FUS Microphase Separation: Regulation by Nucleic Acid Polymers and DNA Repair Proteins

Fused in sarcoma (FUS) is involved in the regulation of RNA and DNA metabolism. FUS participates in the formation of biomolecular condensates driven by phase transition. FUS is prone to self-aggregation and tends to undergo phase transition both with or without nucleic acid polymers. Using dynamic light scattering and fluorescence microscopy, we examined the formation of FUS high-order structures or FUS-rich microphases induced by the presence of RNA, poly(ADP-ribose), ssDNA, or dsDNA and evaluated effects of some nucleic-acid-binding proteins on the phase behavior of FUS–nucleic acid systems. Formation and stability of FUS-rich microphases only partially correlated with FUS’s affinity for a nucleic acid polymer. Some proteins—which directly interact with PAR, RNA, ssDNA, and dsDNA and are possible components of FUS-enriched cellular condensates—disrupted the nucleic-acid-induced assembly of FUS-rich microphases. We found that XRCC1, a DNA repair factor, underwent a microphase separation and formed own microdroplets and coassemblies with FUS in the presence of poly(ADP-ribose). These results probably indicated an important role of nucleic-acid-binding proteins in the regulation of FUS-dependent formation of condensates and imply the possibility of the formation of XRCC1-dependent phase-separated condensates in the cell.     

Oligonucleotide Insecticides for Green Agriculture: Regulatory Role of Contact DNA in Plant–Insect Interactions

Insects vastly outnumber us in terms of species and total biomass, and are among the most efficient and voracious consumers of plants on the planet. As a result, to preserve crops, one of the primary tasks in agriculture has always been the need to control and reduce the number of insect pests. The current use of chemical insecticides leads to the accumulation of xenobiotics in ecosystems and a decreased number of species in those ecosystems, including insects. Sustainable development of human society is impossible without useful insects, so the control of insect pests must be effective and selective at the same time. In this article, we show for the first time a natural way to regulate the number of insect pests based on the use of extracellular double-stranded DNA secreted by the plant Pittosporum tobira. Using a principle similar to one found in nature, we show that the topical application of artificially synthesized short antisense oligonucleotide insecticides (olinscides, DNA insecticides) is an effective and selective way to control the insect Coccus hesperidum. Using contact oligonucleotide insecticide Coccus-11 at a concentration of 100 ng/μL on C. hesperidum larvae resulted in a mortality of 95.59 ± 1.63% within 12 days. Green oligonucleotide insecticides, created by nature and later discovered by humans, demonstrate a new method to control insect pests that is beneficial and safe for macromolecular insect pest management.     

N-Heterocyclic Carbenes and Their Metal Complexes Based on Histidine and Histamine Derivatives of Bacteriopurpurinimide for the Combined Chemo- and Photodynamic Therapy of Cancer

Photodynamic therapy (PDT) is currently regarded as a promising method for the treatment of oncological diseases. However, it involves a number of limitations related to the specific features of the method and the specific characteristics of photosensitizer molecules, including tumor hypoxia, small depth of light penetration into the tumor tissue, and low accumulation sensitivity. These drawbacks can be overcome by combining PDT with other treatment methods, for example, chemotherapy. In this work, we were the first to obtain agents that contain bacteriopurpurinimide as a photodynamic subunit and complexes of gold(I) that implement the chemotherapy effect. To bind the latter agents, N-heterocyclic carbenes (NHC) based on histidine and histamine were obtained. We considered alternative techniques for synthesizing the target conjugates and selected an optimal one that enabled the production of preparative amounts for biological assays. In vitro studies showed that all the compounds obtained exhibited high photoinduced activity. The C-donor Au(I) complexes exhibited the maximum specific activity at longer incubation times compared to the other derivatives, both under exposure to light and without irradiation. In in vivo studies, the presence of histamine in the NHC-derivative of dipropoxy-BPI (7b) had no significant effect on its antitumor action, whereas the Au(I) metal complex of histamine NHC-derivative with BPI (8b) resulted in enhanced antitumor activity and in an increased number of remissions after photodynamic treatment.     

Specific S100 Proteins Bind Tumor Necrosis Factor and Inhibit Its Activity

Tumor necrosis factor (TNF) inhibitors (anti-TNFs) represent a cornerstone of the treatment of various immune-mediated inflammatory diseases and are among the most commercially successful therapeutic agents. Knowledge of TNF binding partners is critical for identification of the factors able to affect clinical efficacy of the anti-TNFs. Here, we report that among eighteen representatives of the multifunctional S100 protein family, only S100A11, S100A12 and S100A13 interact with the soluble form of TNF (sTNF) in vitro. The lowest equilibrium dissociation constants (K_d) for the complexes with monomeric sTNF determined using surface plasmon resonance spectroscopy range from 2 nM to 28 nM. The apparent K_d values for the complexes of multimeric sTNF with S100A11/A12 estimated from fluorimetric titrations are 0.1–0.3 µM. S100A12/A13 suppress the cytotoxic activity of sTNF against Huh-7 cells, as evidenced by the MTT assay. Structural modeling indicates that the sTNF-S100 interactions may interfere with the sTNF recognition by the therapeutic anti-TNFs. Bioinformatics analysis reveals dysregulation of TNF and S100A11/A12/A13 in numerous disorders. Overall, we have shown a novel potential regulatory role of the extracellular forms of specific S100 proteins that may affect the efficacy of anti-TNF treatment in various diseases.     

CRISPR-Cas Controls Cryptic Prophages

The bacterial archetypal adaptive immune system, CRISPR-Cas, is thought to be repressed in the best-studied bacterium, Escherichia coli K-12. We show here that the E. coli CRISPR-Cas system is active and serves to inhibit its nine defective (i.e., cryptic) prophages. Specifically, compared to the wild-type strain, reducing the amounts of specific interfering RNAs (crRNA) decreases growth by 40%, increases cell death by 700%, and prevents persister cell resuscitation. Similar results were obtained by inactivating CRISPR-Cas by deleting the entire 13 spacer region (CRISPR array); hence, CRISPR-Cas serves to inhibit the remaining deleterious effects of these cryptic prophages, most likely through CRISPR array-derived crRNA binding to cryptic prophage mRNA rather than through cleavage of cryptic prophage DNA, i.e., self-targeting. Consistently, four of the 13 E. coli spacers contain complementary regions to the mRNA sequences of seven cryptic prophages, and inactivation of CRISPR-Cas increases the level of mRNA for lysis protein YdfD of cryptic prophage Qin and lysis protein RzoD of cryptic prophage DLP-12. In addition, lysis is clearly seen via transmission electron microscopy when the whole CRISPR-Cas array is deleted, and eliminating spacer #12, which encodes crRNA with complementary regions for DLP-12 (including rzoD), Rac, Qin (including ydfD), and CP4-57 cryptic prophages, also results in growth inhibition and cell lysis. Therefore, we report the novel results that (i) CRISPR-Cas is active in E. coli and (ii) CRISPR-Cas is used to tame cryptic prophages, likely through RNAi, i.e., unlike with active lysogens, active CRISPR-Cas and cryptic prophages may stably co-exist.     

The Reactions of N,N′-Diphenyldithiomalondiamide with Arylmethylidene Meldrum’s Acids

The Michael addition reaction between dithiomalondianilide (N,N′-diphenyldithiomalondiamide) and arylmethylidene Meldrum’s acids, accompanied by subsequent heterocyclization, was investigated along with factors affecting the mixture composition of the obtained products. The plausible mechanism includes the formation of stable Michael adducts which, under the studied conditions, undergo further transformations to yield corresponding N-methylmorpholinium 4-aryl-6-oxo-3-(N-phenylthio-carbamoyl)-1,4,5,6-tetrahydropyridin-2-thiolates and their oxidation derivatives, 4,5-dihydro-3H-[1,2]dithiolo[3,4-b]pyridin-6(7H)-ones. The structure of one such product, N-methylmorpholinium 2,2-dimethyl-5-(1-(2-nitrophenyl)-3-(phenylamino)-2-(N-phenylthiocarbamoyl)-3-thioxopropyl)-4-oxo-4H-1,3-dioxin-6-olate, was confirmed via X-ray crystallography.     

A Pilot Study of Changes in the Level of Catecholamines and the Activity of α-2-Macroglobulin in the Tear Fluid of Patients with Parkinson’s Disease and Parkinsonian Mice

Development of differential and early (preclinical) diagnostics of Parkinson’s disease (PD) is among the priorities in neuroscience. We searched for changes in the level of catecholamines and α-2-macroglobulin activity in the tear fluid (TF) in PD patients at an early clinical stage. It was shown that TF in patients is characterized by an increased level of noradrenaline mainly on the ipsilateral side of pronounced motor symptoms (72%, p = 0.049), a decreased level of adrenaline on both sides (ipsilateral—53%, p = 0.004; contralateral—42%, p = 0.02), and an increased α-2-macroglobulin activity on both sides (ipsilateral—53%, p = 0.03; contralateral—56%, p = 0.037) compared to controls. These changes are considered as potential biomarkers for differential diagnosis. Similar changes in the TF were found in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-treated mice when modeling clinical and preclinical stages of PD. These data show the adequacy of models to the pathogenesis of PD along the selected metabolic pathways, and also suggest that the found TF changes can be considered as potential biomarkers for preclinical diagnosis of PD. In Parkinsonian mice, the level of catecholamines also changes in the lacrimal glands, which makes it possible to consider them as one of the sources of catecholamines in the TF.     

Switching Ion Binding Selectivity of Thiacalix[4]arene Monocrowns at Liquid–Liquid and 2D-Confined Interfaces

Understanding the interaction of ions with organic receptors in confined space is of fundamental importance and could advance nanoelectronics and sensor design. In this work, metal ion complexation of conformationally varied thiacalix[4]monocrowns bearing lower-rim hydroxy (type I), dodecyloxy (type II), or methoxy (type III) fragments was evaluated. At the liquid–liquid interface, alkylated thiacalixcrowns-5(6) selectively extract alkali metal ions according to the induced-fit concept, whereas crown-4 receptors were ineffective due to distortion of the crown-ether cavity, as predicted by quantum-chemical calculations. In type-I ligands, alkali-metal ion extraction by the solvent-accessible crown-ether cavity was prevented, which resulted in competitive Ag⁺ extraction by sulfide bridges. Surprisingly, amphiphilic type-I/II conjugates moderately extracted other metal ions, which was attributed to calixarene aggregation in salt aqueous phase and supported by dynamic light scattering measurements. Cation–monolayer interactions at the air–water interface were monitored by surface pressure/potential measurements and UV/visible reflection–absorption spectroscopy. Topology-varied selectivity was evidenced, towards Sr²⁺ (crown-4), K⁺ (crown-5), and Ag⁺ (crown-6) in type-I receptors and Na⁺ (crown-4), Ca²⁺ (crown-5), and Cs⁺ (crown-6) in type-II receptors. Nuclear magnetic resonance and electronic absorption spectroscopy revealed exocyclic coordination in type-I ligands and cation–π interactions in type-II ligands.     

Novel spray-dried PHA microparticles for antitumor drug release

The production of poly-3-hydroxybutyrate (P3HB) and poly-3-hydroxybutyrate/polyethylene glycol (PEG)-based microparticles, loaded with antitumor drugs paclitaxel (PTX) and 5-Fluorouracil (5-FU) by spray-drying technique, was investigated. The average diameter of microparticles was found to be 3.4?±?0.5?µm and zeta potential was about ?44?mV. The addition of surfactant PEG did not show any effect on the morphological characteristics of the particles. But the chemical structure of drug influenced on the properties. Microparticles had heterogeneous pores on the surface when the hydrophobic PTX was encapsulated. It was established that the addition of surfactant positively influenced on the properties of particles and led to the loading of 5-FU directly into the matrix. This is confirmed by the results of electron microscopy and dynamics of drug release in vitro. As a whole, the release profiles of PTX and 5-FU from composite P3HB/PEG microparticles were less than from P3HB microparticles. The results of the morphological evaluation of Hela cells demonstrated that the use of cytostatic drugs loaded in P3HB microparticles induces morphological changes associated with apoptosis (chromatin condensation, core fragmentation, margination of nucleus). Thus, the obtained results can serve as the basis for the development of new antitumor drugs of prolonged action, intended for various modes of administration.