Search Results (65)

Coronaviruses use discontinuous transcription to generate subgenomic RNAs (sgRNAs) that encode structural and accessory proteins. However, the factors regulating sgRNA abundance in SARS-CoV-2 remain unclear. Here, we combined strand-specific RNA sequencing, RNA–RNA interaction mapping, prediction of RNA folding energies, and targeted mutagenesis to define the regulation of (–) sgRNA synthesis in SARS-CoV-2 infection. We demonstrated that the relative (–) sgRNA abundance across viral genes is stable throughout infection and largely correlates with corresponding (+) sgmRNA levels. Through meta-analysis of published SPLASH data, we found that the frequency of long-range interactions between the 5′ genomic transcription regulatory sequence TRS-Leader and downstream TRS-Body sequences correlates with sgRNA abundance. Notably, the folding energy (ΔG) of these duplexes quantitatively predicts (–) sgRNA transcript levels. Mutations in non-coding regulatory regions that altered the ΔG resulted in corresponding changes in (–) sgRNA expression, suggesting a causal role for TRS duplex stability in transcriptional regulation. Analysis of naturally occurring mutations near regulatory sites further suggests that modulation of duplex stability may also serve as an evolutionary mechanism to fine-tune viral gene expression. Together, our findings identify the pairing stability of TRS-Leader:TRS-Body as a determinant of discontinuous transcription and reveal how RNA pairing potential contributes to the regulation of (–) sgRNA synthesis in SARS-CoV-2. Full article

Background/Objectives: Exposure of cells to ionizing radiation induces isolated DNA lesions, including single-strand breaks, apurinic/apyrimidinic sites, and oxidized bases, as well as clustered damages of different complexity. The latter types of damage are difficult to repair, and the failure to process them accurately and efficiently is related to the induction of mutagenesis, genomic instability, cancer, and aging. Since various types of clustered lesions may occur simultaneously after radiation exposure, leading to a complex architecture of DNA damage, the study of the concomitant formation and the removal kinetics of clustered DNA damage is important to determine the mutagenic and, consequently, the carcinogenic potential of ionizing radiation. Methods: With the aim of capturing real-time coexisting lesion types and assessing the repair kinetics of clustered damages, the simultaneous determination of double-strand breaks, apurinic/apyrimidinic site clusters, and oxypurine clusters induced by γ-irradiation of Saccharomyces cerevisiae yeast cells was performed immediately after exposure and at time intervals during incubation in Liquid Holding Recovery conditions. Results: Ionizing radiation induced lethal and mutagenic events, leading to a dose-dependent linear increase in double-strand breaks, apurinic/apyrimidinic site clusters, and oxypurine clusters. The kinetic study showed that double-strand break frequencies declined during Liquid Holding Recovery, although a transient increase was detected at early time points. At 160 Gy, apurinic/apyrimidinic site clusters repair was evident, whereas at 400 Gy the frequency of damage increased before returning to the initial value at 24 h. In contrast, oxypurine clusters showed no net increase in repaired lesions over 24 h. Conclusions: The complex nature and topological characteristics of ionizing radiation-induced clustered DNA damage may influence lesion processing. Also, ionizing radiation may disrupt redox cellular homeostasis, leading to DNA damage and delayed effects. Full article

This study systematically investigated the genomic alterations in Saccharomyces cerevisiae driven by Replication Factor A (RFA) dosage insufficiency using a promoter-replacement strategy combined with mutation accumulation and whole-genome sequencing. Our findings reveal that transcriptional suppression of RFA2 or RFA3 leads to severe growth inhibition. RFA deficiency induces a distinct mutational spectrum characterized by a high frequency of monosomy and terminal deletions, indicative of severe replication stress. Furthermore, loss of heterozygosity is significantly enriched at centromeres and high-GC regions, underscoring the role of RFA in stabilizing intrinsic genomic barriers. Utilizing an APOBEC3B-induced mutagenesis assay, we demonstrate that RFA insufficiency leads to the extensive accumulation of exposed ssDNA with a distinct bias towards the lagging strand template. Notably, we observed that cells spontaneously inactivate Mismatch Repair (MMR) genes, such as MSH2 and PMS1, to survive RFA-induced stress. This hypermutant phenotype grants a certain degree of growth recovery on Low Galactose (LG) medium. Overall, these findings demonstrate that RFA dosage is a key determinant of genomic integrity and elucidate how repair pathway modulation drives adaptive evolution under replication stress. Full article

Based on PCR with complementary primer pairs and Pfu DNA polymerase, QuickChange site-directed mutagenesis has been widely employed, but its efficiency varies from mutation to mutation. An alternative strategy relies on partially overlapping primer pairs with 3′-overhangs, and this strategy has led to the recent development of P3a and P3b site-directed mutagenesis, in which the use of SuperFi II and Q5 polymerases raises the mutagenesis efficiency to ~100%. It is unclear whether these two DNA polymerases also improve the QuickChange method. Herein, we have evaluated this possibility by engineering 46 mutations on seven expression plasmids, two of which possess extremely GC-rich sequences. As Pfu DNA polymerase is a slow enzyme, its replacement with SuperFi II and Q5 polymerases reduced PCR length. Moreover, the average efficiency for each of the seven plasmids ranged from 48% to 69%, thereby outperforming the original QuickChange method. However, this efficiency is still lower than that from the P3a and P3b methods, supporting the superiority of primer pairs with 3′-overhangs. Analysis of the incorrect plasmids from the improved QuickChange method revealed frequent insertions at primer sites. The insertions were derived from primers and varied from mutation to mutation, with certain sites much more prone to such insertions. In comparison, these insertions occurred at a much lower frequency with the P3a and P3b methods, suggesting that primer pairs with 3′-overhangs enhance mutagenesis efficiency by reducing the likelihood to introduce insertions at primer sites. Thus, this study improves the QuickChange mutagenesis method, supports the superiority of the P3a and P3b methods, and uncovers a novel molecular mechanism by which the efficiency of PCR-based mutagenesis with completely overlapping primer pairs is negatively affected. Full article

Melanocytic tumorigenesis is thought to occur through stepwise genomic evolution from normal skin to nevi and, ultimately, melanoma. To investigate this progression, we performed targeted deep sequencing of a 46-gene panel in matched healthy skin, nevus, and melanoma samples from 15 patients, including 14 complete tissue trios. Mutation burden increased progressively across tissues, with median mutation counts rising from benign skin to nevi and showing the highest levels in melanoma, consistent with cumulative somatic alterations. Canonical MAPK pathway mutations were common: BRAF V600E and NRAS Q61 variants were detected in many nevi and melanomas and were shared between lesions in 8 of 15 patients, providing direct evidence of clonal continuity. Variant allele frequencies for driver and nonsynonymous mutations were higher than those of passenger and synonymous mutations, reflecting selective expansion of functionally relevant clones. UV-signature substitutions were abundant, particularly among synonymous variants, suggesting background mutagenesis without clonal advantage. Melanoma-private mutations in genes such as ARID1A, ARID2, PIK3CA, and CDKN2A indicated additional late events contributing to malignant progression. Overall, this study supports a model in which many melanomas evolve from pre-existing nevi through sequential acquisition and clonal amplification of somatic mutations, while also revealing heterogeneous evolutionary trajectories. Full article

Cas9 with specialized temperature adaptations are essential for broadening the application of CRISPR-based genome editing across diverse biological contexts. Although Cas9 orthologs from thermophilic and mesophilic organisms have been characterized for high- and moderate-temperature applications, cold-active variants remain largely unexplored, limiting genome engineering in low-temperature systems such as aquaculture species. Here, we report the functional characterization of Fp2Cas9, a cold-adapted Type II-C nuclease from Flavobacterium psychrophilum. In vitro assays showed that Fp2Cas9 efficiently cleaves double-stranded DNA with a refined PAM requirement of 5′-SNAAAG-3′, and that its engineered sgRNA scaffold (sgRNA-V2) supports programmable DNA targeting. Notably, Fp2Cas9 retains 75% cleavage efficiency at 5 °C, approximately 2.5-fold higher than SpCas9 under the same conditions, but shows a marked reduction in activity at 35 °C. In vivo, a nuclear-localized variant (2NLS-Fp2Cas9) mediated efficient mutagenesis of the zebrafish slc45a2 gene, yielding ~60% indel frequencies and pigmentation-deficient phenotypes in ~43% of injected embryos. Collectively, these findings establish Fp2Cas9 as a cold-adapted Cas9 with reliable activity at low temperatures. This work adds a valuable tool to the CRISPR-Cas9 toolkit and may facilitate genome editing in cold-water organisms and other low-temperature systems. Full article

Compound T1089—a novel nitrogen mustard based on an indole-3-carboxylic acid derivative (ICAD)—has been synthesized. The ICAD used as the basis for T1089 is a TLR agonist capable of activating an antitumor immune response. This study describes the synthesis method and presents the results of preliminary investigations of this compound. This research included an assessment of acute toxicity in mice, in vivo clastogenic activity evaluated via the bone marrow chromosome aberration (BMCA) test in mice, in vitro cytotoxicity determined by the MTT assay against human lung carcinoma A549 cells, and in vivo antitumor effects (ATEs) in models of conventional chemotherapy (CCT) of solid tumors in mice. The bifunctional alkylating agent cyclophosphamide (CPA) was used as a reference drug. Toxicological studies revealed that T1089 belongs to toxicity class III (moderately toxic), with acute toxicity values (LD₁₆ and LD₅₀) in mice following intraperitoneal (i.p.) administration being 191 and 202 mg/kg, respectively. The alkylating activity and clastogenic potential of T1089 were demonstrated by its effects in the BMCA test, which were comparable to those of CPA. A single i.p. administration of CPA and T1089 at a dose of 0.064 mmol/kg induced similar stimulation of structural mutagenesis associated with DNA strand breaks. The frequency of karyocytes with aberrations increased 20-fold compared to the control, primarily due to a rise in chromatid breaks and fragments, and to a lesser extent, due to an increase in exchange-type aberrations. In vitro cytotoxicity studies indicated differences in the mechanisms of alkylating activity between CPA and T1089. According to the MTT assay, the cytotoxic effects of CPA were observed only at concentrations exceeding 2 mM (IC₅₀ = 4.2 ± 0.3 mM), corresponding to lethal in vivo doses, which is expected since the formation of CPA’s alkylating metabolite requires hepatic microsomal enzymes. In contrast, significant cytotoxic effects of T1089 were observed at much lower concentrations (15–50 μM, IC₅₀ = 33.4 ± 1.3 μM), corresponding to safe in vivo doses. Differences were also observed in the in vivo ATEs of CPA and T1089 in the Ehrlich solid carcinoma (ESC) CCT model. Following seven i.p. administrations at 48 h intervals (33 mg/kg), both compounds exhibited increasing toxicity, manifested as cumulative body weight loss in treated mice. However, despite the aggressive CCT regimen, ESC showed low sensitivity to CPA. The ATE of CPA developed slowly, reaching a significant level only after four injections, and even after seven administrations, tumor inhibition (TI) did not exceed 30%. In contrast, ESC was significantly more sensitive to T1089 under the same CCT conditions. The ATE of T1089 exhibited a cumulative pattern but developed more rapidly and to a greater extent. A significant antitumor effect was observed after just two injections, with maximal efficacy (TI = 53%) achieved after four injections and sustained until the end of the observation period. A high ATE of T1089 was also observed in the B-16 melanoma CCT model. Following six i.p. administrations at 48 h intervals (28 mg/kg), T1089 treatment was associated with minimal toxicity. Despite this mild CCT regimen, melanoma exhibited high sensitivity to T1089. Maximal ATE (TI = 56%) was achieved after two injections, and subsequent administrations maintained a consistently high efficacy (TI = 52–55%) until the end of the study. In summary, preliminary findings demonstrate that T1089 possesses alkylating activity characteristic of bifunctional agents, accompanied by high in vitro cytotoxicity and in vivo ATEs in CCT models (at high doses). Given that the ICAD used as the basis for T1089 is a TLR agonist capable of stimulating antitumor immunity, T1089 can be considered a dual-action alkylating agent with combined antitumor effects. These results justify further investigation of T1089 in conventional and metronomic chemotherapy regimens, particularly in combination with immune checkpoint inhibitors and antitumor vaccines. Full article

Excessive exposure to ultraviolet B (UVB) radiation can lead to skin damage, such as erythema and swelling. Echinacoside is a key effective ingredient of medicinal plant Cistanche deserticola commonly used for therapies and treatments for anti-aging and irradiation-related skin diseases. However, the molecular mechanism underlying the action of echinacoside remains unclear. Here, we report that echinacoside ameliorates UVB-induced skin damage by directly acting on the Ca²⁺-permeable and thermosensitive transient receptor potential vanilloid 3 (TRPV3) channel. Topical application of echinacoside efficaciously suppresses skin lesions induced by UVB radiation in wild-type mice but has no additional benefit in Trpv3 knockout mice. In whole-cell patch clamp recordings, echinacoside selectively inhibits TRPV3 channel currents induced by 2-aminoethoxydiphenyl borate in a concentration-dependent manner with an IC₅₀ value of 21.94 ± 1.28 μM. The single-channel patch clamp results show that echinacoside significantly reduces the open probability and open frequency without significantly altering TRPV3 channel unitary conductance. Molecular docking and site-specific mutagenesis indicate that residue T636 on the p-loop and residue T665 on the S6 segment of TRPV3 are critical for echinacoside binding to TRPV3. Taken together, our findings provide a molecular basis for further studies as use of natural echinacoside in irradiation-related skin care therapy, thus establishing a significant role of the TRPV3 channel in acute skin injury. Full article

Background: Monoclonal antibodies against the sodium-dependent phosphate transporter NaPi2b (SLC34A2) represent a promising approach in the treatment of ovarian and lung cancer. Of particular interest is the potential cancer-specific MX35 epitope of NaPi2b, as it serves as a target for monoclonal antibodies studied at various stages of preclinical and clinical trials. However, variations in the NaPi2b protein structure may limit the efficacy of therapeutic antibodies by affecting the accessibility of the MX35 epitope. Methods: An in silico analysis was performed using data from 101,562 tumor samples. Genomic DNA sequencing was conducted on blood samples from patients with ovarian carcinoma, breast cancer, and renal carcinoma to access the frequency of germline mutations in the SLC34A2 gene region encoding the MX35 epitope. To assess the impact of the selected mutation, we generated a model cell line through site-directed mutagenesis carrying the mutant NaPi2b variant. Results: Using in silico analysis, we identified 17 unique variants in the SLC34A2 gene leading to amino acid substitutions within the MX35 epitope of the NaPi2b. Among these, the most prevalent mutation, c.989C>T, resulting in p.T330M substitution, was detected in 5 out of 64 patients through genomic DNA sequencing. Using site-directed mutagenesis, we created the OVCAR-8/NaPi2b^p.T330M model cell line. L3 (28/1) monoclonal antibodies specific to the MX35 epitope failed to recognize the mutant NaPi2b^p.T330M variant compared to the wild-type of the NaPi2b in both Western blot and confocal microscopy experiments. Conclusions: The obtained data may serve as a basis for predicting the efficacy of monoclonal antibody-based targeted therapy binding to the MX35 epitope of NaPi2b in the treatment of oncological diseases. Full article

The widespread and inappropriate use of antibiotics, for therapeutic and prophylactic purposes, has contributed to a global crisis of rapidly increasing antimicrobial resistance of microorganisms. This resistance is often associated with elevated mutagenesis induced by the presence of antibiotics. Additionally, subinhibitory concentrations of antibiotics can trigger stress responses in bacteria, further exacerbating this problem. In the present study, we investigated the effect of low doses of ciprofloxacin on the induction of the SOS response and the subsequent development of antibiotic resistance in Proteus spp. strains. Our findings revealed an increase in mutation frequencies within the studied strains, accompanied by a significant upregulation of recA expression. These observations were consistent across experiments involving two subinhibitory concentrations of ciprofloxacin. To establish mutation frequencies and assess gene expression changes, we utilized the Rif^S-to-Rif^R forward mutagenesis assay and RT-qPCR analysis, respectively. Furthermore, employing the microdilution method, we demonstrated that these changes could promote cross-resistance to multiple classes of antibiotics in Proteus spp. clinical strains. This, combined with the recurrent nature of Proteus-associated infections, poses a substantial risk of therapeutic failure. In conclusion, exposure to low doses of ciprofloxacin can significantly impact the susceptibility of Proteus bacilli, not only reducing their sensitivity to ciprofloxacin itself but also fostering resistance to other antibiotic classes. These findings underscore the importance of cautious antibiotic use and highlight the potential consequences of subinhibitory antibiotic exposure in clinical and environmental settings. Full article

Chinese kale is a native vegetable from the Brassicaceae family that is grown extensively in Southeast Asia and Southern China. Its low genetic transformation and gene editing efficiency hinder gene function research and molecular biology in Chinese kale. CRISPR/Cas9 is a useful tool for plant genome research due to its rapid development and optimization. This study targeted BocPDSs, (BocPDS1, BocPDS2) to establish an effective CRISPR/Cas9 system in Chinese kale. A tandemly arranged tRNA-sgRNA construct was used to express numerous sgRNAs to induce BocPDS1 and BocPDS2 double and single mutations, with a mutation rate of 61.11%. As predicted, several mutant plants showed an albino phenotype with a harbored mutation in an exon and intron region, highlighting the relevance of the intron. The presence of mutations in the intron region suggests that the cleavage process in Chinese kale, utilizing CRISPR/Cas9 shows a preference for AT-rich regions. The distinct and somewhat redundant functions of BocPDS1 and BocPDS2 are demonstrated by the complete albino phenotype of the double mutants and the mosaic albino phenotype of the individual BocPDS1 and BocPDS2 mutants. Specific gene editing modes, including base deletion, base substitution, and base insertion, were identified in the sequence of the target gene. Among them, short nucleotide insertions were the most common type of insertion, with base insertions having the highest frequency (61.54%). Furthermore, no instances of off-target gene editing were detected. The current work demonstrated that the CRISPR/Cas9 gene editing system, which relies on endogenous tRNA processing, can effectively induce mutagenesis in Chinese kale. This finding establishes a theoretical basis and technical backbone for the more effective implementation of CRISPR/Cas9 gene-editing technology in Chinese kale and Brassica plants. Full article

As a new, safe, and efficient method, Atmospheric-Pressure Room-Temperature Plasma (ARTP) mutagenesis has been widely applied in the field of microbial breeding and industrial applications, but it is rarely used in fish. In this study, ARTP mutagenesis technology was applied for the first time to a common carp strain, Songpu mirror carp (Cyprinus carpio L.), to increase genetic variation in this species. The appropriate experimental conditions were determined to include a radio frequency output power of 160 W and the processing of fertilized eggs for 360 s. The ARTP treatment group had a lower survival rate than the control group. The CV of morphological characters in the ARTP treatment group was significantly higher than that in the control group, and the CV of body weight was the highest (p < 0.05). In addition, the deformity rate in the ARTP treatment group was significantly higher than in the control group (p < 0.05). Individuals with high weight and no deformities were screened within the selection pressure of 1:15 of ARTP treatment group and fed in the same pool with the control group of the same age. The measurement of serum indices showed that, in the ARTP treatment group, TP, ALP, ALB, T-CHO, LDL levels were significantly higher than those in the control group (p < 0.05). Furthermore, the relative expressions of SOD, growth-related genes GH, IGF-I, protein synthesis-related genes TOR and 4EBP1 were significantly higher in the ARTP treatment group than in the control group (p < 0.05). In summary, Songpu mirror carp subjected to ARTP treatment showed a higher growth potential and antioxidant capacity. Full article

Gene mutations linked to diseases like cancer may be caused by exposure to environmental chemicals. The X-linked phosphatidylinositol glycan class A (PIG-A) gene, required for glycosylphosphatidylinositol (GPI) anchor biosynthesis, is a key target locus for in vitro genetic toxicity assays. Various organisms and cell lines may respond differently to genotoxic agents. Here, we compared the mutagenic potential of directly genotoxic ethyl methane sulfonate (EMS) to metabolically activated pro-mutagenic polycyclic aromatic hydrocarbons (PAHs). The two classes of mutagens were compared in an in vitro PIG-A gene mutation test using the metabolically active murine hepatoma Hepa1c1c7 cell line and the human TK6 cell line, which has limited metabolic capability. Determination of cell viability is required for quantifying mutagenicity. Two common cell viability tests, the MTT assay and propidium iodide (PI) staining measured by flow cytometry, were evaluated. The MTT assay overestimated cell viability in adherent cells at high benzo[a]pyrene (B[a]P) exposure concentrations, so PI-based cytotoxicity was used in calculations. The spontaneous mutation rates for TK6 and Hepa1c1c7 cells were 1.87 and 1.57 per million cells per cell cycle, respectively. TK6 cells exposed to 600 µM and 800 µM EMS showed significantly higher mutation frequencies (36 and 47 per million cells per cell cycle, respectively). Exposure to the pro-mutagen benzo[a]pyrene (B[a]P, 10 µM) did not increase mutation frequency in TK6 cells. In Hepa1c1c7 cells, mutation frequencies varied across exposure groups (50, 50, 29, and 81 per million cells per cell cycle when exposed to 10 µM B[a]P, 5-methylcholanthrene (5-MC), chrysene, or 16,000 µM EMS, respectively). We demonstrate that the choice of cytotoxicity assay and cell line can determine the outcome of the Pig-A mutagenesis assay when assessing a specific mutagen. Full article

Natural caffeic acid (CA) and its analogues have been studied for their potential applications in the treatment of various inflammatory and infectious skin diseases. However, the molecular mechanism underlying the effects of the CA remains largely unknown. Here, we report that CA and its two analogues, caffeic acid phenethyl ester (CAPE) and caffeic acid methyl caffeate (CAMC), inhibit TRPV3 currents in their concentration- and structure-dependent manners with IC₅₀ values ranging from 102 to 410 μM. At the single-channel level, CA reduces the channel open probability and open frequency without alteration of unitary conductance. CA selectively inhibits TRPV3 relative to other subtypes of thermo-TRPs, such as TRPA1, TRPV1, TRPV4, and TRPM8. Molecular docking combined with site-specific mutagenesis reveals that a residue T636 in the Pore-loop is critical for CA binding to TRPV3. Further in vivo evaluation shows that CA significantly reverses TRPV3-mediated skin inflammation induced by skin sensitizer carvacrol. Altogether, our findings demonstrate that CA exerts its anti-inflammatory effects by selectively inhibiting TRPV3 through binding to the pocket formed by the Pore-loop and the S6. CA may serve as a lead for further modification and identification of specific TRPV3 channel inhibitors. Full article

As a physical mutagen, carbon ion beam (CIB) irradiation can induce high-frequency mutation, which is user-friendly and environment-friendly in plant breeding. In this study, we resequenced eight mutant lines which were screened out from the progeny of the CIB-irradiated dehulled rice seeds. Among these mutants, CIB induced 135,535 variations, which include single base substitutions (SBSs), and small insertion and deletion (InDels). SBSs are the most abundant mutation, and account for 88% of all variations. Single base conversion is the main type of SBS, and the average ratio of transition and transversion is 1.29, and more than half of the InDels are short-segmented mutation (1–2 bp). A total of 69.2% of the SBSs and InDels induced by CIBs occurred in intergenic regions on the genome. Surprisingly, the average mutation frequency in our study is 9.8 × 10⁻⁵/bp and much higher than that of the previous studies, which may result from the relatively high irradiation dosage and the dehulling of seeds for irradiation. By analyzing the mutation of every 1 Mb in the genome of each mutant strain, we found some unusual high-frequency (HF) mutation regions, where SBSs and InDels colocalized. This study revealed the mutation mechanism of dehulled rice seeds by CIB irradiation on the genome level, which will enrich our understanding of the mutation mechanism of CIB radiation and improve mutagenesis efficiency. Full article