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.

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.

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.

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 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.

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.

Lin28a uses distinct mechanisms of binding to RNA and affects miRNA levels positively and negatively.

Lin28a inhibits the biogenesis of let-7 miRNAs by triggering the polyuridylation and degradation of their precursors by terminal uridylyltransferases TUT4/7 and 3'-5' exoribonuclease Dis3l2, respectively. Previously, we showed that Lin28a also controls the production of neuro-specific miRNA-9 via a polyuridylation-independent mechanism. Here we reveal that the sequences and structural characteristics of pre-let-7 and pre-miRNA-9 are eliciting two distinct modes of binding to Lin28a. We present evidence that Dis3l2 controls miRNA-9 production. Finally, we show that the constitutive expression of untagged Lin28a during neuronal differentiation in vitro positively and negatively affects numerous other miRNAs. Our findings shed light on the role of Lin28a in differentiating cells and on the ways in which one RNA-binding protein can perform multiple roles in the regulation of RNA processing.

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.

Expression of cyclin D1 after treatment with doxorubicin in the HL-60 cell line.

Increased levels of cyclin D1 and amplification of CCND1 gene occur in many types of cancers. We have followed the expression of cyclin D1 after treatment with doxorubicin with reference to cell death and other possible therapeutic implications. The effect of the treatment on the cell cycle, survival, intracellular level (flow cytometry), and intracellular localization of cyclin D1 (fluorescence microscopy) and expression of CCND1 (real-time RT-PCR) was investigated in HL-60 cells. An increase in the fluorescence intensity of cyclin D1 occurred after treatment with 0.15 and 0.3 µM doxorubicin. This tendency was confirmed by real-time RT-PCR. Expression of CCND1 in relation to the reference gene PBGD was increased in cells exposed to 0.15 µM doxorubicin. Concomitantly, some alterations in the regulation of the G0/G1, S, and G2/M checkpoints occurred, accompanied by changes in the polyploid fraction of the population. This was particularly evident at 0.3 µM doxorubicin, at which concentration the rate of cell death was also clearly higher. In conclusion, depending on the concentration used, alterations in cell death and the number of S, G2/M, and polyploid cells may correspond with cyclin D1 levels. This, in turn, may reflect an important role of the protein as one of the possible survival/point-of-no-return regulators dependent on its concentration, which seems especially plausible in the context of more prominent cell death in the above-mentioned fractions of cells.

Arsenic trioxide preferentially induces nonapoptotic cell deaths as well as actin cytoskeleton rearrangement in the CHO AA8 cell line.

INTRODUCTION: The therapeutic effect of arsenic trioxide (ATO, As2O3) has been investigated for many years. However, the precise molecular mechanisms underlying the antitumor activity of ATO are still not fully understood, but seem to depend on cell types, dosage, and duration of exposure. The purpose of this study was to assess the actin cytoskeleton rearrangement during the cell death process induced by arsenic trioxide in the CHO AA8 cells. A better understanding the mechanisms of ATO-action is likely to lead to more rational use of this drug either as monotherapies or in combination with other anticancer agents. MATERIAL AND METHODS: The effect of ATO on actin cytoskeleton was studied in Chinese Hamster Ovary AA8 cell line. Actin was visualized by fluorescence microscopy and phalloidin conjugated to Alexa Fluor® 488. Morphological and ultrastructural alterations in the CHO AA8 cells were evaluated by using light and electron microscope, respectively. For quantitative measurement of cell death, Annexin V-Alexa Fluor® 488 and Propidium Iodide assay was performed. The vital staining of CHO AA8 cells with acridine orange was applied to detect the development of acidic vesicular organelles (AVOs). RESULTS: The performed experiments revealed a dose-dependent decrease in the cell survival. The morphological and ultrastructural features acquired by the cells after ATO-treatment were considered as typical for autophagy and mitotic cell death. As was shown by acridine orange staining, arsenic trioxide treatment increased red fluorescence signals in dose-dependent manner, indicating the development of AVOs, a hallmark of autophagy. Low level of apoptosis was induced in the ATO-treated CHO AA8 cells. Furthermore, the rearrangement of actin filaments associated with cell death process was also detected. CONCLUSIONS: The obtained results suggest that arsenic trioxide preferentially induces nonapoptotic cell deaths, autophagy and mitotic cell death, in p53-deficient CHO AA8 cells. Furthermore, the distinctive patterns of F-actin remodeling after As2O3 treatment were associated with different modes of cell death, confirming that cytoskeleton is a dynamic structure actively involved in the cell death process.

miRNAs in development and pathogenesis of the nervous system.

The human nervous system expresses approximately 70% of all miRNAs (microRNAs). Changing levels of certain ubiquitous and brain-specific miRNAs shape the development and function of the nervous system. It is becoming clear that misexpression of some miRNAs can contribute towards neurodevelopmental disorders. In the present article, we review the current knowledge of the role of miRNAs in development and pathogenesis of the nervous system.

[Involvement of Rho proteins in G1 phase cell cycle progression]. / Udzial bialek Rho w regulacji postepu fazy G1 cyklu komórkowego.

 Rho proteins, including RhoA, Rac1 and Cdc42, are members of the Ras superfamily of monomeric GTP-binding proteins, which are well-known regulators of the cytoskeleton. Numerous studies have shown that an intact cytoskeleton is required for cell cycle progression through G1 phase as well as mitosis and cytokinesis. Because of their role in both cytoskeletal rearrangement and mitogenic signaling, Rho family proteins are key mediators of cell cycle progression. In this paper, we review the current state of knowledge concerning the Rho-dependent signaling pathways that regulate the expression of cell cycle regulatory proteins required for G1 phase progression and S phase entry.

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.

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.

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.

[Cotinine--metabolism, application as a biomarker and the effects on the organism]. / Kotynina--metabolizm, zastosowanie jako biomarker i wplyw na organizm czlowieka.

This review presents the current state of knowledge on cotinine, the major metabolite of nicotine. Special attention is paid to the formation of this compound in the organism, its metabolism, application in diagnostic procedures and evaluation of its in vitro and in vivo activities. For many years, cotinine has been used as a biomarker of exposure to tobacco smoke. Currently, this compound is applied in many other studies including the use of cotinine in the treatment of various diseases. Several years ago, Scott et al. patented therapeutic applications of cotinine in chronic and acute inflammation. Cotinine is an interesting compound with a well-known metabolism; therefore there are suggestions for its application in the diagnosis and treatment of certain diseases. 

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.

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.