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Invertebrates mainly rely on sequence-specific RNA interference (RNAi) to resist viral infections. Increasing studies show that double-stranded RNA (dsRNA) can induce sequence-independent protection and that Dicer-2, the key RNAi player that cleaves long dsRNA into small interfering RNA (siRNA), is necessary for this protection. However, how this protection occurs remains unknown. Herein, we report that it is caused by adenosine triphosphate (ATP)-hydrolysis accompanying the dsRNA-cleavage. Dicer-2 helicase domain is ATP-dependent; therefore, the cleavage consumes ATP. ATP depletion activates adenosine monophosphate-activated protein kinase (Ampk) and induces nuclear localization of Fork head box O (FoxO), a key transcriptional factor for dsRNA-induced genes. siRNAs that do not require processing cannot activate the transcriptional response. This study reveals a unique nonspecific antiviral mechanism other than the specific RNAi in shrimp. This mechanism is functionally similar to, but mechanistically different from, the dsRNA-activated antiviral response in vertebrates and suggests an interesting evolution of innate antiviral immunity. [ABSTRACT FROM AUTHOR]

Scaphoideus titanus Ball (Hemiptera: Cicadellidae) is a major pest for European viticulture due to its high efficiency in the transmission of one of the most destructive pathogens for grapevine, namely flavescence dorée phytoplasmas. Although it plays a major role in spreading this disease, S. titanus is part of a complex epidemiological cycle involving several alternative vectors with variable relevance for phytoplasma spread. Here we provide an updated review on S. titanus monitoring and modelling, as well as the available tools for management of this pest and for limiting phytoplasma transmission and, thus, also spread. Insecticide-based control is examined; additional emphasis is placed on innovative and low-impact control approaches, such as vibrational mating disruption, biocontrol, and methods to reduce vector competence. We also discuss the main emerging challenges to the implementation of effective and sustainable control programs against S. titanus. [ABSTRACT FROM AUTHOR]

BACKGROUND: Implementation of resistance management tools is crucial for the continued efficacy of insect control technologies. An important aspect of insect resistance management (IRM) is the combined or sequential use of different modes‐of‐action to reduce selection pressure and delay evolution of resistance. This is especially important for insect pests with established ability to develop resistance to insecticides, such as the Colorado potato beetle (Leptinotarsa decemlineata, CPB). A new class of insecticides, based on double‐stranded RNA (dsRNA) activating the gene silencing RNA‐interference (RNAi) pathway, are currently under review for regulatory approval and commercial use in the USA against CPB. However, there is no information available on the potential for cross‐resistance between RNAi insecticides and other classes of insecticides used against CPB. Herein, we aim to fill this knowledge gap by capitalizing on the availability of a CPB strain highly resistant to dsRNAs and test its susceptibility to diverse small‐molecule insecticide classes compared to reference dsRNA‐susceptible CPB strains. RESULTS: Differences in activity were observed among the four insecticides tested, with abamectin demonstrating highest activity against all three strains of CPB. However, no differences were observed among the dsRNA‐resistant and susceptible CPB strains for any of the tested compounds. Overall, these results demonstrate lack of cross‐resistance to commonly used chemical insecticides in the dsRNA‐resistant strain of CPB. CONCLUSION: These data support the use of these different insecticide classes along with RNAi‐based insecticides as part of an effective insect resistance management framework aimed at delaying resistance in CPB. © 2023 Society of Chemical Industry. [ABSTRACT FROM AUTHOR]

The purpose of the research is to analyze the literature devoted to the study of the physiological role and functional significance of biologically active substances: FMRFamide-like neuropeptides in the locomotion of root-knot nematodes Meloidogyne incognita, M. minor, M. hapla and M. graminicola using immunological, phylogenetic, molecular and bioinformatic research methods.Results and discussion. The present work shows the importance of endogenous FMRFamide-like neuropeptides (FLPs) in such behavioral reactions of plant nematodes as locomotion, which ensures the vital activity of plant parasites; the functional significance of flp genes in the neurobiology of root-knot nematodes is discussed. It was especially noted that the main physiological and functional characteristics of endogenous FLP in root-knot nematodes were obtained as a result of studies of the functional role of the flp genes encoding these neuropeptides. In the nematodes M. incognita and M. graminicola, components of the peptidergic nervous system were identified in the nervous structures: FMRFamide-like positive immunoreactivity, FLP, flp genes encoding neuropeptides, and G-protein-coupled receptors (GPCR) activated by these neuropeptides. It was shown that the main functional characteristics of endogenous FLPs in nematodes were obtained using one of the methods of reverse genetics, i.e., flp genes knockdown in shadow by means of RNA-interference. It has been established that FLP cause two types of physiological effects on the somatic muscles of root-knot nematodes – stimulation of the locomotor activity of the muscles and its inhibition. In most works, the data obtained on the physiological effects of neuropeptides on the locomotor activity of phytonematodes are considered with a view to their possible use in the development of new targeted anthelmintic drugs.

Bacillus thuringiensis Berliner (Bt) and B. cereus sensu stricto Frankland and Frankland are closely related species of aerobic, spore-forming bacteria included in the B. cereus sensu lato group. This group is one of the most studied, but it remains also the most mysterious species of bacteria. Despite more than a century of research on the features of these ubiquitous bacteria, there are a lot of questionable issues related to their taxonomy, resistance to external influences, endophytic existence, their place in multidimensional relationships in the ecosystem, and many others. The review summarizes current data on the mutualistic relationships of Bt and B. cereus bacteria with plants, the structure of the phytomicrobiomes including Bt and B. cereus, and the abilities of plant-associated and endophytic strains to improve plant resistance to various environmental factors and its productivity. Key findings on the possibility of the use of Cry gene promoter for transcription of the target dsRNA and simultaneous release of pore-forming proteins and provocation of RNA-interference in pest organisms allow us to consider this group of microorganisms as unique tools of genetic engineering and biological control. This will open the prospects for the development and direct change of plant microbiomes, and possibly serve as the basis for the regulation of the entire agroecosystem. [ABSTRACT FROM AUTHOR]

The global climate changes and the consequent increase in the number of soil and air droughts during the vegetation period of grain crops require the development of new strategies to adapt plants to those yield-decreasing stressors. A relevant way of increasing drought-tolerance of cereals is the use of biotechnological methods, particularly RNA interference, which can down-regulate the activity of plants’ genes and increase concentration of stress metabolites that perform osmoprotective functions during drought. We studied the tolerance to soil moisture shortage in transgenic plants of winter wheat with partial suppression of the proline dehydrogenase gene, obtained using the technology of short interfering RNAs. We analyzed physiological and biochemical parameters and structural elements of yield productivity of 4 wild genotypes and their transgenic lines with reduced activity of proline dehydrogenase in the conditions of 7-day drought during the late booting–ear emergence. We determined that the presence of double-stranded RNA suppressor of the proline dehydrogenase gene in transgenic lines led to increase in the level of accumulation of free proline in flag leaves. At the same time, its concentration in transgenic lines was higher than in untransformed plants of the wild genotypes in both drought conditions and conditions of sufficient moisture. We found that against the background of water deficiency, the total chlorophyll content in leaves of plants of transgenic lines was significantly higher, and the ratio of carotenoids to chlorophyll was lower than in plants of the wild genotypes, suggesting mitigation of the negative impact of drought on the plants of transgenic lines. Lacking soil moisture, genetically altered lines of wheat had significantly higher parameters of the structure of grain yield compared with untransformed genotypes. At the same time, we observed genotypic difference according to grain productivity in biotechnological plants. Therefore, the results we obtained confirm the perspectives of using the technology of short interfering RNAs to increase tolerance of winter wheat to water deficiency.

Simple Summary: The increasingly growing demand for palm oil (PO) blankets heavy pressure for sustainable PO production. PO, the world's most efficient oilseed crop, is being used in various applications, including food, cosmetics, and biofuels. However oil palm is always at risk being exposed to Metisa plana infestation, which contributes to the negative impacts on the oil palm plantations and industries. Therefore, conventional pesticides are routinely applied as a control measure in outbreak management. Nevertheless, the perpetual occurrences of M. plana infestation remain, affecting the overall yield productivity. This study uses an integrated bioinformatics approach, including a gene co-expression network and clustering method, to screen, identify, and determine the key genes involved in controlling insect hormone biosynthesis with ecdysone and juvenile hormones among them. The findings provide key regulatory genes (Hnf4, Hr4, MED14, Usp, Tai, and Trr) that could be used as potential targets for gene silencing technologies such as RNA interference (RNAi) desired in understanding M. plana gene function. Metisa plana Walker (Lepidoptera: Psychidae) is a major oil palm pest species distributed across Southeast Asia. M. plana outbreaks are regarded as serious ongoing threats to the oil palm industry due to their ability to significantly reduce fruit yield and subsequent productivity. Currently, conventional pesticide overuses may harm non-target organisms and severely pollute the environment. This study aims to identify key regulatory genes involved in hormone pathways during the third instar larvae stage of M. plana gene co-expression network analysis. A weighted gene co-expression network analysis (WGCNA) was conducted on the M. plana transcriptomes to construct a gene co-expression network. The transcriptome datasets were obtained from different development stages of M. plana, i.e., egg, third instar larvae, pupa, and adult. The network was clustered using the DPClusO algorithm and validated using Fisher's exact test and receiver operating characteristic (ROC) analysis. The clustering analysis was performed on the network and 20 potential regulatory genes (such as MTA1-like, Nub, Grn, and Usp) were identified from ten top-most significant clusters. Pathway enrichment analysis was performed to identify hormone signalling pathways and these pathways were identified, i.e., hormone-mediated signalling, steroid hormone-mediated signalling, and intracellular steroid hormone receptor signalling as well as six regulatory genes Hnf4, Hr4, MED14, Usp, Tai, and Trr. These key regulatory genes have a potential as important targets in future upstream applications and validation studies in the development of biorational pesticides against M. plana and the RNA interference (RNAi) gene silencing method. [ABSTRACT FROM AUTHOR]

Huntington's disease (HD) is an autosomal dominant ailment that affects a larger population. Due to its complex pathology operating at DNA, RNA, and protein levels, it is regarded as a protein-misfolding disease and an expansion repeat disorder. Despite the availability of early genetic diagnostics, disease-modifying treatments are still missing. Importantly, potential therapies are starting to make their way through clinical trials. Still, clinical trials are ongoing to discover potential drugs to relieve HD symptoms. However, now being aware of the root cause, the clinical studies are focused on molecular therapies to target it. The road to success has not been without bumps since a big phase III trial of tominersen was unexpectedly discontinued due to exceeding risks than drug's benefit to the patients. Although the trial's conclusion was disappointing, there is still cause to be optimistic about what this technique may achieve. We have reviewed the present disease-modifying therapies in clinical development for HD and examined the current landscape of developing clinical therapies. We further investigated the pharmaceutical development of Huntington's medicine in the pharma industries and addressed the existing challenges in their therapeutic success. • Antisense oligonucleotides define new era of Huntington' disease therapy. • Tetrabenazine, Pridopidine and Valbenazine are key symptomatic therapeutics for HD. • Biomarin, WVE-003, and AMT-130 are key developing disease-modifying therapy for HD. • Preclinical promises of CRISPR define their potential for future genetic medicines. [ABSTRACT FROM AUTHOR]

RNA interference (RNAi) has potential to become a major tool for integrated management of insect pests of agricultural crops based on sequencespecificity and low doses of rapidly biodegradable dsRNA. Deploying ‘environmental RNAi’ for control of insect vectors of plant pathogens is of increasing interest for combatting emerging plant diseases. Hemipteran insect vectors, including psyllids, are vascular feeders, making their development difficult to control specifically by targeting with pesticidal chemistries. Psyllids transmit “Candidatus Liberibacter solanacearum” the causal organism of potato zebra chip and tomato vein greening diseases, transmitted, respectively, by the potato or tomato psyllid (PoP). Until now, the optimal effective concentration(s) of double-stranded RNA (dsRNA) required for significant gene knockdown and RNAi persistence in PoP have not been determined. The objective of this study was to optimize RNAi in young PoP adults and 3rd instars for screening by oral delivery of dsRNAs. The minimal effective dsRNA concentrations required for robust knockdown and persistence were evaluated by delivering seven concentrations spanning 0.1 ng/μL to 500 ng/μL over post ingestion-access periods (IAP) ranging from 48 h to 12 days. The PoP gene candidates evaluated as targets were vacuolar ATPase subunit A, clathrin heavy chain, and non-fermenting protein 7, which were evaluated for knockdown by qPCR amplification. The minimum and/or the second most effective dsRNA concentration resulting in effective levels of gene knockdown was 100 ng/μL for all three targets. Higher concentrations did not yield further knockdown, indicating potential RISC saturation at the higher doses. Gene silencing post-IAP of 100 ng/μL dsRNA persisted for 3–5 days in adults and nymphs, with the PoP 3rd instar, followed by teneral and mature adults, respectively, exhibiting the most robust RNAiresponse. [ABSTRACT FROM AUTHOR]

The global climate changes and the consequent increase in the number of soil and air droughts during the vegetation period of grain crops require the development of new strategies to adapt plants to those yield-decreasing stressors. A relevant way of increasing droughttolerance of cereals is the use of biotechnological methods, particularly RNA interference, which can down-regulate the activity of plants’ genes and increase concentration of stress metabolites that perform osmoprotective functions during drought. We studied the tolerance to soil moisture shortage in transgenic plants of winter wheat with partial suppression of the proline dehydrogenase gene, obtained using the technology of short interfering RNAs. We analyzed physiological and biochemical parameters and structural elements of yield productivity of 4 wild genotypes and their transgenic lines with reduced activity of proline dehydrogenase in the conditions of 7-day drought during the late booting–ear emergence. We determined that the presence of double-stranded RNA suppressor of the proline dehydrogenase gene in transgenic lines led to increase in the level of accumulation of free proline in flag leaves. At the same time, its concentration in transgenic lines was higher than in untransformed plants of the wild genotypes in both drought conditions and conditions of sufficient moisture. We found that against the background of water deficiency, the total chlorophyll content in leaves of plants of transgenic lines was significantly higher, and the ratio of carotenoids to chlorophyll was lower than in plants of the wild genotypes, suggesting mitigation of the negative impact of drought on the plants of transgenic lines. Lacking soil moisture, genetically altered lines of wheat had significantly higher parameters of the structure of grain yield compared with untransformed genotypes. At the same time, we observed genotypic difference according to grain productivity in biotechnological plants. Therefore, the results we obtained confirm the perspectives of using the technology of short interfering RNAs to increase tolerance of winter wheat to water deficiency. [ABSTRACT FROM AUTHOR]

Infectious myonecrosis virus (IMNV) still causes significant economic and social losses in American and Asian shrimp farming. In this work, we investigated the transcription patterns of Sid-1, Dicer-2 and Argonaute-2 genes from the RNAi mechanism in Penaeus vannamei naturally infected with IMNV, and injected with inoculum containing 1.02 × 10 5 , 1.02 × 10 4 or 1.02 × 10 3 IMNV copies‧μL -1 . We observed that infection with increasing IMNV concentrations affected the transcription levels of these key genes. However, the viral load did not decrease during the experiment. We suggest that changes in Sid-1 mRNA expression could be used as marker of viral replication for evaluating sanitary status in P. vannamei farming., Competing Interests: Declaration of competing interest The authors declare that there are no conflicts of interest., (Copyright © 2024. Published by Elsevier Ltd.)

The demand for RNA-based therapeutics is increasing globally. However, their use is hampered by the lack of safe and effective delivery vehicles. Here, we developed technologies for highly efficient delivery of RNA cargo into programmable extracellular vesicle-mimetic nanovesicles (EMNVs) by fabricating hybrid EMNV-liposomes (Hybs). Tissue targeting is endowed by highly efficient genetic platforms based on truncated CD63 (ΔCD63) or PTGFRN proteins. For the first time we reveal their efficiency in functionalizing EMNVs, resulting in >10-fold enhancement of nanoparticle internalization in vitro and >2-fold in vivo. RNA delivery using Hybs demonstrated efficiency of >85% in human and mouse cell lines. Comparative analysis of EMNVs and Hyb lysosome colocalization and stability suggested that Hybs enter the lysosomal compartment and escape over time, whereas EMNVs primarily avoid it. Finally, we used these technologies to generate liver-targeting Hybs loaded with therapeutic small interfering RNA and demonstrated the robust efficiency of this system in vitro and in vivo. These technologies can be adapted for manufacturing a wide range of next-generation vehicles for highly efficient, safe delivery of RNA into desired organs and tissues for therapeutic and prophylactic applications., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The American Society of Gene and Cell Therapy. Published by Elsevier Inc. All rights reserved.)

RNA interference (RNAi) has potential to become a major tool for integrated management of insect pests of agricultural crops based on sequence-specificity and low doses of rapidly biodegradable dsRNA. Deploying ‘environmental RNAi’ for control of insect vectors of plant pathogens is of increasing interest for combatting emerging plant diseases. Hemipteran insect vectors, including psyllids, are vascular feeders, making their development difficult to control specifically by targeting with pesticidal chemistries. Psyllids transmit “Candidatus Liberibacter solanacearum” the causal organism of potato zebra chip and tomato vein greening diseases, transmitted, respectively, by the potato or tomato psyllid (PoP). Until now, the optimal effective concentration(s) of double-stranded RNA (dsRNA) required for significant gene knockdown and RNAi persistence in PoP have not been determined. The objective of this study was to optimize RNAi in young PoP adults and 3rd instars for screening by oral delivery of dsRNAs. The minimal effective dsRNA concentrations required for robust knockdown and persistence were evaluated by delivering seven concentrations spanning 0.1 ng/μL to 500 ng/μL over post ingestion-access periods (IAP) ranging from 48 h to 12 days. The PoP gene candidates evaluated as targets were vacuolar ATPase subunit A, clathrin heavy chain, and non-fermenting protein 7, which were evaluated for knockdown by qPCR amplification. The minimum and/or the second most effective dsRNA concentration resulting in effective levels of gene knockdown was 100 ng/μL for all three targets. Higher concentrations did not yield further knockdown, indicating potential RISC saturation at the higher doses. Gene silencing post-IAP of 100 ng/μL dsRNA persisted for 3–5 days in adults and nymphs, with the PoP 3rd instar, followed by teneral and mature adults, respectively, exhibiting the most robust RNAi-response.

Resistance to anti-androgen therapy in prostate cancer (PCa) is often driven by genetic and epigenetic aberrations in the androgen receptor (AR) and coregulators that maintain androgen signaling activity. We show that specific small RNAs downregulate expression of multiple essential and androgen receptor-coregulatory genes, leading to potent androgen signaling inhibition and PCa cell death. Expression of different short hairpin/small interfering RNAs (sh-/siRNAs) designed to target TMEFF2 preferentially reduce viability of PCa but not benign cells, and growth of murine xenografts. Surprisingly, this effect is independent of TMEFF2 expression. Transcriptomic and sh/siRNA seed sequence studies indicate that expression of these toxic shRNAs lead to downregulation of androgen receptor-coregulatory and essential genes through mRNA 3′ UTR sequence complementarity to the seed sequence of the toxic shRNAs. These findings reveal a form of the “death induced by survival gene elimination” mechanism in PCa cells that mainly targets AR signaling, and that we have termed androgen network death induced by survival gene elimination (AN-DISE). Our data suggest that AN-DISE may be a novel therapeutic strategy for PCa.

Background: Lumasiran, a sub-cutaneous RNA-interference therapy, has been recently approved for primary hyperoxaluria type 1 (PH1), with doses and intervals according to body weight. Little is known as to its use in infants; the aim of this study was to describe treatment outcome in 3 infants who received lumasiran therapy before 2 years of age. Case–Diagnosis/Treatment: Patient 1 was diagnosed antenatally and received lumasiran from day 9. According to the product information template (PIT), he received monthly lumasiran (3 times at 6 mg/kg, then 3 mg/kg), with hyperhydration and potassium citrate. Despite decreased plasma oxalate levels, persistent normal kidney function, and good tolerance, kidney ultrasound performed after 2 months found nephrocalcinosis, without normalization of urinary oxalate (UOx). The dose was increased back to 6 mg/kg, inducing a normalization in UOx. Nephrocalcinosis started to improve at month 10. Patient 2 was diagnosed at 2.5 months (acute kidney failure); nephrocalcinosis was present from diagnosis. She received monthly lumasiran (6 mg/kg), with progressive decrease in UOx and substantial improvement in kidney function but stable nephrocalcinosis after 9 injections. Patient 3 was diagnosed fortuitously (nephrocalcinosis) at 3.5 months and received lumasiran before genetic diagnosis, leading to decreased UOx and maintenance of normal kidney function. Nephrocalcinosis improved after 5 injections. Conclusions: This report presents the youngest children treated with lumasiran worldwide. Lumasiran seems effective without side effects in infants but does not completely prevent the onset of nephrocalcinosis in the most severe forms. Higher doses than those proposed in the PIT might be required because of hepatic immaturity. [ABSTRACT FROM AUTHOR]

Summary: Because of their importance as chemical mediators, the presence of a rich and varied family of lipoxygenase (LOX) products, collectively named oxylipins, has been investigated thoroughly in diatoms, and the involvement of these products in important processes such as bloom regulation has been postulated. Nevertheless, little information is available on the enzymes and pathways operating in these protists.Exploiting transcriptome data, we identified and characterized a LOX gene, PaLOX, in Pseudo‐nitzschia arenysensis, a marine diatom known to produce different species of oxylipins by stereo‐ and regio‐selective oxidation of eicosapentaenoic acid (EPA) at C12 and C15.PaLOX RNA interference correlated with a decrease of the lipid‐peroxidizing activity and oxylipin synthesis, as well as with a reduction of growth of P. arenysensis. In addition, sequence analysis and structure models of the C‐terminal part of the predicted protein closely fitted with the data for established LOXs from other organisms.The presence in the genome of a single LOX gene, whose downregulation impairs both 12‐ and 15‐oxylipins synthesis, together with the in silico 3D protein modelling suggest that PaLOX encodes for a 12/15S‐LOX with a dual specificity, and provides additional support to the correlation between cell growth and oxylipin biosynthesis in diatoms. [ABSTRACT FROM AUTHOR]

Influenza is a worldwide public health problem. Annually, this infection affects up to 15% of the world population; and about half a million people die from this disease every year. Moreover, influenza A and B viruses tend to garner most of the attention, as these types are a major cause of the epidemics and pandemics. Although the influenza virus primarily affects the respiratory tract, it may also affect the cardiovascular and central nervous systems. Several antiviral drugs, that target various stages of viral reproduction, have been considered effective for the treatment and prevention of influenza, but some virus strains become resistant to these medications. Thus, new strategies and techniques should be developed to overcome the antiviral drug resistance. Recent studies suggest that new drugs based on RNA interference (RNAi) appear to be a promising therapeutic approach that regulates the activity of viral or cellular genes. As it is known, the RNAi is a eukaryotic gene regulatory mechanism that can be triggered by a foreign double-stranded RNA (dsRNA) and results in the cleavage of the target messenger RNA (mRNA). This review discusses the prospects, advantages, and disadvantages of using RNAi in carrying out a specific treatment for influenza infection. However, some viruses confer resistance to small interfering RNAs (siRNA) targeting viral genes. This problem can significantly reduce the effectiveness of RNAi. Therefore, applying siRNAs targeting host cell factors required for influenza virus reproduction can be a way to overcome the antiviral drug resistance.

Invertebrates mainly rely on sequence-specific RNA interference (RNAi) to resist viral infections. Increasing studies show that double-stranded RNA (dsRNA) can induce sequence-independent protection and that Dicer-2, the key RNAi player that cleaves long dsRNA into small interfering RNA (siRNA), is necessary for this protection. However, how this protection occurs remains unknown. Herein, we report that it is caused by adenosine triphosphate (ATP)-hydrolysis accompanying the dsRNA-cleavage. Dicer-2 helicase domain is ATP-dependent; therefore, the cleavage consumes ATP. ATP depletion activates adenosine monophosphate-activated protein kinase (Ampk) and induces nuclear localization of Fork head box O (FoxO), a key transcriptional factor for dsRNA-induced genes. siRNAs that do not require processing cannot activate the transcriptional response. This study reveals a unique nonspecific antiviral mechanism other than the specific RNAi in shrimp. This mechanism is functionally similar to, but mechanistically different from, the dsRNA-activated antiviral response in vertebrates and suggests an interesting evolution of innate antiviral immunity., Competing Interests: Competing interests statement:The authors declare no competing interest.

The application of biological control agents for the management of plant pathogens and pests requires sustainable methods that can increase the global rates of food production and food safety. This strategy has contributed to minimizing various detrimental effects of agrochemicals that not only contaminate our environment, but also present many potential health hazards as well as contributing to climate change. Recent advancement in biotechnology have led to the isolation of novel microorganisms, characterization of their gene products and subsequent cloning in plants with a view to increasing their tolerance to both biotic and abiotic stresses. In addition to improving intimate interactions between microbes and plants, these technological advancements have also allowed manipulation of modes of action of different biological control agents toward enhancing their biocontrol potentials. Therefore, this review emphasizes already existing and recent advancements in molecular techniques that have been adopted in the management of plant diseases and pests for enhanced food security and improved agricultural productivity for providing safe food and a healthy environment. [ABSTRACT FROM AUTHOR]

Here we attempt to reconstruct the sequence of events that led to the formation of three regulatory piRNA clusters, namely, 20A, 38C and flamenco in the Drosophila melanogaster genome. Both the 38C and flamenco clusters include inverted sequences, which potentially form double-stranded RNA hairpins. We present evidence in favor of the well-known hypothesis of piRNA clusters as "transposon traps". According to this model, the presence of the only copy of the transposon in the genome indicates that its expression is suppressed by an RNA-interference mechanism immediately after the mobile element enters the piRNA cluster. We also discuss high the structural variability of piRNAs in Drosophila clusters and cases of horizontal transmobile elements between related species. [ABSTRACT FROM AUTHOR]

RNA-interference (RNAi) is a standard technique for functional genomics in adult mosquitoes. However, RNAi in immature, aquatic mosquito stages has been challenging. Several studies have shown successful larval RNAi, usually in combination with a carrier molecule. Except for one study in malaria mosquito, Anopheles gambiae, none of the previous studies has explored RNAi in mosquito pupae. Even in the study that used RNAi in pupae, double stranded RNA (dsRNA) was introduced by microinjection. Here, we describe a successful method by soaking pupae in water containing dsRNA without any carrier or osmotic challenge. The knockdown persisted into adulthood. We expect that this simple procedure will be useful in the functional analysis of genes that highly express in pupae or newly emerged adults.

The need to minimise the impact of phytosanitary treatments for disease control boosted researchers to implement techniques with less environmental impact. The development of technologies using molecular mechanisms based on the modulation of metabolism by short dsRNA sequences appears promising. The intrinsic fragility of polynucleotides and the high cost of these techniques can be circumvented by nanocarriers that protect the bioactive molecule enabling high efficiency delivery to the leaf surface and extending its half-life. In this work, a specific protocol was developed aiming to assess the best methodological conditions for the synthesis of low-size chitosan nanoparticles (NPs) to be loaded with nucleotides. In particular, NPs have been functionalised with partially purified Green Fluorescent Protein dsRNAs (GFP dsRNA) and their size, surface charge and nucleotide retention capacity were analysed. Final NPs were also stained with FITC and sprayed on Nicotiana benthamiana leaves to assess, by confocal microscopy, both a distribution protocol and the fate of NPs up to 6 days after application. Finally, to confirm the ability of NPs to increase the efficacy of dsRNA interference, specific tests were performed: by means of GFP dsRNA-functionalised NPs, the nucleotide permanence during time was assessed both in vitro on detached wild-type N. benthamiana leaves and in planta; lastly, the inhibition of Botrytis cinerea on single leaves was also evaluated, using a specific fungal sequence (Bc dsRNA) as the NPs' functionalising agent. The encouraging results obtained are promising in the perspective of long-lasting application of innovative treatments based on gene silencing., (© 2023 The Author(s).)

The aim of this study was to investigate the molecular mechanisms underlying the protective effects of hypoxia-inducible factor (HIF) signaling pathway activation in cardiomyocytes under anoxia-reoxygenation (A/R) injury. In this study, rat neonatal cardiomyocytes were pretreated with anti- Hif3A/Hif-3α siRNA or HIF-prolyl hydroxylase inhibitor prior to A/R injury. Our results showed that both HIF3A silencing and HIF-prolyl hydroxylase inhibition effectively increased the cell viability during A/R, led to changes in mRNA expression of HIF1-target genes, and reduced the loss of mitochondrial membrane potential (Δψ m ). Furthermore, application of anti- Hif3a siRNA led to an increase in mRNA expression of Epo , Igf1 , Slc2a1/Glut-1 , and Slc2a4/Glut-4 . Similar results were observed with HIF-prolyl hydroxylase inhibition, which additionally upregulated the mRNA expression of Epor , Tert , and Pdk1 . Hif3a RNA-interference and application of HIF-prolyl hydroxylase inhibitor during A/R modelling led to an increase of Δψ m on 11.5 and 11.9 mV respectively, compared to the control groups. Thus, Hif3a RNA interference and HIF-prolyl hydroxylase inhibition protect cardiomyocytes against A/R injury via the HIF signaling pathway. [ABSTRACT FROM AUTHOR]

Chrysanthemum is a vegetative propagated culture in which viral transmission with planting material is important for its production. Chrysanthemum virus B (CVB) belongs to the viruses that strike this plant culture. Chrysanthemum virus B is found everywhere where chrysanthemum is cultivated. Damage to plants by CVB often leads to a complete loss of floral yield. Chrysanthemum (Chrysanthemum morifolium Ramat cv. White Snowdon) was transformed via Agrobacterium-mediated DNA delivery with the aim of improving resistance to CVB infection. Transformation vectors contain the nucleotide sequence of CVB coat proteins (CP) in sense, antisense, and double sense orientation. The transformative vectors also invert repeats of CVB coat protein gene fragments for the induction of RNA-interference. The transgenic chrysanthemum plants were successfully obtained. The integration of the target sequences in plant genomes was confirmed by polymerase chain reaction (PCR) and Southern blot analyses. Chrysanthemum lines were transformed with antisense, sense, and double sense CVB CP sequences, as well as with hairpin RNA-interference constructs that were assayed for resistance to CVB. Infection of transgenic plants by CVB through the grafting of infected scions shows resistance only among plants with carried double sense (16.7%) and hairpin (12.5%) constructs. The plants transformed by sense and double sense sequences were observed and classified as tolerant. [ABSTRACT FROM AUTHOR]

Background: Small RNA (sRNA) associated gene regulation has been shown to play a significant role during plant-pathogen interaction. In commercial citrus orchards co-infection of Citrus tristeza virus (CTV) and viroids occur naturally. Methods: A next-generation sequencing-based approach was used to study the sRNA and transcriptional response in grapefruit to the co-infection of CTV and Citrus dwarfing viroid. Results: The co-infection resulted in a difference in the expression of a number of sRNA species when comparing healthy and infected plants; the majority of these were derived from transcripts processed in a phased manner. Several RNA transcripts were also differentially expressed, including transcripts derived from two genes, predicted to be under the regulation of sRNAs. These genes are involved in plant hormone systems; one in the abscisic acid, and the other in the cytokinin regulatory pathway. Additional analysis of virus- and viroid-derived small-interfering RNAs (siRNAs) showed areas on the pathogen genomes associated with increased siRNA synthesis. Most interestingly, the starting position of the p23 silencing suppressor's subgenomic RNA generated a siRNA hotspot on the CTV genome. Conclusions: This study showed the involvement of various genes, as well as endogenous and exogenous RNA-derived sRNA species in the plant-defence response. The results highlighted the role of sRNA-directed plant hormone regulation during biotic stress, as well as a counter-response of plants to virus suppressors of RNA-silencing. [ABSTRACT FROM AUTHOR]

RNA-interference is an effective natural mechanism of post-transcriptional modulation of gene expression. RNA-interference mechanism exists as in high eukaryotes both animals and plants as well in lower eukaryotes and viruses. RNA-interference is now used as a powerful tool in the study of functional gene activity, and many essential for fundamental biology results were obtained with this approach. Also it is widely believed that RNA-interference could be used in working out of new therapeutic medicine against malignant, infectious and hereditary diseases. One of the main problems of these developments is search of effective methods of siRNA transfer into the target cells. At the present time different sorts of transfections or viral transduction are used for these purposes. The results of comparison of inhibition of expression of oncogene AML-ETO by synthetic siRNA and by recombinant lentiviruses coding for corresponding shRNA are presented in the article.

Chrysanthemum is a vegetative propagated culture in which viral transmission with planting material is important for its production. Chrysanthemum virus B (CVB) belongs to the viruses that strike this plant culture. Chrysanthemum virus B is found everywhere where chrysanthemum is cultivated. Damage to plants by CVB often leads to a complete loss of floral yield. Chrysanthemum (Chrysanthemum morifolium Ramat cv. White Snowdon) was transformed via Agrobacterium-mediated DNA delivery with the aim of improving resistance to CVB infection. Transformation vectors contain the nucleotide sequence of CVB coat proteins (CP) in sense, antisense, and double sense orientation. The transformative vectors also invert repeats of CVB coat protein gene fragments for the induction of RNA-interference. The transgenic chrysanthemum plants were successfully obtained. The integration of the target sequences in plant genomes was confirmed by polymerase chain reaction (PCR) and Southern blot analyses. Chrysanthemum lines were transformed with antisense, sense, and double sense CVB CP sequences, as well as with hairpin RNA-interference constructs that were assayed for resistance to CVB. Infection of transgenic plants by CVB through the grafting of infected scions shows resistance only among plants with carried double sense (16.7%) and hairpin (12.5%) constructs. The plants transformed by sense and double sense sequences were observed and classified as tolerant.

The nematode Caenorhabditis elegans is a widely used research model for the investigation of metabolism, aging and age-associated diseases. However, when investigating the impact of natural compounds or drugs on those topics, a major confounder is the metabolism of these test substances by live E. coli bacteria, the standard food source of C. elegans. Using paraformaldehyde instead of heat to inactivate E. coli , which allows for high-throughput technologies and better food availability, it is shown here that RNA-interference works equally well, thus demonstrating the absence of considerable interfering modifications of paraformaldehyde with nucleic acids. [ABSTRACT FROM AUTHOR]

Beside the well known nutritional and health benefits, strawberry (Fragaria X ananassa) crop draws increasing attention as plant model system for the Rosaceae family, due to the short generation time, the rapid in vitro regeneration, and to the availability of the genome sequence of F. X ananassa and of the closely related F. vesca species. In the last years, the use of high-throughput sequence technologies provided large amounts of molecular information on the genes possibly related to several biological processes of this crop. Nevertheless, the function of most genes or gene products is still poorly understood and needs investigation. Transient transformation technology provides a powerful tool to study gene function in vivo, avoiding difficult drawbacks that typically affect the stable transformation protocols, such as transformation efficiency, transformants selection and regeneration. In this review we provide an overview of the use of transient expression in the investigation of the function of genes important for strawberry fruit development, defence and nutritional properties. The technical aspects related to an efficient use of this technique are described, and the possible impact and application in strawberry crop improvement are discussed.

After extensive research on radiochemotherapy, 5-year survival rates of children with high risk neuroblastoma still do not exceed 50%, owing to adverse side-effects exemplified by doxorubicin-induced cardiomyopathy. A promising new approach is the combination of conventional therapies with specific modulation of cell signaling pathways promoting therapeutic resistance, such as inhibition of aberrant kinase activity or re-expression of silenced tumor suppressor genes by means of chromatin remodeling. In this regard, we established a system that allows to identify potential drug targets as well as to validate respective candidate inhibitors in high-risk neuroblastoma model cell lines. Cell culture, drug exposure, shRNA-mediated knockdown and phenotype analysis are integrated into an efficient and versatile single well-based protocol. By utilizing this system, we assessed RG108, SGI-1027 and nanaomycin A, three novel DNA methyltransferase inhibitors that have not been tested in neuroblastoma cell lines so far, for their potential of synergistic anti-tumor activity in combination with doxorubicin. We found that, similarly to azacytidine, SGI-1027 and nanaomycin A mediate synergistic growth inhibition with doxorubicin independently of N-Myc status. However, they display high cytotoxicity but lack global DNA demethylation activity. Secondly, we conducted a lentiviral shRNA screen of F-box proteins, key regulators of protein stability, and identified Fbxw11/β-TrCP2 as well as Fbxo5/Emi1 as potential therapeutic targets in neuroblastoma. These results complement existing studies and underline the reliability and versatility of our single well-based protocol. [ABSTRACT FROM AUTHOR]

Beside the well known nutritional and health benefits, strawberry (Fragaria X ananassa) crop draws increasing attention as plant model system for the Rosaceae family, due to the short generation time, the rapid in vitro regeneration, and to the availability of the genome sequence of F. X ananassa and F. vesca species. In the last years, the use of high-throughput sequence technologies provided large amounts of molecular information on the genes possibly related to several biological processes of this crop. Nevertheless, the function of most genes or gene products is still poorly understood and needs investigation. Transient transformation technology provides a powerful tool to study gene function in vivo, avoiding difficult drawbacks that typically affect the stable transformation protocols, such as transformation efficiency, transformants selection, and regeneration. In this review we provide an overview of the use of transient expression in the investigation of the function of genes important for strawberry fruit development, defense and nutritional properties. The technical aspects related to an efficient use of this technique are described, and the possible impact and application in strawberry crop improvement are discussed. [ABSTRACT FROM AUTHOR]

Microdialysis during i.v. drug self-administration (SA) have implicated nucleus accumbens (NAc) shell DA in cocaine and heroin reinforcement. However, this correlative evidence has not been yet substantiated by experimental evidence obtained by studying the effect of selective manipulation of NAc shell DA transmission on cocaine and heroin SA. In order to investigate this issue, DA D1a receptor (D1aR) expression was impaired in the NAc shell and core by locally infusing lentiviral vectors (LV) expressing specific D1aR-siRNAs (LV-siRNAs). Control rats were infused in the same areas with LV expressing GFP. Fifteen days later, rats were trained to acquire i.v. cocaine or heroin self-administration (SA). At the end of behavioral experiments, in order to evaluate the effect of LV-siRNA on D1aR expression, rats were challenged with amphetamine and the brains were processed for immunohistochemical detection of c-Fos and D1aR. Control rats acquired i.v. cocaine and heroin SA. Infusion of LV-siRNAs in the medial NAc shell reduced D1aR density and the number of c-Fos positive nuclei in the NAc shell, while sparing the core, and prevented the acquisition of cocaine, but not heroin SA. In turn, LV-siRNAs infusion in the core reduced D1aR density and the number of c-Fos positive nuclei in the same area, while sparing the shell, and failed to affect acquisition of cocaine. The differential effect of LV impairment of NAc shell D1aR on cocaine and heroin SA indicates that NAc shell DA acting on D1aR specifically mediates cocaine reinforcement. [ABSTRACT FROM AUTHOR]

Honey bee workers display remarkable flexibility in the aging process. This plasticity is closely tied to behavioral maturation. Workers who initiate foraging behavior at earlier ages have shorter lifespans, and much of the variation in total lifespan can be explained by differences in pre-foraging lifespan. Vitellogenin (Vg), a yolk precursor protein, influences worker lifespan both as a regulator of behavioral maturation and through anti-oxidant and immune functions. Experimental reduction of Vg mRNA, and thus Vg protein levels, in wild-type bees results in precocious foraging behavior, decreased lifespan, and increased susceptibility to oxidative damage. We sought to separate the effects of Vg on lifespan due to behavioral maturation from those due to immune and antioxidant function using two selected strains of honey bees that differ in their phenotypic responsiveness to Vg gene knockdown. Surprisingly, we found that lifespans lengthen in the strain described as behaviorally and hormonally insensitive to Vg reduction. We then performed targeted gene expression analyses on genes hypothesized to mediate aging and lifespan: the insulin-like peptides ( Ilp1 and 2 ) and manganese superoxide dismutase ( mnSOD ). The two honey bee Ilps are the most upstream components in the insulin-signaling pathway, which influences lifespan in Drosophila melanogaster and other organisms, while manganese superoxide dismutase encodes an enzyme with antioxidant functions in animals. We found expression differences in the llps in fat body related to behavior ( llp1 and 2 ) and genetic background ( Ilp2 ), but did not find strain by treatment effects. Expression of mnSOD was also affected by behavior and genetic background. Additionally, we observed a differential response to Vg knockdown in fat body expression of mnSOD , suggesting that antioxidant pathways may partially explain the strain-specific lifespan responses to Vg knockdown. [ABSTRACT FROM AUTHOR]

Background To date the therapy for non-small cell lung cancer ( NSCLC) is associated with severe side effects, frustrating outcomes, and does not consider different tumor characteristics. The RNA-interference ( RNAi) pathway represents a potential new approach to treat NSCLC. With small interfering ribonucleic acids ( siRNAs), it is possible to reduce the expression of proliferation-dependent proteins in tumor cells, leading to their apoptosis. We propose that siRNAs could be adapted to the tumor type and may cause fewer side effects than current therapy. Methods Four NSCLC cell lines were cultured under standard conditions and transfected with three different concentrations of siRNAs targeted against the hypoxia-inducible factors 1α and 2α ( HIF1α and HIF2α) and signal transducer and activator of transcription 3 ( STAT3). The expression was observed by quantitative real-time polymerase chain reaction and western blots. For the analysis of cell growth three days after transfection, the cell number was detected using a CASY cell counter system. Results The results of the silencing of the analyzed factors differ in each cell line. Cell growth was significantly reduced in all cell lines after transfection with HIF1α- and STAT3- siRNA. The silencing of HIF2α resulted in a significant effect on cell growth in squamous, and large-cell lung cancer. Conclusions This study shows that the knockdown and viability to siRNA transfection differ in each tumor type according to the used siRNA. This implies that the tumor types differ among themselves and should be treated differently. Therefore, the authors suggest a possible approach to a more personalized treatment of NSCLC. [ABSTRACT FROM AUTHOR]

RNA-interference is an effective natural mechanism of post-transcriptional modulation of gene expression. RNA-interference mechanism exists as in high eukaryotes both animals and plants as well in lower eukaryotes and viruses. RNA-interference is now used as a powerful tool in the study of functional gene activity, and many essential for fundamental biology results were obtained with this approach. also it is widely believed that RNA-interference could be used in working out of new therapeutic medicine against malignant, infectious and hereditary diseases. One of the main problems of these developments is search of effective methods of siRNA transfer into the target cells. at the present time different sorts of transfections or viral transduction are used for these purposes. The results of comparison of inhibition of expression of oncogene AML-ETO by synthetic siRNA and by recombinant lentiviruses coding for corresponding shRNA are presented in the article. [ABSTRACT FROM AUTHOR]

Abstract: Potassium conductance in vascular smooth muscle (VSM) is known to be altered in arterial hypertension. High level of protein kinase C (PKC) activity is a common feature for hypertension of different genesis. The main goal of this study was to investigate the efficacy of the RNA interference (RNAi) technique targeting PKC delta-isoform gene as a possible pharmacological tool to restore vasodilator potential in spontaneously hypertensive rats (SHR). Experimental design of the study comprised RNAi and patch-clamp techniques, RT-PCR analysis and standard acetylcholine test. Total outward currents and acetylcholine-induced endothelium-dependent relaxant responses were blunted in SHR. BKCa alpha subunit mRNA expression in SHR was unchanged whereas KV and KATP mRNA expression appeared significantly increased. PKC inhibitor, chelerythrine (100nM), restored potassium channels activity in SHR. PKC-delta-isoform protein expression and PKC-delta-isoform mRNA expression are 2.5–4 fold increased in VSM from SHR. PKC gene silencing with the short hairpin RNAs (shRNAs)-plasmid delivery system administered intravenously led to an increment in maximal amplitude of acetylcholine-relaxation, restored outward K+ currents and PKC-delta-isoform mRNA and protein expression. Arterial blood pressure in SHR was normalized following shRNAs administration. We conclude that BKCa channels are likely to be the most PKC-dependent member of K+ channels family responsible for vascular hypercontractility in SHR while Kv and KATP channels may constitute a reserve mechanism for the maintenance of vasodilator potential under BKCa channelopathy. It is likely that RNAi technique is a good therapeutic approach to inactivate PKC gene and to normalize vascular functions and high arterial blood pressure in SHR. [Copyright &y& Elsevier]

Highlights: [•] Stability of transgenic bean lines immune (the RNAi concept) to the Bean golden mosaic virus was confirmed for at least 8 generations and after crosses. [•] siRNA signals were detected in leaves and in developing seeds but not cooked grains. [•] The information presented here is relevant for the social acceptance and consumption of GM beans-based food. [•] This study provides a method for detection and identification of these GM beans in the field and market. [Copyright &y& Elsevier]

Background The ability to assess gene function is essential for understanding biological processes. Currently, RNA interference (RNAi) is the only technique available to assess gene function in planarians, in which it has been induced by means of injection of double-stranded RNA (dsRNA), soaking, or ingestion of bacteria expressing dsRNA. Results We describe a simple and robust RNAi protocol, involving in vitro synthesis of dsRNA that is fed to the planarians. Advantages of this protocol include the ability to produce dsRNA from any vector without subcloning, resolution of ambiguities in quantity and quality of input dsRNA, as well as time and ease of application. We have evaluated the logistics of inducing RNAi in planarians using this methodology in careful detail, from the ingestion and processing of dsRNA in the intestine, to timing and efficacy of knockdown in neoblasts, germline, and soma. We also present systematic comparisons of effects of amount, frequency, and mode of dsRNA delivery. Conclusions This method gives robust and reproducible results and is amenable to high-throughput studies. Overall, this RNAi methodology provides a significant advance by combining the strengths of current protocols available for dsRNA delivery in planarians and has the potential to benefit RNAi methods in other systems. Developmental Dynamics 242:718-730, 2013. © 2013 Wiley Periodicals, Inc. [ABSTRACT FROM AUTHOR]

Abstract: RNA interference has been successfully used in adult honeybees, but there are only few reports about abdominal application of dsRNA/siRNA which have reached more distant tissues than the fat body. We studied systemic RNAi in honeybees by injecting fluorescent siRNA of the ubiquitously expressed honeybee homologue of the Glycerol-3-Phosphate Dehydrogenase (amGpdh) into the abdomens of adult bees and followed them by laser scanning microscopy and qPCR. The fat body was the sole tissue emitting fluorescence and showing a decreased gene expression, whereas the siRNA had apparently not reached the other tissues. Therefore, we conclude that certain genes in other tissues than the fat body cannot be easily reached by injecting siRNA into the body cavity. In particular, the lack of amGpdh knock down in ovaries after amGpdh dsRNA injection, supports that in some cases it may be particularly difficult to interfere with gene expression in ovaries by intra-abdominal injection. In these cases alternative inhibition techniques may be required to achieve an organismic non-lethal disruption of transcription. [Copyright &y& Elsevier]