Fluorescent Indicator Research Articles

DYE 10-N-NONYL ACRIDINE ORANGE AS A FLUORESCENT INDICATOR OF THE ACTION OF THE ANTISEPTIC OCTENIDINE ON THE MEMBRANES OF RHODOBACTER SPHAEROIDES CHROMATOPHORES

Increasing volumes of produced antiseptics and disinfectants, which are widely used in medicine, veterinary medicine, the food industry and everyday life, can create a serious environmental problem after use. Previously, under the infl uence of micromolar concentrations of a number of antiseptics, we identifi ed disturbances in the functioning of photosynthetic membranes and phototransforming pigment-protein complexes isolated from them in various representatives of photosynthetic organisms. In this work, to determine the sensitivity of photosynthetic membranes to the action of the cationic antiseptic octenidine, we used chromatophores of the purple nonsulfur bacteria Rhodobacter sphaeroides, labeled with the fluorescent dye 10-N-nonylacridine orange (NAO). It was shown that the binding of NAO to chromatophores is accompanied by a shift in the dye emission maximum from 525 to 640 nm. The “red” fluorescence of NAO associated with chromatophores turned out to be sensitive to the effect of increasing concentrations of octenidine on photosynthetic membranes. We have found antiseptic concentrations which led to the degradation of chromatophore structures and change in the NAO aggregative state. It can be detected by an increase of “green” fluorescence in the emission spectra of the dye. The properties of NAO as a fluorescent indicator of the functional state of photosynthetic membranes and possible changes that can occur in such systems under the influence of a cationic antiseptic are discussed.

Unfolding the role of cell state on stress granule heterogeneity and function in pancreatic cancer.

e16357 Background: A major impediment to cancer therapy is the reality that cancer is a heterogeneous disease. A single tumor can consist of many genotypic or phenotypic profiles, which are marked by degrees of sensitivity to different therapeutics. Identifying and targeting origins of cancer cell heterogeneity is critical for predicting patient outcome and enhancing treatment efficacy. We observe a novel source of cancer cell heterogeneity that involves one of the largest stress-sensing platforms described to date: the stress granule (SG). Stress granules are non-membranous, cytoplasmic organelles that assemble in response to stress, and promote cell survival. Deregulation of SGs has been linked to several pathological conditions, including cancer, neurodegeneration, ischemia, and viral infections. SGs have been shown to promote tumor growth and metastasis, as well as contribute to mechanisms of chemo-resistance. Thus, these signaling hubs are emerging as a potential target in cancer. Methods: To induce SG formation, human pancreatic ductal adenocarcinoma (PDAC) cell lines were treated with sodium arsenite, thapsigargin, or exposed to hypoxic conditions. Immunoflourescent staining of core SG proteins was then conducted. To investigate cell cycle-driven mechanisms of SG heterogeneity, cell cycle stage was identified by expression of a Fluorescent ubiquitination-based cell cycle indicator (Fucci) lentiviral system. To investigate cPLA2’s role in SG formation, the chemical inhibitor Pyrrophenone, an shRNA targeting cPLA2, and an exogenous full-length or cleaved form of cPLA2, were used. In vivo, ES149 cells expressing Luciferase and the Fucci system were orthotopically implanted into C57BL/6 mice. Established tumors were treated every 3 days with Vehicle, Oxaliplatin, Pyrrophenone, or a combination of the latter. Tumor size was tracked via IVIS Spectrum. Fucci flourescence was used to identify cell cycle phase. SGs and cell death were assessed by immunoflourescent staining of tumors. Results: Cancer cells show a high degree of heterogeneity in their production of SGs. Cell cycle phase dictates SG heterogeneity. G2 phase cells have significantly higher levels of SG formation as compared cells in G1/S. This process is regulated by cleavage of the cPLA2 protein. Oxaliplatin administration increases G2 phase content in tumors. Co-treatment with Oxaliplatin and Pyrrophenone significantly decreases SG formation, increases cell death, and leads to tumor regressions in vivo. Conclusions: G2 phase cancer cells develop higher levels of SGs when exposed to stressful stimuli, as compared to G1/S phase counterparts. The cPLA2 protein is cleaved into a catalytically dead form that is present in G1/S phases, but absent in G2 phase. Oxaliplatin treatment in vivo leads to an increase in G2 phase tumor cells. When paired with cPLA2 inhibition, this results in increased cancer cell death and decreased tumor growth.

Development of a genetically encoded NMT indicator for detection of mercury ions based on the green fluorescent protein mNeonGreen and metallothionein II from rat liver

Heavy metals, particularly mercury, rank as some of the most hazardous systemic toxicants known to cause multiple organ damage, even at lower levels of exposure. Its detection in the environment and in the live cells is an actual task. Here, we engineered a novel genetically encoded fluorescent NMT indicator for mercury ions by inserting the metallothionein II domain from rat liver into the bright green-yellow fluorescent protein mNeonGreen, followed by directed molecular evolution of the resulting sensor prototype in bacteria. In solution, the NMT indicator was 1.7-fold brighter than the standard eGFP fluorescent protein and responded to the addition of even 10−18-10−19 M mercury ions by quenching fluorescence with a 5-fold fluorescence response and extremely high affinity to mercury ions characterized by the Kd value of 0.50 ± 0.05 aM. We also characterized the selectivity of the NMT indicator to other metal cations. In cultured mammalian cells, the NMT indicator detected even an extracellular concentration of 0.1 fM mercury ions and achieved a 5.9-fold change in ΔF/F fluorescence intensity.

Mechanism of Purinergic Regulation of Neurotransmission in Mouse Neuromuscular Junction: The Role of Redox Signaling and Lipid Rafts.

Acetylcholine is the main neurotransmitter at the vertebrate neuromuscular junctions (NMJs). ACh exocytosis is precisely modulated by co-transmitter ATP and its metabolites. It is assumed that ATP/ADP effects on ACh release rely on activation of presynaptic Gi protein-coupled P2Y13 receptors. However, downstream signaling mechanism of ATP/ADP-mediated modulation of neuromuscular transmission remains elusive. Using microelectrode recording and fluorescent indicators, the mechanism underlying purinergic regulation was studied in the mouse diaphragm NMJs. Pharmacological stimulation of purinoceptors with ADP decreased synaptic vesicle exocytosis evoked by both low and higher frequency stimulation. This inhibitory action was suppressed by antagonists of P2Y13 receptors (MRS 2211), Ca2+ mobilization (TMB8), protein kinase C (chelerythrine) and NADPH oxidase (VAS2870) as well as antioxidants. This suggests the participation of Ca2+ and reactive oxygen species (ROS) in the ADP-triggered signaling. Indeed, ADP caused an increase in cytosolic Ca2+ with subsequent elevation of ROS levels. The elevation of [Ca2+]in was blocked by MRS 2211 and TMB8, whereas upregulation of ROS was prevented by pertussis toxin (inhibitor of Gi protein) and VAS2870. Targeting the main components of lipid rafts, cholesterol and sphingomyelin, suppressed P2Y13 receptor-dependent attenuation of exocytosis and ADP-induced enhancement of ROS production. Inhibition of P2Y13 receptors decreased ROS production and increased the rate of exocytosis during intense activity. Thus, suppression of neuromuscular transmission by exogenous ADP or endogenous ATP can rely on P2Y13 receptor/Gi protein/Ca2+/protein kinase C/NADPH oxidase/ROS signaling, which is coordinated in a lipid raft-dependent manner.

Dynamic changes in dissolved organic matter during transport of landfill leachate in porous medium.

The dynamic changes in dissolved organic matter (DOM) during the transport of landfill leachate (LL) in porous medium should be explored, considering the high levels of DOM in the LL of municipal solid waste. Column experiments were carried out at 25°C at a Darcy's flux of 0.29cm/h for 2722h to compare the transport of Cl-, ultraviolet absorbance at 254nm (UV254), chemical oxygen demand (COD), and dissolved organic carbon (DOC) in the simulated porous medium by using the CXTFIT2.1 code. Results showed that the convection-dispersion equation (CDE) could describe Cl- transport well. The high levels of λ and D could be highly correlated with the physicochemical properties of the porous medium. The transport of the studied DOM with evident aromatic character could be described appropriately by the CDE model with the first-order reaction assumption, considering the similar variation trends of UV254, COD, and DOC in the effluent during experiments. Specifically, the values of retardation factor (R) were in the following order: DOC > UV254 > COD, whereas the low values of the first-order decay coefficient (k1) for DOC and COD were still higher than that for UV254. High contents of humic-like substances in the DOM with complex toxic components resulted in the natural low removal efficiencies of COD, DOC, and UV254 (≤ 23%), which could be confirmed by the variations of fluorescence index (FI) and humification index (HIX) in the effluent. The results should be helpful in evaluating the environmental risk induced by the LL leakage in a landfill site.

Real-time blood gas analysis using pH, PO2 and PCO2 multifunctional fluorescent capillary sensing system

Real-time blood gas analysis is of great significance in disease diagnosis and treatment. Here we have developed a pH, PO2 and PCO2 multifunctional fluorescent capillary sensing system. The pH, O2 and CO2 fluorescent indicators with same excitation wavelength are fixed into a single capillary tube. The sensing system possesses sensitive fluorescence response to pH, O2 and CO2, and the changes of pH, O2 and CO2 in buffer, in vitro, and in vivo can be detected simultaneously by measuring the fluorescence signals of sensor probe. The PO2, PCO2 and pH of rabbits in four disease models were assessed efficiently by the sensing system, exhibiting similar variation trend with the results by blood gas analyzer. Studies in living rabbit display the reasonable and favorable feasibility of capillary sensing system in real-time arterial blood gas analysis. Overall, the multifunctional fluorescent capillary sensing system exhibits significant potential in real-time arterial blood gas analysis and clinical application of disease diagnosis.

Modulation of the p75NTR during Adolescent Alcohol Exposure Prevents Cholinergic Neuronal Atrophy and Associated Acetylcholine Activity and Behavioral Dysfunction.

Binge alcohol consumption during adolescence can produce lasting deficits in learning and memory while also increasing the susceptibility to substance use disorders. The adolescent intermittent ethanol (AIE) rodent model mimics human adolescent binge drinking and has identified the nucleus basalis magnocellularis (NbM) as a key site of pathology. The NbM is a critical regulator of prefrontal cortical (PFC) cholinergic function and attention. The cholinergic phenotype is controlled pro/mature neurotrophin receptor activation. We sought to determine if p75NTR activity contributes to the loss of cholinergic phenotype in AIE by using a p75NTR modulator (LM11A-31) to inhibit prodegenerative signaling during ethanol exposure. Male and female rats underwent 5 g/kg ethanol (AIE) or water (CON) exposure following 2-day-on 2-day-off cycles from postnatal day 25-57. A subset of these groups also received a protective dose of LM11A-31 (50 mg/kg) during adolescence. Rats were trained on a sustained attention task (SAT) and behaviorally relevant acetylcholine (ACh) activity was recorded in the PFC with a fluorescent indicator (AChGRAB 3.0). AIE produced learning deficits on the SAT, which were spared with LM11A-31. In addition, PFC ACh activity was blunted by AIE, which LM11A-31 corrected. Investigation of NbM ChAT+ and TrkA+ neuronal expression found that AIE led to a reduction of ChAT+TrkA+ neurons, which again LM11A-31 protected. Taken together, these findings demonstrate the p75NTR activity during AIE treatment is a key regulator of cholinergic degeneration.

Optimization of sample preparation, fluorescence- and Raman techniques for environmental microplastics

Microspectroscopic imaging techniques based on spontaneous Raman scattering, Stimulated Raman Scattering (SRS), or fluorescence (with a selective dye) can be used to detect environmental microplastics (MPs) and determine their chemical as well as physical properties. The present study first focuses on optimizing the sample preparation, including a new design for a density separation apparatus and optimization of the Nile Red staining procedure. Tests were carried out with both white and colored reference materials, as well as environmental MPs in a suspended matter sample from the Rhine river. The new ‘MESSY’ system has a mean recovery of 95 ± 5.5 % (three polymer materials, in duplicate). The optimized Nile Red staining allows coarse categorization of MPs into “polar” vs. “non-polar” materials based on their Fluorescence Index (emission wavelength), but fluorescent additives in the polymer can cause misclassification. For unambiguous identification of the polymer type, Raman spectroscopy can be used. Even colored polymers, with or without Nile Red staining, were readily identified by Raman spectroscopy using a red laser (785 nm), except for particles containing carbon black. A Deep-UV Raman microscope (ex = 248.6 nm) was constructed, which allowed identification of all colored plastics, even those pigmented with carbon black. Since unsupervised mapping with spontaneous Raman is very slow, point measurements are preferably used after preselection of particles of interest based on fluorescence imaging. SRS is several orders of magnitude faster than spontaneous Raman mapping but requires multiple scans at different z-heights and at multiple wavenumber settings to detect and identify all particles. The results are expected to contribute to the development of suitable methodologies for the detection and identification of environmental microplastics.

Fabrication of a Cancer Cell Aggregate Culture Device That Facilitates Observations of Nutrient and Oxygen Gradients.

Three-dimensional cell culture spheroids are commonly used for drug evaluation studies because they can produce large quantities of homogeneous cell aggregates. As the spheroids grow, nutrients supplied from outer spheroid regions render the inner spheroid areas hypoxic and hyponutrient, which makes them unobservable through confocal microscopy. In this study, we fabricated a cancer cell aggregate culture device that facilitates the observation of nutrient and oxygen gradients. An alginate gel fiber was created in the cell culture chamber to ensure a flow path for supplying the culture medium. A gradient of nutrients and oxygen was generated by positioning the flow channel close to the edge of the chamber. We devised a fabrication method that uses calcium carbonate as a source of Ca2+ for the gelation of sodium alginate, which has a slow reaction rate. We then cultured a spheroid of HCT116 cells, which were derived from human colorectal carcinoma using a fluorescent ubiquitination-based cell cycle indicator. Fluorescence observation suggested the formation of a hypoxic and hyponutrient region within an area approximately 500 µm away from the alginate gel fiber. This indicates the development of a cancer cell aggregate culture device that enables the observation of different nutrition and oxygen states.

Effects of Akt Activator SC79 on Human M0 Macrophage Phagocytosis and Cytokine Production.

Akt is an important kinase in metabolism. Akt also phosphorylates and activates endothelial and neuronal nitric oxide (NO) synthases (eNOS and nNOS, respectively) expressed in M0 (unpolarized) macrophages. We showed that e/nNOS NO production downstream of bitter taste receptors enhances macrophage phagocytosis. In airway epithelial cells, we also showed that the activation of Akt by a small molecule (SC79) enhances NO production and increases levels of nuclear Nrf2, which reduces IL-8 transcription during concomitant stimulation with Toll-like receptor (TLR) 5 agonist flagellin. We hypothesized that SC79's production of NO in macrophages might likewise enhance phagocytosis and reduce the transcription of some pro-inflammatory cytokines. Using live cell imaging of fluorescent biosensors and indicator dyes, we found that SC79 induces Akt activation, NO production, and downstream cGMP production in primary human M0 macrophages. This was accompanied by a reduction in IL-6, IL-8, and IL-12 production during concomitant stimulation with bacterial lipopolysaccharide, an agonist of pattern recognition receptors including TLR4. Pharmacological inhibitors suggested that this effect was dependent on Akt and Nrf2. Together, these data suggest that several macrophage immune pathways are regulated by SC79 via Akt. A small-molecule Akt activator may be useful in some infection settings, warranting future in vivo studies.

Insights into dissolved organic carbon biodegradation process and influencing factors in shallow lakes in a metropolitan, China

Lakes perform a pivotal role in both regional and global carbon cycles. However, seasonal variations and drivers of dissolved organic carbon (DOC) biodegradability are poorly understood in shallow urban lakes. To address this knowledge gap, a 28-day incubation experiment in urban shallow lakes was carried out to quantify proportion of biodegradable DOC (%BDOC) and temperature sensitivity (Q10 value). %BDOC and Q10 value were significantly higher in the wet season (%BDOC20: 14.48% ± 7.92%, %BDOC30: 17.61% ± 8.98%, Q10:1.32 ± 0.62) over the dry season (%BDOC20: 10.35% ± 6.98%, %BDOC30: 11.59% ± 7.36%, Q10: 1.20 ± 0.65). The absorbance at 350 nm (a350) followed a decreasing-increasing-decreasing trend, while a250 / a365 (E2:E3) had a contrasting trend to a350 during the incubation. Three (dry season) and five (wet season) fluorescent components were captured by parallel factor analysis (PARAFAC). The components C2 (terrestrial humic-like) and C3 (tryptophan-like) were largely biodegraded on day 28 in the wet season. The %BDOC was positively related to total dissolved nitrogen (TDN), total dissolved phosphorus (TDP) and fluorescence index (FI), but negatively related to humification index (HIX) during the dry season. However, in the wet season, the %BDOC was positively related to TDN, TDP and DOC. The results show that the controls of DOC biodegradation shift from the combined DOC quality and nutrients (dry season) to DOC quantity (wet season). The study sheds light on the drivers of BDOC seasonality, and underscores the significance of both nutrients and DOC in understanding carbon-geochemistry within lake ecosystems.

Measurement report: Optical characterization, seasonality, and sources of brown carbon in fine aerosols from Tianjin, North China: year-round observations

Abstract. To investigate the optical characteristics and sources of brown carbon (BrC) in North China, where the atmospheric aerosol loadings are high and have severe impacts on Earth's climate system, we collected fine aerosols (PM2.5) at an urban site in Tianjin over a 1-year period. We measured the ultraviolet (UV) light absorption and excitation emission matrix (EEM) fluorescence of the water-soluble BrC (WSBrC) and the water-insoluble but methanol-soluble BrC (WI-MSBrC) in the PM2.5 using a three-dimensional fluorescence spectrometer. The average light absorption efficiency of both WSBrC (Abs365, WSBrC) and WI-MSBrC (Abs365, WI-MSBrC) at 365 nm was found to be highest in winter (10.4 ± 6.76 and 10.0 ± 5.13 Mm−1, respectively) and distinct from season to season. The averages of the fluorescence index (FI) and the biological index (BIX) of WSBrC were lower in summer than in the other seasons and opposite to that of the humification index (HIX), which implied that the secondary formation and further chemical processing of aerosols were more intensive during the summer period than in the other seasons. However, in winter, the higher HIX together with the higher FI and BIX of WI-MSBrC suggested that the BrC loading was mainly influenced by primary emissions from biomass burning and coal combustion. Based on the EEM, the types of fluorophores in WSBrC were divided into humic-like substances (HULIS), including low-oxygenated and high-oxygenated species and protein-like compounds (PLOM), though mostly PLOM in the WI-MSBrC. The direct radiation absorption by both WSBrC and WI-MSBrC in the range of 300–400 nm accounted for ∼ 40 % of that (SFEAbs, 4.97 ± 2.71 and 7.58 ± 5.75 W g−1, respectively) in the range 300–700 nm.

Activation of stress reactions in the dinophyte microalga Prorocentrum cordatum as a consequence of the toxic effect of ZnO nanoparticles and zinc sulfate

According to the results of the experimental study, the main regularities of changes in morphological, structural-functional and fluorescent indices of P. cordatum were established when zinc oxide nanoparticles ZnO NPs (0.3–6.4 mg L−1) and Zn in form of salt (0.09–0.4 mg L−1) were added to the medium. The studied pollutants have cytotoxic (growth inhibition, development of oxidative stress, destruction of cytoplasmic organelles, disorganization of mitochondria) and genotoxic (changes in the morphology of nuclei, chromatin condensation) effects on microalgae, affecting almost all aspects of cell functioning. Despite the similar mechanism of action of zinc sulfate and ZnO NPs on P. cordatum cells, the negative effect of ZnO NPs is also due to the inhibition of photosynthetic activity of cells (significant decrease in the maximum quantum yield of photosynthesis and electron transport rate), reduction of chlorophyll concentration from 3.5 to 1.8 pg cell−1, as well as mechanical effect on cells: deformation and damage of cell membranes, aggregation of NPs on the cell surface. Apoptosis-like signs of cell death upon exposure to zinc sulfate and ZnO NPs were identified by flow cytometry and laser scanning confocal microscopy methods: changes in cell morphology, cytoplasm retraction, development of oxidative stress, deformation of nuclei, and disorganization of mitochondria. It was shown that the first signs of cell apoptosis appear at 0.02 mg L−1 Zn and 0.6 mg L−1 ZnO NPs after 72 h of exposure. At higher concentrations of pollutants, a dose-dependent decrease in algal enzymatic activity (up to 5 times relative to control) and mitochondrial membrane potential (up to 4 times relative to control), and an increase in the production of reactive oxygen species (up to 4–5 times relative to control) were observed. The results of the presented study contribute to the disclosure of fundamental mechanisms of toxic effects of pollutants and prediction of ways of phototrophic microorganisms reaction to this impact.

A Fiber Sensor for VOC Gas Detection Based on Modulated Aggregation of Fluorescence Indicator

A Fiber Sensor for VOC Gas Detection Based on Modulated Aggregation of Fluorescence Indicator

Phycocyanin-based rapid fluorometric immunoassay for the determination of aflatoxin B1, deoxynivalenol, and zearalenone in food and feed matrices

This study presents a new multiplex lateral flow immunoassay (LFIA) for the detection of aflatoxin B1 (AFB1), deoxynivalenol (DON), and zearalenone (ZEN). The multi-LFIA utilized phycocyanin (PC), a protein with excellent photophysical properties, as a fluorescent indicator for quantifying the three mycotoxins on a lateral flow strip platform. PC and monoclonal antibodies (mAbs) against AFB1, DON, and ZEN were immobilized onto carboxylated latex nanospheres (LNS) through a one-step coupling process. The resulting conjugates with strong red fluorescence were then used for multiplex sensing of AFB1, DON, and ZEN in wheat, corn, and animal feed. The visual results of the assay were obtained using LED UV lights coupled with a smartphone in less than 25 min. Under the optimal conditions, the new multi-LFIA achieved minimum detection limits of 1.04, 1.6, and 2.08 ng/mL for AFB1, 2.2, 6.45, and 2.9 ng/mL for DON, and 1.74, 1.67, and 2.11 ng/mL for ZEN in wheat, corn, and feed, respectively. The proposed method demonstrated high selectivity for AFB1, DON, and ZEN and showed strong correlation with LC-MS/MS results in spiked samples. In summary, the proposed method is feasible, rapid, and could serve as a highly sensitive platform for detecting AFB1, DON, and ZEN and other analytes in a single format.

Fluorescence spectroscopy for tracking microbiological contamination in urban waterbodies

Dissolved organic matter (DOM) plays a crucial role in freshwater ecosystem function. Monitoring of DOM in aquatic environments can be achieved by using fluorescence spectroscopy. Particularly, DOM fluorescence can constitute a signature of microbiological contamination with a potential for high frequency monitoring. However, limited data are available regarding urban waterbodies. This study considers fluorescence data from field campaigns conducted in the Paris metropolitan region: two watercourses (La Villette basin and the river Marne), two stormwater network outlets (SO), and a wastewater treatment plant effluent (WWTP-O). The objectives of the study were to characterize the major fluorescence components in the studied sites, to investigate the impact of local rainfall in such components and to identify a potential fluorescence signature of local microbiological contamination. The components of a PARAFAC model (C1-C7), corresponding to a couple of excitation (ex) and emission (em) wavelengths, and the fluorescence indices HIX and BIX were used for DOM characterization. In parallel, fecal indicator bacteria (FIB) were measured in selected samples. The PARAFAC protein-like components, C6 (ex/em of 280/352 nm) and C7 (ex/em of 305/340 nm), were identified as markers of microbial contamination in the studied sites. In the La Villette basin, where samplings covered a period of more than 2 years, which also included similar numbers of wet and dry weather samples, the protein-like components were significantly higher in wet weather in comparison to dry weather. A positive relationship was obtained between C6 and FIB. In urban rivers, the high frequency monitoring of C6 levels would support the fecal contamination detection in rivers. In addition, it could help targeting specific field campaigns to collect comprehensive dataset of microbiological contamination episodes.

Impact of Hill Fires on Dissolved Organic Matter in Watersheds of Karst Areas Based on Three-Dimensional Fluorescence-Parallel Factor Analysis

Hill fires have the potential to influence dissolved organic matter (DOM) in water bodies, yet fewer studies have investigated the effects of hill fires on DOM within watersheds in karst areas. In this study, we employed the three-dimensional fluorescence-parallel factor analysis (EEM-PARAFAC) method to analyze the DOM fluorescence peaks, component compositions, fluorescence indices, and sources within the water body of the Yuanteng River sub-basin, which was impacted by the hill fire, serving as our primary research focus. The results indicate the presence of three primary fluorescent fractions in the water body of the Yuanteng River: C1, resembling humic acid (fulvic acid); C2, consisting of biopolymers and microbial by-products; and C3, containing proteins such as tyrosine and tryptophan. The Yuanteng River exhibited elevated levels of humus-like substances, diminished concentrations of protein-like substances, and demonstrated higher biogenic, freshness, and humification indices compared to unaffected water samples, reflecting the impact of the hill fire. Elevated levels of exogenous humic acid-like inputs into the waters of the Yuanteng River, along with exogenous inputs of DOM, were primarily influenced by stable, high-molecular-weight organic matter. Additionally, agricultural effluent, domestic sewage, and anthropogenic activities contributed to these inputs to a lesser degree. The impacts of endogenous inputs are mainly related to the restoration of ecosystems. The occurrence of hill fires has significantly influenced the composition of dissolved organic matter in the waters of the Yuanteng River. A comprehensive analysis of the impacts of hill fires on dissolved organic matter in water bodies can serve as a valuable reference for characterizing DOM in the water bodies of the Yuanteng River. Furthermore, it can inform strategies for environmental protection, facilitate the traceability of pollutants in water bodies, and contribute to environmental and ecological restoration efforts following hill fires in the region.

Evaluation of Cell Cycle-Dependent Migration Activity after X-ray Exposure: A Radiobiological Approach for Optimization of Radiotherapy with Cell Cycle-Targeting Agents.

Radiotherapy with cell cycle-specific anticancer agents has become an important option in the control of both primary tumors and metastases. Here, we used image analysis algorithms that enable quick segmentation and tracking to describe a radiobiological approach for the optimized selection of cell cycle-targeting anticancer drugs for radiotherapy. We confirmed cell cycle-synchronization using human cervical cancer HeLa cells expressing a fluorescent ubiquitination-based cell cycle indicator (FUCCI) as a cell cycle-monitoring probe. Cells synchronized in the G1 and G2 phases were irradiated with X rays at 0.5-2 Gy. Each cell was identified using Cellpose, a deep learning-based algorithm for cellular segmentation, and the velocity and direction of migration were analyzed using the TrackMate plugin in Fiji ImageJ. G1 phase synchronized cells showed a dose-dependent decrease in velocity after irradiation, while G2 cells tended to increase their velocity. The migration pattern of all cells appeared to be a random walk model, regardless of the exposure dose. In addition, we used cisplatin to arrest the cell cycle. HeLa-FUCCI cells arrested at the G2 phase via cisplatin treatment showed enhanced cell migration after X-ray exposure. These results indicated that anticancer agents that arrest the cell cycle of cancer cells in a specific phase may enhance cell migration after radiotherapy. Our approach, using cellular segmentation and tracking algorithms, could enhance the radiobiological assessment of cell cycle-specific migration after irradiation to aid in optimizing radiotherapy using cell cycle-targeting agents.

Triple Sensing Modes for Triggered β-Galactosidase Activity Assays Based on Kaempferol-Deduced Silicon Nanoparticles and Biological Imaging of MCF-7 Breast Cancer Cells.

β-Galactosidase (β-Gala) is an essential biomarker enzyme for early detection of breast tumors and cellular senescence. Creating an accurate way to monitor β-Gala activity is critical for biological research and early cancer detection. This work used fluorometric, colorimetric, and paper-based color sensing approaches to determine β-Gala activity effectively. Via the sensing performance, the catalytic activity of β-Gala resulted in silicon nanoparticles (SiNPs), fluorescent indicators obtained via a one-pot hydrothermal process. As a standard enzymatic hydrolysis product of the substrate, kaempferol 3-O-β-d-galactopyranoside (KOβDG) caused the fluorometric signal to be attenuated on kaempferol-silicon nanoparticles (K-SiNPs). The sensing methods demonstrated a satisfactory linear response in sensing β-Gala and a low detection limit. The findings showed the low limit of detection (LOD) as 0.00057 and 0.098 U/mL for fluorometric and colorimetric, respectively. The designed probe was then used to evaluate the catalytic activity of β-Gala in yogurt and human serum, with recoveries ranging from 98.33 to 107.9%. The designed sensing approach was also applied to biological sample analysis. In contrast, breast cancer cells (MCF-7) were used as a model to test the in vitro toxicity and molecular fluorescence imaging potential of K-SiNPs. Hence, our fluorescent K-SiNPs can be used in the clinic to diagnose breast cellular carcinoma, since they can accurately measure the presence of invasive ductal carcinoma in serologic tests.

Large-scale analysis of small molecule-RNA interactions using multiplexed RNA structure libraries

The large-scale analysis of small-molecule binding to diverse RNA structures is key to understanding the required interaction properties and selectivity for developing RNA-binding molecules toward RNA-targeted therapies. Here, we report a new system for performing the large-scale analysis of small molecule–RNA interactions using a multiplexed pull-down assay with RNA structure libraries. The system profiled the RNA-binding landscapes of G-clamp and thiazole orange derivatives, which recognizes an unpaired guanine base and are good probes for fluorescent indicator displacement (FID) assays, respectively. We discuss the binding preferences of these molecules based on their large-scale affinity profiles. In addition, we selected combinations of fluorescent indicators and different ranks of RNA based on the information and screened for RNA-binding molecules using FID. RNAs with high- and intermediate-rank RNA provided reliable results. Our system provides fundamental information about small molecule–RNA interactions and facilitates the discovery of novel RNA-binding molecules.