Effect of Rab18 on liver injury and lipid accumulation by regulating perilipin 2 and peroxisome proliferator-activated receptor gamma in non-alcoholic fatty liver disease.

BACKGROUND AND AIM: Nonalcoholic fatty liver disease (NAFLD) is currently one of the most common chronic liver diseases worldwide, characterized by the presence of lipid droplets. Rab18 is an important lipid droplet protein; however, its effects and mechanisms of action on NAFLD remain unclear. METHODS: Free fatty acid-stimulated AML-12 cells and high-fat diet (HFD)-fed mice were used as NAFLD models. Lentiviruses overexpressing Rab18 (Rab18-OE) or knockdown (Rab18-KD) were used to generate stable cell lines for genetic analysis. Blood serum levels of alanine aminotransferase, aspartate aminotransferase, total cholesterol, triglycerides, high-density lipoprotein cholesterol, low-density lipoprotein cholesterol, glucose, and leptin were measured using a biochemical autoanalyzer. Hematoxylin and eosin staining was performed to detect pathological damage to the liver. Lipid accumulation in the cells was assessed by Oil Red O staining. Target expression was measured using qPCR, western blotting, and immunocytochemistry. RESULTS: Rab18 mRNA and protein expression levels increased in free fatty acid-stimulated AML-12 cells and the livers of HFD-fed mice. Rab18-OE increased lipid accumulation in vitro, which was attenuated by Rab18-KD. In vivo, Rab18-OE augmented liver pathological damage, serum alanine aminotransferase/aspartate aminotransferase activity, and triglyceride, total cholesterol, and low-density lipoprotein levels, whereas Rab18-KD decreased these indicators. Rab18-KD also downregulated blood glucose levels in HFD-fed mice. Mechanistically, Rab18-OE and Rab18-KD regulated the mRNA and protein expression levels of perilipin 2 (PLIN2) and peroxisome proliferator-activated receptor gamma (PPARγ) in vitro and in vivo, respectively. Immunocytochemistry revealed that Rab18 colocalized with PLIN2 and PPARγ in AML-12 cells. CONCLUSION: Rab18 expression was elevated in vitro and in vivo in the NAFLD mouse model. Rab18 regulates PLIN2 and PPARγ expression to exaggerate liver injury and lipid accumulation in patients with NAFLD. Thus, Rab18 may be a crucial protein in this disease and a potential therapeutic target.

Trihalomethane prediction model for water supply system based on machine learning and Log-linear regression.

Laboratory determination of trihalomethanes (THMs) is a very time-consuming task. Therefore, establishing a THMs model using easily obtainable water quality parameters would be very helpful. This study explored the modeling methods of the random forest regression (RFR) model, support vector regression (SVR) model, and Log-linear regression model to predict the concentration of total-trihalomethanes (T-THMs), bromodichloromethane (BDCM), and dibromochloromethane (DBCM), using nine water quality parameters as input variables. The models were developed and tested using a dataset of 175 samples collected from a water treatment plant. The results showed that the RFR model, with the optimal parameter combination, outperformed the Log-linear regression model in predicting the concentration of T-THMs (N25 = 82-88%, rp = 0.70-0.80), while the SVR model performed slightly better than the RFR model in predicting the concentration of BDCM (N25 = 85-98%, rp = 0.70-0.97). The RFR model exhibited superior performance compared to the other two models in predicting the concentration of T-THMs and DBCM. The study concludes that the RFR model is superior overall to the SVR model and Log-linear regression models and could be used to monitor THMs concentration in water supply systems.

Acute Responses of Microorganisms from Membrane Bioreactors in the Presence of NaOCl: Protective Mechanisms of Extracellular Polymeric Substances.

Extracellular polymeric substances (EPS) are key foulants in membrane bioreactors (MBRs). However, their positive functions of protecting microorganisms from environmental stresses, e.g., during in situ hypochlorite chemical cleaning of membranes, have not been adequately elucidated. In this work, we investigated the response of microorganisms in an MBR to various dosages of NaOCl, with a particular emphasis on the mechanistic roles of EPS. Results showed that functional groups in EPS such as the hydroxyl and amino groups were attacked by NaOCl, causing the oxidation of polysaccharides, denaturation of amino acids, damage to protein secondary structure, and transformation of tryptophan protein-like substances to condensed aromatic ring substances. The presence of EPS alleviated the negative impacts on catalase and superoxide dismutase, which in turn reduced the concentration of reactive oxygen species (ROS) in microbial cells. The direct extracellular reaction and the mitigated intracellular oxidative responses facilitated the maintenance of microbial metabolism, as indicated by the quantity of adenosine triphosphate and the activity of dehydrogenase. The reaction with NaOCl also led to the changes of cell integrity and adhesion properties of EPS, which promoted the release of organic matter into bulk solution. Our results systematically demonstrate the protective roles of EPS and the underlying mechanisms in resisting the environmental stress caused by NaOCl, which provides important implications for in situ chemical cleaning in MBRs.

2D Frameworks Self-assembled From a Hydrazone Ligand and Azide Salts: Synthesis, Structures, and Luminescent Property.

Three metal-organic coordination polymers [Cu(L)(N3)]·(H2O)0.25n (1), [Zn(L)(N3)]·(H2O)0.5n (2) and [Cd2(L)2(N3)2(H2O)]n (3) have been synthesized from hydrazone ligand N'-(1-(pyrazin-2-yl)ethylidene)isonicotinohydrazide (HL), NaN3 and corresponding metal nitrates. Complexes were characterized by elemental analysis, IR spectroscopy and single-crystal X-ray diffraction. All three complexes feature 2D coordination network in which L1- acts as NNON tetradentate ligand and azide acts as end-on bridging ligand. In complexes 1 and 2, only intra-sheet hydrogen bonding interactions are found, while the hydrogen bonding interactions between water molecules and host framework result in 3D network for 3. In addition, complexes 2 and 3 exhibited intense fluorescent emissions in the solid state at room temperature.

Associations between novel anthropometric indices and the prevalence of gallstones among 6,848 adults: a cross-sectional study.

Background: Traditional anthropometric measures, including body mass index (BMI), are insufficient for evaluating gallstone risk. This study investigated the association between novel anthropometric indices and gallstone risk among 6,848 participants from the National Health and Nutrition Examination Survey in the United States. Methods: Measures calculated included weight (WT), BMI, waist circumference (WC), waist-to-height ratio (WtHR), conicity index (CI), A Body Shape Index (ABSI), Body Roundness Index (BRI), Abdominal Volume Index (AVI), and Weight-adjusted Waist Index (WWI). Logistic regression and smooth curve fitting assessed the relationships between these indices and gallstones, complemented by receiver operating characteristic (ROC) curve analysis to evaluate their discriminative power. Results: The results indicated significant differences between study groups, with a positive and independent correlation identified between gallstones and all measures except ABSI. Specifically, per 1 SD increase in WC, WT, BMI, WtHR, and AVI was associated with a 57%, 59%, 52%, 53%, and 53% increased risk of gallstones, respectively. Dose-response analysis confirmed a positive correlation between these indices and gallstone risk. ROC analysis highlighted WtHR and BRI as having superior discriminative abilities (AUC = 0.6703). Further, among participants with a BMI < 30 kg/m2, elevated levels of WT, WtHR, CI, BRI, and WWI significantly increased the risk of gallstones (P < 0.001). Likewise, elevated BMI heightened the risk at low levels of WT, WC, WtHR, BRI, AVI, and CI (P < 0.001). Conclusion: This study supports the positive association between various anthropometric indicators and gallstones, recommending that newer anthropometric indices be considered more extensively to enhance gallstone prevention and treatment strategies.

Integrating Tumor and Nodal Radiomics to Predict the Response to Neoadjuvant Chemotherapy and Recurrence Risk for Locally Advanced Gastric Cancer.

AIMS: To develop and evaluate machine learning models using tumor and nodal radiomics features for predicting the response to neoadjuvant chemotherapy (NAC) and recurrence risk in locally advanced gastric cancer (LAGC). BACKGROUND: Early and accurate response prediction is vital to stratify LAGC patients and select proper candidates for NAC. OBJECTIVE: A total of 218 patients with LAGC undergoing NAC followed by gastrectomy were enrolled in our study and were randomly divided into a training cohort (n = 153) and a validation cohort (n = 65). METHODS: We extracted 1316 radiomics features from the volume of interest of the primary lesion and maximal lymph node on venous phase CT images. We built 3 radiomics signatures for distinguishing good responders and poor responders based on tumor radiomics (TR), nodal radiomics (NR), and a combination of the two (TNR), respectively. A nomogram was then developed by integrating the radiomics signature and clinical factors. Kaplan- Meier survival curves were used to evaluate the prognostic value of the nomogram. RESULTS: The TNR signature achieved improved predictive value, with AUCs of 0.755 and 0.744 in the training and validation cohorts. Our proposed nomogram model (TNRN) showed a good performance for GR prediction in the prediction efficacy, calibration ability, and clinical benefit, with AUCs of 0.779 and 0.732 in the training and validation cohorts, superior to the clinical model. Moreover, the TNRN could accurately classify the patients into high-risk and low-risk groups in both training and validation cohorts with regard to postoperative recurrence and metastasis. CONCLUSION: The TNRN performed well in identifying good responders and provided valuable information for predicting progression-free survival time (PFS) in patients with LAGC who underwent NAC.

Multiplexed volumetric CLEM enabled by scFvs provides insights into the cytology of cerebellar cortex.

Mapping neuronal networks is a central focus in neuroscience. While volume electron microscopy (vEM) can reveal the fine structure of neuronal networks (connectomics), it does not provide molecular information to identify cell types or functions. We developed an approach that uses fluorescent single-chain variable fragments (scFvs) to perform multiplexed detergent-free immunolabeling and volumetric-correlated-light-and-electron-microscopy on the same sample. We generated eight fluorescent scFvs targeting brain markers. Six fluorescent probes were imaged in the cerebellum of a female mouse, using confocal microscopy with spectral unmixing, followed by vEM of the same sample. The results provide excellent ultrastructure superimposed with multiple fluorescence channels. Using this approach, we documented a poorly described cell type, two types of mossy fiber terminals, and the subcellular localization of one type of ion channel. Because scFvs can be derived from existing monoclonal antibodies, hundreds of such probes can be generated to enable molecular overlays for connectomic studies.

Biochar Coating as a Cost-Effective Delivery Approach to Promoting Seed Quality, Rice Germination, and Seedling Establishment.

The application of high-quality seeds ensures successful crop establishment, healthy growth, and improved production in both quantity and quality. Recently, biochar-based seed coating has been recognized as a new, effective, and environmentally friendly method to enhance seed quality, seedling uniformity, and nutrient availability. To study the impact of biochar coating on the surface mechanical properties of coated seeds, rice emergence and growth, and related physical and physiological metabolic events, laboratory experiments were performed on two water-saving and drought-resistance rice (WDR) varieties (Huhan1512 and Hanyou73) using biochar formulations with varying contents (20%-60%). The results showed that the appropriate concentration of biochar significantly improved emergence traits and seedling performance of the two rice varieties, compared to the uncoated treatment, and that the optimal percentage of biochar coating was 30% (BC30). On average, across both varieties, BC30 enhanced emergence rate (9.5%), emergence index (42.9%), shoot length (19.5%), root length (23.7%), shoot dry weight (25.1%), and root dry weight (49.8%). The improved germination characteristics and vigorous seedling growth induced by biochar coating were strongly associated with higher water uptake by seeds, increased α-amylase activity and respiration rate, and enhanced accumulation of soluble sugar and soluble protein. Moreover, the evaluation results of mechanical properties related to seed coating quality found that increasing the proportion of biochar in the coating blend decreased the integrity and compressive strength of the coated seeds and reduced the time required for coating disintegration. In conclusion, biochar coating is a cost-effective strategy for enhancing crop seed quality and seedling establishment.

Preserving extracellular space for high-quality optical and ultrastructural studies of whole mammalian brains.

Analysis of brain structure, connectivity, and molecular diversity relies on effective tissue fixation. Conventional tissue fixation causes extracellular space (ECS) loss, complicating the segmentation of cellular objects from electron microscopy datasets. Previous techniques for preserving ECS in mammalian brains utilizing high-pressure perfusion can give inconsistent results owing to variations in the hydrostatic pressure within the vasculature. A more reliable fixation protocol that uniformly preserves the ECS throughout whole brains would greatly benefit a wide range of neuroscience studies. Here, we report a straightforward transcardial perfusion strategy that preserves ECS throughout the whole rodent brain. No special setup is needed besides sequential solution changes, and the protocol offers excellent reproducibility. In addition to better capturing tissue ultrastructure, preservation of ECS has many downstream advantages such as accelerating heavy-metal staining for electron microscopy, improving detergent-free immunohistochemistry for correlated light and electron microscopy, and facilitating lipid removal for tissue clearing.

Multiplexed volumetric CLEM enabled by antibody derivatives provides new insights into the cytology of the mouse cerebellar cortex.

Mapping neuronal networks that underlie behavior has become a central focus in neuroscience. While serial section electron microscopy (ssEM) can reveal the fine structure of neuronal networks (connectomics), it does not provide the molecular information that helps identify cell types or their functional properties. Volumetric correlated light and electron microscopy (vCLEM) combines ssEM and volumetric fluorescence microscopy to incorporate molecular labeling into ssEM datasets. We developed an approach that uses small fluorescent single-chain variable fragment (scFv) immuno-probes to perform multiplexed detergent-free immuno-labeling and ssEM on the same samples. We generated eight such fluorescent scFvs that targeted useful markers for brain studies (green fluorescent protein, glial fibrillary acidic protein, calbindin, parvalbumin, voltage-gated potassium channel subfamily A member 2, vesicular glutamate transporter 1, postsynaptic density protein 95, and neuropeptide Y). To test the vCLEM approach, six different fluorescent probes were imaged in a sample of the cortex of a cerebellar lobule (Crus 1), using confocal microscopy with spectral unmixing, followed by ssEM imaging of the same sample. The results show excellent ultrastructure with superimposition of the multiple fluorescence channels. Using this approach we could document a poorly described cell type in the cerebellum, two types of mossy fiber terminals, and the subcellular localization of one type of ion channel. Because scFvs can be derived from existing monoclonal antibodies, hundreds of such probes can be generated to enable molecular overlays for connectomic studies.

A large-scale volumetric correlated light and electron microscopy study localizes Alzheimer's disease-related molecules in the hippocampus.

Connectomics is a nascent neuroscience field to map and analyze neuronal networks. It provides a new way to investigate abnormalities in brain tissue, including in models of Alzheimer's disease (AD). This age-related disease is associated with alterations in amyloid-ß (Aß) and phosphorylated tau (pTau). These alterations correlate with AD's clinical manifestations, but causal links remain unclear. Therefore, studying these molecular alterations within the context of the local neuronal and glial milieu may provide insight into disease mechanisms. Volume electron microscopy (vEM) is an ideal tool for performing connectomics studies at the ultrastructural level, but localizing specific biomolecules within large-volume vEM data has been challenging. Here we report a volumetric correlated light and electron microscopy (vCLEM) approach using fluorescent nanobodies as immuno-probes to localize Alzheimer's disease-related molecules in a large vEM volume. Three molecules (pTau, Aß, and a marker for activated microglia (CD11b)) were labeled without the need for detergents by three nanobody probes in a sample of the hippocampus of the 3xTg Alzheimer's disease model mouse. Confocal microscopy followed by vEM imaging of the same sample allowed for registration of the location of the molecules within the volume. This dataset revealed several ultrastructural abnormalities regarding the localizations of Aß and pTau in novel locations. For example, two pTau-positive post-synaptic spine-like protrusions innervated by axon terminals were found projecting from the axon initial segment of a pyramidal cell. Three pyramidal neurons with intracellular Aß or pTau were 3D reconstructed. Automatic synapse detection, which is necessary for connectomics analysis, revealed the changes in density and volume of synapses at different distances from an Aß plaque. This vCLEM approach is useful to uncover molecular alterations within large-scale volume electron microscopy data, opening a new connectomics pathway to study Alzheimer's disease and other types of dementia.

Multiplexed volumetric CLEM enabled by antibody derivatives provides new insights into the cytology of the mouse cerebellar cortex.

Mapping neuronal networks that underlie behavior has become a central focus in neuroscience. While serial section electron microscopy (ssEM) can reveal the fine structure of neuronal networks (connectomics), it does not provide the molecular information that helps identify cell types or their functional properties. Volumetric correlated light and electron microscopy (vCLEM) combines ssEM and volumetric fluorescence microscopy to incorporate molecular labeling into ssEM datasets. We developed an approach that uses small fluorescent single-chain variable fragment (scFv) immuno-probes to perform multiplexed detergent-free immuno-labeling and ssEM on the same samples. We generated eight such fluorescent scFvs that targeted useful markers for brain studies (green fluorescent protein, glial fibrillary acidic protein, calbindin, parvalbumin, voltage-gated potassium channel subfamily A member 2, vesicular glutamate transporter 1, postsynaptic density protein 95, and neuropeptide Y). To test the vCLEM approach, six different fluorescent probes were imaged in a sample of the cortex of a cerebellar lobule (Crus 1), using confocal microscopy with spectral unmixing, followed by ssEM imaging of the same sample. The results show excellent ultrastructure with superimposition of the multiple fluorescence channels. Using this approach we could document a poorly described cell type in the cerebellum, two types of mossy fiber terminals, and the subcellular localization of one type of ion channel. Because scFvs can be derived from existing monoclonal antibodies, hundreds of such probes can be generated to enable molecular overlays for connectomic studies.

A cholinergic medial septum input to medial habenula mediates generalization formation and extinction of visual aversion.

Generalization of visual aversion is a critical function of the brain that supports survival, but the underlying neurobiological mechanisms are unclear. We establish a rapid generalization procedure for inducing visual aversion by dynamic stripe images. By using fiber photometry, apoptosis, chemogenetic and optogenetic techniques, and behavioral tests, we find that decreased cholinergic neurons' activity in the medial septum (MS) leads to generalization loss of visual aversion. Strikingly, we identify a projection from MS cholinergic neurons to the medial habenula (MHb) and find that inhibition of the MS→MHb cholinergic circuit disrupts aversion-generalization formation while its continuous activation disrupts subsequent extinction. Further studies show that MS→MHb cholinergic projections modulate the generalization of visual aversion possibly via M1 muscarinic acetylcholine receptors (mAChRs) of downstream neurons coreleasing glutamate and acetylcholine. These findings reveal that the MS→MHb cholinergic circuit is a critical node in aversion-generalization formation and extinction and potentially provides insight into the pathogenesis of affective disorders.

Carotenoid-derived bioactive metabolites shape plant root architecture to adapt to the rhizospheric environments.

Roots are important plant organs for the uptake of water and nutrient elements. Plant root development is finely regulated by endogenous signals and environmental cues, which shapes the root system architecture to optimize the plant growth and adapt to the rhizospheric environments. Carotenoids are precursors of plant hormones strigolactones (SLs) and ABA, as well as multiple bioactive molecules. Numerous studies have demonstrated SLs and ABA as essential regulators of plant root growth and development. In addition, a lot carotenoid-derived bioactive metabolites are recently identified as plant root growth regulators, such as anchorene, ß-cyclocitral, retinal and zaxinone. However, our knowledge on how these metabolites affect the root architecture to cope with various stressors and how they interact with each other during these processes is still quite limited. In the present review, we will briefly introduce the biosynthesis of carotenoid-derived root regulators and elaborate their biological functions on root development and architecture, focusing on their contribution to the rhizospheric environmental adaption of plants.

Innovative sludge pretreatment technology for impurity separation using micromesh.

In order to reduce the impacts on sludge treatment facilities caused by impurities such as fibers, hairs, plastic debris, and coarse sand, an innovative primary sludge pretreatment technology, sludge impurity separator (SIS), was proposed in this study. Non-woven micromesh with pore size of 0.40 mm was used to remove the impurities from primary sludge. Results of lab-scale tests showed that impurity concentration, aeration intensity, and channel gap were the key operation parameters, of which the optimized values were below 25 g/L, 0.8 m3/(m2 min), and 2.5 cm, respectively. In the full-scale SIS with treatment capacity of 300 m3/day, over 88% of impurities could be removed from influent and the cleaning cycle of micromesh was more than 16 days. Economic analysis revealed that the average energy consumption was 1.06 kWh/m3 treated sludge and operation cost was 0.6 yuan/m3 treated sludge.

Influence of fermentation liquid from waste activated sludge on anoxic/oxic- membrane bioreactor performance: Nitrogen removal, membrane fouling and microbial community.

In order to investigate effects of waste activated sludge (WAS) fermentation liquid on anoxic/oxic- membrane bioreactor (A/O-MBR), two A/O-MBRs with and without WAS fermentation liquid addition were operated in parallel. Results show that addition of WAS fermentation liquid clearly improved denitrification efficiency without deterioration of nitrification, while severe membrane fouling occurred. WAS fermentation liquid resulted in an elevated production of proteins and humic acids in bound extracellular polymeric substance (EPS) and release of organic matter with high MW fractions in soluble microbial product (SMP) and loosely bound EPS (LB-EPS). Measurement of deposition rate and fluid structure confirmed increased fouling potential of SMP and LB-EPS. γ-Proteobacteria and Ferruginibacter, which can secrete and export EPS, were also found to be abundant in the MBR with WAS fermentation liquid. It is implied that when WAS fermentation liquid was applied, some operational steps to control membrane fouling should be employed.

A novel, easy and rapid method for constructing yeast two-hybrid vectors using In-Fusion technology.

Yeast two-hybrid systems are powerful tools for analyzing interactions between proteins. Vector construction is an essential step in yeast two-hybrid experiments, which require bait and prey plasmids. In this study, we modified the multiple cloning site sequence of the yeast plasmid pGADT7 by site-directed mutagenesis PCR to generate the pGADT7-In vector, which resulted in an easy and rapid method for constructing yeast two-hybrid vectors using the In-Fusion cloning technique. This method has three key advantages: only one pair of primers and one round of PCR are needed to generate bait and prey plasmids for each gene, it is restriction endonuclease- and ligase-independent, and it is fast and easily performed.

Microbial responses to membrane cleaning using sodium hypochlorite in membrane bioreactors: Cell integrity, key enzymes and intracellular reactive oxygen species.

Sodium hypochlorite (NaClO) is a commonly used reagent for membrane cleaning in membrane bioreactors (MBRs), while it, being a kind of disinfectant (oxidant), may impair viability of microbes or even totally inactivate them upon its diffusion into mixed liquor during membrane cleaning. In this study, we systematically examine the effects of NaClO on microorganisms in terms of microbial cell integrity, metabolism behaviours (key enzymes), and intracellular reactive oxygen species (ROS) under various NaClO concentrations. Different proportions of microbial cells in activated sludge were damaged within several minutes dependent on NaClO dosages (5-50 mg/g-SS), and correspondingly organic matters were released to bulk solution. Inhibition of key enzymes involved in organic matter biodegradation, nitrification and denitrification was observed in the presence of NaClO above 1 mg/g-SS, and thus organic matter and nitrogen removal efficiencies were decreased. It was also demonstrated that intracellular ROS production was increased with the NaClO dosage higher than 1 mg/g-SS, which likely induced further damage to microbial cells.

Membrane bioreactors fed with different COD/N ratio wastewater: impacts on microbial community, microbial products, and membrane fouling.

It is known that an increase of COD/N ratio can result in an enhanced removal of nutrients in membrane bioreactors (MBRs); however, impacts of doing so on membrane filtration performance remain unclear. In this work, comparison of membrane filtration performance, microbial community, and microbial products under low temperature was carried out in anoxic/oxic (A/O) MBRs with COD/N ratios of 9.9 and 5.5 g COD/g N in influent. There was no doubt that an improvement of nitrogen removal under high COD/N ratio was observed; however, severer membrane fouling was found compared to the MBR fed with low COD/N ratio wastewater. The increase of COD/N ratio resulted in an elevated production of humic acids in soluble microbial product (SMP) and carbohydrates, proteins, and humic acids in loosely bound extracellular polymeric substance (LB-EPS). Quartz crystal microbalance with dissipation monitoring (QCM-D) analysis showed that the adsorption capability of SMP and LB-EPS was higher in the MBR with higher COD/N ratio. Four hundred fifty four high-throughput pyrosequencing revealed that the higher COD/N ratio led to the enrichment of Bacteroidetes at phylum level and Azospira, Thauera, Zoogloea, etc. at genus level. Bacteroidetes are considered to potentially release EPS, and Azospira, Thauera, and Zoogloea, etc. have denitrification activity. The change in microbial communities is consistent with MBR performance.

Formation and removal of dissolved organic nitrogen (DON) in membrane bioreactor and conventional activated sludge processes.

Dissolved organic nitrogen (DON) has become a growing concern due to its contribution to eutrophication and nitrogenous disinfection byproducts (N-DBPs) formation. However, information of DON in membrane bioreactors (MBRs) is very limited. In this study, occurrence, transformation and fate of DON in an MBR system were systematically investigated. MBR sludge showed a larger hydrolysis rate of particle organic nitrogen (PON) and also a higher transformation rate of DON to nitrate compared to conventional activated sludge (CAS). For long-term experiments, MBR achieved higher DON removal efficiency at low temperature than CAS; however, at high temperature, the effluent DON concentrations were almost the same in both systems. Batch tests on DON biodegradability showed that DON concentration increased and large molecular weight DON accumulated after 3-h aeration at low temperature, while DON concentration continuously decreased with the increase of aeration time at high temperature. The obtained results provide insights in DON removal in MBRs for meeting increasingly stringent regulations in terms of nitrogen removal.