A novel approach to alleviate acetaminophen-induced hepatotoxicity with hybrid balloon flower root-derived exosome-like nanoparticles (BDEs) with silymarin via inhibition of hepatocyte MAPK pathway and apoptosis.

INTRODUCTION: Balloon flower root-derived exosome-like nanoparticles (BDEs) have recently been proposed as physiologically active molecules with no cytotoxicity. However, the therapeutic effects of drug-induced hepatotoxicity of BDEs have not been elucidated. BDEs contain a large amount of platycodin D, which is widely known to be effective in regulating inflammation and ameliorating systemic toxicity. Thus, the main therapeutic activity of BDEs is attributed to inhibiting the inflammatory response and alleviating toxicity. In this study, we fabricated the hybrid BDEs fused with liposomes containing silymarin (SM) to enhance the synergistic effect on inhibition of acetaminophen-induced hepatotoxicity (APAP). OBJECTIVE: Considering the potential therapeutic effects of BDEs, and the potential to achieve synergistic effects to improve therapeutic outcomes, we constructed hybrid BDEs with a soy lecithin-based liposome loaded with SM. Since liposomes can provide higher thermal stability and have greater structural integrity, these might be more resistant to clearance and enzymatic degradation of drug molecules. METHODS: Hybrid BDEs with liposome-loaded SM (BDEs@lipo-SM) were fabricated by thin-film hydration and extrusion. BDEs@lipo-SM were characterized using dynamic light scattering and high-performance liquid chromatography. After confirmation of the physical properties of BDEs@lipo-SM, various therapeutic properties were evaluated. RESULTS: BDEs@lipo-SM were internalized by hepatocytes and immune cells and significantly decreased mRNA expression of apoptosis and inflammation-relevant cytokines by inhibiting the hepatocyte MAPK pathway. BDEs@lipo-SM significantly induced an increase in glutathione levels and inhibited APAP-induced hepatotoxicity. CONCLUSION: From this study, we know that BDEs are reliable and safe nanovesicles containing natural metabolites derived from balloon flower, and they can facilitate intercellular communication. BDEs are also easily modified to enhance drug loading capacity, targeting effects, and long-term accumulation in vivo. BDEs@lipo-SM have therapeutic benefits for acute liver injury and can alleviate cell death and toxicity. They can be efficiently delivered to the liver and effectively inhibit APAP-induced hepatotoxicity by inhibiting the MAPK signaling pathway and apoptosis, which accelerates liver recovery in the APAP-induced acute liver injury model. These findings highlight that BDEs represent an attractive delivery vehicle for drug delivery.

Efficient Synthesis of Folate-Conjugated Hollow Polymeric Capsules for Accurate Drug Delivery to Cancer Cells.

This study presents an efficient and systematic approach to synthesize bioapplicable porous hollow polymeric capsules (HPCs). The hydroxyl-functionalized nanoporous polymers with hollow capsular shapes could be generated via the moderate Friedel-Crafts reaction without using any hard or soft template. The numerous primitive hydroxyl groups on these HPCs were further converted to carboxyl groups. Owing to the abundance of highly branched carboxyl groups on the surface of the HPCs, biomolecules [such as folic acid (FA)] could be covalently decorated on these organic capsules (FA-HPCs) for drug delivery applications. The intrinsic hollow porosities and specific targeting agent offered a maximum drug encapsulation efficiency of up to 86% and drug release of up to 50% in 30 h in an acidic environment. The in vitro studies against cancer cells demonstrated that FA-HPCs exhibited a more efficient cellular uptake and intracellular doxorubicin release than bare HPCs. This efficient approach to fabricate carbonyl-functionalized hollow organic capsules may open avenues for a new type of morphological-controlled nanoporous polymers for various potential bioengineering applications.

Global Pattern of Microplastics (MPs) in Commercial Food-Grade Salts: Sea Salt as an Indicator of Seawater MP Pollution.

Previous studies have identified microplastics (MPs) in commercial table salts but could not exactly address the origin of the MPs because of several limitations. The present study is based on the hypothesis that commercial sea salts can act as an indicator of MP pollution in the surrounding environment unless the MPs are filtered out during the manufacturing process. A total of 39 different salt brands produced at geospatially different sites, including 28 sea salt brands from 16 countries/regions on six continents, were investigated. A wide range of MP content (in number of MPs per kg of salt; n/kg) was found: 0-1674 n/kg (excluding one outlier of 13 629 n/kg) in sea salts, 0-148 n/kg in rock salt, and 28-462 n/kg in lake salt. Relatively high MP content was identified in sea salts produced in Asian countries/regions. The abundance of MPs in unrefined sea salts ( n = 25) exhibited significant linear correlations with plastic emissions from worldwide rivers ( r2= 0.33; p = 0.003) and with the MP pollution levels in surrounding seawater ( r2= 0.46; p = 0.021) in the published literature. The results indicate that not only is Asia a hot spot of global plastic pollution, as previous studies have suggested, but also that sea salt can be a good indicator of the magnitude of MP pollution in the surrounding marine environment.

Clinical and MR Predictors of Retro-Odontoid Pseudotumor Regression Following Posterior Fixation in Patients with Atlantoaxial Instability.

Purpose: To identify clinical and MR predictors of retro-odontoid pseudotumor (ROP) regression after posterior fixation in patients with atlantoaxial instability. Materials and Methods: We included patients who had undergone posterior fixation for atlantoaxial instability and preoperative and postoperative MR imaging. Patients were classified into two groups according to the degree of ROP regression after posterior fixation: regression (≥ 10% reduction) and no regression (< 10% reduction). Mann-Whitney and Fisher's exact tests were performed to identify the clinical (age and sex) and MR predictors (preoperative ROP thickness, ROP type, MR signal homogeneity of the ROP, spinal cord signal change, spinal cord atrophy, ossified posterior longitudinal ligament, os odontoideum, and atlantodental interval) associated with ROP regression. Results: We retrospectively assessed 11 consecutive patients (7 female; median age, 66 years [range, 31-84 years]). Posterior fixation induced ROP regression in eight (72.7%) patients. Older age and greater preoperative ROP thickness significantly correlated with ROP regression (p = 0.024 and 0.012, respectively). All patients with preoperative ROP thickness > 5 mm exhibited ROP regression. The other variables were not significantly associated with ROP regression. Conclusion: Older age and thicker preoperative ROP are associated with ROP regression after posterior fixation in patients with atlantoaxial instability.

Disruption of early embryonic development in mice by polymethylmethacrylate nanoplastics in an oxidative stress mechanism.

Polymethylmethacrylate (PMMA) has been used in many products, such as acrylic glass, and is estimated to reach 5.7 million tons of production per year by 2028. Thus, nano-sized PMMA particles in the environment are highly likely due to the weathering process. However, information on the hazards of nanoplastics, including PMMA in mammals, especially reproductive toxicity and action mechanism, is scarce. Herein, we investigated the effect of PMMA nanoplastics on the female reproductive system of mice embryos during pre-implantation. The treated plastic particles in embryos (10, 100, and 1000 µg/mL) were endocytosed into the cytoplasm within 30 min, and the blastocyst development and indices of embryo quality were significantly decreased from at 100 µg/mL. Likewise, the transfer of nanoplastic-treated embryos at 100 µg/mL decreased the morula implantation rate on the oviduct of pseudopregnant mice by 70%, calculated by the pregnant individual, and 31.8% by the number of implanted embryos. The PMMA nanoplastics at 100 µg/mL significantly increased the cellular levels of reactive oxygen species in embryos, which was not related to the intrinsic oxidative potential of nanoplastics. This study highlights that the nanoplastics that enter systemic circulation can affect the early stage of embryos. Thus, suitable action mechanisms can be designed to address nanoplastic occurrence.

Paclitaxel-loaded ginsenoside Rg3 liposomes for drug-resistant cancer therapy by dual targeting of the tumor microenvironment and cancer cells.

INTRODUCTION: Inherent or acquired resistance to paclitaxel (PTX) is a pivotal challenge for chemotherapy treatment of multidrug-resistant (MDR) breast cancer. Although various targeted drug-delivery systems, including nanoparticles and liposomes, are effective for MDR cancer treatment, their efficacy is restricted by immunosuppressive tumor microenvironment (TME). METHODS: Ginsenosides Rg3 was used to formulate unique Rg3-based liposomes loaded with PTX to establish Rg3-PTX-LPs, which were prepared by the thin-film hydration method. The stability of the Rg3-PTX-LPs was evaluated by particle size analysis through dynamic light scattering. The active targeting effect of Rg3-based liposomes was examined in an MCF-7/T xenograft model by an in a vivo imaging system. To evaluate the antitumor activity and mechanism of Rg3-PTX-LP, MTT, apoptosis assays, TAM regulation, and TME remodeling were performed in MCF-7/T cells in vitro and in vivo. RESULTS: Rg3-PTX-LPs could specifically distribute to MCF7/T cancer cells and TME simultaneously, mainly through the recognition of GLUT-1. The drug resistance reversing capability and in vivo antitumor effect of Rg3-PTX-LPs were significantly improved compared with conventional cholesterol liposomes. The TME remodeling mechanisms of Rg3-PTX-LPs included inhibiting IL-6/STAT3/p-STAT3 pathway activation to repolarize protumor M2 macrophages to antitumor M1 phenotype, suppressing myeloid-derived suppressor cells (MDSCs), decreasing tumor-associated fibroblasts (TAFs) and collagen fibers in TME, and promoting apoptosis of tumor cells. Hence, through the dual effects of targeting tumor cells and TME remodeling, Rg3-PTX-LPs achieved a high tumor inhibition rate of 90.3%. CONCLUSION: Our multifunctional Rg3-based liposome developed in the present study offered a promising strategy for rescuing the drug resistance tumor treatment.

Differential particle and ion kinetics of silver nanoparticles in the lungs and biotransformation to insoluble silver sulfide.

The measurement of nanoparticles (NPs) in a biological matrix is essential in various toxicity studies. However, the current knowledge has limitations in differentiating particulate and ionic forms and further identification of their biotransformation. Herein, we evaluate the biotransformation and differential lung clearance kinetics of particulate and ionic forms using PEGylated silver NPs (AgNP-PEGs; 47.51 nm) and PEGylated gold NPs (AuNP-PEGs; 11.76 nm). At 0, 3, and 6 h and 1, 3, 7, and 14 days after a single pharyngeal aspiration in mice at 25 µg/mouse, half of the lung is digested by proteinase K (PK) to separate particulates and ions, and the other half is subjected to the acid digestion method for comparison. The quantitative and qualitative evaluation of lung clearance kinetics suggests that AgNP-PEGs are quickly dissolved and transformed into insoluble silver sulfide (Ag2S), which shows a fast-clearing early phase (0 -6 h; particle T1/2: 4.8 h) and slow-clearing late phase (1 -14 days; particle T1/2: 13.20 days). In contrast, AuNP-PEGs were scarcely cleared or biotransformed in the lungs for 14 days. The lung clearance kinetics of AgNPs and biotransformation shown in this study can be informed by the PK digestion method and cannot be obtained using the acid digestion method.

Microplastics in large marine animals stranded in the Republic of Korea.

Microplastics (MPs) are found in every ocean and are frequently ingested by marine animals. This study analyzed MPs in the stomachs and intestines of 12 large marine animals comprising one fin whale (Balaenoptera physalus), seven finless porpoises (Neophocaena asiaeorientalis), two loggerhead turtles (Caretta caretta), one Indo-Pacific bottlenose dolphin (Tursiops aduncus), and one common dolphin (Delphinus delphis) that were stranded off the Republic of Korea between 2019 and 2021. MPs were detected with a mean abundance of 3.42 ± 3.2 items/g and were predominantly of transparent-white, fragment-shaped polypropylene smaller than 200 µm. The abundance of MPs found did not correlate with the biological information (maturity, body length) of the finless porpoises and there were no significant differences in the abundance of MPs between the stomachs and intestines. These results cannot accurately assess the impact of MPs on large marine animals, so further studies are necessary to understand how MPs can potentially affect them.

Toward a long-term monitoring program for seawater plastic pollution in the north Pacific Ocean: Review and global comparison.

Through a literature survey and meta-data analysis, monitoring methods and contamination levels of marine micro- and macroplastics in seawater were compared between the North Pacific and the world's other ocean basins. The minimum cut-off size in sampling and/or analysis of microplastics was crucial to the comparison of monitoring data. The North Pacific was most actively monitored for microplastics and showed comparatively high levels in the global context, while the Mediterranean Sea was most frequently monitored for macroplastics. Of the 65 extracted mean abundances of microplastics in seawater from the North Pacific, two (3.1%) exceeded the lowest predicted no-effect concentration (PNEC) proposed thus far. However, in the context of business-as-usual conditions, the PNEC exceedance probability may be expected to reach 27.7% in the North Pacific in 2100. The abundance of marine plastics in seawater, which reflects the current pollution status and marine organisms' waterborne exposure levels, is a useful indicator for marine plastic pollution. For regional and global assessments of pollution status across space and time, as well as assessment of ecological risk, two microplastic monitoring approaches are recommended along with their key aspects. Although microplastic pollution is closely linked with macroplastics, the monitoring data available for floating macroplastics and more extent to mesoplastics in most ocean basins are limited. A more specific framework for visual macroplastic survey (e.g. fixed minimum cut-off size, along with survey transect width and length according to survey vessel class) is required to facilitate data comparison. With the implementation of standardised methods, increased efforts are required to gather monitoring data for microplastics and-more importantly-floating macroplastics in seawater worldwide.

Inhibition of ganglioside GD1a synthesis suppresses the differentiation of human mesenchymal stem cells into osteoblasts.

In this study, we investigated the regulatory role of ganglioside GD1a in the differentiation of osteoblasts from human mesenchymal stem cells (hMSCs) by using lentivirus-containing short hairpin (sh)RNA to knockdown ST3 ß-galactoside α-2, 3-sialyltransferase 2 (ST3Gal II) mRNA expression. After hMSCs were infected for 72 h with the lentivirus constructed with ST3Gal II shRNAs, the puromycin-resistant cells were selected and subcultured to produce hMSCs with ST3Gal II mRNA knockdown. The hMSCs established from human dental papilla abundantly expressed CD44 and CD105, but not CD45 and CD117. Osteoblasts that differentiated from normal hMSCs showed a significant increase in alkaline phosphatase (ALP) activity and ganglioside GD1a expression level compared with those in hMSCs. Lentiviral infection of hMSCs successfully induced a marked inhibition of ST3Gal II mRNA expression and caused a significant decrease in ALP activity and ganglioside GD1a expression. During osteoblastic differentiation, the increased ALP activity remarkably reduced by suppression of ganglioside GD1a expression by ST3Gal II shRNA. Ganglioside GD1a and ALP were mainly expressed in the cell body of hMSCs and osteoblasts with colocalization. The phosphorylation of extracellular signal-regulated kinases (ERK) 1/2 mitogen-activated protein (MAP) kinase and epidermal growth factor receptor (EGFR) was significantly reduced in the osteoblasts that had differentiated from the hMSCs with ST3Gal II mRNA knockdown. These results suggest that ganglioside GD1a plays an important role in the regulation of osteoblastic differentiation of hMSCs through the activation of ERK 1/2 MAP kinase and EGFR.

Variation and Uncertainty of Microplastics in Commercial Table Salts: Critical Review and Validation.

Increasing concern of human exposure to microplastics (MPs) necessitates an assessment of the quality of MP data relevant to human exposure. In this literature review for table salt, we addressed the variability and uncertainty of MP data caused by different analytical methods among studies. Additionally, validation experiment was conducted to identify and correct uncertainties related to MP size. When combined without validation, salt data in literature (n = 150) showed a wide range of 0-39800 (1386 ±â€¯5477) MPs kg-1. All procedures, including sample treatment, MP identification, and quality assurance were related to this variability. Most serious variability originated from the MP identification methods associated with minimum cut-off size of targeted/measured MPs and the selection of particles identified. When not corrected by size, MP content differed by 10-600 times among MP identification methods, with greatest value from visual observation, followed by FTIR and Raman methods. Meanwhile, there was a significant correlation-regardless of identification method-between logarithmic mean abundances and minimum cut-off sizes. The size-corrected values showed that adults intake up to 19000 MPs ≥10 µm annually via table salt, compared with 5100 MPs that was estimated from uncorrected mean abundance. Our validation experiment also showed the possibility of serious errors being caused by arbitrary selection of "MP-like particles" in spectroscopic analysis, specifically for smaller-sized particles. A combination of unverified data originated from different methods might have failed to adequately produce reliable human health-relevant results, thereby undermining the ability to quantify human risk.

The reactive oxygen species as pathogenic factors of fragmented microplastics to macrophages.

The presence of microplastics in the various food web raised concerns on human health, but little is known about the target cells and mechanism of toxicity of microplastics. In this study, we evaluated the toxicity of microplastics using relevant cell lines to the oral route of exposure. Approximately 100 µm-sized fragment-type polypropylene (PP) and polystyrene (PS) particles were prepared by sieving after pulverization and further applied the accelerated weathering using ultraviolet and heat. Thus, the panel of microplastics includes fresh PP (f-PP), fresh PS (f-PS), weathered PP (w-PP), and weathered PS (w-PS). The spherical PS with a similar size was used as a reference particle. Treatment of all types of PP and PS did not show any toxic effects to the Caco-2 cells and HepG2 cells. However, the treatment of microplastics to THP-1 macrophages showed significant toxicity in the order of f-PS > f-PP > w-PS > w-PP. The weathering process significantly reduced the reactive oxygen species (ROS) generation potential of both microplastics because the weathered microplastics have an increased affinity to bind serum protein which acts as a ROS scavenger. The intrinsic ROS generation potential of microplastics showed a good correlation with the toxicity endpoints including cytotoxicity and pro-inflammatory cytokines in THP-1 macrophages. In conclusion, the results of this study suggest that the target cell type of microplastics via oral administration can be macrophages and the pathogenic factor to THP-1 macrophages is the intrinsic ROS generation potential of microplastics. Nevertheless, the toxic effect of microplastics tested in this study was much less than that of nano-sized particles.

Importance of seasonal sea ice in the western Arctic ocean to the Arctic and global microplastic budgets.

Arctic sea ice entraps microplastics (MP) from seawater and atmosphere and is recognized as sink and transport vector of MPs. However, ice-trapped fraction in the global MP budget, contribution of atmospheric input, and linkage among Arctic basins remain unclear. To assess them, we investigated the number- and mass-based data separated by size and shape geometry for MPs in sea ice, snow, and melt pond water from the western Arctic Ocean (WAO). A significant dependency of MP data on measured cutoff size and geometry was found. For the same size range and geometry, sea ice MPs in WAO ((11.4 ± 9.12) × 103 N m-3 for ≥ 100 µm) were within comparable levels with those in other Arctic basins, but showed closer similarity in polymer and shape compositions between WAO and Arctic Central Basin, indicating the strong linkage of the two basins by the Transpolar Drift. Our budgeting shows that a significant amount of plastic particles ((3.4 ± 2.6) × 1016 N; 280 ± 701 kilotons), which are missed from the global inventory, is trapped in WAO seasonal sea ice, with < 1% snowfall contribution. Our findings highlight that WAO ice zone may play a role as a sink of global MPs as well as a source of Arctic MPs.

Multifunctional ginsenoside Rg3-based liposomes for glioma targeting therapy.

Liposomes have been widely used for targeted drug delivery. However, nonselective distribution, low blood-brain barrier penetration, and the disadvantages of cholesterol greatly limit the application of conventional liposomes in the treatment of brain tumors. In the present study, we aimed to develop a multifunctional ginsenoside Rg3-based liposomal system (Rg3-LPs). Compared to cholesterol liposomes (C-LPs), Rg3-LPs not only significantly improved cellular uptake and penetration across glioma spheroids in vitro, but also remarkably enhanced active glioma targeting and intratumoral diffusion capability in vivo. Paclitaxel-loaded Rg3-LPs (Rg3-PTX-LPs) exhibited a substantially stronger anti-proliferation effect on C6 glioma cells than paclitaxel-loaded C-LPs and re-educated tumor-associated macrophages from the protumor M2 phenotype to the antitumor M1 phenotype in vivo. Rg3-PTX-LPs significantly prolonged median survival time of intracranial C6-bearing mice/rats by activating the immune microenvironment in glioma, facilitating T-cell immune responses with expansion of the CD8+ T-cell population, increasing the M1/M2 ratio, and decreasing regulatory T and myeloid-derived suppressor cells. Together, the results demonstrated that ginsenoside Rg3 is a good alternative for cholesterol in drug delivery liposomes and has a synergistic effect with loaded anticancer drugs. Rg3-PTX-LPs can serve as a multifunctional potential drug for the treatment of glioma.

Commiphora myrrha inhibits itch-associated histamine and IL-31 production in stimulated mast cells.

Commiphora myrrha (Myrrh) is widely recognized for its anti-inflammatory and antimicrobial properties, which are utilized for the treatment of oral ulcers, gingivitis, sinusitis, glomerulonephritis, brucellosis and a variety of skin disorders. The current study aimed to assess whether myrrh modulates itch-associated interleukin (IL)-31 cytokine production and histamine release in stimulated human mast cells (HMC-1). To realize this, molecular biology techniques including real-time quantitiative PCR, western blotting and ELISA were employed. The results indicated that Myrrh successfully suppressed phorbol myristate acetate and calcium ionophore-stimulated mRNA expression, and reduced the production of IL-31 in HMC-1 cells. In addition, myrrh served as a suppressor of extracellular signal-regulated kinase and NF-κB activation, indicating its mechanism in the prevention of HMC-1 cell IL-31 production. Myrrh also prevented the release of histamine in HMC-1 cells. Whilst the present study awaits in vivo support, the pharmacological actions of myrrh provide new indications as to its potential applicability for itch treatment, which cannot be treated with histamine receptor blockers alone.

In Vitro Anti-Inflammatory Activity of Ilex cornuta Extract Mediated by Inhibition of Extracellular Signal-Regulated Kinase Phosphorylation.

Ilex cornuta, commonly known as Chinese holly, is an evergreen shrub from the family Aquifoliaceae, and it is widely distributed in Korea and China. In folk medicine, the leaves of I. cornuta are used for treatment of several disorders, including weakness of the waist and knees, arthrodynia, headache, acute conjunctivitis, toothache, urticaria, rheumatic arthralgia, and cardiovascular diseases. In this study, we investigated the anti-inflammatory effects of an I. cornuta leaf ethanol extract (ILE) and its underlying mechanisms of action. The anti-inflammatory activities of ILE were evaluated in murine RAW 264.7 macrophages, using lipopolysaccharide (LPS) stimulation. ILE treatment-related changes in the production of nitric oxide (NO), prostaglandin E2 (PGE2), and proinflammatory cytokines were also measured. Finally, the expression of signaling molecules involved in inflammatory reactions was also assessed. Pretreatment of macrophages with ILE attenuated the expression of inducible NO synthase and cyclooxygenase-2, resulting in a decrease in NO and PGE2 production. The secretion of proinflammatory cytokines such as interleukin (IL)-6 and IL-1ß was also reduced. Furthermore, ILE reduced extracellular signal-regulated kinases (ERK) phosphorylation, without affecting the inhibitor of kappa Bα and other mitogen-activated protein kinases. Liquid chromatography-tandem mass spectrometry analysis (LC-MS/MS) demonstrated that 1 g of ILE contains 27 mg of kaempferol, 0.3 mg of vanillic acid, and 21 mg of combined amount of isoquercetin and hyperin, among which isoquercetin and kaempferol significantly suppressed IL-6, IL-1ß, and PGE2 production. Our results demonstrated that ILE possesses anti-inflammatory effects mediated through inhibition of ERK phosphorylation.

Fluorescence imaging of spatial location of lipids and proteins during digestion of protein-stabilized oil-in-water emulsions: A simulated gastrointestinal tract study.

A whey protein isolate-rhodamine B conjugate (WPI-RB) was synthesized to visualize changes in the location of a protein emulsifier in oil-in-water emulsions during digestion. An oil-soluble dye (Nile Red) was used to visualize changes in the lipid phase during digestion. Protein-labeled and lipid-labeled emulsions were passed through a simulated gastrointestinal tract consisting of mouth, stomach, and intestinal phases, and changes in protein and lipid location and morphology were monitored using confocal laser scanning microscopy. The proteins remained attached to the lipid droplet surfaces in the mouth and stomach, but formed large aggregates in the small intestine. The lipid droplets were highly flocculated in the mouth, highly coalesced in the stomach, and fully digested in the small intestine. Our results show that conjugation of fluorescence dyes to protein emulsifiers is useful for direct visualization of their location, as well as for understanding the behavior of food emulsions within the gastrointestinal tract.

Development of anode zone using dual-anode system to reduce organic matter crossover in membraneless microbial fuel cells.

To prevent the occurrence of the organic crossover in membraneless microbial fuel cells (ML-MFCs), dual-anode MFC (DA-MFC) was designed from multi-anode concept to ensure anode zone. The anode zone addressed increase the utilization of organic matter in ML-MFCs, as the result, the organic crossover was prevented and performance of MFCs were enhanced. The maximum power of the DA-MFC was 0.46mW, which is about 1.56 times higher than the ML-MFC (0.29mW). Furthermore, the DA-MFC had advantage in correlation of organic substance concentration and dissolved oxygen concentration, and even electric over-potential. In addition, in terms of cathode fouling, the DA-MFC showed clearer surface. Hence, the anode zone should be considered in the advanced ML-MFC for practically use in wastewater treatment process, and also for scale-up of MFCs.