Search Results (151)

Lactic acid bacteria (LAB) are central to food, feed, and health biotechnology, yet their genomes have long resisted rapid, precise manipulation. This review charts the evolution of LAB genome-editing strategies from labor-intensive RecA-dependent double-crossovers to state-of-the-art CRISPR and CRISPR-associated transposase systems. Native homologous recombination, transposon mutagenesis, and phage-derived recombineering opened the door to targeted gene disruption, but low efficiencies and marker footprints limited throughput. Recent phage RecT/RecE-mediated recombineering and CRISPR/Cas counter-selection now enable scar-less point edits, seamless deletions, and multi-kilobase insertions at efficiencies approaching model organisms. Endogenous Cas9 systems, dCas-based CRISPR interference, and CRISPR-guided transposases further extend the toolbox, allowing multiplex knockouts, precise single-base mutations, conditional knockdowns, and payloads up to 10 kb. The remaining hurdles include strain-specific barriers, reliance on selection markers for large edits, and the limited host-range of recombinases. Nevertheless, convergence of phage enzymes, CRISPR counter-selection and high-throughput oligo recombineering is rapidly transforming LAB into versatile chassis for cell-factory and therapeutic applications. Full article

The histone H2A variant H2AZ plays pivotal roles in shaping chromatin architecture and regulating gene expression. We recently identified H2AZ.2 in histone H2A lysine 119 ubiquitination (H2AK119ub)-enriched nucleosomes, but it is not known whether its highly related isoform H2AZ.1 also regulates this modification. In this study, we employed isoform-specific epitope-tagged knock-in mouse embryonic stem cell (ESC) lines to dissect the roles of each isoform in Polycomb Repressive Complex 1 (PRC1)-mediated H2AK119ub. Our results show that H2AZ.1 and H2AZ.2 share highly overlapping genomic binding profiles, both co-localizing extensively with H2AK119ub-enriched loci. The knockdown of either isoform led to reduced H2AK119ub levels; however, the two isoforms appear to function through distinct mechanisms. H2AZ.1 facilitates the recruitment of Ring1B, the catalytic subunit of PRC1, thereby promoting the deposition of H2AK119ub. In contrast, H2AZ.2 does not significantly affect Ring1B recruitment but instead functions as a structural component that stabilizes H2AK119ub-modified nucleosomes. In vitro ubiquitination assays indicate that H2AZ.1-containing nucleosomes serve as more efficient substrates for PRC1-mediated ubiquitination compared to those containing H2AZ.2. Thus, these findings define the distinct mechanisms of the two H2AZ variants in regulated PRC1-mediated H2AK119 ubiquitination and highlight a functional division of labor in epigenetic regulation. Full article

Cellular senescence is a state of the durable cell cycle arrest of dysfunctional cells, which has been associated with the promotion of tumor cell reprogramming into a stem cell state. We previously reported that the mixed lineage kinase (MLK) inhibitor CEP-1347 promotes the differentiation of glioma stem cells (GSCs)—key contributors to glioblastoma recurrence and therapy resistance—into non-stem tumor cells. However, we also noted that CEP-1347–treated GSCs exhibited a morphological change suggestive of senescence. Therefore, we herein investigated whether CEP-1347 induces senescence in GSCs and, consequently, if senescent GSCs may be eliminated using senolytics. Cell death induced by CEP-1347 in combination with senolytic agents or with the knockdown of anti-apoptotic BCL2 family genes, as well as the effects of CEP-1347 on the expression of senescence markers and anti-apoptotic Bcl-2 family proteins, were examined. The results obtained showed that CEP-1347 induced senescence in GSCs accompanied by the increased expression of Bcl-xL. Among the panel of senolytic agents tested, navitoclax, a BH3 mimetic, efficiently induced cell death in GSCs when combined with CEP-1347 at concentrations clinically achievable in the brain. The knockdown of Bcl-xL resulted in more pronounced GSC death in combination with CEP-1347 than that of Bcl-2. These results suggest that combining CEP-1347 with the targeting of Bcl-xL, the expression of which increases with CEP-1347-induced senescence, is a rational approach to ensure the elimination of GSCs, thereby improving the outcomes of glioblastoma treatment. Full article

Background: Inflammatory breast cancer (IBC) is a rare and aggressive subtype of breast cancer that accounts for 1–5% of BC patients and regularly affects women under 40 years of age. Approximately 50% of IBC cases are HER2+ and can be treated with the monoclonal antibody-based therapy Herceptin (trastuzumab). However, resistance to Herceptin develops within a year, and effective second-line targeted therapies are currently unavailable for IBC patients. Lipocalin-2 (LCN2) is a promising therapeutic target for IBC due to its role in promoting tumor invasiveness, angiogenesis, and the inflammatory tumor microenvironment characteristic of IBC. Objective: We developed Herceptin-conjugated liposomes loaded with LCN2-targeted small-interference RNA (siRNA) for HER2+ IBCs. Methods: We synthesized DSPE-PEG(2000)-maleimide-Herceptin in a three-step process and formulated the liposomes together with DOPC, PEG(2000)-PE, cholesterol, and siRNA. Results: Dynamic light scattering confirmed the liposome size distribution, which was 66.7 nm for the Herceptin-conjugated liposome versus 43.0 nm in a non-functionalized liposome. Here, we report efficient internalization of this formulation into HER2+ IBC cells, reducing LCN2 levels by 30% and disrupting tumor emboli formation. RNA sequencing revealed 139 genes that were differentially expressed upon LCN2 knockdown, with 25 canonical pathways identified through Ingenuity Pathway Analysis. Conclusions: These findings suggest that LCN2-targeted siRNA within Herceptin-targeted liposomes represents a promising therapeutic strategy for IBC. Full article

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

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

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

Alternative oxidase (AOX) is a terminal oxidase in the mitochondrial electron transport chain that does not contribute to the generation of ATP. It plays a critical role in maintaining the balance between reactive oxygen species (ROS) production and intracellular redox homeostasis within the mitochondria. In the study, overexpression and knockdown approaches were employed to investigate the function of AOX. AOX-silenced strains (AOXi3 and AOXi25) and AOX-overexpressed strains (OE-AOX2 and OE-AOX21) were constructed. The ROS level and transcription level of the antioxidant-system-related genes, including phosphoglucomutase (pgm) and phosphomannose isomerase (pmi), were differentially upregulated in silenced strains, whereas the opposite effect was observed in the AOX-overexpressed strains. Compared with the wild type (WT), the polysaccharide production of AOXi25 was significantly increased by approximately 38%, while OE-AOX21 was significantly decreased by 80%. Six extracellular polysaccharides (EPSs) were extracted and purified from the WT, OE-AOX21, and AOXi25 strains. These EPSs, consisting of both neutral and acidic polysaccharides, were composed of five different monosaccharides in varying proportions. The average relative molecular masses were 1.68 × 10³, 2.66 × 10³, 1.67 × 10³, 2.42 × 10³, 1.12 × 10³, and 2.35 × 10³ kDa, respectively. Antioxidant assays demonstrated that all EPSs exhibited strong free radical scavenging activity with the acidic polysaccharide from AOXi25 showing the highest efficiency in ABTS⁺ scavenging. These findings highlight the significant role of AOX-derived ROS in regulating polysaccharide synthesis and accumulation in Ganoderma lucidum. Full article

One of the major causes of senescence is oxidative stress caused by ROS, which is mainly generated from dysfunctional mitochondria. Strategies to limit mitochondrial ROS production are considered important for reversing senescence, but effective approaches to reduce them have not yet been developed. In this study, we screened the secondary metabolites that plants produce under oxidative stress and discovered sauchinone as a potential candidate. Sauchinone induced mitochondrial function recovery, enabling efficient electron transport within the electron transport chain (ETC). This led to a decrease in ROS production, a byproduct of inefficient electron transport. The reduction in ROS by sauchinone rejuvenated senescence-associated phenotypes. To understand the underlying mechanism by which sauchinone rejuvenates senescence, we carried out RNA sequencing and found VAMP8 as a key gene. VAMP8 was downregulated by sauchinone. Knockdown of VAMP8 decreased mitochondrial ROS levels and subsequently rejuvenated mitochondrial function, which was similar to the effect of sauchinone. Taken together, these studies revealed a novel mechanism by which sauchinone reduces mitochondrial ROS production by regulating mitochondrial function and VAMP8 expression. Our results open a new avenue for aging research to control senescence by regulating mitochondrial ROS production. Full article

Background/Objectives: Vacuolar (H⁺)-ATPases (V-ATPases) are crucial in several significant biological processes, including intracellular transport, endocytosis, autophagy and protein degradation. However, their role in the growth and development of insects remains largely unknown. This study aimed to explore the molecular and functional properties of V-ATPases in Locusta migratoria. Methods: LmV-ATPase genes were identified based on the locust transcriptome database and bioinformatics analysis. Quantitative reverse-transcription polymerase chain reaction was used to assess the relative expression of LmV-ATPases in different tissues and developmental stages. RNA interference combined with hematoxylin–eosin staining and transmission electron microscopy was used to explore the functions of LmV-ATPases. Results: Ten V-ATPase genes were identified in L. migratoria and were named LmV-ATPase A, B, C, D, E, F, G, c″, d and e, respectively. These genes were highly expressed in the head, integument, gastric caecum, midgut, hindgut, fat body, trachea and ovary. The transcripts of LmV-ATPases were expressed in the developmental stages examined (from the 3rd to 5th instar nymphs). The injection of double-stranded RNA (dsRNA) against each LmV-ATPase induced high silencing efficiency in the 3rd instar nymphs. Knockdown of LmV-ATPases resulted in lethal phenotypes, with visible defects of the wing and cuticle. We further demonstrated that the deformation was caused by the defects of epidermal cells and fewer new cuticles. Conclusions: These findings suggest that LmV-ATPases are required for the wing and cuticle development of L. migratoria, which could be potential targets for the control of locusts. Full article

Madin–Darby Canine Kidney (MDCK) cells are a key cell line for influenza vaccine production, due to their high viral yield and low mutation resistance. In our laboratory, we established a tertiary cell bank (called M60) using a standard MDCK cell line imported from American Type Culture Collection (ATCC) in the USA. Due to their controversial tumourigenicity, we domesticated non-tumourigenic MDCK cells (named CL23) for influenza vaccine production via monoclonal screening in the early stage of this study, and the screened CL23 cells were characterised based on their low proliferative capacity, which had certain limitations in terms of expanding their production during cell resuscitation. It was thus our objective to enhance the proliferation efficiency of MDCK cells for influenza vaccine production following cell resuscitation, with a view to improving the production of non-tumourigenic MDCK cells for vaccines and enhancing the production of influenza virus lysate vaccines from MDCK cells through genetic intervention. We concentrated on the protein thrombosponin-1 (THBS1), which was markedly differentiated in the proteomics data of the two MDCK cells. By integrating this finding with related studies, we were able to ascertain that THBS1 exerts a significant influence on the level of cell proliferation and apoptosis. Consequently, our objective was to investigate the impact of THBS1 expression on MDCK cell apoptosis by verifying the differences in THBS1 expression between the two MDCK cells and by interfering with THBS1 expression in the MDCK cells. We found that the knockdown of THBS1 significantly increased the proliferation and apoptosis of CL23 cells without causing significant changes in cell migration and invasion, and its overexpression significantly decreased the proliferation of M60 cells and increased cell migration, invasion, and apoptosis. In addition, the TGF-β/Smad pathway target genes transforming growth factor-β1 (TGF-β1), mothers against decapentaplegic homolog 2 (Smad2), and mothers against decapentaplegic homolog 3 (Smad3), were significantly down-regulated in CL23 cells after THBS1 knockdown and up-regulated in M60 cells after overexpression, with consistent expression identified at both the mRNA and protein levels. The treatment of cells with TGF-β activators and inhibitors further demonstrated that THBS1 regulated MDCK cell proliferation and apoptosis through the TGF-β/Smad signalling pathway. Finally, we found that THBS1 also regulated H1N1 influenza virus replication. These findings enable a comprehensive understanding of the regulatory mechanisms of THBS1 regarding MDCK cell proliferation and apoptosis functions and the effects of influenza virus replication. Full article

Background/Objectives: Bringing small interfering RNA (siRNA) into the cell cytosol to achieve specific gene silencing is an attractive but also very challenging option for improved therapies. The first step for successful siRNA delivery is the complexation with a permanent cationic or ionizable compound. This protects the negatively charged siRNA and enables transfection through the cell membrane. The current study explores the performance of the innovative, ionizable lipid 2-Tetradecylhexadecanoic acid-(2-bis{[2-(2,6-diamino-1-oxohexyl)amino]ethyl}aminoethyl)-amide (T14diLys), in combination with 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), for siRNA delivery and the impact of the production method (sonication vs. extrusion) on the particle properties. Methods: Liposomes were produced either with sonication or extrusion and characterized. The extruded liposomes were combined with siRNA at different N/P ratios and investigated in terms of size zeta potential, encapsulation efficiency, lipoplex stability against RNase A, and knockdown efficiency using enhanced green fluorescent protein (eGFP)-marked colon adenocarcinoma cells. Results: The liposomes prepared by extrusion were smaller and had a narrower size distribution than the sonicated ones. The combination of siRNA and liposomes at a nitrogen-to-phosphate (N/P) ratio of 5 had optimal particle properties, high encapsulation efficiency, and lipoplex stability. Gene knockdown tests confirmed this assumption. Conclusions: Liposomes produced with extrusion were more reproducible and provided enhanced particle properties. The physicochemical characterization and in vitro experiments showed that an N/P ratio of 5 was the most promising ratio for siRNA delivery. Full article

Culex pipiens is a major vector of pathogens, including West Nile and Usutu viruses, that poses a significant public health risk. Monitoring pyrethroid resistance in mosquito populations is essential for effective vector control. This study aims to evaluate four DNA extraction protocols—QIAsymphony, DNAzol^® Direct reagent, PrepMan^® Ultra Sample Preparation Reagent (USPR), and Chelex^® 100—to identify an optimal method to extract DNA from individual Culex pipiens, as part of a high-throughput surveillance of pyrethroid resistance using Real-Time Genotyping PCR. The target is the L1014F mutation in the voltage-sensitive sodium channel (VSSC) gene, which confers knockdown (kdr) resistance to pyrethroids. Mosquitoes were collected from wintering and summer habitats in Lazio and Tuscany, Italy, and DNA was extracted using the four methods. The quality, quantity, extraction time, and cost of the DNA were compared among the various methods. The PrepMan^® USPR protocol was the most efficient, providing high-quality DNA with a 260/280 purity ratio within the optimal range at the lowest cost and in a short time. This method also demonstrated the highest amplification success rate (77%) in subsequent real-time PCR assays, making it the preferred protocol for large-scale genotyping studies. Full article

Wireworms are the most destructive soil insect pests affecting horticultural crops. The damage often renders them unsuitable for commercial purposes, resulting in substantial economic losses. RNA interference (RNAi) has been broadly used to inhibit gene functions to control insect populations. It employs double-stranded RNA (dsRNA) to knockdown essential genes in target organisms, rendering them incapable of development or survival. Although it is a robust approach, the primary challenges are identifying effective target genes and delivering their dsRNA into wireworms. Thus, the present study established a liquid ingestion methodology that efficiently delivers dsRNA into wireworms. We then investigated the effects of four target genes on wireworm mortality. The highest mortality rate reached 50% when the gene encoding vacuolar ATPase subunit A was targeted. Its transcript content in the fed wireworms was also significantly reduced. The mortality rates of the other three target genes of vacuolar ATPase subunit E, beta-actin, and chitin synthase 1 were 28%, 33%, and 35%, respectively. This is the first report demonstrating an efficient feeding methodology and the silencing of target genes in wireworms. Our findings indicate that RNAi is an effective alternative method for controlling the wireworm pest, and can be used to develop field treatment strategies. Full article

The mammalian target of rapamycin (mTOR), a serine/threonine kinase, promotes cell growth and inhibits autophagy. The following two complexes contain mTOR: mTORC1 with the regulatory associated protein of mTOR (RAPTOR) and mTORC2 with the rapamycin-insensitive companion of mTOR (RICTOR). The phosphatidylinositol 3-kinase (PI3K)/Akt/mTOR signaling pathway is important in the intervertebral disk, which is the largest avascular, hypoxic, low-nutrient organ in the body. To examine gene-silencing therapeutic approaches targeting PI3K/Akt/mTOR signaling in degenerative disk cells, an in vitro comparative study was designed between small interfering RNA (siRNA)-mediated RNA interference (RNAi) and clustered regularly interspaced short palindromic repeat (CRISPR)–CRISPR-associated protein 9 (Cas9) gene editing. Surgically obtained human disk nucleus pulposus cells were transfected with a siRNA or CRISPR–Cas9 plasmid targeting mTOR, RAPTOR, or RICTOR. Both of the approaches specifically suppressed target protein expression; however, the 24-h transfection efficiency differed by 53.8–60.3% for RNAi and 88.1–89.3% for CRISPR–Cas9 (p < 0.0001). Targeting mTOR, RAPTOR, and RICTOR all induced autophagy and inhibited apoptosis, senescence, pyroptosis, and matrix catabolism, with the most prominent effects observed with RAPTOR CRISPR–Cas9. In the time-course analysis, the 168-h suppression ratio of RAPTOR protein expression was 83.2% by CRISPR–Cas9 but only 8.8% by RNAi. While RNAi facilitates transient gene knockdown, CRISPR–Cas9 provides extensive gene knockout. Our findings suggest that RAPTOR/mTORC1 is a potential therapeutic target for degenerative disk disease. Full article