New Opportunities for Electric Fields in Promoting Wound Healing: Collective Electrotaxis.

Significance: It has long been hypothesized that naturally occurring electric fields (EFs) aid wound healing by guiding cell migration. Consequently, the application of EFs has significant potential for promoting wound healing. However, the mechanisms underlying the cellular response to EFs remain unclear. Recent Advances: Although the directed migration of isolated single cells under EFs has been studied for decades, only recently has experimental evidence demonstrated the distinct collective migration of large sheets of keratinocytes and corneal epithelial cells in response to applied EFs. Accumulating evidence suggests that the emergent properties of cell groups in response to EF guidance offer new opportunities for EF-assisted directional migration. Critical Issues: In this review, we provide an overview of the field of collective electrotaxis, highlighting key advances made in recent years. We also discuss advanced engineering strategies utilized to manipulate collective electrotaxis. Future Directions: We outline a series of unanswered questions in this field and propose potential applications of collective electrotaxis in developing electrical stimulation technologies for wound healing.

5Z-7-Oxozaenol attenuates cuprizone-induced demyelination in mice through microglia polarization regulation.

INTRODUCTION: Demyelination is a key factor in axonal degeneration and neural loss, leading to disability in multiple sclerosis (MS) patients. Transforming growth factor beta activated kinase 1 (TAK1) is a critical molecule involved in immune and inflammatory signaling pathways. Knockout of microglia TAK1 can inhibit autoimmune inflammation of the brain and spinal cord and improve the outcome of MS. However, it is unclear whether inhibiting TAK1 can alleviate demyelination. METHODS: Eight-week-old male c57bl/6j mice were randomly divided into five groups: (a) the control group, (b) the group treated with cuprizone (CPZ) only, (c) the group treated with 5Z-7-Oxozaenol (OZ) only, and (d) the group treated with both cuprizone and 15 µg/30 µg OZ. Demyelination in the mice of this study was induced by administration of CPZ (ig) at a daily dose of 400 mg/kg for consecutive 5 weeks. OZ was intraperitoneally administered at mentioned doses twice a week, starting from week 3 after beginning cuprizone treatment. Histology, rotarod test, grasping test, pole test, Western blot, RT-PCR, and ELISA were used to evaluate corpus callosum demyelination, behavioral impairment, oligodendrocyte differentiation, TAK1 signaling pathway expression, microglia, and related cytokines. RESULTS: Our results demonstrated that OZ protected against myelin loss and behavior impairment caused by CPZ. Additionally, OZ rescued the loss of oligodendrocytes in CPZ-induced mice. OZ inhibited the activation of JNK, p65, and p38 pathways, transformed M1 polarized microglia into M2 phenotype, and increased brain-derived neurotrophic factor (BDNF) expression to attenuate demyelination in CPZ-treated mice. Furthermore, OZ reduced the expression of proinflammatory cytokines and increases anti-inflammatory cytokines in CPZ-treated mice. CONCLUSION: These findings suggest that inhibiting TAK1 may be an effective approach for treating demyelinating diseases.

A mitochondria-targeting dihydroartemisinin derivative as a reactive oxygen species -based immunogenic cell death inducer.

Immunogenic cell death (ICD) can activate the anticancer immune response and its occurrence requires high reliance on oxidative stress. Inducing mitochondrial reactive oxygen species (ROS) is a desirable capability for ICD inducers. However, in the category of ICD-associated drugs, numerous reported ICD inducers are a series of anthracyclines and weak in ICD induction. Herein, a mitochondria-targeting dihydroartemisinin derivative (T-D) was synthesized by conjugating triphenylphosphonium (TPP) to dihydroartemisinin (DHA). T-D can selectively accumulate in mitochondria to trigger ROS generation, leading to the loss of mitochondrial membrane potential (ΔΨm) and ER stress. Notably, T-D exhibits far more potent ICD-inducing properties than its parent compound. In vivo, T-D-treated breast cancer cell vaccine inhibits metastasis to the lungs and tumor growth. These results indicate that T-D is an excellent ROS-based ICD inducer with the specific function of trigging vigorous ROS in mitochondria and sets an example for incorporating artemisinin-based drugs into the ICD field.

Rapid Analysis of Compounds from Piperis Herba and Piperis Kadsurae Caulis and Their Differences Using High-Resolution Liquid-Mass Spectrometry and Molecular Network Binding Antioxidant Activity.

There is a serious mixing of Piperis Herba and Piperis Kadsurae Caulis in various parts of China due to the similar traits of lianas, and there is a lack of systematic research on the compound and activity evaluation of the two. Likewise, the differences in compounds brought about by the distribution of origin also need to be investigated. In this study, high-resolution liquid-mass spectrometry (UPLC-Q-Zeno-TOF-MS/MS) was used to analyze samples of Piperis Herba from five origins and Piperis Kadsurae Caulis from five origins, with three batches collected from each origin. The compounds were identified based on precise molecular weights, secondary fragments, and an online database combined with node-to-node associations of the molecular network. The t-test was used to screen and analyze the differential compounds between the two. Finally, the preliminary evaluation of antioxidant activity of the two herbs was carried out using DPPH and ABTS free radical scavenging assays. The results showed that a total of 72 compounds were identified and deduced in the two Chinese medicines. These compounds included 54 amide alkaloids and 18 other compounds, such as flavonoid glycosides. The amide alkaloids among them were then classified, and the cleavage pathways in positive ion mode were summarized. Based on the p-value of the t-test, 32 differential compounds were screened out, and it was found that the compounds of Piperis Herba were richer and possessed a broader spectrum of antioxidant activity, thus realizing a multilevel distinction between Piperis Herba and Piperis Kadsurae Caulis. This study provides a preliminary reference for promoting standardization and comprehensive quality research of the resources of Piperis Herba using Piperis Kadsurae Caulis as a reference.

Revealing discrepancies and drivers in the impact of lomefloxacin on groundwater denitrification throughout microbial community growth and succession.

The coexistence of antibiotics and nitrates has raised great concern about antibiotic's impact on denitrification. However, conflicting results in these studies are very puzzling, possibly due to differences in microbial succession stages. This study investigated the effects of the high-priority urgent antibiotic, lomefloxacin (LOM), on groundwater denitrification throughout microbial growth and succession. The results demonstrated that LOM's impact on denitrification varied significantly across three successional stages, with the most pronounced effects exhibited in the initial stage (53.8% promotion at 100 ng/L-LOM, 84.6% inhibition at 100 µg/L-LOM), followed by the decline stage (13.3-18.2% inhibition), while no effect in the stable stage. Hence, a distinct pattern encompassing susceptibility, insusceptibility, and sub-susceptibility in LOM's impact on denitrification was discovered. Microbial metabolism and environment variation drove the pattern, with bacterial numbers and antibiotic resistance as primary influencers (22.5% and 15.3%, p < 0.01), followed by carbon metabolism and microbial community (5.0% and 3.68%, p < 0.01). The structural equation model confirmed results reliability. Bacterial numbers and resistance influenced susceptibility by regulating compensation and bacteriostasis, while carbon metabolism and microbial community impacted energy, electron transfer, and gene composition. These findings provide valuable insights into the complex interplay between antibiotics and denitrification patterns in groundwater.

Exploring the supramolecular chemistry of cyclopropeniums: halogen-bonding-induced electrostatic assembly of polymers.

Exploring new noncovalent synthons for supramolecular assembly is essential for material innovation. Accordingly, we herein report a unique type of cyclopropenium-based supramolecular motif and demonstrate its applications to polymer self-assembly. Because of the "ion pair strain" effect, trisaminocyclopropenium iodides complex strongly with fluoroiodobenzene derivatives, forming stable adducts. Crystal structure analysis reveals that halogen-bonding between the iodide anion and the iodo substituent of the fluoroiodobenzene is the driving force for the formation of these electrostatically complexed adducts. Such halogen-bonding-induced electrostatic interactions were further successfully applied to drive the assembly of polymers in solution, on surfaces, and in bulk, demonstrating their potential for constructing supramolecular polymeric materials.

GPX4 inhibition synergistically boosts mitochondria targeting nanoartemisinin-induced apoptosis/ferroptosis combination cancer therapy.

Artemisinin, originally used for its antimalarial activity, has received much attention in recent years for cancer therapy. The anticancer mechanisms of artemisinin are complicated and debatable. Challenges in the delivery of artemisinin also persist because the anticancer effect of artemisinin alone is often not satisfactory when used with traditional nanocarriers. We herein report the mitochondrial delivery of artemisinin with extremely high anticancer capacity. The action mode of artemisinin in the mitochondria of cancer cells includes heme-participating and oxygen-independent conversion of artemisinin into a carbon-centered radical, which is partly converted into ROS in the presence of molecular oxygen. We reveal that artemisinin alone in the mitochondria can induce strong cancer cell apoptosis. In addition, due to the weak inhibition of GPX4 activity by artemisinin, weak ferroptosis is also observed. We further discover that GPX4 activity in MCF-7 cells is greatly inhibited by RSL3 to synergistically enhance the anticancer capacity of artemisinin via enhancing ferroptosis. The synergistic anticancer activity of artemisinin and RSL3 in the mitochondria not only improves cancer cell-killing ability, but also inhibits the re-proliferation of residual cancer cells. This study provides a new insight into developing highly efficient and practical artemisinin nanomedicines for cancer therapy.

Tumor-targeted AIE polymeric micelles mediated immunogenic sonodynamic therapy inhibits cancer growth and metastasis.

Aggregation-induced emission luminogens (AIEgens) exhibit potent sonosensitivity in nanocarriers compared with conventional organic sonosensitizers owing to the strong fluorescence emission in the aggregated state. However, the premature drug leakage and ineffective tumor targeting of current AIE nanosonosensitizers critically restrict their clinical applications. Here, an AIEgen-based sonosensitizer (AIE/Biotin-M) with excellent sonosensitivity was developed by assembling salicylaldazine-based amphiphilic polymers (AIE-1) and 4T1 tumor-targeting amphiphilic polymers (DSPE-PEG-Biotin) for the effective delivery of salicylaldazine to 4T1 tumor tissues, aiming to mediate immunogenic SDT. In vitro, AIE/Biotin-M were highly stable and generated plentiful singlet oxygen (1O2) under ultrasound (US) irradiation. After AIE/Biotin-M targeted accumulation in the tumor, upon US irradiation, the generation of 1O2 not only led to cancer cell death, but also elicited a systemically immune response by causing the immunogenic cell death (ICD) of cancer cells. In addition to mediating SDT, AIE/Biotin-M could chelate and reduce Fe3+, Cu2+ and Zn2+ by salicylaldazine for inhibiting neovascularization in tumor tissues. Ultimately, AIE/Biotin-M systemically inhibited tumor growth and metastasis upon US irradiation. This study presents a facile approach to the development of AIE nanosonosensitizers for cancer SDT.

Combination effects of tebuconazole with Bacillus subtilis to control rice false smut and the related synergistic mechanism.

BACKGROUND: Management of rice false smut is based mainly on chemical control, which poses many safety and environmental challenges. The other option, biological control with biofungicides, does not have such problems but is not as reliable because of low systemic ability, and lower and unstable efficacy. Therefore, it is necessary to combine application of chemical fungicides and biological control agents (BCAs) and elucidate their synergistic mechanism. RESULTS: A combination of tebuconazole and a proven BCA, Bacillus subtilis H158, was evaluated for control of rice false smut. Tebuconazole at low application rates stimulated growth of B. subtilis, prolonged the effective period of B. subtilis by enhancing its persistence on the surface of rice plants, accelerated biofilm formation of the BCA to facilitate colonization, promoted induced systemic resistance in rice by regulating defense-related enzymes and genes, and reduced the natural resistance of the pathogen by suppressing the key gene for fungal resistance to tebuconazole. However, at high application rates, tebuconazole had adverse effects on these factors and showed antagonistic combination effects with B. subtilis. The combination of B. subtilis with tebuconazole at low application rates showed great synergistic effects, but at high application rates showed only antagonistic effects in field experiments over two consecutive years. CONCLUSION: The combination of B. subtilis with tebuconazole had significant synergistic effects at low application rates. The synergistic effects are the result of multiple mechanisms involved in BCA, rice and the fungal pathogen. © 2022 Society of Chemical Industry.

Promoting halogen-bonding catalyzed living radical polymerization through ion-pair strain.

Discovering efficient catalysts is highly desired in expanding the application of halogen-bonding catalysis. We herein report our findings on applying triaminocyclopropenium (TAC) iodides as highly potent catalysts for halogen-bonding catalyzed living radical polymerization. Promoted by the unique effect of ion-pair strain between the TAC cation and the iodide anion, the TAC iodides showed high catalytic efficiency in the halogen-bonding catalysis toward radical generation, and surpassed the previously reported organic iodide catalysts. With the TAC iodide as catalyst, radical polymerization with a living feature was successfully realized, which shows general applicability with a variety of monomers and produced block copolymers. In addition, the TAC-iodides also showed promising feasibility in catalyzing the radical depolymerization of iodo-terminated polymethacrylates. Noteworthily, the catalytic capacity of the TAC iodides is demonstrated to be closely related to the electronic properties of the TAC cation, which offers a molecular platform for further catalyst screening and optimization.

A bacterially expressed triple-type chimeric vaccine against human papillomavirus types 51, 69, and 26.

Persistent infection of high-risk human papillomavirus (HPV) is a leading cause of some cancers, including cervical cancer. However, with over 20 carcinogenic HPV types, it is difficult to design a multivalent vaccine that can offer complete protection. Here, we describe the design and optimization of a HPV51/69/26 triple-type chimeric virus-like particle (VLP) for vaccine development. Using E. coli and a serial N-terminal truncation strategy, we created double- and triple-type chimeric VLPs through loop-swapping at equivalent surface loops. The lead candidate, H69-51BC-26FG, conferred similar particulate properties as that of its parental VLPs and comparable immunogenicity against HPV51, -69 and -26. When produced in a GMP-like facility, these H69-51BC-26FG VLPs were verified to have excellent qualities for the development of a multivalent HPV vaccine. This study showcases an amenable way to create a single VLP using type-specific epitope clustering for the design of a triple-type vaccine.

Effect of Qiangdi 863 Nanosynergids Treated Water, Nitrogen, Phosphorous and Potassium Fertilizers on Rice Growth Physiology and Grain Quality.

Nanotechnology is an emerging technique that helps in solving the biotic and abiotic agricultural issues leading to enhance crop productivity. Therefore, it was hypothesized to check the effect of Qiangdi 863 nano synergids biological-assisted growth apparatus and nitrogen, phosphorous, and potassium (NPK) fertilizers improving rice germination, early growth, physiology, and yield. An experiment was performed on five rice varieties for three consecutive years (2017-2019). The nanosynergids-treated water (NTW) significantly improved the speed of germination (25.3, 35.6, and 32.3%), final emergence percentage (100%) and seed emergence energy percentage (80, 95, and 90%), radical (1.25, 1.7, and 2.35 cm) and plumule growth (1.29, 1.24, and 1.66 cm), soil plant analysis development (46, 45, and 47), antioxidant enzymatic activities, such as catalase activity (34,376 µg-1FW h-1, 33,264 µg-1FW h-1, and 34,453 µg-1F W h-1), superoxide dismutase (18,456 µg-1F W h-1, 19,445 µg-1F W h-1, and 19,954 µg-1F W h-1), peroxide (745 Ug-1F W, 734 Ug-1F W, and 752 Ug-1F W), production and declined malondialdehyde (4.5 µmolg-1F W, 5.1 µmolg-1F W, and 4.2 µmolg-1F W) for all years respectively in KSK 133. The application of nano-treated irrigated water enriched the biomass of rice seedlings. The overall nano synergid treatments successfully enhanced the endogenous hormones as salicylic acid (6,016.27 p mol/L, 5823.22 p mol/L, and 5922.12 p mol/L), jasmonates (JA) (5,175.6 p mol/L, 4231 p mol/L, and 5014.21 p mol/L) brassinosteroids (BR) (618.2 p mol/L, 546.83 p mol/L, and 582.1 p mol/L) quantification and yield 1000 grain weight (22.3, 22, and 23.2 g) of KSK 133. Hence, the overall results proved that NTW could effectively enhance the early growth and yield of rice varieties.

Genome-Wide Identification and Functional Analysis of the bZIP Transcription Factor Family in Rice Bakanae Disease Pathogen, Fusarium fujikuroi.

Fungal basic leucine zipper (bZIP) proteins play a vital role in biological processes such as growth, biotic/abiotic stress responses, nutrient utilization, and invasion. In this study, genome-wide identification of bZIP genes in the fungus Fusarium fujikuroi, the pathogen of bakanae disease, was carried out. Forty-four genes encoding bZIP transcription factors (TFs) from the genome of F. fujikuroi (FfbZIP) were identified and functionally characterized. Structures, domains, and phylogenetic relationships of the sequences were analyzed by bioinformatic approaches. Based on the phylogenetic relationships with the FfbZIP proteins of eight other fungi, the bZIP genes can be divided into six groups (A-F). The additional conserved motifs have been identified and their possible functions were predicted. To analyze functions of the bZIP genes, 11 FfbZIPs were selected according to different motifs they contained and were knocked out by genetic recombination. Results of the characteristic studies revealed that these FfbZIPs were involved in oxygen stress, osmotic stress, cell wall selection pressure, cellulose utilization, cell wall penetration, and pathogenicity. In conclusion, this study enhanced understandings of the evolution and regulatory mechanism of the FfbZIPs in fungal growth, abiotic/biotic stress resistance, and pathogenicity, which could be the reference for other fungal bZIP studies.

The Velvet Protein UvVEA Regulates Conidiation and Chlamydospore Formation in Ustilaginoidea virens.

Rice false smut, caused by Ustilaginoidea virens, is a serious disease of rice worldwide, severely reducing the quantity and quality of rice production. The conserved fungal velvet proteins are global regulators of diverse cellular processes. We identified and functionally characterized two velvet genes, UvVEA and UvVELB, in U. virens. The deletion of these genes affected the conidiation of U. virens but had no effect on the virulence of this pathogen. Interestingly, the ΔUvVEA mutants appeared in the form of smaller false smut balls with a reduced number of chlamydospores compared with the wide-type strains. In addition, the deletion of UvVEA affected the expression of some transmembrane transport genes during chlamydospore formation and rice false smut balls development. Furthermore, the ΔUvVEA mutants were shown to be defective in the utilization of glucose. These findings proved the regulatory mechanism underlying the formation of rice false smut balls and chlamydospores and provided a basis for the further exploration of the mechanism of these processes.

Validation of housekeeping genes as internal controls for gene expression studies on biofilm formation in Bacillus velezensis.

Bacillus velezensis is an important bacterium widely applied in agriculture and industry, and biofilms play critical roles in its environmental tolerance. The appropriate choice of reference genes is essential for key gene expression studies. Multiple internal control genes were selected and validated from the 21 housekeeping genes of B. velezensis by expression stability evaluation during biofilm formation and were used to study the expression of key genes involved in the process. The results showed that pyk, gyrA, recA, and gyrB were stably expressed, and the expression of pyk was the most stable during biofilm formation. A pair of two genes, pyk and gyrA, provided high-quality data when used as internal controls, and the combination of three genes, pyk, gyrA, and recA, was even better. The expression levels of pyk, gyrA, and recA approximated those of five key genes, abrB, epsD, kinC, sinR, and tasA, in biofilm formation, meeting the requirements of ideal internal control genes. The expression patterns of 5 key genes were studied with 16S, pyk, the pair of 2 genes, pyk and gyrA, and the combination of 3 genes, pyk, gyrA, and recA, as internal controls during the biofilm formation process. The results proved that pyk was a suitable internal control, as were the pair of 2 genes, pyk and gyrA, and the combination of 3 genes, pyk, gyrA, and recA. This study provided genes and gene combinations which were validated as suitable internal controls for gene expression studies, especially those on the mechanism of biofilm formation in B. velezensis or even other Bacillus spp. KEY POINTS: • Reference genes is necessary for gene expression study in biofilm formation of Bacillus velezensis • Pyk and 2 gene combinations were selected and validated from 21 common used genes • Expression of key genes in biofilm formation was normalized with the selected internal controls.

SUN-Family Protein UvSUN1 Regulates the Development and Virulence of Ustilaginoidea virens.

Ustilaginoidea virens, the causal agent of rice false smut disease, is an important plant pathogen that causes severe quantitative and qualitative losses in rice worldwide. UvSUN1 is the only member of Group-I SUN family proteins in U. virens. In this work, the role of UvSUN1 in different aspects of the U. virens biology was studied by phenotypic analysis of Uvsun1 knockout strains. We identified that UvSUN1 was expressed during both conidial germination and the infection of rice. Disruption of the Uvsun1 gene affected the hyphal growth, conidiation, morphology of hyphae and conidia, adhesion and virulence. We also found that UvSUN1 is involved in the production of toxic compounds, which are able to inhibit elongation of the germinated seeds. Moreover, RNA-seq data showed that knockout of Uvsun1 resulted in misregulation of a subset of genes involved in signal recognition and transduction system, glycometabolism, cell wall integrity, and secondary metabolism. Collectively, this study reveals that Uvsun1 is required for growth, cell wall integrity and pathogenicity of U. virens, thereby providing new insights into the function of SUN family proteins in the growth and pathogenesis of this pathogen.

Short-term exposure to nitrogen dioxide and mortality: A systematic review and meta-analysis.

BACKGROUND: Ambient air pollution has been characterized as a leading cause of mortality worldwide and has been associated with cardiovascular and respiratory diseases. There is increasing evidence that short-term exposure to nitrogen dioxide (NO2), is related to adverse health effects and mortality. METHODS: We conducted a systematic review of short-term NO2 and daily mortality, which were indexed in PubMed and Embase up to June 2021. We calculated random-effects estimates by different continents and globally, and tested for heterogeneity and publication bias. RESULTS: We included 87 articles in our quantitative analysis. NO2 and all-cause as well as cause-specific mortality were positively associated in the main analysis. For all-cause mortality, a 10 ppb increase in NO2 was associated with a 1.58% (95%CI 1.28%-1.88%, I2 = 96.3%, Eggers' test p < 0.01, N = 57) increase in the risk of death. For cause-specific mortality, a 10 ppb increase in NO2 was associated with a 1.72% (95%CI 1.41%-2.04%, I2 = 87.4%, Eggers' test p < 0.01, N = 42) increase in cardiovascular mortality and a 2.05% (95%CI 1.52%-2.59%, I2 = 78.5%, Eggers' test p < 0.01, N = 38) increase in respiratory mortality. In the sensitivity analysis, the meta-estimates for all-cause mortality, cardiovascular and respiratory mortality were nearly identical. The heterogeneity would decline to varying degrees through regional and study-design stratification. CONCLUSIONS: This study provides evidence of an association between short-term exposure to NO2, a proxy for traffic-sourced air pollutants, and all-cause, cardiovascular and respiratory mortality.

SiC-Coated Carbon Nanotubes with Enhanced Oxidation Resistance and Stable Dielectric Properties.

Carbon nanotubes (CNTs) coated with SiC coating was successfully prepared by pyrolysis of polycarbosilane (PCS) used as a precursor. The function of pyrolysis temperature on the oxidation resistance and the dielectric properties of CNTs/SiC were studied in X-band. The results demonstrate that the obtained dense SiC film can prevent the oxidation of CNTs when the pyrolysis temperature reaches 600 °C. Correspondingly, after heat treatment is at 400 °C for 200 h, the mass loss of P-600 is less than 1.86%, and the real and imaginary parts of the dielectric constant nearly keep constant (ε' from 14.2 to 14, and ε″ from 5.7 to 5.5). SiC-coated CNTs have a better oxidation resistance than pristine CNTs. Therefore, this work, with a facile preparation process, enhances the oxidation resistance of CNTs at high temperature for a long time and maintains a stable dielectric property, which means CNTs/SiC composites can be good candidates for applications in the field of high-temperature absorbers.

Dual-step irradiation strategy to sequentially destroy singlet oxygen-responsive polymeric micelles and boost photodynamic cancer therapy.

Nanotechnology provides a powerful tool to overcome many disadvantages of small-molecule photosensitizers for photodynamic cancer therapy, such as hydrophobicity, rapid blood clearance, low accumulation in tumor tissue and low cell penetration, etc. The occurrence of quench in photosensitizer-loaded nanoparticle greatly downregulates the ability to generate singlet oxygen with light irradiation. Stimuli-responsive nanocarriers can improve the efficacy of PDT to a certain extent. However, insufficient release of photosensitizer from either endogenous or exogenous stimuli responsive nanocarriers in the short period of light irradiation restricts full usage of the photosensitizer delivered into cancer cells. We here report a dual-step light irradiation strategy to enhance the efficacy of cancer PDT. Ce6 as a photosensitizer is loaded in singlet oxygen-sensitive micelles (Ce6-M) via self-assembly of amphiphilic polymer mPEG2000-TK-C16. After co-incubation of Ce6-M with cancer cells or i.v. injection of Ce6-M, cancer cells or tumor tissues are irradiated with light for a short time to trigger Ce6 release, and 2 h later, re-irradiated for relatively long time. The sufficient release of Ce6 in the period between twice light irradiation significantly improves the generation of singlet oxygen, leading to more efficient cancer therapeutic effects of dual-step irradiation than that of single-step irradiation for the same total irradiation time.