Structure-Based Discovery of High-Affinity Small Molecule Ligands and Development of Tool Probes to Study the Role of Chitinase-3-Like Protein 1.

Chitinase-3-like-1 (CHI3L1), also known as YKL-40, is a glycoprotein linked to inflammation, fibrosis, and cancer. This study explored CHI3L1's interactions with various oligosaccharides using microscale thermophoresis (MST) and AlphaScreen (AS). These investigations guided the development of high-throughput screening assays to assess interference of small molecules in binding between CHI3L1 and biotinylated small molecules or heparan sulfate-based probes. Small molecule binders of YKL-40 were identified in our chitotriosidase inhibitors library with MST and confirmed through X-ray crystallography. Based on cocrystal structures of potent hit compounds with CHI3L1, small molecule probes 19 and 20 were designed for an AS assay. Structure-based optimization led to compounds 30 and 31 with nanomolar activities and drug-like properties. Additionally, an orthogonal AS assay using biotinylated heparan sulfate as a probe was developed. The compounds' affinity showed a significant correlation in both assays. These screening tools and compounds offer novel avenues for investigating the role of CHI3L1.

Structure of human TRPM8 channel.

TRPM8 is a non-selective cation channel permeable to both monovalent and divalent cations that is activated by multiple factors, such as temperature, voltage, pressure, and changes in osmolality. It is a therapeutic target for anticancer drug development, and its modulators can be utilized for several pathological conditions. Here, we present a cryo-electron microscopy structure of a human TRPM8 channel in the closed state that was solved at 2.7 Å resolution. Our structure comprises the most complete model of the N-terminal pre-melastatin homology region. We also visualized several lipids that are bound by the protein and modeled how the human channel interacts with icilin. Analyses of pore helices in available TRPM structures showed that all these structures can be grouped into different closed, desensitized and open state conformations based on the register of the pore helix S6 which positions particular amino acid residues at the channel constriction.

Arginase 1/2 Inhibitor OATD-02: From Discovery to First-in-man Setup in Cancer Immunotherapy.

Pharmacologic inhibition of the controlling immunity pathway enzymes arginases 1 and 2 (ARG1 and ARG2) is a promising strategy for cancer immunotherapy. Here, we report the discovery and development of OATD-02, an orally bioavailable, potent arginases inhibitor. The unique pharmacologic properties of OATD-02 are evidenced by targeting intracellular ARG1 and ARG2, as well as long drug-target residence time, moderate to high volume of distribution, and low clearance, which may jointly provide a weapon against arginase-related tumor immunosuppression and ARG2-dependent tumor cell growth. OATD-02 monotherapy had an antitumor effect in multiple tumor models and enhanced an efficacy of the other immunomodulators. Completed nonclinical studies and human pharmacokinetic predictions indicate a feasible therapeutic window and allow for proposing a dose range for the first-in-human clinical study in patients with cancer. SIGNIFICANCE: We have developed an orally available, small-molecule intracellular arginase 1 and 2 inhibitor as a potential enhancer in cancer immunotherapy. Because of its favorable pharmacologic properties shown in nonclinical studies, OATD-02 abolishes tumor immunosuppression induced by both arginases, making it a promising drug candidate entering clinical trials.

Morphology and immunohistochemical characteristics of the otic ganglion in the chinchilla (Chinchilla laniger Molina).

INTRODUCTION: The available literature provides relatively little information on the morphology of the autonomic head ganglia in rodents including their neurochemical codding. MATERIAL AND METHODS: Morphological investigations of the otic ganglion of the chinchilla were performed using the modified acetylcholinesterase method. The cellular structure was investigated with histological techniques and neurochemical properties were studied with the double-labelling immunofluorescence method. RESULTS: Macromorphological investigations allowed the otic ganglion to be identified as a compact, oval agglomeration of neurons and nerve fibers. Multidimensional cross-sections revealed densely arranged neuronal perikarya and two populations of nerve cells differing in size were distinguished. The large cells (40-50 µm) accounted for about 80% of the neurons in the cross-sections. Moreover, a small number of intraganglionic nerve fibers was observed. Immunohistochemical staining revealed that over 85% of the neuronal cell bodies in the otic ganglion contained immunoreactivity to VAChT or ChAT. VIP-immunoreactive perikarya comprised approximately 10% of the ganglionic cells. Double staining revealed the presence of VAChT+ and NOS+ neurons which amounted to about 45% of the nerve cells in the otic ganglion. NOS+ only perikarya comprised approx. 15% of all the neurons. Immunoreactivity to enkephalins, substance P, somatostatin, and galanin was expressed in single nerve cell bodies and nerve fibers except numerous substance P+ intraganglionic nerve fibers. Some of them were stained also for CGRP. Single neurons stained for tyroxine hydroxylase. CONCLUSIONS: Our results, compared with findings in other rodent species suggest the existence of interspecies differences in the morphology, cellular structure, and immunohistochemical properties of the head autonomic ganglia in mammals.

The right coronary artery in the heart of chinchilla (Chinchilla laniger Molina).

The pattern of normal coronary vascularization in a mammalian heart includes the presence of both right and left coronary arteries. According to the literature data, the presence of single major coronary arteries is mainly related to cardiac abnormalities. Previously it has been reported that the right coronary artery is absent in the coronary vascularization of the heart in the chinchilla. Our research was carried out on thirty chinchillas (Chinchilla laniger Molina). The coronary vessels were filled with colored latex to render them visible. The examinations were supplemented additionally with the use of microcomputed tomography with arterial contrast. Our study demonstrates its undoubtedly presence of the right coronary artery. In all subjects the right coronary artery was present, as was the left coronary artery. Two types of right coronary artery were found. Our results indicate that the normal pattern of coronary vascularization of heart in chinchilla includes both the right and left coronary arteries. An open question remains the presence of single coronary artery is a normal pattern of cardiac arterial vascularization in chinchilla.

Osteoarthritis in past human populations from Radom (14th-17th and 18th-19th century).

Osteoarthritis (OA) is a widespread skeletal condition in the historical population, but it still raises many methodological and interpretative problems. The present study aimed to examine the osteoarthritic changes (osteophytes, porosity, eburnation) in the skeletal material from Radom (14th-19th century) (Poland), enriching knowledge about osteoarthritis and its prevalence in the past. Additionally, a comparison of OA changes prevalence in two chronological periods (the population from Radom during the 14th-17th century versus the 18th-19th century) was done. In the Late Medieval (14th-17th century) population from Radom, osteoarthritic changes were observed in 22% of individuals (males, 18%; females, 29%) and in the Modern Period Radom (18th-19th century) in 25% individuals (males, 25.7%; females, 26.5%). In both skeletal samples, the greatest number of OA changes was recorded in the hip and elbow. Knee and ankle were the least affected joints. Osteophytes were the most frequently observed type of lesions, while eburnation was the least frequent. Although the higher prevalence of osteoarthritis in the Modern Period in Radom is noted, the differences are not statistically significant. Taking the multifactorial etiology of osteoarthritic changes, and the fact that osteoarthritis, as a single indicator of health, could not tell much about the overall lifestyle of past human populations, one must be caution when drawing unambiguous conclusions according to the simple, linear effect of environmental changes on osteoarthritic changes formation.

Distribution and neurochemical characteristic of the cardiac nerve structures in the heart of chinchilla (Chinchilla laniger Molina).

INTRODUCTION: The heart innervation is made up of plexo-ganglionic formation containing sympathetic, parasympathetic, and sensory components. We examined the distribution and neurochemical coding of the ganglia and nerve fibers in the chinchilla's heart. MATERIAL AND METHODS: The heart sections of 10 male and 10 female adult chinchillas were processed in accordance with the thiocholine method for acetylcholine esterase (AChE), and the SPG method for detecting the presence of adrenergic fibers was applied. The routine technique of immunohistochemical (IHC) staining with primary antibodies directed against ChAT, VAChT, DbH, TH, CART, NPY, VIP, GAL and SOM was used. The secondary antibodies were conjugated with Alexa Fluor 488 and Alexa Fluor 555 fluorophores. RESULTS: The epicardium contained ganglia and nerve fibers, the myocardium had a few ganglion neurocytes and nerve fibers, and the endocardium contained only nerve fibers. In the epicardium, AChE-positive fibers were more prevalent than SPG-positive fibers. All the ganglion cells were immunopositive for ChAT and VAChT. Some cells also had a positive reaction to DbH and TH. Fibers containing cholinergic and adrenergic markers were numerous, while many of them were ChAT/DbH- and VAChT/TH-positive. CART/NPY and CART/VIP, as well as CART and GAL, were observed to be colocalized in ganglion neurocytes, as well as in individual cells. The nerve fibers were found to contain all the neurotransmitters we tested for, as well as the following co-occurrences: ChAT/DbH, VAChT/TH, CART/NPY, CART/VIP, CART/GAL, and CART/SOM. CONCLUSIONS: Our analysis of the neurochemical profile of the nerve structures in chinchilla's heart showed that, despite interspecies differences, the general pattern of the distribution of autonomic nervous system structures is similar to that of other mammals' species, including humans.

Structures of Substrate Complexes of Foamy Viral Protease-Reverse Transcriptase.

Reverse transcriptases (RTs) use their DNA polymerase and RNase H activities to catalyze the conversion of single-stranded RNA to double-stranded DNA (dsDNA), a crucial process for the replication of retroviruses. Foamy viruses (FVs) possess a unique RT, which is a fusion with the protease (PR) domain. The mechanism of substrate binding by this enzyme has been unknown. Here, we report a crystal structure of monomeric full-length marmoset FV (MFV) PR-RT in complex with an RNA/DNA hybrid substrate. We also describe a structure of MFV PR-RT with an RNase H deletion in complex with a dsDNA substrate in which the enzyme forms an asymmetric homodimer. Cryo-electron microscopy reconstruction of the full-length MFV PR-RT-dsDNA complex confirmed the dimeric architecture. These findings represent the first structural description of nucleic acid binding by a foamy viral RT and demonstrate its ability to change its oligomeric state depending on the type of bound nucleic acid. IMPORTANCE Reverse transcriptases (RTs) are intriguing enzymes converting single-stranded RNA to dsDNA. Their activity is essential for retroviruses, which are divided into two subfamilies differing significantly in their life cycles: Orthoretrovirinae and Spumaretrovirinae. The latter family is much more ancient and comprises five genera. A unique feature of foamy viral RTs is that they contain N-terminal protease (PR) domains, which are not present in orthoretroviral enzymes. So far, no structural information for full-length foamy viral PR-RT interacting with nucleic substrates has been reported. Here, we present crystal and cryo-electron microscopy structures of marmoset foamy virus (MFV) PR-RT. These structures revealed the mode of binding of RNA/DNA and dsDNA substrates. Moreover, unexpectedly, the structures and biochemical data showed that foamy viral PR-RT can adopt both a monomeric configuration, which is observed in our structures in the presence of an RNA/DNA hybrid, and an asymmetric dimer arrangement, which we observed in the presence of dsDNA.

Structural Insights into the Interaction of Clinically Relevant Phosphorothioate mRNA Cap Analogs with Translation Initiation Factor 4E Reveal Stabilization via Electrostatic Thio-Effect.

mRNA-based therapies and vaccines constitute a disruptive technology with the potential to revolutionize modern medicine. Chemically modified 5' cap structures have provided access to mRNAs with superior translational properties that could benefit the currently flourishing mRNA field. Prime examples of compounds that enhance mRNA properties are antireverse cap analog diastereomers that contain an O-to-S substitution within the ß-phosphate (ß-S-ARCA D1 and D2), where D1 is used in clinically investigated mRNA vaccines. The compounds were previously found to have high affinity for eukaryotic translation initiation factor 4E (eIF4E) and augment translation in vitro and in vivo. However, the molecular basis for the beneficial "thio-effect" remains unclear. Here, we employed multiple biophysical techniques and captured 11 cap analog-eIF4E crystallographic structures to investigate the consequences of the ß-O-to-S or -Se substitution on the interaction with eIF4E. We determined the SP/RP configurations of ß-S-ARCA and related compounds and obtained structural insights into the binding. Unexpectedly, in both stereoisomers, the ß-S/Se atom occupies the same binding cavity between Lys162 and Arg157, indicating that the key driving force for complex stabilization is the interaction of negatively charged S/Se with positively charged amino acids. This was observed for all structural variants of the cap and required significantly different conformations of the triphosphate for each diastereomer. This finding explains why both ß-S-ARCA diastereomers have higher affinity for eIF4E than unmodified caps. Binding affinities determined for di-, tri-, and oligonucleotide cap analogs suggested that the "thio-effect" was preserved in longer RNAs. Our observations broaden the understanding of thiophosphate biochemistry and enable the rational design of translationally active mRNAs and eIF4E-targeting drugs.

Changes in the Expression of TLR2 During the Intestinal Phase of Trichinellosis.

INTRODUCTION: Toll-like receptors (TLRs) play an important role in fast activation of the immune response to a variety of pathogens, including parasites. In this study, we focused on TLR2, because this receptor is one of the best known and most frequently analysed members of the TLR family. The aim of this study was to assess the effect of Trichinella spiralis on expression of TLR2 during the intestinal stage of infection. MATERIAL AND METHODS: The experimental material consisted of isolates prepared from the intestines (jejunum and colon) of BALB/c mice infected with T. spiralis taken at 4, 8, and 16 days post infection. RESULTS: Our results based on quantitative real-time PCR showed that the mRNA level for TLR2 was statistically significantly higher in the jejuna of mice infected with T. spiralis than in this tissue of uninfected mice. In addition, the presence of TLR2 protein in the intestinal phase of trichinellosis was confirmed by a strong positive immunohistochemical reaction. CONCLUSION: Our results indicate that infection with T. spiralis changes the expression of TLR2 in the small intestine of the mouse host and suggest a contribution of these receptors to the host defence mechanisms during experimental trichinellosis.

Cholinergic and adrenergic innervation of the pancreas in chinchilla (Chinchilla Laniger Molina).

INTRODUCTION: Cholinergic and adrenergic innervation of the pancreas in chinchilla (Chinchilla Laniger Molina) was examined in this study. The pancreas is both an exocrine and endocrine gland with autonomic and sensory innervation presented by the numerous nerve fibers and small agglomerations of nerve cells. MATERIAL AND METHODS: Investigations were performed on 16 adult chinchillas of both sexes. The material was collected immediately after death of the animals. Histochemical methods: AChE and SPG were used, in addition to routine technique of single and double immunohistochemical (IHC) staining using whole mount specimens and freezing sections with a thickness of 8 to 12 µm. In the immunofluorescence staining, primary antibodies directed against markers used to identify cholinergic - ChAT and VAChT, and adrenergic - DbH and TH neurons. Secondary antibodies were coupled to Alexa Fluor 488 and Alexa Fluor 555 fluorophores. RESULTS: Histochemical studies (AChE) revealed that chinchilla pancreatic cholinergic innervation consisted of ganglionic neurocytes and numerous nerve fibers. These structures are located in the parenchyma of the exocrine part of the organ in close proximity to blood vessels and are present within the walls of the pancreatic ducts and interstitial connective tissue. A delicate fiber network around the Langerhans islets was also observed. The most numerous cholinergic structures were found in the head and tail, and the least numbers were found in the body of the pancreas. The SPG method revealed that adrenergic fibers form a network in the adventitia of blood vessels, and individual fibers run throughout the pancreatic parenchyma. Moreover, adrenergic nerve fibers were observed around the ganglionic neurocytes. This innervation was similar in all parts of the investigated organ. IHC investigations allowed observations of both the cholinergic and adrenergic activities of autonomic nerve structures. Additionally, using ChAT/DbH double staining, colocalization of these substances was observed in the fibers of the pancreatic parenchyma that passed through the cholinergic ganglia. Colocalization of VAChT and TH was found in nerve fibers of the exocrine part, in the walls of blood vessels, and in individual nerve cells. Colocalization of ChAT/DbH and VAChT/TH was observed in the single nerve cells and in the small (2-3 cell) ganglia. ChAT- and DbH-immunopositive nerve fibers were found in the area of the islets of Langerhans. CONCLUSIONS: The results indicate a more intense cholinergic innervation of the chinchilla's pancreas, which is represented by both ganglia and nerve fibers, while adrenergic structures are mainly represented by fibers and only single neurocytes. This arrangement of the investigated structures in this species may imply a major role for hormonal control of exocrine secretion in rodents.

Structural bases of peptidoglycan recognition by lysostaphin SH3b domain.

Staphylococcus simulans lysostaphin cleaves pentaglycine cross-bridges between stem peptides in the peptidoglycan of susceptible staphylococci, including S. aureus. This enzyme consists of an N-terminal catalytic domain and a cell wall binding domain (SH3b), which anchors the protein to peptidoglycan. Although structures of SH3bs from lysostaphin are available, the binding modes of peptidoglycan to these domains are still unclear. We have solved the crystal structure of the lysostaphin SH3b domain in complex with a pentaglycine peptide representing the peptidoglycan cross-bridge. The structure identifies a groove between ß1 and ß2 strands as the pentaglycine binding site. The structure suggests that pentaglycine specificity of the SH3b arises partially directly by steric exclusion of Cß atoms in the ligand and partially indirectly due to the selection of main chain conformations that are easily accessible for glycine, but not other amino acid residues. We have revealed further interactions of SH3b with the stem peptides with the support of bioinformatics tools. Based on the structural data we have attempted engineering of the domain specificity and have investigated the relevance of the introduced substitutions on the domain binding and specificity, also in the contexts of the mature lysostaphin and of its bacteriolytic activity.

The modulatory effect of Artemisia annua L. on toll-like receptor expression in Acanthamoeba infected mouse lungs.

The genus Acanthamoeba, which may cause different infections in humans, occurs widely in the environment. Lung inflammation caused by these parasites induces pulmonary pathological changes such as pulmonary necrosis, peribronchial plasma cell infiltration, moderate desquamation of alveolar cells and partial destruction of bronchial epithelial cells, and presence of numerous trophozoites and cysts among inflammatory cells. The aim of this study was to assess the influence of plant extracts from Artemisia annua L. on expression of the toll-like receptors TLR2 and TLR4 in lungs of mice with acanthamoebiasis. A. annua, which belongs to the family Asteraceae, is an annual plant that grows wild in Asia. In this study, statistically significant changes of expression of TLR2 and TLR4 were demonstrated. In the lungs of infected mice after application of extract from A. annua the expression of TLRs was observed mainly in bronchial epithelial cells, pneumocytes (to a lesser extent during the outbreak of infection), and in the course of high general TLR expression. TLR4 in particular was also visible in stromal cells of lung parenchyma. In conclusion, we confirmed that a plant extract of A. annua has a modulatory effect on components of the immune system such as TLR2 and TLR4.

5'-Phosphorothiolate Dinucleotide Cap Analogues: Reagents for Messenger RNA Modification and Potent Small-Molecular Inhibitors of Decapping Enzymes.

The 5' cap consists of 7-methylguanosine (m7G) linked by a 5'-5'-triphosphate bridge to messenger RNA (mRNA) and acts as the master regulator of mRNA turnover and translation initiation in eukaryotes. Cap analogues that influence mRNA translation and turnover (either as small molecules or as part of an RNA transcript) are valuable tools for studying gene expression, which is often also of therapeutic relevance. Here, we synthesized a series of 15 dinucleotide cap (m7GpppG) analogues containing a 5'-phosphorothiolate (5'-PSL) moiety (i.e., an O-to-S substitution within the 5'-phosphoester) and studied their biological properties in the context of three major cap-binding proteins: translation initiation factor 4E (eIF4E) and two decapping enzymes, DcpS and Dcp2. While the 5'-PSL moiety was neutral or slightly stabilizing for cap interactions with eIF4E, it significantly influenced susceptibility to decapping. Replacing the γ-phosphoester with the 5'-PSL moiety (γ-PSL) prevented ß-γ-pyrophosphate bond cleavage by DcpS and conferred strong inhibitory properties. Combining the γ-PSL moiety with α-PSL and ß-phosphorothioate (PS) moiety afforded first cap-derived hDcpS inhibitor with low nanomolar potency. Susceptibility to Dcp2 and translational properties were studied after incorporation of the new analogues into mRNA transcripts by RNA polymerase. Transcripts containing the γ-PSL moiety were resistant to cleavage by Dcp2. Surprisingly, superior translational properties were observed for mRNAs containing the α-PSL moiety, which were Dcp2-susceptible. The overall protein expression measured in HeLa cells for this mRNA was comparable to mRNA capped with the translation augmenting ß-PS analogue reported previously. Overall, our study highlights 5'-PSL as a synthetically accessible cap modification, which, depending on the substitution site, can either reduce susceptibility to decapping or confer superior translational properties on the mRNA. The 5'-PSL-analogues may find application as reagents for the preparation of efficiently expressed mRNA or for investigation of the role of decapping enzymes in mRNA processing or neuromuscular disorders associated with decapping.

Structural analysis of mtEXO mitochondrial RNA degradosome reveals tight coupling of nuclease and helicase components.

Nuclease and helicase activities play pivotal roles in various aspects of RNA processing and degradation. These two activities are often present in multi-subunit complexes from nucleic acid metabolism. In the mitochondrial exoribonuclease complex (mtEXO) both enzymatic activities are tightly coupled making it an excellent minimal system to study helicase-exoribonuclease coordination. mtEXO is composed of Dss1 3'-to-5' exoribonuclease and Suv3 helicase. It is the master regulator of mitochondrial gene expression in yeast. Here, we present the structure of mtEXO and a description of its mechanism of action. The crystal structure of Dss1 reveals domains that are responsible for interactions with Suv3. Importantly, these interactions are compatible with the conformational changes of Suv3 domains during the helicase cycle. We demonstrate that mtEXO is an intimate complex which forms an RNA-binding channel spanning its entire structure, with Suv3 helicase feeding the 3' end of the RNA toward the active site of Dss1.

Influence of Artemisia annua L. on toll-like receptor expression in brain of mice infected with Acanthamoeba sp.

The treatment of acanthamoebiasis is a still a problem. Our previous studies showed that the application of extracts from Artemisia annua L. significantly prolonged the survival of mice infected by Acanthamoeba. This plant has medicinal properties in the treatment of human parasitic diseases. The aim of this study was to evaluate the effects of A. annua on expression of Toll-like receptors (TLRs) 2 and 4 in brain of mice with Acanthamoeba infection. Mice were infected with Acanthamoeba sp. strain Ac309 (KY203908) by intranasal inoculation without and after application of A. annua extract. The administration of extract from A. annua significantly reduced the level of expression of TLR2 and modified the level of expression of TLR4. A. annua extract is a natural substance that is well tolerated in animals and may be considered as a combination therapy in treatment of acanthamoebiasis. Our study suggested that A. annua extract may be used as an alternative therapeutic tool.

Trichinella Spiralis: Impact on the Expression of Toll-like Receptor 4 (TLR4) Gene During the Intestinal Phase of Experimental Trichinellosis.

INTRODUCTION: Toll-like receptors (TLRs) play a key role in the rapid activation of the innate immune response to a variety of pathogens. The aim of this study was to evaluate the effect of Trichinella spiralis infection on the level of expression of the tlr4 gene in mouse intestines during the intestinal phase of experimental trichinellosis. MATERIAL AND METHODS: The experimental material consisted of the small and large intestines of BALB/c mice infected with Trichinella spiralis sampled at 4, 8, and 16 days post infection (dpi). RESULTS: A statistically significant increase was demonstrated in the tlr4 mRNA level isolated from the infected mice jejunum at 4, 8, and 16 dpi over the uninfected control. Moreover, at 4, 8, and 16 dpi in the jejunum of infected mice, a strong positive reaction for the presence of TLR4 protein compared with that of uninfected mice was observed. CONCLUSION: Infection with T. spiralis changes the expression of the tlr4 gene in the small intestine of the mouse host.

The siRNA suppressor RTL1 is redox-regulated through glutathionylation of a conserved cysteine in the double-stranded-RNA-binding domain.

RNase III enzymes cleave double stranded (ds)RNA. This is an essential step for regulating the processing of mRNA, rRNA, snoRNA and other small RNAs, including siRNA and miRNA. Arabidopsis thaliana encodes nine RNase III: four DICER-LIKE (DCL) and five RNASE THREE LIKE (RTL). To better understand the molecular functions of RNase III in plants we developed a biochemical assay using RTL1 as a model. We show that RTL1 does not degrade dsRNA randomly, but recognizes specific duplex sequences to direct accurate cleavage. Furthermore, we demonstrate that RNase III and dsRNA binding domains (dsRBD) are both required for dsRNA cleavage. Interestingly, the four DCL and the three RTL that carry dsRBD share a conserved cysteine (C230 in Arabidopsis RTL1) in their dsRBD. C230 is essential for RTL1 and DCL1 activities and is subjected to post-transcriptional modification. Indeed, under oxidizing conditions, glutathionylation of C230 inhibits RTL1 cleavage activity in a reversible manner involving glutaredoxins. We conclude that the redox state of the dsRBD ensures a fine-tune regulation of dsRNA processing by plant RNase III.

mRNA cap analogues substituted in the tetraphosphate chain with CX2: identification of O-to-CCl2 as the first bridging modification that confers resistance to decapping without impairing translation.

Analogues of the mRNA 5'-cap are useful tools for studying mRNA translation and degradation, with emerging potential applications in novel therapeutic interventions including gene therapy. We report the synthesis of novel mono- and dinucleotide cap analogues containing dihalogenmethylenebisphosphonate moiety (i.e. one of the bridging O atom substituted with CCl2 or CF2) and their properties in the context of cellular translational and decapping machineries, compared to phosphate-unmodified and previously reported CH2-substituted caps. The analogues were bound tightly to eukaryotic translation initiation factor 4E (eIF4E), with CCl2-substituted analogues having the highest affinity. When incorporated into mRNA, the CCl2-substituted dinucleotide most efficiently promoted cap-dependent translation. Moreover, the CCl2-analogues were potent inhibitors of translation in rabbit reticulocyte lysate. The crystal structure of eIF4E in complex with the CCl2-analogue revealed a significantly different ligand conformation compared to that of the unmodified cap analogue, which likely contributes to the improved binding. Both CCl2- and CF2- analogues showed lower susceptibility to hydrolysis by the decapping scavenger enzyme (DcpS) and, when incorporated into RNA, conferred stability against major cellular decapping enzyme (Dcp2) to transcripts. Furthermore, the use of difluoromethylene cap analogues was exemplified by the development of 19F NMR assays for DcpS activity and eIF4E binding.

Polycyclic aromatic hydrocarbons connected with Schiff base linkers: Experimental and theoretical photophysical characterization and electrochemical properties.

A series of polyaromatic hydrocarbons with anthracene, phenanthrene and pyrene units connected with Schiff base junctions were synthesized via condensation of p-phenylenediamine and hydrazine with selected aldehydes. The effect of both hydrocarbon structures and presence of N-N- or phenyl- linked diimines on properties of the prepared azines and azomethines was analyzed. The obtained compounds were soluble in common organic solvents and melted in the range of 226-317°C. Their photophysical and electrochemical properties were investigated by UV-vis, photoluminescence spectroscopies and cyclic voltammetry (CV), respectively. Moreover, a density functional theory (DFT) was applied for calculation of their electronic and geometric structures as well as absorption and emission spectra. Additionally, their electron acceptor activity was preliminary tested in photovoltaic experiment.