FUS (Fused in Sarcoma) protein is a crucial RNA-binding protein whose liquid-liquid phase separation (LLPS) behavior plays a key role in cellular physiological functions and the pathogenesis of neurodegenerative diseases. This review systematically summarizes the molecular mechanisms and physiological-pathological functions of FUS condensates, with a particular focus on the applications and challenges of fluorescence imaging techniques in resolving the dynamic processes of FUS phase separation. The integration of appropriate fluorescent labelling strategies (such as fluorescent protein fusion and self-labelling tag techniques) with advanced microscopic imaging technologies (e.g., confocal and super-resolution microscopy) has enabled nanoscale dynamic analysis of FUS biomolecular condensates both in vitro and within living cells, including the dynamic regulation of FUS and the liquid-to-solid transition process, which may first form a shell on the droplet surface. Furthermore, this review discusses the functions of FUS under various stimulatory conditions and the potential mechanisms underlying disease initiation. However, challenges such as labelling specificity, phototoxicity, spatiotemporal resolution limitations, and the integration of multiple regulatory factors in living environments remain to be addressed. Finally, this article prospects future technological directions, including in situ labelling, biosensors, and artificial intelligence-based analysis, aiming to provide new insights for a deeper understanding of FUS phase separation mechanisms and the treatment of related diseases.

The liquid-to-solid transition of fused in sarcoma (FUS) condensates is a key molecular event in neurodegenerative diseases. However, in living cells, the spatiotemporal evolution of internal substructures arising from local protein concentration differences during early condensate solidification remains poorly understood. Existing fluorescent probes largely rely on microenvironmental signals, confounding protein localization with environmental fluctuations. In this study, we found that the Halo-Rho-A2 probe is environmentally insensitive and capable of rapid Halo-tag labeling, allowing its fluorescence intensity directly reflecting protein spatial distribution. By combining this probe with Halo-tag technology and conventional confocal imaging, we tracked in real time localization changes within FUS condensates in living cells. Intensity heatmaps resolved the spatial distribution of FUS protein inside droplets, revealing three distinct internal substructures: uniform, locally enriched, and fully enriched. Fluorescence recovery after photobleaching (FRAP) experiments confirmed that these three states correspond to liquid, intermediate, and solid-like states, respectively. Real-time tracking revealed that fully enriched droplets form through a dynamic process of "surface enrichment–fusion-induced disassembly–surface reassembly". Using the percentage of fully enriched droplets as a quantitative indicator, we systematically evaluated the effects of NaCl stimulation time and pathogenic FUS mutations (P525L, G156E) on the solidification process. This method requires no complex equipment and converts the complex liquid-to-solid transition into statistically comparable morphological parameters using only standard fluorescence imaging. Collectively, this study provides a new tool and conceptual framework for directly resolving substructures within live-cell FUS condensates, offering insights into early mechanisms of liquid-to-solid transition and the development of intervention strategies.

Red fluorescent proteins with large Stokes shift (LSS-RFPs) are advantageous for multicolor imaging applications that allow simultaneous visualizations of multiple biological events. But it is difficult to develop LSS-RFPs by extending the emission wavelength of RFPs to far-red region. Here, we employed F & ouml;rster resonance energy transfer (FRET) strategy to engineer the far-red fluorescent proteins with large Stokes shift. LSS-mApple and LSS-mCherry were constructed by fusing HaloTag to mApple and mCherry, allowing the fluorophore TMSiR to be connected to these RFPs. FRET between RFPs and TMSiR enabled them to apply the excitation of donor RFPs to emit far-red fluorescence of acceptor TMSiR. The Stokes shifts of LSS-mApple and LSS-mCherry were 97 nm and 75 nm, respectively. The high FRET efficiency of LSSmCherry (EFRET = 83.7 %) can greatly reduce the fluorescence from the donor channel, which did not affect co-imaging with mCherry. In addition, LSS-mCherry also showed excellent photostability ( t1/2 = 449.3 s), enabling stable confocal fluorescence imaging for 15 min under continuous strong excitation. Furthermore, LSS-mCherry was applied for fluorescence labeling and imaging of the nucleus, mitochondria, lysosomes, and endoplasmic reticulum in living cells. Finally, we applied LSS-mCherry to perform multi-color bioimaging of 2-4 channels, and there was no obvious crosstalk between these channels. (c) 2025 Published by Elsevier B.V. on behalf of Chinese Chemical Society and Institute of Materia Medica, Chinese Academy of Medical Sciences.

A gas-liquid pulse-modulated plasma with bubbling coupled with plasma-derived heat system was developed for efficient degradation of octadecylamine (ODA) and 4-dodecylmorpholine (DMP). The structure-activity relationship between high-voltage electrode structures and the degradation activity of ODA and DMP was delved into using fluid dynamics simulations. The crucial role of bubbling is evident in the enhancement of ODA and DMP degradation efficiencies by 100% and 47% within 30 min, respectively. ODA and DMP degradation efficiencies reached 96% and 100% with treatment of 30 min, respectively, accompanied by a high mineralization efficiency of 80 %. An in-depth analysis was performed on the evolution of the physicochemical properties of the reaction solution during degradation. The plasma coupled with plasma-derived heat system exhibited remarkable synergistic effects, achieving synergistic intensities of 0.96 for ODA and 1.28 for DMP, with energy efficiencies increased by 31% and 16%. Analysis of the evolution of total nitrogen and nitrogen-containing species revealed that ODA and DMP degradation commenced with the decomposition of amino groups, ultimately converting them into NO 3 and NH+4 . Quenching tests and electron spin resonance characterization confirmed the generation of center dot OH, center dot O 2 and 1O2, with center dot OH playing a pivotal role in the degradation process. Furthermore, the possible degradation pathways of ODA and DMP were elucidated by the theoretical calculations and gas chromatographymass spectrometry results, respectively. The toxicity evaluation of degradation intermediates demonstrated a consistent decrease in toxicity during the degradation process. This study advances our understanding of the synergistic potential of plasma-plasma-derived heat systems for pollutant degradation and paves the way for the development of more effective pollutant degradation technologies.

By incorporating hybrid FRET pairs with RFPs as energy donors and TMSiR as the acceptor, the intersystem crossing (ISC) of RFPs is suppressed via FRET, leading to enhanced photostability of the RFPs.

The continuous mutation and rapid spread of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have led to the ineffectiveness of many antiviral drugs targeting the original strain. To keep pace with the virus' evolutionary speed, there is a crucial need for the development of rapid, cost-effective, and efficient inhibitor screening methods. In this study, we created a novel approach based on fluorescence resonance energy transfer (FRET) technology for in vitro detection of inhibitors targeting the interaction between the SARS-CoV-2 spike protein RBD (s-RBD) and the virus receptor angiotensin-converting enzyme 2 (ACE2). Utilizing crystallographic insights into the s-RBD/ACE2 interaction, we modified ACE2 by fusing SNAP tag to its N-terminus (resulting in SA740) and Halo tag to s-RBD's C-terminus (producing R525H and R541H), thereby ensuring the proximity (<10 nm) of labeled FRET dyes. We found that relative to the R541H fusion protein, R525H exhibited higher FRET efficiency, which attributed to the shortened distance between FRET dyes due to the truncation of s-RBD. Utilizing the sensitive FRET effect between SA740 and R525H, we evaluated its efficacy in detecting inhibitors of SARS-CoV-2 entry in solution and live cells. Ultimately, this FRET-based detection method was demonstrated high sensitivity, rapidity, and simplicity in solution and held promise for high-throughput screening of SARS-CoV-2 inhibitors.

Pseudomonas aeruginosa is a highly adaptable Gram-negative pathogen known for its remarkable ability of forming biofilms. Understanding the environmental cues and regulatory mechanisms that drive biofilm formation is essential for developing effective control strategies. In this study, we screened 57 clinical and environmental P. aeruginosa isolates and discovered that a universal environmental cue, temperature downshift from host-associated 37 °C to room temperature (21 °C), significantly promotes biofilm formation in 63% of the strains. Using the ATCC 27853 strain as a model, we demonstrate that this enhancement results from increased production of the Psl exopolysaccharides at lower temperature. LC-MS/MS analysis revealed elevated levels of the secondary messenger c-di-GMP, a key regulator of the motile-to-sessile transition, at room temperature. Through screening a mutant library targeting 18 c-di-GMP metabolic enzymes, we identified the diguanylate cyclase SiaD within the SiaABCD signaling and functional module as a principal driver of c-di-GMP elevation and biofilm promotion. Further investigation showed that the entire SiaABCD module, especially the signal-sensing domain of SiaA, mediates the temperature-dependent response. Integrating lipidomics with genetics and physiological assays, we show that a temperature downshift triggers rapid membrane perturbations that activate the SiaABCD signaling module, thereby increasing Psl production to strengthen surface adhesion and drive robust biofilm formation. These findings establish temperature downshift as a previously unrecognized physiological cue that promotes biofilm formation in P. aeruginosa and define an adaptive regulatory pathway linking specific environmental stresses of membrane perturbation to dedicated c-di-GMP signaling module, paving the way for new strategies to disrupt biofilm-associated infections and transmission.

MicroRNA-21 (miR-21) is a significant tumor marker for early cancer screening. Hybridization chain reaction (HCR) as an enzyme-free signal amplification method has been used for intracellular miR-21 detection in living cells, but it is limited by unclear reaction details and prolonged detection time. In this study, by effectively regulating the HCR reaction conditions, we achieved rapid visualization of endogenous miR-21 in live cells. A pair of HCR DNA hairpins H1 and H2 was designed, and the effects of reaction concentration, the type of HCR products, ionic selectivity, and the reaction order of H1 and H2 on the HCR process were investigated. We discovered that the cascade reaction produced by HCR in cells primarily formed six distinct assemblies and was dependent on different concentrations of Na+ and Mg2+ salts. Importantly, it was found that the addition sequence of the two hairpin DNAs was closely related to the speed of the cascading reaction in living cells. Adding H1 first and then H2 resulted in stronger detection signals in a relatively short time. By elucidating these details, we carried out the rapid fluorescence colocalization detection of endogenous miR-21 in living cells.

Fluorescent dyes are indispensable chemical tools for protein labeling, yet their utility in live-cell imaging remains constrained by background fluorescence from off-target interactions. A chemigenetic strategy is presented that integrates synthetic dye chemistry with genetically encoded fluorescence to achieve high-fidelity, wash-free imaging of target proteins. By leveraging Förster resonance energy transfer (FRET) between fluorescent proteins (FPs) and Halo-tag dyes, a system is engineered where fluorescence emission depends on FPs donor excitation, ensuring only dyes bound to target protein are fluorescent, while nonspecifically bound dyes remain dark, enhancing the signal-to-noise ratio (SNR). This approach is implemented by fusing Halo-tag with FPs (sfGFP, mCherry) to create FRET pairs with Halo dyes (O-Rho, Si-Rho), yielding chemigenetic fluorophores (GLH-O, CLH-Si) with improved SNR even at low expression levels. By optimizing FRET efficiency, the developed probes CH-Si are combined with standard FPs and commercial dyes, and achieve four-color structured illumination microscopy (SIM) imaging of target proteins, and track the mitochondria interactions with endoplasmic reticulum, and lysosomes. Furthermore, by incorporating the ultra-stable mStayGold as donor, a photostable fluorophore (SLH-O) capable of long-term super-resolution imaging is developed. This versatile strategy combines chemical and genetic tools, offering a generalizable platform for high-precision studies of protein and cellular processes.

Malachite Green (MG) and its fluorogen-activating protein (FAP) pair are valuable tools for live-cell and super-resolution fluorescence imaging due to their unique near-infrared absorption and signal enhancement. However, the low brightness and photostability of MG have limited its use in dynamic imaging. In this study, we introduce a novel derivative, azetidinly Malachite Green (Aze-MG), which enhances the brightness of the MG-FAP complex by 2.6-fold. This enhancement is achieved by replacing the N,N-dimethylamino group in MG with an azetidine group, which suppresses the twisted intramolecular charge transfer (TICT) effect, leading to improved quantum yield and photostability. Additionally, the reduced binding affinity of Aze-MG for FAP enables a buffering strategy, allowing the reversible exchange of photobleached fluorogens with free fluorogens, thereby ensuring stable fluorescence over time. This combination of improved brightness and buffering capability makes Aze-MG an ideal probe for live-cell and dynamic SIM imaging.

We developed the fluorogenic SNAP probe BGAN-8C to monitor protein degradation. It exhibited a 6-fold fluorescence enhancement upon binding with SNAP-tag. After the SNAP protein is degraded, the fluorescence of the released probe is quenched due to aggregation. BGAN-8C was successfully employed to monitor the degradation of mODC in live cells, offering a new tool for studying protein dynamics.

Increased levels of octadecylamine (ODA) and 4-dodecylmorpholine (DMP) in the aqueous, primarily utilized as flotation agents in the worldwide production of potash fertilizer, imperil the stability of ecosystems and the downstream production of high-end chemicals. Nevertheless, there is a dearth of exhaustive studies pertaining to the elimination of ODA and DMP. Herein, the Ti/SnO2-Sb/beta-PbO2 anode was fabricated by a thermal decomposition-electrodeposition technique for the electrocatalytic degradation of ODA and DMP. The degradation efficiency of ODA and DMP can achieve complete degradation, reaching 100%, after treatment periods of 10 min and 120 min, respectively. Meanwhile, the TOC removal efficiency of ODA and DMP is up to 85% and 61% at 30 min and 120 min, respectively. In particular, sulphate exhibits inhibitory degradation activity of ODA and DMP in comparison to chloride salt. The recycling and accelerated lifetime tests indicate excellent stability and recyclability of the Ti/SnO2-Sb/beta-PbO2 electrode. The mechanism of electrocatalytic degradation involves indirect electrochemical oxidation mediated by free radicals. The primary reactive species responsible for the degradation of DMP and ODA, as determined through scavenger quenching experiments and ESR, are center dot OH and Cl center dot. The degradation of the ODA and DMP commences with the elimination of N element, leading to the formation of the NO3-. The carbon chain subsequently undergoes the breakdown. The degradation pathways of ODA and DMP were also proposed based on the GC-MS and H-NMR analyses, respectively. Moreover, the Ti/SnO2-Sb/beta-PbO2 anode performed excellently in the removal of ODA and DMP in real samples, TOC removal from ODA and DMP in natural samples was 56% and 52%, respectively, within 180 min. This study provides the first exploration of electrocatalytic degradation mechanisms and pathways of ODA and DMP based on the Ti/SnO2-Sb/beta-PbO2 anode.

The highly efficient sieving of Cs+ from natural brine resources has garnered considerable attention for the extraction of cesium raw materials. However, for the trade-off between ion rejection and water permeability, it remains challenging for graphene oxide (GO) membranes to improve efficient sieving of Cs+ with a large water permeance. Here, we have successfully addressed this challenge by fabricating a GO membrane that incorporates crown ether (CE) through pi-pi and CH-pi interactions with GO sheets. This CE intercalation has resulted in a significantly enhanced Cs+ sieving capability from salt lake brines, concurrently improving water permeance. The unique Cs+ selective cavity structure of the CE has enabled the GO membrane to exhibit a Cs+ rejection rate of 94.4 % and a water permeance of 15.8 L m(-2) h(-1) bar(-1) at a Cs+ concentration of 0.150 mmol/L (similar to 20 ppm). Compared to the pure GO membrane, these results represent an improvement of over 70 % in Cs+ rejection and more than 230 % in water permeance. Interestingly, we have also observed that this GO membrane exhibits a low Na+ system consisting of two components, the separation factor of Cs+/Na+ reaches up to 5.0, which is significant in monovalent ionic membrane separations. These significant discoveries provide a promising strategy for the highly efficient extraction and concentration detection of Cs+ from complex ion solutions.

Fluorescent dyes are the main fluorophore for protein labeling and fluorescent imaging in living cells. However, due to the inevitable nonspecific binding of the dye to non-targets within the cell, background signals are generated, which severely affect the imaging quality. Here, we endow the dye with genic fluorescence by introducing a fluorescent proteins FRET pair, such that only the dye bound to the target protein emits fluorescence (λex = FPs, λem = Dyes), while nonspecifically bound dye remains non-fluorescent. This significantly improves the signal-to-noise ratio (SNR) in wash-free imaging. This strategy is achieved by fusing the Halo-tag to fluorescent proteins (sfGFP, mCherry) that can generate FRET with Halo dyes O-Rho and Si-Rho. The FPs serve as the donor, and the FRET mechanism imparts genic fluorescence property to O-Rho and Si-Rho. Since the excitation wavelength of the donor (λex = FPs) cannot excite the unspecifically dye fluorescence, background fluorescence interference is reduced. We further improved the SNR by increasing the FRET efficiency between the FPs and dyes. The system was used for four-color super-resolution imaging of target proteins and dynamic SIM tracking of mitochondria. In addition, the genic FRET fluorophore constructed with the ultra-stable mStayGold showed significant photobleaching resistance and can be used for long-term dynamic super-resolution imaging. Theoretically, by selecting appropriate FRET pairs, this method can improve the SNR of any fluorophore, providing a new labeling strategy for live-cell wash-free imaging. ### Competing Interest Statement The authors have declared no competing interest.

Rubidium (Rb) and cesium (Cs) have important applications in highly technical fields. Salt lakes contain huge reserves of Rb and Cs with industrial significance, which can be utilized after extraction. In this study, a composite magnetic adsorbent (Fe3O4@ZIF-8@AMP, AMP = ammonium phosphomolybdate) was prepared and its adsorption properties for Rb+ and Cs+ were studied in simulated and practical brine. The structure of the adsorbent was characterized by SEM, XRD, N2 adsorption-desorption, FT-IR, and vibrating sample magnetometer (VSM). The adsorbent had good adsorption affinity for Rb+ and Cs+. The Langmuir model and pseudo-second-order dynamics described the adsorbing isotherm and kinetic dates, respectively. The adsorption capacity and adsorption rate of Fe3O4@ZIF-8@AMP were increased by 1.86- and 2.5-fold compared with those of powdered crystal AMP, owing to the large specific surface area and high dispersibility of the adsorbent in the solution. The adsorbent was rapidly separated from the solution within 17 s using an applied magnetic field owing to the good magnetic properties. The composite adsorbent selectively adsorbed Rb+ and Cs+ from the practical brine even in the presence of a large number of coexisting ions. The promising adsorbent can be used to extract Rb+ and Cs+ from aqueous solutions.

The escalating health threats posed by bacteria and the pressing issue of antibiotic resistance underscore the urgent need for selective detection technologies and antimicrobial reagents. Fluorescein's biocompatibility renders it the preferred fluorescent dye for in vivo imaging. This study reveals that compounds from the aliphatic chain-derived fluorescein series (BMP-alkyl) not only selectively stain and fluorescently image Gram-positive bacteria but also demonstrate the ability to rapidly eliminate them. The discovery of fluorescein's antibacterial properties is poised to have applications in antibiotic research due to its excellent biocompatibility. These amphiphilic BMP-alkyl compounds were easily synthesized through a one-step amidation process involving dicarboxyfluorescein and long-chain fatty amines. In aqueous solutions, the formed aggregates exhibited complete fluorescence quenching. Upon encountering the Gram-positive bacterial membrane, BMP-alkyl compounds underwent depolymerization, resulting in a single fluorescent molecule and the activation of fluorescence. Subsequently, as these amphiphilic fluoresceins continuously combined and entered the bacterial interior, the potential for bacterial membrane depolarization, membrane structure damage, and ultimately bacterial death ensued.

In this study, super-resolution structured illumination microscope (SIM) was used to analyze molecular mechanism of endocytic acidification inhibitors in the SARS-CoV-2 pandemic, such as Chloroquine (CQ), Hydroxychloroquine (HCQ) and Bafilomycin A1 (BafA1). We fluorescently labeled the SARS-CoV-2 RBD and its receptor ACE2 protein with small molecule dyes. Utilizing SIM imaging, the real-time impact of inhibitors (BafA1, CQ, HCQ, Dynasore) on the RBD-ACE2 endocytotic process was dynamically tracked in living cells. Initially, the protein activity of RBD and ACE2 was ensured after being labeled. And then our findings revealed that these inhibitors could inhibit the internalization and degradation of RBD-ACE2 to varying degrees. Among them, 100 nM BafA1 exhibited the most satisfactory endocytotic inhibition (similar to 63.9 %) and protein degradation inhibition (similar to 97.7 %). And it could inhibit the fusion between endocytic vesicles in the living cells. Additionally, Dynasore, a widely recognized dynein inhibitor, also demonstrated cell acidification inhibition effects. Together, these inhibitors collectively hinder SARS-CoV-2 infection by inhibiting both the viral internalization and RNA release. The comprehensive evaluation of pharmacological mechanisms through super-resolution fluorescence imaging has laid a crucial theoretical foundation for the development of potential drugs to treat COVID-19.

Octadecylamine is a flotation collector commonly used to produce potassium chloride by cold crystallization-positive flotation, and its dosage will significantly affect the flotation separation efficiency. In addition, octadecylamine will adsorb on the surface of potassium chloride and inevitably remain in potassium chloride products, which is inconducive to developing high-purity potassium salt products. To meet the requirement of determination of octadecamide potency in potassium chloride, an extraction spectrophotometry was developed, based on the principle of van der Waals force and hydrogen bonding between octadecylamine and bromophenol blue with butyl acetate as extractant and bromophenol blue sodium salt as chromogenic agent. The effects of solution pH, dodecyl morpholine, co-existing salt, equilibrium time, and the amount of chromogenic agent on the concentration measurement of octachylamine were investigated. The results show that when the solution pH increases from 3 to 9, the absorbance of the extract decreases because the complexation between octachylamine and bromophenol blue weakens. The polarity of the complex molecules increases. When the solution pH is less than 5, dodecyl morpholine and bromophenol blue can also form colored complexes. While pH is between 6 and 9, the absorbance tends to zero. The ionic strength of the coexisting solutions of potassium chloride, sodium chloride, potassium sulfate and magnesium chloride increases, weakening the hydrogen bonding. Still, the salting-out effect of the complex molecules is enhanced, and the trace Li+, NH4+ and B in the solution have little effect on the absorbance. The absorbance does not conform to Lambert-Beer law when the amount of chromogenic agent is too large. Besides, equilibrium time has little effect on the absorbance. The determination conditions in potassium chloride solution are as follows: The solution pH is 6, the ionic strength is 1 mol . L, and the amount of 2 mmol. L-1 chromogenic agent is 0. 5 mL. 5 mL butyl acetate is added to the 25 mL aqueous solution adjusted by buffer after 5 min of reaction for extraction. After 2 min of delamination equilibrium, the absorbance of the extraction solution is tested at 458 nm. The working curve is A=0.049 49c+ 0. 066 24 (R-2 = 0. 992 3, epsilon=1. 33 X 10(4) L . mol(-1) . cm(-1), 0 similar to 10 mg . L-1). The relative standard deviation of this method was 0. 33%similar to 2. 63%, the mean relative error was 0. 90%, and the mean relative error of the system of octadecylamine and dodecylmorpholine was -0. 25%. The content of octadecamide in the filter liquor derived from washing potassium chloride after positive flotation was 8. 66 mg . L-1 and recoveres were 95. 5% 106% by this method. The extraction spectrophotometry has been proven to be appropriate for detecting the concentration of octadecamide in the production of potassic fertilizer in salt lakes.

More than 70% of the potash fertilizer globally is produced by the froth flotation process, in which 4-dodecylmorpholine (DMP) serves as a reverse flotation agent. As the potash fertilizer production rapidly rises, the increased DMP levels in discharged brine pose a threat to the production of high-value chemicals. In this paper, composite particles of basic magnesium sulfate@TiO2 (BMS@TiO2) were prepared using a simple and mild loading method. These particles were utilized for the adsorption and photocatalytic degradation of DMP in brine. Compared with normal powdered materials, the granular BMS@TiO2 in this study can be easily separated from liquid, and the degradation intermediates will not enter the brine without causing secondary pollution. BMS@TiO2 consists of 5·1·7 phase (5Mg(OH)2·MgSO4·7H2O) whisker clusters embedding 2.3% TiO2. The adsorption equilibrium of DMP on BMS@TiO2 particles was achieved through hydrogen bonding and pore interception with the adsorption capacity of approximately 5 mg g−1 after 6 h. The photodegradation efficiency of DMP adsorbed on BMS@TiO2 reached about 92% within 16 h, which is compared with that of pure TiO2 nanoparticles. Additionally, excellent stability and recyclability of BMS@TiO2 were also observed in five cycle tests of adsorption and photocatalytic degradation of DMP, and the possible photocatalytic degradation pathways and mechanism of DMP are proposed following molecular electrostatic potential analysis. This work provides a sustainable and environmentally friendly approach for eliminating organic micropollutants from water environments.