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.

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.

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.

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.

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.

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.

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.