Background:Regulatory T cells (Tregs) suppress antitumor immunity in ovarian cancer (OC) and are promising targets for immunotherapy. However, the heterogeneity and regulatory mechanisms of tumor-infiltrating Tregs (TI-Tregs) remain poorly defined. Here, we aim to delineate TI-Treg programs to identify potential therapeutic targets. Methods:CD4⁺CD25⁺CD127⁻ Tregs from OC, adjacent tissues, and peripheral blood were profiled by single-cell RNA sequencing and spatial transcriptomics, with regulatory networks inferred using SCENIC. Functional and mechanistic studies of SOX4 were performed using hypoxic/tumor-conditioned models, CRISPR-Cas9 perturbation, ectopic overexpression, Cut&Tag profiling, and oxidative phosphorylation (OXPHOS) inhibition. Results:We identified nine transcriptionally distinct Treg subsets, revealing a highly activated and immunosuppressive state among TI-Tregs. These TI-Tregs exhibited strong co-expression of TNFRSF4, TNFRSF9, TNFRSF18, and CTLA4, and their increased intratumoral abundance was independently associated with poorer overall survival. SCENIC analysis identified SOX4 as the top regulon defining TI-Treg identity, with spatial transcriptomics revealing SOX4⁺ Tregs forming an immunoregulatory barrier at tumor margins. This phenotypic identity was robustly induced by the hypoxic microenvironment and TCR stimulation in a well-established murine OC model. Mechanistically, SOX4 transactivated MT1X to promote mitochondrial fitness and OXPHOS. Consistently, Cut&Tag profiling showed reduced chromatin accessibility at OXPHOS-related loci following SOX4 depletion. Furthermore, CRISPR-Cas9-mediated disruption of SOX4 reduced FOXP3 expression and other suppressive markers, whereas SOX4 overexpression enhanced FOXP3 expression in an OXPHOS-dependent manner. Notably, pharmacological inhibition of OXPHOS abolished this effect. Conclusions:SOX4 is a central regulator of TI-Treg suppressive function and metabolic fitness, representing a promising therapeutic target for OC.

PYL receptor proteins are essential for enhancing plant resilience to extreme environments; however, their functions in woody plants-particularly in response to osmotic stress-remain poorly understood. In this study, we identified and comprehensively characterized 14 PYL genes in Populus trichocarpa. PtrPYL11, a core member, showed up-regulation following both ABA and dehydration treatments, suggesting that it may act as a positive regulator of osmotic stress responses by mediating the expression of ABA-related genes. Specifically, PtrPYL11-overexpressing poplar lines exhibited stronger osmotic stress tolerance than the wild type. Transcriptome analysis revealed that PtrPYL11 may indirectly regulate the expression of core transcription factors such as DOF1 and TCP9, mediating the ABA-signaling pathway in poplar. This study systematically characterizes the molecular features of the PtrPYL gene family and clarifies the role of PtrPYL11 in poplar osmotic stress responses. These findings not only lay a foundation for further exploration of its biological functions but also provide valuable genetic resources for breeding drought-tolerant forest tree varieties through genetic engineering.

Polymer-doped chiral organic afterglow (COA) materials represent an emerging frontier in photonics, yet their development is constrained by weak hydrogen bond interactions and limited spectral diversity. Herein, a supramolecular engineering strategy utilizing phosphonic acid-derived directional hydrogen bond networks is proposed to construct COA materials. Leveraging the tetrahedral coordination geometry and dual proton-donor functionality of phosphonic acid derivatives, a robust three-dimensional hydrogen bond network is formed with polyvinyl alcohol, yielding blue afterglow emission with a lifetime of 3.05 s, a photoluminescence quantum yield of 33.3%, and enhanced thermal stability. Structural and computational analyses reveal that near-linear hydrogen bond geometry and orbital hybridization synergistically enhance the hydrogen bond strength while enabling chiral amplification by an interfacial chiral polylactic acid coating. Furthermore, through efficient phosphorescence energy transfer, multicolor COA emissions are achieved in stacked polymeric films, exhibiting dissymmetry factors up to 0.03 and afterglow emissions across the visible spectra, allowing the development of customizable encryption inks with spatiotemporal resolved chiroptical signatures for multiple applications. This work not only thoroughly investigates the modulation of hydrogen bonds on afterglow properties but also provides a fundamental understanding of non-covalent interactions in organic optoelectronics.

Single nucleotide variation (SNV), as a key biomarker for disease diagnosis and personalized treatment, faces challenges in rapid and accurate detection. This study developed a single-tube accelerated recognition of SNVs strategy named STAR-CRISPR, which could accomplish SNV detection within only 20 min. This method integrated isothermal amplification and CRISPR/Cas12b cleavage system in one pot, and results could be directly identified by the naked eye. This method could accurately distinguish single-base differences, and could detect as low as 1% mutations against high background interference. We verified the proposed method by testing 70 clinical samples of idiopathic chronic pancreatitis, pancreatic cancer and acute myeloid leukemia. Results showed 100% consistency with next-generation sequencing results, demonstrating good accuracy and reliability of the proposed method. To further facilitate point-of-care diagnosis, we developed integrated miniature microfluidic chips, which greatly simplified sample identification and enabled logical interpretation of results. The combined STAR-CRISPR and microfluidic platform not only identifies SNVs but also supports simultaneous visual genotyping of wild-type, homozygous, and heterozygous mutations. Consequently, the proposed strategy is accurate, rapid, and versatile, holding significant potential for next-generation molecular diagnostics.

Organismal genome stability is constantly challenged by endogenous and exogenous genotoxins, and DNA damage response (DDR) is a classical cell-autonomous mechanism for counteracting such damage. It remains unclear how somatic cells with DDR deficiencies maintain genomic integrity under stress. This study investigated whether inter-tissue communication, particularly from the reproductive system, coordinates DNA damage repair in repair-deficient cells. Using Caenorhabditis elegans, in which certain upstream DDR signaling components are transcriptionally repressed in somatic cells, we examined UV-induced neurotoxicity through behavioral assays (head thrashes, body bends, foraging, and salt chemotaxis). These deficits were associated with preferential impairment of dopaminergic, glutamatergic, and serotonergic reporter readouts, along with heterogeneous transcriptional remodeling of neurotransmitter pathway and pan-neuronal synaptic genes. Deficiencies in upstream DDR signaling (ATM/ATR), whether systemic or germline-specific, significantly exacerbated UV-induced neurobehavioral and transcriptional deficits. In contrast, germline-specific loss of nucleotide excision repair (NER) machinery had no significant effect, highlighting a unique inter-tissue signaling role for upstream DDR. Furthermore, CPR-4 was identified as a key mediator of this inter gonad–soma signaling. Germline DDR activation regulated cpr-4 expression, and loss of CPR-4 abolished UV-induced transcriptional activation of the somatic NER pathway and heightened behavioral sensitivity. Our study revealed a novel germline DDR to somatic tissue signaling axis essential for organismal stress resilience and proposed a new regulatory model centered on gonad–soma signaling.

Pathogens pose significant threats to biosecurity and environmental health due to their potential for widespread outbreaks. Effective pathogen detection requires methods that are rapid, sensitive, specific, and informative. Here, we proposed a multiplex visual detection system that integrated ultrafast polymerase chain reaction (PCR) and molecular beacons, allowing the simultaneous detection of three pathogens in a one-pot reaction. The ultrafast PCR protocol employed cycles of just 7 s each, allowing the entire process-from sampling to result-to be completed within only 10 min. Molecular beacons hybridized with target sequences during ultrafast PCR, generating fluorescence signals that are visually detectable without specialized equipment. Additionally, we developed a compact, portable cartridge integrated with online software for fluorescence visualization and direct result output, eliminating the need for bulky instruments and specialized personnel, thereby facilitating point-of-care testing (POCT). The method demonstrated high specificity and sensitivity, with a limit of detection (LOD) as low as 23 copies per reaction. It achieved a 100

Rapid and reliable nucleic acid detection methods are essential in clinical diagnostics and biotechnology. The clustered regularly interspaced short palindromic repeats (CRISPR) system is emerging as a next-generation nucleic acid detection technology, offering versatility, convenience and rapid detection. However, CRISPR methods are significantly limited by the protospacer adjacent motif (PAM) sequence, and achieving a one-pot reaction for detecting single nucleotide variations (SNVs) within a short time still remains challenging. Here, we developed a comprehensive method for screening PAM sequences, which significantly expands the CRISPR detection scope. Additionally, we also proposed a one-pot CRISPR method, termed "SIMPLE", capable of identifying SNVs within 30 min. We applied the SIMPLE method to the clinical diagnostics of drug-resistant bacteria and the screening of cancer hotspot mutations. The SIMPLE method successfully detected drug-resistant bacteria mediated by canonical PAM TTN sequence with a sensitivity of 10 copies per reaction and achieved 100

DNA double-strand breaks (DSBs) repair via POLQ-mediated alternative end-joining (Alt-EJ) is error-prone and mutagenic. However, Alt-EJ is often inhibited by classical nonhomologous end-joining (C-NHEJ) or homologous recombination, the precise impact of Alt-EJ on plant genome instability remains unclear. Here, we employed carbon-ion beam (CIB) which induce complex DSBs to bias cellular repair strategies toward Alt-EJ; additionally, a specific genetic background of C-NHEJ deficiency (lig4-4) Arabidopsis thaliana line and the POLQ-deficient (teb-3 and teb-8) were combined to further amplify the mutagenic effects of CIB mediated by Alt-EJ. The lig4-4 exhibited higher sensitivity to CIB than POLQ-deficient lines. teb-8 exhibited constitutive DNA damage response (DDR), whereas DDR in lig4-4 was strictly induced by CIB. At genome scale, lig4-4 showed substantial changes in the insertion and deletion (InDels) mutation profile, with a higher proportion and larger size of InDels as well as greater microhomology dependence than wild-type. In contrast, teb-8 showed moderate changes, including increased single-base InDels and complex mutations, but lacking >30 bp InDels. Loss-of-function in LIG4 and POLQ resulted in a higher proportion of high-impact genome mutations than wild-type even at lower doses. These findings offered essential insights for the development of a novel repair pathway-driven heavy ion beam mutagenesis system.

Abstract While cancer cells overexpress lactate dehydrogenase A (LDHA) to support glycolytic flux and lactate production, the role of LDHB—which preferentially catalyzes lactate oxidation—remains unclear. Here, we demonstrate that LDHB, but not LDHA, is essential for mitotic progression in cancers. During mitosis, CDK1 phosphorylates LDHA at threonine 18, reducing its incorporation into the lactate dehydrogenase (LDH) tetramer. This results in LDHB-enriched tetramers that shift catalytic activity toward lactate oxidation, converting lactate and NAD⁺ into pyruvate and NADH. The generated NADH fuels oxidative phosphorylation and ATP production, thereby sustaining mitosis. Notably, LDHA-T18 phosphorylation occurs exclusively in tumor tissues. Our findings reveal a tumor-specific mechanism in which CDK1 reprograms LDH isoenzyme composition to direct lactate toward NADH production, ensuring energy homeostasis during mitosis. This underscores the therapeutic necessity of targeting both LDHA and LDHB in cancer.

Ralstonia solanacearum is a destructive soil-borne pathogen that causes severe bacterial wilt (BW) disease in peppers. Phytohormone signaling pathways, including those mediated by jasmonic acid (JA), are crucial to plant defense responses against pathogen attacks. Nevertheless, the function of JA in the resistance response of pepper to R. solanacearum is unclear. Therefore, RNA sequencing (RNA-seq) and phytohormone determination experiments were performed to compare the dynamic transcriptome changes and differences in JA and salicylic acid (SA) contents between the resistant pepper line BVRC1 and the susceptible pepper line BVRC25 during R. solanacearum infection. The number of differentially expressed genes (DEGs) was greater in BVRC1 than in BVRC25 at 12, 24, 48, and 72 h post-infection. JA concentrations were also markedly elevated in BVRC1 in response to R. solanacearum. Four distinct co-expression modules and hub genes were identified using weighted gene co-expression network analysis. Exogenous application of JA significantly delayed the onset of R. solanacearum infection and reduced symptom severity in BVRC25. RNA-seq was performed on the resistant pepper line BVRC1 at 24 h post exogenous JA application. GO analysis revealed DEGs enriched in the cell wall biosynthesis-related pathway of BVRC1 after JA treatment. Notably, a pathogenesis-related protein, Capana08g002211, exhibited common up-regulated patterns in response to JA treatment and R. solanacearum infection. A yeast-2-hybrid assay and luciferase complementation imaging assay demonstrated that Capana08g002211 interacted with type III effector (RipTPS). These results demonstrate that the pepper JA-mediated cell wall synthesis pathway participates in the defense response to R. solanacearum.

Background:Zika virus (ZIKV) infection can result in severe neurological complications, yet no approved antiviral treatments are currently available. Ginseng, a medicinal herb extensively utilized in Asian traditional medicine, has demonstrated efficacy against various diseases, which has sparked interest in exploring its potential antiviral properties for the treatment of ZIKV. Methods:We evaluated the antiviral effects of ginsenoside Rb2 (G-Rb2) in human neuronal cell lines (SK-N-SH and CCF-STTG) and in a lethal ZIKV-infected mouse model. The antiviral efficacy was assessed using bioluminescence imaging with a NanoLuc luciferase reporter ZIKV. In vitro assays were conducted to determine the direct impact of G-Rb2 on ZIKV, while surface plasmon resonance (SPR) was employed to analyze its interaction with ZIKV envelope proteins and viral particles. Results:G-Rb2 (200 μM) significantly inhibited ZIKV infection in vitro and protected mice from ZIKV-induced mortality. Bioluminescence imaging validated its antiviral efficacy. In vitro studies demonstrated that incubation with G-Rb2 reduced viral infectivity, and SPR analysis confirmed direct binding between G-Rb2 and ZIKV components. Conclusion:G-Rb2 effectively inhibits ZIKV infection both in vitro and in vivo, presumably through direct interaction with viral particles. Given the accessibility of ginseng and its established processing methods, G-Rb2 emerges as a promising candidate for the treatment of ZIKV in humans. Further research is warranted to elucidate its mechanisms of action and evaluate its clinical potential.

Introduction: Epstein–Barr virus (EBV), the first identified human oncogenic gamma-herpesvirus, establishes lifelong persistent infection through a dual-phase viral life cycle: latent infection and lytic replication. Studies have shown that DNA methylation inhibits the activity of gene promoters through CpG site methylation, which is the core epigenetic mechanism by which EBV regulates the latent/lytic state. The viral genome is highly methylated at specific sites in EBV-infected cancer cells and latent infection cell lines, whereas the methylation level is lower in cells undergoing lytic replication and viral particles. Therefore, methylation profiling of EBV genomes can distinguish viral states: low methylation correlates with lytic replication, and high methylation indicates latency. Despite this mechanistic insight, no direct, cost-effective, or technically simple method exists to distinguish the latent infection EBV genome and the lytic replication state. Methods: We have established an ultrasensitive platform for the detection of DNA methylation utilizing RPA-mediated CRISPR/Cas13a technology. This innovative strategy synergistically integrates the high specificity of BstUI/HhaI restriction endonucleases in the digestion of unmethylated cytosines with the robust signal amplification efficiencies of the RPA and CRISPR/Cas13a systems. This activated Cas13a indiscriminately degrades nearby fluorescent RNA reporters, thereby enabling sensitive detection of DNA methylation events. The EBV-positive cell line Raji (in a latent cycle) was treated with drugs and then reactivated to enter the replication cycle. DNA within the cells before and after treatment was collected and quantitatively analyzed directly using a methyl-sensitive CRISPR biosensor. The clinical samples used were as follows: EBV-positive saliva from healthy subjects (n=10) and whole blood from patients with primary EBV infection (n=9). Results: To validate the feasibility of the methyl-CRISPR biosensor, we initially employed model double-stranded DNA constructs with and without methylation to demonstrate that digestion with BstUI/HhaI enzymes was feasible and could rapidly trigger RPA. We subsequently optimized the reaction components and time parameters, confirming its ability to detect methylation-specific targets within 0.5 h. Furthermore, the practical applicability of the method was evaluated using serum samples from EBV-infected individuals, which yielded acceptable results, with recovery rates ranging from 91.3% to 96.9%. Collectively, these findings unequivocally demonstrate that the methyl-CRISPR biosensor provides a viable solution for high-sensitivity DNA methylation analysis while also proving effective for methylation detection in practical clinical applications. Following validation through DNA modeling, we selected four EBV genomic loci (BZLF1, BALF5, LF2, and BDLF2) harboring CCGG motifs for comparative analysis of their methylation status. The fluorescence signal intensity ratios for the methylation markers in the nonactivated cells were 1000-fold higher than those in the reactivated samples across all four loci. This differential methylation pattern, detectable via methyl-CRISPR biosensor-based targeting, demonstrates the ability of this system to quantify latent versus lytic EBV DNA proportions in infected cells. These results underscore the utility of locus-specific methylation profiling as a biomarker for viral replication state discrimination in oncogenic herpesvirus research. Blood samples from nine patients with primary EBV infection were analyzed. Most of the tested genomic regions of EBV exhibited high methylation levels, indicating that the majority of the EBV genomes detected in the peripheral blood during primary infection were in a latent state. In PTLD patients, elevated methylation indices across multiple EBV genomic regions further confirmed the presence of latent EBV DNA. This finding suggests that the observed high viral loads in these patients result from the proliferation of cells harboring latent viral genomes. Conclusions: Overall, our study demonstrates that the methyl-CRISPR biosensor offers a convenient methodology for discriminating between latent and reactivation EBV genomes within biological specimens. We propose that this approach enables rapid, facile, and cost-effective differentiation of the EBV latency status, thereby providing a valuable basis for guiding clinical management and therapeutic strategies.

The performance of CRISPR-mediated genome editing near inverted repeats (IRs) potentially results in chromosomal translocations and other catastrophic rearrangements. However, the extent of this risk may be significantly underestimated because current reporter systems focus solely on site-specific translocations. Here, trans-acting reporter systems in Escherichia coli are developed to detect nontargeted translocations. Markedly increased frequency of translocations following CRISPR-Cas9 activation is observed, with the magnitude determined primarily by the length of the IRs and the proximity between Cas9 target sites and IRs. These translocations arise through a combination of intramolecular single-strand annealing and alternative end-joining mechanisms. Furthermore, it is discovered that introducing segments homologous to IR loci can substantially mitigate nontargeted translocations without significantly compromising CRISPR-Cas9-mediated editing. The study provides valuable insights into the genetic risks associated with CRISPR technologies and suggests a viable strategy for developing genetically safer CRISPR systems.

Rationale: The ultrasensitive and accurate detection of driver mutations is critical for early cancer screening and precision medicine. Current methods face challenges in balancing sensitivity, specificity, and speed, which limits their clinical utility. Therefore, a rapid, sensitive, and specific method is essential for detecting cancer-related SNPs. Methods: This study introduces SPEAR (Specific Point mutation Evaluation via CRISPR-Cas Assisted Recognition), a novel methodology combining NEAR (Nicking Enzyme Amplification Reaction) isothermal amplification with SNP-specific recognition by Cas12b RNP in a one-pot configuration to detect cancer-related single nucleotide polymorphisms (SNPs). SPEAR leverages the power of NEAR isothermal amplification to efficiently amplify target DNA, followed by Cas12b RNP for SNP-specific recognition. This integrated approach ensures a rapid and precise mutation detection system in a single reaction. Results: The method was applied to blood samples for the detection of cancer-related mutations, with results obtained in approximately 30 min. The SPEAR enables detection of gene mutations at the single-molecule level and it can detect targets at a 0.1% ratio despite strong background interference. The method exhibits single-base resolution specificity, allowing for the detection of multiple SNPs in a single reaction. It outperforms first-generation sequencing (FGS) in both convenience and sensitivity, while remaining compatible with next-generation sequencing (NGS). Conclusion: SPEAR offers a rapid, sensitive, and convenient approach to detect cancer-related SNPs, with significant potential for clinical applications, including real-time detection and molecular diagnostics in precision medicine.

The plant hormone jasmonic acid (JA) is pivotal in regulating plant growth and defense mechanisms. Despite extensive research on the JA signaling network, the integration of other factors with JAZ (jasmonate ZIM domain) proteins to modulate JA signaling in response to diverse herbivore attacks remains unclear. In this study, we employed molecular biology and multi-omics techniques to identify an E3 ubiquitin ligase, PtrRZFP4. This E3 ubiquitin ligase exerts a positive regulatory effect on the JA signaling pathway, yet demonstrates differential responses to distinct insect types. We found that upon insect attack, the expression of PtrRZFP4 transcripts is upregulated. In addition, PtrRZFP4 is able to interact with PtrJAZ2 and promote the ubiquitination and degradation of PtrJAZ2 protein. Furthermore, PtrRZFP4 activates the jasmonic acid (JA) signaling pathway at both the transcriptional and metabolic levels, triggering the synthesis of a large number of secondary metabolites related to insect resistance, such as terpenoids and alkaloids. This ultimately enhances the plant's defense against insect herbivory. The larvae of the specialist insect showed a strong preference for feeding on the leaves of transgenic poplar overexpressing PtrRZFP4 (OX-PtrRZFP4), which contain elevated levels of secondary metabolites. In contrast, the larvae of the generalist insect avoid leaves with increased secondary metabolite levels. However, PtrJAZ2 exhibits an opposite function to PtrRZFP4 in the resistance of poplar trees to different herbivores. Therefore, our study uncovers the significant role of the PtrRZFP4-PtrJAZ2 module in plant JA signaling and resistance to insect herbivory, highlighting its potential for biotechnological applications in improving herbivore resistance in forest trees.

To establish a rapid detection method for norovirus GII.2 genotype, this study employed reverse transcription recombinase polymerase amplification (RT-RPA) combined with CRISPR/Cas12a and lateral flow strip (RT-RPA-Cas12a-LFS). Here, the genome of norovirus GII.2 genotype was compared to identify highly conserved sequences, facilitating the design of RT-RPA primers and crRNA specific to the conserved regions of norovirus GII.2. Subsequently, the reaction parameters of RT-RPA were optimized and evaluated using agar-gel electrophoresis and LFS. The results indicate that the conserved sequences of norovirus GII.2 were successfully amplified through RT-RPA at 37°C for 25 minutes. Additionally, CRISPR/Cas12a-mediated cleavage detection was achieved through LFS at 37°C within 10 minutes using the amplification products as templates. Including the isothermal amplification reaction time, the total time is 35 minutes. The established RT-RPA-Cas12a-LFS method demonstrated specific detection of norovirus GII.2, yielding negative results for other viral genomes, and exhibited an excellent detection limit of 10 copies/μl. The RT-RPA-Cas12a-LFS method was further compared with qRT-PCR by analyzing 60 food-contaminated samples. The positive conformity rate was 100%, the negative conformity rate was 95.45%, and the overall conformity rate reached 98.33%. This detection method for norovirus GII.2 genotype is cost-effective, highly sensitive, specific, and easy to operate, offering a promising technical solution for field-based detection of the norovirus GII.2 genotype.

The clinical diagnosis of pathogen infectious diseases increasingly requires sensitive and rapid RNA detection technologies. The RNA-guided clustered regularly interspaced short palindromic repeats (CRISPR)/Cas13a system has shown immense potential in molecular diagnostics due to its trans-cleavage activity. However, most Cas13a-based detection methods require an amplicon transcription step, and the multi-step open-tube operations are prone to contamination, limiting their widespread application. Here, we propose an ultrasensitive (single-copy range, similar to aM) and rapid (within 40 min) isothermal one-pot RNA detection platform, termed SATCAS (Simultaneous Amplification and Testing platform based on Cas13a). This method effectively distinguishes viable bacteria (0%-100%) under constant total bacterial conditions, demonstrating its robustness and universality. SATCAS excels in identifying single nucleotide polymorphisms (SNPs), particularly detecting 0.5% drug-resistant mutations. We validated SATCAS by detecting infections in biological samples from 68 HBV, 23 EBV, and 48 SARS-CoV-2 patients, achieving 100% sensitivity, 92.86% specificity, and 97.06% accuracy in HBV infection testing. We anticipate that SATCAS has broad application potential in the early diagnosis, subtyping, drug resistance detection, and point-of-care monitoring of pathogen infectious diseases.

OBJECTIVE:Hepatocellular carcinoma (HCC) is the third leading cause of cancer-associated death worldwide. As a first-line drug for advanced HCC treatment, lenvatinib faces a significant hurdle due to the development of both intrinsic and acquired resistance among patients, and the underlying mechanism remains largely unknown. The present study aims to identify the pivotal gene responsible for lenvatinib resistance in HCC, explore the potential molecular mechanism, and propose combinatorial therapeutic targets for HCC management.METHODS:Cell viability and colony formation assays were conducted to evaluate the sensitivity of cells to lenvatinib and dicoumarol. RNA-Seq was used to determine the differences in transcriptome between parental cells and lenvatinib-resistant (LR) cells. The upregulated genes were analyzed by GO and KEGG analyses. Then, qPCR and Western blotting were employed to determine the relative gene expression levels. Afterwards, the intracellular reactive oxygen species (ROS) and apoptosis were detected by flow cytometry.RESULTS:PLC-LR and Hep3B-LR were established. There was a total of 116 significantly upregulated genes common to both LR cell lines. The GO and KEGG analyses indicated that these genes were involved in oxidoreductase and dehydrogenase activities, and reactive oxygen species pathways. Notably, NAD(P)H:quinone oxidoreductase 1 (NQO1) was highly expressed in LR cells, and was involved in the lenvatinib resistance. The high expression of NQO1 decreased the production of ROS induced by lenvatinib, and subsequently suppressed the apoptosis. The combination of lenvatinib and NQO1 inhibitor, dicoumarol, reversed the resistance of LR cells.CONCLUSION:The high NQO1 expression in HCC cells impedes the lenvatinib-induced apoptosis by regulating the ROS levels, thereby promoting lenvatinib resistance in HCC cells.

Pneumocystis jirovecii is a prevalent opportunistic fungal pathogen that can lead to life-threatening Pneumocystis pneumonia in immunocompromised individuals. Given that timely and accurate diagnosis is essential for initiating prompt treatment and enhancing patient outcomes, it is vital to develop a rapid, simple, and sensitive method for P. jirovecii detection. Herein, we exploited a novel detection method for P. jirovecii by combining recombinase polymerase amplification (RPA) of nucleic acids isothermal amplification and the trans cleavage activity of Cas12a. The factors influencing the efficiency of RPA and Cas12a-mediated trans cleavage reaction, such as RPA primer, crRNA, the ratio of crRNA to Cas12a and ssDNA reporter concentration, were optimized. Our RPA-Cas12a-based fluorescent assay can be completed within 30–40 min, comprising a 25–30 min RPA reaction and a 5–10 min trans cleavage reaction. It can achieve a lower detection threshold of 0.5 copies/µL of target DNA with high specificity. Moreover, our RPA-Cas12a-based fluorescent method was examined using 30 artificial samples and demonstrated high accuracy with a diagnostic accuracy of 93.33

Versatile, informative, sensitive, and specific nucleic acid detection plays a crucial role in point-of-care pathogen testing, genotyping, and disease monitoring. In this study, we present a novel one-pot Cas12b-based method coupled with the "Green-Yellow-Red" strategy for multiplex detection. By integrating RT-LAMP amplification and Cas12b cleavage in a single tube, the entire detection process can be completed within 1 h. Our proposed method exhibits high specificity, enabling the discrimination of single-base mutations with detection sensitivity approaching single molecule levels. Additionally, the fluorescent results can be directly observed by the naked eye or automatically analyzed using our custom-designed software Result Analyzer. To realize point-of-care detection, we developed a portable cartridge capable of both heating and fluorescence excitation. In a clinical evaluation involving 20 potentially SARS-CoV-2-infected samples, our method achieved a 100% positive detection rate when compared to standard RT-PCR. Furthermore, the identification of SARS-CoV-2 variants using our method yielded results that were consistent with the sequencing results. Notably, our proposed method demonstrates excellent transferability, allowing for the simultaneous detection of various pathogens and the identification of mutations as low as 0.5% amidst a high background interference. These findings highlight the tremendous potential of our developed method for molecular diagnostics.