Search Results (63)

Background: Upland cotton (Gossypium hirsutum) is a major natural fiber crop and an important model for studying genome evolution and gene function in polyploid plants. However, its large and highly redundant genome presents substantial challenges for efficient and coordinated multiplex genome editing. Methods: Here, we developed a high-efficiency CRISPR/Cas12a-based multiplex genome editing system in cotton by integrating a tRNA–crRNA polycistronic expression strategy with a Bean yellow dwarf virus (BeYDV)-derived replicon. Results: This platform enabled coordinated expression of multiple crRNAs and simultaneous targeting of 16 loci within a centromere-proximal region of chromosome D03 (18.65–24.47 Mb). In individual transgenic lines, up to 10 target sites were edited concurrently, with nine targets exhibiting editing efficiencies above 56% and the highest efficiency reaching 96.46%. High-density multiplex editing predominantly induced small insertions and deletions at target loci. Notably, edited plants exhibited reduced growth and pronounced cytological abnormalities, including chromosome bridges, lagging chromosomes, and abnormal meiotic products. Transcriptome analysis revealed widespread dysregulation of genes involved in chromosome segregation and cell cycle regulation. Despite these functional perturbations, HiFi long-read sequencing detected no large-scale chromosomal rearrangements, indicating that genome instability arises from cumulative local perturbations rather than global structural alterations. Conclusions: Together, our results establish an efficient multiplex genome editing platform in cotton and highlight potential constraints of high-density editing on genome stability in complex plant genomes. Full article

Endogenous estrogens are implicated in carcinogenesis through both estrogen receptor-mediated cell proliferation and the direct genotoxicity of reactive metabolites. Oxidative metabolism of estrogens produces catechol estrogens that are further converted to electrophilic ortho-quinones capable of alkylating DNA. The prevailing model of mutagenesis proposes that these N3Ade and N7Gua adducts depurinate to form abasic sites that induce mutations initiating hormone-related cancers. However, the mutation spectrum observed in experimental data is inconsistent with this mechanism, and synthetic studies of estrogen-DNA adducts have relied on acidic conditions that artificially promote depurination, leaving stable N7-dG lesions poorly understood. To address this, we synthesized stable N7-dG catechol and estrone adducts using 2′-fluorinated deoxyguanosine, a modification that inhibits N-glycosidic bond cleavage. ROESY 2D NMR spectroscopy revealed through-space correlations consistent with a preferred anti-conformation in solution, supported by molecular modeling. Structural analysis suggests that these cationic aryl adducts likely preserve the Watson–Crick base pairing edge but may promote tautomerization capable of altering base pairing and generating G-to-A mutations. These findings provide the first synthesized stable models of N7-dG estrogen adducts and may support an alternative mechanism of estrogen-induced mutagenesis independent of depurination, enabling future biochemical investigations of related DNA repair and mutagenesis. Full article

CRISPR-Cas12a enables rapid and specific detection of PCR/LAMP (loop-mediated isothermal amplification) reaction products; however, this approach often requires open-tube manipulation, rendering it prone to cross-contamination. Here, we developed a novel one-pot reaction system that eliminated carryover contamination and facilitated endpoint detection using a CRISPR/Cas12a-based system. We leveraged the dependence of the CRISPR-Cas12a cleavage system on the protospacer-adjacent motif (PAM) to design PCR/LAMP primers that incorporated the PAM site (TTT) into amplified DNA. Pre-incubation of Cas12a with crRNA1 and crRNA2 using PCR/LAMP resulted in efficient cleavage of cross-contaminating DNA, while the target gene remained intact due to the lack of PAM sites. Furthermore, a Cas12a-detection complex (comprising Cas12a, crRNA3, trehalose, and the ssDNA probe) pre-stored on the lid was introduced to mix with the PCR/LAMP amplicons, which triggered the non-specific cleavage of fluorescent probes for direct visual detection under a blue LED instrument. This method effectively degraded up to 10⁶ copies of carryover contaminants within one hour, demonstrating the potential of one-pot detection methods in complex samples. Full article

Bactrocera dorsalis Hendel is a devastating invasive pest that costs billions of dollars in agricultural losses worldwide. Current control strategies rely heavily on male-specific attractants such as methyl eugenol, which are less effective against females, underscoring the need for female-targeted control approaches. Here, we investigated the molecular mechanisms underlying female attraction to cis-linalool oxide by functionally characterizing the odorant receptor OR45a, identifying it as a molecular target for female-oriented pest management. We conducted spatiotemporal expression analysis of OR45a in response to cis-linalool oxide, followed by RNAi and behavioral assays. Phylogenetic analysis of OR45a orthologs from 10 Dipteran species, combined with structural topology prediction and solvent-accessible surface area (ASA) analysis, helped identify functional domains and residues. Site-directed mutagenesis and two-electrode voltage clamp (TEVC) recordings validated receptor–ligand interactions. Results showed that OR45a was specifically upregulated in antennae, with peak expression at 10 days post-eclosion, coinciding with oviposition periods. RNAi significantly reduced OR45a transcript levels and female behavioral responses to cis-linalool oxide. Phylogenetic analysis showed that OR45a is highly conserved within Tephritidae but diverges from Drosophilidae, with closest similarity to Anastrepha ludens, indicating ecological specialization. Structural modeling predicted a canonical seven-transmembrane architecture with three extracellular loops forming the ligand-binding pocket. Among five key residues identified, Leu122 and Ile146 were essential for ligand recognition, while Tyr107 contributed to protein stability. These findings reveal a female-specific odorant receptor mechanism in B. dorsalis and provide molecular targets for OR45a-based attractants, addressing a critical gap in female-focused pest management. Full article

Over the past few decades, Fc fragment-conjugated proteins, such as Protein A, have been extensively utilized across a range of applications, including antibody purification, site-specific immobilization of antibodies, and the development of biosensing platforms. In this study, building upon our group prior research, we designed and screened an affinity DNA functional ligand (A-DNAFL) and experimentally validated its binding affinity (K_D = 6.59 × 10⁻⁸) toward mouse IgG antibodies, whose binding performance was comparable to that of protein A. Systematic evaluations were performed to assess the binding efficiency under varying pH levels and ionic strength conditions. Optimal antibody immobilization was achieved in PBST-B buffer under physiological pH 7.2–7.4 and containing approximately 154 mM Na⁺ and 4 mM K⁺. Two competitive binding assays confirmed that the A-DNAFL binds to the Fc fragment of murine IgG antibody. Furthermore, molecular docking simulations were employed to investigate the interaction mode, revealing key residues involved in binding as well as the contributions of hydrogen bonding and hydrophobic interactions to complex stabilization. Leveraging these insights, A-DNAFL was utilized as a tool for oriented immobilization of antibodies on the sensing interface, enabling the construction of an immunosensor for ricin detection. Following optimization of immobilization parameters, the biosensor exhibited a detection limit of 30.5 ng/mL with the linear regression equation is lg(Response) = 0.329 lg(C_ricin) − 2.027 (N = 9, R = 0.938, p < 0.001)—representing a 64-fold improvement compared to conventional protein A-based methods. The system demonstrated robust resistance to nonspecific interference. Sensing interface reusability was also evaluated, showing only 8.55% signal reduction after two regeneration cycles, indicating that glycine effectively elutes bound antibodies while preserving sensor activity. In summary, the A-DNAFL presented in this study represents a novel antibody-directed immobilization material that serves as a promising alternative to protein A. It offers several advantages, including high modifiability, low production cost, and a relatively small molecular weight. These features collectively contribute to its broad application potential in biosensing, antibody purification, and other areas of life science research. Full article

Background/Objectives: DNA interstrand cross-links (ICLs) mark one of the most deleterious lesions that can preclude strand separation required for essential cellular processes. Efforts to discover ICL-inducing agents and endogenous substrates for ICL repair pathways have led to the identification of structurally diverse ICLs produced by reactive aldehydes and abasic sites, among others. While several studies point to UV rays as ICL-inducing agents, UV ray-induced ICL formation from biologically relevant DNA lesions has been rarely reported. We conjectured that solar radiation-induced reactive oxygen species may give rise to ICLs via further oxidation of DNA lesions with lower redox potential (e.g., 8-oxoadenine (oxoA)). Here, we present the discovery of ICL production via light-induced modification of the major oxidative adenine lesion oxoA. Methods/Results: In the absence of a photosensitizer, both UVC and UVB rays, but not UVA and visible rays, trigger the formation of oxoA-G ICLs, albeit in low yields. By contrast, the inclusion of the naturally occurring photosensitizer riboflavin in the cross-linking reaction makes UVA and visible rays readily generate oxoA-G ICLs, suggesting solar radiation facilitates the formation of oxoA ICLs in vivo. Conclusions: The plausible oxoA-G ICL formation mechanism concerns the further oxidation of oxoA into an iminoquinone, followed by the nucleophilic attack of the opposite guanine on the iminoquinone. OxoA-G ICLs represent rare examples of ICLs produced by photosensitization. These results will contribute to the discovery of a novel form of ICLs induced by solar radiation. Full article

Acinetobacter baumannii (A. baumannii), a very common pathogen, poses a significant public health threat due to its antibiotic resistance and long survival in healthcare environments. Both A. baumannii and carbapenem-resistant A. baumannii (CRAB) can spread through the air, increasing infection risks. Therefore, monitoring their presence in the air is of great significance, especially in hospitals. Herein, we developed a Chelex-100-LAMP-CRISPR/Cas12a (CLC) platform including DNA release and nucleic acid test. Combined with a wet cyclone sampler, the platform can detect airborne A. baumannii and its most common carbapenem-resistant gene, bla_OXA-23, within 70 min. This CLC platform has also been proven to have a detection limit of 6 × 10² CFU of CRAB per test through simulated air samples. Moreover, this platform was also used to test five actual air samples from a tertiary hospital, and the results achieved perfect concordance with sequencing data, validating the platform’s accuracy and reliability. Therefore, the CLC platform showed great potential for the rapid, on-site detection of airborne A. baumannii and its carbapenem-resistant gene bla_OXA-23, offering a valuable tool for infection control in healthcare environments. Full article

Fusarium fujikuroi is the primary causal agent of rice bakanae disease, which can lead to substantial yield losses. Developing a rapid, highly specific, and accurate method for detecting F. fujikuroi is crucial for effective surveillance, prevention, and control of rice bakanae disease. In this study, a novel detection assay, RPA-Cas12a-F, was developed by integrating recombinase polymerase amplification (RPA) and Cas12a for the detection of F. fujikuroi. This assay demonstrated a limit of detection (LOD) of 1 copy/μL of reference plasmid or 0.1 fg/μL of F. fujikuroi genomic DNA (gDNA). Furthermore, to enable on-site detection, the RPA-Cas12a technique was combined with a lateral flow strip (LFS) for visual readout, thereby developing the RPA-Cas12a-LFS assay. The LOD of the RPA-Cas12a-LFS assay was 1000 copies/μL of plasmid or 10 fg/μL of F. fujikuroi gDNA. The RPA-Cas12a-based assays developed in this study enable rapid, highly accurate, sensitive, and specific detection of F. fujikuroi, making them a promising tool for on-site detection without the need for expensive equipment and time-consuming methodologies. Full article

The apurinic/apyrimidinic site (AP site) is a highly mutagenic and cytotoxic DNA lesion. Normally, AP sites are removed from DNA by base excision repair (BER). Methoxyamine (MOX), a BER inhibitor currently under clinical trials as a tumor sensitizer, forms adducts with AP sites (AP-MOX) resistant to the key BER enzyme, AP endonuclease. As AP-MOX remains unrepaired, translesion DNA synthesis is expected to be the main mechanism of cellular response to this lesion. However, the mutagenic potential of AP-MOX is still unclear. Here, we compare the blocking and mutagenic properties of AP-MOX and the natural AP site for major eukaryotic DNA polymerases involved in translesion synthesis: DNA polymerases η, ι, ζ, Rev1, and primase–polymerase PrimPol. The miscoding properties of both abasic lesions remained mostly the same for each studied enzyme. In contrast, the blocking properties of AP-MOX compared to the AP site were DNA polymerase specific. Pol η and PrimPol bypassed both lesions with the same efficiency. The bypass of AP-MOX by Pol ι was 15-fold lower than that of the AP site. On the contrary, Rev1 bypassed AP-MOX 5-fold better than the AP site. Together, our data suggest that Rev1 is best suited to support synthesis across AP-MOX in human cells. Full article

Hyperthermophilic archaea such as Pyrococcus furiosus survive under very aggressive environmental conditions by occupying niches inaccessible to representatives of other domains of life. The ability to survive such severe living conditions must be ensured by extraordinarily efficient mechanisms of DNA processing, including repair. Therefore, in this study, we compared kinetics of conformational changes of DNA Endonuclease Q from P. furiosus during its interaction with various DNA substrates containing an analog of an apurinic/apyrimidinic site (F-site), hypoxanthine, uracil, 5,6-dihydrouracil, the α-anomer of adenosine, or 1,N⁶-ethenoadenosine. Our examination of DNA cleavage activity and fluorescence time courses characterizing conformational changes of the dye-labeled DNA substrates during the interaction with EndoQ revealed that the enzyme induces multiple conformational changes of DNA in the course of binding. Moreover, the obtained data suggested that the formation of the enzyme–substrate complex can proceed through dissimilar kinetic pathways, resulting in different types of DNA conformational changes, which probably allow the enzyme to perform its biological function at an extreme temperature. Full article

Avian leukosis viruses (ALVs) include a group of avian retroviruses primarily associated with neoplastic diseases in poultry, commonly referred to as avian leukosis. Belonging to different subgroups based on their envelope properties, ALV subgroups A, B, and J (ALV-A, ALV-B, and ALV-J) are the most widespread in poultry populations. Early identification and removal of virus-shedding birds from infected flocks are essential for the ALVs’ eradication. Therefore, the development of rapid, accurate, simple-to-use, and cost effective on-site diagnostic methods for the detection of ALV subgroups is very important. Cas13a, an RNA-guided RNA endonuclease that cleaves target single-stranded RNA, also exhibits non-specific endonuclease activity on any bystander RNA in close proximity. The distinct trans-cleavage activity of Cas13 has been exploited in the molecular diagnosis of multiple pathogens including several viruses. Here, we describe the development and application of a highly sensitive Cas13a-based molecular test for the specific detection of proviral DNA of ALV-A, B, and J subgroups. Prokaryotically expressed LwaCas13a, purified through ion exchange and size-exclusion chromatography, was combined with recombinase polymerase amplification (RPA) and T7 transcription to establish the SHERLOCK (specific high-sensitivity enzymatic reporter unlocking) molecular detection system for the detection of proviral DNA of ALV-A/B/J subgroups. This novel method that needs less sample input with a short turnaround time is based on isothermal detection at 37 °C with a color-based lateral flow readout. The detection limit of the assay for ALV-A/B/J subgroups was 50 copies with no cross reactivity with ALV-C/D/E subgroups and other avian oncogenic viruses such as reticuloendotheliosis virus (REV) and Marek’s disease virus (MDV). The development and evaluation of a highly sensitive and specific visual method of detection of ALV-A/B/J nucleic acids using CRISPR-Cas13a described here will help in ALV detection in eradication programs. Full article

Chronic variable mild stress (CVS) in rats is a well-established paradigm for inducing depressive-like behaviors and has been utilized extensively to explore potential therapeutic interventions for depression. While the behavioral and neurobiological effects of CVS have been extensively studied, its impact on myocardial function remains largely unexplored. To induce the CVS model, rats were exposed to various stressors over 40 days. Behavioral assessments confirmed depressive-like behavior. Biochemical analyses revealed alterations in myocardial metabolism, including changes in NAD+ and NADP+, and NADPH concentrations. Free amino acid analysis indicated disturbances in myocardial amino acid metabolism. Evaluation of oxidative DNA damage demonstrated an increased number of abasic sites in the DNA of rats exposed to CVS. Molecular analysis showed significant changes in gene expression associated with glucose metabolism, oxidative stress, and cardiac remodeling pathways. Histological staining revealed minor morphological changes in the myocardium of CVS-exposed rats, including increased acidophilicity of cells, collagen deposition surrounding blood vessels, and glycogen accumulation. This study provides novel insights into the impact of chronic stress on myocardial function and metabolism, highlighting potential mechanisms linking depression and cardiovascular diseases. Understanding these mechanisms may aid in the development of targeted therapeutic strategies to mitigate the adverse cardiovascular effects of depression. Full article

Many efforts are continuously undertaken to develop glucose-sensitive biomaterials able of controlling glucose levels in the body and self-regulating insulin delivery. Hydrogels that swell or shrink as a function of the environmental free glucose content are suitable systems for monitoring blood glucose, delivering insulin doses adapted to the glucose concentration. In this context, the development of sensors based on reversible binding to glucose molecules represents a continuous challenge. Concanavalin A (Con A) is a bioactive protein isolated from sword bean plants (Canavalia ensiformis) and contains four sugar-binding sites. The high affinity for reversibly and specifically binding glucose and mannose makes Con A as a suitable natural receptor for the development of smart glucose-responsive materials. During the last few years, Con A was used to develop smart materials, such as hydrogels, microgels, nanoparticles and films, for producing glucose biosensors or drug delivery devices. This review is focused on Con A-based materials suitable in the diagnosis and therapeutics of diabetes. A brief outlook on glucose-derived theranostics of cancer is also presented. Full article

Apurinic/apyrimidinic (AP) sites are abundant DNA lesions generated both by spontaneous base loss and as intermediates of base excision DNA repair. In human cells, they are normally repaired by an essential AP endonuclease, APE1, encoded by the APEX1 gene. Other enzymes can cleave AP sites by either hydrolysis or β-elimination in vitro, but it is not clear whether they provide the second line of defense in living cells. Here, we studied AP site repairs in APEX1 knockout derivatives of HEK293FT cells using a reporter system based on transcriptional mutagenesis in the enhanced green fluorescent protein gene. Despite an apparent lack of AP site-processing activity in vitro, the cells efficiently repaired the tetrahydrofuran AP site analog resistant to β-elimination. This ability persisted even when the second AP endonuclease homolog, APE2, was also knocked out. Moreover, APEX1 null cells were able to repair uracil, a DNA lesion that is removed via the formation of an AP site. If AP site hydrolysis was chemically blocked, the uracil repair required the presence of NTHL1, an enzyme that catalyzes β-elimination. Our results suggest that human cells possess at least two back-up AP site repair pathways, one of which is NTHL1-dependent. Full article

Orientia tsutsugamushi is responsible for causing scrub typhus (ST) and is the leading cause of acute encephalitis syndrome (AES) in AES patients. A rapid and sensitive method to detect scrub typhus on-site is essential for the timely deployment of control measures. In the current study, we developed a rapid, sensitive, and instrument-free lateral flow assay (LFA) detection method based on CRISPR/Cas12a technology for diagnosing ST (named LoCIST). The method is completed in three steps: first, harnessing the ability of recombinase polymerase for isothermal amplification of the target gene; second, CRISPR/Cas12a-based recognition of the target; and third, end-point detection by LFA. The detection limit of LoCIST was found to be one gene copy of ST genomic DNA per reaction, and the process was complete within an hour. In 81 clinical samples, the assay showed no cross-reactivity with other rickettsial DNA and was 100% consistent with PCR detection of ST. LoCIST demonstrated 97.6% sensitivity and 100% specificity. Overall, the LoCIST offers a novel alternative for the portable, simple, sensitive, and specific detection of ST, and it may help prevent and control AES outbreaks due to ST. In conclusion, LoCIST does not require specialized equipment and poses a potential for future applications as a point-of-care diagnostic. Full article