Search Results (473)

Microbial contamination in aquatic environments poses severe threats to aquaculture sustainability, ecological balance and public health. Traditional culture-based detection methods, while standardized, are time-consuming and labor-intensive, often failing to meet the urgent need for rapid on-site monitoring required to prevent disease outbreaks and manage water quality effectively. By integrating latest research advances (2020–2025), this study reviews advances in rapid detection technologies for aquatic microorganisms, including the evolution of nucleic acid amplification strategies, with a focused comparison of the analytical sensitivity and field deployability of quantitative polymerase chain reaction (qPCR) and mainstream isothermal amplification techniques (loop-mediated isothermal amplification, LAMP; recombinase polymerase amplification, RPA). Furthermore, this study reports on the emergence of Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)-associated protein (Cas) systems as next-generation diagnostic tools, highlighting their integration with microfluidic Lab-on-a-Chip (LOC) platforms to achieve attomolar sensitivity. We also consider the application of portable nanopore sequencing for real-time pathogen identification and the growing role of Artificial Intelligence (AI) in analyzing complex diagnostic datasets. Advanced molecular methods have achieved significant reductions in time consumption—from days to less than one hour—while challenges regarding sample preparation and environmental matrix inhibition remain. The future of aquatic monitoring lies in integrated, automated systems that combine the specificity of CRISPR-Cas diagnostics with the connectivity of IoT-enabled biosensors. Comparative analysis indicates that isothermal amplification methods (LAMP, RPA) coupled with CRISPR-Cas systems offer the optimal balance of sensitivity, speed, and field deployability for point-of-care aquaculture diagnostics, while qPCR/dPCR remain indispensable for quantitative regulatory applications. We propose a structured technology selection framework to guide researchers and practitioners in choosing appropriate detection modalities based on specific sensitivity, cost, throughput, and deployment requirements. Full article

Telomerase reverse transcriptase subunit (TERT) is a key factor involved in telomere maintenance and genome stability, and the decline in its expression is closely related to cellular senescence. In this study, we established TERT monoallelic knockout (TERT+/−) and TERT overexpression (TERT-Over) cell lines in porcine iliac artery endothelial cells (PIEC) using CRISPR/Cas9 and PiggyBac systems to compare the effects of TERT monoallelic knockout versus overexpression on cellular biology. TERT expression and telomere length were assessed via qPCR and Western blot analysis. Cellular proliferation and senescence were evaluated using CCK-8 assays, cell cycle analysis, and SA-β-gal staining. Furthermore, the expression of key genes involved in cell proliferation, metabolism, and related signaling pathways was quantified using q-PCR. The results showed that the TERT mRNA level and telomere length decreased in TERT+/− cells. Meanwhile, we also observed that TERT+/− cells exhibited G1 phase arrest in the cell cycle, with suppressed proliferation and increased SA-β-gal-positive cells. This was accompanied by downregulation of cell cycle and proliferation-related genes, including c-Myc, the E2F family, and Ki-67, as well as downregulation of cell metabolism-related genes, including HIF1α, HK2, GLUT1, the SMAD family, FOXO1, and ATF4. In addition, cytochrome C was downregulated, suggesting activation of mitochondrial apoptotic signaling. Together, these findings indicate impaired proliferative and metabolic activity and are consistent with cellular senescence associated with telomere shortening. In TERT-overexpressing cells, the TERT gene expression and telomere length increase, cell proliferation accelerates, and the survival rate significantly increases under H₂O₂ treatment. This indicated that the overexpression of TERT can enhance resistance to oxidative stress, thus showing a kind of anti-aging phenotype. In conclusion, TERT monoallelic knockout induces cellular senescence-associated phenotypes in porcine endothelial cells, whereas TERT overexpression enhances proliferation and resistance to oxidative stress under the experimental conditions used in this study. The two porcine cell models established here may provide useful experimental materials for studying aging-related mechanisms and evaluating anti-aging interventions in large animals. Further studies are needed to directly determine their effects on cellular replicative lifespan. Full article

Lactococcus garvieae is a major bacterial pathogen responsible for lactococcosis outbreaks in aquaculture, resulting in substantial economic losses worldwide. Accurate identification of L. garvieae remains challenging because of its genetic similarity to other Lactococcus species and the limited field applicability of many existing molecular diagnostic methods. Therefore, there is an urgent need for a rapid, highly specific, and field-deployable analytical method that enables accurate identification of L. garvieae outside conventional laboratory settings. In this study, a one-pot analytical strategy integrating enzymatic recombinase amplification (ERA) with CRISPR/Cas12a detection was developed, enabling fluorescence or lateral flow dipstick (LFD) readouts within a single closed reaction tube. The one-pot ERA-CRISPR/Cas12a assays achieved a detection limit of 10 copies/reaction. When combined with a rapid DNA release protocol, qualitative detection could be completed within 50 min without the need for sophisticated instrumentation. In parallel, a TaqMan quantitative PCR assay was established as an analytical benchmark, exhibiting a detection limit of 20 copies/reaction with high linearity and good reproducibility. Clinical evaluation using 136 diseased fish samples demonstrated full concordance between the one-pot ERA-CRISPR/Cas12a and qPCR assays, with both methods achieving a positive detection rate of 23.5% (32/136). In addition, the ERA-CRISPR/Cas12a platform was successfully validated under simulated field conditions using a portable reaction device. This study presents a rapid and field-deployable CRISPR-based platform for the early detection and epidemiological surveillance of lactococcosis. Full article

Sexual reproduction is the predominant mode of reproduction in animals, and spermatogenesis is the fundamental step in this process. As the model organism for lepidopteran, the silkworm Bombyx mori exhibits typical dichotomous spermatogenesis, producing both nucleated (eupyrene) and anucleate (apyrene) sperm. Leucylaminopeptidases (LAPs), members of the M17 metalloprotease family, are characterized by their ability to cleave leucine residues from the N-terminus of peptides. In addition to this canonical function, they have been implicated in male fertility in mammals and Diptera. Nevertheless, whether LAPs are required for dimorphic spermatogenesis in Lepidoptera remains to be clarified. Here, we demonstrated that Sperm-Leucylaminopeptidase (S-LAP) plays vital roles in the silkworm eupyrene sperm development. Similar to the testis-specific expression pattern of eight S-LAPs in Drosophila melanogaster, BmS-LAP was also predominantly expressed in testis. Depletion of BmS-LAP via CRISPR/Cas9 system resulted in male sterility, while the fertility of female mutant was unaffected. Notably, male mutants displayed severe defects in the formation and migration of eupyrene sperm, whereas apyrene sperm development appeared normal. In addition, RNA-seq and qRT-PCR analyses demonstrated that spermatogenesis defects were associated with energy metabolism and flagellar assembly. Our study provides the first evidence that LAP is necessary for dimorphic spermatogenesis in Lepidopteran, offering new insights into the molecular basis of male infertility. Full article

Fascioliasis, a globally prevalent zoonosis, severely threatens public health and livestock security. Current diagnostic approaches, hindered by the need for sophisticated instrumentation and specialized expertise, are inadequate for on-site surveillance in resource-constrained settings. This study developed a rapid, visual detection assay for Fasciola hepatica via recombinase-aided amplification (RAA) integrated with CRISPR/Cas12b, addressing critical equipment and operational constraints. Targeting a specific mitochondrial DNA fragment of F. hepatica, recombinant plasmid standards were constructed, RAA primers and sgRNA optimized, and three detection modalities (real-time fluorescence, UV lamp, test strip) integrated. Clinical validation against PCR demonstrated 45 min turnaround time, F. hepatica-specific positivity, and real-time fluorescence sensitivity of 2.6 copies/μL. Results showed high concordance with PCR and qPCR, with substantially reduced assay duration and streamlined workflow. This highly sensitive, specific, multi-visualized method overcomes limitations of conventional techniques, offering an efficient, field-deployable tool for fascioliasis surveillance and control in grassroots and pastoral regions. Full article

Background/Objectives: Canonical microRNAs possess a 5′ phosphate required for Argonaute binding and activity. However, prior work identified an unphosphorylated, inactive nuclear pool of the important radiation-responsive microRNA, miR-34, that is rapidly phosphorylated and activated in response to ionizing radiation (IR). Here, we extend this work and investigate the role of paraspeckles, a phase-separated nuclear sub-compartment, and their association with the localization of unphosphorylated miR-34a. Methods: Mass spectrometry was performed to identify interacting partners of unphosphorylated mir-34. CRISPR-mediated deletion of the paraspeckle NEAT1_2 triple helix motif was performed to create an A549 cell line lacking paraspeckles (dTH). Activity and expression of mir-34a post-irradiation were evaluated by qRT-PCR and luciferase assays comparing dTH and wild-type (WT) A549 cell lines. In situ hybridization (ISH) was performed to evaluate mir-34a localization before and after IR, comparing dTH and WT cell lines. Results: Mass spectrometry identified paraspeckle proteins as significantly enriched interacting partners of unphosphorylated mir-34 mimics. By qRT-PCR and luciferase assays, we found that paraspeckle loss prevented radiation-induced early activation of unphosphorylated mir-34a. We found no difference in radiation-induced transcription of pri-miR-34a, but early processing to pre-miR-34a appeared delayed. ISH confirmed that loss of paraspeckles altered the nuclear localization of miR-34a before and after IR. Conclusions: These data suggest that paraspeckles are associated with nuclear localization and early radiation-responsive activation of unphosphorylated miR-34a. This suggests a coordinated nuclear sequestration of this important miR in its unphosphorylated state to enable an enhanced radiation response. Full article

The modulation of plant responses to abscisic acid (ABA) and/or abiotic stresses can be manipulated by the expression of ABA-responsive genes, which is affected by phytohormone ABA. While some ABA-responsive genes have been shown to regulate plant responses to ABA and/or abiotic stresses, the functions of numerous ABA-responsive genes remain unknown. Therefore, characterizing these unstudied genes would provide a practical way to identify novel regulators of plant adaptations to ABA and/or abiotic stresses. Here, we characterized four closely related unstudied ABA-responsive genes in Arabidopsis thaliana, named Arabidopsis thaliana ABA-up regulated genes (AtAUGs). We found that ABA treatment induces AtAUGs expression level, and our results in transfected protoplasts show that AtAUGs exhibit nucleus localization and downregulate the co-transfected reporter expression level. The results of ABA sensitivity assays, including seed germination, cotyledon greening, and root extension assay show that transgenic plants overexpressing AtAUGs had increased sensitivity, but ataugs mutants generated by isolating T-DNA insertion lines or through CRISPR/Cas9 gene-editing of AtAUGs had decreased sensitivity; in addition, the greatest decrease in ABA sensitivity was observed in the ataug1 ataug2 ataug3 ataug4 (ataug1234) quadruple mutants. The qRT-PCR results show that the expression levels of several Type 2C Protein Phosphatase (PP2C) genes, the key negative regulator genes of ABA signaling including PP2CA, Hypersensitive to ABA 1 (HAB1), HAB2, Highly ABA-Induced PP2C protein 3 (HAI3), ABA-Hypersensitive Germination 1 (AHG1), and ABA Insensitive 2 (ABI2) decreased in 35S:AtAUGs transgenic plants, but increased in the ataug1234 quadruple mutants. Taken together, these results suggest that AtAUGs are ABA-responsive genes, and AtAUGs positively regulate ABA responses in a redundant manner, by downregulating the expression of crucial negative regulator genes in ABA signaling. Full article

In recent years, pigeon rotavirus A (PiRVA) infection, an important emerging disease, has posed a major threat to the healthy development of the pigeon industry and public health. Therefore, developing an accurate, rapid and convenient detection method for this virus is vital for monitoring and early diagnosis of the disease. In this study, on the basis of the ORF sequence characteristics of the PiRVA VP6 gene, crRNA and reverse transcription recombinase-aided amplification (RT-RAA) primers were designed. On the basis of the CRISPR/Cas12a system, for the first time, the RT-RAA-CRISPR/Cas12a rapid detection method of PiRVA was established by combining RT-RAA and lateral flow strips. This method could specifically detect PiRVA, and there was no cross-reaction with other common viruses originating from pigeons. The minimum detection limit was 16.8 copies/μL, and the results of the intrabatch and interbatch repeated tests were consistent. Moreover, the method established in this study and the previously established common PCR method were used to analyse 56 clinical tissue samples from racing pigeons and domestic pigeons collected in 2025. The positive rates of racing pigeon and domestic pigeon samples detected by PCR were 17.6% and 12.8%, respectively, and the positive rates of racing pigeon and meat pigeon samples detected by the RT-RAA-CRISPR/Cas12a method were 23.5% and 17.9%, respectively, indicating that PiRVA infection occurs in both racing pigeon and domestic pigeon populations in China. In summary, the PiRVA RT-RAA-CRISPR/Cas12a detection method established in this study has good specificity, sensitivity, and reproducibility, and allows visualization of the results, which can be used for field applications. This study provides technical support for epidemiological surveillance and etiological research on PiRVA. Full article

Molecular point-of-care testing (POCT) for respiratory infections has undergone remarkable advancement over the past two decades, driven by technological innovation and urgent clinical needs highlighted by the COVID-19 pandemic. This comprehensive systematic review was conducted following PRISMA 2020 guidelines, synthesizing evidence from 254 peer-reviewed studies published between 2006 and 2026, with detailed analysis of the 30 most relevant papers selected through a rigorous four-stage screening process. The review examines the evolution of molecular POCT technologies, including reverse transcription polymerase chain reaction (RT-PCR), loop-mediated isothermal amplification (LAMP), recombinase polymerase amplification (RPA), and CRISPR-based detection systems. Key findings demonstrate that modern molecular POCT platforms achieve diagnostic performance comparable to laboratory-based testing, with sensitivities ranging from 88% to 100% and specificities from 98% to 100%, while delivering results in 15 to 80 min. These technologies enable rapid, accurate detection of major respiratory pathogens, including SARS-CoV-2, influenza A/B, respiratory syncytial virus (RSV), and atypical bacteria. The integration of microfluidic systems, portable devices, and smartphone-based analysis has expanded access to testing in resource-limited settings, emergency departments, and wearable platforms. This review provides critical insights for clinicians, researchers, and policymakers regarding the current state, clinical applications, and future directions of molecular POCT for respiratory infections. 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

Bacilus cereus and Pseudomonas fluorescens are major foodborne psychrotrophic bacteria posing global health and economic risks. B. cereus has a 23.8% food prevalence worldwide. P. fluorescens is a leading cause of spoilage in refrigerated products. Their rapid detection is crucial for food safety. However, existing detection methods often rely on open-tube operations, risking aerosol contamination. In this study, we developed two independent one-tube RPA-CRISPR/Cas12a visual detection assays for B. cereus and P. fluorescens. Using a physical separation design, the recombinase polymerase amplification (RPA) and CRISPR/Cas12a detection were pre-assembled in a single reaction tube. After incubation, a brief centrifugation combined the components for enclosed detection. This step is compatible with portable mini-centrifuges. The assays can be completed within 40 min at 37 °C, with results visualized directly under blue light. Both assays demonstrated good specificity against six common non-target pathogens. The visual detection limits were 5.1 × 10¹ copies/μL for B. cereus and 2.1 × 10¹ copies/μL for P. fluorescens. Each assay was applied to 14 types of real-world food samples (naturally contaminated and uncontaminated, confirmed by PCR), achieving 100% concordance with conventional PCR. The one-tube assays are tailored for psychrotrophic bacteria in refrigerated foods. They minimize aerosol contamination risk and provide a reliable solution for on-site cold-chain food safety monitoring. Full article

Triple-negative breast cancer (TNBC) lacks effective targeted therapies, and the mechanisms by which developmental endothelial locus-1 (EDIL3/Del-1) promotes TNBC remain incompletely defined. We profiled Del-1 and AMPK subunits in TNBC cell lines by RT-PCR and immunoblotting, performed functional assays in CRISPR/Cas9 Del-1 knockout and AMPKβ-manipulated cells, and evaluated AMPKβ in early-stage TNBC tumors using tissue microarrays (TMA) (immunohistochemistry; n = 100) and AMPKβ2 mRNA quantification. Del-1 and AMPKβ were enriched in TNBC cells, most prominently in the mesenchymal stem-like subtype, whereas AMPKα levels were relatively stable. Increased Del-1 and activated AMPKβ enhanced proliferation and invasion, while Del-1 deletion reduced AMPKβ expression and suppressed tumor-promoting phenotypes. Mechanistically, Del-1 increased AMPKβ phosphorylation at serine 108, and a phospho-mimetic AMPKβ mutant further amplified oncogenic effects. In the pilot TMA study, high AMPKβ protein expression showed a trend toward poorer DFS in Kaplan–Meier analysis, while multivariate analysis identified high AMPKβ protein expression as an independent factor associated with poorer DFS in patients with early TNBC. These data support AMPKβ as a key mediator of Del-1-driven signaling and suggest AMPKβ could be a therapeutic target in aggressive TNBC subsets. Full article

Hepatocellular carcinoma (HCC) remains a leading cause of cancer-related death, yet adequate in vitro models mimicking the tumor immune microenvironment (TIME) are rare. Specifically, the role of the transcription factor nuclear factor erythroid 2-related factor 2 (Nrf2) in modulating interactions between tumor cells and tumor-associated macrophages (TAMs) is not fully understood. We established a 3D multicellular tumor spheroid (MCT) model using murine N-HCC25 cells with CRISPR/Cas9-mediated knockouts of Nrf2 and its negative regulator Kelch-like ECH-associated protein 1 (Keap1), the latter mimicking constitutive activation. N-HCC25 cells were co-cultured with bone marrow-derived macrophages (BMDMs) isolated from wild-type and Nrf2-knockout C57BL/6J mice. We compared co-culture setups (conditioned media, transwell systems, direct contact) using RT-qPCR, flow cytometry, and invasion assays. 3D spheroid systems better preserved stemness than 2D cultures and revealed functional Nrf2-dependent effects such as increased Vegf-α secretion in Keap1-deficient spheroids. Among the different co-cultivation models, the most profound effects were observed in the MCT model. Macrophages successfully integrated into the spheroids and triggered invasive outgrowth, whereas MCTs containing Nrf2-deficient macrophages displayed markedly reduced tumor spheroid growth and lower programmed cell death ligand-1 expression. These findings demonstrate that Nrf2 signaling in macrophages fosters an immunosuppressive and pro-invasive microenvironment. The established MCT model provides a suitable platform to further unravel Nrf2-dependent mechanisms in the HCC TIME. Full article

The small molecule SR8278 was initially identified as an antagonist of the REV-ERB (reverse c-ERBAa) nuclear receptor proteins, which play important roles in metabolism and circadian rhythms. Though SR8278 has been shown to have beneficial physiological effects in a variety of different preclinical disease contexts, its impact on gene expression and cell proliferation in keratinocytes has not previously been examined. We therefore carried out an RNA-seq analysis and found that genes involved in the G1/S transition of the cell cycle were significantly impacted by SR8278 treatment, and these effects were confirmed at both the RNA and protein level by RT-qPCR and Western blotting, respectively. Cell proliferation assays showed that SR8278 slowed cell growth but did not induce genotoxic stress or apoptosis. Finally, the use of CRISPR/Cas9 genome editing and siRNA-mediated disruption of REV-ERB gene expression showed that the loss of the REV-ERB proteins did not impact the effect of SR8278 on gene expression and cell proliferation. We conclude that the anti-proliferative effects of SR8278 are not mediated by the REV-ERB proteins, and, thus, care should be taken when interpreting studies involving this compound unless complementary genetic approaches are also shown, particularly in studies involving cell proliferation. Full article

As a tropical fruit of considerable economic importance, passionfruit (Passiflora edulis Sims) is extensively cultivated in the tropical and subtropical regions of China; however, the widespread incidence of viral diseases has significantly hampered the safety of its production. Rapid, sensitive, and visual detection of plant viruses is essential to effectively prevent and manage these viral diseases. In this study, we developed a visual detection system (displaying under blue light or UV) utilizing reverse transcription recombinase-aided amplification (RT-RAA) in conjunction with CRISPR/Cas12a to detect three viruses harmful to passionfruit production: telosma mosaic virus (TelMV), East Asian passiflora virus (EAPV), and passiflora mottle virus (PaMoV). Within this system, the optimal primer concentration for the RT-RAA reaction was determined to be 0.4 μM for all three viruses, with an optimal reaction temperature of 37 °C. The optimal reaction times were established as 20 min for TelMV, 15 min for EAPV, and 30 min for PaMoV. The entire detection process could be completed within 30 min without the need for sophisticated equipment or instruments. For TelMV and EAPV, the detection system demonstrated the capability to detect samples at a dilution of 10⁶, representing an approximately 10⁴-fold improvement over RT-PCR, while for PaMoV, it could identify samples at a dilution of 10⁶, representing an approximately 10²-fold improvement over traditional RT-PCR methods. These results confirm the successful development of the CRISPR/Cas12a-based detection systems. Subsequently, the system was applied for in situ detection of the three target viruses in field settings, yielding results that were fully consistent with laboratory-based RT-PCR assays, a consistency which underscores the system’s strong potential for field application in detecting important crop viruses. Full article