Noncanonical Wnt signaling maintains hematopoietic stem cells in the niche.

Wnt signaling is involved in self-renewal and maintenance of hematopoietic stem cells (HSCs); however, the particular role of noncanonical Wnt signaling in regulating HSCs in vivo is largely unknown. Here, we show Flamingo (Fmi) and Frizzled (Fz) 8, members of noncanonical Wnt signaling, both express in and functionally maintain quiescent long-term HSCs. Fmi regulates Fz8 distribution at the interface between HSCs and N-cadherin(+) osteoblasts (N-cad(+)OBs that enrich osteoprogenitors) in the niche. We further found that N-cad(+)OBs predominantly express noncanonical Wnt ligands and inhibitors of canonical Wnt signaling under homeostasis. Under stress, noncanonical Wnt signaling is attenuated and canonical Wnt signaling is enhanced in activation of HSCs. Mechanistically, noncanonical Wnt signaling mediated by Fz8 suppresses the Ca(2+)-NFAT- IFNγ pathway, directly or indirectly through the CDC42-CK1α complex and also antagonizes canonical Wnt signaling in HSCs. Taken together, our findings demonstrate that noncanonical Wnt signaling maintains quiescent long-term HSCs through Fmi and Fz8 interaction in the niche.

Rational Synthesis of Functionalized Covalent Organic Frameworks via Four-Component Reaction.

The construction of function-oriented covalent organic frameworks (COFs) remains a challenge as it requires simultaneous consideration of diversified structures, robust linkage, and tailorable functionalities. Herein, we report the rational synthesis of functionalized COFs via a four-component reaction strategy. Through the four-component Debus-Radziszewski reaction, 11 N-substituted imidazole-based COFs with diversified structures were facilely constructed from readily available building blocks. By forming the N-substituted imidazole linkage, these synthesized COFs displayed ultrastability toward strong acids and base. Moreover, the four components reaction allows the rational synthesis of COFs with tailorable functionalities. As an example, the phosphonate-functionalized COF (LZU-530) was rationally constructed for the efficient adsorption of uranium(VI). The uranium(VI) uptake of LZU-530 reaches up to 95 mg·g-1 in 2 M HNO3, which is the highest uptake of the existing organic porous materials under such harsh conditions. Our results highlight the use of multicomponent reaction for the rational synthesis of robust and functionalized COFs toward targeted applications.

Melatonin in cancer biology: pathways, derivatives, and the promise of targeted delivery.

Melatonin, historically recognized for its primary role in regulating circadian rhythms, has expanded its influence particularly due to its wide range of biological activities. It has firmly established itself in cancer research. To highlight its versatility, we delved into how melatonin interacts with key signaling pathways, such as the Wnt/ß-Catenin, PI3K, and NF-κB pathways, which play foundational roles in tumor development and progression. Notably, melatonin can intricately modulate these pathways, potentially affecting various cellular functions such as apoptosis, metastasis, and immunity. Additionally, a comprehensive review of current clinical studies provides a dual perspective. These studies confirm melatonin's potential in cancer management but also underscore its inherent limitations, particularly its limited bioavailability, which often relegates it to a supplementary role in treatments. Despite this limitation, there is an ongoing quest for innovative solutions and current advancements include the development of melatonin derivatives and cutting-edge delivery systems. By synthesizing the past, present, and future, this review provides a detailed overview of melatonin's evolving role in oncology, positioning it as a potential cornerstone in future cancer therapeutics.

Smaller Aerobic Granules Significantly Reduce N2O Production by Ammonia-Oxidizing Bacteria: Evidences from Biochemical and Isotopic Analyses.

The mitigation of nitrous oxide (N2O) is of primary significance to offset carbon footprints in aerobic granular sludge (AGS) systems. However, a significant knowledge gap still exists regarding the N2O production mechanism and its pathway contribution. To address this issue, the impact of varying granule sizes, dissolved oxygen (DO), and nitrite (NO2-) levels on N2O production by ammonia-oxidizing bacteria (AOB) during nitrification in AGS systems was comprehensively investigated. Biochemical and isotopic experiments revealed that increasing DO or decreasing NO2- levels reduced N2O emission factors (by 13.8 or 19.5%) and production rates (by 0.08 or 0.35 mg/g VSS/h) via weakening the role of the AOB denitrification pathway since increasing DO competed for more electrons required for AOB denitrification. Smaller granules (0.5 mm) preferred to diminish N2O production via enhancing the role of NH2OH pathway (i.e., 59.4-100% in the absence of NO2-), while larger granules (2.0 mm) induced conspicuously higher N2O production via the AOB denitrification pathway (approximately 100% at higher NO2- levels). Nitrifying AGS systems with a unified size of 0.5 mm achieved 42% N2O footprint reduction compared with the system with mixed sizes (0.5-2.0 mm) under optimal conditions (DO = 3.0 mg-O2/L and NO2- = 0 mg-N/L).

Modeling Free Nitrous Acid Inhibition on the Removal of Nitrogen and Atenolol during Sidestream Partial Nitritation Processes.

Sidestream serves as an important reservoir collecting pharmaceuticals from sludge. However, the knowledge on sidestream pharmaceutical removal is still insufficient. In this work, atenolol biodegradation during sidestream partial nitritation (PN) processes characterized by high free nitrous acid (FNA) accumulation was modeled. To describe the FNA inhibition on ammonia oxidation and atenolol removal, Vadivelu-type and Hellinga-type inhibition kinetics were introduced into the model framework. Four inhibitory parameters along with four biodegradation kinetic parameters were calibrated and validated separately with eight sets of batch experimental data and 60 days' PN reactor operational data. The developed model could accurately reproduce the dynamics of nitrogen and atenolol. The model prediction further revealed that atenolol biodegradation efficiencies by ammonia-oxidizing bacteria (AOB)-induced cometabolism, AOB-induced metabolism, and heterotrophic bacteria-induced biodegradation were 0, ∼ 60, and ∼35% in the absence of ammonium and FNA; ∼ 14, ∼ 29, and ∼28% at 0.03 mg-N L-1 FNA; and 7, 15, and 5% at 0.19 mg-N L-1 FNA. Model simulation showed that the nitritation efficiency of ∼99% and atenolol removal efficiency of 57.5% in the PN process could be achieved simultaneously by controlling pH at 8.5, while 89.2% total nitrogen and 57.1% atenolol were removed to the maximum at pH of 7.0 in PN coupling with the anammox process. The pH-based operational strategy to regulate FNA levels was mathematically demonstrated to be effective for achieving the simultaneous removal of nitrogen and atenolol in PN-based sidestream processes.

Rational design of a two-dimensional high-temperature ferromagnet from HCP cobalt.

Cobalt has the highest Curie temperature (Tc) among the elemental ferromagnetic metals and has a hexagonal close-packed (HCP) structure at room temperature. In this study, HCP Co was thinned to the thickness of several (n) unit cells along the c-axis and then passivated by halogen atoms, thus being named Co2nX2 (X = F, Cl, Br and I). For Co2X2 and Co3X2, all of them are not only kinetically but also thermodynamically stable from the viewpoint of the phonon spectra and molecular dynamics. Similar to HCP Co, two-dimensional (2D) Co2F2, Co2Cl2 and Co3X2 (X = Cl, Br and I) are still ferromagnetic metals within the Stoner model but Co2X2 (X = Br and I) is a ferromagnetic half-metal with the coexistence of the metallic behavior for one spin and the insulating behavior for the other spin. Taking into account the spin-orbital coupling (SOC), the easy-magnetization axis is within the plane where the magnetization is isotropic, making it look like a 2D XY magnet. Applying a critical biaxial strain could lead to an easy-magnetization axis changing from the in-plane to the out-of-plane direction. Finally, we use classical Monte Carlo simulations to estimate the Curie temperature (Tc) which is as high as 957 and 510 K for Co2F2 and Co2Cl2, respectively, because of the strong direct exchange interaction. Different from being obtained by mechanical or liquid exfoliation from van der Waals layered structures, our study opens up new possibilities to search for novel 2D ferromagnets from the elemental ferromagnets and provides opportunities for realizing realistic ultra-thin spintronic devices.

[Circadian rhythm and health: dialogue between traditional Chinese medicine and modern medicine].

Circadian rhythm refers to the daily rhythmic variations in an organism. The irregular lifestyles of modern humans have led to a high incidence of chronic diseases, highlighting an inseparable relationship between disrupted circadian rhythm and disease development. TCM has long discussed rhythmic variations, with records dating back to the Yellow Emperor's Inner Canon(Huang Di Nei Jing), which laid a rich theoretical foundation for the research on circadian rhythm. Modern medical research has provided a more comprehensive explanation of its molecular mechanisms. This article integrated the current understanding of circadian rhythm in both Chinese and western medicine, emphasizing the crucial relationship between rhythm regulation and disease treatment. By highlighting the interdisciplinary nature of the two fields, it offers new directions for exploring the field of chronomedicine.

Free Nitrous Acid Inhibits Atenolol Removal during the Sidestream Partial Nitritation Process through Regulating Microbial-Induced Metabolic Types.

Limited studies have attempted to evaluate pharmaceutical removal during the sidestream partial nitritation (PN) process. In this work, atenolol biodegradation by PN cultures was investigated by maintaining ammonium and pH at different levels. For the first time, free nitrous acid (FNA), other than ammonium, pH, and free ammonia, was demonstrated to inhibit atenolol removal, with biodegradation efficiencies of ∼98, ∼67, and ∼28% within 6 days at average FNA levels of 0, 0.03, and 0.19 mg-N L-1, respectively. Ammonia-oxidizing bacteria (AOB)-induced metabolism was predominant despite varying FNA concentrations. In the absence of ammonium/FNA, atenolol was mostly biodegraded via AOB-induced metabolism (65%) and heterotroph-induced metabolism (33%). AOB-induced metabolism was largely inhibited (down to 29%) at 0.03 mg-N L-1 FNA, while ∼27 and ∼11% were degraded via heterotroph-induced metabolism and AOB-induced cometabolism, respectively. Higher FNA (0.19 mg-N L-1) substantially reduced atenolol biodegradation via heterotroph-induced metabolism (4%), AOB-induced metabolism (16%), and AOB-induced cometabolism (8%). Newly identified products and pathways were related to metabolic types and FNA levels: (i) deamination and decarbonylation (AOB-induced cometabolism, 0.03 mg-N L-1 FNA); (ii) deamination from atenolol acid (heterotrophic biodegradation); and (iii) nitro-substitution (reaction with nitrite). This suggests limiting FNA to realize simultaneous nitrogen and pharmaceutical removal during the sidestream process.

Cometabolic biodegradation of antibiotics by ammonia oxidizing microorganisms during wastewater treatment processes.

Studies on antibiotic removal during wastewater treatment processes are crucial since their release into the environment could bring potential threats to human health and ecosystem. Cometabolic biodegradation of antibiotics by ammonia oxidizing microorganisms (AOMs) has received special attentions due to the enhanced removal of antibiotics during nitrification processes. However, the interactions between antibiotics and AOMs are less well-elucidated. In this review, the recent research proceedings on cometabolic biodegradation of antibiotics by AOMs were summarized. Ammonia oxidizing bacteria (AOB), ammonia oxidizing archaea (AOA) and complete ammonia oxidizers (comammox) played significant roles in both nitrification and cometabolic biodegradation of antibiotics. Antibiotics at varying concentrations might pose inhibiting or stimulating effect on AOMs, influencing the microbial activity, community abundance and ammonia monooxygenase subunit A gene expression level. AOMs-induced cometabolic biodegradation products were analyzed as well as the corresponding pathways for each type of antibiotics. The effects of ammonium availability, initial antibiotic concentration, sludge retention time and temperature were assessed on the cometabolic biodegradation efficiencies of antibiotics. This work might provide further insights into the fate and removal of antibiotics during nitrification processes.

Enhanced biodegradation of ciprofloxacin by enriched nitrifying sludge: assessment of removal pathways and microbial responses.

Antibiotics are mostly collected by sewage systems, but not completely removed within wastewater treatment plants. Their release to aquatic environment poses a great threat to public health. This study evaluated the removal of a widely used fluoroquinolone antibiotic, ciprofloxacin, in enriched nitrifying culture through a series of experiments by controlling ammonium concentrations and inhibiting functional microorganisms. The removal efficiency of ciprofloxacin at an initial concentration of 50 µg L-1 reached 81.86 ± 3.21% in the presence of ammonium, while only 22.83 ± 8.22% of ciprofloxacin was removed in its absence. A positive linear correlation was found between the ammonia oxidation rate (AOR) and ciprofloxacin biodegradation rate. These jointly confirmed the importance of the AOB-induced cometabolism in ciprofloxacin biodegradation, with adsorption and metabolic degradation pathways playing minor roles. The continuous exposure of AOB to ciprofloxacin led to decreases of ammonia monooxygenase (AMO) activities and AOR. The antibacterial effects of ciprofloxacin and its biodegradation products were further evaluated and the results revealed that biodegradation products of ciprofloxacin exhibited less toxicity compared to the parent compound, implying the potential application of cometabolism in alleviation of antimicrobial activity. The findings provided new insights into the AOB-induced cometabolic biodegradation of fluoroquinolone antibiotics.

Effectiveness of the attachment position in molar intrusion with clear aligners: a finite element study.

OBJECTIVE: To evaluate the biomechanical effects of different attachments' position for maxillary molar intrusion with clear aligner treatment by finite element analysis. METHODS: Cone-beam computed tomography images of a patient with supra-eruption of the maxillary second molars were selected to construct three-dimensional models of the maxilla, periodontal ligaments, dentition, and clear aligner. The models were divided into four groups depending on the attachment location on the first molar: (1) no attachment (NA), (2) buccal attachment (BA), (3) palatal attachment (PA), and (4) bucco-palatal attachment (BPA). After applying an intrusion of 0.2 mm on the second molar, displacements and stress distributions of the teeth, aligner, and periodontal ligament were analyzed with the finite element software. RESULTS: All groups displayed equivalent movement patterns of aligners. The NA and BA groups showed buccal tipping of the second molar, while the PA group showed palatal tipping. The BPA group had the highest intruding value and the lowest buccal/palatal tipping value. All groups showed mesial tipping of the second molar. Stress distribution in the periodontal ligament strongly correlated with the attachment position. The BPA group showed the best stress distribution. CONCLUSION: Combined BA and PA could effectively prevent buccal and palatal tipping and showed the best efficiency in intruding the second molar. The second molar showed an unavoidable tendency to tip mesially, regardless of the attachment position.

Anaerobic Oxidation of Methane Coupled with Dissimilatory Nitrate Reduction to Ammonium Fuels Anaerobic Ammonium Oxidation.

Nitrate/nitrite-dependent anaerobic methane oxidation (n-DAMO) is critical for mitigating methane emission and returning reactive nitrogen to the atmosphere. The genomes of n-DAMO archaea show that they have the potential to couple anaerobic oxidation of methane to dissimilatory nitrate reduction to ammonium (DNRA). However, physiological details of DNRA for n-DAMO archaea were not reported yet. This work demonstrated n-DAMO archaea coupling the anaerobic oxidation of methane to DNRA, which fueled Anammox in a methane-fed membrane biofilm reactor with nitrate as only electron acceptor. Microelectrode analysis revealed that ammonium accumulated where nitrite built up in the biofilm. Ammonium production and significant upregulation of gene expression for DNRA were detected in suspended n-DAMO culture with nitrite exposure, indicating that nitrite triggered DNRA by n-DAMO archaea. 15N-labeling batch experiments revealed that n-DAMO archaea produced ammonium from nitrate rather than from external nitrite. Localized gradients of nitrite produced by n-DAMO archaea in biofilms induced ammonium production via the DNRA process, which promoted nitrite consumption by Anammox bacteria and in turn helped n-DAMO archaea resist stress from nitrite. As biofilms predominate in various ecosystems, anaerobic oxidation of methane coupled with DNRA could be an important link between the global carbon and nitrogen cycles that should be investigated in future research.

Pyrimidazole-Based Covalent Organic Frameworks: Integrating Functionality and Ultrastability via Isocyanide Chemistry.

Development of new chemistry to simultaneously meet the demands for topology, connectivity, and functionality is highly desired in the research area of covalent organic frameworks (COFs). We explore herein the isocyanide chemistry so as to establish a facile paradigm to integrate functionality and ultrastability in COFs. Using the representative Groebke-Blackburn-Bienaymé (GBB) reaction based on isocyanide chemistry, we are able to construct a series of pyrimidazole-based COFs in one step from isocyanide, aminopyridine, and aldehyde monomers. Diversified functionalities have been bottom-up integrated by the simple replacement of readily available 2-aminopyridine monomers. Meanwhile, the ubiquitous formation of fused imidazole rings within the frameworks has guaranteed their ultrastability. In view of the rich synthetic possibilities provided by isocyanide chemistry, we expect that this contribution opens up a new avenue toward the divergent construction of robust COFs for practical applications.

Return-Sludge Treatment with Endogenous Free Nitrous Acid Limits Nitrate Production and N2O Emission for Mainstream Partial Nitritation/Anammox.

Nitrite oxidizing bacteria (NOB) and nitrous oxide (N2O) hinder the development of mainstream partial nitritation/anammox. To overcome these, endogenous free ammonia (FA) and free nitrous acid (FNA), which can be produced in the sidestream, were used for return-sludge treatment for two integrated-film activated sludge reactors containing biomass in flocs and on carriers. The repeated exposure of biomass from one reactor to FA shocks had a limited impact on NOB suppression but inhibited anammox bacteria (AnAOB). In the other reactor, repeated FNA shocks to the separated flocs failed to limit the system's nitrate production since NOB activity was still high on the biofilms attached to the unexposed carriers. In contrast, the repeated FNA treatment of flocs and carriers favored aerobic ammonium-oxidizing bacteria (AerAOB) over NOB activity with AnAOB negligibly affected. It was further revealed that return-sludge treatment with higher FNA levels led to lower N2O emissions under similar effluent nitrite concentrations. On this basis, weekly 4 h FNA shocks of 2.0 mg of HNO2-N/L were identified as an optimal and realistic treatment, which not only enabled nitrogen removal efficiencies of ∼65% at nitrogen removal rates of ∼130 mg of N/L/d (20 °C) but also yielded the lowest cost and carbon footprint.

Denitrifying Anaerobic Methane Oxidation and Anammox Process in a Membrane Aerated Membrane Bioreactor: Kinetic Evaluation and Optimization.

Denitrifying anaerobic methane oxidation (DAMO) coupled to anaerobic ammonium oxidation (anammox) is a promising technology for complete nitrogen removal with economic and environmental benefit. In this work, a model framework integrating DAMO and anammox process was constructed based on suspended-growth systems. The proposed model was calibrated and validated using experimental data from a sequencing batch reactor and a membrane aerated membrane bioreactor (MAMBR). The model managed to describe removal rates of ammonium (NH4+), nitrite (NO2-), and total nitrogen, as well as biomass changes of DAMO archaea, DAMO bacteria, and anaerobic ammonium oxidizing bacteria (AnAOB) in both reactors. The estimated parameter values revealed that DAMO archaea possessed properties of faster growth and higher biomass yield in suspended-growth systems compared to those in attached-growth systems (e.g., biofilm). Model simulation demonstrated that solid retention time (SRT) was effective in washing out DAMO bacteria, but retaining DAMO archaea and AnAOB in the MAMBR. The optimal SRT and nitritation efficiency (the ratio of the NO2- to the sum of NH4+ and NO2- in the MAMBR influent) were simulated so that 99% of total nitrogen was removed to meet the discharge standard. MAMBR further suggested to be operated with SRT between 15 and 30 days so that the optimal nitritation efficiency could be minimized to 49% for cost savings.

Constructing Robust Covalent Organic Frameworks via Multicomponent Reactions.

Methodology development of robust linkages is fundamentally important for the synthesis and application of covalent organic frameworks (COFs). We report herein a new strategy based on multicomponent reactions (MCRs) to construct ultrastable COFs. With the one-pot formation of five covalent bonds in each cyclic joint, a series of imidazole-linked COFs were robustly constructed through the Debus-Radziszewski MCR from three easily available components. By reaching a higher level of complexity and precision in covalent assembly, this research explores a new direction in integrating sophisticated reversible/irreversible reactions to construct crystalline porous frameworks.

Membrane-Binding Adhesive Particulates Enhance the Viability and Paracrine Function of Mesenchymal Cells for Cell-Based Therapy.

Understanding the fundamental cell-material interactions is essential to designing functional materials for biomedical applications. Although mesenchymal stromal cells (MSCs) are known to secrete cytokines and exosomes that are effective to treat degenerative diseases, the inherent property of biomaterials to modulate the therapeutic function of MSCs remains to be investigated. Here, a multivalent cell-membrane adhesive conjugate was generated through polyamindoamine (PAMAM) and an oligopeptide, IKVAV, and the conjugate was further complexed with hyaluronic acid (HA). The adhesive particulates were used to coat the surface of adipose-derived mesenchymal stromal cells (Ad-MSCs) and studied in the MSC spheroid culture. The analysis showed that the adhesive complexes formed via PAMAM conjugates and HA significantly promoted the proliferation and the gene expression of pro-angiogenesis cytokines in MSCs; the production of anti-inflammatory miRNAs in exosomes could also be elevated. The transplantation of the Ad-MSCs primed with PAMAM-IKVAV/HA composite particulates in a rat myocardial infarction model further demonstrated the beneficial effects of membrane-binding materials on improving the cell retention and tissue angiogenesis. The new function of membrane-binding adhesive materials potentially provides useful ways to improve cell-based therapy.

Substrate Diffusion within Biofilms Significantly Influencing the Electron Competition during Denitrification.

A common and long-existing operational issue of wastewater denitrification is the unexpected accumulation of nitrite (NO2-) that could suppress the activity of various microorganisms involved in biological wastewater treatment process and nitrous oxide (N2O) that could emit as a potent greenhouse gas. Recently, it has been confirmed that the accumulation of these denitrification intermediates in biological wastewater treatment process is greatly influenced by the electron competition between the four denitrification steps. However, little is known about this in biofilm systems. In this work, we applied a mathematical model that links carbon oxidation and nitrogen reduction processes through a pool of electron carriers, to assess electron competition in denitrifying biofilms. Simulations were performed comprehensively at seven combinations of electron acceptor addition scheme (i.e., simultaneous addition of one, two or three among nitrate (NO3-), NO2-, and N2O) to compare the effect of electron competition on NO3-, NO2- and N2O reduction. Overall, the effects of substrate loading, biofilm thickness and effective diffusion coefficients on electron competition are not always intuitive. Model simulations show that electron competition was intensified due to the substrate load limitation (from 120 to 20 mg COD/L) and increasing biofilm thicknesses (from 0.1 to 1.6 mm) in most cases, where electrons were prioritized to nitrite reductase because of the insufficient electron donor availability in the biofilm. In contrast, increasing effective diffusion coefficients did not pose a significant effect on electron competition and only increased electrons distributed to nitrite reductase when both NO2- and N2O are added.

Maternal imprinting at the H19-Igf2 locus maintains adult haematopoietic stem cell quiescence.

The epigenetic regulation of imprinted genes by monoallelic DNA methylation of either maternal or paternal alleles is critical for embryonic growth and development. Imprinted genes were recently shown to be expressed in mammalian adult stem cells to support self-renewal of neural and lung stem cells; however, a role for imprinting per se in adult stem cells remains elusive. Here we show upregulation of growth-restricting imprinted genes, including in the H19-Igf2 locus, in long-term haematopoietic stem cells and their downregulation upon haematopoietic stem cell activation and proliferation. A differentially methylated region upstream of H19 (H19-DMR), serving as the imprinting control region, determines the reciprocal expression of H19 from the maternal allele and Igf2 from the paternal allele. In addition, H19 serves as a source of miR-675, which restricts Igf1r expression. We demonstrate that conditional deletion of the maternal but not the paternal H19-DMR reduces adult haematopoietic stem cell quiescence, a state required for long-term maintenance of haematopoietic stem cells, and compromises haematopoietic stem cell function. Maternal-specific H19-DMR deletion results in activation of the Igf2-Igfr1 pathway, as shown by the translocation of phosphorylated FoxO3 (an inactive form) from nucleus to cytoplasm and the release of FoxO3-mediated cell cycle arrest, thus leading to increased activation, proliferation and eventual exhaustion of haematopoietic stem cells. Mechanistically, maternal-specific H19-DMR deletion leads to Igf2 upregulation and increased translation of Igf1r, which is normally suppressed by H19-derived miR-675. Similarly, genetic inactivation of Igf1r partly rescues the H19-DMR deletion phenotype. Our work establishes a new role for this unique form of epigenetic control at the H19-Igf2 locus in maintaining adult stem cells.

Heterotrophic denitrifiers growing on soluble microbial products contribute to nitrous oxide production in anammox biofilm: Model evaluation.

In this work, a model framework was constructed to assess and predict nitrous oxide (N2O) production, substrate and microbe interactions in an anammox biofilm bioreactor. The anammox kinetics were extended by including kinetics of autotrophic soluble microbial products (SMP) formation, which consisted of utilization-associated products (UAP) and biomass-associated products (BAP). Heterotrophic bacteria growing on UAP, BAP and decay released substance (SS) were modelled to perform four-step sequential reductions from nitrate to dinitrogen gas. N2O was modelled as an intermidiate of heterotrophic denitrification via three pathways with UAP, BAP and SS as the electron donors. The developed model framework was evaluated using long-term operational data from an anammox biofilm reactor and satisfactorily reproduced effluent nitrogen and SMP as well as N2O emission factors under different operational conditions. The modeling results revealed that N2O was mainly produced with UAP as the electron donor while BAP and SS play minor roles. Heterotrophic denitrifiers growing on UAP would significantly contribute to N2O emission from anammox biofilm reactor even though heterotrophs only account for a relatively small fraction of active biomass in the anammox biofilm. Comprehensive simulations were conducted to investigate the effects of N loading rate and biofilm thickness, which indicated that maintaining a low N loading rate and a thick biofilm thickness were essential for high total nitrogen removal efficiency and low N2O emission.