Search Results (207)

The invertebrate neuropeptide F (NPF) signaling plays versatile roles in diverse biological activities and processes. Still, whether and how it mediates feeding and digestion in Pomacea canaliculate remain gaps in our knowledge. Herein, we first identified and characterized PcNPFR via bioinformatics analysis in P. canaliculate, which is a polyphagous herbivore with a voracious appetite that causes devastating damages to ecosystem functioning and services in colonized ranges. Double stranded RNA (dsRNA)-based RNA interference (RNAi) and exogenous rescue were utilized to decipher and substantiate underlying mechanisms whereby NPFR executed its modulatory functions. Multiple sequence alignment and phylogeny indicated that PcNPFR harbored typical seven transmembrane domains (7 TMD) and belonged to rhodopsin-like GPCRs, with amino acid sequence sharing 27.61–63.75% homology to orthologues. Spatio-temporal expression profiles revealed the lowest abundance of PcNPFR occurred in pleopod tissues and the egg stage, while it peaked in male snails and testes. Quantitative real-time PCR (qRT-PCR) analysis showed that 4 µg dsNPFR and 10⁻⁶ M trNPF (NPFR agonist) were optimal doses to exert silencing and rescue effects, accordingly with sampling time at 3 days post treatments. Moreover, the dsNPFR injection (4 µg) at 1/3/5/7 day/s delivered silencing efficiency of 32.20–74.01%. After 3 days upon dsNPFR knockdown (4 µg), mRNA levels of ILP7/InR/Akt/PI3Kc/PI3K_R were significantly downregulated compared to dsGFP controls, except FOXO substantially upregulated at both transcript and translation levels. In addition, the activities of alpha-amylase, protease and lipase were significantly suppressed, accompanied by decreased leaf area consumption, attenuated feeding behavior and diminished feeding rate. Moreover, expression trends were opposite and proxies were partially or fully restored to baseline levels post exogenous compensation of trNPF, suggesting phenotypes specifically attributable to PcNPFR RNAi but not off-target effects. PcNPFR is implicated in both feeding and digestion by modulating the ISP pathway and digestive enzyme activities. It may serve as a promising molecular target for RNAi-based antifeedants to manage P. canaliculate invasion. Full article

Plant viruses have been traditionally considered pathogens restricted to plant hosts. However, recent studies have shown that some plant viruses can infect and replicate in filamentous fungi and oomycetes, suggesting that their host range is broader than previously thought, and that their ecological interactions are more complex. In this study, we investigated the ability of the well-characterized positive-sense RNA plant virus Tobacco mosaic virus (TMV) to replicate in four major phytopathogenic fungi from different taxonomic groups: Botrytis cinerea, Fusarium oxysporum f. sp. lycopersici, Verticillium dahliae, and Monilinia fructicola. Using a recombinant TMV-based vector expressing a green fluorescent protein (TMV-GFP-1056) as reporter, we demonstrated that TMV can enter, replicate, and persist within the mycelia of B. cinerea and V. dahliae—at least through the first subculture. However, it cannot replicate in F. oxysporum f. sp. lycopersici and M. fructicola. RNA interference (RNAi) is a conserved eukaryotic epigenetic mechanism that provides an efficient defence against viruses. We explored the role of RNAi in the interaction between TMV and the mycelia of V. dahliae and B. cinerea. Our results revealed a strong induction of the Dicer-like 1 and Argonaute 1 genes, which are key compounds of the RNA silencing pathway. This RNAi-based response impaired TMV-GFP replication in both fungi. Notably, despite viral replication and RNAi activation, the virulence of V. dahliae and B. cinerea on their respective host plants remained unaffected. These findings reinforce the emerging recognition of cross-kingdom virus transmission and interactions, which likely play a crucial role in pathogen ecology and viral evolution. Understanding these virus–fungus interactions not only sheds light on RNAi interference silencing mechanisms but also suggests that plant viruses like TMV could serve as simple and effective tools for functional genomic studies in fungi, such as in V. dahliae and B. cinerea. Full article

Grapholita molesta is a globally significant fruit pest. Females achieve maximal reproductive output through efficient sperm utilization following a single copulation. Post-mating maturation of eupyrene sperm is a critical step in reproductive success. Here, we report that a male accessory gland-derived serine protease (named GmAGSP1) is essential for this process. GmAGSP1 was only distantly related to other identified sperm-activating SPs, and its transcript was highly expressed in the AG at 48 h after emergence. RNAi-mediated knockdown of GmAGSP1 in males did not affect courtship rate, copulation duration, or mating frequency, whereas male fertility decreased significantly. Mating with GmAGSP1-knockdown males markedly impaired eupyrene sperm maturation in the spermatophores, with phenotypes including failure of eupyrene sperm bundles to dissociate normally and marked reduction in viability of the dissociated eupyrene sperm. Finally, untargeted metabolomic analysis preliminarily demonstrated marked alterations in multiple metabolic pathways within the spermatophore following mating with GmAGSP1-knockdown males. This study advances our understanding of the regulatory mechanism of “sperm activation in the spermatophore’s metabolic microenvironment mediated by male AG-derived SP” while providing critical insights for the development of novel genetic control strategies targeting G. molesta. Full article

The functional role of MAPKK genes in potato (Solanum tuberosum L.) under high-temperature stress remains unexplored, despite their critical importance in stress signaling and yield protection. We characterized StMAPKK1, a novel group D MAPKK localized to plasma membrane/cytoplasm. Quantitative real-time polymerase chain reaction (qRT-PCR) revealed cultivar-specific upregulation in potato (‘Atlantic’ and ‘Desiree’) leaves under heat stress (25 °C, 30 °C, and 35 °C). Transgenic lines overexpressing (OE) StMAPKK1 exhibited elevated antioxidant enzyme activity, including ascorbate peroxidase (APX), catalase (CAT), superoxide dismutase (SOD), and peroxidase (POD), mitigating oxidative damage. Increased proline and chlorophyll accumulation and reduced oxidative stress markers, hydrogen peroxide (H₂O₂) and malondialdehyde (MDA), indicate improved cellular redox homeostasis. The upregulation of key antioxidant and heat stress-responsive genes (StAPX, StCAT1/2, StPOD12/47, StFeSOD2/3, StMnSOD, StCuZnSOD1/2, StHSFA3 and StHSP20/70/90) strengthened the enzymatic defense system, enhanced thermotolerance, and improved photosynthetic efficiency, with significant improvements in net photosynthetic rate (Pn), transpiration rate (E), and stomatal conductance (Gs) under heat stress (35 °C) in StMAPKK1-OE plants. Superior growth and biomass (plant height, plant and its root fresh and dry weights, and tuber yield) accumulation, confirming the positive role of StMAPKK1 in thermotolerance. Conversely, RNA interference (RNAi)-mediated suppression of StMAPKK1 led to a reduction in enzymatic activity, proline content, and chlorophyll levels, exacerbating oxidative stress. Downregulation of antioxidant-related genes impaired ROS scavenging capacity and declines in photosynthetic efficiency, growth, and biomass, accompanied by elevated H₂O₂ and MDA accumulation, highlighting the essential role of StMAPKK1 in heat stress adaptation. These findings highlight StMAPKK1’s potential as a key genetic target for breeding heat-tolerant potato varieties, offering a foundation for improving crop resilience in warming climates. Full article

RNA interference (RNAi) has emerged as a promising tool for controlling fungal plant pathogens, offering a targeted and environmentally friendly alternative to traditional chemical fungicides. Botrytis cinerea, the causative agent of gray mold disease, serves as a model and plant pathogen for investigating RNAi-based strategies due to its wide host range and economic impact. This review synthesizes current knowledge on RNAi mechanisms in B. cinerea, and that several factors influence the efficacy of RNAi in B. cinerea, including the stability and uptake of double-stranded RNAs (dsRNAs), the efficiency of RNA processing machinery, and environmental conditions. Furthermore, RNAi responses can vary significantly across strains, developmental stages, and infection modes, underscoring the complexity of fungal responses. With this review, I also aim to present the field trials reported so far, underscoring the practicality of RNAi. This review identifies current hotspots and outlines future directions for deploying RNAi as a sustainable control strategy against fungal pathogens. Full article

Self-assembling protein nanocages (SAPNs) are distinct natural structures formed by the self-assembly of identical subunits, providing a highly efficient platform and a novel strategy for vaccine development and RNAi therapy. Their internal cavity allows for precise cargo encapsulation, while the externally modifiable surface supports multivalent antigen presentation, thereby enhancing stability, targeted delivery, and immune activation. In addition to serving as stable subunit vaccines with multivalent antigen display, SAPNs can be incorporated into mRNA vaccines (SAPN-RNA vaccines) by pre-fusing with the antigen. This strategy stabilizes secreted antigenic proteins with prolonged presentation to the immune system, and improves vaccine efficacy while reducing off-target effects and minimizing required doses. Additionally, SAPNs can overcome cellular uptake barriers, enhance DNA vaccine efficacy, and enable the co-delivery of antigens and adjuvants. Functionalization with adjuvants or targeting ligands further improves their immunostimulatory properties and specificity. The SAPN-RNAi strategy optimizes siRNA delivery by promoting lysosomal escape, enhancing targeted uptake, and protecting siRNA from degradation through SAPN encapsulation. This review examines the structural and functional properties of protein nanocages and their applications in vaccine design and RNAi delivery, emphasizing their synergistic effects, and exploring current progress, challenges, and future directions. In conclusion, SAPNs represent a versatile multifunctional platform with broad applicability across subunit, mRNA and DNA vaccines, adjuvant co-delivery, and RNAi therapeutics, with significant potential against viral infections. Full article

Background: RNA interference (RNAi) is a powerful tool that can target many proteins without the expensive and time-consuming drug development studies. However, due to the challenges in delivering RNA molecules, the potential impact of RNAi approaches is yet to be fully realized in clinical settings. Lipid nanoparticles (LNPs) have been the most successful delivery system for nucleic acids, but targeted delivery to a solid tumor still eludes the developed LNPs. We hypothesized that specially designed low-molecular-weight PEIs can partially or completely replace the ionizable lipids for more accommodating vehicles due to the structural flexibility offered by polymers, which could lead to safer and more efficient nucleic acid delivery. Methods: To achieve this, we first optimized the LNP formulations as a point of reference for three outcomes: cellular uptake, cytotoxicity, and silencing efficiency. Using a response surface methodology (Design Expert), we optimized siRNA delivery by varying mole fractions of lipid components. Leveraging the optimal LNP formulation, we integrated specifically designed cationic polymers as partial or complete replacements for the ionizable lipid. This methodological approach, incorporating optimal combined designs and response surface methodologies, refined the LPNPs to an optimal efficiency. Results: Our data revealed that DOPE and Dlin-MC3-DMA contributed to higher efficiency in selected breast cancer cells over DSPC and ALC-0315 as neutral and ionizable lipids, respectively, based on the software analysis and direct comparative experiments. Incorporation of selected polymers enhanced the cellular internalization significantly, which in some formulations resulted in higher efficiency. Conclusions: These findings offer a framework for the rational design of LPNPs, that could enhance the passive targeting and silencing efficiency in cancer treatment and broader applications for RNAi-based strategies. Full article

The large growth in the global population requires new solutions for the control of harmful insects that compete for our food. Changing regulatory requirements and public perception, together with the continuous evolution of resistance to conventional insecticides, also require, in addition to innovative molecules with different modes of action, new non-chemical control strategies that can help maintain efficient integrated pest management programs. The last 30 years have inaugurated a new era characterised by the discovery of new mechanisms of action and new chemical families. Although European programs also promote a green deal in the crop protection sector, the existing thorough regulations slow down its spread and the adoption of new products. In light of these changes, this review will describe in more detail the dynamics of discovery and registration of new conventional insecticides and the difficulties that the agrochemical industries encounter. Subsequently, the different innovative control strategies alternative to conventional insecticides based on natural substances of different origin, entomopathogenic microorganisms, semiochemical and semiophysical compounds, and classical and augmentative biological control will be described. The advantages of these green strategies will be illustrated and also the constrains to their diffusion and commercialisation. Finally, the main biotechnological discoveries will be described, from transgenic plants to symbiotic control, classical genetic control, and, more recently, control based on insect genomic transformation or on RNAi. These new biotechnologies can revolutionise the sector despite some constrains related to the regulatory restrictions present in different countries. Full article

Sclerotinia sclerotiorum is a globally widespread and vast destructive plant pathogenic fungus that causes significant yield losses in crops. Due to the lack of effective resistant germplasm resources, the control of diseases caused by S. sclerotiorum largely relies on chemical fungicides. However, excessive use of these chemicals not only causes environmental concerns but also leads to the increased development of resistance in S. sclerotiorum. In contrast, trans-kingdom sRNA silencing-based technologies, such as host-induced gene silencing (HIGS) and spray-induced gene silencing (SIGS), offer novel, effective, and environmentally friendly methods for the management of S. sclerotiorum infection. This review summarizes recent advances in the identification of S. sclerotiorum pathogenic genes, target gene selection, categories, and application of trans-kingdom RNA interference (RNAi) technologies targeting this pathogen. Although some challenges, including off-target effects and the efficiency of external sRNA uptake, exist, recent findings have proposed solutions for further improvement. Combined with the latest developments in CRISPR/Cas gene editing and other technologies, trans-kingdom RNAi has significant potential to become a crucial tool in the control of sclerotinia stem rot (SSR), mitigating the impact of S. sclerotiorum on crop production. Full article

In planta RNAi or host-induced gene silencing (HIGS) has undergone significant advancements that have rendered it efficient and stable at the transgenerational level in plants for regulating host genes and targeting genes of insect pests and plant pathogens. Similarly, topical RNAi or spray-induced gene silencing (SIGS) has garnered considerable attention as an environmentally sustainable, selective, and alternative approach to chemical control of insect pests and plant pathogens. Several biotechnology companies and startups have focused their efforts on RNAi-based solutions for topical application in agriculture. Nevertheless, further technological advancements are required to enhance the efficacy of topical RNAi in agriculture, including improved dsRNA delivery systems, better target gene selection, and addressing biosafety regulatory issues. Herein, this review discusses key advances and bottlenecks in RNAi, and summarizes successful applications of these RNAi-based technologies in agriculture focusing on in planta and topical RNAi to control insect pests and plant pathogens. Furthermore, this review delves into the patenting landscape, biosafety considerations, risk evaluations, and the current regulatory status of RNAi in Latin America. Finally, it explores the contributions of RNAi to plant science, food production, and fostering a more sustainable form of agriculture. Full article

Metabolic syndrome (MetS) is a subclinical disease, resulting in increased risk of type 2 diabetes (T2D), cardiovascular diseases, cancer, and mortality. Dynamical network biomarkers (DNB) theory has been developed to provide early-warning signals of the disease state during a preclinical stage. To improve the efficiency of DNB analysis for the target genes discovery, the DNB intervention analysis based on the control theory has been proposed. However, its biological validation in a specific disease such as MetS remains unexplored. Herein, we identified eight candidate genes from adipose tissue of MetS model mice at the preclinical stage by the DNB intervention analysis. Using Drosophila, we conducted RNAi-mediated knockdown screening of these candidate genes and identified vasa (also known as DDX4), encoding a DEAD-box RNA helicase, as a fat metabolism-associated gene. Fat body-specific knockdown of vasa abrogated high-fat diet (HFD)-induced enhancement of starvation resistance through up-regulation of triglyceride lipase. We also confirmed that DDX4 expressing adipocytes are increased in HFD-fed mice and high BMI patients using the public datasets. These results prove the potential of the DNB intervention analysis to search the therapeutic targets for diseases at the preclinical stage. Full article

Transfection is a fundamental method in biomedical research to study intracellular molecular mechanisms by manipulating target protein expression. Various methods have been developed to deliver nucleic acids into the cells of interest in vitro, with chemical transfection by cationic lipids being the most widely used for RNA interference (RNAi). However, translating these in vitro results into in vivo remains a significant challenge. In this study, we established an ex vivo transfection model using cationic lipids in human whole blood. Three different lipid-based reagents were evaluated regarding toxicity, transfection efficiency, and immunogenicity across leukocyte populations using spectral flow cytometry. CD14⁺ monocytes were identified as the primary population to be transfected by cationic lipids in whole blood. To assess immunogenicity, the monocyte-specific activation markers CD80 and human leukocyte antigen DR isotype (HLA-DR) were analyzed upon transfection. Our results demonstrated that Lipofectamine RNAiMAX outperforms the other two reagents, showing low toxicity and high transfection efficiency in combination with a minimal potential for monocyte activation. Functional knockdown experiments using siRNA targeting CIITA and the microRNA mir-3972 targeting HLA-DRA showed dose-dependent suppression in HLA-DR expression. This study provides the framework for preliminary testing of RNAi in a physiologically relevant ex vivo model, enabling assessment of key endpoints such as toxicity, transfection efficiency, and immune activation potential of gene delivery systems. Full article

Background: Polymeric nanoparticles have been explored as efficient tools for siRNA delivery to induce RNAi-mediated gene knockdown. Chemical modifications of polyethylenimines (PEI) enhance nanoparticle efficacy and biocompatibility. Their in vivo use, however, benefits from prior analyses in relevant in vitro 3D conditions. Methods: We utilize a 3D ALI cell culture model for testing the biological activities and toxicities of a set of different PEI-based nanoparticles with different chemical modifications. This also includes a novel, fluoroalkyl-modified PEI. Reporter gene knockdown is directly compared to 2D cell culture. In parallel, biocompatibility is assessed by measuring cell viability and lactate dehydrogenase (LDH) release. Results: Knockdown efficacies in the 3D ALI model are dependent on the chemical modification and complex preparation conditions. Results only correlate in part with gene knockdown in 2D cell culture, identifying nanoparticle penetration and cellular internalization under 3D conditions as important parameters. The 3D ALI cell culture is also suitable for the quantitative determination of nanoparticle effects on cell viability and acute toxicity, with biocompatibility benefitting from PEI modifications. Conclusions: The 3D ALI cell model allows for a more realistic assessment of biological nanoparticle effects. A novel fluoroalkyl-modified PEI is described. Optimal preparations of PEI-based nanoparticles for siRNA delivery and gene knockdown are identified. Full article

Background: Neurological disorders such as Alzheimer’s disease (AD), Parkinson’s disease (PD), stroke, and spinal cord injury (SCI) are significant global health challenges due to their complex pathology and limited therapeutic options. Conventional treatments often fail to efficiently cross the blood–brain barrier (BBB), leading to poor bioavailability and systemic toxicity. This narrative review explores the potential of nanomedicine in addressing these limitations and advancing targeted therapies for neural disorders. Methods: This review examines recent studies on the use of engineered nanoparticles (NPs), including liposomes, dendrimers, micelles, and nanogels, for targeted drug delivery and multifunctional theranostics in neural diseases. It evaluates their role in promoting axon regeneration, reducing neuroinflammation, and repairing neural damage. Additionally, innovative applications in gene therapy and RNA-based treatments, such as CRISPR-Cas9 and RNA interference (RNAi), are discussed. Challenges related to toxicity, scalability, affordability, and regulatory barriers are highlighted, along with potential strategies to address these issues. Results: Nanoparticles have shown significant promise in crossing the BBB, delivering therapeutic agents to neural tissues, and minimizing off-target effects. Emerging applications in gene and RNA-based therapies demonstrate their versatility in addressing disease-specific challenges. However, unresolved issues such as long-term safety, manufacturing scalability, and cost continue to pose challenges. Conclusions: Nanomedicine offers a promising approach to overcoming current limitations in the treatment of neural disorders. This review emphasizes the need for continued interdisciplinary efforts to address translational barriers and highlights the potential for nanomedicine to improve the outcomes and quality of life for patients with neural disorders, stroke, and SCI. Full article

Psoriasis is a chronic inflammatory disease with a complex pathogenesis, influenced by various factors involving environment, genes, and immunity. The main symptoms of psoriasis include erythema, scales, itching, etc. At present, therapeutic drugs for psoriasis are continually evolving towards enhancing treatment efficacy and reducing side effects. Firstly, the pathogenesis and characteristics of psoriasis were summarized. Then, the types and benefits of topical therapy were introduced, such as the aspects of avoiding systemic toxic effects, first pass effect, and gastrointestinal reactions with accelerating the onset time of the drugs and improving its efficacy, and were compared to systemic drugs. In the case of methotrexate, cyclosporin A, Janus kinase (JAK) inhibitors, and phosphodiesterase-4 (PDE-4) inhibitors, this review had a further discussion on the improvement and translation of these molecules from systemic therapy to topical therapy in clinical practice. To further augment the limitation of skin permeability, nanotechnology and novel topical drug delivery system including nanomedicines, hydrogels, ionic liquids, and microneedles were elaborated for psoriasis management. Also, exploration of topical targeting pathogenic genes through small interfering RNA (siRNA) using nanoparticles and ionic liquids (ILs) is of great significance for long-term treatment in psoriasis. Taken together, the development of numerous topical delivery platforms is expected to achieve enhanced penetration, and precise and efficient delivery of small molecule and RNA interference (RNAi) therapeutics in psoriasis with clinical translation prospects. Full article