Tissue-Specific Knockdown of Genes of the Argonaute Family Modulates Lifespan and Radioresistance in Drosophila melanogaster

Small RNAs are essential to coordinate many cellular processes, including the regulation of gene expression patterns, the prevention of genomic instability, and the suppression of the mutagenic transposon activity. These processes determine the aging, longevity, and sensitivity of cells and an organism to stress factors (particularly, ionizing radiation). The biogenesis and activity of small RNAs are provided by proteins of the Argonaute family. These proteins participate in the processing of small RNA precursors and the formation of an RNA-induced silencing complex. However, the role of Argonaute proteins in regulating lifespan and radioresistance remains poorly explored. We studied the effect of knockdown of Argonaute genes (AGO1, AGO2, AGO3, piwi) in various tissues on the Drosophila melanogaster lifespan and survival after the γ-irradiation at a dose of 700 Gy. In most cases, these parameters are reduced or did not change significantly in flies with tissue-specific RNA interference. Surprisingly, piwi knockdown in both the fat body and the nervous system causes a lifespan increase. But changes in radioresistance depend on the tissue in which the gene was knocked out. In addition, analysis of changes in retrotransposon levels and expression of stress response genes allow us to determine associated molecular mechanisms.     

Insulin and α-Tocopherol Enhance the Protective Effect of Each Other on Brain Cortical Neurons under Oxidative Stress Conditions and in Rat Two-Vessel Forebrain Ischemia/Reperfusion Injury

Clinical trials show that insulin administered intranasally is a promising drug to treat neurodegenerative diseases, but at high doses its use may result in cerebral insulin resistance. Identifying compounds which could enhance the protective effects of insulin, may be helpful to reduce its effective dose. Our aim was thus to study the efficiency of combined use of insulin and α-tocopherol (α-T) to increase the viability of cultured cortical neurons under oxidative stress conditions and to normalize the metabolic disturbances caused by free radical reaction activation in brain cortex of rats with two-vessel forebrain ischemia/reperfusion injury. Immunoblotting, flow cytometry, colorimetric, and fluorometric techniques were used. α-T enhanced the protective and antioxidative effects of insulin on neurons in oxidative stress, their effects were additive. At the late stages of oxidative stress, the combined action of insulin and α-T increased Akt-kinase activity, inactivated GSK-3beta and normalized ERK1/2 activity in cortical neurons, it was more effective than either drug action. In the brain cortex, ischemia/reperfusion increased the lipid peroxidation product content and caused Na⁺,K⁺-ATPase oxidative inactivation. Co-administration of insulin (intranasally, 0.25 IU/rat) and α-T (orally, 50 mg/kg) led to a more pronounced normalization of the levels of Schiff bases, conjugated dienes and trienes and Na⁺,K⁺-ATPase activity than administration of each drug alone. Thus, α-T enhances the protective effects of insulin on cultured cortical neurons in oxidative stress and in the brain cortex of rats with cerebral ischemia/reperfusion injury.     

The Effects of Separate and Combined Treatment of Male Rats with Type 2 Diabetes with Metformin and Orthosteric and Allosteric Agonists of Luteinizing Hormone Receptor on Steroidogenesis and Spermatogenesis

In men with type 2 diabetes mellitus (T2DM), steroidogenesis and spermatogenesis are impaired. Metformin and the agonists of luteinizing hormone/human chorionic gonadotropin(hCG)-receptor (LH/hCG-R) (hCG, low-molecular-weight allosteric LH/hCG-R-agonists) can be used to restore them. The aim was to study effectiveness of separate and combined administration of metformin, hCG and 5-amino-N-tert-butyl-2-(methylsulfanyl)-4-(3-(nicotinamido)phenyl)thieno[2,3-d]pyrimidine-6-carboxamide (TP3) on steroidogenesis and spermatogenesis in male rats with T2DM. hCG (15 IU/rat/day) and TP3 (15 mg/kg/day) were injected in the last five days of five-week metformin treatment (120 mg/kg/day). Metformin improved testicular steroidogenesis and spermatogenesis and restored LH/hCG-R-expression. Compared to control, in T2DM, hCG stimulated steroidogenesis and StAR-gene expression less effectively and, after five-day administration, reduced LH/hCG-R-expression, while TP3 effects changed weaker. In co-administration of metformin and LH/hCG-R-agonists, on the first day, stimulating effects of LH/hCG-R-agonists on testosterone levels and hCG-stimulated expression of StAR- and CYP17A1-genes were increased, but on the 3–5th day, they disappeared. This was due to reduced LH/hCG-R-gene expression and increased aromatase-catalyzed estradiol production. With co-administration, LH/hCG-R-agonists did not contribute to improving spermatogenesis, induced by metformin. Thus, in T2DM, metformin and LH/hCG-R-agonists restore steroidogenesis and spermatogenesis, with metformin being more effective in restoring spermatogenesis, and their co-administration improves LH/hCG-R-agonist-stimulating testicular steroidogenesis in acute but not chronic administration.     

��-Tocopherol at Nanomolar Concentration Protects PC12 Cells from Hydrogen Peroxide-Induced Death and Modulates Protein Kinase Activities

The aim of this work was to compare protective and anti-apoptotic effects of α-tocopherol at nanomolar and micromolar concentrations against 0.2 mM H₂O₂-induced toxicity in the PC12 neuronal cell line and to reveal protein kinases that contribute to α-tocopherol protective action. The protection by 100 nM α-tocopherol against H₂O₂-induced PC12 cell death was pronounced if the time of pre-incubation with α-tocopherol was 3–18 h. For the first time, the protective effect of α-tocopherol was shown to depend on its concentration in the nanomolar range (1 nM < 10 nM < 100 nM), if the pre-incubation time was 18 h. Nanomolar and micromolar α-tocopherol decreased the number of PC12 cells in late apoptosis induced by H₂O₂ to the same extent if pre-incubation time was 18 h. Immunoblotting data showed that α-tocopherol markedly diminished the time of maximal activation of extracellular signal-regulated kinase 1/2 (ERK 1/2) and protein kinase B (Akt)-induced in PC12 cells by H₂O₂. Inhibitors of MEK 1/2, PI 3-kinase and protein kinase C (PKC) diminished the protective effect of α-tocopherol against H₂O₂-initiated toxicity if the pre-incubation time was long. The modulation of ERK 1/2, Akt and PKC activities appears to participate in the protection by α-tocopherol against H₂O₂-induced death of PC12 cells. The data obtained suggest that inhibition by α-tocopherol in late stage ERK 1/2 and Akt activation induced by H₂O₂ in PC12 cells makes contribution to its protective effect, while total inhibition of these enzymes is not protective.     

Isolation and Characterization of Homologically Expressed Methanol Dehydrogenase from Methylorubrum extorquens AM1 for the Development of Bioelectrocatalytical Systems

(Ca²⁺)-dependent pyrroloquinolinequinone (PQQ)-dependent methanol dehydrogenase (MDH) (EC: 1.1.2.7) is one of the key enzymes of primary C1-compound metabolism in methylotrophy. PQQ-MDH is a promising catalyst for electrochemical biosensors and biofuel cells. However, the large-scale use of PQQ-MDH in bioelectrocatalysis is not possible due to the low yield of the native enzyme. Homologously overexpressed MDH was obtained from methylotrophic bacterium Methylorubrum extorquens AM1 by cloning the gene of only one subunit, mxaF. The His-tagged enzyme was easily purified by immobilized metal ion affinity chromatography (36% yield). A multimeric form (α6β6) of recombinant PQQ-MDH possessing enzymatic activity (0.54 U/mg) and high stability was demonstrated for the first time. pH-optimum of the purified protein was about 9–10; the enzyme was activated by ammonium ions. It had the highest affinity toward methanol (K_M = 0.36 mM). The recombinant MDH was used for the fabrication of an amperometric biosensor. Its linear range for methanol concentrations was 0.002–0.1 mM, the detection limit was 0.7 µM. The properties of the invented biosensor are competitive to the analogs, meaning that this enzyme is a promising catalyst for industrial methanol biosensors. The developed simplified technology for PQQ-MDH production opens up new opportunities for the development of bioelectrocatalytic systems.     

Coating of polylactide films by chitosan: Comparison of methods

Control over biomaterials surface characteristics through surface modification or deposition of coatings is one of the key aspects of tissue engineering. This work was aimed to evaluate an effectiveness of various methods of chitosan-coating formations onto polylactide films using a number of techniques, such as vacuum deposition by electron-beam sputtering, chemical entrapment method, and electrospray procedure. Differently coated films were studied in terms of surface morphology (scanning electron and atomic-force microscopy), chemical structure (FTIR and X-ray photoelectron spectroscopy), and hydrophilic/hydrophobic balance (goniometry). The effect of coating technique on homogeneity of chitosan distribution over the substrate surface was evaluated using genipin and fluorescein isothiocyanate labeling as well as FTIR-microscopy. © 2019 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2020 , 137, 48287.     

Linear and Non-Linear Optical Imaging of Cancer Cells with Silicon Nanoparticles

New approaches for visualisation of silicon nanoparticles (SiNPs) in cancer cells are realised by means of the linear and nonlinear optics in vitro. Aqueous colloidal solutions of SiNPs with sizes of about 10–40 nm obtained by ultrasound grinding of silicon nanowires were introduced into breast cancer cells (MCF-7 cell line). Further, the time-varying nanoparticles enclosed in cell structures were visualised by high-resolution structured illumination microscopy (HR-SIM) and micro-Raman spectroscopy. Additionally, the nonlinear optical methods of two-photon excited fluorescence (TPEF) and coherent anti-Stokes Raman scattering (CARS) with infrared laser excitation were applied to study the localisation of SiNPs in cells. Advantages of the nonlinear methods, such as rapid imaging, which prevents cells from overheating and larger penetration depth compared to the single-photon excited HR-SIM, are discussed. The obtained results reveal new perspectives of the multimodal visualisation and precise detection of the uptake of biodegradable non-toxic SiNPs by cancer cells and they are discussed in view of future applications for the optical diagnostics of cancer tumours.     

Low temperature solution-phase growth of ZnSe and ZnSe/CdSe core/shell nanowires

High quality ZnSe nanowires (NWs) and complementary ZnSe/CdSe core/shell species have been synthesized using a recently developed solution-liquid-solid (SLS) growth technique. In particular, bismuth salts as opposed to pre-synthesized Bi or Au/Bi nanoparticles have been used to grow NWs at low temperatures in solution. Resulting wires are characterized using transmission electron microscopy and possess mean ensemble diameters between 15 and 28 nm with accompanying lengths ranging from 4-10 μm. Subsequent solution-based overcoating chemistry results in ZnSe wires covered with CdSe nanocrystals. By varying the shell's growth time, different thicknesses can be obtained and range from 8 to 21 nm. More interestingly, the mean constituent CdSe nanocrystal diameter can be varied and results in size-dependent shell emission spectra.