and equal to several hundreds of Bq/kg but it is connected with the mineralogical composition of proppants. The significant enhancement of Ra-226 and Ra-228 activity concentrations after fracking process was observed in the case of return fracking fluids, but the radium isotopes content in these fluids is comparable with that in waste waters from copper and coal mines in Poland.

Determination of the self-attenuation based on the sample composition in gamma-ray spectrometry of 210Pb: requirements for the scope of chemical analyses.

In the presented paper analysis of sensitivity of self-attenuation correction Cs to the accuracy of chemical composition analysis is presented. The analyses were done by means of Monte Carlo simulation for cylindrical samples and for four sample materials: peat, water, ash and soil. For each of these materials the major elements were selected whose determination in the analysed material is necessary. For the remaining elements threshold levels of their concentration were determined-if expected element concentration in a sample exceeds this value, its determination is indispensable, assuming the accuracy of Cs determination at 3 %.