Revolutionizing aquatic eco-environmental monitoring: Utilizing the RPA-Cas-FQ detection platform for zooplankton.

The integration of recombinase polymerase amplification (RPA) with CRISPR/Cas technology has revolutionized molecular diagnostics and pathogen detection due to its unparalleled sensitivity and trans-cleavage ability. However, its potential in the ecological and environmental monitoring scenarios for aquatic ecosystems remains largely unexplored, particularly in accurate qualitative/quantitative detection, and its actual performance in handling complex real environmental samples. Using zooplankton as a model, we have successfully optimized the RPA-CRISPR/Cas12a fluorescence detection platform (RPA-Cas-FQ), providing several crucial "technical tips". Our findings indicate the sensitivity of CRISPR/Cas12a alone is 5 × 109 copies/reaction, which can be dramatically increased to 5 copies/reaction when combined with RPA. The optimized RPA-Cas-FQ enables reliable qualitative and semi-quantitative detection within 50 min, and exhibits a good linear relationship between fluorescence intensity and DNA concentration (R2 = 0.956-0.974***). Additionally, we developed a rapid and straightforward identification procedure for single zooplankton by incorporating heat-lysis and DNA-barcode techniques. We evaluated the platform's effectiveness using real environmental DNA (eDNA) samples from the Three Gorges Reservoir, confirming its practicality. The eDNA-RPA-Cas-FQ demonstrated strong consistency (Kappa = 0.43***) with eDNA-Metabarcoding in detecting species presence/absence in the reservoir. Furthermore, the two semi-quantitative eDNA technologies showed a strong positive correlation (R2 = 0.58-0.87***). This platform also has the potential to monitor environmental pollutants by selecting appropriate indicator species. The novel insights and methodologies presented in this study represent a significant advancement in meeting the complex needs of aquatic ecosystem protection and monitoring.

Micro(nano)plastics in commercial foods: A review of their characterization and potential hazards to human health.

Micro (nano)plastics (MNPs) are pollutants of worldwide concern for their ubiquitous environmental presence and associated impacts. The higher consumption of MNPs contaminated commercial food can cause potential adverse human health effects. This review highlights the evidence of MNPs in commercial food items and summarizes different sampling, extraction, and digestion techniques for the isolation of MNPs, such as oxidizing digestion, enzymatic digestion, alkaline digestion and acidic digestion. Various methods for the characterization and quantification of microplastics (MPs) are also compared, including µ-Raman spectroscopy, µ-Fourier transform infrared spectroscopy (FTIR), thermal analysis and Scanning electron microscopy with energy-dispersive X-ray spectroscopy (SEM-EDX). Finally, we share our concerns about the risks of MNPs to human health through the consumption of commercial seafood. The knowledge of the potential human health impacts at a subcellular or molecular level of consuming mariculture products contaminated with MNPs is still limited. Moreover, MNPs are somewhat limited, hard to measure, and still contentious. Due to the nutritional significance of fish consumption, the risk of exposure to MNPs and the associated health effects are of the utmost importance.

Organochlorine pesticides (OCPs) in freshwater resources of Pakistan: A review on occurrence, spatial distribution and associated human health and ecological risk assessment.

The extensive use of organochlorine pesticides (OCPs) has resulted in the widespread contamination of different environmental matrices in Pakistan. Freshwater bodies are also prone to OCPs contamination as they receive agricultural and industrial runoff from different sources. In the present study, the data regarding OCPs' fate and distribution in freshwater resources of Pakistan was reviewed and associated risks to human and ecological health were assessed. Among all the OCPs, DDTs were more prevalent with the highest mean concentration of 2290 ng/L observed in River Ravi (Lahore and Sahiwal District). Human health risk assessment showed a higher risk to the children with high Hazard Quotient (HQ) values ranging between 4.1 × 10-9- 295 for Aldrin. The River Ravi (Lahore and Sahiwal District), the River Sutlej (Kasur & Bahawalpur District), and the River Kabul (Nowshehra District) were categorized as high-risk water bodies based on Hazard Index (HI) and Non-Cancer Risk (CRI) index values > 10. Ecological risk assessment revealed a higher risk posed to invertebrate species from DDT exposure. In summary, this review highlights the occurrence and distribution of OCPs and their associated human health and ecological risks in freshwater bodies of Pakistan and also contributes to signifying the need for proper management and regulation of banned pesticides and future research perspectives.

Current understanding of gliomagenesis: from model to mechanism.

Glioma, a kind of central nervous system (CNS) tumor, is hard to cure and accounts for 32% of all CNS tumors. Establishing a stable glioma model is critically important to investigate the underlying molecular mechanisms involved in tumorigenesis and tumor progression. Various core signaling pathways have been identified in gliomagenesis, such as RTK/RAS/PI3K, TP53, and RB1. Traditional methods of establishing glioma animal models have included chemical induction, xenotransplantation, and genetic modifications (RCAS/t-va system, Cre-loxP, and TALENs). Recently, CRISPR/Cas9 has emerged as an efficient gene editing tool with high germline transmission and has extended the scope of stable and efficient glioma models that can be generated. Therefore, this review will highlight the documented evidence about the molecular characteristics, critical genetic markers, and signaling pathways responsible for gliomagenesis and progression. Moreover, methods of establishing glioma models using gene editing techniques and therapeutic aspects will be discussed. Finally, the prospect of applying gene editing in glioma by using CRISPR/Cas9 strategy and future research directions to establish a stable glioma model are also included in this review. In-depth knowledge of glioma signaling pathways and use of CRISPR/Cas9 can greatly assist in the development of a stable, efficient, and spontaneous glioma model, which can ultimately improve the effectiveness of therapeutic responses and cure glioma patients.

Surface state dominated and carbon core coordinated red-emitting carbon dots for the detection of Cr2O72- and cell imaging.

Cr(VI) as a toxic heavy metal ion can easily enter into the body through drinking or eating and cause liver and kidney diseases as well as cancer. Considering its high biological toxicity and adverse effects on human body, it is desirable to develop a probe to monitor its level in the environment. Herein, a high-efficiency fluorescent nanoprobe based on red emissive carbon dots (R-CDs) was established through a convenient solvothermal strategy. The as-prepared CDs with excitation-independency had the fixed emission wavelength at 627 nm when the excitation wavelength was 560 nm. Further study manifested that the new surface state formed by nitrogen and sulfur doping and the increased conjugated system established through dehydration and carbonization were the main reasons for the fluorescence redshift. In this system, these R-CDs as a fluorescent probe exhibited high specificity and sensitivity to Cr2O72- with the linear range of 4-40 µΜ and the limit of detection could reach 80.00 nM. The quenching of these CDs by Cr2O72- was efficiently induced through a static quenching process. Meanwhile, the obtained CDs could enter into HeLa cells through endocytosis and exhibit bright red fluorescence in cells under a confocal laser scanning microscope. Thus, this work provided a promising probe not only for detecting Cr(VI) in natural environment but also for imaging in cells.

Generation and application of a novel high-throughput detection based on RPA-CRISPR technique to sensitively monitor pathogenic microorganisms in the environment.

Staphylococcus aureus (S. aureus) is an important opportunistic human and animal pathogen that can cause a wide diversity of infections. Due to its environmental health risks, it is crucial to establish a time-saving, high-throughput, and highly sensitive technique for water quality surveillance. In this study, we developed a novel method to detect S. aureus in the water environment based on recombinase polymerase amplification (RPA) and CRISPR/Cas12a. This method utilizes isothermal amplification of nucleic acids and the trans-cleavage activity of the CRISPR/Cas12a system to generate fluorescence signals with a single-stranded DNA-fluorophore-quencher (ssDNA-FQ) reporter and a naked-eye detected lateral flow assay (LFA). Our RPA-CRISPR/Cas12a detection system can reduce the detection time to 35 min and enhance the high-throughput detection threshold to ≥5 copies of pathogen DNA, which is more sensitive than that of reported. Moreover, in the lower reaches of the Jialing River in Chongqing, China, 10 water samples from the mainstream and 7 ones from tributaries were successfully monitored S. aureus for less than 35 min using RPA-CRISPR/Cas12a detection system. Taken together, a novel high-throughput RPA-CRISPR detection was established and firstly applied for sensitively monitoring S. aureus in the natural water environment.

Tissue distribution and endocrine disruption effects of chronic exposure to pharmaceuticals and personal care products mixture at environmentally relevant concentrations in zebrafish.

Pharmaceuticals and personal care products (PPCPs) as emerging contaminants are ubiquitously present in the aquatic environment. Using in vivo and in silico techniques, this study aims to elucidate tissue distribution and endocrine disruption effects of chronic exposure (120 days) to PPCP mixture at environmentally relevant concentrations (ERCs) in adult zebrafish. Results from UHPLC-MS/MS analyses showed elevated distribution of PPCPs in zebrafish tissues in the order of liver > gonad > brain. Upregulation of steroid hormone receptors, both gonadotropin, and steroidogenic genes perturb the HPG axis pathway in females, while male fish exhibited significantly downregulated expressions of vtg, cyp17, and 17ßhsd genes with inhibited fecundity. The Spearman correlation indicated a significant positive relationship between PPCPs bioaccumulation and mRNA levels of HPG axis genes. In silico molecular docking (MD) revealed specific amino acid residues of PPCPs binding with zebrafish estrogen receptors. Furthermore, the strongest binding energies of sulfamethoxazole, carbamazepine, and triclosan were discovered in erα and erß estrogen receptors, confirming PPCPs' xenoestrogenic behavior. To summarize, chronic exposure to ERCs resulted in a high accumulation of PPCPs in the liver and gonad tissues of adult zebrafish, as well as associated perturbed genetic responses. As a result, strict environmental regulations for the disposal of PPCPs should be ensured to protect ecological and public health.

Functional kinomics establishes a critical node of volume-sensitive cation-Cl- cotransporter regulation in the mammalian brain.

Cell volume homeostasis requires the dynamically regulated transport of ions across the plasmalemma. While the ensemble of ion transport proteins involved in cell volume regulation is well established, the molecular coordinators of their activities remain poorly characterized. We utilized a functional kinomics approach including a kinome-wide siRNA-phosphoproteomic screen, a high-content kinase inhibitor screen, and a kinase trapping-Orbitrap mass spectroscopy screen to systematically identify essential kinase regulators of KCC3 Thr991/Thr1048 phosphorylation - a key signaling event in cell swelling-induced regulatory volume decrease (RVD). In the mammalian brain, we found the Cl--sensitive WNK3-SPAK kinase complex, required for cell shrinkage-induced regulatory volume decrease (RVI) via the stimulatory phosphorylation of NKCC1 (Thr203/Thr207/Thr212), is also essential for the inhibitory phosphorylation of KCC3 (Thr991/Thr1048). This is mediated in vivo by an interaction between the CCT domain in SPAK and RFXV/I domains in WNK3 and NKCC1/KCC3. Accordingly, genetic or pharmacologic WNK3-SPAK inhibition prevents cell swelling in response to osmotic stress and ameliorates post-ischemic brain swelling through a simultaneous inhibition of NKCC1-mediated Cl- uptake and stimulation of KCC3-mediated Cl- extrusion. We conclude that WNK3-SPAK is an integral component of the long-sought "Cl-/volume-sensitive kinase" of the cation-Cl- cotransporters, and functions as a molecular rheostat of cell volume in the mammalian brain.