A CD25×TIGIT bispecific antibody induces anti-tumor activity through selective intratumoral Treg cell depletion.

Intratumoral regulatory T cells (Tregs) express high levels of CD25 and TIGIT, which are also recognized as markers of effector T cell (Teff) activation. Targeting these molecules each alone with monoclonal antibodies (mAbs) poses a risk of concurrently depleting both Teffs and peripheral Tregs, thereby compromising the effectiveness and selectivity of intratumoral Treg depletion. Here, leveraging the increased abundance of CD25+ TIGIT+ double-positive Tregs in the solid tumor microenvironment (but not in peripheral tissues), we explore the feasibility of using a CD25×TIGIT bispecific antibody (bsAb) to selectively deplete intratumoral Tregs. We initially constructed a bsAb co-targeting mouse CD25 and TIGIT, NSWm7210, and found that NSWm7210 conferred enhanced intratumoral Treg depletion, Teff activation, and tumor suppression as compared to the parental monotherapies in mouse models. We subsequently constructed a bsAb co-targeting human CD25 and TIGIT (NSWh7216), which preferentially eliminated CD25+ TIGIT+ double-positive cells over single-positive cells in vitro. NSWh7216 exhibited enhanced anti-tumor activity without toxicity of peripheral Tregs in CD25 humanized mice compared to the parental monotherapies. Our study illustrates the use of CD25×TIGIT bsAbs as effective agents against solid tumors based on selective depletion of intratumoral Tregs.

Impact of polystyrene nanoplastics on apoptosis and inflammation in zebrafish larvae: Insights from reactive oxygen species perspective.

In recent years, there has been a growing focus on the toxicity and mortality induced by nanoplastics (NPs) in aquatic organisms. However, studies investigating mechanisms underlying oxidative stress (OS), apoptosis, and inflammation induced by NPs in fish remain limited. This study observed that polystyrene NPs (PS-NPs) were accumulated into zebrafish larvae and zebrafish embryonic fibroblast (ZF4 cells), accompanied by the occurrence of pathological damage both at the cellular and tissue-organ level. Additionally, the transcriptional up-regulation of NADPH oxidases (NOXs) and subsequent excessive generation of reactive oxygen species (ROS) resulted in notable changes in the relative mRNA and protein expression levels associated with antioxidant oxidase systems in larvae. Furthermore, the study identified the impact of NPs on mitochondrial ultrastructural, resulting in mitochondrial depolarization and downregulation of mRNA expression related to the electron transport chain due to excessive ROS generation. Short-term exposure to NPs also triggered apoptosis and inflammation in zebrafish larvae, evident from significant up-regulation in mRNA expressions of proapoptotic factors and NF-κB proinflammatory signaling pathway, as well as increased transcription and protein levels of pro-inflammatory factors in larvae. Inhibition of intracellular excessive ROS effectively reduced the induction of apoptosis, NF-κB P65 nuclear migration levels, and cytokine secretion, underscoring OS as a pivotal factor throughout the process of apoptosis and inflammatory responses induced by NPs. This research significantly advances our comprehension of biological effects and underlying mechanisms of NPs in freshwater fish.

Applications and perspectives of tumor organoids in radiobiology (Review).

Radiotherapy exhibits significant versatility and efficacy in cancer treatment, thereby playing a crucial role in the field of oncology. However, there remains an urgent need for extensive research on various aspects of radiotherapy, including target selection, damage repair and its combination with immunotherapy. Particularly, the development of in vitro models to replicate in vivo tumor lesion responses is vital. The present study provides a thorough review of the establishment and application of tumor organoids in radiotherapy, aiming to explore their potential impact on cancer treatment.

An anti-CD98 antibody displaying pH-dependent Fc-mediated tumour-specific activity against multiple cancers in CD98-humanized mice.

The cell-surface glycoprotein CD98-a subunit of the LAT1/CD98 amino acid transporter-is an attractive target for cancer immunotherapies, but its widespread expression has hampered the development of CD98-targeting antibody therapeutics. Here we report that an anti-CD98 antibody, identified via the screening of phage-display libraries of CD98 single-chain variable fragments with mutated complementarity-determining regions, preserves the physiological function of CD98 and elicits broad-spectrum crystallizable-fragment (Fc)-mediated anti-tumour activity (requiring Fcγ receptors for immunoglobulins, macrophages, dendritic cells and CD8+ T cells, as well as other components of the innate and adaptive immune systems) in multiple xenograft and syngeneic tumour models established in CD98-humanized mice. We also show that a variant of the anti-CD98 antibody with pH-dependent binding, generated by solving the structure of the antibody-CD98 complex, displayed enhanced tumour-specific activity and pharmacokinetics. pH-dependent antibody variants targeting widely expressed antigens may lead to superior therapeutic outcomes.

A cross-reactive pH-dependent EGFR antibody with improved tumor selectivity and penetration obtained by structure-guided engineering.

The clinical use of anti-EGFR antibody-based cancer therapy has been limited by antibody-EGFR binding in normal tissues, so developing pH-dependent anti-EGFR antibodies that selectively bind with EGFR in tumors-by taking advantage of the acidity of tumor microenvironment relative to normal tissues-may overcome these limitations. Here, we generated pH-dependent anti-EGFR antibodies with cross-species reactivity for human and mouse EGFR, and we demonstrate that pH-dependent antibodies exhibit tumor-selective binding by binding strongly to EGFR under acidic conditions (pH 6.5) but binding weakly under neutral (pH 7.4) conditions. Based on screening a non-immune human antibody library and antibody affinity maturation, we initially generated antibodies with cross-species reactivity for human and mouse EGFR. A structure model was subsequently constructed and interrogated for hotspots affecting pH-dependent binding, which supported development of a cross-reactive pH-dependent anti-EGFR antibody, G532. Compared with its non-pH-dependent antibody variant, G532 exhibits improved tumor selectivity, tumor penetration, and antitumor activity. Thus, beyond showing that pH-dependent anti-EGFR antibodies can overcome multiple limitations with antibody-based cancer therapies targeting EGFR, our study illustrates a structure-guided antibody-antigen binding pH-dependency engineering strategy to enhance antibody tumor selectivity and tumor penetration, which can inform the future development of antibody-based cancer therapies targeting other ubiquitously expressed molecules.

Brain organoids for addressing COVID-19 challenge.

COVID-19 is a systemic disease involving multiple organs, and clinically, patients have symptoms of neurological damage to varying degrees. However, we do not have a clear understanding of the relationship between neurological manifestations and viral infection due to the limitations of current in vitro study models. Brain organoids, formed by the differentiation of stem cells under 3D culture conditions, can mimic the structure of tiny cell clusters with neurodevelopmental features in different patients. The paper reviewed the history of brain organoids development, the study of the mechanism of viral infection, the inflammatory response associated with neurological damage, the detection of antiviral drugs, and combined microarray technology to affirm the status of the brain organoid models in the study of COVID-19. In addition, our study continuously improved the model in combination with emerging technologies, to lay a theoretical foundation for clinical application and academic research.

Haemorrhagic complications following cataract and vitreoretinal surgery with sub-Tenon's block in patients receiving non-vitamin K oral anticoagulant agents: A prospective audit.

There is a lack of data to support either continuation or interruption of non-vitamin K oral anticoagulants for cataract and vitreoretinal surgery. A prospective audit was undertaken of 291 patients undergoing cataract surgery or vitreoretinal surgery, predominantly under sub-Tenon's block, while continuing these agents. The median time from last non-vitamin K oral anticoagulant dose to the insertion of sub-Tenon's block was five hours. No patient required emergency reversal of anticoagulation. There were no sight-threatening complications in the immediate perioperative period, although two vitreoretinal patients (3.8%) had a moderate haemorrhagic complication on day five, and two cataract patients (0.8%) had a minor haemorrhagic complication on days one and 14 postoperatively. Despite continuing their non-vitamin K oral anticoagulants, three (1%) cataract patients had a moderate thromboembolic complication within the 30-day postoperative period. The risk of haemorrhagic complications associated with continuation of anticoagulation with non-vitamin K oral anticoagulants for cataract and vitreoretinal surgery is low, and this audit supports the continuation of non-vitamin K oral anticoagulants for our patients having cataract and vitreoretinal surgery.

Facile Preparation and Dye Adsorption Performance of Poly(N-isopropylacrylamide-co-acrylic acid)/Molybdenum Disulfide Composite Hydrogels.

Using N-isopropylacrylamide (NIPAM) and acrylic acid (AAc) as monomers, N,N'-methylenebisacrylamide (MBA) as a cross-linking agent, and molybdenum disulfide (MoS2) as functional particles, a P(NIPAM-co-AAc)/MoS2 composite hydrogel was prepared by free radical polymerization initiated by ultraviolet light. The results of Fourier transform infrared spectroscopy, Raman spectroscopy, and scanning electron microscopy show that MoS2 has been successfully introduced into the P(NIPAM-co-AAc) system, and the obtained composite hydrogel has a porous network structure. Studies on the swelling property and dye adsorption performance show that the addition of MoS2 can increase the swelling ratio of P(NIPAM-co-AAc) hydrogels to a certain extent and can significantly improve the ability of the P(NIPAM-co-AAc) hydrogel to adsorb methylene blue (MB). The adsorption process of MB by the composite hydrogels conforms to the pseudo-second-order kinetics and the Langmuir isotherm adsorption models. The estimated equilibrium adsorption capacity (Q m) using the Langmuir isotherm model can reach 1258 mg/g, mainly due to the electrostatic interaction between the negatively charged groups -COO- and MoS2 particles on the network structure and the positively charged dye MB. The adsorption of MB by P(NIPAM-co-AAc)/MoS2 composite hydrogels depends on the temperature during adsorption. Compared with room temperature, a high temperature of 40 °C above the poly(N-isopropylacrylamide) (PNIPAM) phase transition temperature (∼32 °C) leads to a decreased adsorption capacity of the P(NIPAM-co-AAc)/MoS2 composite hydrogel for MB due to the enhanced hydrophobic properties of the network structure and the decrease of the swelling ratio. The prepared hydrogel material can be used as a good adsorbent for dyes, which is promising in wastewater treatment.

Preparation and Laser Marking Properties of Poly(propylene)/Molybdenum Sulfide Composite Materials.

In this study, using molybdenum sulfide (MoS2) as laser-sensitive particles and poly(propylene) (PP) as the matrix resin, laser-markable PP/MoS2 composite materials with different MoS2 contents ranging from 0.005 to 0.2% were prepared by melt-blending. A comprehensive analysis of the laser marking performance of PP/MoS2 composites was carried out by controlling the content of laser additives, laser current intensity, and the scanning speed of laser marking. The color difference test shows that the best laser marking performance of the composite can be obtained at the MoS2 content of 0.02 wt %. The surface morphology of the PP/MoS2 composite material was observed after laser marking using a metallographic microscope, an optical microscope, and a scanning electron microscope (SEM). During the laser marking process, the laser energy was absorbed and converted into heat energy to cause high-temperature melting, pyrolysis, and carbonization of PP on the surface of the PP/MoS2 composite material. The black marking from carbonized materials was formed in contrast to the white matrix. Using X-ray diffraction (XRD), Fourier transform infrared (FT-IR) spectroscopy, and Raman spectroscopy, the composite materials before and after laser marking were tested and characterized. The PP/MoS2 composite material was pyrolyzed to form amorphous carbonized materials. The effect of the laser-sensitive MoS2 additive on the mechanical properties of composite materials was investigated. The results show that the PP/MoS2 composite has the best laser marking property when the MoS2 loading content is 0.02 wt %, the laser marking current intensity is 11 A, and the laser marking speed is 800 mm/s, leading to a clear and high-contrast marking pattern.

Construction of Self-Assembled Polyelectrolyte/Cationic Microgel Multilayers and Their Interaction with Anionic Dyes Using Quartz Crystal Microbalance and Atomic Force Microscopy.

This study aimed to reveal the interaction between self-assembled multilayers and dye molecules in the environment, which is closely related to the multilayers' stable performance and service life. In this work, the pH-responsive poly (N-isopropylacrylamide-co-2-(dimethylamino) ethyl methacrylate) microgels were prepared by free-radical copolymerization and self-assembled with sodium alginate (SA) into multilayers by the layer-by-layer deposition method. Quartz crystal microbalance (QCM) and atomic force microscopy (AFM) results confirmed the construction of multilayers and the absorbed mass, resulting in a decrease in the frequency shift of the QCM sensor and the deposition of microgel particles on its surface. The interaction between the self-assembled SA/microgel multilayers and anionic dyes in the aqueous solution was further investigated by QCM, and it was found that the electrostatic attraction between dyes and microgels deposited on the QCM sensor surface was much larger than that of the microgels with SA in multilayers, leading to the release of the microgels from the self-assembled structure and a mass loss ratio of 27.6%. AFM observation of the multilayer morphology exposed to dyes showed that 29% of the microgels was peeled off, and the corresponding microgel imprints were generated on the surface. In contrast, the shape and size of the remaining self-assembled microgel particles did not change.

Effect of the Trifunctional Chain Extender on Intrinsic Viscosity, Crystallization Behavior, and Mechanical Properties of Poly(Ethylene Terephthalate).

In this work, poly(ethylene terephthalate) (PET) chain-extending products with different molecular weights were prepared by reactive extrusion using isocyanate trimer (C-HK) as the trifunctional chain extender. The effect of the chain extender C-HK on the intrinsic viscosity, melt flow property, crystallization behavior, crystallization morphology, and mechanical property of PET was investigated. The results showed that when the content of the chain extender was increased from 0.6 to 1.4 wt%, the viscosity average molecular weight of PET was effectively increased from 2.36 × 104 to 5.46 × 104 g·mol-1. After the chain extending, the crystallinity and the time of semicrystallization of PET were significantly decreased. After the isothermal crystallization at 220 °C for 5 min, the spherulites formed by pure PET became larger. With the increase in molecular weight of PET after chain extension, its spherulite size was significantly decreased without changing the crystalline structure. The chain-extended PET also exhibited more excellent bending-resistant and impact-resistant properties. While the tensile strength of PET after chain extension was slightly decreased, the bending strength was increased by a maximum value of 56.8%, and the impact strength was increased by a maximum value of five times.

Surface Laser-Marking and Mechanical Properties of Acrylonitrile-Butadiene-Styrene Copolymer Composites with Organically Modified Montmorillonite.

In this study, organically modified montmorillonite (OMMT) was prepared by modifying MMT with a cationic surfactant cetyltrimethylammonium bromide (CTAB). The obtained OMMT of different loading contents (1, 2, 4, 6, and 8 wt %) was melt-blended with poly(acrylonitrile-co-butadiene-co-styrene) (ABS) to prepare a series of ABS/OMMT composites, which were laser marked using a neodymium-doped yttrium aluminum garnet (Nd:YAG) laser beam of 1064 nm under different laser current processes. X-ray diffraction (XRD), color difference spectrometer, optical microscope, water contact angle tests, scanning electron microscope (SEM), and Raman spectroscopy were carried out to characterize the morphology, structure, and properties of the laser-patterned ABS composites. The effects of the addition of OMMT and the laser marking process on the mechanical properties of ABS/OMMT composites were investigated through mechanical property tests. The results show that the obtained ABS/OMMT composites have enhanced laser marking performance, compared to the ABS. When the OMMT content is 2 wt % and the laser current intensity is 9 A, the marking on ABS composites has the highest contrast (ΔE = 36.38) and sharpness, and the quick response (QR) code fabricated can be scanned and identified with a mobile app. SEM and water contact angle tests showed that the holes, narrow cracks, and irregular protrusion are formed on the composite surface after laser marking, resulting in a more hydrophobic surface and an increased water contact angle. Raman spectroscopy and XRD indicate that OMMT can absorb the near-infrared laser energy, undergo photo thermal conversion, and cause the pyrolysis and carbonization of ABS to form black marking, and the crystal structure itself does not change significantly. When the 2 wt % of OMMT is loaded, the tensile strength, elongation at break, and impact strength of ABS/OMMT are increased by 15, 20, and 14%, respectively, compared to ABS. Compared with the unmarked ABS/OMMT, the defects including holes and cracks generated on the surface of the marked one lead to the decreased mechanical property. The desirable combination of high contrast laser marking performance and mechanical properties can be achieved at an OMMT loading content of 2 wt % and a laser current intensity of 9 A. This research work provides a simple, economical, and environmentally friendly method for laser marking of engineering materials such as ABS.

Facile preparation of polyacrylamide/chitosan/Fe3O4 composite hydrogels for effective removal of methylene blue from aqueous solution.

In this study, Fe3O4 magnetic nanoparticles were synthesized in situ in the polyacrylamide/chitosan (PAAm/CS) hydrogel networks. The obtained hydrogels are characterized by Scanning electron microscopy (SEM), Transmission electron microscopy (TEM), X-ray diffraction (XRD), and Raman spectroscopy. The results confirm that the three-dimensional network structure of the hydrogels is incorporated with Fe3O4 nanoparticles. The adsorption properties of PAAm/CS/Fe3O4 hydrogels for methylene blue (MB) in aqueous solution were studied using Ultraviolet and visible spectrophotometry (UV-vis). The results show that when compared to PAAm/CS hydrogels, PAAm/CS/Fe3O4 hydrogels can adsorb MB with higher adsorption capacities of approximately 1603 mg/g, and the kinetics and isotherm models of the adsorption process could be better described by the pseudo-first order model and Langmuir isotherm model, respectively. Due to the facile preparation, high adsorption capacity, and low cost, the PAAm/CS/Fe3O4 hydrogels are good adsorbents for MB and exhibit significant potential in the treatment of sewage.

Facile Fabrication of High-Contrast and Light-Colored Marking on Dark Thermoplastic Polyurethane Materials.

In this work, using ferroferric oxide (Fe3O4) and zirconium oxide (ZrO2) as laser-sensitive particles and thermoplastic polyurethane (TPU) as the matrix resin, a series of TPU/Fe3O4/ZrO2 composites were prepared by melt blending, and the effect of the laser marking additive content, composition, and laser marking parameters on the laser marking properties of composites was investigated. The laser marking mechanism of Fe3O4/ZrO2 additives and the role of each component in TPU laser marking were studied by metallographic microscopy, color difference test, scanning electron microscopy, and Raman spectroscopy. Fe3O4 nanoparticles as a laser sensitizer component, on the one hand, can act as a pigment to make the TPU substrate black and, on the other hand, can absorb laser energy to contribute to the formation of laser markings on TPU composite surfaces. In addition, the introduction of ZrO2 nanoparticles can help absorb the laser energy, while the contrast can be improved to enhance the laser marking performance of the TPU composite. Through thermogravimetric analysis, the changes in the thermally stable properties of TPU composites before and after laser marking were investigated, and the results indicated that Fe3O4/ZrO2 nanoparticles can absorb the laser energy, causing melting and pyrolysis of the TPU backbone at a high temperature, to produce a gaseous product resulting in foaming. Finally, the high-contrast and light-colored markings were formed on the black TPU composite surface. This work provides a facile method for producing high-contrast and light-colored markings on the dark TPU composite surface.

[Threshold Flux and Membrane Fouling Analysis of the Hybrid Pre-ozonation and CNTs Membrane Modification Process].

Polyvinylidene fluoride (PVDF) hollow fiber ultrafiltration membranes were modified with carbon nanotubes (CNT). Hybrid pre-ozonation and CNT modification were investigated by experimentally manipulating the ozonation process, threshold flux, and membrane fouling. The results showed that the threshold fluxes of the unmodified membrane and hybrid process were 45 L·(m2·h)-1 and 81 L·(m2·h)-1, respectively. Additionally, the fouling rate of the hybrid process was about 0.00137 kPa·min-1·L-1·m2·h, which was notably lower compared to other process. The results showed that the filtration volume under threshold flux was higher than that under critical flux with the same CNT loading mass and ozone dosage. This comparison indicated that membrane fouling was alleviated under threshold flux and that the corresponding operation period was extended. Through the carbon balance experiment, the fouling capacity and recoverability improved remarkably after CNT modification. Additionally, ozonation could enhance the recoverability of membranes. The hybrid process examined in this study could dramatically improve the permeability and extend the operation time of the ultrafiltration membrane.

TesG is a type I secretion effector of Pseudomonas aeruginosa that suppresses the host immune response during chronic infection.

Pseudomonas aeruginosa is a versatile Gram-negative pathogen with intricate intracellular regulatory networks that enable it to adapt to and flourish in a variety of biotic and abiotic habitats. However, the mechanism permitting the persistent survival of P. aeruginosa within host tissues and causing chronic symptoms still remains largely elusive. By using in situ RNA sequencing, here we show that P. aeruginosa adopts different metabolic pathways and virulence repertoires to dominate the progression of acute and chronic lung infections. Notably, a virulence factor named TesG, which is controlled by the vital quorum-sensing system and secreted by the downstream type I secretion system, can suppress the host inflammatory response and facilitate the development of chronic lung infection. Mechanically, TesG can enter the intracellular compartment of macrophages through clathrin-mediated endocytosis, competitively inhibit the activity of eukaryotic small GTPase and thus suppress subsequent neutrophil influx, cell cytoskeletal rearrangement of macrophages and the secretion of cytokines and chemokines. Therefore, the identification of TesG in this study reveals a type I secretion apparatus of P. aeruginosa that functions during the host-pathogen interaction, and may open an avenue for the further mechanistic study of chronic respiratory diseases and the development of antibacterial therapy.

Structural and Functional Insights into PpgL, a Metal-Independent ß-Propeller Gluconolactonase That Contributes to Pseudomonas aeruginosa Virulence.

Biofilm formation is a critical determinant in the pathopoiesis of Pseudomonas aeruginosa It could significantly increase bacterial resistance to drugs and host defense. Thus, inhibition of biofilm matrix production could be regarded as a promising attempt to prevent colonization of P. aeruginosa and the subsequent infection. PpgL, a periplasmic gluconolactonase, has been reported to be involved in P. aeruginosa quorum-sensing (QS) system regulation. However, the detailed function and catalysis mechanism remain elusive. Here, the crystal structure of PpgL is described in the current study, along with biochemical analysis, revealing that PpgL is a typical ß-propeller enzyme with unique metal-independent lactone hydrolysis activity. Consequently, comparative analysis of seven-bladed propeller lactone-catalyzing enzymes and mutagenesis studies identify the critical sites which contribute to the diverse catalytic and substrate recognition functions. In addition, the reduced biofilm formation and attenuated invasion phenotype resulting from deletion of ppgL confirm the importance of PpgL in P. aeruginosa pathogenesis. These results suggest that PpgL is a potential target for developing new agents against the diseases caused by P. aeruginosa.

Identification of Glycine Receptor α3 as a Colchicine-Binding Protein.

Colchicine (Col) is considered a kind of highly effective alkaloid for preventing and treating acute gout attacks (flares). However, little is known about the underlying mechanism of Col in pain treatment. We have previously developed a customized virtual target identification method, termed IFPTarget, for small-molecule target identification. In this study, by using IFPTarget and ligand similarity ensemble approach (SEA), we show that the glycine receptor alpha 3 (GlyRα3), which play a key role in the processing of inflammatory pain, is a potential target of Col. Moreover, Col binds directly to the GlyRα3 as determined by the immunoprecipitation and bio-layer interferometry assays using the synthesized Col-biotin conjugate (linked Col and biotin with polyethylene glycol). These results suggest that GlyRα3 may mediate Col-induced suppression of inflammatory pain. However, whether GlyRα3 is the functional target of Col and serves as potential therapeutic target in gouty arthritis requires further investigations.

Resonant-type piezoelectric inertial linear motor based on the optimization of a dual stage tuning fork transducer.

A dual stage tuning fork transducer (DSTFT) is designed as a stator for a resonant-type inertial linear motor. The first- and second-layer resonant frequencies of DSTFT are automatically adjusted with a ratio of 1:2 by using an ANSYS optimization design algorithm, and a resonant-type sawtooth-shaped mechanical waveform is generated by composing the two resonant vibrations of DSTFT. An inertial linear motor prototype is fabricated and tested. Experimental results confirmed the effectiveness of the designed transducer. The no-load maximum speed is 21.5 mm/s with a driving voltage of 67.2 Vp-p at a base frequency of 2831 Hz. The linear speed is 10.5 mm/s, and the drag load is 0.02 N at a preload force of 1 N and a driving voltage of 114 Vp-p for the base frequency. The movement direction could be reversed by changing the driving voltage phase.

[Effect of Hybrid Process of Pre-ozonation and CNT Modification on Hollow Fiber Membrane Fouling Control].

Polyvinylidene fluoride (PVDF) hollow fiber ultrafiltration membranes were modified with carbon nanotube (CNT). Combined with the ozonation process, the effect of the hybrid pre-ozonation and CNT modification on fouling alleviation was investigated. The impacts of CNT loading mass and ozone dosage on the variation of flux and antifouling ability of the membrane modules were evaluated. Under a critical flux of 144 L·(m2·h)-1, CNT loading mass of 3 g·m-2, and ozone dosage(O3/DOC) of 0.22 mg·mg-1, the results revealed that the filtration volume of the hybrid process was promoted to 850 L·m-2, which was about 4.5 times higher than that of the original unmodified membrane. With a flux of 18 L·(m2·h)-1 and 15 day operation, the filtration volume was promoted to 3000 L·m-2, which was 10 times that of the unmodified membrane. The fouling membrane surface was observed using confocal laser scanning electron microscopy (CLSM). The results demonstrated that more living bacteria were present on the membrane surface of the unmodified membrane, which showed a rapid transmembrane pressure (TMP) increase. Both pre-ozonation and CNT modification decreased the total amount of microorganisms and the amount of the living bacteria as well, which mitigated the increase in TMP. After pre-ozonation, the presence of a CNT layer on the membrane surface further decreased the number of living bacteria. Although the CNT layer captured some dead bacteria, it had no obvious relationship with the increase in TMP.