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Introduction: Members of the family Geminiviridae have been reported to infect either a monocot plant or a dicot plant, but not both. This study reports a geminivirus, Wheat Dwarf India Virus (WDIV), first identified in wheat, that is capable of infecting both monocot and dicot plants and acting as a viral vector. Objectives: This study was aimed at developing a broad host range viral vector system for reverse genetics and genome editing. Methods: Here we used a wheat isolate of WDIV and Ageratum yellow leaf curl betasatellite (AYLCB) for infectivity assays and vector development. We performed Agrobacterium-mediated inoculation of WDIV and AYLCB in wheat, oat, barley, corn, soybean, and tobacco. To examine the potential of WDIV to act as a viral vector, we modified the WDIV genome and cloned DNA fragments of the phytoene desaturase (PDS) genes from wheat and tobacco, separately. For gene editing experiments, tobacco lines expressing Cas9 were infiltrated with a WDIV-based vector carrying gRNA targeting the PDS gene. Results: About 80 to 90% of plants inoculated with infectious clones of WDIV alone or WDIV together with AYLCB showed mild symptoms, whereas some plants showed more prominent symptoms. WDIV and AYLCB were detected in the systemically infected leaves of all the plant species. Furthermore, the inoculation of the WDIV vector carrying PDS fragments induced silencing of the PDS gene in both wheat and tobacco plants. We also observed high-efficiency genome editing in the Cas9-expressing tobacco plants that were inoculated with WDIV vector-carrying gRNA. Conclusion: Detection of WDIV in naturally infected wheat, barley, and sugarcane in the field and its ability to systemically infect wheat, oat, barley, corn, soybean, and tobacco under laboratory conditions, provides compelling evidence that WDIV is the first geminivirus identified with the capability of infecting both monocot and dicot plant species. The wide host range of WDIV can be exploited for developing a single vector system for high-throughput genome editing in many plant species.

Crouzon syndrome is a rare genetic craniofacial malformation caused by heterozygous gain-of-function mutations in the FGFR2 gene. The resulting constitutive activation of the FGFR2 signaling causes the premature osteogenic differentiation of calvarial mesenchymal stromal cells in skull sutures, leading to early suture ossification. Craniectomy is the gold standard treatment, being invasive and burdened by complications. To address these issues, we developed personalized allele-specific (AS) small interfering RNA (siRNA) to knockdown the expression of the FGFR2 mutant allele in Crouzon patient-derived suture cells. The selected therapeutic siRNA mitigated FGFR2 cascade downregulating phosphorylation of FGFR2 (48%) and of its key effector ERK1/2 (77%) as RUNX2 protein levels (34%). This effect was confirmed by the reduced osteogenic commitment and differentiation of treated cells, evidenced by decreased expression of osteogenic marker genes and a 5-fold decrease in mineralized matrix deposition. We developed a highly biocompatible delivery system for siRNAs, based on human recombinant ferritin nanoparticles (NPs), combining cell targeting with improved nucleic acid encapsulation and endosomal escape properties. We demonstrated the ability of these NPs to deliver and release siRNAs within target cells, sustaining their inhibitory and AS effects. Here, we show that ferritin-mediated AS FGFR2 knockdown by siRNA represents a suitable strategy to dampen FGFR2 overactivation in patients’ cells.

Hepatitis B virus (HBV) and its associated chronic liver disease pose a significant health hazard. Chronic HBV infection is a major contributor to the development of liver cirrhosis, liver fibrosis, and liver cancer. This study focused on ISG12a and its inhibitory effect in Huh7 cells on HBV gene expression and replication. The mammalian hepatoma cells Huh7 were transfected with the pHBV1.3 vector (pCAGGS) or HA-tagged ISG12a to determine the overexpression, to test this hypothesis further, we did knockdown or silencing by transducing Huh7 cells with lentiviruses containing shRNAs targeting ISG12a or scramble control shRNA (shControl). The expression of ISG12a was evaluated through western blot analysis. The HBsAg and HBeAg secretion in the Huh7 cells’ culture media was examined using an ELISA test. The qRT-PCR confirmed the mRNA and 3.5 mRNA Kb of ISG12a. The HBV total RNA was extracted and evaluated through a Northern blot. The isolated DNA was detected through qPCR. Additionally, a mechanistic study of enhancer II (EnhII/Cp) was examined through luciferase reporter testing. Our research findings indicate that ISG12a, an interferon-stimulated gene (ISG), plays a crucial role in suppressing the replication and gene expression of HBV. According to our study using the Huh7 cell system, overexpression of ISG12a resulted in a notable reduction in HBV protein levels as well as intracellular core-associated DNA and RNA levels. On the other hand, silencing ISG12a in Huh7 cells resulted in increased HBV RNA transcripts, DNA, and secreted proteins, indicating that ISG12a plays a role in suppressing HBV replication. Additionally, the research revealed that ISG12a inhibits the function of the EnhII/Cp promoter, which results in reduced HBV gene expression. The EnhII/Cp promoter is involved in regulating HBV gene expression, and the study showed that ISG12a restricts its activity. ISG12a may have a regulatory role in controlling the expression of HBV genes. These findings highlight the importance of ISG12a in HBV gene expression and provide valuable insights for understanding its antiviral function.

Introduction: During tissue repair or regeneration, several bioactive molecules are released and interact with each other and act as complex additives or inhibitors for tissue reconstruction. In this study, the bone-healing effects of the combination treatment with tumor necrosis factor-α (TNF-α) inhibition, vascular endothelial growth factor A (VEGF-A) and bone morphogenetic protein-7 (BMP-7) release by gene silencing, and gene transfection with calcium phosphate nanoparticles (CaP) in the rat femoral head was histologically, morphologically, and biochemically evaluated. Methods: A triple-functionalized paste of CaP carrying plasmid DNA encoding for BMP-7 and for VEGF), and siRNA against TNF-α was developed and denoted as CaP3mix. To compare the effects of 3mixCaP, CaP with plasmid DNA encoding BMP-7, VEGF, or siRNA encoding TNF-α was prepared and denoted as CaP/PEI/pBMP-7/SiO2, CaP/PEI/pVEGF/SiO2, or CaP/PEI/siRNA-TNF-α/SiO2, respectively. The bone healing in bone defects in the rat femoral head was investigated after 10 and 21 days of implantation. Results: The levels of bone formation-related markers OCN, Runx2, and SP7 increased at the protein and gene levels in 3mixCaP after 10 days, and 3mixCaP significantly accelerated bone healing compared with the other treatments after 21 days of implantation. Conclusion: The triple-functionalized CaP paste loading plasmid DNA encoding BMP-7 and VEGF and siRNA encoding TNF-α is a promising bioactive material for bone tissue repair.

Strigolactones (SLs) and hydrogen sulfide (H2S) have both been shown to play important roles in plant growth, development, and environmental adaption. At present, there was no reported on the mechanism by which SLs might participate in H2S-induced salt tolerance. In this study, tomato (Solanum lycopersicum L. ‘Micro-Tom’) was used to investigate the role of SLs and H2S and their relationship under salt stress. Our results show that 25 µM H2S donor sodium hydrosulfide (NaHS) and 15 µM SLs synthetic analogue GR24 significantly promoted tomato seedling growth under salt stress. TIS108 (3 µM, a SLs synthesis inhibitor) and hypotaurine (HT, 300 µM, a H2S scavenger) inhibited the positive role of NaHS and GR24 under salt stress, respectively. Meanwhile, NaHS treatment significantly increased endogenous SL content, the activity of SL synthesis-related enzymes CCD7 and CCD8, and the expression of SL synthesis-related genes (SlD27, SlD14, SlMAX1, and SlMAX2) under salt stress. Further, after silencing a SL synthesis-related gene SlD27, the relieving role of NaHS in salt stress was basically eliminated. Silencing of SlD27 decreased endogenous SL content, while NaHS did not enhance the endogenous SL content in SlD27-silencing seedling. Therefore, H2S might enhance salt tolerance in tomato seedlings by up-regulating the expression of SL synthesis-related gene SlD27.

Inherited retinal dystrophies caused by dominant mutations in photoreceptor (PR) cell expressed genes are a major cause of irreversible vision loss. Oligonucleotide therapy has been of interest in diseases that conventional medicine cannot target. In the early days, small interfering RNAs (siRNAs) were explored in clinical trials for retinal disorders with limited success due to a lack of stability and efficient cellular delivery. Thus, an unmet need exists to identify siRNA chemistry that targets PR cell expressed genes. Here, we evaluated 12 different fully chemically modified siRNA configurations, where the valency and conjugate structure were systematically altered. The impact on retinal distribution following intravitreal delivery was examined. We found that the increase in valency (tetravalent siRNA) supports the best PR accumulation. A single intravitreal administration induces multimonths efficacy in rodent and porcine retinas while demonstrating a good safety profile. The data suggest that this configuration can treat retinal diseases caused by PR cell expressed genes with 1–2 intravitreal injections per year.

Background: The Asian citrus psyllid, Diaphorina citri, is a vector of the plant pathogen Candidatus Liberibacter asiaticus (CLas), the causal agent of Huanglongbing disease. HLB represents the main threat to the citrus industry around the world due to its fast spreading, high infectivity, and incurability. An alternative for the control of this plague in Rutaceae is the implementation of RNA interference (RNAi). Here, we propose a novel method to produce small interfering RNA (siRNA) in Escherichia coli. The method includes a reporter system to visually demonstrate dsRNA expression. We produced and tested siRNAs against three D. citri genes: Abnormal Wing Disk (AWD), Superoxide dismutase 1 (SOD), and Wingless (WNT). These genes play key roles in psyllid development and maturity, and thus, represent promising targets for potential vector control. Results: The blue coloration in bacterial cultures was easily observable, and it corroborated the generation of the dsRNA that is in the same transcript. We evaluated the dosage efficiency of siRNA using the AWD siRNA. The RNAi treatment was evaluated, and all the siRNAs tested were able to induce silencing (-3.05 for AWD, -2.60 for SOD and -2.57 for WNT). Conclusions: The novel bacterial plasmid effectively produces siRNAs, and the blue color reporter is visually facilitated to check the expression of each dsRNA. Treating D. citri with the produced siRNAs resulted in a decrease in gene expression. In addition, psyllid mortality was observed, being the highest when treated with WNT-siRNA. Our results suggest the potential of siRNA treatment as a method for controlling Huanglongbing.How to cite: Rueda-Silva JC, González-Campos LI, Durán-Armenta LF, et al. Novel bacterial plasmid produces small interfering RNAs (siRNAs) that induce effective gene silencing in the Asian citrus psyllid Diaphorina citri. Electron J Biotechnol 2023;64. https://doi.org/10.1016/j.ejbt.2023.03.006.

Sickle cell disease (SCD) is due to a mutation in the β-globin gene causing production of the toxic sickle hemoglobin (HbS; α2βS2). Transplantation of autologous hematopoietic stem and progenitor cells (HSPCs) transduced with lentiviral vectors (LVs) expressing an anti-sickling β-globin (βAS) is a promising treatment; however, it is only partially effective, and patients still present elevated HbS levels. Here, we developed a bifunctional LV expressing βAS3-globin and an artificial microRNA (amiRNA) specifically downregulating βS-globin expression with the aim of reducing HbS levels and favoring βAS3 incorporation into Hb tetramers. Efficient transduction of SCD HSPCs by the bifunctional LV led to a substantial decrease of βS-globin transcripts in HSPC-derived erythroid cells, a significant reduction of HbS+ red cells, and effective correction of the sickling phenotype, outperforming βAS gene addition and BCL11A gene silencing strategies. The bifunctional LV showed a standard integration profile, and neither HSPC viability, engraftment, and multilineage differentiation nor the erythroid transcriptome and miRNAome were affected by the treatment, confirming the safety of this therapeutic strategy. In conclusion, the combination of gene addition and gene silencing strategies can improve the efficacy of current LV-based therapeutic approaches without increasing the mutagenic vector load, thus representing a novel treatment for SCD.

Myotonic dystrophy type 1 (DM1) is a neuromuscular disease that originates from an expansion of CTG microsatellites in the 3′ untranslated region of the DMPK gene, thus leading to the expression of transcripts containing expanded CUG repeats (CUGexp). The pathophysiology is explained by a toxic RNA gain of function where CUGexp RNAs form nuclear aggregates that sequester and alter the function of MBNL splicing factors, triggering splicing misregulation linked to the DM1 symptoms. There is currently no cure for DM1, and most therapeutic strategies aim at eliminating CUGexp-DMPK transcripts. Here, we investigate a DMPK-promoter silencing strategy using CRISPR interference as a new alternative approach. Different sgRNAs targeting the DMPK promoter are evaluated in DM1 patient muscle cells. The most effective guides allowed us to reduce the level of DMPK transcripts and CUGexp-RNA aggregates up to 80%. The CUGexp-DMPK repression corrects the overall transcriptome, including spliceopathy, and reverses a physiological parameter in DM1 muscle cells. Its action is specific and restricted to the DMPK gene, as confirmed by genome-wide expression analysis. Altogether, our findings highlight DMPK-promoter silencing by CRISPRi as a promising therapeutic approach for DM1.

CRISPR-mediated epigenome editing enables gene expression regulation without changing the underlying DNA sequence, and thus has vast potential for basic research and gene therapy. Effective selection of a single guide RNA (sgRNA) with high on-target efficiency and specificity would facilitate the application of epigenome editing tools. Here we performed an extensive analysis of CRISPR-mediated epigenome editing tools on thousands of experimentally examined on-target sites and established EpiCas-DL, a deep learning framework to optimize sgRNA design for gene silencing or activation. EpiCas-DL achieves high accuracy in sgRNA activity prediction for targeted gene silencing or activation and outperforms other available in silico methods. In addition, EpiCas-DL also identifies both epigenetic and sequence features that affect sgRNA efficacy in gene silencing and activation, facilitating the application of epigenome editing for research and therapy. EpiCas-DL is available at http://www.sunlab.fun:3838/EpiCas-DL.

Abstract The spread of HIV is on the rise and has become a global issue, especially for underdeveloped and developing countries. This is due to the fact that HIV majorly occurs asymptomatically and is implausible for early diagnosis. Recent advances in research and science have enabled the investigation of a new potential treatment involving gene-based therapy, known as RNA interference (RNAi) that will direct gene silencing and further compensate for natural variants and viral mutants. Several types of small regulatory RNA are discussed in this present study, including microRNA (miRNA), small interfering RNA (siRNA), and short hairpin RNA (shRNA). This paper examines the mechanism of RNAi as a viable HIV therapy, using a minimum of four shRNAs to target both dispensable host components (CCR5) and viral genes (Gag, Env, Tat, Pol I, Pol II and Vif). Moreover, a multiplexed mechanism of shRNAs and miRNA is known to be effective in preventing viral escape due to mutation as the miRNA develops a general polycistronic platform for the expression of a large amount of shRNA-miRs. Several administration methods as well as the advantages of this RNAi treatment are also discussed in this study. The administration methods include (1) ex vivo delivery with the help of viral vectors, nanoparticles, and electroporation, (2) nonspecific in vivo delivery using non-viral carriers including liposomes, dendrimers and aptamers, as well as (3) targeted delivery that uses antibodies, modified nanoparticles, nucleic acid aptamers, and tissue-specific serotypes of AAV. Moreover, the advantages of this treatment are related to the effectiveness in silencing the HIV gene, which is more compatible compared to other gene therapy treatments, such as ZFN, TALEN, and CRISPR/Cas9.

Chorein is variably expressed in different cancer cells. In various non-malignant cell types, this protein regulates cytoskeleton microstructure and signaling. However, the role of chorein in regulating the actin cytoskeleton in tumor cells remains elusive. Here, we investigated chorein expression in various breast tumor cells and the involvement of this protein in microfilament organization. We used laser scanning microscopy to analyze microfilaments architecture, Triton X-100 fractionation to quantify G and Total actin levels, and quantitative RT-PCR to assess chorein gene transcription. We show that in line with previous observations, the less differentiated MCF7 breast cancer cells exhibited the highest relative expression of chorein compared to MDA-MB231 and T47D cell lines. Contrastingly, in less differentiated ZF rhabdomyosarcoma cells expressing high chorein levels, silencing of this protein was followed by clear depolymerization of actin microfilaments as apparent from IF morphological analysis. Quantification of G- and F-actin levels by Triton X-100 fractionation that revealed a significant increase in this ratio fully supported this finding. These results disclose that chorein is highly expressed in less differentiated tumor cells. In addition, silencing of this protein induces significant structural disorganization of the actin network, providing clear evidence that chorein regulates microfilament cytoskeleton architecture in tumor cells of higher malignant potential.

The human malaria parasite Plasmodium falciparum represses transcription of the gene encoding AP2-G, which is the master regulator of germ cell differentiation, via heterochromatin condensation following histone H3 lysine 9 trimethylation (H3K9me3). Although H3K9me3-marked heterochromatin is typically constitutive and its establishment depends on the RNA interference (RNAi) pathway in fission yeast centromeres, malaria parasites lack molecular members essential for RNAi. We developed a strategy to assess heterochromatin establishment on artificial chromosomes introduced into P. falciparum. We show that a particular DNA sequence in the AP2-G promoter is able to induce de novo H3K9me3 nucleosome deposition. In addition, we also found that the AP2-G promoter contains a distinct element required in maintenance of the repression memory. Thus, we speculate that malaria parasites have evolutionarily acquired a sequence-dependent establishment system of non-constitutive, i.e. facultative, H3K9me3-marked heterochromatin.

Abstract Background Cancer is a group of diseases involving abnormal cell growth with the potential to invade or spread to other parts of the body. It is unlikely that there will ever be a single cure for cancer, but the development of molecular biology and cell biology has brought new options for cancer treatment. Our research group found in the preliminary experiments that AAs exhibited significant anti-tumor activity. Studies also showed that AAs exhibited varying degrees of downregulation effects on the expression of the NDUFV2 gene in the MCF-7/ADR and SMMC-7721/ADR cell lines. However, there is no relevant report on the role of this gene expression during the growth process of drug-resistant tumor cells. To address possible objections, this paper aims to investigate the effect of NDUFV2 gene silencing on the proliferation of the MCF-7/ADR and SMMC-7721/ADR cell lines. Results The interfering plasmids pPLK/GFP+Puro-NDUFV2 shRNA-3 and shRNA-2 inhibited the NDUFV2 gene and protein expression most significantly in MCF-7/ADR and SMMC-7721/ADR cells, respectively. NDUFV2 gene silencing could effectively inhibit the proliferation of both cell lines. The inhibition rates for MCF-7/ADR were 67.31%, 73.02%, and 69.76% at 24 h, 48 h, and 72 h, while that for SMMC-7721/ADR were 68.89%, 71.97%, and 74.40% at 24 h, 48 h, and 72 h, respectively. The inhibition rate of SMMC-7721/ADR cell proliferation was positively correlated with time. Conclusions Interference with the NDUFV2 gene may significantly inhibit the proliferation of MCF-7/ADR and SMMC-7721/ADR cells. This study is the pioneer to investigate that the NDUFV2 gene has been associated with the activity of inhibiting tumor cell proliferation, suggesting that the NDUFV2 gene may become a potential target for the study of tumor genesis and the development of antineoplastic drugs.

Antisense gapmer oligonucleotides containing phosphoryl guanidine (PG) groups, e.g., 1,3-dimethylimidazolidin-2-imine, at three to five internucleotidic positions adjacent to the 3′ and 5′ ends were prepared via the Staudinger chemistry, which is compatible with conditions of standard automated solid-phase phosphoramidite synthesis for phosphodiester and, notably, phosphorothioate linkages, and allows one to design a variety of gapmeric structures with alternating linkages, and deoxyribose or 2′-O-methylribose backbone. PG modifications increased nuclease resistance in serum-containing medium for more than 21 days. Replacing two internucleotidic phosphates by PG groups in phosphorothioate-modified oligonucleotides did not decrease their cellular uptake in the absence of lipid carriers. Increasing the number of PG groups from two to seven per oligonucleotide reduced their ability to enter the cells in the carrier-free mode. Cationic liposomes provided similar delivery efficiency of both partially PG-modified and unmodified oligonucleotides. PG-gapmers were designed containing three to four PG groups at both wings and a central “window” of seven deoxynucleotides with either phosphodiester or phosphorothioate linkages targeted to MDR1 mRNA providing multiple drug resistance of tumor cells. Gapmers efficiently silenced MDR1 mRNA and restored the sensitivity of tumor cells to chemotherapeutics. Thus, PG-gapmers can be considered as novel, promising types of antisense oligonucleotides for targeting biologically relevant RNAs.

We present the influence of treating progeroid fibroblasts with two modified antisense oligonucleotides (ONs) on the nuclear envelope. Two modified ONs were designed to block ribosome binding during translation and spliceosome binding at the cryptic splice site. We analysed the changes in the nuclear morphology of progeria cell nuclei after repetitive transfection with modified ONs as a physical analysis tool for estimating alteration of the gene expression at the protein level. Confocal microscopy was used to image the nuclei, and the nuclear lobulations were quantified to study the changes in the morphology of the nuclear envelope upon treatment. PCR was used to identify the changes in the expression of lamin A and progerin after antisense treatment at the RNA level. We found a significant decrease in the number of nuclear envelope lobulations and a lower progerin expression in progeria cells after transfection with modified ONs.

Recognition that the most abundant class of genes present in the human genome are those producing long noncoding RNA (lncRNA) has hyped research on this category of transcripts. One such prototypical RNA, Xist, has particularly fueled interest. Initially characterized for its specific expression from the inactive X (Xi), recent studies have uncovered the molecular mechanisms underlying its essential role in the initiation of X-chromosome inactivation, from its exquisitely precise transcriptional regulation to the plethora of protein interactors forming the Xist ribonucleoprotein (RNP) that mediate its gene silencing activity. Here, we will discuss the recent advances that have broadened our knowledge of Xist functions, challenging classical models and revealing unsuspected, unconventional actions of its RNP., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Background Aims: Gene-silencing by small interfering RNA (siRNA) is an attractive therapy to regulate cancer death, tumor recurrence or metastasis. Because siRNAs are easily degraded, it is necessary to develop transport and delivery systems to achieve efficient tumor targeting. Self-nanoemulsifying systems (SNEDDS) have been successfully used for pDNA transport and delivery, so they may be useful for siRNA. The aim of this work is to introduce siRNA-RAD51 into a SNEDDS prepared with Phospholipon-90G, Labrafil-M1944-CS and Cremophor-RH40 and evaluate its efficacy in preventing homologous recombination of DNA double-strand breaks caused by photodynamic therapy (PDT) and ionizing radiation (IR)., Methods: The siRNA-RAD51 was loaded into SNEDDS using chitosan. Transfection capacity was estimated by comparison with Lipofectamine-2000., Results: SNEDDS(siRNA-RAD51) induced gene silencing effect on the therapies evaluated by cell viability and clonogenic assays using T47D breast cancer cells., Conclusions: SNEDDS(siRNA-RAD51) shown to be an effective siRNA-delivery system to decrease cellular resistance in PDT or IR., Competing Interests: Declaration of Competing Interest The authors have no commercial, proprietary or financial interest in the products or companies described in this article., (Copyright © 2024 International Society for Cell & Gene Therapy. Published by Elsevier Inc. All rights reserved.)

Angiogenesis significantly drives tumor progression, and the functions of vascular endothelial cells are influenced by various factors. Tumor cells are characterized by abnormal sialylation, and their dynamic balance depends on sialyltransferases and sialidases. NEU3 is a plasma membrane-associated sialidase, vital for the regulation of cell surface sialylation. Our study revealed that, NEU3 is the most abundantly expressed among the four sialidase subtypes in EA.hy926 cells. Silencing NEU3 expression resulted in cell apoptosis and reduced proliferation, highlighting its crucial function in the regulation of cell activity. Subsequent experiments using transwell and tube formation assays demonstrated that the inhibition of NEU3 expression suppressed cell migration and angiogenesis. RNA sequencing analysis further elucidated that altering NEU3 expression in EA.hy926 cells impacts the Wnt/β-Catenin signaling pathway and c-Myc levels, thereby modulating cellular survival and migration capacity and exerting a regulatory effect on angiogenesis. These findings suggest that targeting NEU3 in the vascular endothelium may represent a promising strategy for anti-angiogenic therapy in tumors., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Xue Ma reports financial support was provided by Science and Technology Department of Sichuan Province. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier Inc.)

Glioblastoma (GB) is a cancerous brain tumor that originates from glial cells and leads to thousands of deaths each year and a five-year survival of only 6.8 %. Treatments for GB include surgery, chemotherapy, radiation, and immunotherapy. GB is an incurable fatal disease, necessitating the development of innovative strategies to find a developing effective therapy. Genetic therapies may be crucial in treating GB by identifying the mutations and amplifications of multiple genes, which drive its proliferation and spread. Use of small interfering RNAs (siRNAs) provides a novel technology used to suppress the genes associated with disease, which forms a basis for targeted therapy in GB and its stem cell population, which are recognized for their ability to develop resistance to chemotherapy and tumorigenic capabilities. This review examines the use of siRNAs in GB, emphasizing their effectiveness in suppressing key oncogenes and signaling pathways associated with tumor development, invasion, stemness, and resistance to standard treatments. siRNA-based gene silencing is a promising approach for developing targeted therapeutics against GB and associated stem cell populations, potentially enhancing patient outcomes and survival rates in this devastating disease., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

The nuclear receptor-binding SET domain protein (NSD) gene family encodes histone methyltransferases that mono- and di-methylate lysine 36 on histone H3 (H3K36). Here, we examine the effects of NSD loss-of-function on transcription and heterochromatin formation in Drosophila to elucidate the role of NSD in chromatin structure regulation. Transcriptome analysis showed that NSD deletion activated more genes on chromosome 4, predominantly heterochromatic, than on other chromosomes. We further analyzed the position-effect variegation of fly eyes due to mini-white (mw + ) transgenes inserted at various chromosomal loci and found that NSD deletion promoted mw +  transgene expression on chromosome 4. Additionally, NSD deletion reduced the binding of heterochromatin markers HP1a and H3K9 to chromosome 4. These findings suggest that NSD deletion disrupts chromosome 4 heterochromatin structure by reducing HP1a binding, implying NSD's role as an epigenetic regulator of chromosome 4 silencing., Competing Interests: Declaration of competing interest The authors declare that they have no competing financial interests or personal relationships that may have influenced the work reported in this study., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Purpose: Colorectal cancer (CRC) is recognized as the third most common form of malignancy, with the liver frequently serving as the main site for metastasis. Anoikis resistance (AR) is critical in colorectal cancer liver metastases (CRLM). Fatty acid synthase (FASN), essential in lipid synthesis, mediates AR in many cancers. The present research examines the function of FASN in ERK1/2-mediated AR in CRLM and evaluates its therapeutic potential., Methods: We performed scratch and migration experiment to evaluate the migration capacity of the LoVo cells. Flow cytometry was employed to identify cell apoptosis. The levels of FASN, p-ERK1/2, and proteins related to apoptosis was analyzed by Western blot. The mRNA level of FASN was determined by q-PCR after FASN silencing. In addition, we used an intrasplenic liver metastasis model of nude to assess the effect of FASN on CRLM., Results: In vitro experiments showed that after FASN silencing, the cell apoptosis rate was increased, migration capability was notably decreased, the expression of p-ERK1/2, the proteins related to anti-apoptotic were significantly decreased, and the proteins related to apoptosis were significantly increased. In vivo experiments showed that AR significantly increased the number of liver metastatic foci, whereas FASN silencing significantly inhibited CRLM., Conclusion: These results suggest that FASN silencing suppressed AR through the ERK 1/2 pathway, which in turn suppressed CRLM., Competing Interests: Declaration of competing interest All authors declare that they have no competing interests., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Hypoxia inducible factors (HIFs) are heterodimeric proteins that belong to a small group of transcription factors, which mainly regulates transcription of genes under hypoxic conditions. Particularly, oxygen sensing subunit of HIF-1α is a predominant subtype that heterodimerizes with oxygen-independent HIF-1β subunit, to trigger the transcription of hypoxia responsive genes. Due to poor supply of blood and rapid division of cancerous cells, tumor microenvironment exhibits low oxygen condition and therefore increased levels of HIF-1α. One of the promising therapeutic strategies to cancer is modulation of HIF-1α signaling pathway. Small interfering RNA (siRNA) mediated downregulation of HIF-1α has been reported to prevent growth and progression of various types of cancer and holds great promise in the cancer treatment. In this study, computational approaches were used to design potential siRNAs targeting HIF-1α and investigate their interaction with the human argonaute-2 (hAgo2). Molecular dynamic simulation of HIF-1α siRNAs-hAgo2 complexes revealed key interactions required for the efficient binding of guide strand to hAgo2 protein. Two siRNAs (S2 and S5) exhibiting strong binding with hAgo2 were further considered. Subsequently, we transfected the MCF-7 cell line with both standard HIF-1α and our designed siRNAs (S2 and S5). Following transfection, translation changes in the MCF-7 cells were assessed through western blotting. S2 and S5 efficiently reduced the expression of HIF-1α in hypoxic conditions. The aim of the present study is to understand the siRNA-hAgo2 interaction. It is also focused on the desiging of effective siRNA against HIF-1α., Competing Interests: Declaration of competing interest There is no conflict of interest., (Copyright © 2024. Published by Elsevier B.V.)

Human stem cell-derived models have emerged as an important platform to study tissue differentiation and disease mechanisms. Those models could capitalize on biochemical and cell biological methodologies such as omics, autophagy, and organelle dynamics. However, epigenetic silencing in stem cells creates a barrier to apply genetically encoded tools. Here we investigate the molecular mechanisms underlying exogenously expressed gene silencing by employing multiple commonly used promoters in human induced pluripotent stem cells (iPSCs), glioblastoma cells (GBM), and embryonic kidney cells (HEK). We discover that all promoters tested are highly methylated on the CpG island regions with lower protein expression in iPSCs, as compared to non-iPSCs. Elongation factor 1 alpha short (EF1α short or EFS) promoter, which has fewer CpG island number compared to the other promoters, can drive relatively higher gene expression in iPSCs, despite CpG methylation. Adding a minimal A2 ubiquitous chromatin opening element (minimal A2 UCOE or miniUCOE) upstream of a promoter inhibits CpG methylation and enhances gene expression in iPSCs. Our results demonstrate stem cell type-specific epigenetic modification of transgenic promoter region and provide useful information for designing anti-silencing strategies to increase transgene expression in iPSCs., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial or personal interests that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Traditionally, antiviral drugs target viral enzymes and or structural proteins, identified through large drug screens or rational drug design. The screening, chemical optimisation, small animal toxicity studies and clinical trials mean time to market is long for a new compound, and in the event of a novel virus or pandemic, weeks, and months matter. Small interfering RNAs (siRNAs) as a gene silencing platform is an alluring alternative. SiRNAs are now approved for use in the clinic to treat a range of diseases, are cost effective, scalable, and can be easily programmed to target any viral target in a matter of days. Despite the large number of preclinical studies that clearly show siRNAs are highly effective antivirals this has not translated into clinical success with no products on the market. This review provides a comprehensive overview of both the clinical and preclinical work in this area and outlines the challenges the field faces going forward that need to be addressed in order to see siRNA antivirals become a clinical reality., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Crown Copyright © 2024. Published by Elsevier B.V. All rights reserved.)

The application of exogenous RNA for gene-silencing strategies has gained significant traction in agriculture, offering a highly efficient and eco-friendly alternative to conventional plant protection methods. This success has been driven by advances in biotechnology, from the design of long double-stranded RNA (dsRNA) and small interfering RNA (siRNA) molecules to the development of nanocarrier systems that address the challenge of RNA delivery into plant cells. In particular, polymer-based nanocarriers have emerged as a promising solution for enhancing the stability and delivery efficiency of RNA molecules. This review provides a comprehensive overview of the current state of research on the use of polymeric nanocarriers in RNA interference (RNAi) systems for crop protection. It examines key technological developments that have enabled the effective delivery of dsRNA/siRNA to target organisms, with a focus on the unique advantages polymers offer as carriers. Recent studies highlight significant progress in the preparation, characterization, and application of polymeric nanocarriers for RNA encapsulation and delivery. The review also explores the environmental and health challenges posed by these technologies, emphasizing the need for sustainable approaches in their development. Specifically, the production of nanocarriers must adhere to the principles of green chemistry, prioritizing chemical modification routes that reduce harmful residues, such as toxic solvents. Finally, this paper discusses both the current challenges and future prospects of using polymer-based nanocarriers in sustainable agriculture, offering critical insights into their potential to transform crop protection through RNAi technologies., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Co-delivery of chemical drugs and nucleic acids has attracted a great interest recently for treatment of inflammatory diseases. Dasatinib (DB), a tyrosine kinase inhibitor with anti-cancer effects, and Interferon Regulatory Factor 5 (IRF5) siRNA have shown anti-inflammatory effects. In the present study, a novel redox-responsive polymeric micelle was designed for co-delivery of DB and IRF5 siRNA-expressing plasmid (psiRF5) to enhance anti-inflammatory effects on macrophages. psiRF5 was condensed efficiently to redox-responsive micelles of DB-conjugated chitosan (CN) composed of disulfide bond, from different molecular weights of CN to form CN-SS-DB/psiRF5 micelles. The micelles with optimum N/P ratios had particle sizes of 287.8 and 245.4 nm and positive zeta potentials. The disulfide bond bearing micelles showed a redox-responsive drug release, protected the plasmid from being dissociated or degraded in exposure with heparin, serum and DNase I, and significantly enhanced the transfection efficiency and IRF5-gene silencing compared to naked psiRF5. The optimum micelles exhibited a dramatic reduction in IRF5 expression and revealed a notably higher anti-inflammatory effect than either DB or psiRF5, as indicated by more IL-10 and less IL-6 and TNF-α production by LPS-stimulated RAW264.7 macrophages incubated with the co-delivery system. The resultant nanocarriers might be promising for more effective treatment of inflammatory diseases., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

The endoplasmic reticulum (ER) is an important organelle in eukaryotic cells, responsible for a range of biological functions such as the secretion, modification and folding of proteins, maintaining Ca 2+ homeostasis and the synthesis of steroids/lipids, secreted proteins and membrane proteins. When cells are affected by internal or external factors, including abnormal energy metabolism, disrupted Ca 2+ balance, altered glycosylation, drug toxicity, and so on, the unfolded or misfolded proteins accumulate in the ER, leading to the unfolded protein response (UPR) and ER stress. The abnormal ER stress has been reported to be involved in various pathological processes. MicroRNAs (miRNAs) are non-coding RNAs with the length of approximately 19-25 nucleotides. They control the expression of multiple genes through posttranscriptional gene silencing in eukaryotes or some viruses. Increasing evidence indicates that miRNAs are involved in various cellular functions and biological processes, such as cell proliferation and differentiation, growth and development, and metabolic homeostasis. Hence, miRNAs participate in multiple pathological processes. Recently, many studies have shown that miRNAs play an important role by regulating ER stress in ischemia-reperfusion (I/R) injury, but the relevant mechanisms are not fully understood. In this review, we reviewed the current understanding of ER stress, as well as the biogenesis and function of miRNAs, and focused on the role of miRNAs regulation of ER stress in I/R injury, with the aim of providing new targets for the treatment of I/R injury., Competing Interests: Declaration of competing interest The authors declare that there are no conflicts of interest., (Copyright © 2024 Elsevier B.V. All rights reserved.)

The emergence of long noncoding RNAs (lncRNAs) was proved to be crucial to the aggravation of triple negative breast cancer (TNBC), a fatal female malignancy. LINC00668 was unveiled as an overexpressed lncRNA in TNBC previously. However, its exact function and whether it functioned in TNBC development needs to be ascertained. To explore this, qRT-PCR was used to detect its dysregulation in TNBC cells. Biological functions of LINC00668 were determined through loss-of-function experiments. Bioinformatics analysis was utilized to predict the downstream modulatory genes of LINC00668. Dual-luciferase reporter assay plus RNA immunoprecipitation analysis, quantitative PCR analysis, and rescue assays were employed for the exploration of potential action of mode in competitive endogenous RNA (ceRNA) network. It was revealed that LINC00668 was upregulated and its depletion resulted in impeded proliferation and migration of TNBC cells. Bioinformatics analysis and mechanical assays uncovered that LINC00668 sponged miR-518c-3p to facilitate WDR1 level in TNBC. Furthermore, rescue experiments demonstrated that LINC00668/miR-518c-3p pathway contributed to TNBC cell proliferation and migration in the form of WDR1 dependency. Overall our study might discover a vital clue for the cure of lncRNA-directed treatment for TNBC patients., Competing Interests: Declaration of competing interest The authors declare no competing interests., (Copyright © 2024 Elsevier B.V. All rights reserved.)

After fiber, cottonseed is the second most important by-product of cotton production. However, high concentrations of toxic free gossypol deposited in the glands of the cottonseed greatly hamper its effective usage as food or feed. Here, we developed a cotton line with edible cottonseed by specifically silencing the endogenous expression of GoPGF in the seeds, which led to a glandless phenotype with an ultra-low gossypol content in the seeds and nearly normal gossypol in other parts of the plants. This engineered cotton maintains normal resistance to insect pests, but the gossypol content in the seeds dropped by 98%, and thus, it can be consumed directly as food. The trait of a low gossypol content in the cottonseeds was stable and heritable, while the protein, oil content, and fiber yield or quality were nearly unchanged compared to the transgenic receptor W0. In addition, comparative transcriptome analysis showed that down-regulated genes in the ovules of the glandless cotton were enriched in terpenoid biosynthesis, indicating the underlying relationship between gland formation and gossypol biosynthesis. These results pave the way for the comprehensive utilization of cotton as a fiber, oil, and feed crop in the future.

Transcription factors (TFs) orchestrate the regulation of cellular gene expression and thereby determine cell functionality. In this study, we analyzed the distribution of TFs containing domains, which named as ZnFTFs, both in ascomycete and basidiomycete fungi. We found that ZnFTFs were widely distributed in these fungal species, but there was more expansion of the ZnFTF class in Ascomycota than Basidiomycota. We identified 40 ZnFTFs in Ustilaginoidea virens, and demonstrated the involvement of UvZnFTF1 in vegetative growth, conidiation, pigment biosynthesis and pathogenicity. RNA-Seq analysis suggested that UvZnFTF1 may regulate different nutrient metabolism pathways, the production of secondary metabolites, and the expression of pathogen-host interaction genes and secreted protein- encoding genes. Analysis of the distribution of different fungal TFs in U. virens further demonstrated that UvZnFTFs make up a large TF family and may play essential biological roles in U. virens.

Graphene oxide (GO) has emerged as a potential drug delivery vector. For siRNA delivery, GO should be modified to endow it with gene delivery ability and targeting effect. However, the cationic materials used previously usually had greater toxicity. In this study, GO was modified with a non-toxicity cationic material (chitosan) and a tumor specific monoclonal antibody (anti-EpCAM) for the delivery of survivin-siRNA (GCE/siRNA). And the vector (GCE) prepared was proved with excellent biosafety and tumor targeting effect. The GCE exhibited superior performance in loading siRNA, maintained stability in different solutions and showed excellent protection effect for survivin-siRNA in vitro. The gene silencing results in vitro showed that the mRNA level and protein level were down-regulated by 48.24% ± 2.50% and 44.12% ± 3.03%, respectively, which was equal with positive control (P > 0.05). It was also demonstrated that GCE/siRNA had a strong antitumor effect in vitro, which was attributed to the efficient antiproliferation, and migration and invasion inhibition effect of GCE/siRNA. The results in vivo indicated that GCE could accumulate siRNA in tumor tissues. The tumor inhibition rate of GCE/siRNA 54.74% ± 5.51% was significantly higher than control 4.87% ± 8.49%. Moreover, GCE/siRNA showed no toxicity for blood and main organs, suggesting that it is a biosafety carrier for gene delivery. Taken together, this study provides a novel design strategy for gene delivery system and siRNA formulation.

T cells that are gene-modified with tumor-specific T cell receptors are a promising treatment for metastatic melanoma patients. In a clinical trial, we treated seven metastatic melanoma patients with autologous T cells transduced to express a tyrosinase-reactive T cell receptor (TCR) (TIL 1383I) and a truncated CD34 molecule as a selection marker. We followed transgene expression in the TCR-transduced T cells after infusion and observed that both lentiviral- and retroviral-transduced T cells lost transgene expression over time, so that by 4 weeks post-transfer, few T cells expressed either lentiviral or retroviral transgenes. Transgene expression was reactivated by stimulation with anti-CD3/anti-CD28 beads and cytokines. TCR-transduced T cell lentiviral and retroviral transgene expression was also downregulated in vitro when T cells were cultured without cytokines. Transduced T cells cultured with interleukin (IL)-15 maintained transgene expression. Culturing gene-modified T cells in the presence of histone deacetylase (HDAC) inhibitors maintained transgene expression and functional TCR-transduced T cell responses to tumor. These results implicate epigenetic processes in the loss of transgene expression in lentiviral- and retroviral-transduced T cells.

Idiopathic pulmonary fibrosis (IPF) is a chronic disease in which the lungs become irreversibly scarred, leading to declining lung function. As currently available drugs do not cure IPF, there remains a great medical need for more effective treatments. Perhaps this need could be addressed by gene therapies, which offer powerful and versatile ways to attenuate a wide range of processes involved in fibrosis. Despite the potential benefits of gene therapy, no one has reviewed the current state of knowledge regarding its application for treating IPF. We therefore analyzed publications that reported the use of gene therapies to treat pulmonary fibrosis in animals, as clinical studies have not been published yet. In this review, we first provide an introduction on the pathophysiology of IPF and the most well-established gene therapy approaches. We then present a comprehensive evaluation of published animal studies, after which we provide recommendations for future research to address challenges with respect to the selection and use of animal models as well as the development of delivery vectors and dosage forms. Addressing these considerations will bring gene therapies one step closer to clinical testing and thus closer to patients.

Breast cancer is a challenging health issue in women and the second-leading cause of mortality around world. In this review, we addressed beneficial role of siRNA to control breast cancer. Various limitations of siRNA based therapeutic approaches, innate challenges siRNA delivery, in vitro and in vivo findings have been highlighted to focus recent advancement to control breast cancer. The current approaches and comparative assessment against conventional chemotherapeutics methods have been thoroughly investigated for the conclusive outcomes. Various nanocomposites and nanomedicines with or without surface functionalization have played a major role for convincing impact of siRNA delivery for anticipated therapeutic efficacy by stabilization and protecting bio-active siRNA in in-vitro and in-vivo condition. Various targeted genes for siRNA knockdown were reported to control breast cancer by tuning its biological performance in nanocarrier systems. Conclusively, siRNA loaded nanocarriers are promising option to control breast cancer compared to conventional (druggable) and mAbs (monoclonal antibodies) based therapy.

Despite promise in preventing peritendinous adhesion, electrospun membranes face many challenges related to their complex fabrication process, untargeted/uncontrolled drug delivery and consequently low therapeutical effect. Here, a micro-and nano-environment dual-modulated barrier membrane (MNBM) with on-demand gene delivery capability is presented. Our MNBM is developed by first preparing extracellular signal-regulated kinase-2 (ERK2) siRNA-loaded gelatin methacryloyl (GelMA) nanogels via facile nano-emulsification technique, then incorporating these nanogels into poly-L-lactic acid (PLLA) fibers via simple blending electrospinning. The GelMA nanogels offer a nano-niche for ERK2-siRNA encapsulation and allow for a nano-environment controlled siRNA release by readily tuning the GelMA concentrations during nano-emulsification, while the resultant MNBM can mediate a micro-environment controlled siRNA delivery in response to the matrix metalloproteinase-2 (MMP-2) enriched micro-environment at the tendon repair site. Such MNBM can not only biologically orchestrate fibroblast behaviors by silencing the target gene expression, but also physically shield the tendon from extrinsic cell/tissue invasion. This study provides a proof-of-concept of anti-adhesion barrier membrane as an intelligent gene delivery system to offer a spatiotemporal and biophysical dual control over tendon recovery according to disease state and ensure long-term therapeutic efficacy. We envision such MNBM represents a promising therapeutic platform with great efficacy to achieve adhesion-free tendon repair.

Sugar beet is vulnerable to rhizomania as the most destructive viral disease. Two selected events of transgenic sugar beet carrying cassettes inducing RNA silencing mechanism, 219-T3:S3-13.2 (S3) and 6018-T3:S6-44 (S6), were shown to inhibit propagation of Beet Necrotic Yellow Vein Virus, the causative agent. As a method for signifying the substantial equivalence, we analyzed the levels of some metabolites through LC-MS in order to demonstrate possible unintended changes in the leaves of the transgenic events. There was no significant difference in the concentrations of examined key metabolites but cis-aconitate and fructose-1,6-bisphosphatase which were decreased in S3. Also, ATP was reduced in both genetically modified sugar beets. Among free amino acids, only glycine level in S6 was increased compared to the wild plant, while the production levels of 5 and 12 ones were increased in S3 compared to S6 event and the wild type plants, respectively.

Oncolytic adenoviruses are being developed as new anti-cancer agents. Their efficacy can be improved by incorporating RNA interference (RNAi) molecules. RNAi molecules can be expressed in various precursor formats. The aim of this study was to determine the most effective format. To this end, we constructed three Δ24-type oncolytic adenoviruses, with human microRNA-1 (miR-1) expression cassettes in short hairpin RNA (shRNA), precursor microRNA (pre-miRNA), and primary miRNA (pri-miRNA) format, respectively. The viruses were compared for virus replication, mature miR-1 expression, and target gene silencing in cancer cells. Incorporation of the cassettes had only minor effects on virus replication. Mature miR-1 expression from the pri-miRNA format reached on average 100-fold higher levels than from the other two formats. This expression remained stable upon long-term virus propagation. Infection with the pri-miR-1-expressing virus silenced the validated miR-1 targets FOXP1 and MET. Drosha knockout almost completely abrogated mature miR-1 expression, confirming that processing of adenovirus-encoded pri-miR-1 was dependent on the host cell miRNA machinery. Using simple in vitro recombination cloning, a similar virus expressing miR-26b was made and shown to silence the validated miR-26b target PTGS2. We thus provide a platform for construction of oncolytic adenoviruses with high expression of RNAi molecules of choice.

Diabetic wounds are difficult to treat due to multiple causes, including reduced blood flow and bacterial infections. Reduced blood flow is associated with overexpression of prostaglandin transporter (PGT) gene, induced by hyperglycaemia which causing poor vascularization and healing of the wound. Recently, gold nanoparticles (AuNPs) have been biosynthesized using cold and hot sclerotium of Lignosus rhinocerotis extracts (CLRE and HLRE, respectively) and capped with chitosan (CS) to produce biocompatible antibacterial nanocomposites. The AuNPs have shown to produce biostatic effects against selected gram positive and negative bacteria. Therefore, in this study, a dual therapy for diabetic wound consisting Dicer subtract small interfering RNA (DsiRNA) and AuNPs was developed to improve vascularization by inhibiting PGT gene expression and preventing bacterial infection, respectively. The nanocomposites were incorporated into thermoresponsive gel, made of pluronic and polyethylene glycol. The particle size of AuNPs synthesized using CLRE (AuNPs-CLRE) and HLRE (AuNPs-HLRE) was 202 ± 49 and 190 ± 31 nm, respectively with positive surface charge (+30 to + 45 mV). The thermoresponsive gels containing DsiRNA-AuNPs gelled at 32 ± 1 °C and released the active agents in sufficient amount with good texture and rheological profiles for topical application. DsiRNA-AuNPs and those incorporated into thermoresponsive pluronic gels demonstrated high cell viability, proliferation and cell migration rate via in vitro cultured cells of human dermal fibroblasts, indicating their non-cytotoxicity and wound healing properties. Taken together, the thermoresponsive gels are expected to be useful as a potential dressing that promotes healing of diabetic wounds.

The soybean cyst nematode (SCN; Heterodera glycines Ichinohe) is a significant agricultural pest that causes extensive damage to soybean production worldwide. Second-stage juveniles (J2s) of the SCN migrate through the soil and infest the roots of host plants in response to certain chemical substances secreted from the host roots. Therefore, controlling SCN chemotaxis could be an effective strategy for its management. In the present study, we identified the Hg-gpa-3d gene, which encodes the G protein alpha subunit, as a key regulator of SCN chemotaxis. Gene silencing of Hg-gpa-3d reduced the attraction of SCN J2s to host roots, as well as to nitrate ions, a chemoattractant recognized through a mechanism different from that of host recognition. However, silencing of Hg-gpa-3d did not affect avoidance behavior towards unpleasant temperatures or stylet protrusion. These results suggest that Hg-gpa-3d is a crucial gene in the regulation of SCN chemotaxis and provide new insights into the chemotactic mechanisms of the SCN., Competing Interests: Declaration of competing interest The authors have no conflict of interest to declare., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

RNA interference (RNAi) technology has emerged as a pivotal strategy in sustainable pest management, offering a targeted approach that significantly mitigates the environmental and health risks associated with traditional insecticides. Originally implemented through genetically modified organisms (GMOs) to produce specific RNAi constructs, the technology has evolved in response to public and regulatory concerns over GMOs. This evolution has spurred the development of non-transgenic RNAi applications such as spray-induced gene silencing (SIGS), which employs double-stranded RNA (dsRNA) to silence pest genes directly without altering the plant's genetic makeup. Despite its advantages in specificity and reduced ecological footprint, SIGS faces significant obstacles, particularly the instability of dsRNA in field conditions, which limits its practical efficacy. To overcome these limitations, innovative delivery mechanisms have been developed. These include nanotechnology-based systems, minicells, and nanovesicles, which are designed to protect dsRNA from degradation and enhance its delivery to target organisms. While these advancements have improved the stability and application efficiency of dsRNA, comprehensive assessments of their environmental safety and the potential for increased exposure risks to non-target organisms remain incomplete. This comprehensive review aims to elucidate the environmental fate of dsRNA and evaluate the potential risks associated with its widespread application on non-target organisms, encompassing soil microorganisms, beneficial insects, host plants, and mammals. The objective is to establish a more refined framework for RNAi risk assessment within environmental and ecotoxicological contexts, thereby fostering the development of safer, non-transgenic RNAi-based pest control strategies., Competing Interests: Declaration of competing interest The authors declare that no conflict of interest exists regarding the completion and publication of this article., (Copyright © 2024 Elsevier B.V. All rights reserved.)

The Ribosomal protein L18 (RPL18) protein gene family plays an important role in plant growth, development and stress response. Although the RPL18 genes have been identified in several plant species, the RPL18 gene family in wheat (Triticum aestivum) is still unexplored. This study found 8 TaRPL18 genes, each of which has a significantly different gene sequence length and is evenly distributed on the chromosome; Additionally, these proteins have similar physicochemical characteristics as well as secondary and tertiary structures. 17 RPL18 genes in 4 species (wheat, Arabidopsis, rice, and maize) were classified into 5 groups, and the TaRPL18 genes within the same group showed similar structures and conserved motifs. Analysis of the cis-acting elements in the TaRPL18 genes promoter regions revealed the presence of developmental and stress-responsive elements in the majority of the genes. Through yeast two-hybrid (Y2H) experiments, it was confirmed that the powdery mildew resistance protein TaPm46 physically interacts with the Class IV TaRPL18-1. Functional analysis indicated that TaRPL18-1-silenced wheat plants show reduced resistance to powdery mildew compared to the wild type (WT), with decreased expression levels of PAL and PPO genes, and increased expression levels of the PR gene. The findings of this study provide a basis for clarifying the function of the TaRPL18 genes and will be useful for the selection of disease-resistant varieties of wheat., Competing Interests: Declaration of competing interest The authors declare that they have no conflict of interest., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Although RNAi-based pest management holds great potential as an alternative to traditional chemical control, its efficiency is restricted by dsRNA instability and limited cellular uptake. Using nanomaterials to facilitate dsRNA delivery has shown promise in solving these challenges. In this study, we firstly used RNAi to investigate the role of the juvenile hormone and ecdysteroid signaling pathways genes in reproduction of Spodoptera frugiperda, the fall armyworm. Females in knocked-down treatments of any of the Met, EcR, and USP genes had greatly reduced fertility with the most pronounced inhibitory effects on oviposition observed following EcR knockdown, and thus the dsEcR could be a candidate target for RNAi-based oviposition inhibitory agency. Then a combinatorial spray-induced and nanocarrier-delivered gene silencing (SI-NDGS) approach that targeted EcR was conducted. At 72 h post-spay, the transcript levels of EcR and the oviposition were successfully reduced and inhibited. These findings support the groundwork for further developing novel RNAi-based pest management strategies for S. frugiperda., Competing Interests: Declaration of competing interest The authors have declared no conflict of interest., (Copyright © 2024. Published by Elsevier B.V.)

The pine wood nematode Bursaphelenchus xylophilus is a highly invasive species responsible for the widespread pine wilt disease. Double-stranded RNA (dsRNA) biopesticides represent a novel strategy for controlling plant-parasitic nematodes. The B. xylophilus arginine kinase (BxAK) features a conserved ATP-binding domain and exhibits nematode-specific divergence in the phylogenetic tree. Notably, whole-mount in situ hybridization signals are evident in the nematode head and middle sections, particularly in the juvenile stage before sex differentiation. In this study, we developed a novel dsRNA-like small interfering RNA (siRNA) assembly that specifically targets BxAK and presents highly nematicidal effects. The RNA interference (RNAi) efficiency achieved a 95.9 % reduction in second-stage juveniles. In bioassays, the median lethal concentrations of this siRNA assembly against B. xylophilus were 168.5 ng/μl for juveniles and 603.8 ng/μl for adults within 48 h. Moreover, transcriptomic results revealed significantly downregulated expression levels of genes related to metabolism and development, suggesting that the mode of action of BxAK silencing is related to disruptions in energy homeostasis and juvenile development. In conclusion, BxAK is a molecular target for controlling B. xylophilus, and our siRNA assembly significantly enhances RNAi efficiency and lowers the lethal concentration required, making it a promising candidate for future biocontrol applications., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

The prognostic value of Runt-related transcription factor 2 (Runx2) and its involvement in cell growth and motility have been reported in patients diagnosed with renal cell carcinoma (RCC). Since Runx2 may have the potential to be a target for the purpose of antitumor intervention, there is an urgent need to gain insight into its oncogenic properties. Using human 786-O, Caki-1 and ACHN RCC cells as models, the silencing of cellular Runx2 expression brought about a reduction in cyclin D1 and β-catenin expression, cell growth and migration without any significant cell death. Runx2-silenced cells turned into apoptosis vulnerable in the presence of ABT-737, a BH3 mimetic Bcl-2 inhibitor. Data from biochemical and molecular studies have revealed a positive correlation between Runx2 expression and Akt phosphorylation, Mcl-1 expression, and fibronectin expression. Results of genetic silencing studies have indicated the potential involvement of Mcl-1 and fibronectin in the decision of RCC cell ABT-737 resistance and sensitivity. The regulatory roles of the PI3K/Akt axis in the expression of Mcl-1 and fibronectin were suggested by means of the results taken from experiments involving pharmacological study of the PI3K/Akt. Since overexpression and prognostic roles of Runx2, activated Akt, Mcl-1, fibronectin, cyclin D1, and β-catenin have been revealed in RCC, it is important to explore the precise mechanisms underlying Runx2 oncogenic effects. Although the linking details between Runx2 and PI3K/Akt have yet to be identified, our findings suggest that Mcl-1 and fibronectin are downstream effectors of Runx2 via a regulatory axis of the PI3K/Akt and their promotion of cell growth, migration, and ABT-737 resistance in RCC cells., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Phytohormones play crucial roles in regulation of plant growth and tolerance to abiotic stresses. The 2-oxoglutarate-dependent dioxygenase (2OGD) superfamily responds to hormone biosynthesis and metabolism in plants. However, the Nt2OGD family in tobacco has not been fully explored. In this study, we identify 126 members of the Nt2OGD family, and 60 of them are involved in hormone biosynthesis and metabolism process (Nt2OGD-Hs), including 1-aminocyclopropane-1-carboxylic acid oxidases (ACO), dioxygenases for auxin oxidation (DAO), gibberellin (GA) 20-oxidases and 3-oxidases (GA20ox and GA3ox), carbon-19 and carbon-20 GA 2-oxidases (C19-GA2ox and C20-GA2ox), lateral branching oxidoreductases (LBO), jasmonate-induced oxygenases (JOX), downy mildew resistant 6, and DMR6-like oxygenases (DMR6/DLO). Gene duplication analysis suggests the segmental duplication and whole genome duplication (WGD) might be a potential mechanism for the expansion of this family. Expression analysis reveals that most of Nt2OGD-Hs show tissue-specific expression patterns, and some of them respond to environmental conditions. Of Nt2OGD-Hs, the expression of NtJOX3 and NtJOX5, which are involved in JA metabolism, exhibits remarkable changes during drought treatments. Silencing of NtJOX3 or NtJOX5 increases tobacco tolerance to drought stress. Furthermore, knocking out OsJOX3 and OsJOX4, respectively in rice, result in high tolerance to drought. Taken together, our work comprehensively identifies the Nt2OGD family in tobacco and provides new insights into roles of the JA pathway in drought tolerance in plants., Competing Interests: Declaration of competing interest All authors have declared that they have no conflicts of interest to this work. We declare that we have no commercial and personal relationships with other people or organizations that can inappropriately influence our work., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Background and Aims: Atherosclerosis (AS), an arterial vasculature disease, is characterized by abnormal lipid accumulation and inflammatory response. ADP ribosylation factor like GTPase 11 (ARL11) is linked to multifarious processes in cells. This study aims to clarify the underlying mechanism of ARL11 in AS., Methods: ApoE -/- mice fed with high-fat diet were used as mouse model of AS. Gene expression in AS was determined by mRNA-sequencing. ARL11 expression was detected by real-time PCR, Western blot and immunofluorescence. M1 polarization of macrophages was indicated by TNF-α and IL-6 levels as detected with ELISA, and iNOS expression determined by real-time PCR and Western blot. The role of ARL11 during AS was explored through loss-of-function analysis., Results: There were 1301 upregulated and 1110 downregulated genes during AS. These differentially expressed genes (DEGs) were mainly enriched in pathways and terms which are involved in inflammation. Moreover, Arl11 was highly expressed in AS models. Downregulation of Arl11 decreased lipid deposition and atherosclerotic plaques in the aortas of AS mice, and declined inflammatory cytokines and M1 polarization of macrophages induced by IFN-γ. Furthermore, ARL11 interacted with JAK2 and p-JAK2 and modulated their degradation, thus inhibiting the activation of JAK2/STAT1 pathway., Conclusions: ARL11 promoted the development of AS via interacting with JAK2 and activating JAK2/STAT1 pathway. Thus, silencing ARL11 may prevent the process of AS and be a novel way to treat AS., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Epiregulin (EREG) is a member of the epidermal growth factor (EGF) family. An increasing body of evidence has demonstrated the pivotal role of EREG in the pathogenesis and progression of various malignancies. However, the clinical significance and biological role of EREG in pancreatic ductal adenocarcinoma (PDAC) have yet to be fully elucidated. We found that EREG is highly expressed in PDAC tissues compared with paracancerous tissues through public databases and clinical samples. High EREG expression predicted worse overall survival (OS) and recurrence-free survival (RFS) in patients with PDAC. Multivariate analysis revealed that EREG can serve as an independent prognostic indicator. In addition, EREG silencing inhibited PDAC cell proliferation, migration, progression, altered cell cycle, facilitated apoptosis in vitro and suppressed tumor growth in vivo. Conversely, EREG overexpression facilitated the proliferation, migration, and invasion in PaTu-8988 t cell. Through transcriptome sequencing and experimental verification, we found EREG mediates PDAC tumorigenesis through ERK/p38 MAPK signaling pathway. Moreover, we found EREG expression is closely related to PD-L1 expression in PDAC tissues and cells. Therefore, EREG is expected to be a prospective prognostic and therapeutic marker for PDAC., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Lesion mimic mutants (LMMs) refer to the spontaneous formation of disease-like spots on leaves without any obvious pathogen infection. The LMM genes can regulate plant immunity, thus promoting the defense of crops against pathogens. However, there is a lack of systematic understanding of the regulatory mechanism of LMMs in wheat. This study identified a wheat LMM TaCAT2, a homolog of the Arabidopsis CAT2. The prediction of the cis-regulatory element revealed that TaCAT2 was involved in the response of plants to various hormones and stresses. RT-qPCR analysis indicated that TaCAT2 was significantly up-regulated by NaCl, drought, and Fusarium graminearum infection. Fluorescence microscopy showed that the TaCAT2 was localized to the peroxisome. Overexpression of TaCAT2 enhanced plant resistance to Phytophthora infestation and F. graminearum by constitutionally activating SA and JA pathways. VIGS of TaCAT2 enhanced the sensitivity of wheat to F. graminearum. Further, TaCAT2 enhanced stress resistance by scavenging the excessive ROS and increasing the activities of antioxidative enzymes. This study lays the basis for the functional identification of TaCAT2 and its applicability in the disease resistance of wheat., Competing Interests: Declaration of competing interest We declare that we have no financial, personal and competing relationships with other people or organizations., (Copyright © 2024. Published by Elsevier B.V.)

The RNA interference (RNAi)-based gene silencing technique has enormous potential as a non-chemical and eco-friendly alternative to hazardous pesticides. This study reports a spray-induced gene silencing (SIGS) approach for managing Thrips palmi by lowering survival and offspring development. Vacuolar ATP synthases (V-ATPases) are responsible for survival, egg-laying, and viability of eggs in insects. In the current study, T. palmi V-ATPase-B was targeted to suppress the pest population by spray-on application of double-stranded RNA (dsRNA). Silencing of V-ATPase-B was first validated by oral administration of dsV-ATPase-B. The expression of V-ATPase-B was reduced by 5.40-fold post-dsRNA feeding leading to increased mortality (57.03 %) and reduced reproductive fitness (67.73 %). Spray-on application of naked dsV-ATPase-B at concentrations of 3.0 μg/mL and 5.0 μg/mL effectively suppressed the population by 30.00 % and 43.33 %, respectively. The expression of the target gene was downregulated by up to 4.24-fold. Two consecutive sprays at a concentration of 5.0 μg/mL provided substantial protection against the fresh release of T. palmi for up to 10 days. The spray-on application of dsV-ATPase-B would be an eco-friendly alternative for managing T. palmi populations thereby reducing crop damage and limiting the spread of orthotospoviruses., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)