Single-Atom Iridium Nanozyme-Based Persistent Luminescence Nanoparticles for Multimodal Imaging-Guided Combination Tumor Therapy.

Persistent luminescence nanoparticles (PLNPs) can achieve autofluorescence-free afterglow imaging, while near-infrared (NIR) emission realizes deep tissue imaging. Nanozymes integrate the merits of nanomaterials and enzyme-mimicking activities with simple preparation. Here PLNPs are prepared of Zn1.2Ga1.6Ge0.2O4:Cr0.0075 with NIR emission at 700 nm. The PLNPs are then incubated with IrCl3 solution, and the nanoparticles are collected and annealed at 750 °C to obtain iridium@PLNPs. Iridium is observed on the PLNPs at the atomic level as a single-atom nanozyme with peroxidase-like catalytic activity, photothermal conversion, and computed tomography (CT) contrast capability. After coating with exosome membrane (EM), the Ir@PLNPs@EM composite exhibits long-lasting NIR luminescence, peroxidase-like catalytic activity, photothermal conversion, and CT contrast capability, with the targeting capability and biocompatibility from EM. Thus, NIR afterglow/photothermal/CT trimodal imaging-guided photothermal-chemodynamic combination therapy is realized as validated with the in vitro and in vivo inhibition of tumor growth, while toxicity and side effects are avoided as drug-free treatment. This work offers a promising avenue for advanced single-atom nanozyme@PLNPs to promote the development of nanozymes and PLNPs for clinical applications.

Comprehensive comparison of water quality risk and microbial ecology between new and old cast iron pipe distribution systems.

The effects of cast iron pipe corrosion on water quality risk and microbial ecology in drinking water distribution systems (DWDSs) were investigated. It was found that trihalomethane (THMs) concentration and antibiotic resistance genes (ARGs) increased sharply in the old DWDSs. Under the same residual chlorine concentration conditions, the adenosine triphosphate concentration in the effluent of old DWDSs (Eff-old) was significantly higher than that in the effluent of new DWDSs. Moreover, stronger bioflocculation ability and weaker hydrophobicity coexisted in the extracellular polymeric substances of Eff-old, meanwhile, iron particles could be well inserted into the structure of the biofilms to enhance the mechanical strength and stability of the biofilms, hence enhancing the formation of THMs. Old DWDSs significantly influenced the microbial community of bulk water and triggered stronger microbial antioxidant systems response, resulting in higher ARGs abundance. Corroded cast iron pipes induced a unique interaction system of biofilms, chlorine, and corrosion products. Therefore, as the age of cast iron pipes increases, the fluctuation of water quality and microbial ecology should be paid more attention to maintain the safety of tap water.

Nano-Proteolysis Targeting Chimeras (Nano-PROTACs) in Cancer Therapy.

Proteolysis-targeting chimeras (PROTACs) are heterobifunctional molecules that have the capability to induce specific protein degradation. While playing a revolutionary role in effectively degrading the protein of interest (POI), PROTACs encounter certain limitations that impede their clinical translation. These limitations encompass off-target effects, inadequate cell membrane permeability, and the hook effect. The advent of nanotechnology presents a promising avenue to surmount the challenges associated with conventional PROTACs. The utilization of nano-proteolysis targeting chimeras (nano-PROTACs) holds the potential to enhance specific tissue accumulation, augment membrane permeability, and enable controlled release. Consequently, this approach has the capacity to significantly enhance the controllable degradation of target proteins. Additionally, they enable a synergistic effect by combining with other therapeutic strategies. This review comprehensively summarizes the structural basis, advantages, and limitations of PROTACs. Furthermore, it highlights the latest advancements in nanosystems engineered for delivering PROTACs, as well as the development of nano-sized PROTACs employing nanocarriers as linkers. Moreover, it delves into the underlying principles of nanotechnology tailored specifically for PROTACs, alongside the current prospects of clinical research. In conclusion, the integration of nanotechnology into PROTACs harbors vast potential in enhancing the anti-tumor treatment response and expediting clinical translation.

Phospholipid-Anchored Ligand Conjugation on Extracellular Vesicles for Enhanced Cancer Targeting.

Extracellular vesicles (EVs) are recognized as potential candidates for next-generation drug delivery systems. However, the inherent cancer-targeting efficiency is unsatisfactory, necessitating surface modification to attach cell-binding ligands. By utilizing phospholipase D from Streptomyces in combination with maleimide-containing primary alcohol, the authors successfully anchored ligands onto milk-derived EVs (mEVs), overcoming the issues of ligand leakage or functional alteration seen in traditional methods. Quantitative nano-flow cytometry demonstrated that over 90% of mEVs are effectively modified with hundreds to thousands of ligands. The resulting mEV formulations exhibited remarkable long-term stability in conjugation proportion, ligand number, size distribution, and particle concentration, even after months of storage. It is further shown that conjugating transferrin onto mEVs significantly enhanced cellular uptake and induced pronounced cytotoxic effects when loaded with paclitaxel. Overall, this study presents a highly efficient, stable, cost-effective, and scalable ligand conjugation approach, offering a promising strategy for targeted drug delivery of EVs.

Comparison of Lipid Composition between Quasipaa spinosa Oil and Rana catesbeiana Oil and Its Effect on Lipid Accumulation in Caenorhabditis elegans.

Frog oil has been recognized for its nutritional and medicinal value. However, there is limited research on the role of frog oil in preventing obesity. In this study, we aimed to investigate the lipid composition of Quasipaa spinosa oil (QSO) and Rana catesbeiana oil (RCO) using lipidomics analysis. We compared the lipid accumulation effects of these two kinds of frog oils and soybean oil (SO) in Caenorhabditis elegans (C. elegans). Additionally, we determined the gene expression related to lipid metabolism and used the nhr-49 mutant (RB1716) and sir-2.1 mutant (VC199) for validation experiments. The results showed that the lipid composition of QSO and RCO was significantly different (p < 0.05), and QSO was rich in more polyunsaturated fatty acids (PUFAs). After feeding C. elegans, the lipid accumulation of the QSO group was the lowest among the three dietary oil groups. In addition, compared with RCO and SO, QSO significantly inhibited the production of malondialdehyde (MDA) and increased the activity of superoxide dismutase (SOD). The effects of three kinds of dietary oils on the fatty acid composition of C. elegans were significantly different. Compared with SO and RCO, QSO significantly up-regulated (p < 0.05) the expression of sir-2.1 and ech-1 genes. The results showed that QSO might reduce lipid accumulation through the SIRT1 and nuclear hormone signaling pathways. Such a situation was verified experimentally by the nhr-49 mutant (RB1716) and sir-2.1 mutant (VC199). This study proposed a new functional oil, laying the groundwork for developing functional foods from Quasipaa spinosa.

Preparation and identification of antioxidant peptides from Quasipaa spinosa skin through two-step enzymatic hydrolysis and molecular simulation.

Frog skin, a by-product of Quasipaa Spinosa farming, is rich in protein and potentially a valuable raw material for obtaining antioxidant peptides. This study used papain combined with acid protease to digest frog skin in a two-step enzymatic hydrolysis method. Based on a single factor and response surface experiments, experimental conditions were optimized, and the degree of hydrolysis was 30 %. A frog skin hydrolysate (QSPH-Ⅰ-3) was obtained following ultrafiltration and gel filtration chromatography. IC50 for DPPH, ABTS, and hydroxyl radical scavenging capacities were 1.68 ± 0.05, 1.20 ± 0.14 and 1.55 ± 0.11 mg/mL, respectively. Peptide sequences (17) were analyzed and, through molecular docking, peptides with low binding energies for KEAP1 were identified, which might affect the NRF2-KEAP1 pathway. These findings suggest protein hydrolysates and antioxidant peptide derivatives might be used in functional foods.

Debittering of Emblica (Phyllanthus emblica L.) fruit powder: Preparation and biological activity.

Emblica, also known as Phyllanthus emblica L., is a drug homologous food that is rich in polyphenols with various biological activities. However, its bitterness and astringency pose a significant challenge to its utilization in food products. In this study, we aimed to identify the optimal conditions for debittering Emblica. Our findings revealed that the best debittering conditions were: temperature = 50 °C, pH = 4, α-l-rhamnosidase concentration 200 U/g, and time = 5 h. High-performance liquid chromatography (HPLC) and molecular docking analysis revealed that enzymatic hydrolysis partially removed bitterness compounds. The results of antioxidant activity, xanthine oxidase, and α-glucosidase inhibitory activity assays confirmed that the Emblica fruit powder still exhibited good biological activity after enzymatic debitterization. Moreover, gastric fluids treatment might contribute to the above enhancing effect of enzymatic hydrolysates of Emblica. This study provided a theoretical basis for promoting the processing and utilization of Emblica fruit powder, as well as understanding its biological activity.

Synergistic effects of trace sulfadiazine and corrosion scales on disinfection by-product formation in bulk water of cast iron pipe.

The effects of trace sulfadiazine (SDZ) and cast-iron corrosion scales on the disinfection by-product (DBP) formation in drinking water distribution systems (DWDSs) were investigated. The results show that under the synergistic effect of trace SDZ (10 µg/L) and magnetite (Fe3O4), higher DBP concentration occurred in the bulk water with the transmission and distribution of the drinking water. Microbial metabolism-related substances, one of the important DBP precursors, increased under the SDZ/Fe3O4 condition. It was found that Fe3O4 induced a faster microbial extracellular electron transport (EET) pathway, resulting in a higher microbial regrowth activity. On the other hand, the rate of chlorine consumption was quite high, and the enhanced microbial EET based on Fe3O4 eliminated the need for microorganisms to secrete excessive extracellular polymeric substances (EPS). More importantly, EPS could be continuously secreted due to the higher microbial activity. Finally, high reactivity between EPS and chlorine disinfectant resulted in the continuous formation of DBPs, higher chlorine consumption, and lower EPS content. Therefore, more attention should be paid to the trace antibiotics polluted water sources and cast-iron corrosion scale composition in the future. This study reveals the synergistic effects of trace antibiotics and corrosion scales on the DBP formation in DWDSs, which has important theoretical significance for the DBP control of tap water.

miR-494-5p mediates the antioxidant activity of EPA by targeting the mitochondrial elongation factor 1 gene MIEF1 in HepG2 cells.

Eicosapentaenoic acid (EPA) shows antioxidant activity, which may be attributed to its regulatory effect on microRNA expression. Our preliminary study indicated that EPA upregulated miR-494-5p, which was possibly involved in the regulation of cellular stress responses. The current study aimed to address whether miR-494-5p was targeted by EPA to regulate cellular oxidative stress and its possible functional mechanism. The results showed that miR-494-5p mediated the antioxidant effect of EPA and miR-494-5p reduction deteriorated EPA-induced increase in the cellular antioxidant capacity of HepG2 cells. Moreover, the mitochondrial elongation factor 1 (MIEF1) gene was a target gene of miR-494-5p. Both miR-494-5p overexpression and MIEF1 knockdown significantly enhanced cellular antioxidant capacity, as indicated by a reduction in the reactive oxygen species level and an increase in the total cellular antioxidant capacity, along with enhancing antioxidant enzymes. Thus, miR-494-5p and MIEF1 had opposite effects on cellular antioxidant capacity. Furthermore, their regulatory effects on oxidative stress may have been attributed to modulation of mitochondrial function, biogenesis and homeostasis. Taken together, the findings indicated that miR-494-5p mediated EPA activity and promoted cellular antioxidant capacity by inhibiting the expression of MIEF1, which further modulated mitochondrial structure and activity. This study may provide novel insights into the post-translational regulation of antioxidation reactions, which involves the coordinated control of mitochondria.

Intelligent Labeling of Tumor Lesions Based on Positron Emission Tomography/Computed Tomography.

Positron emission tomography/computed tomography (PET/CT) plays a vital role in diagnosing tumors. However, PET/CT imaging relies primarily on manual interpretation and labeling by medical professionals. An enormous workload will affect the training samples' construction for deep learning. The labeling of tumor lesions in PET/CT images involves the intersection of computer graphics and medicine, such as registration, a fusion of medical images, and labeling of lesions. This paper extends the linear interpolation, enhances it in a specific area of the PET image, and uses the outer frame scaling of the PET/CT image and the least-squares residual affine method. The PET and CT images are subjected to wavelet transformation and then synthesized in proportion to form a PET/CT fusion image. According to the absorption of 18F-FDG (fluoro deoxy glucose) SUV in the PET image, the professionals randomly select a point in the focus area in the fusion image, and the system will automatically select the seed point of the focus area to delineate the tumor focus with the regional growth method. Finally, the focus delineated on the PET and CT fusion images is automatically mapped to CT images in the form of polygons, and rectangular segmentation and labeling are formed. This study took the actual PET/CT of patients with lymphatic cancer as an example. The semiautomatic labeling of the system and the manual labeling of imaging specialists were compared and verified. The recognition rate was 93.35%, and the misjudgment rate was 6.52%.

Contribution of extracellular polymeric substances and microbial community on the safety of drinking water quality: By mean of Cu/activated carbon biofiltration.

Change in water quality was investigated with laboratory-scale ozone-biological activated carbon filters using copper-modified granular activated carbon (Cu/GAC) and unmodified granular activated carbon (GAC). In the first seven days of the experimental period, Cu/GAC removed organic matter more efficiently owing to its enhanced adsorption capacity. As the running time increased, the amount of disinfection by-products (DBPs), dissolved organic carbon, and extracellular polymeric substances (EPS) increased sharply in the effluent of the Cu/GAC filter (CCW). More importantly, the EPS suspended in the CCW exhibited weaker flocculating efficiency and hydrophobicity, causing more active chemical reactions between chlorine and EPS substances. The copper species significantly limited the microbial biomass (0.01 nmol/L adenosine triphosphate) but stimulated the secretion of significant amounts of EPS by microorganisms for self-protection. Furthermore, the microbial community in the bulk water was successfully shaped by Cu/GAC, resulting in a continuous supply of EPS-derived DBP precursors and a sharp rise in chlorine consumption in the downstream drinking water distribution. Therefore, use of modified GAC materials, similar to Cu/GAC, as carrier materials for biological activated carbon (BAC) treatment remains controversial, despite enhanced pollutant adsorption capacity. This is the first study to reveal the mechanism of BAC-modified materials for water quality stability. The study potentially contributes to a comprehensive understanding of the effects of biofilm transformation and microbial community succession on drinking water quality. These results showed that tap water safety risks could be reduced by improving BAC pretreatment in drinking water treatment plants.

Effects of cast iron pipe corrosion on nitrogenous disinfection by-products formation in drinking water distribution systems via interaction among iron particles, biofilms, and chlorine.

The effects of cast iron pipe corrosion on nitrogenous disinfection by-products formation (N-DBPs) in drinking water distribution systems (DWDSs) were investigated. The results verified that in the effluent of corroded DWDSs simulated by annular reactors with corroded cast iron coupons, typical N-DBPs, including haloacetamides, halonitromethanes, and haloacetonitriles, increased significantly compared with the influent of DWDSs. In addition, more dissolved organic carbon, adenosine triphosphate, and iron particles were simultaneously detected in the bulk water of corroded DWDSs, thereby indicating that abundant iron particles acted as a "protective umbrella" for microorganisms. Under the condition of corroded DWDSs, the extracellular polymeric substances gradually exhibited distinct characteristics, including a higher content and lower flocculation efficiency, thereby resulting in a large supply of N-DBPs precursors. Corroded cast iron pipes, equivalent to a unique microbial interface, induced completely distinct microbial community structures and metabolic functions in DWDSs, thereby enhancing the formation of N-DBPs. This is the first study to successfully reveal the interactions among iron particles, biofilms, and chlorine in DWDSs, which may help to fully understand the biofilm transformation and microbial community succession in DWDSs.

Active cargo loading into extracellular vesicles: Highlights the heterogeneous encapsulation behaviour.

Extracellular vesicles (EVs) have demonstrated unique advantages in serving as nanocarriers for drug delivery, yet the cargo encapsulation efficiency is far from expectation, especially for hydrophilic chemotherapeutic drugs. Besides, the intrinsic heterogeneity of EVs renders it difficult to evaluate drug encapsulation behaviour. Inspired by the active drug loading strategy of liposomal nanomedicines, here we report the development of a method, named "Sonication and Extrusion-assisted Active Loading" (SEAL), for effective and stable drug encapsulation of EVs. Using doxorubicin-loaded milk-derived EVs (Dox-mEVs) as the model system, sonication was applied to temporarily permeabilize the membrane, facilitating the influx of ammonium sulfate solution into the lumen to establish the transmembrane ion gradient essential for active loading. Along with extrusion to downsize large mEVs, homogenize particle size and reshape the nonspherical or multilamellar vesicles, SEAL showed around 10-fold enhancement of drug encapsulation efficiency compared with passive loading. Single-particle analysis by nano-flow cytometry was further employed to reveal the heterogeneous encapsulation behaviour of Dox-mEVs which would otherwise be overlooked by bulk-based approaches. Correlation analysis between doxorubicin auto-fluorescence and the fluorescence of a lipophilic dye DiD suggested that only the lipid-enclosed particles were actively loadable. Meanwhile, immunofluorescence analysis revealed that more than 85% of the casein positive particles was doxorubicin free. These findings further inspired the development of the lipid-probe- and immuno-mediated magnetic isolation techniques to selectively remove the contaminants of non-lipid enclosed particles and casein assemblies, respectively. Finally, the intracellular assessments confirmed the superior performance of SEAL-prepared mEV formulations, and demonstrated the impact of encapsulation heterogeneity on therapeutic outcome. The as-developed cargo-loading approach and nano-flow cytometry-based characterization method will provide an instructive insight in the development of EV-based delivery systems.

General and mild modification of food-derived extracellular vesicles for enhanced cell targeting.

Food-derived extracellular vesicles (FDEVs) have attracted increasing attention as potential delivery vehicles for therapeutic agents due to their desirable features such as excellent biocompatibility, easy accessibility and cost effectiveness. However, the intrinsic targeting capability of FDEVs is unsatisfactory compared to artificial nanoparticles or other source-derived EVs, which calls for efficient surface engineering strategies to equip them with specific ligands. Here we report a general and mild modification method via reduction of disulfide groups to maleimide reactive thiols. Taking milk-derived EVs (mEVs) as a model system, we demonstrated the feasibility for tethering various ligands on the surface without compromising the vesicular structures. Building an ultra-sensitive nano-flow cytometer (nFCM), the heterogeneous nature of the functionalized samples was revealed, and a magnetic separation approach was proposed accordingly to remove the as-observed non-EV particles. The cellular uptake and cytotoxicity experiments provided direct evidence showing an enhanced cell targeting and cargo delivery capability of the ligand conjugated mEVs. In addition, the in vivo imaging further proved the applicability of transferrin conjugation for increased tumor enrichment of mEVs. Collectively, this general and mild ligand conjugation method enables an efficient surface functionalization of FDEVs, which is of vital importance for enhanced targeting delivery.

Rapid and quantitative in vitro analysis of mitochondrial fusion and its interplay with apoptosis.

Mitochondrial fusion is essential to maintain genomic stability and physiological functions of mitochondria. Since mitochondrial fusion and fission work in concert to regulate mitochondrial morphology and functions, it has been challenging to quantitatively measure the direct roles of mitochondrial fusion in apoptosis and cancer progression. Here, we report the development of a high-throughput in vitro method to quantify mitochondrial fusion through single mitochondria analysis by a laboratory-built nano-flow cytometer (nFCM). Isolated mitochondria expressing green fluorescent protein (GFP-mito) or discosoma red fluorescent protein (DsRed-mito) were mixed together, induced to fuse, and analyzed by nFCM. A particle exhibiting both green and red fluorescence was identified as an event of heterotypic fusion, and the efficiency of heterotypic fusion was used as a surrogate of overall fusion efficiency. The as-developed method was applied to reveal the interplay between mitochondrial fusion and apoptosis without the interference of fission. We show that cytosolic components promoted mitochondrial fusion, and this upregulation was diminished during apoptosis. Combined with the translocation of Bid and Bax from cytosol to mitochondria, these findings suggest that cytosolic pro-apoptotic Bcl-2 family proteins could be the positive mediators of mitochondrial fusion. On the other hand, fusion also renders mitochondria more resistant to membrane potential collapse upon apoptosis induction. Our data suggest that disruption of mitochondrial fusion could be a potent strategy for cancer therapy. Furthermore, the as-developed method offers an effective approach to identify fusion inhibitors, including betulinic acid and antimycin A, giving reasons for their powerful utility in cancer treatment.

Protein Profiling and Sizing of Extracellular Vesicles from Colorectal Cancer Patients via Flow Cytometry.

Extracellular vesicles (EVs) have stimulated considerable scientific and clinical interest, yet protein profiling and sizing of individual EVs remains challenging due to their small particle size, low abundance of proteins, and overall heterogeneity. Building upon a laboratory-built high-sensitivity flow cytometer (HSFCM), we report here a rapid approach for quantitative multiparameter analysis of single EVs down to 40 nm with an analysis rate up to 10 000 particles per minute. Statistically robust particle size distribution was acquired in minutes with a resolution and profile well matched with those of cryo-TEM measurements. Subpopulations of EVs expressing CD9, CD63, and/or CD81 were quantified upon immunofluorescent staining. When HSFCM was used to analyze blood samples, a significantly elevated level of CD147-positive EVs was identified in colorectal cancer patients compared to healthy controls (P < 0.001). HSFCM provides a sensitive and rapid platform for surface protein profiling and sizing of individual EVs, which could greatly aid the understanding of EV-mediated intercellular communication and the development of advanced diagnostic and therapeutic strategies.

Quantification of protein copy number in single mitochondria: The Bcl-2 family proteins.

Bcl-2 family proteins, represented by antiapoptotic protein Bcl-2 and proapoptotic protein Bax, are key regulators of mitochondria-mediated apoptosis pathway. To build a quantitative model of how Bcl-2 family protein interactions control mitochondrial outer membrane permeabilization and subsequent cytochrome c release, it is essential to know the number of proteins in individual mitochondria. Here, we report an effective method to quantify the copy number and distribution of proteins in single mitochondria via immunofluorescent labeling and sensitive detection by a laboratory-built high sensitivity flow cytometer (HSFCM). Mitochondria isolated from HeLa cells were stained with Alexa Fluor 488 (AF488)-labeled monoclonal antibodies specifically targeting Bcl-2 or Bax and with nucleic acid dye. A series of fluorescent nanospheres with fluorescence intensity calibrated in the unit of molecules of equivalent soluble fluorochrome (MESF)-AF488 were used to construct a calibration curve for converting the immunofluorescence of a single mitochondrion to the number of antibodies bound to it and then to the number of proteins per mitochondrion. Under the normal condition, the measured mean copy numbers were 1300 and 220 per mitochondrion for Bcl-2 and Bax, respectively. A significant variation in protein copy number was identified, which ranged from 130 to 6000 (2.5-97.5%) for Bcl-2 and from 65 to 700 (2.5-97.5%) for Bax, respectively. We observed an approximately 4.4 fold increase of Bax copy number per mitochondrion upon 9h of apoptosis stimulation while the abundance of Bcl-2 remained almost unchanged. To the best of our knowledge, this is the first report of Bcl-2 family protein copy number and variance in single mitochondria. Collectively, we demonstrate that the HSFCM-based immunoassay provides a rapid and sensitive method for determining protein copy number distribution in single mitochondria.