Search Results (165)

Background/Objectives: Pancreatic ductal adenocarcinoma (PDAC) is a lethal disease with limited treatment options. Patients are treated with DNA damaging chemotherapies which act by inducing DNA damage in rapidly dividing tumor cells. Unfortunately, these tumors frequently develop treatment resistance, underscoring the need to understand resistance mechanisms in order to develop better treatment strategies. DNA damage response (DDR) detects and repairs DNA damage, and the DDR pathway has been shown to contribute to chemoresistance. Another factor known to drive chemoresistance in PDAC is the dense stroma, composed of extracellular matrix proteins secreted by cancer-associated fibroblasts (CAFs). Our recent work identified a CAF-induced resistance mechanism involving N-myc downstream regulated gene 1 (NDRG1). CAF-induced signaling resulted in the phosphorylation of NDRG1 and NDRG1-dependent DNA repair and protection from chemotherapies. Loss of NDRG1 resulted in increased chemotherapy-induced DNA damage and decreased replication fork speed and recovery. Methods: To gain insight into the molecular mechanism of NDRG1-mediated DNA repair and replication, we performed a BioID screen to identify binding partners of NDRG1. We further assessed the mechanistic roles of the identified interaction partners on DNA repair using DNA replication and repair assays such as the Comet assay and DNA fiber assays. Results: Our BioID screen identified meiotic recombination 11 (MRE11) protein, a nuclease involved in DDR, as a putative NDRG1 interacting protein. Interaction between MRE11 and NDRG1 was enriched during the late S/early G2 cell cycle phases and under replication stress. However, this interaction is likely indirect as the interaction only occurred in a cellular context and not with in vitro purified proteins. Blocking NDRG1 phosphorylation or blocking MRE11 exonuclease activity both resulted in protection of newly synthesized DNA at stalled replication forks. In NDRG1 knockout cells, blocking MRE11 led to decreased protection of nascent DNA, suggesting that NDRG1 and MRE11 may be acting in the same pathway and that NDRG1 is required for MRE11’s activity at stalled forks. Conclusions: In summary, our work has uncovered a protein complex between NDRG1 and MRE11 that may play a key role in chemoresistance due to its role in the processing of stalled replication forks. Full article

Spray-induced gene silencing (SIGS) represents a transformative paradigm in sustainable pest management, utilizing the exogenous application of double-stranded RNA (dsRNA) to achieve sequence-specific silencing of essential genes in arthropod pests. Unlike transgenic approaches, sprayable RNA interference (RNAi) biopesticides offer superior versatility across crop systems, flexible application timing, and a more favorable regulatory and public acceptance profile. The 2023 U.S. EPA registration of Ledprona, the first sprayable dsRNA biopesticide targeting Leptinotarsa decemlineata, marks a significant milestone toward the commercialization of non-transformative RNAi technologies. Despite the milestone, large-scale field deployment faces critical bottlenecks, primarily environmental instability, enzymatic degradation by nucleases, and variable cellular uptake across pest taxa. This review critically analyzes the mechanistic basis of spray-applied RNAi and synthesizes the recent technological breakthroughs designed to overcome physiological and environmental barriers. We highlight advanced delivery strategies, including nuclease inhibitor co-application, liposome encapsulation, and nanomaterial-based formulations that enhance persistence on plant foliage and uptake efficiency. Furthermore, we discuss how innovations in microbial fermentation have drastically reduced synthesis costs, rendering industrial-scale production economically viable. Finally, we outline the roadmap for broad adoption, addressing essential factors such as biosafety assessment, environmental fate, resistance management protocols, and the path toward cost-effective manufacturing. Full article

Agrobacterium tumefaciens, a destructive pathogen causing crown gall disease, results in substantial agricultural losses. Traditional chemical and existing biocontrol methods are limited by environmental pollution, pesticide resistance, and low efficacy, while bacteriophages emerge as a promising alternative due to their high host specificity, environmental compatibility, and low resistance risk. In this study, we isolated and characterized a lytic phage (PAT-A) targeting A. tumefaciens, evaluating its biological traits, genomic features, and biocontrol potential. The host strain A. tumefaciens CL-1 was isolated from cherry crown gall tissue and identified by 16S rDNA sequencing. Phage PAT-A was recovered from orchard soil via the double-layer agar method, showing a tadpole-shaped morphology (60 nm head diameter, 30 nm tail length) under transmission electron microscopy (TEM). Nucleic acid analysis confirmed a double-stranded DNA genome, susceptible to DNase I but resistant to RNase A and Mung Bean Nuclease. PAT-A exhibited an optimal MOI of 0.01, tolerated wide pH and temperature ranges, but was sensitive to UV (titer declined after 15 min of irradiation) and chloroform (8% survival at a 5% concentration). Whole-genome sequencing revealed a 44,828 bp genome with a compact structure, and phylogenetic/collinearity analyses placed it in the Atuphduvirus genus (Autographiviridae). Biocontrol experiments on tobacco plants demonstrated that PAT-A significantly reduced crown gall incidence. Specifically, simultaneous inoculation of PAT-A and A. tumefaciens CL-1 resulted in the lowest tumor incidence (12.0%), while pre-inoculation of PAT-A 2 days before pathogen exposure achieved an incidence rate of 33.3%. In conclusion, PAT-A is a novel strictly lytic phage with favorable biological properties and potent biocontrol efficacy against A. tumefaciens, enriching phage resources for crown gall management and supporting phage-based agricultural biocontrol strategies. Full article

Glioblastoma (GBM) is the most aggressive primary cancer with poor survival. In the absence of an effective treatment and a high probability of recurrence, new therapeutic approaches are urgently needed. This study focused on targeting microRNA-10b (miR-10b) highly expressed in GBM cells that has been identified as one of the key drivers of GBM progression. Inhibiting miR-10b using antisense oligonucleotides (ASOs) has shown promise, but its delivery is challenging due to short circulation half-life, degradation by nucleases, and limited blood–brain barrier (BBB) permeability. To overcome these barriers, we employed a magnetic nanoparticle (MN) platform to deliver anti-miR-10b ASOs (MN-anti-miR10b). In addition to serving as a delivery vehicle, these nanoparticles can be used for monitoring delivery using magnetic resonance imaging (MRI). In therapeutic studies in orthotopic models of GBM presented here we used MN-anti-miR10b as well as TTX-MC138, a clinically tested anti-miR10b nanotherapeutic now in Phase I trials in patients with solid (non-GBM) cancers. Both formulations showed efficient delivery, as demonstrated by imaging and improved survival, leading to target inhibition and increased apoptosis. This approach may offer a novel strategy for delivering therapeutics to GBM and improving patient outcomes in one of the most aggressive and treatment-resistant forms of brain cancer. Full article

Antisense inhibition of gene expression is usually achieved using nuclease-resistant oligonucleotide analogs that promote mRNA degradation through RNase H or RNase P, or by steric hindrance of translation. Bridge nucleic acids (BNAs) are nucleotide analogs available in a few chemical variants. We evaluated gapmers composed of an oligodeoxynucleotide flanked by BNA residues in a BNA₅-DNA₈-BNA₄ configuration, using the available variants: the original locked nucleic acid (LNA; 2′-O,4′-methylene bridge), cET (2′-O,4′-ethyl bridge), cMOE (2′-O,4′-methoxyethyl bridge), and BNANC (2′-O,4′-aminomethylene bridge). These gapmers were tested in vitro for their ability to induce cleavage of the model aac(6′)-Ib mRNA. All gapmers complementary to a previously identified region suitable for interaction with antisense oligomers induced RNase H-mediated degradation. Instead, only the LNA-containing gapmer also elicited RNase P-dependent cleavage, demonstrating dual RNA- and DNA-mimicking capability. In vitro coupled transcription–translation assays using cell lysates or reconstituted systems confirmed inhibition of expression and ruled out steric hindrance as the mechanism. In contrast, gapmers targeting the ribosome-binding site strongly inhibited expression by steric hindrance. These findings demonstrate that LNA-containing gapmers can exert their effects through multiple mechanisms, depending on the targeted mRNA region, thereby supporting their potential for synergistic inhibition of gene expression. Full article

Genome editing using site-directed nucleases (SDNs), particularly with the CRISPR/Cas9 system, has emerged as a powerful platform for aquaculture innovation, enabling precise, heritable, and non-transgenic modifications that enhance productivity, sustainability, and animal welfare. This review synthesizes molecular, regulatory, ecological, and societal perspectives to highlight global advances in genome-edited fish and their transition from laboratory research to field applications. To date, over forty aquatic species have been successfully edited to improve traits such as growth, disease resistance, pigmentation, and reproductive control. Notably, market-approved SDN-1 fish lines, including mstn-knockout red seabream and Nile tilapia, and lepr-edited tiger puffer and olive flounder, have demonstrated improved productivity; however, publicly available welfare data remain limited. These examples illustrate how product-based SDN-1 regulatory frameworks in Japan, Argentina, and Brazil enable commercialization while ensuring biosafety. Nonetheless, limited field trials and regulatory heterogeneity continue to hinder global harmonization. Major challenges include the development of standardized welfare metrics, assessment of multigenerational stability, evaluation of ecological risks, and transparent data sharing. To address these gaps, a structured reporting checklist is proposed to guide consistent molecular validation, welfare assessment, biosafety containment, and data transparency. Genome editing through SDN-based precision, coupled with ethical governance, represents a crucial step toward sustainable, resilient, and publicly trusted aquaculture systems. Full article

Background/Objectives: Heterofunctional cationic polyester dendrimers derived from a 2-(bromomethyl)-2-(hydroxymethyl)propane-1,3-diol (BHP-diol) based AB₂C monomer were evaluated as efficient and biodegradable nonviral carriers for siRNA delivery. Methods: These dendrimers feature dual internal and external charge architectures, enabling precise control of charge distribution and siRNA interaction strength. Results: They achieved complete siRNA complexation at nitrogen-to-phosphate (N/P) ratios of 0.50–2.14 and provided up to 93% RNase protection, outperforming amino-functional scaffolds based on 2,2-bis(methylol)propionic acid (bis-MPA). In human (T98G) and murine (GL261) glioblastoma cells, the dendrimers exhibited minimal cytotoxicity while achieving 52–61% target protein knockdown, a two- to three-fold improvement over conventional polyester dendrimers, and approaching the silencing efficiency of the commercial Interferin^® reagent. Conclusions: The combination of high complexation efficiency, strong nuclease resistance, and excellent biocompatibility establishes these heterofunctional dendrimers as a new generation of precisely tunable, biodegradable vectors for therapeutic siRNA delivery. Full article

Introduction: Staphylococcus aureus is a high-priority foodborne pathogen contributing to several food poisoning outbreaks. Methicillin- and vancomycin-resistant S. aureus (MRSA and VRSA), pose significant public health concerns due to their potential for serious illness, antibiotic resistance, and transmission within both healthcare and community settings. These bacteria can cause numerous infections, ranging from skin and soft tissue infections to life-threatening conditions like bloodstream infections, pneumonia, and endocarditis. Although several publications are concerned with Staphylococcus aureus contamination in ready-to-eat (RTE) food products, little published data is available about its prevalence in pizza, which is widely distributed and consumed worldwide. Methods: The current study is intended to determine the prevalence, virulence genes, and antimicrobial resistance profiles of S. aureus in three hundred ready-to-eat pizza samples (100 each of meat, chicken, and canned tuna pizzas) collected from different restaurants in Mansoura City, Egypt. The typical colonies on Baird–Parker selective agar supplemented with egg yolk tellurite emulsion were counted and further confirmed based on Gram staining, coagulase testing, catalase testing, carbohydrate fermentation, and thermostable nuclease production. The genomic DNA of the confirmed coagulase-positive isolates was prepared and subjected to PCR analyses for detecting the nuc gene, mecA (methicillin resistance gene), and vancomycin resistance gene (vanA), as well as six selected S. aureus virulence genes: sea, seb, sec, sed, hla, and tsst. The antimicrobial resistance profile of the S. aureus isolates was determined against 16 antimicrobial agents belonging to six classes using the agar disc diffusion method according to the Clinical and Laboratory Standards Institute guidelines (CLSI), except for oxacillin and vancomycin, which were assessed using the MIC test. Results: The results revealed that 56% (56/100), 56% (56/100), and 40% (40/100) of chicken, meat, and canned tuna pizzas were positive for S. aureus, with an overall prevalence of 50.7% (152/300). All 560 isolates (100%) were verified as S. aureus based on molecular confirmation of the nuc gene. Interestingly, 48.6% (272/560) and 8.6% (48/560) of the isolates tested were identified as methicillin- and vancomycin-resistant S. aureus (MRSA and VRSA) through detection of mecA and vanA genes, respectively. Among the S. aureus isolates tested, the hla gene was detected in 87.1% (488/560), while the enterotoxin genes sea, seb, sec, and sed were identified in 50% (280/560), 78.6% (440/560), 9.8% (55/560), and 24.5% (137/560) of isolates, respectively. All recovered isolates (n = 560) were classified as multidrug-resistant and were resistant to penicillin, oxacillin, and ampicillin. Moreover, 77% (431/560), 24% (134/560), 8% (45/560), and 8.6% (48/560) of isolates were resistant to cefotaxime, ciprofloxacin, azithromycin, and vancomycin, respectively. Conclusions: The current study emphasizes that ready-to-eat pizza is highly contaminated with multidrug-resistant S. aureus, highlighting the urgent need for rationalizing antibiotic use in both veterinary and human medicine to prevent the transmission of resistant bacteria through the food chain. Additionally, strict adherence to good hygienic practices throughout all stages of the food chain is essential to minimize overall contamination and enhance food safety. Full article

Circular RNAs (circRNAs) have emerged as a transformative class of RNA therapeutics, distinguished by their closed-loop structure conferring nuclease resistance, reduced immunogenicity, and sustained translational activity. While challenges in pharmacokinetic control and manufacturing standardization require resolution, emerging synergies between computational design tools and modular delivery platforms are accelerating clinical translation. In this review, we synthesize recent advances in circRNA therapeutics, with a focused analysis of their stability and immunogenic properties in vaccine and drug development. Notably, key synthesis strategies, delivery platforms, and AI-driven optimization methods enabling scalable production are discussed. Moreover, we summarize preclinical and emerging clinical studies that underscore the potential of circRNA in vaccine development and protein replacement therapies. As both a promising expression vehicle and programmable regulatory molecule, circRNA represents a versatile platform poised to advance next-generation biologics and precision medicine. Full article

Pest management is undergoing a transformative shift with the development of the cutting-edge antisense technologies: RNA interference (RNAi), contact unmodified antisense DNA biotechnology (CUADb), and the CRISPR-associated proteins (CRISPR/Cas). These approaches function by facilitating sequence-specific pairing of nucleic acids followed by nuclease-mediated cleavage, offering exceptional precision for targeted pest control. While RNA-guided mechanisms such as RNAi and CRISPR/Cas were initially characterized in non-insect systems, primarily as innate defenses against viral infections, the DNA-guided CUADb pathway was first identified in insect pests as a functional pest control strategy. Its broader role in ribosomal RNA (rRNA) biogenesis was recognized later. Together, these discoveries have revealed an entirely new dimension of gene regulation, with profound implications for sustainable pest management. Despite sharing a common principle of sequence-specific targeting RNAi, CUADb, and CRISPR/Cas differ in several key aspects, including their mechanisms of action, target specificity, and applicability. Rather than serving as universal solutions, each technology is likely to be optimally effective against specific pest groups. Moreover, these technologies allow for rapid adaptation of control strategies to overcome target-site resistance, ensuring long-term efficacy. This review summarizes the core functional characteristics, potential applications, and current limitations of each antisense technology, emphasizing their complementary roles in advancing environmentally sustainable pest control. By integrating foundational biological discoveries with applied innovations, this work provides a new perspectives on incorporating antisense-based strategies into next-generation integrated pest management systems. Full article

In recent years, global public health security has encountered significant challenges, with infectious diseases accounting for approximately 25% of global mortality annually. The worldwide pandemic instigated by the novel coronavirus, alongside the persistent threats posed by Ebola, influenza, and multidrug-resistant bacteria, has severely compromised human health, economic development, and social stability. Within this context, the development of rapid and precise pathogen detection technologies has emerged as a critical frontline defense for epidemic prevention and control, serving as a pivotal component in the implementation of the “early detection, early isolation, and early treatment” strategy. The Argonaute (Ago) protein, recognized as a programmable and target-specific activated nuclease, has demonstrated substantial potential in the realm of nucleic acid detection due to its distinctive biological properties, garnering considerable attention. In this study, we delineate the structural characteristics of Ago proteins and elucidate the mechanism underlying their nuclease activity. Furthermore, we review the principles of nucleic acid detection based on Argonaute and provide a comprehensive analysis of recent advancements in related detection systems. Additionally, we compare the advantages of detection based on Argonaute with other detection methodologies. Through a comprehensive analysis, we aim to provide a robust theoretical foundation and an advanced technical reference for the development of new-generation nucleic acid detection platforms with high sensitivity and high specificity. Full article

T40214 (STAT) and its recently investigated analogue STATB are G-quadruplex (G4) forming aptamers characterized by an unusually high percentage of C. The therapeutic potential of T40214 relies on its ability to inhibit the signalling pathway of STAT3, a protein frequently overexpressed in tumor cells. STAT adopts a dimeric 5′-5′ end-stacked quadruplex structure, characterized by parallel strands, three G-tetrads and three propeller-shaped loops formed by a cytidine residue. STATB folds in a very similar structure, apart from an additional cytidine bulge loop. Many studies suggest that thermal stability and topology of G4 can be significantly affected by C methylation, thus resulting in altered interaction of G4-binding proteins with these structures. Considering this, two series of STAT and STATB analogues containing a single 5-methyl-2′-deoxycytidine (mC) residue instead of canonical C nucleotide in the loop have been prepared and investigated by a combination of spectroscopic and electrophoretic techniques. CD, NMR and PAGE data clearly indicate that all derivatives adopt dimeric G4 strictly similar to that assumed by parent aptamers, but with higher stabilities. Furthermore, the resistance to nucleases and the antiproliferative activity of these mC-containing derivatives against HCT116 (human colorectal carcinoma) and T24 (human bladder carcinoma) cell lines have been evaluated. In most of the cases, STAT and STATB derivatives inhibit cell proliferation to different extents, although to a lesser degree than the unmodified parent sequences. All the data highlight the key role of the loops and indicate mC as a useful tool to contribute favorably to the stability of G4-forming aptamers without alteration of their topology, required for the biological activity. Full article

Background: Most newly developed anticancer treatments trigger tumor cell death through apoptosis, for which involvement of caspases activity is essential. However, numerous mutations in apoptotic pathways that lead to cancer and favor resistance to apoptosis are known; most are related to caspase-dependent apoptosis pathways and thus have low efficacy. To overcome these limitations, we constructed a novel chimeric protein, GnRH-AIF, using a gonadotropin-releasing hormone (GnRH) analog as a targeting moiety and the apoptosis-inducing factor (AIF) in its cleaved form as a killing moiety, fused at the cDNA level. AIF has a crucial role in the caspase-independent apoptotic pathway. A wide variety of solid tumors overexpress GnRH-receptors (GnRH-R) that are targeted by the new GnRH-AIF chimeric protein. Methods and Results: In this study, we constructed, expressed, and highly purified GnRH-AIF chimeric proteins. We demonstrated the ability of the chimera to enter and specifically and very efficiently kill solid cancer cell lines overexpressing GnRH-R. Most importantly, upon its entry, GnRH-AIFs translocate to the nucleus where it causes DNA fragmentation leading to a direct caspase-independent apoptotic death. As AIFs lack nuclease activity, our findings also emphasize that cell death induced by GnRH-AIF is dependent on the presence of the ENDOG and PPIA proteins, known to participate in the formation of a DNA–degradosome complex. Finally, we demonstrated the high anti-tumor efficacy of the GnRH-AIF ex vivo, in a human, colon cancer organoid model. Conclusions: Our study shows the potential of using a GnRH-AIF chimeric protein as a novel approach to treat solid cancers that overexpress GnRH-R, via a caspase-independent apoptotic pathway. Full article

This review summarizes the research progress in the co-delivery of natural products (NPs) and small RNAs in cancer therapy. NPs such as paclitaxel, camptothecin, and curcumin possess multi-target antitumor effects, but their applications are limited by drug resistance and non-specific distribution. Small RNAs can achieve precise antitumor effects through gene regulation, yet their delivery efficiency is low, and they are prone to degradation by nucleases. Nanomaterial-based drug delivery systems (nano-DDSs) provide an efficient platform for the co-delivery of both, which can enhance the targeting of their delivery and improve the synergistic antitumor effects simultaneously. The mechanisms of the antitumor action of natural compounds and small RNAs, the design and application of nanocarriers, and the latest research progress in co-delivery systems are introduced in detail in this paper. The application prospects of the co-delivery of natural compounds and small RNAs in cancer therapy are also discussed. Full article

Microbes that survive transport to and in the stratosphere endure extremes of low temperature, atmospheric pressure, and relative humidity, as well as high fluxes in ultraviolet radiation (UVR). The high atmosphere thus provides an ideal environment to explore the genetic and physiological determinants conveying high tolerance to desiccation and UVR. In this study, we examined Curtobacterium aetherium L6-1, an actinobacterium obtained from stratospheric aerosol sampling that displays high resistance to desiccation and UVR. We found that its phylogenetic relatives are resistant to desiccation, but only C. aetherium displayed a high tolerance to UVR. Comparative genome analysis and directed evolution experiments implicated genes encoding photolyase, DNA nucleases and helicases, and catalases as responsible for UVR resistance in C. aetherium. Differential gene expression analysis revealed the upregulation of DNA repair and stress response mechanisms when cells were exposed to UVR, while genes encoding sugar transporters, sugar metabolism enzymes, and antioxidants were induced upon desiccation. Based on changes in gene expression as a function of water content, C. aetherium can modulate its metabolism through transcriptional regulation at very low moisture levels (X_w < 0.25 g H₂O per gram dry weight). Uncovering the genetic underpinnings of desiccation and UVR resistance in C. aetherium provides new insights into how bacterial DNA repair and antioxidant mechanisms function to exhibit traits at the extreme ends of phenotypic distributions. Full article