Synthetic heparan sulfate standards and machine learning facilitate the development of solid-state nanopore analysis.

The application of solid-state (SS) nanopore devices to single-molecule nucleic acid sequencing has been challenging. Thus, the early successes in applying SS nanopore devices to the more difficult class of biopolymer, glycosaminoglycans (GAGs), have been surprising, motivating us to examine the potential use of an SS nanopore to analyze synthetic heparan sulfate GAG chains of controlled composition and sequence prepared through a promising, recently developed chemoenzymatic route. A minimal representation of the nanopore data, using only signal magnitude and duration, revealed, by eye and image recognition algorithms, clear differences between the signals generated by four synthetic GAGs. By subsequent machine learning, it was possible to determine disaccharide and even monosaccharide composition of these four synthetic GAGs using as few as 500 events, corresponding to a zeptomole of sample. These data suggest that ultrasensitive GAG analysis may be possible using SS nanopore detection and well-characterized molecular training sets.

Over One Million DNA and Protein Events Through Ultra-Stable Chemically-Tuned Solid-State Nanopores.

Stability, long lifetime, resilience against clogging, low noise, and low cost are five critical cornerstones of solid-state nanopore technology. Here, a fabrication protocol is described wherein >1 million events are obtained from a single solid-state nanopore with both DNA and protein at the highest available lowpass filter (LPF, 100 kHz) of the Axopatch 200B-the highest event count mentioned in literature. Moreover, a total of ≈8.1 million events are reported in this work encompassing the two analyte classes. With the 100 kHz LPF, the temporally attenuated population is negligible while with the more ubiquitous 10 kHz, ≈91% of the events are attenuated. With DNA experiments, the pores are operational for hours (typically >7 h) while the average pore growth is merely ≈0.16 ± 0.1 nm h-1 . The current noise is exceptionally stable with traces typically showing <10 pA h-1 increase in noise. Furthermore, a real-time method to clean and revive pores clogged with analyte with the added benefit of minimal pore growth during cleaning (< 5% of the original diameter) is showcased. The enormity of the data collected herein presents a significant advancement to solid-state pore performance and will be useful for future ventures such as machine learning where large amounts of pristine data are a prerequisite.

Biofouling potential of surface-enhanced Raman scattering-based seawater quality sensors by Ulva spp.

This study investigated the biofouling potential of surface-enhanced Raman scattering (SERS)-based sensor materials in the context of marine environments. Uncoated and monolithic commercial gold (Au) silicon nanopillar array SERS substrates, Au-coated carbon black nanoparticle (AuCB NP) substrates, uncoated and Au sputter-coated in-house SERS, and uncoated and Au sputter-coated glass controls were tested for biofouling potential using Ulva spp. as model biofouling organisms. The mean percentages of Ulva spp. zoospores that adhered per mm2 (×103) on the uncoated and coated Au silicon nanopillar array, AuCB NP, uncoated and Au sputter-coated in-house, and uncoated and Au sputter-coated glass substrates were 10.28%, 5.45%, 10.49%, 3.25%, 24.84%, 12.86% and 7.78%, respectively. Results indicated that surface properties such as hydrophobicity, roughness, Au sputter-coating and the presence of micro-refuges on nano- and microstructured substrates were critical to the biofouling formation.

A Closer Look at Delayed Primary Cleft Surgery and Unrepaired Cleft Lip and/or Palate in 5 UK Cleft Centers.

BACKGROUND: There may be many reasons for delays to primary cleft surgery. Our aim was to investigate the age of children undergoing primary cleft lip or primary cleft palate repair in 5 cleft centers within the United Kingdom. Identify the reasons for delayed primary cleft lip repair (beyond 6 months) and delayed primary palate repair (beyond 13 months). Identify children who had a cleft lip and/or palate (CL±P) that was intentionally unrepaired and the reasons for this. METHODS: A retrospective, multicenter review of patients born with a CL±P between December 1, 2012, and December 31, 2016. Three regional cleft centers, comprising of 5 cleft administrative units in the United Kingdom participated. RESULTS: In all, 1826 patients with CL±P were identified. Of them, 120 patients had delayed lip repair, outside the expected standard of 183 days. And, 178 patients in total had delayed palate repair, outside the expected standard of 396 days. Twenty (1%) patients had an unrepaired cleft palate. CONCLUSIONS: This large retrospective review highlights variations between centers regarding the timing of lip and palate surgery and details the reasons stated for delayed primary surgery. A small number of patients with an unrepaired cleft palate were identified. All had complex medical problems or comorbidities listed as a reason for the decision not to operate and 50% had a syndromic diagnosis. The number of patients receiving delayed surgery due to comorbidities, being underweight or prematurity, highlights the importance of the cleft specialist nurse and pediatrician within the cleft multidisciplinary team.

Beyond nanopore sizing: improving solid-state single-molecule sensing performance, lifetime, and analyte scope for omics by targeting surface chemistry during fabrication.

Solid-state nanopores (SSNs) are single-molecule resolution sensors with a growing footprint in real-time bio-polymer profiling-most prominently, but far from exclusively, DNA sequencing. SSNs accessibility has increased with the advent of controlled dielectric breakdown (CDB), but severe fundamental challenges remain: drifts in open-pore current and (irreversible) analyte sticking. These behaviors impede basic research and device development for commercial applications and can be dramatically exacerbated by the chemical complexity and physical property diversity of different analytes. We demonstrate a SSN fabrication approach attentive to nanopore surface chemistry during pore formation, and thus create nanopores in silicon nitride (SiNx) capable of sensing a wide analyte scope-nucleic acid (double-stranded DNA), protein (holo-human serum transferrin) and glycan (maltodextrin). In contrast to SiNx pores fabricated without this comprehensive approach, the pores are Ohmic in electrolyte, have extremely stable open-pore current during analyte translocation (>1 h) over a broad range of pore diameters ([Formula: see text]3- ∼30 nm) with spontaneous current correction (if current deviation occurs), and higher responsiveness (i.e. inter-event frequency) to negatively charged analytes (∼6.5 × in case of DNA). These pores were fabricated by modifying CDB with a chemical additive-sodium hypochlorite-that resulted in dramatically different nanopore surface chemistry including ∼3 orders of magnitude weaker Ka (acid dissociation constant of the surface chargeable head-groups) compared to CDB pores which is inextricably linked with significant improvements in nanopore performance with respect to CDB pores.

Chemically tailoring nanopores for single-molecule sensing and glycomics.

A nanopore can be fairly-but uncharitably-described as simply a nanofluidic channel through a thin membrane. Even this simple structural description holds utility and underpins a range of applications. Yet significant excitement for nanopore science is more readily ignited by the role of nanopores as enabling tools for biomedical science. Nanopore techniques offer single-molecule sensing without the need for chemical labelling, since in most nanopore implementations, matter is its own label through its size, charge, and chemical functionality. Nanopores have achieved considerable prominence for single-molecule DNA sequencing. The predominance of this application, though, can overshadow their established use for nanoparticle characterization and burgeoning use for protein analysis, among other application areas. Analyte scope continues to be expanded, and with increasing analyte complexity, success will increasingly hinge on control over nanopore surface chemistry to tune the nanopore, itself, and to moderate analyte transport. Carbohydrates are emerging as the latest high-profile target of nanopore science. Their tremendous chemical and structural complexity means that they challenge conventional chemical analysis methods and thus present a compelling target for unique nanopore characterization capabilities. Furthermore, they offer molecular diversity for probing nanopore operation and sensing mechanisms. This article thus focuses on two roles of chemistry in nanopore science: its use to provide exquisite control over nanopore performance, and how analyte properties can place stringent demands on nanopore chemistry. Expanding the horizons of nanopore science requires increasing consideration of the role of chemistry and increasing sophistication in the realm of chemical control over this nanoscale milieu.

Conductance-based profiling of nanopores: Accommodating fabrication irregularities.

Solid-state nanopores are nanoscale channels through otherwise impermeable membranes. Single molecules or particles can be passed through electrolyte-filled nanopores by, e.g. electrophoresis, and then detected through the resulting physical displacement of ions within the nanopore. Nanopore size, shape, and surface chemistry must be carefully controlled, and on extremely challenging <10 nm-length scales. We previously developed a framework to characterize nanopores from the time-dependent changes in their conductance as they are being formed through solution-phase nanofabrication processes with the appeal of ease and accessibility. We revisited this simulation work, confirmed the suitability of the basic conductance equation using the results of time-dependent experimental conductance measurements during nanopore fabrication by Yanagi et al., and then deliberately relaxed the model constraints to allow for (i) the presence of defects; and (ii) the formation of two small pores instead of one larger one. Our simulations demonstrated that the time-dependent conductance formalism supports the detection and characterization of defects, as well as the determination of pore number, but with implementation performance depending on the measurement context and results. In some cases, the ability to discriminate numerically between the correct and incorrect nanopore profiles was slight, but with accompanying differences in candidate nanopore dimensions that could yield to post-fabrication conductance profiling, or be used as convenient uncertainty bounds. Time-dependent nanopore conductance thus offers insight into nanopore structure and function, even in the presence of fabrication defects.

Ribosomal protein S6 kinase 1 signaling in prefrontal cortex controls depressive behavior.

Current treatments for major depressive disorder (MDD) have a time lag and are ineffective for a large number of patients. Development of novel pharmacological therapies requires a comprehensive understanding of the molecular events that contribute to MDD pathophysiology. Recent evidence points toward aberrant activity of synaptic proteins as a critical contributing factor. In the present studies, we used viral-mediated gene transfer to target a key mediator of activity-dependent synaptic protein synthesis downstream of mechanistic target of rapamycin complex 1 (mTORC1) known as p70 S6 kinase 1 (S6K1). Targeted delivery of two mutants of S6K1, constitutively active or dominant-negative, to the medial prefrontal cortex (mPFC) of rats allowed control of the mTORC1/S6K1 translational pathway. Our results demonstrate that increased expression of S6K1 in the mPFC produces antidepressant effects in the forced swim test without altering locomotor activity. Moreover, expression of active S6K1 in the mPFC blocked the anhedonia caused by chronic stress, resulting in a state of stress resilience. This antidepressant response was associated with increased neuronal complexity caused by enhanced S6K1 activity. Conversely, expression of dominant-negative S6K1 in the mPFC resulted in prodepressive behavior in the forced swim test and was sufficient to cause anhedonia in the absence of chronic stress exposure. Together, these data demonstrate a critical role for S6K1 activity in depressive behaviors, and suggest that pathways downstream of mTORC1 may underlie the pathophysiology and treatment of MDD.

A comparison of SERS and MEF of rhodamine 6G on a gold substrate.

Rhodamine 6G is spin-cast onto gold surfaces and the reflectance, emission, excitation, and SERS spectra are reported. Electron microscopy shows that the particle sizes of the gold are uniform for all preparations. Reflection spectra demonstrate the spectroscopic signature for Rh6G aggregates for thicker films and that the gold plasmon band shifts due to the refractive index change on the surface. The intensity of the SERS spectra increases with increasing surface coverage but the change is nonlinear between submonolayer and multilayer surface densities. The SERS resonance frequencies are unchanged as a function of Rh6G thickness, indicating that there is no coupling between Rh6G molecules in the ground state. The emission spectra behave unexpectedly as a function of Rh6G coverage. At submonolayer coverage the emission is relatively strong, decreases as the surface density increases to a monolayer, and then increases as the Rh6G thickness increases. Excitation spectra demonstrate that the emitting species at low surface density is monomeric but for thicker layers the moiety responsible for emission is Rh6G excited state aggregates. For the thicker films, the Rh6G acts as its own dielectric layer for metal enhanced fluorescence of the aggregates, which is the first example of a system where the fluorophore acts as its own dielectric for metal enhanced fluorescence. The intensity of the aggregate emission on gold intensity is three times of that found when Rh6G is deposited on glass. The gold induces emission in the Rh6G excited state aggregates that are quenched in the absence of the plasmon field.

Rapid antidepressant actions of scopolamine: Role of medial prefrontal cortex and M1-subtype muscarinic acetylcholine receptors.

Clinical studies demonstrate that scopolamine, a non-selective muscarinic acetylcholine receptor (mAchR) antagonist, produces rapid therapeutic effects in depressed patients, and preclinical studies report that the actions of scopolamine require glutamate receptor activation and the mechanistic target of rapamycin complex 1 (mTORC1). The present study extends these findings to determine the role of the medial prefrontal cortex (mPFC) and specific muscarinic acetylcholine receptor (M-AchR) subtypes in the actions of scopolamine. The administration of scopolamine increases the activity marker Fos in the mPFC, including the infralimbic (IL) and prelimbic (PrL) subregions. Microinfusions of scopolamine into either the IL or the PrL produced significant antidepressant responses in the forced swim test, and neuronal silencing of IL or PrL blocked the antidepressant effects of systemic scopolamine. The results also demonstrate that the systemic administration of a selective M1-AChR antagonist, VU0255035, produced an antidepressant response and stimulated mTORC1 signaling in the PFC, similar to the actions of scopolamine. Finally, we used a chronic unpredictable stress model as a more rigorous test of rapid antidepressant actions and found that a single dose of scopolamine or VU0255035 blocked the anhedonic response caused by CUS, an effect that requires the chronic administration of typical antidepressants. Taken together, these findings indicate that mPFC is a critical mediator of the behavioral actions of scopolamine and identify the M1-AChR as a therapeutic target for the development of novel and selective rapid-acting antidepressants.

BDNF release is required for the behavioral actions of ketamine.

BACKGROUND: Recent studies demonstrate that the rapid antidepressant ketamine increases spine number and function in the medial prefrontal cortex (mPFC), and that these effects are dependent on activation of glutamate α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptors and brain-derived neurotrophic factor (BDNF). In vitro studies also show that activation of AMPA receptors stimulates BNDF release via activation of L-type voltage-dependent calcium channels (VDCC). METHODS: Based on this evidence, we examined the role of BDNF release and the impact of L-type VDCCs on the behavioral actions of ketamine. RESULTS: The results demonstrate that infusion of a neutralizing BDNF antibody into the mPFC blocks the behavioral effects of ketamine in the forced swim test (FST). In addition, we show that pretreatment with nifedipine or verapamil, two structurally-different L-type calcium channel antagonists, blocks the behavioral effects of ketamine in the FST. Finally, we show that ketamine treatment stimulates BDNF release in primary cortical neurons and that this effect is blocked by inhibition of AMPA receptors or L-type VDCCs. CONCLUSIONS: Taken together, these results indicate that the antidepressant effects of ketamine are mediated by activation of L-type VDCCs and the release of BDNF. They further elucidate the cellular mechanisms underlying this novel rapid-acting antidepressant.

Editors' Choice-Perspective-Deciphering the Glycan Kryptos by Solid-State Nanopore Single-Molecule Sensing: A Call for Integrated Advancements Across Glyco- and Nanopore Science.

Glycans, or complex carbohydrates, are information-rich biopolymers critical to many biological processes and with considerable importance in pharmaceutical therapeutics. Our understanding, though, is limited compared to other biomolecules such as DNA and proteins. The greater complexity of glycan structure and the limitations of conventional chemical analysis methods hinder glycan studies. Auspiciously, nanopore single-molecule sensors-commercially available for DNA sequencing-hold great promise as a tool for enabling and advancing glycan analysis. We focus on two key areas to advance nanopore glycan characterization: molecular surface coatings to enhance nanopore performance including by molecular recognition, and high-quality glycan chemical standards for training.

Comprehensive assessment of microplastics in Australian biosolids: Abundance, seasonal variation and potential transport to agroecosystems.

Striving towards a circular economy, the application of treated sewage sludge (biosolids) to land is an opportunity to improve the condition of the soil and add essential nutrients, in turn reducing the need for fertilisers. However, there is an increasing concern about microplastic (MP) contamination of biosolids and their transport to terrestrial ecosystems. In Australia, agriculture is the largest biosolids end-user, however, there is limited understanding of MPs in Australian biosolids. Also, while the method to isolate MPs from biosolid is established, a need to extract and analyse MPs more efficiently is still pressing. In this study, we comprehensively quantified and characterised MPs in 146 biosolids samples collected from thirteen wastewater treatment plants (WWTPs) including different seasons. We have optimised an oxidative-enzymatic purification method to overcome current limitations for MP identification in complex samples and accurately report MPs in biosolids. This method enabled removal of >93 % of dry weight of organic material and greatly facilitated the MPs instrumental analysis. The concentration of MPs (>20 µm) in all biosolids samples ranged from 11 to 150 MPs/g dry weight. Abundance of MPs was affected by seasons with higher abundance of MPs usually found during cold and wet seasons. Despite seasonal variations, polyethylene terephthalate, polyurethane and polymethyl methacrylate were the most abundant polymers. Smaller MPs (20 to 200 µm) comprised >70 % of all detected MPs with a clear negative linear relationship observed between MP size and abundance. Per capita concentration of MPs in biosolids across all studied WWTPs was 0.7 to 21 g MPs per person per year. Therefore, biosolids are an important sink and source of MPs to agroecosystems, emphasising the need to more comprehensively understand the fate, impact and risks associated with MPs on agricultural soils.

Coupling Partial Nitritation, Anammox and n-DAMO in a membrane aerated biofilm reactor for simultaneous dissolved methane and nitrogen removal.

Anaerobic technologies with downstream autotrophic nitrogen removal have been proposed to enhance bioenergy recovery and transform a wastewater treatment plant from an energy consumer to an energy exporter. However, approximately 20-50 % of the produced methane is dissolved in the anaerobically treated effluent and is easily stripped into the atmosphere in the downstream aerobic process, contributing to the release of greenhouse gas emissions. This study aims to develop a solution to beneficially utilize dissolved methane to support high-level nitrogen removal from anaerobically treated mainstream wastewater. A novel technology, integrating Partial Nitritation, Anammox and Methane-dependent nitrite/nitrate reduction (i.e. PNAM) was demonstrated in a membrane-aerated biofilm reactor (MABR). With the feeding of ∼50 mg NH4+-N/L and ∼20 mg/L dissolved methane at a hydraulic retention time of 15 h, around 90 % of nitrogen and ∼100 % of dissolved methane can be removed together in the MABR. Microbial community characterization revealed that ammonia-oxidizing bacteria (AOB), nitrite-oxidizing bacteria (NOB), anammox bacteria, nitrite/nitrate-dependent anaerobic methane oxidation microorganisms (n-DAMO bacteria and archaea) and aerobic methanotrophs co-existed in the established biofilm. Batch tests confirmed the active microbial pathways and showed that AOB, anammox bacteria and n-DAMO microbes were jointly responsible for the nitrogen removal, and dissolved methane was mainly removed by the n-DAMO process, with aerobic methane oxidation making a minor contribution. In addition, the established system was robust against dynamic changes in influent composition. The study provides a promising technology for the simultaneous removal of dissolved methane and nitrogen from domestic wastewater, which can support the transformation of wastewater treatment from an energy- and carbon-intensive process, to one that is energy- and carbon-neutral.

Phosphodiesterase 11A in brain is enriched in ventral hippocampus and deletion causes psychiatric disease-related phenotypes.

Phosphodiesterase 11A (PDE11A) is the most recently identified family of phosphodiesterases (PDEs), the only known enzymes to break down cyclic nucleotides. The tissue expression profile of this dual specificity PDE is controversial, and little is understood of its biological function, particularly in the brain. We seek here to determine if PDE11A is expressed in the brain and to understand its function, using PDE11A(-/-) knockout (KO) mice. We show that PDE11A mRNA and protein are largely restricted to hippocampus CA1, subiculum, and the amygdalohippocampal area, with a two- to threefold enrichment in the ventral vs. dorsal hippocampus, equal distribution between cytosolic and membrane fractions, and increasing levels of protein expression from postnatal day 7 through adulthood. Interestingly, PDE11A KO mice show subtle psychiatric-disease-related deficits, including hyperactivity in an open field, increased sensitivity to the glutamate N-methyl-D-aspartate receptor antagonist MK-801, as well as deficits in social behaviors (social odor recognition memory and social avoidance). In addition, PDE11A KO mice show enlarged lateral ventricles and increased activity in CA1 (as per increased Arc mRNA), phenotypes associated with psychiatric disease. The increased sensitivity to MK-801 exhibited by PDE11A KO mice may be explained by the biochemical dysregulation observed around the glutamate alpha-amino-3-hydroxy-5-methyl-4-isozazolepropionic (AMPA) receptor, including decreased levels of phosphorylated-GluR1 at Ser845 and the prototypical transmembrane AMPA-receptor-associated proteins stargazin (gamma2) and gamma8. Together, our data provide convincing evidence that PDE11A expression is restricted in the brain but plays a significant role in regulating brain function.

Probing nanopore surface chemistry through real-time measurements of nanopore conductance response to pH changes.

We developed a flow cell apparatus and method for streamlined, real-time measurements of nanopore conductance (G) in response to pH changes. By time-resolving the measurements of interfacial kinetics, we were able to probe nanopore surface coating presence and properties more thoroughly than in our previous work. Nanopores have emerged as a prominent tool for single-molecule sensing, characterization, and sequencing of DNA, proteins, and carbohydrates. Nanopore surface chemistry affects analyte passage, signal characteristics, and sensor lifetime through a range of electrostatic, electrokinetic, and chemical phenomena, and optimizing nanopore surface chemistry has become increasingly important. Our work makes nanopore surface chemistry characterizations more accessible as a complement to routine single-pH conductance measurements used to infer nanopore size. We detail the design and operation of the apparatus and discuss the trends in G and capacitance. Characteristic G vs pH curves matching those obtained in previous work could be obtained with the addition of time-resolved interfacial kinetic information. We characterized native and chemically functionalized (carboxylated) silicon nitride (SiNx) nanopores, illustrating how the method can inform of thin film compositions, interfacial kinetics, and nanoscale chemical phenomena.

N-Heterocyclic carbene-based gold etchants.

N-Heterocyclic carbenes (NHCs) are an emerging alternative to thiols for the formation of stable self-assembled monolayers (SAMs) on gold. We examined several different species that have been used to produce NHC-based monolayers on gold, namely 1,3-diisopropyl-5-nitrobenzimidazolium iodide, 1,3-diisopropyl-5-nitrobenzimidazolium hydrogen carbonate, bis(1,3-diisopropyl-5-nitrobenzimidazolium)gold(I) iodide, and 1,3-diisopropyl-5-nitrobenzimidazole-2-ylidene. Contrary to expectation, solutions containing the first two species in tetrahydrofuran and dichloromethane caused visible loss of gold from thin-film-coated glass slides. The use of toluene solutions of all species resulted in no apparent dissolution of gold. We present scanning electron micrographs and elemental imaging analyses by energy dispersive X-ray spectroscopy to examine the effect of solutions of each species on the gold film. This work highlights the risk of unwanted etching during some routes to NHC-based surface functionalization but also the potential for deliberate etching, with the outcome determined by choice of chemically synthesized organic species and solvent.

Deep learning in wastewater treatment: a critical review.

Modeling wastewater processes supports tasks such as process prediction, soft sensing, data analysis and computer assisted design of wastewater systems. Wastewater treatment processes are large, complex processes, with multiple controlling mechanisms, a high degree of disturbance variability and non-linear (generally stable) behavior with multiple internal recycle loops. Semi-mechanistic biochemical models currently dominate research and application, with data-driven deep learning models emerging as an alternative and supplementary approach. But these modeling approaches have grown in separate communities of research and practice, and so there is limited appreciation of the strengths, weaknesses, contrasts and similarities between the methods. This review addresses that gap by providing a detailed guide to deep learning methods and their application to wastewater process modeling. The review is aimed at wastewater modeling experts who are familiar with established mechanistic modeling approach, and are curious about the opportunities and challenges afforded by deep learning methods. We conclude with a discussion and needs analysis on the value of different ways of modeling wastewater processes and open research problems.

One-year stable pilot-scale operation demonstrates high flexibility of mainstream anammox application.

Mainstream nitrogen removal via anammox is widely recognized as a promising wastewater treatment process. However, its application is challenging at large scale due to unstable suppression of nitrite-oxidizing bacteria (NOB). In this study, a pilot-scale mainstream anammox process was implemented in an Integrated Fixed-film Activated Sludge (IFAS) configuration. Stable operation with robust NOB suppression was maintained for over one year. This was achieved through integration of three key control strategies: i) low dissolved oxygen (DO = 0.4 ± 0.2 mg O2/L), ii) regular free nitrous acid (FNA)-based sludge treatment, and iii) residual ammonium concentration control (NH4 + with a setpoint of ∼8 mg N/L). Activity tests and FISH demonstrated that NOB barely survived in sludge flocs and were inhibited in biofilms. Despite receiving organic-deficient wastewater from a pilot-scale High-Rate Activated Sludge (HRAS) system as the feed, the system maintained a stable effluent total nitrogen concentration mostly below 10 mg N/L, which was attributed to the successful retention of anammox bacteria. This study successfully demonstrated large-scale long-term mainstream anammox application and generated new practical knowledge for NOB control and anammox retention.