How Does the Sample Preparation of Phytophthora infestans Mycelium Affect the Quality of Isolated RNA?

RNA isolation from fungi and fungus-like organisms is not an easy task. Active endogenous RNases quickly hydrolyze RNA after the sample collection, and the thick cell wall prevents inhibitors from penetrating the cells. Therefore, the initial collection and grinding steps may be crucial for the total RNA isolation from the mycelium. When isolating RNA from Phytophthora infestans, we varied the grinding time of the Tissue Lyser and used TRIzol and beta-mercaptoethanol to inhibit the RNase. In addition, we tested the mortar and pestle grinding of mycelium in liquid nitrogen, with this method showing the most consistent results. During the sample grinding with the Tissue Lyser device, adding an RNase inhibitor proved to be a prerequisite, and the best results were achieved using TRIzol. We considered ten different combinations of grinding conditions and isolation methods. The classical combination of a mortar and pestle, followed by TRIzol, has proved to be the most efficient.

Stabilization of Octahedral Metal Halide Clusters by Host-Guest Complexation with γ-Cyclodextrin: Toward Nontoxic Luminescent Compounds.

γ-Cyclodextrin (γ-CD) interacts in aqueous solution with octahedral halide clusters Na2[{M6X8}Cl6] (M = Mo, W; X = Br, I) to form robust inclusion supramolecular complexes [{M6X8}Cl6@2γ-CD]2-. Single-crystal X-ray diffraction analyses revealed two conformational organizations within the adduct depending on the nature of the inner halide X within the {M6X8} core. Using 35Cl NMR and UV-vis as complementary techniques, the kinetics of the hydrolysis process were shown to increase with the following order: {W6I8} < {W6Br8} ≈ {Mo6I8} < {Mo6Br8}. The complexation with γ-CD drastically enhances the hydrolytic stability of luminescent [{M6X8}Cl6]2- cluster-based units, which was quantitatively proved by the same techniques. The resulting host-guest complexation provides a protective shell against contact with water and offers promising horizons for octahedral clusters in biology as revealed by the low dark cytotoxicity and cellular uptake.

Anti-Inflammatory Activity of Soluble Epoxide Hydrolase Inhibitors Based on Selenoureas Bearing an Adamantane Moiety.

The inhibitory potency of the series of inhibitors of the soluble epoxide hydrolase (sEH) based on the selenourea moiety and containing adamantane and aromatic lipophilic groups ranges from 34.3 nM to 1.2 µM. The most active compound 5d possesses aliphatic spacers between the selenourea group and lipophilic fragments. Synthesized compounds were tested against the LPS-induced activation of primary murine macrophages. The most prominent anti-inflammatory activity, defined as a suppression of nitric oxide synthesis by LPS-stimulated macrophages, was demonstrated for compounds 4a and 5b. The cytotoxicity of the obtained substances was studied using human neuroblastoma and fibroblast cell cultures. Using these cell assays, the cytotoxic concentration for 4a was 4.7-18.4 times higher than the effective anti-inflammatory concentration. The genotoxicity and the ability to induce oxidative stress was studied using bacterial lux-biosensors. Substance 4a does not exhibit genotoxic properties, but it can cause oxidative stress at concentrations above 50 µM. Put together, the data showed the efficacy and safety of compound 4a.

Recent Advances in the Development of Exogenous dsRNA for the Induction of RNA Interference in Cancer Therapy.

Selective regulation of gene expression by means of RNA interference has revolutionized molecular biology. This approach is not only used in fundamental studies on the roles of particular genes in the functioning of various organisms, but also possesses practical applications. A variety of methods are being developed based on gene silencing using dsRNA-for protecting agricultural plants from various pathogens, controlling insect reproduction, and therapeutic techniques related to the oncological disease treatment. One of the main problems in this research area is the successful delivery of exogenous dsRNA into cells, as this can be greatly affected by the localization or origin of tumor. This overview is dedicated to describing the latest advances in the development of various transport agents for the delivery of dsRNA fragments for gene silencing, with an emphasis on cancer treatment.

Foveolar Müller Cells of the Pied Flycatcher: Morphology and Distribution of Intermediate Filaments Regarding Cell Transparency.

Specialized intermediate filaments (IFs) have critical importance for the clearness and uncommon transparency of vertebrate lens fiber cells, although the physical mechanisms involved are poorly understood. Recently, an unusual low-scattering light transport was also described in retinal Müller cells. Exploring the function of IFs in Müller cells, we have studied the morphology and distribution pattern of IFs and other cytoskeletal filaments inside the Müller cell main processes in the foveolar part of the avian (pied flycatcher) retina. We found that some IFs surrounded by globular nanoparticles (that we suggest are crystallines) are present in almost every part of the Müller cells that span the retina, including the microvilli. Unlike IFs implicated in the mechanical architecture of the cell, these IFs are not connected to any specific cellular membranes. Instead, they are organized into bundles, passing inside the cell from the endfeet to the photoreceptor, following the geometry of the processes, and repeatedly circumventing numerous obstacles. We believe that the presently reported data effectively confirm that the model of nanooptical channels built of the IFs may provide a viable explanation of Müller cell transparency.

Exogenous dsRNA-Induced Silencing of the Phytophthora infestans Elicitin Genes inf1 and inf4 Suppresses Its Pathogenicity on Potato Plants.

Phytophthora infestans, an Oomycete pathogen, has a devastating impact on potato agriculture, leading to the extensive use of chemical fungicides to prevent its outbreaks. Spraying double-stranded RNAs to suppress specific genes of the pathogen via the RNA interference (RNAi) pathway may provide an environmentally friendly alternative to chemicals. However, this novel approach will require various target genes and application strategies to be tested. Using the L4440 backbone, we have designed two plasmids to express dsRNA targeting inf1 and inf4 genes of P. infestans that are known to contribute to the disease development at different stages. The dsRNA produced by the bacteria was tested on potato explants and demonstrated a statistically significant reduction in lesions five days after inoculation compared to water treatment. The study results allow us to consider our approach to be promising for potato late blight control.

Metabolic rate, sleep duration, and body temperature in evolution of mammals and birds: the influence of geological time of principal groups divergence.

This study contains an analysis of basal metabolic rate (BMR) in 1817 endothermic species. The aim was to establish how metabolic scaling varies between the main groups of endotherms during evolution. The data for all the considered groups were combined and the common exponent in the allometric relationship between the BMR and body weight was established as b = 0.7248. Reduced to the common slope, the relative metabolic rate forms the following series: Neognathae - Passeriformes - 1.00, Neognathae - Non-Passeriformes - 0.75, Palaeognathae - 0.53, Eutheria - 0.57, Marsupialia - 0.44, and Monotremata - 0.26. The main finding is that the metabolic rate in the six main groups of mammals and birds consistently increases as the geological time of the group's divergence approaches the present. In parallel, the average body temperature in the group rises, the duration of sleep decreases and the duration of activity increases. BMR in a taxon correlates with its evolutionary age: the later a clade diverged, the higher is its metabolic rate and the longer is its activity period; group exponents decrease as group divergence nears present times while with increase metabolic rate during activity, they not only do not decrease but can increase. Sleep duration in mammals was on average 40% longer than in birds while BMR, in contrast, was 40% higher in birds. The evolution of metabolic scaling, body temperature, sleep duration, and activity during the development of endothermic life forms is demonstrated, allowing for a better understanding of the underlying principles of endothermy formation.

Evolution of metabolic scaling among the tetrapod: effect of phylogeny, the geologic time of class formation, and uniformity of species within a class.

The metabolic scaling in the animal has been discussed for over 90 years, but no consensus has been reached. Our analysis of 2126 species of vertebrates reveals a significant allometric exponent heterogeneity. We show that classes of terrestrial vertebrates exhibit the evolution of metabolic scaling. Both the allometric coefficient "a" and the allometric exponent "b" change naturally, but differently depending on the geological time of group formation. The allometric coefficient "a" shows the measure of the evolutionary development of systems that forms resting metabolism in animals. Endothermic classes, such as birds and mammals, have a metabolic rate that is in an order of magnitude higher than that in ectothermic classes, including amphibians and reptiles. In the terrestrial vertebrate phylogeny, we find that the metabolic scaling is characterized by 3 main allometric exponent values: b = 3/4 (mammals), b > 3/4 (ectotherms, such as amphibians and reptiles), and b < 3/4 (birds). The heterogeneity of the allometric exponent is a natural phenomenon associated with the general evolution of vertebrates. The scaling factor decreases depending on both the external design and the size (birds vs mammals) of the animal. The metabolic rate and uniformity of species within a class increase as the geological start date of formation of the class approaches the present time. The higher the mass-specific standard metabolic rate in the class, the slower metabolic rate grows with increasing body size in this class. Our results lay the groundwork for further exploration of the evolutionary and ecological aspects of the development of metabolic scaling in animals.

Metabolic Scaling in Birds and Mammals: How Taxon Divergence Time, Phylogeny, and Metabolic Rate Affect the Relationship between Scaling Exponents and Intercepts.

Analysis of metabolic scaling in currently living endothermic animal species allowed us to show how the relationship between body mass and the basal metabolic rate (BMR) has evolved in the history of endothermic vertebrates. We compared six taxonomic groups according to their energetic characteristics and the time of evolutionary divergence. We transformed the slope of the regression lines to the common value and analyzed three criteria for comparing BMR of different taxa regardless of body size. Correlation between average field metabolic rate (FMR) of the group and its average BMR was shown. We evaluated the efficiency of self-maintenance in ordinary life (defined BMR/FMR) in six main groups of endotherms. Our study has shown that metabolic scaling in the main groups of endothermic animals correlates with their evolutionary age: the younger the group, the higher the metabolic rate, but the rate increases more slowly with increasing body weight. We found negative linear relationship for scaling exponents and the allometric coefficient in five groups of endotherms: in units of mL O2/h per g, in relative units of allometric coefficients, and also in level or scaling elevation. Mammals that diverged from the main vertebrate stem earlier have a higher "b" exponent than later divergent birds. A new approach using three criteria for comparing BMR of different taxa regardless of body mass will be useful for many biological size-scaling relationships that follow the power function.

A protocol for recruiting and analyzing the disease-oriented Russian disc degeneration study (RuDDS) biobank for functional omics studies of lumbar disc degeneration.

Lumbar intervertebral disc degeneration (DD) disease is one of the main risk factors for low back pain and a leading cause of population absenteeism and disability worldwide. Despite a variety of biological studies, lumbar DD is not yet fully understood, partially because there are only few studies that use systematic and integrative approaches. This urges the need for studies that integrate different omics (including genomics and transcriptomics) measured on samples within a single cohort. This protocol describes a disease-oriented Russian disc degeneration study (RuDDS) biobank recruitment and analyses aimed to facilitate further omics studies of lumbar DD integrating genomic, transcriptomic and glycomic data. A total of 1,100 participants aged over 18 with available lumbar MRI scans, medical histories and biological material (whole blood, plasma and intervertebral disc tissue samples from surgically treated patients) will be enrolled during the three-year period from two Russian clinical centers. Whole blood, plasma and disc tissue specimens will be used for genotyping with genome-wide SNP-arrays, glycome profiling and RNA sequencing, respectively. Omics data will be further used for a genome-wide association study of lumbar DD with in silico functional annotation, analysis of plasma glycome and lumbar DD disease interactions and transcriptomic data analysis including an investigation of differential expression patterns associated with lumbar DD disease. Statistical tests applied in each of the analyses will meet the standard criteria specific to the attributed study field. In a long term, the results of the study will expand fundamental knowledge about lumbar DD development and contribute to the elaboration of novel personalized approaches for disease prediction and therapy. Additionally to the lumbar disc degeneration study, a RuDDS cohort could be used for other genetic studies, as it will have unique omics data. Trial registration number NCT04600544.

Method for the Isolation of "RNA-seq-Quality" RNA from Human Intervertebral Discs after Mortar and Pestle Homogenization.

The problem of isolating high-quality total RNA from intervertebral discs has no recognized solution yet. This is due to the extremely low content of live cells in the samples and the voluminous intercellular matrix. A variety of published protocols focused on isolating RNA from articular cartilage have recommended the use of expensive equipment, enzymatic matrix cleavage, or cell culture. In our study, we used a combination of the traditional QIAzol protocol (Qiagen, Germany) and RNEasy column purification (Qiagen, Germany) to obtain high-quality RNA from post-surgical intervertebral disc fragments. Only a mortar and a pestle were used for grinding, making our method particularly accessible. The isolated RNA with a RIN of ~7 is suitable for studying the expression profile of chondrocytes in situ. RNA-seq analysis of three samples demonstrated cell type ratios to be mostly relevant to intervertebral disc tissues, with over 70% of the chondrocytes of the three subtypes having an admixture of blood-related cells.

Investigation of cytotoxic and antioxidative activity of 1,2,3-triazolyl-modified furocoumarins and 2,3-dihydrofurocoumarins.

High biological activity of natural furocoumarins is often linked to a series of adverse side effects, e.g., genotoxicity. This makes it desirable to develop semi-synthetic derivatives with reduced negative activity while retaining or even enhancing the positive properties. Previously, we have studied the genotoxic activity of a library of twenty-one 1,2,3-triazolyl-modified furocoumarins and 2,3-dihydrofurocoumarins and identified modifications that minimize the negative properties. In the current article, we report on an investigation into the cytotoxic activity of the same library. We have aimed to rank the substances in order of the severity of their cytotoxicity and therefore to predict, with the use of statistical processing, the most promising substituents for the furocoumarin scaffold.

Phytophthora infestans: An Overview of Methods and Attempts to Combat Late Blight.

Phytophthora infestans (Mont.) de Bary is one of the main pathogens in the agricultural sector. The most affected are the Solanaceae species, with the potato (Solanum tuberosum) and the tomato (Solanum lycopersicum) being of great agricultural importance. Ornamental Solanaceae can also host the pests Petunia spp., Calibrachoa spp., as well as the wild species Solanum dulcamara, Solanum sarrachoides, etc. Annual crop losses caused by this pathogen are highly significant. Although the interaction between P. infestans and the potato has been investigated for a long time, further studies are still needed. This review summarises the basic approaches in the fight against the late blight over the past 20 years and includes four sections devoted to methods of control: (1) fungicides; (2) R-gene-based resistance of potato species; (3) RNA interference approaches; (4) other approaches to control P. infestans. Based on the latest advances, we have provided a description of the significant advantages and disadvantages of each approach.

Clinical benefits of single vs repeated courses of mesenchymal stem cell therapy in epilepsy patients.

PURPOSE: Epilepsy is defined as "drug-resistant" when existing anti-epileptic drugs (AED) are found to have minimal to no effect on patient's condition. Therefore the search and testing of new treatment strategies is warranted. This study focuses on the effects of autologous mesenchymal stem cells (MSC) in drug-resistant epilepsy patients within a Phase I/II open-label registered clinical trial NCT02497443. MATERIALS/METHODS: A total of 67 patients was included (29 males, 38 females, mean age 33 ± 1.3 yo). The patients received either standard treatment with AEDs, or AEDs supplemented with one or two courses of therapy with autologous bone marrow-derived MSCs expanded in vitro. MSC therapy courses were 6 months apart, and each course consisted of two cell injections: an intravenous infusion of MSCs, followed within 1 week by an intrathecal injection. Primary outcome of the study was safety, secondary outcome was efficacy in terms of seizure frequency reduction and response to treatment. RESULTS: MSC injections proved safe and did not cause any severe side effects. In MSC group (n = 34), 61.7% patients responded to therapy at 6 months timepoint (p < 0.01 vs control, n = 33), and the number rose to 76.5% by 12 months timepoint. Decrease in anxiety and depression scores and paroxysmal epileptiform activity was observed in MSC group based on HADS and EEG, respectively, and MMSE score has also improved. Another observation was that concomitant administration of levetiracetam, but not other AEDs, correlated significantly with the success of MSC therapy. Second course of MSC therapy facilitated further reduction in seizure count and epileptiform EEG activity (p < 0.05 vs single course). CONCLUSIONS: Application of autologous mesenchymal stem cell-based therapy in patients with pharmacoresistant epilepsy demonstrated significant anticonvulsant potential. This effect lasted for at least 1 year, with repeated administration of MSCs conveying additional clinical benefit.

Electron microscopy study of the central retinal fovea in Pied flycatcher: evidence of a mechanism of light energy transmission through the retina.

We present unique ultrastructural data on avian retinal cells. Presently and earlier (Zueva et al., 2016) we explored distribution of intermediate filaments (IFs) in retinal cells of the Pied flycatcher (Ficedula hypoleuca, Passeriformes, Aves) in the central foveolar zone. This retinal zone only contains single and double cone photoreceptors. Previously we found that continuous IFs span Müller cells (MC) lengthwise from the retinal inner limiting membrane (ILM) layer up to the outer limiting membrane (OLM) layer. Here we describe long cylindrical bundles of IFs (IFBs) inside the cone inner segments (CIS) adjoining the cone plasma membrane, with these IFBs following along the cone lengthwise, and surrounding the cone at equal spacing one from the other. Double cones form a combined unit, wherein they are separated by their respective plasma membranes. Double cones thus have a common external ring of IFBs, surrounding both cone components. In the layer of cilia, the IFBs that continue into the cone outer segment (COS) follow on to the cone apical tip along the direction of incident light, with single IFs separating from the IFB, touching, and sometimes passing in-between the light-sensitive lamellae of the COS. These new data support our previous hypothesis on the quantum mechanism of light energy propagation through the vertebrate retina (Zueva et al., 2016, 2019).

Quantum mechanism of light energy propagation through an avian retina.

Taking into account the ultrastructure of the Pied Flycatcher foveal retina reported earlier and the earlier reported properties of Müller cell (MC) intermediate filaments (IFs) isolated from vertebrate retina, we proposed a quantum mechanism (QM) of light energy transfer from the inner limiting membrane level to visual pigments in the photoreceptor cells. This mechanism involves electronic excitation energy transfer in a donor-acceptor system, with the IFs excited by photons acting as energy donors, and visual pigments in the photoreceptor cells acting as energy acceptors. It was shown earlier that IFs with diameter 10 nm and length 117 µm isolated from vertebrate eye retina demonstrate properties of light energy guide, where exciton propagates along such IFs from MC endfeet area to photoreceptor cell area. The energy is mostly transferred via the contact exchange quantum mechanism. Our estimates demonstrate that energy transfer efficiencies in such systems may exceed 80-90%. Thus, the presently developed quantum mechanism of light energy transfer in the inverted retina complements the generally accepted classic optical mechanism and the mechanism whereby Müller cells transmit light like optical fibers. The proposed QM of light energy transfer in the inverted retina explains the high image contrast achieved in photopic conditions by an avian eye, being probably also active in other vertebrates.

Quantum mechanism of light transmission by the intermediate filaments in some specialized optically transparent cells.

Some very transparent cells in the optical tract of vertebrates, such as the lens fiber cells, possess certain types of specialized intermediate filaments (IFs) that have essential significance for their transparency. The exact mechanism describing why the IFs are so important for transparency is unknown. Recently, transparency was described also in the retinal Müller cells (MCs). We report that the main processes of the MCs contain bundles of long specialized IFs, each about 10 nm in diameter; most likely, these filaments are the channels providing light transmission to the photoreceptor cells in mammalian and avian retinas. We interpret the transmission of light in such channels using the notions of quantum confinement, describing energy transport in structures with electroconductive walls and diameter much smaller than the wavelength of the respective photons. Model calculations produce photon transmission efficiency in such channels exceeding 0.8, in optimized geometry. We infer that protein molecules make up the channels, proposing a qualitative mechanism of light transmission by such structures. The developed model may be used to describe light transmission by the IFs in any transparent cells.

Errata: Quantum mechanism of light transmission by the intermediate filaments in some specialized optically transparent cells.

[This corrects the article DOI: 10.1117/1.NPh.4.1.011005.].

Spectral selectivity model for light transmission by the intermediate filaments in Müller cells.

Presently we continue our studies of the quantum mechanism of light energy transmission in the form of excitons by axisymmetric nanostructures with electrically conductive walls. Using our theoretical model, we analyzed the light energy transmission by biopolymers forming optical channels within retinal Müller cells. There are specialized intermediate filaments (IF) 10-18nm in diameter, built of electrically conductive polypeptides. Presently, we analyzed the spectral selectivity of these nanostructures. We found that their transmission spectrum depends on their diameter and wall thickness. We also considered the classical approach, comparing the results with those predicted by the quantum mechanism. We performed experimental measurements on model quantum waveguides, made of rectangular nanometer-thick chromium (Cr) tracks. The optical spectrum of such waveguides varied with their thickness. We compared the experimental absorption/transmission spectra with those predicted by our model, with good agreement between the two. We report that the observed spectra may be explained by the same mechanisms as operating in metal nanolayers. Both the models and the experiment show that Cr nanotracks have high light transmission efficiency in a narrow spectral range, with the spectral maximum dependent on the layer thickness. Therefore, a set of intermediate filaments with different geometries may provide light transmission over the entire visible spectrum with a very high (~90%) efficiency. Thus, we believe that high contrast and visual resolution in daylight are provided by the quantum mechanism of energy transfer in the form of excitons, whereas the ultimate retinal sensitivity of the night vision is provided by the classical mechanism of photons transmitted by the Müller cell light-guides.

On the Role of the Blood Vessel Endothelial Microvilli in the Blood Flow in Small Capillaries.

Endothelial microvilli that protrude into the capillary lumen, although invisible in the optical microscopy, may play an important role in the blood flow control in the capillaries. Because of the plug effects, the width of the gap between the capillary wall and the blood cell is especially critical for the blood flow dynamics in capillaries, while microvilli located on the capillary wall can easily control the velocity of the blood flow. We report that microvilli in the capillaries of different vertebrate species have similar characteristics and density, suggesting similarities between the respective regulation mechanisms. A simplified physical model of the capillary effective diameter control by the microvilli is presented.