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Selfish genetic elements contribute to hybrid incompatibility and bias or drive their own transmission1,2. Chromosomal drive typically functions in asymmetric female meiosis, whereas gene drive is normally post-meiotic and typically found in males. Here, using single-molecule and single-pollen genome sequencing, we describe Teosinte Pollen Drive, an instance of gene drive in hybrids between maize (Zea mays ssp. mays) and teosinte mexicana (Z. mays ssp. mexicana) that depends on RNA interference (RNAi). 22-nucleotide small RNAs from a non-coding RNA hairpin in mexicana depend on Dicer-like 2 (Dcl2) and target Teosinte Drive Responder 1 (Tdr1), which encodes a lipase required for pollen viability. Dcl2, Tdr1 and the hairpin are in tight pseudolinkage on chromosome 5, but only when transmitted through the male. Introgression of mexicana into early cultivated maize is thought to have been critical to its geographical dispersal throughout the Americas3, and a tightly linked inversion in mexicana spans a major domestication sweep in modern maize4. A survey of maize traditional varieties and sympatric populations of teosinte mexicana reveals correlated patterns of admixture among unlinked genes required for RNAi on at least four chromosomes that are also subject to gene drive in pollen from synthetic hybrids. Teosinte Pollen Drive probably had a major role in maize domestication and diversification, and offers an explanation for the widespread abundance of self small RNAs in the germ lines of plants and animals.

The clinical use of RNA interference (RNAi) molecular mechanisms has introduced a novel, growing class of RNA therapeutics capable of treating diseases by controlling target gene expression at the posttranscriptional level. With the newly approved nedosiran (Rivfloza), there are now six RNAi-based therapeutics approved by the United States Food and Drug Administration (FDA). Interestingly, five of the six FDA-approved small interfering RNA (siRNA) therapeutics [patisiran (Onpattro), lumasiran (Oxlumo), inclisiran (Leqvio), vutrisiran (Amvuttra), and nedosiran] were revealed to act on the 3'-untranslated regions of target mRNAs, instead of coding sequences, thereby following the common mechanistic action of genome-derived microRNAs (miRNA). Furthermore, three of the FDA-approved siRNA therapeutics [patisiran, givosiran (Givlaari), and nedosiran] induce target mRNA degradation or cleavage via near-complete rather than complete base-pair complementarity. These features along with previous findings confound the currently held characteristics to distinguish siRNAs and miRNAs or biosimilars, of which all converge in the RNAi regulatory pathway action. Herein, we discuss the RNAi mechanism of action and current criteria for distinguishing between miRNAs and siRNAs while summarizing the common and unique chemistry and molecular pharmacology of the six FDA-approved siRNA therapeutics. The term "RNAi" therapeutics, as used previously, provides a coherently unified nomenclature for broader RNAi forms as well as the growing number of therapeutic siRNAs and miRNAs or biosimilars that best aligns with current pharmacological nomenclature by mechanism of action. SIGNIFICANCE STATEMENT: The common and unique chemistry and molecular pharmacology of six FDA-approved siRNA therapeutics are summarized, in which nedosiran is newly approved. We point out rather a surprisingly mechanistic action as miRNAs for five siRNA therapeutics and discuss the differences and similarities between siRNAs and miRNAs that supports using a general and unified term "RNAi" therapeutics to align with current drug nomenclature criteria in pharmacology based on mechanism of action and embraces broader forms and growing number of novel RNAi therapeutics.

Synthetic biology constitutes a scientific domain focused on intentional redesign of organisms to confer novel functionalities or create new products through strategic engineering of their genetic makeup. Leveraging the inherent capabilities of nature, one may address challenges across diverse sectors including medicine. Inspired by this concept, we have developed an innovative bioengineering platform, enabling high-yield and large-scale production of biological small interfering RNA (BioRNA/siRNA) agents via bacterial fermentation. Herein, we show that with the use of a new tRNA fused pre-miRNA carrier, we can produce various forms of BioRNA/siRNA agents within living host cells. We report a high-level overexpression of nine target BioRNA/siRNA molecules at 100% success rate, yielding 3-10 mg of BioRNA/siRNA per 0.25 L of bacterial culture with high purity (>98%) and low endotoxin (

Genome-derived microRNAs (miRNAs or miRs) govern posttranscriptional gene regulation and play important roles in various cellular processes and disease progression. While chemo-engineered miRNA mimics or biosimilars made in vitro are widely available and used, miRNA agents produced in vivo are emerging to closely recapitulate natural miRNA species for research. Our recent work has demonstrated the success of high-yield, in vivo production of recombinant miRNAs by using human tRNA (htRNA) fused precursor miRNA (pre-miR) carriers. In this study, we aim to compare the production of bioengineered RNA (BioRNA) molecules with glycyl versus leucyl htRNA fused hsa-pre-miR-34a carriers, namely, BioRNAGly and BioRNALeu, respectively, and perform the initial functional assessment. We designed, cloned, overexpressed, and purified a total of 48 new BioRNA/miRNAs, and overall expression levels, final yields, and purities were revealed to be comparable between BioRNAGly and BioRNALeu molecules. Meanwhile, the two versions of BioRNA/miRNAs showed similar activities to inhibit non-small cell lung cancer cell viability. Interestingly, functional analyses using model BioRNA/miR-7-5p demonstrated that BioRNAGly/miR-7-5p exhibited greater efficiency to regulate a known target gene expression (EGFR) than BioRNALeu/miR-7-5p, consistent with miR-7-5p levels released in cells. Moreover, BioRNAGly/miR-7-5p showed comparable or slightly greater activities to modulate MRP1 and VDAC1 expression, compared with miRCURY LNA miR-7-5p mimic. Computational modeling illustrated overall comparable 3D structures for exemplary BioRNA/miRNAs with noticeable differences in htRNA species and payload miRNAs. These findings support the utility of hybrid htRNA/hsa-pre-miR-34a as reliable carriers for RNA molecular bioengineering, and the resultant BioRNAs serve as functional biologic RNAs for research and development.

Global control of infectious diseases depends on the continuous development and deployment of diverse vaccination strategies. Currently available live-attenuated and killed virus vaccines typically take a week or longer to activate specific protection by the adaptive immunity. The mosquito-transmitted Nodamura virus (NoV) is attenuated in mice by mutations that prevent expression of the B2 viral suppressor of RNA interference (VSR) and consequently, drastically enhance in vivo production of the virus-targeting small-interfering RNAs. We reported recently that 2 d after immunization with live-attenuated VSR-disabled NoV (NoVΔB2), neonatal mice become fully protected against lethal NoV challenge and develop no detectable infection. Using Rag1-/- mice that produce no mature B and T lymphocytes as a model, here we examined the hypothesis that adaptive immunity is dispensable for the RNAi-based protective immunity activated by NoVΔB2 immunization. We show that immunization of both neonatal and adult Rag1-/- mice with live but not killed NoVΔB2 induces full protection against NoV challenge at 2 or 14 d postimmunization. Moreover, NoVΔB2-induced protective antiviral immunity is virus-specific and remains effective in adult Rag1-/- mice 42 and 90 d after a single-shot immunization. We conclude that immunization with the live-attenuated VSR-disabled RNA virus vaccine activates rapid and long-lasting protective immunity against lethal challenges by a distinct mechanism independent of the adaptive immunity mediated by B and T cells. Future studies are warranted to determine whether additional animal and human viruses attenuated by VSR inactivation induce similar protective immunity in healthy and adaptive immunity-compromised individuals.

Lowering low-density lipoprotein cholesterol (LDL-C) is a cornerstone of reducing risk for atherosclerotic cardiovascular disease. Despite the approval of nonstatin therapies for LDL-C lowering over the past 2 decades, these medications are underused, and most patients are still not at guideline-recommended LDL-C goals. Barriers include poor adherence, clinical inertia, concern for side effects, cost, and complex prior authorization processes. With atherosclerotic cardiovascular disease-related mortality increasing globally, there remains a need for additional therapeutic options for lowering LDL-C as part of an atherosclerotic cardiovascular disease prevention strategy. Following the identification of PCSK9 (proprotein convertase subtilisin/kexin type 9) as a promising therapeutic target, inclisiran was developed using the natural process of RNA interference for robust, sustained prevention of hepatic PCSK9 synthesis. Twice-yearly maintenance subcutaneous inclisiran (following initial loading doses at Day 1 and Day 90) reduces circulating LDL-C levels by ≈50% versus placebo when added to maximally tolerated statins. Long-term safety and tolerability of inclisiran have been assessed, with studies underway to evaluate the effects of inclisiran on cardiovascular outcomes and to provide additional safety and effectiveness data. In 2021,

Abstract\nHIGHLIGHTSThe convoluted relationships between plants, viruses, and arthropod vectors housing bacterial endosymbionts are pivotal in the spread of harmful plant viral diseases. Endosymbionts play key roles in: manipulating host responses, influencing insect resistance to pesticides, shaping insect evolution, and bolstering virus acquisition, retention, and transmission. This interplay presents an innovative approach for developing sustainable strategies to manage plant diseases. Recent progress in targeting specific endosymbionts through genetic modifications, biotechnological advancements, and RNA interference shows potential for curbing viral spread and disease progression. Additionally, employing synthetic biology techniques like CRISPR/Cas9 to engineer endosymbionts and disrupt crucial interactions necessary for viral transmission in arthropod vectors holds promise for effective control measures. In this review, these obligate and facultative bacterial cruxes have been discussed to elaborate on their mechanistic involvement in the regulation and/or inhibition of tripartite pathways of interactions. Furthermore, we provide an in-depth understanding of endosymbionts’ synergistic and antagonistic effects on: insect biology, plant immunity, and virus acquisition and transmission. Finally, we point out open questions for future research and provide research directions concerning the deployment of genetically engineered symbionts to affect plant-virus-vector interactions for sustainable disease management. By addressing existing knowledge gaps and charting future research paths, a deeper comprehension of the role of endosymbionts in plant-virus-vector interactions can pave the way for innovative and successful disease management strategies. The exploration of antiviral therapies, paratransgenesis, and pathogen-blocking tactics using engineered endosymbionts introduces pioneering solutions for lessening the impact of plant viral diseases and green pest management.The tripartite interactions among plants, viruses, and arthropod vectors with bacterial endosymbionts play a crucial role in the spread of plant viral diseases.Plant-virus-vector interactions have been extensively studied; however, the role of arthropod endosymbionts remains largely unexplored necessitating further investigation.This comprehensive review delves into the mechanisms by which obligate and facultative bacterial symbionts influence the regulation of interactions within the plant-virus-vector pathosystem.The impact of endosymbionts on: plant immunity, insect biology, vector ecology, and evolutionary pathways is explored, emphasizing their role in virus transmission.Sustainable disease management strategies targeting specific endosymbionts can disrupt viral transmission pathways by utilizing advanced biotechnological tools like RNA interference (RNAi) and CRISPR/Cas9. [ABSTRACT FROM AUTHOR]

RNA interference (RNAi) is one of the important defense responses against viral infection, but its mechanism and impact remain unclear in mycovirus infections. In our study, reverse genetics and virus-derived small RNA sequencing were used to show the antiviral responses of RNAi components in Aspergillus flavus infected with Aspergillus flavus partitivirus 1 (AfPV1). qRT-PCR revealed that AfPV1 infection induced the expression of the RNAi components in A. flavus compared with noninfected A. flavus. Knock mutants of each RNAi component were generated, but the mutants did not exhibit any obvious phenotypic changes compared with the A. flavus parental strain. However, after AfPV1 inoculation, production of AfPV1 was significantly less than in the parental strain. Furthermore, sporulation was greater in each AfPV1-infected mutant compared with the AfPV1-infected parental A. flavus. We also investigated the sensitivity of virus-free and AfPV1-infected RNAi mutants and the parental strain to cell wall stress, osmotic stress, genotoxic stress, and oxidative stress. The mutants of DCLs and AGOs infected by AfPV1 displayed more changes than RDRP mutants in response to the first three stresses. Small RNA sequencing analysis suggested that AfPV1 infection reduced the number of unique reads of sRNA in A. flavus , although there were many vsiRNA derived from the AfPV1 genome. GO term and KEGG pathway analyses revealed that the functions of sRNA affected by AfPV1 infection were closely related to vacuole production. These results provide a better understanding of the functional role of RNAi in the impact of AfPV1 on the hypovirulence of A. flavus. [ABSTRACT FROM AUTHOR]

Aims: Aurora kinase A (AURKA) has been implicated in promoting myeloid and renal fibrosis. This study aimed to investigate the impact and underlying mechanism of AURKA on liver fibrosis and to assess the therapeutic potential of MLN8237, a small-molecule AURKA inhibitor, in preventing liver fibrosis in mice. Methods: The research used bioinformatics analysis and immunohistochemistry staining on fibrotic liver tissues from human and mouse models to assess AURKA expression. The cellular localization of AURKA was determined through double immunofluorescence staining in human fibrotic liver tissues and primary mouse hepatic stellate cells. RNA interference and AURKA antagonism were used to examine the effects of AURKA on liver fibrosis, while RNA-sequencing, qRT-PCR, and western blotting were employed to elucidate the potential molecular mechanisms of AURKA on hepatic stellate cell activation. Results: The results showed that AURKA was positively correlated with the progression of liver fibrosis and was predominantly expressed in activated HSCs. Silencing AURKA inhibited HSC activation and proliferation, and induced HSC apoptosis, effects that were similar to those observed with MLN8237 treatment. Additionally, silencing AURKA suppressed the glycogen synthase kinase-3β/β-catenin signaling pathway. Pharmacological inhibition of AURKA phosphorylation also resulted in reduced liver fibrosis in vivo. Conclusion: In conclusion, AURKA may promote HSC activation and liver fibrosis through the Wnt/β-catenin pathway, suggesting its potential as a therapeutic target for liver fibrosis. [ABSTRACT FROM AUTHOR]

AbstractPurposeMethodsResultsConclusionPterygium is a common ocular surface disease characterized by a high recurrence rate and unknown etiology.In this study, we investigated the upregulation of matrix metalloproteinase genes, including MMP1, MMP2, MMP3, MMP7, MMP9, MMP11, MMP12, MMP13, MMP23B, and MMP28, in pterygium tissue using RNA sequencing, Western blotting, and immunohistochemistry.Employing the MEME tool, we identified a conserved DNA motif within the promoter regions of these matrix metalloproteinase genes. Mass spectrometry analysis revealed an interaction between the cohesin complex and this motif. Disrupting the cohesin complex through RNA interference of RAD21 cohesin complex component or structural maintenance of chromosomes 3 in primary pterygial fibroblasts led to decreased matrix metalloproteinase gene expression and reduced recruitment of twist family bHLH transcription factor 1 and transcription factor 4 to matrix metalloproteinase gene promoters.Overall, our findings suggest a novel epigenetic mechanism regulating matrix metalloproteinase transcription in pterygium. [ABSTRACT FROM AUTHOR]

Background: Odorant binding proteins (OBPs) initiate the process of odorant perception. Numerous investigations have demonstrated that OBPs bind a broad variety of chemicals and are more likely to carry pheromones or odor molecules with high binding affinities. However, few studies have investigated its effects on insect behavior. Previously, we found that AmelOBP4 has a significantly higher expression in the heads of foragers than that of nurses regardless of their ages, revealing its importance in foraging behaviour of the honey bee. RNA interference (RNAi) is the induction of sequence specific gene silencing by double-stranded RNA (dsRNA), it is a powerful tool that makes gene inactivation possible in organisms that were not amenable to genetic analysis before. Results: In this study, we found that AmelOBP4 had high expression levels in the antennae of both nurses and foragers, and could be successfully inhibited by feeding double stranded RNA of AmelOBP4 (dsAmelOBP4). Foragers with inhibited AmelOBP4 showed significantly lower sugar responsiveness than control bees, and also significantly reduced EAG response to plant volatiles of nonanal, linalool and 1-Octen-3ol. On the other hand, nurses with inhibited AmelOBP4 showed significantly reduced EAG response to brood pheromone of ethyl oleate, methyl linoleate, methyl palmitate and β-ocimene. Finally, the Y-tube choice assay showed nurses only exhibited a significantly reduced preference to ethyl oleate, but foragers exhibited significantly reduced preference to all these three plant volatiles. Conclusions: The findings of our study suggested that AmelOBP4 plays an important role in the odorant binding process, especially in modulating olfactory behaviour in workers. Our results provide a foundation for exploring the olfactory mechanism of Apis mellifera. [ABSTRACT FROM AUTHOR]

AbstractThe field of biomedical science has witnessed another milestone with the advent of RNA-based therapeutics. This review explores three major RNA molecules, namely: messenger RNA (mRNA), RNA interference technology (RNAi), and Antisense Oligonucleotide (ASO), and analyses U.S. Food and Drug Administration drugs from 14 RNA-based pharmaceutical companies in terms of targeted genes, diseases and types, clinical trials and status, the mode of delivery, and the year of production. Many of such drugs are clinically approved or pending approval by the U.S. Food and Drug Administration (FDA) alongside other leading drugs agencies. Regarding diseases, this article emphasizes cancer therapy, genetic diseases, viral infections, and two categories of drug delivery systems include viral vectors and nanoparticles. Despite the tremendous progress made, key issues associated with these delivery systems are stability, off-target activities of RNA payloads, efficiency in cellular uptake, and the innovative need for engineering techniques for modifications. This review emphasizes the transformative potential of RNA therapeutics and the role of innovative technologies in addressing clinical needs, paving the way for a new era in precision medicine. [ABSTRACT FROM AUTHOR]

Background: Fusarium head blight (FHB) is a major disease affecting cereal crops including wheat, barley, rye, oats and maize. Its predominant causal agent is the ascomycete fungus Fusarium graminearum, which infects the spikes and thereby reduces grain yield and quality. The frequency and severity of FHB epidemics has increased in recent years, threatening global food security. Spray-induced gene silencing (SIGS) is an alternative technique for tackling this devastating disease through foliar spraying with exogenous double-stranded RNA (dsRNA) to silence specific pathogen genes via RNA interference. This has the advantage of avoiding transgenic approaches, but several aspects of the technology require further development to make it a viable field-level management tool. One such existing knowledge gap is how dsRNA spraying affects the microbiota of the host plants. Results: We found that the diversity, structure and composition of the bacterial microbiota are subject to changes depending on dsRNA targeted and host studied, while the fungal microbiota in the phyllosphere remained relatively unchanged upon spraying with dsRNA. Analyses of fungal co-occurrence patterns also showed that F. graminearum established itself among the fungal communities through negative interactions with neighbouring fungi. Through these analyses, we have also found bacterial and fungal genera ubiquitous in the phyllosphere, irrespective of dsRNA treatment. These results suggest that although rarer and less abundant microbial species change upon dsRNA spray, the ubiquitous bacterial and fungal components of the phyllosphere in wheat and barley remain unchanged. Conclusion: We show for the first time the effects of exogenous dsRNA spraying on bacterial and fungal communities in the wheat and barley phyllospheres using a high-throughput amplicon sequencing approach. The results obtained further validate the safety and target-specificity of SIGS and emphasize its potential as an environmentally friendly option for managing Fusarium head blight in wheat and barley. [ABSTRACT FROM AUTHOR]

The stress response following burns may be a crucial factor in keloid formation, yet the underlying pathological mechanisms remain to be elucidated. This study initially investigated how heat shock factor 1 (HSF1) and heat shock proteins (HSPs) within the heat shock pathway influence keloid fibrosis, providing insights into the role of the heat shock response in keloid development. This study aims to further elucidate the role of the heat shock pathway in keloid fibrosis and investigate the specific function of HSF1 within this pathway. This study focused on human keloid fibroblasts, examining the expression and regulatory role of HSF1 on HSPs under heat stress using immunohistochemistry, RNA interference, real-time fluorescent PCR, and Western blotting techniques. HSF1 was overexpressed in keloid fibroblasts and tissues compared to normal skin, and heat stress could further enhance HSF1 expression in both keloid tissues and fibroblasts. Functional inhibition of HSF1 significantly affected the expression of downstream HSPs in keloid fibroblasts, ultimately leading to the inhibition of keloid fibrosis. The heat shock pathway plays a crucial role in keloid fibrosis, with HSF1 as a key regulator influencing the expression of HSPs. Heat stress treatment of keloid fibroblasts offers an approach for investigating keloid formation. [ABSTRACT FROM AUTHOR]

Background: Triple-negative breast cancer (TNBC) is among the most aggressive forms of breast cancer, characterized by a dismal prognosis. In the absence of drug-targetable receptors, chemotherapy remains the sole systemic treatment alternative. Recent advancements in immunotherapy, particularly immune checkpoint inhibitors (ICIs) that target programmed death 1/programmed death ligand 1 (PD-1/PD-L1) and cytotoxic T lymphocyte associated antigen 4 (CTLA-4), have provided renewed optimism for the treatment of patients with TNBC. Prior research has indicated that the expression level of the cell polarity protein discs large homolog 5 (DLG5) correlates with the malignant progression and prognosis of breast cancer; nevertheless, its influence on PD-L1 expression and its function in immunotherapy for TNBC require further investigation. Methods: The hypoxia cell model was established by simulating the cell hypoxic microenvironment in the human SUM159 and MDA-MB-231 cell lines using cobalt II chloride (CoCl2). A combination of PD-L1 inhibitors and DLG5 RNA interference techniques was used, along with various methods including cell counting kit-8 (CCK-8), colony formation, wound healing, transwell migration, reverse transcription-quantitative real-time PCR (RT-qPCR), immunofluorescence, immunohistochemical staining (IHC), expression analysis from datasets and western blotting. These methods were employed to evaluate changes in cell proliferation, migration, and the expression levels of PD-L1 and DLG5. Additionally, the correlation between the expression of PD-L1 and DLG5 in clinical samples was analyzed. Results: (1) In vitro experiments, a cellular hypoxia model was effectively established utilizing 150 µM CoCl₂. Under these conditions, cell clone formation, invasiveness, and migration rate were all significantly inhibited. (2) The expression levels of DLG5 and PD-L1 were significantly increased in both MDA-MB-231 and SUM159 cells following treatment with 150 µM CoCl₂. (3) Silencing DLG5 resulted in a considerable upregulation of PD-L1 expression in MDA-MB-231 and SUM159 cells under normoxic circumstances, but it was markedly downregulated under hypoxic settings. Inhibition of PD-L1 expression resulted in a considerable increase in DLG5 expression under normoxic conditions, but it decreased under hypoxic conditions. Correlation research demonstrated an inverse association between the expression of DLG5 and PD-L1 in TNBC tissues. Conclusion: This study provides new theoretical evidence and potential therapeutic targets for the immunotherapy strategies of TNBC, holding significant clinical application value. [ABSTRACT FROM AUTHOR]

Feeding and molting are particularly important physiological processes for insects, and it has been reported that neuropeptides are involved in the nervous regulation of these 2 processes. Sulfakinin (SK) is an important neuropeptide that is widely distributed among insects and plays a pivotal role in regulating feeding, courtship, aggression, and locomotion. In this study, we investigated the involvement of SK in feeding and molting on a highly notorious pest insect, the fall armyworm, Spodoptera frugiperda. SK transcript levels were found in all larval stages and there was a predominant expression of SK in the brain of 5th instar larvae. By immunostaining, SK was detected in 2 pairs of cells in the median protocerebrum. But during prolonged periods of starvation, there was a significant reduction in SK messenger RNA levels; however, subsequent refeeding led to a notable increase. To investigate the role of SK in feeding and molting, SK was silenced in S. frugiperda larvae through RNA interference. This resulted in a significant increase in food intake, weight gain, and the molting process happened more rapidly in the double‐stranded SK‐treated larvae compared to the controls. Conversely, injection of sulfated SK peptide (sSK) caused opposite effects. Interestingly, SK‐knockdown in larvae resulted in increased levels of 20‐hydroxyecdysone and also of the expression of some of it signaling pathway genes. Altogether, this study highlights the important role played by SK in regulating feeding and molting in S. frugiperda. [ABSTRACT FROM AUTHOR]

Double-stranded RNA (dsRNA) has emerged as key player in gene silencing for the past two decades. Tailor-made dsRNA is now recognized a versatile raw material, suitable for a wide range of applications in biopesticide formulations, including insect control to pesticide resistance management. The mechanism of RNA interference (RNAi) acts at the messenger RNA (mRNA) level, utilizing a sequence-dependent approach that makes it unique in term of effectiveness and specificity compared to conventional agrochemicals. Two primary categories of small RNAs, known as short interfering RNAs (siRNAs) and microRNAs (miRNAs), function in both somatic and germline lineages in a broad range of eukaryotic species to regulate endogenous genes and to defend the genome from invasive nucleic acids. Furthermore, the application of RNAi in crop protection can be achieved by employing plant-incorporated protectants through plant transformation, but also by non-transformative strategies such as the use of formulations of sprayable RNAs as direct control agents, resistance factor repressors or developmental disruptors. This review explores the agricultural applications of RNAi, delving into its successes in pest-insect control and considering its broader potential for managing plant pathogens, nematodes, and pests. Additionally, the use of RNAi as a tool for addressing pesticide-resistant weeds and insects is reviewed, along with an evaluation of production costs and environmental implications. [ABSTRACT FROM AUTHOR]

Simple Summary: Studies have shown that cytochrome P450s (CYPs) in the antennae of insects are involved in the entire process of olfactory recognition of odor compounds. In this study, through transcriptome technology and RT-qPCR, we identified CYPs that are specifically and highly expressed in the antennae of Solenopsis invicta worker ant (SinvCYP6K1 and SinvCYP4V2). Results from RNA interference (RNAi) combined with electroantennogram (EAG) and behavioral experiments demonstrated that SinvCYP6K1 and SinvCYP4V2 are involved in the recognition process of 2-ethyl-3,6(5)-dimethylpyrazine by S. invicta worker ant. Insects have highly developed olfactory systems in which cytochrome P450s (CYPs) were involved as odor-degrading enzymes throughout the olfactory recognition of odor compounds by insects to avoid continuous stimulation of signaling molecules and thus damage to the olfactory nervous. To understand whether the highly expressed CYPs in the antennae play an olfactory function in Solenopsis invicta worker, in this study, we find six highly expressed antennal CYPs from the transcriptome of S. invicta. Multiple sequence alignment and phylogenetic analysis divided them into two families: the CYP3 family (SinvCYP6K1, SinvCYP6K1-1) and the CYP4 family (SinvCYP4C1, SinvCYP4C1-1, SinvCYP4C1-2, SinvCYP4V2). The expression patterns of these six CYPs were analyzed by RT-qPCR, which revealed that SinvCYP6K1 and SinvCYP4V2 were only highly expressed in the antennae of adult workers. The expression of SinvCYP6K1 and SinvCYP4V2 in workers was markedly diminished after feeding with dsRNA. The electroantennography (EAG) assay demonstrated that the silencing of either SinvCYP6K1 or SinvCYP4V2 resulted in a notable reduction in the EAG response of workers to 2-ethyl-3,6(5)-dimethylpyrazine (EDMP). Furthermore, the trajectory behavior assay showed that the worker's range and speed of movement in response to EDMP significant decreased after the silencing of SinvCYP6K1 and SinvCYP4V2. The findings indicated that both SinvCYP6K1 and SinvCYP4V2 were implicated in the recognition of EDMP by S. invicta. [ABSTRACT FROM AUTHOR]

Background/Objectives: The balanced regulation of innate immunity plays essential roles in rhizobial infection and the establishment and maintenance of symbiosis. The evolutionarily conserved cell death suppressor Bax inhibitor-1 plays dual roles in nodule symbiosis, providing a valuable clue in balancing immunity and symbiosis, while it remains largely unexplored in the legume Lotus japonicus. Methods/Results: In the present report, the BI-1 gene family of L. japonicus was identified and characterized. We identified 6 BI-1 genes that translate into peptides containing 240–255 amino acids with different structural characteristics and isoelectric points. We performed phylogenetic analyses and detected evolutionary conservation and divergence among BI-1 proteins from L. japonicus, Glycine max, Medicago truncatula, Arabidopsis thaliana, and Oryza sativa. Expression profiles among different roots indicated that the inoculation of MAFF303099 significantly increased the expression of most of the L. japonicus BI-1 family genes. We down-regulated the transcripts of LjBI-1a by RNA interference and observed that LjBI-1a promotes nodulation and nodule formation. Conclusions: These discoveries shed light on the functions of BI-1 genes in L. japonicus, and simultaneously emphasize the potential application of LjBI-1a in enhancing the symbiotic nitrogen fixation ability of legumes. [ABSTRACT FROM AUTHOR]

Although genome-wide association studies have identified dozens of loci associated with colorectal cancer (CRC) susceptibility, the causal genes or risk variants within these loci and their biological functions often remain elusive. Recently, the genomic locus 12p13.32, with the tag single-nucleotide polymorphism rs10774214, was identified as a crucial CRC risk locus in Asian populations. However, the functional mechanism of this region has not been fully elucidated. Here, we applied a high-throughput RNA interference approach in CRC cell lines to interrogate the function of genes in this genomic region. Multiple genes were found to affect cell functions, with CCND2 having the most significant effect as an oncogene. Moreover, overexpressed CCND2 could promote CRC cell proliferation. Subsequently, by integrating a fine-mapping analysis and multi-ancestry large-scale population cohorts consisting of 14 358 CRC cases and 34 251 healthy controls, we identified a regulatory variant rs4477507-T that contributed to an increased CRC risk in populations from China (odds ratio = 1.16, 95% confidence interval = 1.11–1.22, P  = 4.45 × 10−10) and Europe (odds ratio = 1.17, 95% confidence interval = 1.12–1.21, P  = 1.65 × 10−14). Functional characterization of the variant demonstrated that it could act as an allele-specific enhancer to distally facilitate the expression of CCND2 mediated by the transcription factor TEAD4. Overall, our study underscores the essential role of CCND2 in CRC development and delineates its regulatory mechanism mediated by rs4477507, establishing an epidemiological and biological link between genetic variation and CRC pathogenesis. [ABSTRACT FROM AUTHOR]

Functional cure of chronic hepatitis B (CHB)—defined as sustained seroclearance of hepatitis B surface antigen (HBsAg) with unquantifiable hepatitis B virus (HBV) DNA at 24 weeks off treatment, is an optimal treatment endpoint. Nonetheless, it cannot be consistently attained by current treatment modalities. RNA interference (RNAi) is a novel treatment strategy using small-interfering RNA (siRNA) or antisense oligonucleotide (ASO) to target HBV post-transcriptional RNA, in turn suppressing viral protein production and replication. Hence, RNAi has indirect effects in promoting host immune reconstitution against HBV. Multiple RNAi therapeutics have entered phase II/III clinical trials, demonstrating potent, dose-dependent, and sustainable effects in suppressing HBsAg. Incidences of HBsAg seroclearance, particularly with the use of ASO, have also been documented. The combination of RNAi with other antivirals/immunomodulators (e.g. pegylated interferon), have shown promising results in potentiating RNAi effects and enhancing treatment durability. Further research will be required to establish predictors of response, optimal treatment protocols, and long-term outcomes in patients on RNAi. RNAi therapeutics have shown promising results and will likely be the keystone of future HBV treatment. [ABSTRACT FROM AUTHOR]

BACKGROUND Transthyretin amyloidosis with cardiomyopathy (ATTR-CM) is a progressive, fatal disease. Vutrisiran, a subcutaneously administered RNA interference therapeutic agent, inhibits the production of hepatic transthyretin. METHODS In this double-blind, randomized trial, we assigned parents with ATTR-CM in a 1:1 ratio to receive vutrisiran (25 mg) or placebo every 12 weeks for up to 36 months. The primary end point was a composite of death from any cause and recurrent cardiovascular events. Secondary end points included death from any cause, the change from baseline in the distance covered on the 6-minute walk test, and the change from baseline in the Kansas City Cardiomyopathy Questionnaire-Overall Summary (KCCQ-OS) score. The efficacy end points were assessed in the overall population and in the monotherapy population (the patients who were not receiving tafamidis at baseline) and were tested hierarchically. RESULTS A total of 655 patients underwent randomization; 326 were assigned to receive vutrisiran and 329 to receive placebo. Vutrisiran treatment led to a lower risk of death from any cause and recurrent cardiovascular events than placebo (hazard ratio in the overall population, 0.72; 95% confidence interval [CI], 0.56 to 0.93; P=0.01; hazard ratio in the monotherapy population, 0.67; 95% CI, 0.49 to 0.93; P=0.02) and a lower risk of death from any cause through 42 months (hazard ratio in the overall population, 0.65; 95% CI, 0.46 to 0.90; P=0.01). Among the patients in the overall population, 125 in the vutrisiran group and 159 in the placebo group had at least one primary end-point event. In the overall population, treatment with vutrisiran resulted in less of a decline in the distance covered on the 6-minute walk test than placebo (least-squares mean difference, 26.5 m; 95% CI, 13.4 to 39.6; P<0.001) and less of a decline in the KCCQ-OS score (least-squares mean difference, 5.8 points; 95% CI, 2.4 to 9.2; P<0.001). Similar benefits were observed in the monotherapy population. The incidence of adverse events was similar in the two groups (99% in the vutrisiran group and 98% in the placebo group); serious adverse events occurred in 62% of the patients in the vutrisiran group and in 67% of those in the placebo group. CONCLUSIONS Among patients with ATTR-CM, treatment with vutrisiran led to a lower risk of death from any cause and cardiovascular events than placebo and preserved functional capacity and quality of life. [ABSTRACT FROM AUTHOR]

Virus-derived small interfering RNAs (vsiRNAs) have been widely recognized to play an antiviral immunity role. However, it is unclear whether vsiRNAs can also play a positive role in viral infection. Here, we characterized three highly abundant vsiRNAs mapped to the genomic termini of rice stripe virus (RSV), a negative-strand RNA virus transmitted by insect vectors. The three vsiRNAs shared 11 nucleotides due to the conservative genomic termini and were likely generated from viral terminal panhandle structure, depending on both Dicer1 and Dicer2 in insects. In addition to targeting viral RNAs in a miRNA-like manner, the three vsiRNAs coordinately downregulated the expression of DOPA decarboxylase, thereby suppressing the prophenoloxidase immune reaction in insect vectors. In vsiRNA-silenced transgenic rice, the viral titer significantly decreased, indicating that these vsiRNAs promote RSV replication in rice. This study elucidates a unique function of vsiRNAs derived from the conserved panhandle structure of negative-strand RNA viruses in enhancing viral infection. Author summary: RNA interference (RNAi) is one of the important innate immune systems against viruses in insects. Virus-derived small interfering RNAs (vsiRNAs) have been widely recognized to play an antiviral immunity role. Here, we find that three vsiRNAs generated from the terminal panhandle structure of a rice virus promote viral infection in both insect vectors and host plants. These vsiRNAs coordinately target DOPA decarboxylase to inhibit melanization immune reaction in insect vectors. The vsiRNA-silenced transgenic rice exhibits an antiviral phenotype. Our work reveals a conserved positive effect of vsiRNAs from negative-strand RNA viruses on viral infection. [ABSTRACT FROM AUTHOR]

Sodium taurocholate co-transporting polypeptide (NTCP) has been identified as an entry receptor for hepatitis B virus (HBV), but the molecular events of the viral post-endocytosis steps remain obscure. In this study, we discovered that manganese (Mn) could strongly inhibit HBV infection in NTCP-reconstituted HepG2 cells without affecting viral replication. We therefore profiled the antiviral effects of Mn2+ in an attempt to elucidate the regulatory mechanisms involved in early HBV infection. Intriguingly, Mn2+ conspicuously stimulated lysosomal activity, as evidenced by hyperactivation of mTORC1 and increased endo/lysosomal acidity. After HBV-triggered internalization, the NTCP receptor was sorted to late endosomal compartments by the ESCRT machinery in concert with the invading virion. The establishment of HBV infection was found to be independent of lysosomal fusion-driven late endosome maturation; Mn2+-induced lysosomal hyperfunction virtually impaired infection, suggesting that virions may gain cytosolic access directly from late endosomes. In contrast, suppression of lysosomal activity substantially enhanced HBV infection. Prolonged mTORC1 inactivation facilitated viral infection by depleting lysosomes and accelerating endocytic transport of virions. Notably, treatment with the natural steroidal alkaloid tomatidine recapitulated the effects of Mn2+ in stimulating lysosomal activity and exhibited potent anti-HBV activity in HepG2-NTCP cells and in proliferating human hepatocyte organoids. These findings provide new insights into the post-endocytosis events of HBV infection. The negative regulation of early HBV infection by endo/lysosomal activity makes it a promising target for antiviral therapies. Author summary: Despite the identification of NTCP as a functional HBV receptor over a decade ago, the later stages of the HBV entry process remain poorly understood. Inspired by a previous publication, we tested the possible effect of manganese (Mn) on HBV infectivity. To our surprise, although Mn2+ did not induce a type I interferon response, it was still able to inhibit HBV infectivity in HepG2-NTCP cells. Using loss-of-function assays with pharmacological inhibitors and RNA interference, we found that after internalization, HBV and the NTCP receptor are co-sorted via the ESCRT machinery into the late endosome, where the virions achieve endosomal escape. Notably, endosome-lysosome fusion is not required for HBV infection. In contrast, lysosomal hyperfunction, induced by Mn2+ or the natural steroidal alkaloid tomatidine, impairs de novo HBV infection, while the use of lysosomal inhibitors facilitates this process. The suppressive impact of lysosomal activity on the early HBV infection renders it a promising target for antiviral therapies. [ABSTRACT FROM AUTHOR]